US20030229216A1 - Constitutively activated human G protein coupled receptors - Google Patents

Constitutively activated human G protein coupled receptors Download PDF

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US20030229216A1
US20030229216A1 US10/417,820 US41782003A US2003229216A1 US 20030229216 A1 US20030229216 A1 US 20030229216A1 US 41782003 A US41782003 A US 41782003A US 2003229216 A1 US2003229216 A1 US 2003229216A1
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Ruoping Chen
Chen Liaw
Kevin Lowitz
Derek Chalmers
Dominic Behan
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Arena Pharmaceuticals Inc
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Arena Pharmaceuticals Inc
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Priority claimed from US09/170,496 external-priority patent/US6555339B1/en
Application filed by Arena Pharmaceuticals Inc filed Critical Arena Pharmaceuticals Inc
Priority to US10/417,820 priority Critical patent/US20030229216A1/en
Assigned to ARENA PHARMACEUTICALS, INC. reassignment ARENA PHARMACEUTICALS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BEHAN, DOMINIC P., CHALMERS, DEREK T., CHEN, RUOPING, LIAW, CHEN W., LOWITZ, KEVIN P.
Priority to US10/723,955 priority patent/US8440391B2/en
Publication of US20030229216A1 publication Critical patent/US20030229216A1/en
Priority to US11/603,356 priority patent/US20070072248A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Provisional No. 60/136,439 filed May 28, 1999; U.S. Provisional No. 60/136,567, filed May 28, 1999; U.S. Provisional No. 60/137,127, filed May 28, 1999; U.S. Provisional No. 60/137,131, filed May 28, 1999; U.S. Provisional No. 60/141,448, filed Jun. 29, 1999 claiming benefit of U.S. Provisional No. 60/136,437, filed May 28, 1999; U.S. Provisional No. 60/156,633, filed Sep. 29, 1999; U.S. Provisional No. 60/156,555, filed Sep. 29, 1999; U.S. Provisional No. 60/156,634, filed Sep. 29, 1999; U.S. Provisional No.
  • the invention disclosed in this patent document relates to transmembrane receptors, and more particularly to human G protein-coupled receptors, and specifically to GPCRs that have been altered to establish or enhance constitutive activity of the receptor.
  • the altered GPCRs are used for the direct identification of candidate compounds as receptor agonists, inverse agonists or partial agonists having potential applicability as therapeutic agents.
  • GPCR G protein-coupled receptor
  • Receptors including GPCRs, for which the endogenous ligand has been identified are referred to as “known” receptors, while receptors for which the endogenous ligand has not been identified are referred to as “orphan” receptors.
  • GPCRs represent an important area for the development of pharmaceutical products: from approximately 20 of the 100 known GPCRs, 60% of all prescription pharmaceuticals have been developed.
  • GPCRs share a common structural motif. All these receptors have seven sequences of between 22 to 24 hydrophobic amino acids that form seven alpha helices, each of which spans the membrane (each span is identified by number, i.e., transmembrane-1 (TM-1), transmebrane-2 (TM-2), etc.).
  • the transmembrane helices are joined by strands of amino acids between transmembrane-2 and transmembrane-3, transmembrane-4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the exterior, or “extracellular” side, of the cell membrane (these are referred to as “extracellular” regions 1, 2 and 3 (EC-1, EC-2 and EC-3), respectively).
  • transmembrane helices are also joined by strands of amino acids between transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the interior, or “intracellular” side, of the cell membrane (these are referred to as “intracellular” regions 1, 2 and 3 (IC-1, IC-2 and IC-3), respectively).
  • the “carboxy” (“C”) terminus of the receptor lies in the intracellular space within the cell, and the “amino” (“N”) terminus of the receptor lies in the extracellular space outside of the cell.
  • GPCRs are “promiscuous” with respect to G proteins, i.e., that a GPCR can interact with more than one G protein. See, Kenakin, T., 43 Life Sciences 1095 (1988). Although other G proteins exist, currently, Gq, Gs, Gi, Gz and Go are G proteins that have been identified. Endogenous ligand-activated GPCR coupling with the G-protein begins a signaling cascade process (referred to as “signal transduction”). Under normal conditions, signal transduction ultimately results in cellular activation or cellular inhibition. It is thought that the IC-3 loop as well as the carboxy terminus of the receptor interact with the G protein.
  • GPCRs exist in the cell membrane in equilibrium between two different conformations: an “inactive” state and an “active” state.
  • a receptor in an inactive state is unable to link to the intracellular signaling transduction pathway to produce a biological response.
  • Changing the receptor conformation to the active state allows linkage to the transduction pathway (via the G-protein) and produces a biological response.
  • a receptor may be stabilized in an active state by an endogenous ligand or a compound such as a drug.
  • Recent discoveries including but not exclusively limited to modifications to the amino acid sequence of the receptor, provide means other than endogenous ligands or drugs to promote and stabilize the receptor in the active state conformation. These means effectively stabilize the receptor in an active state by simulating the effect of an endogenous ligand binding to the receptor. Stabilization by such ligand-independent means is termed “constitutive receptor activation.”
  • FIG. 1 is a representation of 8 ⁇ CRE-Luc reporter plasmid (see, Example 4(c)3.)
  • FIGS. 2A and 2B are graphic representations of the results of ATP and ADP binding to endogenous TDAG8 ( 2 A) and comparisons in serum and serum free media ( 2 B).
  • FIG. 3 is a graphic representation of the comparative signaling results of CMV versus the GPCR Fusion Protein H9(F236K):Gs ⁇ .
  • AGONISTS shall mean materials (e.g., ligands, candidate compounds) that activate the intracellular response when they bind to the receptor, or enhance GTP binding to membranes.
  • AMINO ACID ABBREVIATIONS used herein are set out in Table A: TABLE A ALANINE ALA A ARGININE ARG R ASPARAGINE ASN N ASPARTIC ACID ASP D CYSTEINE CYS C GLUTAMIC ACID GLU E GLUTAMINE GLN Q GLYCINE GLY G HISTIDINE HIS H ISOLEUCINE ILE I LEUCINE LEU L LYSINE LYS K METHIONINE MET M PHENYLALANINE PHE F PROLINE PRO P SERINE SER S THREONINE THR T TRYPTOPHAN TRP W TYROSINE TYR Y VALINE VAL V
  • PARTIAL AGONISTS shall mean materials (e.g., ligands, candidate compounds) that activate the intracellular response when they bind to the receptor to a lesser degree/extent than do agonists, or enhance GTP binding to membranes to a lesser degree/extent than do agonists.
  • ANTAGONIST shall mean materials (e.g., ligands, candidate compounds) that competitively bind to the receptor at the same site as the agonists but which do not activate the intracellular response initiated by the active form of the receptor, and can thereby inhibit the intracellular responses by agonists or partial agonists.
  • ANTAGONISTS do not diminish the baseline intracellular response in the absence of an agonist or partial agonist.
  • CANDIDATE COMPOUND shall mean a molecule (for example, and not limitation, a chemical compound) that is amenable to a screening technique.
  • the phrase “candidate compound” does not include compounds which were publicly known to be compounds selected from the group consisting of inverse agonist, agonist or antagonist to a receptor, as previously determined by an indirect identification process (“indirectly identified compound”); more preferably, not including an indirectly identified compound which has previously been determined to have therapeutic efficacy in at least one mammal; and, most preferably, not including an indirectly identified compound which has previously been determined to have therapeutic utility in humans.
  • COMPOSITION means a material comprising at least one component; a “pharmaceutical composition” is an example of a composition.
  • COMPOUND EFFICACY shall mean a measurement of the ability of a compound to inhibit or stimulate receptor functionality, as opposed to receptor binding affinity. Exemplary means of detecting compound efficacy are disclosed in the Example section of this patent document.
  • CODON shall mean a grouping of three nucleotides (or equivalents to nucleotides) which generally comprise a nucleoside (adenosine (A), guanosine (G), cytidine (C), uridine (U) and thymidine (T)) coupled to a phosphate group and which, when translated, encodes an amino acid.
  • A adenosine
  • G guanosine
  • C cytidine
  • U uridine
  • T thymidine
  • CONSTITUTIVELY ACTIVATED RECEPTOR shall mean a receptor subject to constitutive receptor activation.
  • a constitutively activated receptor can be endogenous or non-endogenous.
  • CONSTITUTIVE RECEPTOR ACTIVATION shall mean stabilization of a receptor in the active state by means other than binding of the receptor with its endogenous ligand or a chemical equivalent thereof.
  • CONTACT or CONTACTING shall mean bringing at least two moieties together, whether in an in vitro system or an in vivo system.
  • DIRECTLY IDENTIFYING or DIRECTLY IDENTIFIED in relationship to the phrase “candidate compound”, shall mean the screening of a candidate compound against a constitutively activated receptor, preferably a constitutively activated orphan receptor, and most preferably against a constitutively activated G protein-coupled cell surface orphan receptor, and assessing the compound efficacy of such compound.
  • This phrase is, under no circumstances, to be interpreted or understood to be encompassed by or to encompass the phrase “indirectly identifying” or “indirectly identified.”
  • ENDOGENOUS shall mean a material that a mammal naturally produces.
  • ENDOGENOUS in reference to, for example and not limitation, the term “receptor,” shall mean that which is naturally produced by a mammal (for example, and not limitation, a human) or a virus.
  • the term NON-ENDOGENOUS in this context shall mean that which is not naturally produced by a mammal (for example, and not limitation, a human) or a virus.
  • a receptor which is not constitutively active in its endogenous form, but when manipulated becomes constitutively active is most preferably referred to herein as a “non-endogenous, constitutively activated receptor.”
  • Both terms can be utilized to describe both “in vivo” and “in vitro” systems.
  • the endogenous or non-endogenous receptor may be in reference to an in vitro screening system.
  • screening of a candidate compound by means of an in vivo system is viable.
  • G PROTEIN COUPLED RECEPTOR FUSION PROTEIN and GPCR FUSION PROTEIN in the context of the invention disclosed herein, each mean a non-endogenous protein comprising an endogenous, constitutively activate GPCR or a non-endogenous, constitutively activated GPCR fused to at least one G protein, most preferably the alpha ( ⁇ ) subunit of such G protein (this being the subunit that binds GTP), with the G protein preferably being of the same type as the G protein that naturally couples with endogenous orphan GPCR.
  • Gs ⁇ is the predominate G protein that couples with the GPCR
  • a GPCR Fusion Protein based upon the specific GPCR would be a non-endogenous protein comprising the GPCR fused to Gs ⁇ ; in some circumstances, as will be set forth below, a non-predominant G protein can be fused to the GPCR.
  • the G protein can be fused directly to the c-terminus of the constitutively active GPCR or there may be spacers between the two.
  • HOST CELL shall mean a cell capable of having a Plasmid and/or Vector incorporated therein.
  • a Plasmid is typically replicated as a autonomous molecule as the Host Cell replicates (generally, the Plasmid is thereafter isolated for introduction into a eukaryotic Host Cell); in the case of a eukaryotic Host Cell, a Plasmid is integrated into the cellular DNA of the Host Cell such that when the eukaryotic Host Cell replicates, the Plasmid replicates.
  • the Host Cell is eukaryotic, more preferably, mammalian, and most preferably selected from the group consisting of 293, 293T and COS-7 cells.
  • INDIRECTLY IDENTIFYING or INDIRECTLY IDENTIFIED means the traditional approach to the drug discovery process involving identification of an endogenous ligand specific for an endogenous receptor, screening of candidate compounds against the receptor for determination of those which interfere and/or compete with the ligand-receptor interaction, and assessing the efficacy of the compound for affecting at least one second messenger pathway associated with the activated receptor.
  • INHIBIT or INHIBITING in relationship to the term “response” shall mean that a response is decreased or prevented in the presence of a compound as opposed to in the absence of the compound.
  • INVERSE AGONISTS shall mean materials (e.g., ligand, candidate compound) which bind to either the endogenous form of the receptor or to the constitutively activated form of the receptor, and which inhibit the baseline intracellular response initiated by the active form of the receptor below the normal base level of activity which is observed in the absence of agonists or partial agonists, or decrease GTP binding to membranes.
  • the baseline intracellular response is inhibited in the presence of the inverse agonist by at least 30%, more preferably by at least 50%, and most preferably by at least 75%, as compared with the baseline response in the absence of the inverse agonist.
  • KNOWN RECEPTOR shall mean an endogenous receptor for which the endogenous ligand specific for that receptor has been identified.
  • LIGAND shall mean an endogenous, naturally occurring molecule specific for an endogenous, naturally occurring receptor.
  • MUTANT or MUTATION in reference to an endogenous receptor's nucleic acid and/or amino acid sequence shall mean a specified change or changes to such endogenous sequences such that a mutated form of an endogenous, non-constitutively activated receptor evidences constitutive activation of the receptor.
  • a subsequent mutated form of a human receptor is considered to be equivalent to a first mutation of the human receptor if (a) the level of constitutive activation of the subsequent mutated form of a human receptor is substantially the same as that evidenced by the first mutation of the receptor; and (b) the percent sequence (amino acid and/or nucleic acid) homology between the subsequent mutated form of the receptor and the first mutation of the receptor is at least about 80%, more preferably at least about 90% and most preferably at least 95%.
  • the percent sequence homology should be at least 98%.
  • NON-ORPHAN RECEPTOR shall mean an endogenous naturally occurring molecule specific for an endogenous naturally occurring ligand wherein the binding of a ligand to a receptor activates an intracellular signaling pathway.
  • ORPHAN RECEPTOR shall mean an endogenous receptor for which the endogenous ligand specific for that receptor has not been identified or is not known.
  • PHARMACEUTICAL COMPOSITION shall mean a composition comprising at least one active ingredient, whereby the composition is amenable to investigation for a specified, efficacious outcome in a mammal (for example, and not limitation, a human).
  • a mammal for example, and not limitation, a human.
  • PLASMID shall mean the combination of a Vector and cDNA.
  • a Plasmid is introduced into a Host Cell for the purposes of replication and/or expression of the cDNA as a protein.
  • STIMULATE or STIMULATING in relationship to the term “response” shall mean that a response is increased in the presence of a compound as opposed to in the absence of the compound.
  • VECTOR in reference to cDNA shall mean a circular DNA capable of incorporating at least one cDNA and capable of incorporation into a Host Cell.
  • any search for therapeutic compounds should start by screening compounds against the ligand-independent active state.
  • Receptor homology is useful in terms of gaining an appreciation of a role of the receptors within the human body. As the patent document progresses, we will disclose techniques for mutating these receptors to establish non-endogenous, constitutively activated versions of these receptors.
  • Screening candidate compounds against a non-endogenous, constitutively activated version of the human GPCRs disclosed herein allows for the direct identification of candidate compounds which act at this cell surface receptor, without requiring use of the receptor's endogenous ligand.
  • By determining areas within the body where the endogenous version of human GPCRs disclosed herein is expressed and/or over-expressed it is possible to determine related disease/disorder states which are associated with the expression and/or over-expression of the receptor; such an approach is disclosed in this patent document.
  • inverse agonists to the non-endogenous, constitutively activated GPCR can be identified by the methodologies of this invention.
  • Such inverse agonists are ideal candidates as lead compounds in drug discovery programs for treating diseases related to this receptor.
  • a search for diseases and disorders associated with the GPCR is relevant. For example, scanning both diseased and normal tissue samples for the presence of the GPCR now becomes more than an academic exercise or one which might be pursued along the path of identifying an endogenous ligand to the specific GPCR. Tissue scans can be conducted across a broad range of healthy and diseased tissues.
  • tissue scans provide a preferred first step in associating a specific receptor with a disease and/or disorder. See, for example, co-pending application (docket number ARE-0050) for exemplary dot-blot and RT-PCR results of several of the GPCRs disclosed herein.
  • the DNA sequence of the human GPCR is used to make a probe for (a) dot-blot analysis against tissue-mRNA, and/or (b) RT-PCR identification of the expression of the receptor in tissue samples.
  • the presence of a receptor in a tissue source, or a diseased tissue, or the presence of the receptor at elevated concentrations in diseased tissue compared to a normal tissue can be preferably utilized to identify a correlation with a treatment regimen, including but not limited to, a disease associated with that disease.
  • Receptors can equally well be localized to regions of organs by this technique. Based on the known functions of the specific tissues to which the receptor is localized, the putative functional role of the receptor can be deduced.
  • G protein receptor When a G protein receptor becomes constitutively active, it binds to a G protein (e.g., Gq, Gs, Gi, Gz, Go) and stimulates the binding of GTP to the G protein. The G protein then acts as a GTPase and slowly hydrolyzes the GTP to GDP, whereby the receptor, under normal conditions, becomes deactivated. However, constitutively activated receptors continue to exchange GDP to GTP.
  • a non-hydrolyzable analog of GTP [ 35 S]GTP ⁇ S, can be used to monitor enhanced binding to membranes which express constitutively activated receptors. It is reported that [ 35 S]GTP ⁇ S can be used to monitor G protein coupling to membranes in the absence and presence of ligand.
  • candidate compounds are identified using the “generic” G protein-coupled receptor assay (i.e., an assay to select compounds that are agonists, partial agonists, or inverse agonists), further screening to confirm that the compounds have interacted at the receptor site is preferred.
  • a compound identified by the “generic” assay may not bind to the receptor, but may instead merely “uncouple” the G protein from the intracellular domain.
  • Gs stimulates the enzyme adenylyl cyclase. Gi (and Gz and Go), on the other hand, inhibit this enzyme. Adenylyl cyclase catalyzes the conversion of ATP to cAMP; thus, constitutively activated GPCRs that couple the Gs protein are associated with increased cellular levels of cAMP. On the other hand, constitutively activated GPCRs that couple Gi (or Gz, Go) protein are associated with decreased cellular levels of cAMP. See, generally, “Indirect Mechanisms of Synaptic Transmission,” Chpt. 8, From Neuron To Brain (3 rd Ed.) Nichols, J. G. et al eds. Sinauer Associates, Inc. (1992).
  • assays that detect cAMP can be utilized to determine if a candidate compound is, e.g., an inverse agonist to the receptor (i.e., such a compound would decrease the levels of cAMP).
  • a candidate compound e.g., an inverse agonist to the receptor
  • a variety of approaches known in the art for measuring cAMP can be utilized; a most preferred approach relies upon the use of anti-cAMP antibodies in an ELISA-based format.
  • Another type of assay that can be utilized is a whole cell second messenger reporter system assay. Promoters on genes drive the expression of the proteins that a particular gene encodes.
  • Cyclic AMP drives gene expression by promoting the binding of a cAMP-responsive DNA binding protein or transcription factor (CREB) that then binds to the promoter at specific sites called cAMP response elements and drives the expression of the gene.
  • Reporter systems can be constructed which have a promoter containing multiple cAMP response elements before the reporter gene, e.g., ⁇ -galactosidase or luciferase.
  • a constitutively activated Gs-linked receptor causes the accumulation of cAMP that then activates the gene and expression of the reporter protein.
  • the reporter protein such as ⁇ -galactosidase or luciferase can then be detected using standard biochemical assays (Chen et al. 1995).
  • Gq and Go are associated with activation of the enzyme phospholipase C, which in turn hydrolyzes the phospholipid PIP 2 , releasing two intracellular messengers: diacycloglycerol (DAG) and inistol 1,4,5-triphoisphate (IP 3 ). Increased accumulation of IP 3 is associated with activation of Gq- and Go-associated receptors.
  • DAG diacycloglycerol
  • IP 3 inistol 1,4,5-triphoisphate
  • Assays that detect IP 3 accumulation can be utilized to determine if a candidate compound is, e.g., an inverse agonist to a Gq- or Go-associated receptor (i.e., such a compound would decrease the levels of IP 3 ).
  • Gq-associated receptors can also been examined using an AP1 reporter assay in that Gq-dependent phospholipase C causes activation of genes containing AP1 elements; thus, activated Gq-associated receptors will evidence an increase in the expression of such genes, whereby inverse agonists thereto will evidence a decrease in such expression, and agonists will evidence an increase in such expression.
  • Commercially available assays for such detection are available.
  • an endogenous, constitutively activate orphan GPCR or a non-endogenous, constitutively activated orphan GPCR for use in screening of candidate compounds for the direct identification of inverse agonists, agonists and partial agonists provide an interesting screening challenge in that, by definition, the receptor is active even in the absence of an endogenous ligand bound thereto.
  • the non-endogenous receptor in the presence of a candidate compound and the non-endogenous receptor in the absence of that compound with an aim of such a differentiation to allow for an understanding as to whether such compound may be an inverse agonist, agonist, partial agonist or have no affect on such a receptor, it is preferred that an approach be utilized that can enhance such differentiation.
  • a preferred approach is the use of a GPCR Fusion Protein.
  • this signal be enhanced such that in the presence of, e.g., an inverse agonist to the receptor, it is more likely that it will be able to more readily differentiate, particularly in the context of screening, between the receptor when it is contacted with the inverse agonist.
  • the GPCR Fusion Protein is intended to enhance the efficacy of G protein coupling with the non-endogenous GPCR.
  • the GPCR Fusion Protein is preferred for screening with a non-endogenous, constitutively activated GPCR because such an approach increases the signal that is most preferably utilized in such screening techniques. This is important in facilitating a significant “signal to noise” ratio; such a significant ratio is import preferred for the screening of candidate compounds as disclosed herein.
  • GPCR Fusion Protein The construction of a construct useful for expression of a GPCR Fusion Protein is within the purview of those having ordinary skill in the art. Commercially available expression vectors and systems offer a variety of approaches that can fit the particular needs of an investigator. The criteria of importance for such a GPCR Fusion Protein construct is that the endogenous GPCR sequence and the G protein sequence both be in-frame (preferably, the sequence for the endogenous GPCR is upstream of the G protein sequence) and that the “stop” codon of the GPCR must be deleted or replaced such that upon expression of the GPCR, the G protein can also be expressed.
  • the GPCR can be linked directly to the G protein, or there can be spacer residues between the two (preferably, no more than about 12, although this number can be readily ascertained by one of ordinary skill in the art).
  • spacer residues preferably, no more than about 12, although this number can be readily ascertained by one of ordinary skill in the art.
  • the G protein that couples to the non-endogenous GPCR will have been identified prior to the creation of the GPCR Fusion Protein construct.
  • a construct comprising the sequence of the G protein i.e., a universal G protein construct
  • a construct comprising the sequence of the G protein be available for insertion of an endogenous GPCR sequence therein; this provides for efficiency in the context of large-scale screening of a variety of different endogenous GPCRs having different sequences.
  • a Gz coupled receptor such as H9
  • a GPCR Fusion Protein can be established that utilizes a Gs fusion protein—we believe that such a fusion construct, upon expression, “drives” or “forces” the non-endogenous GPCR to couple with, e.g., Gs rather than the “natural” Gz protein, such that a cyclase-based assay can be established.
  • Gi, Gz and Go coupled receptors we prefer that that when a GPCR Fusion Protein is used and the assay is based upon detection of adenyl cyclase activity, that the fusion construct be established with Gs (or an equivalent G protein that stimulates the formation of the enzyme adenylyl cyclase).
  • Candidate compounds selected for further development can be formulated into pharmaceutical compositions using techniques well known to those in the art. Suitable pharmaceutically-acceptable carriers are available to those in the art; for example, see Remington's Pharmaceutical Sciences, 16 th Edition, 1980, Mack Publishing Co., (Oslo et al., eds.)
  • the non-endogenous versions the human GPCRs disclosed herein may be for the direct identification of candidate compounds as inverse agonists, agonists or partial agonists (preferably for use as pharmaceutical agents), these versions of human GPCRs can also be utilized in research settings.
  • in vitro and in vivo systems incorporating GPCRs can be utilized to further elucidate and understand the roles these receptors play in the human condition, both normal and diseased, as well as understanding the role of constitutive activation as it applies to understanding the signaling cascade.
  • non-endogenous human GPCRs are useful as a research tool in that, because of their unique features, non-endogenous human GPCRs can be used to understand the role of these receptors in the human body before the endogenous ligand therefor is identified.
  • Other uses of the disclosed receptors will become apparent to those in the art based upon, inter alia, a review of this patent document.
  • sequence cassettes from one sequence to another (e.g. from rat receptor to human receptor or from human receptor A to human receptor B) is generally predicated upon sequence alignment techniques whereby the sequences are aligned in an effort to determine areas of commonality.
  • the mutational approach disclosed herein does not rely upon this approach but is instead based upon an algorithmic approach and a positional distance from a conserved proline residue located within the TM6 region of human GPCRs. Once this approach is secured, those in the art are credited with the ability to make minor modifications thereto to achieve substantially the same results (i.e., constitutive activation) disclosed herein. Such modified approaches are considered within the purview of this disclosure
  • Mouse EST clone 1179426 was used to obtain a human genomic clone containing all but three amino acid G2A coding sequences.
  • the 5′ of this coding sequence was obtained by using 5′RACE, and the template for PCR was Clontech's Human Spleen Marathon-ReadyTM cDNA.
  • the disclosed human G2A was amplified by PCR using the G2A cDNA specific primers for the first and second round PCR as shown in SEQ.ID.NO.: 41 and SEQ.ID.NO.: 42 as follows: 5′-CTGTGTACAGCAGTTCGCAGAGTG-3′ (SEQ.ID.NO.:41; 1 st round PCR) 5′-GAGTGCCAGGCAGAGCAGGTAGAC-3′. (SEQ.ID.NO.:42; second round PCR)
  • PCR was performed using Advantage GC Polymerase Kit (Clontech; manufacturing instructions will be followed), at 94° C. for 30 sec followed by 5 cycles of 94° C. for 5 sec and 72° C. for 4 min; and 30 cycles of 94° for 5 sec and 70° for 4 min.
  • An approximate PCR fragment was purified from agarose gel, digested with Hind III and Xba I and cloned into the expression vector pRC/CMV2 (Invitrogen).
  • the cloned-insert was sequenced using the T7 SequenaseTM kit (USB Amersham; manufacturer instructions followed) and the sequence was compared with the presented sequence. Expression of the human G2A was detected by probing an RNA dot blot (Clontech; manufacturer instructions followed) with the P 32 -labeled fragment.
  • PCR was performed using primers based upon the 5′ sequence flanking the initiation codon found in CHN9 and the 3′ sequence around the termination codon found in the LTB4R 5′ untranslated region.
  • the 5′ primer sequence utilized was as follows: 5′-CCCGAATTCCTGCTTGCTCCCAGCTTGGCCC-3′ (SEQ.ID.NO.: 43; sense) and 5′-TGTGGATCCTGCTGTCAAAGGTCCCATTCCGG-3′. (SEQ.ID.NO.: 44; antisense)
  • PCR was performed using thymus cDNA as a template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 uM of each primer, and 0.2 mM of each 4 nucleotides.
  • the cycle condition was 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 1 min and 10 sec.
  • a 1.1 kb fragment consistent with the predicted size was obtained from PCR. This PCR fragment was subcloned into pCMV (see below) and sequenced (see, SEQ.ID.NO.: 35).
  • RUP4 was cloned by RT-PCR with human brain cDNA (Clontech) as templates: (SEQ.ID.NO.: 45; sense) 5′-TCACAATGCTAGGTGTGGTC-3′ and (SEQ.ID.NO.: 46; antisense) 5′-TGCATAGACAATGGGATTACAG-3′.
  • PCR was performed using TaqPlus PrecisionTM polymerase (Stratagene; manufacturing instructions followed) by the following cycles: 94° C. for 2 min; 94° C. 30 sec; 55° C. for 30 sec, 72° C. for 45 sec, and 72° C. for 10 min. Cycles 2 through 4 were repeated 30 times.
  • PCR products were separated on a 1% agarose gel and a 500 bp PCR fragment was isolated and cloned into the pCRII-TOPOTM vector (Invitrogen) and sequenced using the T7 DNA SequenaseTM kit (Amsham) and the SP6/T7 primers (Stratagene). Sequence analysis revealed that the PCR fragment was indeed an alternatively spliced form of A1307658 having a continuous open reading frame with similarity to other GPCRs.
  • [0092] were used for 3′- and 5′-RACE PCR with a human brain Marathon-ReadyTM cDNA (Clontech, Cat# 7400-1) as template, according to manufacture's instructions.
  • DNA fragments generated by the RACE PCR were cloned into the pCRII-TOPOTM vector (Invitrogen) and sequenced using the SP6/T7 primers (Stratagene) and some internal primers.
  • the 3′ RACE product contained a poly(A) tail and a completed open reading frame ending at a TAA stop codon.
  • the 5′ RACE product contained an incomplete 5′ end; i.e., the ATG initiation codon was not present.
  • oligo 3 and the following primer: 5′-GCAATGCAGGTCATAGTGAGC-3′ (SEQ.ID.NO.: 52; oligo 5) were used for the second round of 5′ race PCR and the PCR products were analyzed as above.
  • a third round of 5′ race PCR was carried out utilizing antisense primers: (SEQ.ID.NO.: 53; oligo 6) 5′-TGGAGCATGGTGACGGGAATGCAGAAG-3′ and (SEQ.ID.NO.: 54; oligo 7) 5′-GTGATGAGCAGGTCACTGAGCGCCAAG-3′.
  • RUP5 The full length RUP5 was cloned by RT-PCR using a sense primer upstream from ATG, the initiation codon (SEQ.ID.NO.: 57), and an antisense primer containing TCA as the stop codon (SEQ.ID.NO.: 58), which had the following sequences: 5′-ACTCCGTGTCCAGCAGGACTCTG-3′ (SEQ.ID.NO.: 57) 5′-TGCGTGTTCCTGGACCCTCACGTG-3′ (SEQ.ID.NO.: 58)
  • AdvantageTM cDNA polymerase (Clontech) was used for the amplification in a 50 ul reaction by the following cycle with step 2 through step 4 repeated 30 times: 94° C. for 30 sec; 94° for 15 sec; 69° for 40 sec; 72° C. for 3 min; and 72° C. fro 6 min.
  • a 1.4 kb PCR fragment was isolated and cloned with the pCRII-TOPOTM vector (Invitrogen) and completely sequenced using the T7 DNA SequenaseTM kit (Amsham). See, SEQ.ID.NO.: 9.
  • RUP6 The full length RUP6 was cloned by RT-PCR using primers: (SEQ.ID.NO.: 59) 5′-CAGGCCTTGGATTTTAATGTCAGGGATGG-3′ and (SEQ.ID.NO.: 60) 5′-GGAGAGTCAGCTCTGAAAGAATTCAGG-3′;
  • RUP7 was cloned by RT-PCR using primers: (SEQ.ID.NO.: 61; sense) 5′-TGATGTGATGCCAGATACTAATAGCAC-3′ and (SEQ.ID.NO.: 62; antisense) 5′-CCTGATTCATTTAGGTGAGATTGAGAC-3′
  • AdvantageTM cDNA polymerase (Clontech) was used for the amplification in a 50 ul reaction by the following cycle with step 2 to step 4 repeated 30 times: 94° C. for 2 minutes; 94° C. for 15 seconds; 60° C. for 20 seconds; 72° C. for 2 minutes; 72° C. for 10 minutes.
  • a 1.25 Kb PCR fragment was isolated and cloned into the pCRII-TOPOTM vector (Invitrogen) and completely sequenced using the ABI Big Dye TerminatorTM kit (P.E. Biosystem). See, SEQ.ID.NO.: 13.
  • AT1 human angiotensin II type 1 receptor
  • genomic DNA as template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 ⁇ M of each primer, and 0.2 mM of each 4 nucleotides.
  • the cycle condition was 30 cycles of 94° C. for 1 min, 55° C. for 1 min and 72° C. for 1.5 min.
  • the 5′ PCR primer contains a HindIII site with the sequence:
  • the 3′ primer contains a BamHI site with the following sequence:
  • PCR was performed by combining two PCR fragments, using human genomic cDNA as template and rTth poymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 uM of each primer, and 0.2 mM of each 4 nucleotides.
  • the cycle condition for each PCR reaction was 30 cycles of 94° C. for 1 min, 62° C. for 1 min and 72° C. for 2 min.
  • the first fragment was amplified with the 5′ PCR primer that contained an end site with the following sequence:
  • the second PCR fragment was amplified with a 5′ primer having the following sequence:
  • the two fragments were used as templates to amplify GPR38, using SEQ.ID.NO.: 67 and SEQ.ID.NO.: 70 as primers (using the above-noted cycle conditions).
  • the resulting 1.44 kb PCR fragment was digested with BamHI and cloned into Blunt-BamHI site of pCMV expression vector.
  • PCR was performed using human genomic cDNA as template and rTth poymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 uM of each primer, and 0.2 mM of each 4 nucleotides.
  • the cycle condition for each PCR reaction was 30 cycles of 94° C. for 1 min, 54° C. for 1 min and 72° C. for 1.5 min.
  • the 5′ PCR contained an EcoRI site with the sequence:
  • the 1.0 kb PCR fragment was digest with EcoRI and BamHI and cloned into EcoRI-BamHI site of pCMV expression vector. Nucleic acid (SEQ.ID.NO.: 73) and amino acid (SEQ.ID.NO.: 74) sequences for human MC4 were thereafter determined.
  • PCR was performed using human stomach cDNA as template and rTth poymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 uM of each primer, and 0.2 mM of each 4 nucleotides.
  • the cycle condition for each PCR reaction was 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 1 min and 30 sec.
  • the 5′ PCR contained a HindIII site with the sequence:
  • PCR was performed using genomic DNA as template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 ⁇ M of each primer, and 0.2 mM of each 4 nucleotides.
  • the cycle condition was 30 cycles of 94° C. for 1 min, 56° C. for 1 min and 72° C. for 1 min and 20 sec.
  • the 5′PCR primer contained a HindIII site with the following sequence:
  • the 3′ primer contained a BamHI site with the following sequence:
  • the resulting 1.1 kb PCR fragment was digested with HindIII and BamHI and cloned into HindIII-BamHI site of pCMV expression vector.
  • Three resulting clones sequenced contained three potential polymorphisms involving changes of amino acid 43 from Pro to Ala, amino acid 97 from Lys to Asn and amino acid 130 from Ile to Phe.
  • Nucleic acid (SEQ.ID.NO.: 81) and amino acid (SEQ.ID.NO.: 82) sequences for human TDAG8 were thereafter determined.
  • PCR was performed using pituitary cDNA as template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 ⁇ M of each primer, and 0.2 mM of each 4 nucleotides.
  • the cycle condition was 30 cycles of 94° C. for 1 min, 62° C. for 1 min and 72° C. for 2 min.
  • the 5′ PCR primer contained a HindIII site with the following sequence:
  • the 3′ primer contained a BamHI site with the following sequence:
  • H9 contained three potential polymorphisms involving changes of amino acid P320S, S493N and amino acid G448A. Nucleic acid (SEQ.ID.NO.: 139) and amino acid (SEQ.ID.NO.: 140) sequences for human H9 were thereafter determined and verified.
  • Preparation of non-endogenous human GPCRs may be accomplished on human GPCRs using Transformer Site-DirectedTM Mutagenesis Kit (Clontech) according to the manufacturer instructions.
  • Two mutagenesis primers are utilized, most preferably a lysine mutagenesis oligonucleotide that creates the lysine mutation, and a selection marker oligonucleotide.
  • codon mutation to be incorporated into the human GPCR is also noted, in standard form (Table E): TABLE E Receptor Identifier Codon Mutation hARE-3 F313K hARE-4 V233K hARE-5 A240K hGPCR14 L257K hGPCR27 C283K hARE-1 E232K hARE-2 G285K hPPR1 L239K hG2A K232A hRUP3 L224K hRUP5 A236K hRUP6 N267K hRUP7 A302K hCHN4 V236K hMC4 A244K hCHN3 S284K hCHN6 L352K hCHN8 N235K hCHN9 G223K hCHN10 L231K hH9 F236K
  • Preparation of a non-endogenous, constitutively activated human AT1 receptor was accomplished by creating an F239K mutation (see, SEQ.ID.NO.: 89 for nucleic acid sequence, and SEQ.ID.NO.: 90 for amino acid sequence). Mutagenesis was performed using Transformer Site-Directed MutagenesisTM Kit (Clontech) according to the to manufacturer's instructions.
  • the two mutagenesis primers were used, a lysine mutagenesis oligonucleotide (SEQ.ID.NO.: 91) and a selection marker oligonucleotide (SEQ.ID.NO.: 92), which had the following sequences: (SEQ.ID.NO.: 91) 5′-CCAAGAAATGATGATATTAAAAAGATAATTATGGC-3′ (SEQ.ID.NO.: 92) 5′-CTCCTTCGGTCCTCCTATCGTTGTCAGAAGT-3′, respectively.
  • Preparation of a non-endogenous human AT1 receptor was also accomplished by creating an N111A mutation (see, SEQ.ID.NO.: 93 for nucleic acid sequence, and SEQ.ID.NO.: 94 for amino acid sequence).
  • Two PCR reactions were performed using pfu polymerase (Stratagene) with the buffer system provided by the manufacturer, supplemented with 10% DMSO, 0.25 ⁇ M of each primer, and 0.5 mM of each 4 nucleotides.
  • the 5′ PCR sense primer used had the following sequence:
  • the resulting 400 bp PCR fragment was digested with HindIII site and subcloned into HindIII-SmaI site of pCMV vector (5′ construct).
  • the 3′ PCR sense primer used had the following sequence:
  • the resulting 880 bp PCR fragment was digested with BamHI and inserted into Pst (blunted by T4 polymerase) and BamHI site of 5′ construct to generated the full length N111A construct.
  • the cycle condition was 25 cycles of 94° C. for 1 min, 60° C. for 1 min and 72° C. for 1 min (5′ PCR) or 1.5 min (3′ PCR).
  • Preparation of a non-endogenous, constitutively activated human AT1 was accomplished by creating an AT2K255IC3 “domain swap” mutation (see, SEQ.ID.NO.: 99 for nucleic acid sequence, and SEQ.ID.NO.: 100 for amino acid sequence). Restriction sites flanking IC3 of AT1 were generated to facilitate replacement of the IC3 with corresponding IC3 from angiotensin 11 type 2 receptor (AT2). This was accomplished by performing two PCR reactions. A 5′ PCR fragment (Fragment A) encoded from the 5′ untranslated region to the beginning of IC3 was generated by utilizing SEQ.ID.NO.: 63 as sense primer and the following sequence:
  • a 3′ PCR fragment (Fragment B) encoding from the end of IC3 to the 3′ untranslated region was generated by using the following sequence:
  • sense primer and SEQ.ID.NO. 64 as antisense primer.
  • the PCR condition was 30 cycles of 94° C. for 1 min, 55° C. for 1 min and 72 ° C for 1.5 min using endogenous AT1 cDNA clone as template and pfu polymerase (Stratagene), with the buffer systems provided by the manufacturer, supplemented with 10% DMSO, 0.25 ⁇ M of each primer, and 0.5 mM of each 4 nucleotides.
  • Fragment A (720 bp) was digested with HindIII and EcoRI and subcloned.
  • Fragment B was digested with BamHI and subcloned into pCMV vector with an EcoRI site 5′ to the cloned PCR fragment.
  • Fragment C was inserted in front of Fragment B through EcoRI and AflIII site. The resulting clone was then ligated with the Fragment A through the EcoRI site to generate AT1 with AT2K255IC3.
  • Preparation of a non-endogenous human AT1 receptor was also accomplished by creating an A243+ mutation (see, SEQ.ID.NO.: 105 for nucleic acid sequence, and SEQ.ID.NO.: 106 for amino acid sequence).
  • An A243+ mutation was constructed using the following PCR based strategy: Two PCR reactions was performed using pfu polymerase (Stratagene) with the buffer system provided by the manufacturer supplemented with 10% DMSO, 0.25 ⁇ M of each primer, and 0.5 mM of each 4 nucleotides.
  • the 5′ PCR sense primer utilized had the following sequence:
  • the 3′ PCR sense primer utilized had the following sequence:
  • the cycle condition was 25 cycles of 94° C for 1 min, 54° C. for 1 min and 72° C. for 1.5 min. An aliquot of the 5′ and 3′ PCR were then used as co-template to perform secondary PCR using the 5′ PCR sense primer and 3′ PCR antisense primer.
  • the PCR condition was the same as primary PCR except the extention time was 2.5 min.
  • the resulting PCR fragment was digested with HindIII and BamHI and subcloned into pCMV vector. (See, SEQ.ID.NO.: 105)
  • the first PCR fragment (1 kb) was amplified by using SEQ.ID.NO.: 75 and an antisense primer comprising a V322K mutation:
  • the second PCR fragment (0.44 kb) was amplified by using a sense primer comprising the V322K mutation:
  • the two resulting PCR fragments were then used as template for amplifying CCKB comprising V332K, using SEQ.ID.NO.: 75 and SEQ.ID.NO.: 76 and the above-noted system and conditions.
  • the resulting 1.44 kb PCR fragment containing the V332K mutation was digested with HindIII and EcoRI and cloned into HindIII-EcoRI site of pCMV expression vector. (See, SEQ.ID.NO.: 111).
  • Preparation of non-endogenous human GPCRs can also be accomplished by using QuikChangeTM Site-DirectedTM Mutagenesis Kit (Stratagene, according to manufacturer's instructions). Endogenous GPCR is preferably used as a template and two mutagenesis primers utilized, as well as, most preferably, a lysine mutagenesis oligonucleotide and a selection marker oligonucleotide (included in kit).
  • mammalian cells Although a variety of cells are available to the art for the expression of proteins, it is most preferred that mammalian cells be utilized. The primary reason for this is predicated upon practicalities, i.e., utilization of, e.g., yeast cells for the expression of a GPCR, while possible, introduces into the protocol a non-mammalian cell which may not (indeed, in the case of yeast, does not) include the receptor-coupling, genetic-mechanism and secretary pathways that have evolved for mammalian systems—thus, results obtained in non-mammalian cells, while of potential use, are not as preferred as that obtained from mammalian cells.
  • COS-7, 293 and 293T cells are particularly preferred, although the specific mammalian cell utilized can be predicated upon the particular needs of the artisan.
  • tube A was prepared by mixing 20 ⁇ g DNA (e.g., pCMV vector; pCMV vector with receptor cDNA, etc.) in 1.2 ml serum free DMEM (Irvine Scientific, Irvine, Calif.); tube B was prepared by mixing 120 ⁇ l lipofectamine (Gibco BRL) in 1.2 ml serum free DMEM. Tubes A and B were admixed by inversions (several times), followed by incubation at room temperature for 30-45 min. The admixture is referred to as the “transfection mixture”.
  • Plated 293T cells were washed with 1 ⁇ PBS, followed by addition of 10 ml serum free DMEM. 2.4 ml of the transfection mixture were added to the cells, followed by incubation for 4 hrs at 37° C./5% CO 2 . The transfection mixture was removed by aspiration, followed by the addition of 25 ml of DMEM/10% Fetal Bovine Serum. Cells were incubated at 37° C./5% CO 2 . After 72 hr incubation, cells were harvested and utilized for analysis.
  • a G protein-coupled receptor When a G protein-coupled receptor is in its active state, either as a result of ligand binding or constitutive activation, the receptor couples to a G protein and stimulates the release of GDP and subsequent binding of GTP to the G protein.
  • the alpha subunit of the G protein-receptor complex acts as a GTPase and slowly hydrolyzes the GTP to GDP, at which point the receptor normally is deactivated. Constitutively activated receptors continue to exchange GDP for GTP.
  • the non-hydrolyzable GTP analog, [ 35 S]GTP ⁇ S can be utilized to demonstrate enhanced binding of [ 35 S]GTP ⁇ S to membranes expressing constitutively activated receptors.
  • the assay utilizes the ability of G protein coupled receptors to stimulate [ 35 S]GTP ⁇ S binding to membranes expressing the relevant receptors.
  • the assay can, therefore, be used in the direct identification method to screen candidate compounds to known, orphan and constitutively activated G protein-coupled receptors.
  • the assay is generic and has application to drug discovery at all G protein-coupled receptors.
  • the [ 35 S]GTP ⁇ S assay can be incubated in 20 mM HEPES and between 1 and about 20 mM MgCl 2 (this amount can be adjusted for optimization of results, although 20 mM is preferred) pH 7.4, binding buffer with between about 0.3 and about 1.2 nM [ 35 S]GTP ⁇ S (this amount can be adjusted for optimization of results, although 1.2 is preferred) and 12.5 to 75 ⁇ g membrane protein (e.g, COS-7 cells expressing the receptor; this amount can be adjusted for optimization, although 75 ⁇ g is preferred) and 1 ⁇ M GDP (this amount can be changed for optimization) for 1 hour.
  • membrane protein e.g, COS-7 cells expressing the receptor; this amount can be adjusted for optimization, although 75 ⁇ g is preferred
  • 1 ⁇ M GDP this amount can be changed for optimization
  • Wheatgerm agglutinin beads (25 ⁇ l; Amersham) should then be added and the mixture incubated for another 30 minutes at room temperature. The tubes are then centrifuged at 1500 ⁇ g for 5 minutes at room temperature and then counted in a scintillation counter.
  • Flash platesTM and WallacTM scintistrips may be utilized to format a high throughput [ 35 S]GTP ⁇ S binding assay.
  • the assay can be utilized for known GPCRs to simultaneously monitor tritiated ligand binding to the receptor at the same time as monitoring the efficacy via [ 35 S]GTP ⁇ S binding. This is possible because the Wallac beta counter can switch energy windows to look at both tritium and 35 S-labeled probes.
  • This assay may also be used to detect other types of membrane activation events resulting in receptor activation.
  • the assay may be used to monitor 32 P phosphorylation of a variety of receptors (both G protein coupled and tyrosine kinase receptors).
  • receptors both G protein coupled and tyrosine kinase receptors.
  • the assay also has utility for measuring ligand binding to receptors using radioactively labeled ligands.
  • the scintistrip label comes into proximity with the radiolabeled ligand resulting in activation and detection.
  • a Flash PlateTM Adenylyl Cyclase kit (New England Nuclear; Cat. No. SMP004A) designed for cell-based assays can be modified for use with crude plasma membranes.
  • the Flash Plate wells contain a scintillant coating which also contains a specific antibody recognizing cAMP.
  • the cAMP generated in the wells was quantitated by a direct competition for binding of radioactive cAMP tracer to the cAMP antibody. The following serves as a brief protocol for the measurement of changes in cAMP levels in membranes that express the receptors.
  • Transfected cells are harvested approximately three days after transfection.
  • Membranes were prepared by homogenization of suspended cells in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCl 2 . Homogenization is performed on ice using a Brinkman PolytronTM for approximately 10 seconds. The resulting homogenate is centrifuged at 49,000 ⁇ g for 15 minutes at 4° C. The resulting pellet is then resuspended in buffer containing 20 mM HEPES, pH 7.4 and 0.1 mM EDTA, homogenized for 10 seconds, followed by centrifugation at 49,000 ⁇ g for 15 minutes at 4° C. The resulting pellet can be stored at ⁇ 80° C. until utilized.
  • the membrane pellet On the day of measurement, the membrane pellet is slowly thawed at room temperature, resuspended in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCL 2 (these amounts can be optimized, although the values listed herein are preferred), to yield a final protein concentration of 0.60 mg/ml (the resuspended membranes were placed on ice until use).
  • cAMP standards and Detection Buffer comprising 2 ⁇ Ci of tracer [ 125 I cAMP (100 ⁇ l] to 11 ml Detection Buffer) are prepared and maintained in accordance with the manufacturer's instructions.
  • Assay Buffer is prepared fresh for screening and contained 20 mM HEPES, pH 7.4, 10 mM MgCl 2 , 20 mM (Sigma), 0.1 units/ml creatine phosphokinase (Sigma), 50 ⁇ M GTP (Sigma), and 0.2 mM ATP (Sigma); Assay Buffer can be stored on ice until utilized.
  • the assay is initiated by addition of 50 ul of assay buffer followed by addition of 50 ul of membrane suspension to the NEN Flash Plate.
  • the resultant assay mixture is incubated for 60 minutes at room temperature followed by addition of 100 ul of detection buffer. Plates are then incubated an additional 2-4 hours followed by counting in a Wallac MicroBetaTM scintillation counter. Values of cAMP/well are extrapolated from a standard cAMP curve that is contained within each assay plate.
  • a method to detect Gs stimulation depends on the known property of the transcription factor CREB, which is activated in a cAMP-dependent manner.
  • a PathDetectTM CREB trans-Reporting System (Stratagene, Catalogue #219010) can utilized to assay for Gs coupled activity in 293 or 293T cells. Cells are transfected with the plasmids components of this above system and the indicated expression plasmid encoding endogenous or mutant receptor using a Mammalian Transfection Kit (Stratagene, Catalogue #200285) according to the manufacturer's instructions.
  • pFR-Luc luciferase reporter plasmid containing Gal4 recognition sequences
  • 40 ng pFA2-CREB Gal4-CREB fusion protein containing the Gal4 DNA-binding domain
  • 80 ng pCMV-receptor expression plasmid comprising the receptor
  • 20 ng CMV-SEAP secreted alkaline phosphatase expression plasmid; alkaline phosphatase activity is measured in the media of transfected cells to control for variations in transfection efficiency between samples
  • Half of the precipitate is equally distributed over 3 wells in a 96-well plate, kept on the cells overnight, and replaced with fresh medium the following morning. Forty-eight (48) hr after the start of the transfection, cells are treated and assayed for, e.g., luciferase activity
  • a method to detect Gq stimulation depends on the known property of Gq-dependent phospholipase C to cause the activation of genes containing AP1 elements in their promoter.
  • a PathdetectTM AP-1 cis-Reporting System (Stratagene, Catalogue #219073) can be utilized following the protocol set forth above with respect to the CREB reporter assay, except that the components of the calcium phosphate precipitate were 410 ng pAP1-Luc, 80 ng pCMV-receptor expression plasmid, and 20 ng CMV-SEAP.
  • 293 and 293T cells are plated-out on 96 well plates at a density of 2 ⁇ 10 4 cells per well and were transfected using Lipofectamine Reagent (BRL) the following day according to manufacturer instructions.
  • a DNA/lipid mixture is prepared for each 6-well transfection as follows: 260 ng of plasmid DNA in 100 ⁇ l of DMEM were gently mixed with 2 ⁇ l of lipid in 100 ⁇ l of DMEM (the 260 ng of plasmid DNA consisted of 200 ng of a 8 ⁇ CRE-Luc reporter plasmid (see below and FIG.
  • the 8 ⁇ CRE-Luc reporter plasmid was prepared as follows: vector SRIF- ⁇ -gal was obtained by cloning the rat somatostatin promoter ( ⁇ 71/+51) at BglV-HindIII site in the p ⁇ gal-Basic Vector (Clontech).
  • cAMP response element Eight (8) copies of cAMP response element were obtained by PCR from an adenovirus template AdpCF126CCRE8 (see, 7 Human Gene Therapy 1883 (1996)) and cloned into the SRIF- ⁇ -gal vector at the Kpn-BglV site, resulting in the 8 ⁇ CRE- ⁇ -gal reporter vector.
  • the 8 ⁇ CRE-Luc reporter plasmid was generated by replacing the beta-galactosidase gene in the 8 ⁇ CRE- ⁇ -gal reporter vector with the luciferase gene obtained from the pGL3-basic vector (Promega) at the HindIII-BamHI site. Following 30 min.
  • the DNA/lipid mixture was diluted with 400 ⁇ l of DMEM and 100 ⁇ l of the diluted mixture was added to each well. 100 ⁇ l of DMEM with 10% FCS were added to each well after a 4 hr incubation in a cell culture incubator. The following day the transfected cells were changed with 200 ⁇ l/well of DMEM with 10% FCS. Eight (8) hours later, the wells were changed to 100 ⁇ l/well of DMEM without phenol red, after one wash with PBS. Luciferase activity were measured the next day using the LucLiteTM reporter gene assay kit (Packard) following manufacturer instructions and read on a 1450 MicroBetaTM scintillation and luminescence counter (Wallac).
  • LucLiteTM reporter gene assay kit Packard
  • Gq-dependent phospholipase C One method to detect Gq stimulation depends on the known property of Gq-dependent phospholipase C to cause the activation of genes containing serum response factors in their promoter.
  • a PathdetectTM SRF-Luc-Reporting System (Stratagene) can be utilized to assay for Gq coupled activity in, e.g., COS7 cells. Cells are transfected with the plasmid components of the system and the indicated expression plasmid encoding endogenous or non-endogenous GPCR using a Mammalian TransfectionTM Kit (Stratagene, Catalogue #200285) according to the manufacturer's instructions.
  • 410 ng SRF-Luc, 80 ng pCMV-receptor expression plasmid and 20 ng CMV-SEAP secreted alkaline phosphatase expression plasmid; alkaline phosphatase activity is measured in the media of transfected cells to control for variations in transfection efficiency between samples
  • CMV-SEAP secreted alkaline phosphatase expression plasmid; alkaline phosphatase activity is measured in the media of transfected cells to control for variations in transfection efficiency between samples
  • Cells are then lysed and assayed for luciferase activity using a LucliteTM Kit (Packard, Cat. #6016911) and “Trilux 1450 Microbeta” liquid scintillation and luminescence counter (Wallac) as per the manufacturer's instructions.
  • the data can be analyzed using GraphPad PrismTM 2.0a (GraphPad Software Inc.).
  • cells comprising the receptors can be plated onto 24 well plates, usually 1 ⁇ 10 5 cells/well (although his umber can be optimized.
  • cells can be transfected by firstly mixing 0.25 ug DNA in 50 ul serum free DMEM/well and 2 ul lipofectamine in 50 ⁇ l serumfree DMEM/well. The solutions are gently mixed and incubated for 15-30 min at room temperature. Cells are washed with 0.5 ml PBS and 400 ⁇ l of serum free media is mixed with the transfection media and added to the cells.
  • the cells are then incubated for 3-4 hrs at 37° C./5% CO 2 and then the transfection media is removed and replaced with 1 ml/well of regular growth media.
  • the cells are labeled with 3 H-myo-inositol. Briefly, the media is removed and the cells are washed with 0.5 ml PBS. Then 0.5 ml inositol-free/serum free media (GIBCO BRL) is added/well with 0.25 ⁇ Ci of 3 H-myo-inositol/well and the cells are incubated for 16-18 hrs o/n at 37° C./5% CO 2 .
  • GEBCO BRL inositol-free/serum free media
  • the cells are washed with 0.5 ml PBS and 0.45 ml of assay medium is added containing inositol-free/serum free media 10 ⁇ M pargyline 10 mM lithium chloride or 0.4 ml of assay medium and 50 ul of 10 ⁇ ketanserin (ket) to final concentration of 10 ⁇ M.
  • the cells are then incubated for 30 min at 37° C.
  • the cells are then washed with 0.5 ml PBSand 200 ul of fresh/icecold stop solution (1M KOH; 18 mM Na-borate; 3.8 mM EDTA) is added/well.
  • the solution is kept on ice for 5-10 min or until cells were lysed and then neutralized by 200 ⁇ l of fresh/ice cold neutralization sol. (7.5% HCL).
  • the lysate is then transferred into 1.5 ml eppendorf tubes and 1 ml of chloroform/methanol (1:2) is added/tube.
  • the solution is vortexed for 15 sec and the upper phase is applied to a Biorad AG1-X8TM anion exchange resin (100-200 mesh). Firstly, the resin is washed with water at 1:1.25 W/V and 0.9 ml of upper phase is loaded onto the column.
  • the column is washed with 10 mls of 5 mM myo-inositol and 10 ml of 5 mM Na-borate/60 mM Na-formate.
  • the inositol tris phosphates are eluted into scintillation vials containing 10 ml of scintillation cocktail with 2 ml of 0.1 M formic acid/1 M ammonium formate.
  • the columns are regenerated by washing with 10 ml of 0.1 M formic acid/3M ammonium formate and rinsed twice with dd H 2 O and stored at 4° C. in water.
  • the media with serum contained the following: 10% Fetal Bovine Serum (Hyclone #SH30071.03), 1% of 100 mM Sodium Pyruvate (Irvine Scientific #9334),1% of20 mM L-Glutamine (Irvine Scientific #9317), and 1% of Penicillin-Streptomycin solution (Irvine Scientific #9366).
  • a 96-well Adenylyl Cyclase Activation FlashplateTM was used (NEN: #SMP004A).
  • 50 ul of the standards for the assay were added to the plate, in duplicate, ranging from concentrations of 50 pmol to zero pmol cAMP per well.
  • the standard cAMP (NEN: #SMP004A) was reconstituted in water, and serial dilutions were made using 1 ⁇ PBS (Irvine Scientific: #9240).
  • 50 ul of the stimulation buffer (NEN: #SMP004A) was added to all wells.
  • the media was aspirated and the cells washed once with 1 ⁇ PBS. Then 5 ml of 1 ⁇ PBS was added to the cells along with 3 ml of cell dissociation buffer (Sigma: #C-1544). The detached cells were transferred to a centrifuge tube and centrifuged at room temperature for five minutes. The supernatant was removed and the cell pellet was resuspended in an appropriate amount of 1 ⁇ PBS to obtain a final concentration of 2 ⁇ 10 6 cells per milliliter. To the wells containing the compound, 50 ul of the cells in 1 ⁇ PBS (1 ⁇ 10 5 cells/well) were added. The plate was incubated on a shaker for 15 minutes at room temperature. The detection buffer containing the tracer cAMP was prepared.
  • FIG. 2A ATP and ADP bind to endogenous TDAG8 resulting in an increase of cAMP of about 59% and about 55% respectively.
  • FIG. 2B evidences ATP and ADP binding to endogenous TDAG8 where endogenous TDAG8 was transfected and grown in serum and serum-free medium. ATP binding to endogenous TDAG8 grown in serum media evidences an increase in cAMP of about 65%, compared to the endogenous TDAG8 with no compounds; in serum-free media there was an increase of about 68%. ADP binding to endogenous TDAG8 in serum evidences about a 61% increase, while in serum-free ADP binding evidences an increase of about 62% increase. ATP and ADP bind to endogenous TDAG8 with an EC50 value of 139.8 uM and 120.5 uM, respectively (data not shown).
  • the modified pcDNA3.1( ⁇ ) containing the rat Gs ⁇ gene at HindIII sequence was then verified; this vector was now available as a “universal” Gs ⁇ protein vector.
  • the pcDNA3.1( ⁇ ) vector contains a variety of well-known restriction sites upstream of the HindIII site, thus beneficially providing the ability to insert, upstream of the Gs protein, the coding sequence of an endogenous, constitutively active GPCR.
  • This same approach can be utilized to create other “universal” G protein vectors, and, of course, other commercially available or proprietary vectors known to the artisan can be utilized—the important criteria is that the sequence for the GPCR be upstream and in-frame with that of the G protein.
  • TDAG8 couples via Gs
  • H9 couples via Gz.
  • fusion to Gs ⁇ was accomplished.
  • a TDAG8(1225K)-Gs ⁇ Fusion Protein construct was made as follows: primers were designed as follows: 5′-gatcTCTAGAATGAACAGCACATGTATTGAAG-3′ (SEQ.ID.NO.: 125; sense) 5′-ctagGGTACCCGCTCAAGGACCTCTAATTCCATAG-3′ (SEQ.ID.NO.: 126; antisense)
  • Nucleotides in lower caps are included as spacers in the restriction sites between the G protein and TDAG8.
  • PCR was then utilized to secure the respective receptor sequences for fusion within the Gs ⁇ universal vector disclosed above, using the following protocol for each: 100 ng cDNA for TDAG8 was added to separate tubes containing 2 ul of each primer (sense and anti-sense), 3 uL of 10 mM dNTPs, 10 uL of 10 ⁇ TaqPlusTM Precision buffer, 1 uL of TaqPlusTM Precision polymerase (Stratagene: #600211), and 80 uL of water. Reaction temperatures and cycle times for TDAG8 were as follows: the initial denaturing step was done it 94° C. for five minutes, and a cycle of 94° C. for 30 seconds; 55° C. for 30 seconds; 72° C.
  • PCR product for was run on a 1% agarose gel and then purified (data not shown).
  • the purified product was digested with XbaI and KpnI (New England Biolabs) and the desired inserts purified and ligated into the Gs universal vector at the respective restriction site.
  • the positive clones was isolated following transformation and determined by restriction enzyme digest; expression using 293 cells was accomplished following the protocol set forth infra. Each positive clone for TDAG8:Gs-Fusion Protein was sequenced to verify correctness.
  • H9(F236K)-Gs ⁇ Fusion Protein construct was made as follows: primers were designed as follows: 5′-TTAgatatcGGGGCCCACCCTAGCGGT-3′ (SEQ.ID.NO.: 145; sense) 5′-ggtaccCCCACAGCCATTTCATCAGGATC-3′. (SEQ.ID.NO.: 146; antisense)
  • Nucleotides in lower caps are included as spacers in the restriction sites between the G protein and H9.
  • the sense and anti-sense primers included the restriction sites for EcoRV and KpnI, respectively such that spacers (attributed to the restriction sites) exists between the G protein and H9.
  • PCR was then utilized to secure the respective receptor sequences for fusion within the Gs ⁇ universal vector disclosed above, using the following protocol for each: 80 ng cDNA for H9 was added to separate tubes containing 100 ng of each primer (sense and anti-sense), and 45 uL of PCR SupermixTM (Gibco-Brl, LifeTech) (50 ul total reaction volume). Reaction temperatures and cycle times for H9 were as follows: the initial denaturing step was done it 94° C. for one, and a cycle of 94° C. for 30 seconds; 55° C. for 30 seconds; 72° C. for two minutes. A final extension time was done at 72° C. for seven minutes.
  • Binding Buffer consisted of 10 mM HEPES, 100 mM NaCl and 10 mM MgCl (ph 7.4).
  • Regeneration Buffer was prepared in Binding Buffer and consisted of 20 mM phosphocreatine, 20U creatine phosphokinase, 20 uM GTP, 0.2 mM ATP, and 0.6 mM IBMX.
  • cAMP Standards were prepared in Binding Buffer as follows: cAMP Stock Added to indicted Final Assay Concentration (5,000 pmol/ml in amount of Binding (50 ul into 100 ul) 2 ml H 2 O) in ul Buffer to achieve indicated pmol/well A 250 1 ml 50 B 500 of A 500 ul 25 C 500 of B 500 ul 12.5 D 500 of C 750 ul 5.0 E 500 of D 500 ul 2.5 F 500 of E 500 ul 1.25 G 500 of F 750 ul 0.5
  • Frozen membranes (both pCMV as control and the non-endogenous H(-Gs Fusion Protein) were thawed (on ice at room temperature until in solution). Membranes were homogenized with a polytron until in suspension (2 ⁇ 15 seconds). Membrane protein concentration was determined using the Bradford Assay Protocol (see infra). Membrane concentration was diluted to 0.5 mg/ml in Regeneration Buffer (final assay concentration—25 ug/well). Thereafter, 50 ul of Binding Buffer was added to each well. For control, 50 ul/well of cAMP standard was added to wells 11 and 12 A-G, with Binding Buffer alone to 12H (on the 96-well format).
  • Protocol Direct Identification of Inverse Agonists and Agonists Using [ 35 S]GTP ⁇ S
  • a GPCR Fusion Protein as disclosed above, is also utilized with a non-endogenous, constitutively activated GPCR.
  • intra-assay variation appears to be substantially stabilized, whereby an effective signal-to-noise ratio is obtained. This has the beneficial result of allowing for a more robust identification of candidate compounds.
  • a GPCR Fusion Protein be used and that when utilized, the following assay protocols be utilized.
  • Membranes comprising the non-endogenous, constitutively active orphan GPCR Fusion Protein of interest and for use in the direct identification of candidate compounds as inverse agonists, agonists or partial agonists are preferably prepared as follows:
  • “Membrane Scrape Buffer” is comprised of 20 mM HEPES and 10 mM EDTA, pH 7.4; “Membrane Wash Buffer” is comprised of 20 mM HEPES and 0.1 mM EDTA, pH 7.4; “Binding Buffer” is comprised of 20 mM HEPES, 100 mM NaCl, and 10 mM MgCl 2 , pH 7.4
  • protein concentration of the membranes is determined using the Bradford Protein Assay (protein can be diluted to about 1.5 mg/ml, aliquoted and frozen ( ⁇ 80° C.) for later use; when frozen, protocol for use is as follows: on the day of the assay, frozen Membrane Protein is thawed at room temperature, followed by vortex and then homogenized with a polytron at about 12 ⁇ 1,000 rpm for about 5-10 seconds; it is noted that for multiple preparations, the homogenizer should be thoroughly cleaned between homoginezation of different preparations).
  • Binding Buffer (as per above); Bradford Dye Reagent; Bradford Protein Standard are utilized, following manufacturer instructions (Biorad, cat. no. 500-0006).
  • Duplicate tubes are prepared, one including the membrane, and one as a control “blank”. Each contained 800 ul Binding Buffer. Thereafter, 10 ul of Bradford Protein Standard (1 mg/ml) is added to each tube, and 10 ul of membrane Protein is then added to just one tube (not the blank). Thereafter, 200 ul of Bradford Dye Reagent is added to each tube, followed by vortex of each. After five (5) minutes, the tubes were re-vortexed and the material therein is transferred to cuvettes. The cuvettes are then read using a CECIL 3041 spectrophotometer, at wavelength 595.
  • GDP Buffer consists of 37.5 ml Binding Buffer and 2 mg GDP (Sigma, cat. no. G-7127), followed by a series of dilutions in Binding Buffer to obtain 0.2 uM GDP (final concentration of GDP in each well was 0.1 uM GDP); each well comprising a candidate compound, has a final volume of 200 ul consisting of 100 ul GDP Buffer (final concentration, 0.1 uM GDP), 50 ul Membrane Protein in Binding Buffer, and 50 ul [ 35 S]GTP ⁇ S (0.6 nM) in Binding Buffer (2.5 ul [ 35 S]GTP ⁇ S per 10 ml Binding Buffer).
  • Candidate compounds are preferably screened using a 96-well plate format (these can be frozen at ⁇ 80° C.).
  • Membrane Protein or membranes with expression vector excluding the GPCR Fusion Protein, as control), are homogenized briefly until in suspension. Protein concentration is then determined using the Bradford Protein Assay set forth above. Membrane Protein (and control) is then diluted to 0.25 mg/ml in Binding Buffer (final assay concentration, 12.5 ug/well). Thereafter, 100 ul GDP Buffer is added to each well of a Wallac ScintistripTM (Wallac).
  • a 5 ul pin-tool is then used to transfer 5 ul of a candidate compound into such well (i.e., 5 ul in total assay volume of 200 ul is a 1:40 ratio such that the final screening concentration of the candidate compound is 10 uM).
  • the pin tool should be rinsed in three reservoirs comprising water (1 ⁇ ), ethanol (1 ⁇ ) and water (2 ⁇ )—excess liquid should be shaken from the tool after each rinse and dried with paper and kimwipes.
  • 50 ul of Membrane Protein is added to each well (a control well comprising membranes without the GPCR Fusion Protein is also utilized), and pre-incubated for 5-10 minutes at room temperature.
  • Binding Buffer 50 ul of [ 35 S]GTP ⁇ S (0.6 nM) in Binding Buffer is added to each well, followed by incubation on a shaker for 60 minutes at room temperature (again, in this example, plates were covered with foil). The assay is then stopped by spinning of the plates at 4000 RPM for 15 minutes at 22° C. The plates are then aspirated with an 8 channel manifold and sealed with plate covers. The plates are then read on a Wallacc 1450 using setting “Prot. #37” (as per manufacturer instructions).
  • the preferred confirmation assay is a cyclase-based assay.
  • a modified Flash PlateTM Adenylyl Cyclase kit (New England Nuclear; Cat. No. SMP004A) is preferably utilized for confirmation of candidate compounds directly identified as inverse agonists and agonists to non-endogenous, constitutively activated orphan GPCRs in accordance with the following protocol.
  • Transfected cells are harvested approximately three days after transfection.
  • Membranes are prepared by homogenization of suspended cells in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCl 2 . Homogenization is performed on ice using a Brinkman PolytronTM for approximately 10 seconds. The resulting homogenate is centrifuged at 49,000 ⁇ g for 15 minutes at 4° C. The resulting pellet is then resuspended in buffer containing 20 mM HEPES, pH 7.4 and 0.1 mM EDTA, homogenized for 10 seconds, followed by centrifugation at 49,000 ⁇ g for 15 minutes at 4° C. The resulting pellet can be stored at ⁇ 80° C. until utilized.
  • the membrane pellet On the day of direct identification screening, the membrane pellet is slowly thawed at room temperature, resuspended in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCL2, to yield a final protein concentration of 0.60 mg/ml (the resuspended membranes are placed on ice until use).
  • cAMP standards and Detection Buffer comprising 2 ⁇ Ci of tracer [ 125 I ]cAMP (100 ⁇ l] to 11 ml Detection Buffer) are prepared and maintained in accordance with the manufacturer's instructions.
  • Assay Buffer is prepared fresh for screening and contained 20 mM HEPES, pH 7.4, 10 mM MgCl 2 , 20 mM phospocreatine (Sigma), 0.1 units/ml creatine phosphokinase (Sigma), 50 ⁇ M GTP (Sigma), and 0.2 mM ATP (Sigma); Assay Buffer can be stored on ice until utilized.
  • Candidate compounds identified as per above are added, preferably, to 96-well plate wells (3 ⁇ l/well; 12 ⁇ M final assay concentration), together with 40 ⁇ l Membrane Protein (30 ⁇ g/well) and 50 ⁇ l of Assay Buffer. This admixture is then incubated for 30 minutes at room temperature, with gentle shaking.
  • the vector utilized be pCMV.
  • This vector was deposited with the American Type Culture Collection (ATCC) on Oct. 13, 1998 (10801 University Boulevard., Manassas, Va. 20110-2209 USA) under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure. The DNA was tested by the ATCC and determined to be. The ATCC has assigned the following deposit number to pCMV: ATCC #20335 1.

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Abstract

The invention disclosed in this patent document relates to transmembrane receptors, more particularly to a human G protein-coupled receptor for which the endogenous ligand is unknown (“orphan GPCR receptors”), and most particularly to mutated (non-endogenous) versions of the human GPCRs for evidence of constitutive activity.

Description

  • This patent application is a continuation-in-part of, and claims priority from, U.S. Ser. No. 09/170,496, filed with the United States Patent and Trademark Office on Oct. 13, 1998. This application also claims the benefit of priority from the following provisional applications, all filed via U.S. Express Mail with the United States Patent and Trademark Office on the indicated dates: U.S. Provisional No. 60/110,060, filed Nov. 27, 1998; U.S. Provisional No. 60/120,416, filed Feb. 16, 1999; U.S. Provisional No. 60/121,852, filed Feb. 26, 1999 claiming benefit of U.S. Provisional No. 60/109,213, filed Nov. 20, 1998; U.S. Provisional No. 60/123,944, filed Mar. 12, 1999; U.S. Provisional No. 60/123,945, filed Mar. 12, 1999; U.S. Provisional No. 60/123,948, filed Mar. 12, 1999; U.S. Provisional No. 60/123,951, filed Mar. 12, 1999; U.S. Provisional No. 60/123,946, filed Mar. 12, 1999; U.S. Provisional No. 60/123,949, filed Mar. 12, 1999; U.S. Provisional No. 60/152,524, filed Sep. 3, 1999, claiming benefit of U.S. Provisional No. 60/151,114, filed Aug. 27, 1999 and U.S. Provisional No. 60/108,029, filed Nov. 12, 1998; U.S. Provisional No. 60/136,436, filed May 28, 1999; U.S. Provisional No. 60/136,439, filed May 28, 1999; U.S. Provisional No. 60/136,567, filed May 28, 1999; U.S. Provisional No. 60/137,127, filed May 28, 1999; U.S. Provisional No. 60/137,131, filed May 28, 1999; U.S. Provisional No. 60/141,448, filed Jun. 29, 1999 claiming benefit of U.S. Provisional No. 60/136,437, filed May 28, 1999; U.S. Provisional No. 60/156,633, filed Sep. 29, 1999; U.S. Provisional No. 60/156,555, filed Sep. 29, 1999; U.S. Provisional No. 60/156,634, filed Sep. 29, 1999; U.S. Provisional No. ______ (Arena Pharmaceuticals, Inc. docket number: CHN10-1), filed Sep. 29, 1999; U.S. Provisional No. ______ (Arena Pharmaceuticals, Inc. docket number: RUP6-1), filed Oct. 1, 1999; U.S. Provisional No. ______ (Arena Pharmaceuticals, Inc. docket number: RUP7-1), filed Oct. 1, 1999; U.S. Provisional No. ______ (Arena Pharmaceuticals, Inc. docket number: CHN6-1), filed Oct. 1, 1999; U.S. Provisional No. ______ (Arena Pharmaceuticals, Inc. docket number: RUP5-1), filed Oct. 1, 1999; and U.S. Provisional No. ______ (Arena Pharmaceuticals, Inc. docket number: CHN9-1), filed Oct. 1, 1999. This application is also related to co-pending U.S. Ser. No. _______ (Woodcock, Washburn, Kurtz, Makiewicz & Norris, LLP docket number AREN-0050), filed on Oct. 12, 1999 (via U.S. Express Mail) and U.S. Ser. No. 09/364,425, filled on Jul. 30, 1999, both incorporated herein by reference. Each of the foregoing applications are incorporated by reference herein in their entirety.[0001]
    • FIELD OF THE INVENTION
  • The invention disclosed in this patent document relates to transmembrane receptors, and more particularly to human G protein-coupled receptors, and specifically to GPCRs that have been altered to establish or enhance constitutive activity of the receptor. Preferably, the altered GPCRs are used for the direct identification of candidate compounds as receptor agonists, inverse agonists or partial agonists having potential applicability as therapeutic agents. [0002]
    • BACKGROUND OF THE INVENTION
  • Although a number of receptor classes exist in humans, by far the most abundant and therapeutically relevant is represented by the G protein-coupled receptor (GPCR or GPCRs) class. It is estimated that there are some 100,000 genes within the human genome, and of these, approximately 2%, or 2,000 genes, are estimated to code for GPCRs. Receptors, including GPCRs, for which the endogenous ligand has been identified are referred to as “known” receptors, while receptors for which the endogenous ligand has not been identified are referred to as “orphan” receptors. GPCRs represent an important area for the development of pharmaceutical products: from approximately 20 of the 100 known GPCRs, 60% of all prescription pharmaceuticals have been developed. [0003]
  • GPCRs share a common structural motif. All these receptors have seven sequences of between 22 to 24 hydrophobic amino acids that form seven alpha helices, each of which spans the membrane (each span is identified by number, i.e., transmembrane-1 (TM-1), transmebrane-2 (TM-2), etc.). The transmembrane helices are joined by strands of amino acids between transmembrane-2 and transmembrane-3, transmembrane-4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the exterior, or “extracellular” side, of the cell membrane (these are referred to as “extracellular” [0004] regions 1, 2 and 3 (EC-1, EC-2 and EC-3), respectively). The transmembrane helices are also joined by strands of amino acids between transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the interior, or “intracellular” side, of the cell membrane (these are referred to as “intracellular” regions 1, 2 and 3 (IC-1, IC-2 and IC-3), respectively). The “carboxy” (“C”) terminus of the receptor lies in the intracellular space within the cell, and the “amino” (“N”) terminus of the receptor lies in the extracellular space outside of the cell.
  • Generally, when an endogenous ligand binds with the receptor (often referred to as “activation” of the receptor), there is a change in the conformation of the intracellular region that allows for coupling between the intracellular region and an intracellular “G-protein.” It has been reported that GPCRs are “promiscuous” with respect to G proteins, i.e., that a GPCR can interact with more than one G protein. See, Kenakin, T., 43 [0005] Life Sciences 1095 (1988). Although other G proteins exist, currently, Gq, Gs, Gi, Gz and Go are G proteins that have been identified. Endogenous ligand-activated GPCR coupling with the G-protein begins a signaling cascade process (referred to as “signal transduction”). Under normal conditions, signal transduction ultimately results in cellular activation or cellular inhibition. It is thought that the IC-3 loop as well as the carboxy terminus of the receptor interact with the G protein.
  • Under physiological conditions, GPCRs exist in the cell membrane in equilibrium between two different conformations: an “inactive” state and an “active” state. A receptor in an inactive state is unable to link to the intracellular signaling transduction pathway to produce a biological response. Changing the receptor conformation to the active state allows linkage to the transduction pathway (via the G-protein) and produces a biological response. [0006]
  • A receptor may be stabilized in an active state by an endogenous ligand or a compound such as a drug. Recent discoveries, including but not exclusively limited to modifications to the amino acid sequence of the receptor, provide means other than endogenous ligands or drugs to promote and stabilize the receptor in the active state conformation. These means effectively stabilize the receptor in an active state by simulating the effect of an endogenous ligand binding to the receptor. Stabilization by such ligand-independent means is termed “constitutive receptor activation.”[0007]
    • SUMMARY OF THE INVENTION
  • Disclosed herein are non-endogenous versions of endogenous, human GPCRs and uses thereof.[0008]
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 is a representation of 8×CRE-Luc reporter plasmid (see, Example 4(c)3.) [0009]
  • FIGS. 2A and 2B are graphic representations of the results of ATP and ADP binding to endogenous TDAG8 ([0010] 2A) and comparisons in serum and serum free media (2B).
  • FIG. 3 is a graphic representation of the comparative signaling results of CMV versus the GPCR Fusion Protein H9(F236K):Gsα.[0011]
    • DETAILED DESCRIPTION
  • The scientific literature that has evolved around receptors has adopted a number of terms to refer to ligands having various effects on receptors. For clarity and consistency, the following definitions will be used throughout this patent document. To the extent that these definitions conflict with other definitions for these terms, the following definitions shall control: [0012]
  • AGONISTS shall mean materials (e.g., ligands, candidate compounds) that activate the intracellular response when they bind to the receptor, or enhance GTP binding to membranes. [0013]
  • AMINO ACID ABBREVIATIONS used herein are set out in Table A: [0014]
    TABLE A
    ALANINE ALA A
    ARGININE ARG R
    ASPARAGINE ASN N
    ASPARTIC ACID ASP D
    CYSTEINE CYS C
    GLUTAMIC ACID GLU E
    GLUTAMINE GLN Q
    GLYCINE GLY G
    HISTIDINE HIS H
    ISOLEUCINE ILE I
    LEUCINE LEU L
    LYSINE LYS K
    METHIONINE MET M
    PHENYLALANINE PHE F
    PROLINE PRO P
    SERINE SER S
    THREONINE THR T
    TRYPTOPHAN TRP W
    TYROSINE TYR Y
    VALINE VAL V
  • PARTIAL AGONISTS shall mean materials (e.g., ligands, candidate compounds) that activate the intracellular response when they bind to the receptor to a lesser degree/extent than do agonists, or enhance GTP binding to membranes to a lesser degree/extent than do agonists. [0015]
  • ANTAGONIST shall mean materials (e.g., ligands, candidate compounds) that competitively bind to the receptor at the same site as the agonists but which do not activate the intracellular response initiated by the active form of the receptor, and can thereby inhibit the intracellular responses by agonists or partial agonists. ANTAGONISTS do not diminish the baseline intracellular response in the absence of an agonist or partial agonist. [0016]
  • CANDIDATE COMPOUND shall mean a molecule (for example, and not limitation, a chemical compound) that is amenable to a screening technique. Preferably, the phrase “candidate compound” does not include compounds which were publicly known to be compounds selected from the group consisting of inverse agonist, agonist or antagonist to a receptor, as previously determined by an indirect identification process (“indirectly identified compound”); more preferably, not including an indirectly identified compound which has previously been determined to have therapeutic efficacy in at least one mammal; and, most preferably, not including an indirectly identified compound which has previously been determined to have therapeutic utility in humans. [0017]
  • COMPOSITION means a material comprising at least one component; a “pharmaceutical composition” is an example of a composition. [0018]
  • COMPOUND EFFICACY shall mean a measurement of the ability of a compound to inhibit or stimulate receptor functionality, as opposed to receptor binding affinity. Exemplary means of detecting compound efficacy are disclosed in the Example section of this patent document. [0019]
  • CODON shall mean a grouping of three nucleotides (or equivalents to nucleotides) which generally comprise a nucleoside (adenosine (A), guanosine (G), cytidine (C), uridine (U) and thymidine (T)) coupled to a phosphate group and which, when translated, encodes an amino acid. [0020]
  • CONSTITUTIVELY ACTIVATED RECEPTOR shall mean a receptor subject to constitutive receptor activation. A constitutively activated receptor can be endogenous or non-endogenous. [0021]
  • CONSTITUTIVE RECEPTOR ACTIVATION shall mean stabilization of a receptor in the active state by means other than binding of the receptor with its endogenous ligand or a chemical equivalent thereof. [0022]
  • CONTACT or CONTACTING shall mean bringing at least two moieties together, whether in an in vitro system or an in vivo system. [0023]
  • DIRECTLY IDENTIFYING or DIRECTLY IDENTIFIED, in relationship to the phrase “candidate compound”, shall mean the screening of a candidate compound against a constitutively activated receptor, preferably a constitutively activated orphan receptor, and most preferably against a constitutively activated G protein-coupled cell surface orphan receptor, and assessing the compound efficacy of such compound. This phrase is, under no circumstances, to be interpreted or understood to be encompassed by or to encompass the phrase “indirectly identifying” or “indirectly identified.”[0024]
  • ENDOGENOUS shall mean a material that a mammal naturally produces. ENDOGENOUS in reference to, for example and not limitation, the term “receptor,” shall mean that which is naturally produced by a mammal (for example, and not limitation, a human) or a virus. By contrast, the term NON-ENDOGENOUS in this context shall mean that which is not naturally produced by a mammal (for example, and not limitation, a human) or a virus. For example, and not limitation, a receptor which is not constitutively active in its endogenous form, but when manipulated becomes constitutively active, is most preferably referred to herein as a “non-endogenous, constitutively activated receptor.” Both terms can be utilized to describe both “in vivo” and “in vitro” systems. For example, and not limitation, in a screening approach, the endogenous or non-endogenous receptor may be in reference to an in vitro screening system. As a further example and not limitation, where the genome of a mammal has been manipulated to include a non-endogenous constitutively activated receptor, screening of a candidate compound by means of an in vivo system is viable. [0025]
  • G PROTEIN COUPLED RECEPTOR FUSION PROTEIN and GPCR FUSION PROTEIN, in the context of the invention disclosed herein, each mean a non-endogenous protein comprising an endogenous, constitutively activate GPCR or a non-endogenous, constitutively activated GPCR fused to at least one G protein, most preferably the alpha (α) subunit of such G protein (this being the subunit that binds GTP), with the G protein preferably being of the same type as the G protein that naturally couples with endogenous orphan GPCR. For example, and not limitation, in an endogenous state, if the G protein “Gsα” is the predominate G protein that couples with the GPCR, a GPCR Fusion Protein based upon the specific GPCR would be a non-endogenous protein comprising the GPCR fused to Gsα; in some circumstances, as will be set forth below, a non-predominant G protein can be fused to the GPCR. The G protein can be fused directly to the c-terminus of the constitutively active GPCR or there may be spacers between the two. [0026]
  • HOST CELL shall mean a cell capable of having a Plasmid and/or Vector incorporated therein. In the case of a prokaryotic Host Cell, a Plasmid is typically replicated as a autonomous molecule as the Host Cell replicates (generally, the Plasmid is thereafter isolated for introduction into a eukaryotic Host Cell); in the case of a eukaryotic Host Cell, a Plasmid is integrated into the cellular DNA of the Host Cell such that when the eukaryotic Host Cell replicates, the Plasmid replicates. Preferably, for the purposes of the invention disclosed herein, the Host Cell is eukaryotic, more preferably, mammalian, and most preferably selected from the group consisting of 293, 293T and COS-7 cells. [0027]
  • INDIRECTLY IDENTIFYING or INDIRECTLY IDENTIFIED means the traditional approach to the drug discovery process involving identification of an endogenous ligand specific for an endogenous receptor, screening of candidate compounds against the receptor for determination of those which interfere and/or compete with the ligand-receptor interaction, and assessing the efficacy of the compound for affecting at least one second messenger pathway associated with the activated receptor. [0028]
  • INHIBIT or INHIBITING, in relationship to the term “response” shall mean that a response is decreased or prevented in the presence of a compound as opposed to in the absence of the compound. [0029]
  • INVERSE AGONISTS shall mean materials (e.g., ligand, candidate compound) which bind to either the endogenous form of the receptor or to the constitutively activated form of the receptor, and which inhibit the baseline intracellular response initiated by the active form of the receptor below the normal base level of activity which is observed in the absence of agonists or partial agonists, or decrease GTP binding to membranes. Preferably, the baseline intracellular response is inhibited in the presence of the inverse agonist by at least 30%, more preferably by at least 50%, and most preferably by at least 75%, as compared with the baseline response in the absence of the inverse agonist. [0030]
  • KNOWN RECEPTOR shall mean an endogenous receptor for which the endogenous ligand specific for that receptor has been identified. [0031]
  • LIGAND shall mean an endogenous, naturally occurring molecule specific for an endogenous, naturally occurring receptor. [0032]
  • MUTANT or MUTATION in reference to an endogenous receptor's nucleic acid and/or amino acid sequence shall mean a specified change or changes to such endogenous sequences such that a mutated form of an endogenous, non-constitutively activated receptor evidences constitutive activation of the receptor. In terms of equivalents to specific sequences, a subsequent mutated form of a human receptor is considered to be equivalent to a first mutation of the human receptor if (a) the level of constitutive activation of the subsequent mutated form of a human receptor is substantially the same as that evidenced by the first mutation of the receptor; and (b) the percent sequence (amino acid and/or nucleic acid) homology between the subsequent mutated form of the receptor and the first mutation of the receptor is at least about 80%, more preferably at least about 90% and most preferably at least 95%. Ideally, and owing to the fact that the most preferred cassettes disclosed herein for achieving constitutive activation includes a single amino acid and/or codon change between the endogenous and the non-endogenous forms of the GPCR, the percent sequence homology should be at least 98%. [0033]
  • NON-ORPHAN RECEPTOR shall mean an endogenous naturally occurring molecule specific for an endogenous naturally occurring ligand wherein the binding of a ligand to a receptor activates an intracellular signaling pathway. [0034]
  • ORPHAN RECEPTOR shall mean an endogenous receptor for which the endogenous ligand specific for that receptor has not been identified or is not known. [0035]
  • PHARMACEUTICAL COMPOSITION shall mean a composition comprising at least one active ingredient, whereby the composition is amenable to investigation for a specified, efficacious outcome in a mammal (for example, and not limitation, a human). Those of ordinary skill in the art will understand and appreciate the techniques appropriate for determining whether an active ingredient has a desired efficacious outcome based upon the needs of the artisan. [0036]
  • PLASMID shall mean the combination of a Vector and cDNA. Generally, a Plasmid is introduced into a Host Cell for the purposes of replication and/or expression of the cDNA as a protein. [0037]
  • STIMULATE or STIMULATING, in relationship to the term “response” shall mean that a response is increased in the presence of a compound as opposed to in the absence of the compound. [0038]
  • VECTOR in reference to cDNA shall mean a circular DNA capable of incorporating at least one cDNA and capable of incorporation into a Host Cell. [0039]
  • The order of the following sections is set forth for presentational efficiency and is not intended, nor should be construed, as a limitation on the disclosure or the claims to follow. [0040]
  • A. Introduction [0041]
  • The traditional study of receptors has always proceeded from the a priori assumption (historically based) that the endogenous ligand must first be identified before discovery could proceed to find antagonists and other molecules that could affect the receptor. Even in cases where an antagonist might have been known first, the search immediately extended to looking for the endogenous ligand. This mode of thinking has persisted in receptor research even after the discovery of constitutively activated receptors. What has not been heretofore recognized is that it is the active state of the receptor that is most useful for discovering agonists, partial agonists, and inverse agonists of the receptor. For those diseases which result from an overly active receptor or an under-active receptor, what is desired in a therapeutic drug is a compound which acts to diminish the active state of a receptor or enhance the activity of the receptor, respectively, not necessarily a drug which is an antagonist to the endogenous ligand. This is because a compound that reduces or enhances the activity of the active receptor state need not bind at the same site as the endogenous ligand. Thus, as taught by a method of this invention, any search for therapeutic compounds should start by screening compounds against the ligand-independent active state. [0042]
  • B. Identification of Human GPCRs [0043]
  • The efforts of the Human Genome project has led to the identification of a plethora of information regarding nucleic acid sequences located within the human genome; it has been the case in this endeavor that genetic sequence information has been made available without an understanding or recognition as to whether or not any particular genomic sequence does or may contain open-reading frame information that translate human proteins. Several methods of identifying nucleic acid sequences within the human genome are within the purview of those having ordinary skill in the art. For example, and not limitation, a variety of human GPCRs, disclosed herein, were discovered by reviewing the GenBank™ database, while other GPCRs were discovered by utilizing a nucleic acid sequence of a GPCR, previously sequenced, to conduct a BLAST™ search of the EST database. Table B, below, lists several endogenous GPCRs that we have discovered, along with a GPCR's respective homologous receptor. [0044]
    TABLE B
    Open Reference to
    Disclosed Reading Per Cent Homologous
    Human Accession Frame Homology GPCR
    Orphan Number (Base To Designated (Accession
    GPCRs Identified Pairs) GPCR No.)
    hARE-3 AL033379 1,260 bp 52.3% LPA-R U92642
    hARE-4 AC006087 1,119 bp 36% P2Y5 AF000546
    hARE-5 AC006255 1,104 bp 32% Oryzias D43633
    latipes
    hGPR27 AA775870 1,128 bp
    hARE-1 A1090920   999 bp 43% D13626
    KIAA0001
    hARE-2 AA359504 1,122 bp 53% GPR27
    hPPR1 H67224 1,053 bp 39% EBI1 L31581
    hG2A AA754702 1,113 bp 31% GPR4 L36148
    hRUP3 AL035423 1,005 bp 30% 2133653
    Drosophila
    melanogaster
    hRUP4 AI307658 1,296 bp 32% pNPGPR NP_004876
    28% and 29% AAC41276
    Zebra fish Ya and
    and Yb, AAB94616
    respectively
    hRUP5 AC005849 1,413 bp 25% DEZ Q99788
    23% FMLPR P21462
    hRUP6 AC005871 1,245 bp 48% GPR66 NP_006047
    hRUP7 AC007922 1,173 bp 43% H3R AF140538
    hCHN3 EST 36581 1,113 bp 53% GPR27
    hCHN4 AA804531 1,077 bp 32% thrombin 4503637
    hCHN6 EST 2134670 1,503 bp 36% edg-1 NP_001391
    hCHN8 EST 764455 1,029 bp 47% D13626
    KIAA0001
    hCHN9 EST 1541536 1,077 bp 41% LTB4R NM_000752
    hCHN10 EST 1365839 1,055 bp 35% P2Y NM_002563
  • Receptor homology is useful in terms of gaining an appreciation of a role of the receptors within the human body. As the patent document progresses, we will disclose techniques for mutating these receptors to establish non-endogenous, constitutively activated versions of these receptors. [0045]
  • The techniques disclosed herein have also been applied to other human, orphan GPCRs known to the art, as will be apparent as the patent document progresses. [0046]
  • C. Receptor Screening [0047]
  • Screening candidate compounds against a non-endogenous, constitutively activated version of the human GPCRs disclosed herein allows for the direct identification of candidate compounds which act at this cell surface receptor, without requiring use of the receptor's endogenous ligand. By determining areas within the body where the endogenous version of human GPCRs disclosed herein is expressed and/or over-expressed, it is possible to determine related disease/disorder states which are associated with the expression and/or over-expression of the receptor; such an approach is disclosed in this patent document. [0048]
  • With respect to creation of a mutation that may evidence constitutive activation of the human GPCR disclosed herein is based upon the distance from the proline residue at which is presumed to be located within TM6 of the GPCR; this algorithmic technique is disclosed in co-pending and commonly assigned patent document U.S. Ser. No. 09/170,496, incorporated herein by reference. The algorithmic technique is not predicated upon traditional sequence “alignment” but rather a specified distance from the aforementioned TM6 proline residue. By mutating the amino acid residue located 16 amino acid residues from this residue (presumably located in the IC3 region of the receptor) to, most preferably, a lysine residue, such activation may be obtained. Other amino acid residues may be useful in the mutation at this position to achieve this objective. [0049]
  • D. Disease/Disorder Identification and/or Selection [0050]
  • As will be set forth in greater detail below, most preferably inverse agonists to the non-endogenous, constitutively activated GPCR can be identified by the methodologies of this invention. Such inverse agonists are ideal candidates as lead compounds in drug discovery programs for treating diseases related to this receptor. Because of the ability to directly identify inverse agonists to the GPCR, thereby allowing for the development of pharmaceutical compositions, a search for diseases and disorders associated with the GPCR is relevant. For example, scanning both diseased and normal tissue samples for the presence of the GPCR now becomes more than an academic exercise or one which might be pursued along the path of identifying an endogenous ligand to the specific GPCR. Tissue scans can be conducted across a broad range of healthy and diseased tissues. Such tissue scans provide a preferred first step in associating a specific receptor with a disease and/or disorder. See, for example, co-pending application (docket number ARE-0050) for exemplary dot-blot and RT-PCR results of several of the GPCRs disclosed herein. [0051]
  • Preferably, the DNA sequence of the human GPCR is used to make a probe for (a) dot-blot analysis against tissue-mRNA, and/or (b) RT-PCR identification of the expression of the receptor in tissue samples. The presence of a receptor in a tissue source, or a diseased tissue, or the presence of the receptor at elevated concentrations in diseased tissue compared to a normal tissue, can be preferably utilized to identify a correlation with a treatment regimen, including but not limited to, a disease associated with that disease. Receptors can equally well be localized to regions of organs by this technique. Based on the known functions of the specific tissues to which the receptor is localized, the putative functional role of the receptor can be deduced. [0052]
  • E. Screening of Candidate Compounds [0053]
  • 1. Generic GPCR Screening Assay Techniques [0054]
  • When a G protein receptor becomes constitutively active, it binds to a G protein (e.g., Gq, Gs, Gi, Gz, Go) and stimulates the binding of GTP to the G protein. The G protein then acts as a GTPase and slowly hydrolyzes the GTP to GDP, whereby the receptor, under normal conditions, becomes deactivated. However, constitutively activated receptors continue to exchange GDP to GTP. A non-hydrolyzable analog of GTP, [[0055] 35S]GTPγS, can be used to monitor enhanced binding to membranes which express constitutively activated receptors. It is reported that [35S]GTPγS can be used to monitor G protein coupling to membranes in the absence and presence of ligand. An example of this monitoring, among other examples well-known and available to those in the art, was reported by Traynor and Nahorski in 1995. The preferred use of this assay system is for initial screening of candidate compounds because the system is generically applicable to all G protein-coupled receptors regardless of the particular G protein that interacts with the intracellular domain of the receptor.
  • 2. Specific GPCR Screening Assay Techniques [0056]
  • Once candidate compounds are identified using the “generic” G protein-coupled receptor assay (i.e., an assay to select compounds that are agonists, partial agonists, or inverse agonists), further screening to confirm that the compounds have interacted at the receptor site is preferred. For example, a compound identified by the “generic” assay may not bind to the receptor, but may instead merely “uncouple” the G protein from the intracellular domain. [0057]
  • a. Gs, Gz and Gi. [0058]
  • Gs stimulates the enzyme adenylyl cyclase. Gi (and Gz and Go), on the other hand, inhibit this enzyme. Adenylyl cyclase catalyzes the conversion of ATP to cAMP; thus, constitutively activated GPCRs that couple the Gs protein are associated with increased cellular levels of cAMP. On the other hand, constitutively activated GPCRs that couple Gi (or Gz, Go) protein are associated with decreased cellular levels of cAMP. See, generally, “Indirect Mechanisms of Synaptic Transmission,” Chpt. 8, [0059] From Neuron To Brain (3rd Ed.) Nichols, J. G. et al eds. Sinauer Associates, Inc. (1992). Thus, assays that detect cAMP can be utilized to determine if a candidate compound is, e.g., an inverse agonist to the receptor (i.e., such a compound would decrease the levels of cAMP). A variety of approaches known in the art for measuring cAMP can be utilized; a most preferred approach relies upon the use of anti-cAMP antibodies in an ELISA-based format. Another type of assay that can be utilized is a whole cell second messenger reporter system assay. Promoters on genes drive the expression of the proteins that a particular gene encodes. Cyclic AMP drives gene expression by promoting the binding of a cAMP-responsive DNA binding protein or transcription factor (CREB) that then binds to the promoter at specific sites called cAMP response elements and drives the expression of the gene. Reporter systems can be constructed which have a promoter containing multiple cAMP response elements before the reporter gene, e.g., β-galactosidase or luciferase. Thus, a constitutively activated Gs-linked receptor causes the accumulation of cAMP that then activates the gene and expression of the reporter protein. The reporter protein such as β-galactosidase or luciferase can then be detected using standard biochemical assays (Chen et al. 1995).
  • b. Go and Gq. [0060]
  • Gq and Go are associated with activation of the enzyme phospholipase C, which in turn hydrolyzes the phospholipid PIP[0061] 2, releasing two intracellular messengers: diacycloglycerol (DAG) and inistol 1,4,5-triphoisphate (IP3). Increased accumulation of IP3 is associated with activation of Gq- and Go-associated receptors. See, generally, “Indirect Mechanisms of Synaptic Transmission,” Chpt. 8, From Neuron To Brain (3rd Ed.) Nichols, J. G. et al eds. Sinauer Associates, Inc. (1992). Assays that detect IP3 accumulation can be utilized to determine if a candidate compound is, e.g., an inverse agonist to a Gq- or Go-associated receptor (i.e., such a compound would decrease the levels of IP3). Gq-associated receptors can also been examined using an AP1 reporter assay in that Gq-dependent phospholipase C causes activation of genes containing AP1 elements; thus, activated Gq-associated receptors will evidence an increase in the expression of such genes, whereby inverse agonists thereto will evidence a decrease in such expression, and agonists will evidence an increase in such expression. Commercially available assays for such detection are available.
  • 3. GPCR Fusion Protein [0062]
  • The use of an endogenous, constitutively activate orphan GPCR or a non-endogenous, constitutively activated orphan GPCR, for use in screening of candidate compounds for the direct identification of inverse agonists, agonists and partial agonists provide an interesting screening challenge in that, by definition, the receptor is active even in the absence of an endogenous ligand bound thereto. Thus, in order to differentiate between, e.g., the non-endogenous receptor in the presence of a candidate compound and the non-endogenous receptor in the absence of that compound, with an aim of such a differentiation to allow for an understanding as to whether such compound may be an inverse agonist, agonist, partial agonist or have no affect on such a receptor, it is preferred that an approach be utilized that can enhance such differentiation. A preferred approach is the use of a GPCR Fusion Protein. [0063]
  • Generally, once it is determined that a non-endogenous orphan GPCR has been constitutively activated using the assay techniques set forth above (as well as others), it is possible to determine the predominant G protein that couples with the endogenous GPCR. Coupling of the G protein to the GPCR provides a signaling pathway that can be assessed. Because it is most preferred that screening take place by use of a mammalian expression system, such a system will be expected to have endogenous G protein therein. Thus, by definition, in such a system, the non-endogenous, constitutively activated orphan GPCR will continuously signal. In this regard, it is preferred that this signal be enhanced such that in the presence of, e.g., an inverse agonist to the receptor, it is more likely that it will be able to more readily differentiate, particularly in the context of screening, between the receptor when it is contacted with the inverse agonist. [0064]
  • The GPCR Fusion Protein is intended to enhance the efficacy of G protein coupling with the non-endogenous GPCR. The GPCR Fusion Protein is preferred for screening with a non-endogenous, constitutively activated GPCR because such an approach increases the signal that is most preferably utilized in such screening techniques. This is important in facilitating a significant “signal to noise” ratio; such a significant ratio is import preferred for the screening of candidate compounds as disclosed herein. [0065]
  • The construction of a construct useful for expression of a GPCR Fusion Protein is within the purview of those having ordinary skill in the art. Commercially available expression vectors and systems offer a variety of approaches that can fit the particular needs of an investigator. The criteria of importance for such a GPCR Fusion Protein construct is that the endogenous GPCR sequence and the G protein sequence both be in-frame (preferably, the sequence for the endogenous GPCR is upstream of the G protein sequence) and that the “stop” codon of the GPCR must be deleted or replaced such that upon expression of the GPCR, the G protein can also be expressed. The GPCR can be linked directly to the G protein, or there can be spacer residues between the two (preferably, no more than about 12, although this number can be readily ascertained by one of ordinary skill in the art). We have a preference (based upon convenience) of use of a spacer in that some restriction sites that are not used will, effectively, upon expression, become a spacer. Most preferably, the G protein that couples to the non-endogenous GPCR will have been identified prior to the creation of the GPCR Fusion Protein construct. Because there are only a few G proteins that have been identified, it is preferred that a construct comprising the sequence of the G protein (i.e., a universal G protein construct) be available for insertion of an endogenous GPCR sequence therein; this provides for efficiency in the context of large-scale screening of a variety of different endogenous GPCRs having different sequences. [0066]
  • As noted above, constitutively activated GPCRs that couple to Gi, Gz and Go are expected to inhibit the formation of cAMP making assays based upon these types of GPCRs challenging (i.e., the cAMP signal decreases upon activation thus making the direct identification of, e.g, inverse agonists (which would further decrease this signal), interesting). As will be disclosed herein, we have ascertained that for these types of receptors, it is possible to create a GPCR Fusion Protein that is not based upon the endogenous GPCR's endogenous G protein, in an effort to establish a viable cyclase-based assay. Thus, for example, a Gz coupled receptor such as H9, a GPCR Fusion Protein can be established that utilizes a Gs fusion protein—we believe that such a fusion construct, upon expression, “drives” or “forces” the non-endogenous GPCR to couple with, e.g., Gs rather than the “natural” Gz protein, such that a cyclase-based assay can be established. Thus, for Gi, Gz and Go coupled receptors, we prefer that that when a GPCR Fusion Protein is used and the assay is based upon detection of adenyl cyclase activity, that the fusion construct be established with Gs (or an equivalent G protein that stimulates the formation of the enzyme adenylyl cyclase). [0067]
  • F. Medicinal Chemistry [0068]
  • Generally, but not always, direct identification of candidate compounds is preferably conducted in conjunction with compounds generated via combinatorial chemistry techniques, whereby thousands of compounds are randomly prepared for such analysis. Generally, the results of such screening will be compounds having unique core structures; thereafter, these compounds are preferably subjected to additional chemical modification around a preferred core structure(s) to further enhance the medicinal properties thereof. Such techniques are known to those in the art and will not be addressed in detail in this patent document. [0069]
  • G. Pharmaceutical Compositions [0070]
  • Candidate compounds selected for further development can be formulated into pharmaceutical compositions using techniques well known to those in the art. Suitable pharmaceutically-acceptable carriers are available to those in the art; for example, see Remington's Pharmaceutical Sciences, 16[0071] th Edition, 1980, Mack Publishing Co., (Oslo et al., eds.)
  • H. Other Utility [0072]
  • Although a preferred use of the non-endogenous versions the human GPCRs disclosed herein may be for the direct identification of candidate compounds as inverse agonists, agonists or partial agonists (preferably for use as pharmaceutical agents), these versions of human GPCRs can also be utilized in research settings. For example, in vitro and in vivo systems incorporating GPCRs can be utilized to further elucidate and understand the roles these receptors play in the human condition, both normal and diseased, as well as understanding the role of constitutive activation as it applies to understanding the signaling cascade. The value in non-endogenous human GPCRs is that their utility as a research tool is enhanced in that, because of their unique features, non-endogenous human GPCRs can be used to understand the role of these receptors in the human body before the endogenous ligand therefor is identified. Other uses of the disclosed receptors will become apparent to those in the art based upon, inter alia, a review of this patent document. [0073]
    • EXAMPLES
  • The following examples are presented for purposes of elucidation, and not limitation, of the present invention. While specific nucleic acid and amino acid sequences are disclosed herein, those of ordinary skill in the art are credited with the ability to make minor modifications to these sequences while achieving the same or substantially similar results reported below. The traditional approach to application or understanding of sequence cassettes from one sequence to another (e.g. from rat receptor to human receptor or from human receptor A to human receptor B) is generally predicated upon sequence alignment techniques whereby the sequences are aligned in an effort to determine areas of commonality. The mutational approach disclosed herein does not rely upon this approach but is instead based upon an algorithmic approach and a positional distance from a conserved proline residue located within the TM6 region of human GPCRs. Once this approach is secured, those in the art are credited with the ability to make minor modifications thereto to achieve substantially the same results (i.e., constitutive activation) disclosed herein. Such modified approaches are considered within the purview of this disclosure [0074]
    • Example 1
  • Endogenous Human GPCRs [0075]
  • 1. Identification of Human GPCRs [0076]
  • Certain of the disclosed endogenous human GPCRs were identified based upon a review of the GenBank™ database information. While searching the database, the following cDNA clones were identified as evidenced below (Table C). [0077]
    TABLE C
    Open
    Disclosed Complete Reading Nucleic Amino
    Human DNA Frame Acid Acid
    Orphan Accession Sequence (Base SEQ.ID. SEQ.ID.
    GPCRs Number (Base Pairs) Pairs) NO. NO.
    hARE-3 AL033379 111,389 bp 1,260 bp 1 2
    hARE-4 AC006087 226,925 bp 1,119 bp 3 4
    hARE-5 AC006255 127,605 bp 1,104 bp 5 6
    hRUP3 AL035423 140,094 bp 1,005 bp 7 8
    hRUP5 AC005849 169,144 bp 1,413 bp 9 10
    hRUP6 AC005871 218,807 bp 1,245 bp 11 12
    hRUP7 AC007922 158,858 bp 1,173 bp 13 14
  • Other disclosed endogenous human GPCRs were identified by conducting a BLAST™ search of EST database (dbest) using the following EST clones as query sequences. The following EST clones identified were then used as a probe to screen a human genomic library (Table D). [0078]
    TABLE D
    Open
    Disclosed Reading Nucleic Amino
    Human EST Clone/ Frame Acid Acid
    Orphan Query Accession No. (Base SEQ. SEQ.
    GPCRs (Sequence) Identified Phairs) ID.NO. ID.NO.
    hGPCR27 Mouse AA775870 1,125 bp 17 18
    GPCR27
    hARE-1 TDAG 1689643   999 bp 19 20
    AI090920
    hARE-2 GPCR27 68530 1,122 bp 21 22
    AA359504
    hPPR1 Bovine 238667 1,053 bp 23 24
    PPR1 H67224
    hG2A Mouse See Example 1,113 bp 25 26
    1179426 2(a), below
    hCHN3 N.A. EST 36581 1,113 bp 27 28
    (full length)
    hCHN4 TDAG 1184934 1,077 bp 29 30
    AA804531
    hCHN6 N.A. EST 2134670 1,503 bp 31 32
    (full length)
    hCHN8 KIAA0001 EST 764455 1,029 bp 33 34
    hCHN 9 1365839 EST 1541536 1,077 bp 35 36
    hCHN10 Mouse EST Human 1,005 bp 37 38
    1365839 1365839
    hRUP4 N.A. AI307658 1,296 bp 39 40
  • 2. Full Length Cloning [0079]
  • a. Human G2A [0080]
  • Mouse EST clone 1179426 was used to obtain a human genomic clone containing all but three amino acid G2A coding sequences. The 5′ of this coding sequence was obtained by using 5′RACE, and the template for PCR was Clontech's Human Spleen Marathon-Ready™ cDNA. The disclosed human G2A was amplified by PCR using the G2A cDNA specific primers for the first and second round PCR as shown in SEQ.ID.NO.: 41 and SEQ.ID.NO.: 42 as follows: [0081]
    5′-CTGTGTACAGCAGTTCGCAGAGTG-3′ (SEQ.ID.NO.:41; 1st round PCR)
    5′-GAGTGCCAGGCAGAGCAGGTAGAC-3′. (SEQ.ID.NO.:42; second round PCR)
  • PCR was performed using Advantage GC Polymerase Kit (Clontech; manufacturing instructions will be followed), at 94° C. for 30 sec followed by 5 cycles of 94° C. for 5 sec and 72° C. for 4 min; and 30 cycles of 94° for 5 sec and 70° for 4 min. An approximate PCR fragment was purified from agarose gel, digested with Hind III and Xba I and cloned into the expression vector pRC/CMV2 (Invitrogen). The cloned-insert was sequenced using the T7 Sequenase™ kit (USB Amersham; manufacturer instructions followed) and the sequence was compared with the presented sequence. Expression of the human G2A was detected by probing an RNA dot blot (Clontech; manufacturer instructions followed) with the P[0082] 32-labeled fragment.
  • b. CHN9 [0083]
  • Sequencing of the EST clone 1541536 showed CHN9 to be a partial cDNA clone having only an initiation codon; i.e., the termination codon was missing. When CHN9 was used to blast against data base (nr), the 3′ sequence of CHN9 was 100% homologous to the 5′ untranslated region of the leukotriene B4 receptor cDNA, which contained a termination codon in the frame with CHN9 coding sequence. To determine whether the 5′ untranslated region of LTB4R cDNA was the 3′ sequence of CHN9, PCR was performed using primers based upon the 5′ sequence flanking the initiation codon found in CHN9 and the 3′ sequence around the termination codon found in the [0084] LTB4R 5′ untranslated region. The 5′ primer sequence utilized was as follows:
    5′-CCCGAATTCCTGCTTGCTCCCAGCTTGGCCC-3′ (SEQ.ID.NO.: 43; sense)
    and
    5′-TGTGGATCCTGCTGTCAAAGGTCCCATTCCGG-3′. (SEQ.ID.NO.: 44; antisense)
  • PCR was performed using thymus cDNA as a template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 uM of each primer, and 0.2 mM of each 4 nucleotides. The cycle condition was 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 1 min and 10 sec. A 1.1 kb fragment consistent with the predicted size was obtained from PCR. This PCR fragment was subcloned into pCMV (see below) and sequenced (see, SEQ.ID.NO.: 35). [0085]
  • c. RUP4 [0086]
  • The full length RUP4 was cloned by RT-PCR with human brain cDNA (Clontech) as templates: [0087]
    (SEQ.ID.NO.: 45; sense)
    5′-TCACAATGCTAGGTGTGGTC-3′
    and
    (SEQ.ID.NO.: 46; antisense)
    5′-TGCATAGACAATGGGATTACAG-3′.
  • PCR was performed using TaqPlus Precision™ polymerase (Stratagene; manufacturing instructions followed) by the following cycles: 94° C. for 2 min; 94° C. 30 sec; 55° C. for 30 sec, 72° C. for 45 sec, and 72° C. for 10 min. [0088] Cycles 2 through 4 were repeated 30 times.
  • The PCR products were separated on a 1% agarose gel and a 500 bp PCR fragment was isolated and cloned into the pCRII-TOPO™ vector (Invitrogen) and sequenced using the T7 DNA Sequenase™ kit (Amsham) and the SP6/T7 primers (Stratagene). Sequence analysis revealed that the PCR fragment was indeed an alternatively spliced form of A1307658 having a continuous open reading frame with similarity to other GPCRs. The completed sequence of this PCR fragment was as follows: [0089]
    (SEQ.ID.NO.: 47)
    5′-TCACAATGCTAGGTGTGGTCTGGCTGGTGGCAGTCATCGTAGGATCA
    CCCATGTGGCACGTGCAACAACTTGAGATCAAATATGACTTCCTATATGA
    AAAGGAACACATCTGCTGCTTAAGAGTGGACCAGCCCTGTGCACCAGAAG
    ATCTACACCACCTTCATCCTTGTCATCCTCTTCCTCCTGCCTCTTATGGT
    GATGCTTATTCTGTACGTAAAATTGGTTATGAACTTTGGATAAAGAAAAG
    AGTTGGGGATGGTTCAGTGCTTCGAACTATTCATGGAAAAGAAATGTCCA
    AAATAGCCAGGAAGAAGAAACGAGCTGTCATTATATGATGGTGACAGTGG
    TGGCTCTCTTTGCTGTGTGCTGGGCACCATTCCATGTTGTCCATATGATG
    ATTGAATACAGTAATTTTGAAAAGGAATATGATGATGTCACAATCAAGAT
    GATTTTTGCTATCGTGCAAATTATTGGATTTTCCAACTCCATCTGTAATC
    CCATTGTCTATGCA-3′
  • Based on the above sequence, two sense oligonucleotide primer sets: [0090]
    (SEQ.ID.NO.: 48; oligo 1)
    5′-CTGCTTAGAAGAGTGGACCAG-3′,
    (SEQ.ID.NO.: 49; oligo 2)
    5′-CTGTGCACCAGAAGATCTACAC-3′
    and
  • two antisense oligonucleotide primer sets: [0091]
    (SEQ.ID.NO.: 50; oligo 3)
    5′-CAAGGATGAAGGTGGTGTAGA-3′
    (SEQ.ID.NO.: 51; oligo 4)
    5′-GTGTAGATCTTCTGGTGCACAGG-3′
  • were used for 3′- and 5′-RACE PCR with a human brain Marathon-Ready™ cDNA (Clontech, Cat# 7400-1) as template, according to manufacture's instructions. DNA fragments generated by the RACE PCR were cloned into the pCRII-TOPO™ vector (Invitrogen) and sequenced using the SP6/T7 primers (Stratagene) and some internal primers. The 3′ RACE product contained a poly(A) tail and a completed open reading frame ending at a TAA stop codon. The 5′ RACE product contained an incomplete 5′ end; i.e., the ATG initiation codon was not present. [0092]
  • Based on the new 5′ sequence, [0093] oligo 3 and the following primer: 5′-GCAATGCAGGTCATAGTGAGC-3′ (SEQ.ID.NO.: 52; oligo 5) were used for the second round of 5′ race PCR and the PCR products were analyzed as above. A third round of 5′ race PCR was carried out utilizing antisense primers:
    (SEQ.ID.NO.: 53; oligo 6)
    5′-TGGAGCATGGTGACGGGAATGCAGAAG-3′
    and
    (SEQ.ID.NO.: 54; oligo 7)
    5′-GTGATGAGCAGGTCACTGAGCGCCAAG-3′.
  • The sequence of the 5′ RACE PCR products revealed the presence of the initiation codon ATG, and further round of 5′ race PCR did not generate any more 5′ sequence. The completed 5′ sequence was confirmed by RT-PCR using [0094] sense primer 5′-GCAATGCAGGCGCTTAACATTAC-3′ (SEQ.ID.NO.: 55; oligo 8) and oligo 4 as primers and sequence analysis of the 650 bp PCR product generated from human brain and heart cDNA templates (Clontech, Cat# 7404-1). The completed 3′ sequence was confirmed by RT-PCR using oligo 2 and the following antisense primer: 5′-TTGGGTTACAATCTGAAGGGCA-3′ (SEQ.ID.NO.: 56; oligo 9) and sequence analysis of the 670 bp PCR product generated from human brain and heart cDNA templates. (Clontech, Cat# 7404-1).
  • d. RUP5 [0095]
  • The full length RUP5 was cloned by RT-PCR using a sense primer upstream from ATG, the initiation codon (SEQ.ID.NO.: 57), and an antisense primer containing TCA as the stop codon (SEQ.ID.NO.: 58), which had the following sequences: [0096]
    5′-ACTCCGTGTCCAGCAGGACTCTG-3′ (SEQ.ID.NO.: 57)
    5′-TGCGTGTTCCTGGACCCTCACGTG-3′ (SEQ.ID.NO.: 58)
  • and human peripheral leukocyte cDNA (Clontech) as a template. Advantage™ cDNA polymerase (Clontech) was used for the amplification in a 50 ul reaction by the following cycle with [0097] step 2 through step 4 repeated 30 times: 94° C. for 30 sec; 94° for 15 sec; 69° for 40 sec; 72° C. for 3 min; and 72° C. fro 6 min. A 1.4 kb PCR fragment was isolated and cloned with the pCRII-TOPO™ vector (Invitrogen) and completely sequenced using the T7 DNA Sequenase™ kit (Amsham). See, SEQ.ID.NO.: 9.
  • e. RUP6 [0098]
  • The full length RUP6 was cloned by RT-PCR using primers: [0099]
    (SEQ.ID.NO.: 59)
    5′-CAGGCCTTGGATTTTAATGTCAGGGATGG-3′
    and
    (SEQ.ID.NO.: 60)
    5′-GGAGAGTCAGCTCTGAAAGAATTCAGG-3′;
  • and human thymus Marathon-Ready™ cDNA (Clontech) as a template. Advantage cDNA polymerase (Clontech, according to manufacturer's instructions) was used for the amplification in a 50 ul reaction by the following cycle: 94° C. for 30sec; 94° C. for 5 sec; 66° C. for 40 sec; 72° C. for 2.5 sec and 72° C. for 7 min. [0100] Cycles 2 through 4 were repeated 30 times. A 1.3 Kb PCR fragment was isolated and cloned into the pCRII-TOPO™ vector (Invitrogen) and completely sequenced (see, SEQ.ID.NO.: 11) using the ABI Big Dye Terminator™ kit (P.E. Biosystem).
  • f. RUP7 [0101]
  • The full length RUP7 was cloned by RT-PCR using primers: [0102]
    (SEQ.ID.NO.: 61; sense)
    5′-TGATGTGATGCCAGATACTAATAGCAC-3′
    and
    (SEQ.ID.NO.: 62; antisense)
    5′-CCTGATTCATTTAGGTGAGATTGAGAC-3′
  • and human peripheral leukocyte cDNA (Clontech) as a template. Advantage™ cDNA polymerase (Clontech) was used for the amplification in a 50 ul reaction by the following cycle with [0103] step 2 to step 4 repeated 30 times: 94° C. for 2 minutes; 94° C. for 15 seconds; 60° C. for 20 seconds; 72° C. for 2 minutes; 72° C. for 10 minutes. A 1.25 Kb PCR fragment was isolated and cloned into the pCRII-TOPO™ vector (Invitrogen) and completely sequenced using the ABI Big Dye Terminator™ kit (P.E. Biosystem). See, SEQ.ID.NO.: 13.
  • 3. [0104] Angiotensin II Type 1 Receptor (“AT1”)
  • The endogenous human angiotensin II [0105] type 1 receptor (“AT1”) was obtained by PCR using genomic DNA as template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 μM of each primer, and 0.2 mM of each 4 nucleotides. The cycle condition was 30 cycles of 94° C. for 1 min, 55° C. for 1 min and 72° C. for 1.5 min. The 5′ PCR primer contains a HindIII site with the sequence:
  • 5′-CCCAAGCTTCCCCAGGTGTATTTGAT-3′ (SEQ.ID.NO.: 63) [0106]
  • and the 3′ primer contains a BamHI site with the following sequence: [0107]
  • 5′-GTTGGATCCACATAATGCATTTTCTC-3′ (SEQ.ID.NO.: 64). [0108]
  • The resulting 1.3 kb PCR fragment was digested with HindIII and BamHI and cloned into HindIII-BamHI site of pCMV expression vector. The cDNA clone was fully sequenced. Nucleic acid (SEQ.ID.NO.: 65) and amino acid (SEQ.ID.NO.: 66) sequences for human AT1 were thereafter determined and verified. [0109]
  • 4. GPR38 [0110]
  • To obtain GPR38, PCR was performed by combining two PCR fragments, using human genomic cDNA as template and rTth poymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 uM of each primer, and 0.2 mM of each 4 nucleotides. The cycle condition for each PCR reaction was 30 cycles of 94° C. for 1 min, 62° C. for 1 min and 72° C. for 2 min. [0111]
  • The first fragment was amplified with the 5′ PCR primer that contained an end site with the following sequence: [0112]
  • 5′-ACCATGGGCAGCCCCTGGAACGGCAGC-3′ (SEQ.ID.NO.: 67) [0113]
  • and a 3′ primer having the following sequence: [0114]
  • 5′-AGAACCACCACCAGCAGGACGCGGACGGTCTGCCGGTGG-3′ (SEQ.ID.NO.: 68). [0115]
  • The second PCR fragment was amplified with a 5′ primer having the following sequence: [0116]
  • 5′-GTCCGCGTCCTGCTGGTGGTGGTTCTGGCATTATAATT-3′ (SEQ.ID.NO.: 69) [0117]
  • and a 3′ primer that contained a BamHI site and having the following sequence: [0118]
  • 5′-CCTGGATCCTTATCCCATCGTCTCACGTTAGC-3′ (SEQ.ID.NO.: 70). [0119]
  • The two fragments were used as templates to amplify GPR38, using SEQ.ID.NO.: 67 and SEQ.ID.NO.: 70 as primers (using the above-noted cycle conditions). The resulting 1.44 kb PCR fragment was digested with BamHI and cloned into Blunt-BamHI site of pCMV expression vector. [0120]
  • 5. MC4 [0121]
  • To obtain MC4, PCR was performed using human genomic cDNA as template and rTth poymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 uM of each primer, and 0.2 mM of each 4 nucleotides. The cycle condition for each PCR reaction was 30 cycles of 94° C. for 1 min, 54° C. for 1 min and 72° C. for 1.5 min. The 5′ PCR contained an EcoRI site with the sequence: [0122]
  • 5′-CTGGAATTCTCCTGCCAGCATGGTGA-3′ (SEQ.ID.NO.: 71) [0123]
  • and the 3′ primer contained a BamHI site with the sequence: [0124]
  • 5′-GCAGGATCCTATATTGCGTGCTCTGTCCCC′-3 (SEQ.ID.NO.: 72). [0125]
  • The 1.0 kb PCR fragment was digest with EcoRI and BamHI and cloned into EcoRI-BamHI site of pCMV expression vector. Nucleic acid (SEQ.ID.NO.: 73) and amino acid (SEQ.ID.NO.: 74) sequences for human MC4 were thereafter determined. [0126]
  • 6. CCKB [0127]
  • To obtain CCKB, PCR was performed using human stomach cDNA as template and rTth poymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 uM of each primer, and 0.2 mM of each 4 nucleotides. The cycle condition for each PCR reaction was 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 1 min and 30 sec. The 5′ PCR contained a HindIII site with the sequence: [0128]
  • 5′-CCGAAGCTTCGAGCTGAGTAAGGCGGCGGGCT-3′ (SEQ.ID.NO.: 75) [0129]
  • and the 3′ primer contained an EcoRI site with the sequence: [0130]
  • 5′-GTGGAATTCATTTGCCCTGCCTCAACCCCCA-3 (SEQ.ID.NO.: 76). The resulting 1.44 kb PCR fragment was digest with HindIII and EcoRI and cloned into HindIII-EcoRI site of pCMV expression vector. Nucleic acid (SEQ.ID.NO.: 77) and amino acid (SEQ.ID.NO.: 78) sequences for human CCKB were thereafter determined. [0131]
  • 7. TDAG8 [0132]
  • To obtain TDAG8, PCR was performed using genomic DNA as template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 μM of each primer, and 0.2 mM of each 4 nucleotides. The cycle condition was 30 cycles of 94° C. for 1 min, 56° C. for 1 min and 72° C. for 1 min and 20 sec. The 5′PCR primer contained a HindIII site with the following sequence: [0133]
  • 5′-TGCAAGCTTAAAAAGGAAAAAATGAACAGC-3′ (SEQ.ID.NO.: 79) [0134]
  • and the 3′ primer contained a BamHI site with the following sequence: [0135]
  • 5′-TAAGGATCCCTTCCCTTCAAAACATCCTTG-3′ (SEQ.ID.NO.: 80). [0136]
  • The resulting 1.1 kb PCR fragment was digested with HindIII and BamHI and cloned into HindIII-BamHI site of pCMV expression vector. Three resulting clones sequenced contained three potential polymorphisms involving changes of amino acid 43 from Pro to Ala, amino acid 97 from Lys to Asn and amino acid 130 from Ile to Phe. Nucleic acid (SEQ.ID.NO.: 81) and amino acid (SEQ.ID.NO.: 82) sequences for human TDAG8 were thereafter determined. [0137]
  • 8. H9 [0138]
  • To obtain H9, PCR was performed using pituitary cDNA as template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 μM of each primer, and 0.2 mM of each 4 nucleotides. The cycle condition was 30 cycles of 94° C. for 1 min, 62° C. for 1 min and 72° C. for 2 min. The 5′ PCR primer contained a HindIII site with the following sequence: [0139]
  • 5′-GGAAAGCTTAACGATCCCCAGGAGCAACAT-3′ (SEQ.ID.NO.: 15) [0140]
  • and the 3′ primer contained a BamHI site with the following sequence: [0141]
  • 5′-CTGGGATCCTACGAGAGCATTTTCACACAG-3′ (SEQ.ID.NO.: 16). [0142]
  • The resulting 1.9 kb PCR fragment was digested with HindIII and BamHI and cloned into HindIII-BamHI site of pCMV expression vector. H9 contained three potential polymorphisms involving changes of amino acid P320S, S493N and amino acid G448A. Nucleic acid (SEQ.ID.NO.: 139) and amino acid (SEQ.ID.NO.: 140) sequences for human H9 were thereafter determined and verified. [0143]
    • Example 2
  • Preparation of Non-Endogenous, Constitutively Activated GPCRs [0144]
  • Those skilled in the art are credited with the ability to select techniques for mutation of a nucleic acid sequence. Presented below are approaches utilized to create non-endogenous versions of several of the human GPCRs disclosed above. The mutations disclosed below are based upon an algorithmic approach whereby the 16[0145] th amino acid (located in the IC3 region of the GPCR) from a conserved proline residue (located in the TM6 region of the GPCR, near the TM6/IC3 interface) is mutated, most preferably to a lysine amino acid residue.
  • 1. Tranformer Site-Directed™ Mutagenesis [0146]
  • Preparation of non-endogenous human GPCRs may be accomplished on human GPCRs using Transformer Site-Directed™ Mutagenesis Kit (Clontech) according to the manufacturer instructions. Two mutagenesis primers are utilized, most preferably a lysine mutagenesis oligonucleotide that creates the lysine mutation, and a selection marker oligonucleotide. For convenience, the codon mutation to be incorporated into the human GPCR is also noted, in standard form (Table E): [0147]
    TABLE E
    Receptor Identifier Codon Mutation
    hARE-3 F313K
    hARE-4 V233K
    hARE-5 A240K
    hGPCR14 L257K
    hGPCR27 C283K
    hARE-1 E232K
    hARE-2 G285K
    hPPR1 L239K
    hG2A K232A
    hRUP3 L224K
    hRUP5 A236K
    hRUP6 N267K
    hRUP7 A302K
    hCHN4 V236K
    hMC4 A244K
    hCHN3 S284K
    hCHN6 L352K
    hCHN8 N235K
    hCHN9 G223K
    hCHN10 L231K
    hH9 F236K
  • The following GPCRs were mutated according with the above method using the designated sequence primers (Table F). [0148]
    TABLE F
    Lysine Mutagenesis
    (SEQ.ID.NO.) Selection Marker
    Receptor Codon
    5′-3′ orientation, mutation (SEQ.ID.NO.)
    Identifier Mutation sequence underlined 5′-3′ orientation
    hRUP4 V272K CAGGAAGAAGAAACGAGC CACTGTCACCATCATAATG
    TGTCATTATGATGGTGACA ACAGCTCGTTTCTTCTTCC
    GTG (83) TG (84)
    hAT1 see below alternative approach; see below alternative approach; see below
    hGPR38 V297K GGCCACCGGCAGACCAAA CTCCTTCGGTCCTCCTATC
    CGCGTCCTGCTG (85) GTTGTCAGAAGT (86)
    hCCKB V332K alternative approach; see below alternative approach; see below
    hTDAG8 I225K GGAAAAGAAGAGAATCAA CTCCTTCGGTCCTCCTATC
    AAAACTACTTGTCAGCATC GTTGTCAGAAGT (88)
    (87)
    hH9 F236K GCTGAGGTTCGCAATAAAC CTCCTTCGGTCCTCCTATC
    TAACCATGTTTGTG (143) GTTGTCAGAAGT (144)
    hMC4 A244K GCCAATATGAAGGGAAAA CTCCTTCGGTCCTCCTATC
    ATTACCTTGACCATC (137) GTTGTCAGAAGT (138)
  • The non-endogenous human GPCRs were then sequenced and the derived and verified nucleic acid and amino acid sequences are listed in the accompanying “Sequence Listing” appendix to this patent document, as summarized in Table G below: [0149]
    TABLE G
    Non Endogenous Human Amino Acid Sequence
    GPCR Nucleic Acid Sequence Listing Listing
    hRUP4 SEQ.ID.NO.: 127 SEQ.ID.NO.: 128
    (V272K)
    hAT1 (see alternative approaches (see alternative approaches,
    (see alternative approaches below) below)
    below)
    hGPR38 SEQ.ID.NO.: 129 SEQ.ID.NO.: 130
    (V297K)
    hCCKB SEQ.ID.NO.: 131 SEQ.ID.NO.: 132
    (V332K)
    HTDAG8 SEQ.ID.NO.: 133 SEQ.ID.NO.: 134
    (I225K)
    hH9 SEQ.ID.NO.: 141 SEQ.ID.NO.: 142
    (F236K)
    hMC4 SEQ.ID.NO.: 135 SEQ.ID.NO.: 136
    (A244K)
  • 2. Alternative Approaches for Creation of Non-Endogenous Human GPCRs [0150]
  • a. AT1 [0151]
  • 1. F239K Mutation [0152]
  • Preparation of a non-endogenous, constitutively activated human AT1 receptor was accomplished by creating an F239K mutation (see, SEQ.ID.NO.: 89 for nucleic acid sequence, and SEQ.ID.NO.: 90 for amino acid sequence). Mutagenesis was performed using Transformer Site-Directed Mutagenesis™ Kit (Clontech) according to the to manufacturer's instructions. The two mutagenesis primers were used, a lysine mutagenesis oligonucleotide (SEQ.ID.NO.: 91) and a selection marker oligonucleotide (SEQ.ID.NO.: 92), which had the following sequences: [0153]
    (SEQ.ID.NO.: 91)
    5′-CCAAGAAATGATGATATTAAAAAGATAATTATGGC-3′
    (SEQ.ID.NO.: 92)
    5′-CTCCTTCGGTCCTCCTATCGTTGTCAGAAGT-3′,
    respectively.
  • 2. N111A Mutation [0154]
  • Preparation of a non-endogenous human AT1 receptor was also accomplished by creating an N111A mutation (see, SEQ.ID.NO.: 93 for nucleic acid sequence, and SEQ.ID.NO.: 94 for amino acid sequence). Two PCR reactions were performed using pfu polymerase (Stratagene) with the buffer system provided by the manufacturer, supplemented with 10% DMSO, 0.25 μM of each primer, and 0.5 mM of each 4 nucleotides. The 5′ PCR sense primer used had the following sequence: [0155]
  • 5′-CCCAAGCTTCCCCAGGTGTATTTGAT-3′ (SEQ.ID.NO.: 95) [0156]
  • and the antisense primer had the following sequence: [0157]
  • 5′-CCTGCAGGCGAAACTGACTCTGGCTGAAG-3′ (SEQ.ID.NO.: 96). [0158]
  • The resulting 400 bp PCR fragment was digested with HindIII site and subcloned into HindIII-SmaI site of pCMV vector (5′ construct). The 3′ PCR sense primer used had the following sequence: [0159]
  • 5′-CTGTACGCTAGTGTGTTTCTACTCACGTGTCTCAGCATTGAT-3′ (SEQ.ID.NO.: 97) [0160]
  • and the antisense primer had the following sequence: [0161]
  • 5′-GTTGGATCCACATAATGCATTTTCTC-3′ (SEQ.ID.NO.: 98) [0162]
  • The resulting 880 bp PCR fragment was digested with BamHI and inserted into Pst (blunted by T4 polymerase) and BamHI site of 5′ construct to generated the full length N111A construct. The cycle condition was 25 cycles of 94° C. for 1 min, 60° C. for 1 min and 72° C. for 1 min (5′ PCR) or 1.5 min (3′ PCR). [0163]
  • 3. AT2K255IC3 Mutation [0164]
  • Preparation of a non-endogenous, constitutively activated human AT1 was accomplished by creating an AT2K255IC3 “domain swap” mutation (see, SEQ.ID.NO.: 99 for nucleic acid sequence, and SEQ.ID.NO.: 100 for amino acid sequence). Restriction sites flanking IC3 of AT1 were generated to facilitate replacement of the IC3 with corresponding IC3 from [0165] angiotensin 11 type 2 receptor (AT2). This was accomplished by performing two PCR reactions. A 5′ PCR fragment (Fragment A) encoded from the 5′ untranslated region to the beginning of IC3 was generated by utilizing SEQ.ID.NO.: 63 as sense primer and the following sequence:
  • 5′-TCCGAATTCCAAAATAACTTGTAAGAATGATCAGAAA-3′ (SEQ.ID.NO.: 101) [0166]
  • as antisense primer. A 3′ PCR fragment (Fragment B) encoding from the end of IC3 to the 3′ untranslated region was generated by using the following sequence: [0167]
  • 5′-AGATCTTAAGAAGATAATATGGCAATTGTGCT-3′ (SEQ.ID.NO.: 102) [0168]
  • as sense primer and SEQ.ID.NO.: 64 as antisense primer. The PCR condition was 30 cycles of 94° C. for 1 min, 55° C. for 1 min and 72 ° C for 1.5 min using endogenous AT1 cDNA clone as template and pfu polymerase (Stratagene), with the buffer systems provided by the manufacturer, supplemented with 10% DMSO, 0.25 μM of each primer, and 0.5 mM of each 4 nucleotides. Fragment A (720 bp) was digested with HindIII and EcoRI and subcloned. Fragment B was digested with BamHI and subcloned into pCMV vector with an [0169] EcoRI site 5′ to the cloned PCR fragment.
  • The DNA fragment (Fragment C) encoding IC3 of AT2 with a L255K mutation and containing an EcoRI cohesive end at 5′ and a AflII cohesive end at 3′, was generated by annealing 2 synthetic oligonucleotides having the following sequences: [0170]
    (sense; SEQ.ID.NO.: 103)
    5′AATTCGAAAACACTTACTGAAGACGAATAGCTATGGGAAGAACAGGATAACCCGTGACCAAG-3′
    (antisense; SEQ.ID.NO.: 104)
    5′TTAACTTGGTCACGGGTTATCCTGTTCTTCCCATAGCTATTCGTCTTCAGTAAGTGTTTTCG-3′.
  • Fragment C was inserted in front of Fragment B through EcoRI and AflIII site. The resulting clone was then ligated with the Fragment A through the EcoRI site to generate AT1 with AT2K255IC3. [0171]
  • 4. A243+ Mutation [0172]
  • Preparation of a non-endogenous human AT1 receptor was also accomplished by creating an A243+ mutation (see, SEQ.ID.NO.: 105 for nucleic acid sequence, and SEQ.ID.NO.: 106 for amino acid sequence). An A243+ mutation was constructed using the following PCR based strategy: Two PCR reactions was performed using pfu polymerase (Stratagene) with the buffer system provided by the manufacturer supplemented with 10% DMSO, 0.25 μM of each primer, and 0.5 mM of each 4 nucleotides. The 5′ PCR sense primer utilized had the following sequence: [0173]
  • 5′-CCCAAGCTTCCCCAGGTGTATTTGAT-3′ (SEQ.ID.NO.: 107) [0174]
  • and the antisense primer had the following sequence: [0175]
  • 5′-AAGCACAATTGCTGCATAATTATCTTAAAAATATCATC-3′ (SEQ.ID.NO.: 108). [0176]
  • The 3′ PCR sense primer utilized had the following sequence: [0177]
  • 5′-AAGATAATTATGGCAGCAATTGTGCTTTTCTTTTTCTT-3′ (SEQ.ID.NO.: 109) [0178]
  • containing the Ala insertion and antisense primer: [0179]
  • 5′-GTTGGATCCACATAATGCATTTTCTC-3′ (SEQ.ID.NO.: 110). [0180]
  • The cycle condition was 25 cycles of 94° C for 1 min, 54° C. for 1 min and 72° C. for 1.5 min. An aliquot of the 5′ and 3′ PCR were then used as co-template to perform secondary PCR using the 5′ PCR sense primer and 3′ PCR antisense primer. The PCR condition was the same as primary PCR except the extention time was 2.5 min. The resulting PCR fragment was digested with HindIII and BamHI and subcloned into pCMV vector. (See, SEQ.ID.NO.: 105) [0181]
  • 4. CCKB [0182]
  • Preparation of the non-endogenous, constitutively activated human CCKB receptor was accomplished by creating a V322K mutation (see, SEQ.ID.NO.: 111 for nucleic acid sequence and SEQ.ID.NO.: 112 for amino acid sequence). Mutagenesis was performed by PCR via amplification using the wildtype CCKB from Example 1. [0183]
  • The first PCR fragment (1 kb) was amplified by using SEQ.ID.NO.: 75 and an antisense primer comprising a V322K mutation: [0184]
  • 5′-CAGCAGCATGCGCTTCACGCGCTTCTTAGCCCAG-3′ (SEQ.ID.NO.: 113). [0185]
  • The second PCR fragment (0.44 kb) was amplified by using a sense primer comprising the V322K mutation: [0186]
  • 5′-AGAAGCGCGTGAAGCGCATGCTGCTGGTGATCGTT-3′ (SEQ.ID.NO.: 114) and SEQ.ID.NO.: 76. [0187]
  • The two resulting PCR fragments were then used as template for amplifying CCKB comprising V332K, using SEQ.ID.NO.: 75 and SEQ.ID.NO.: 76 and the above-noted system and conditions. The resulting 1.44 kb PCR fragment containing the V332K mutation was digested with HindIII and EcoRI and cloned into HindIII-EcoRI site of pCMV expression vector. (See, SEQ.ID.NO.: 111). [0188]
  • 3. QuikChange™ Site-Directed™ Mutagenesis [0189]
  • Preparation of non-endogenous human GPCRs can also be accomplished by using QuikChange™ Site-Directed™ Mutagenesis Kit (Stratagene, according to manufacturer's instructions). Endogenous GPCR is preferably used as a template and two mutagenesis primers utilized, as well as, most preferably, a lysine mutagenesis oligonucleotide and a selection marker oligonucleotide (included in kit). For convenience, the codon mutation incorporated into the human GPCR and the respective oligonucleotides are noted, in standard form (Table H): [0190]
    TABLE H
    Lysine Mutagenesis
    (SEQ.ID.NO.) Selection Marker
    Receptor Codon
    5′-3′ orientation, (SEQ.ID.NO.)
    Identifier Mutation mutation underlined 5′-3′ orientation
    hCHN3 S284K ATGGAGAAAAGAATCAAAAGAA TATATAGAACATTCTTTT
    TGTTCTATATA (115) GATTCTTTTCTCCAT
    (116)
    hCHN6 L352K CGCTCTCTGGCCTTGAAGCGCAC GCTGAGCGTGCGCTTCA
    GCTCAGC (117) AGGCCAGAGAGCG (118)
    hCHN8 N235K CCCAGGAAAAAGGTGAAAGTCA GAAAACTTTGACTTTCA
    AAGTTTTC (119) CCTTTTTCCTGGG (120)
    hCHN9 G223K GGGGCGCGGGTGAAACGGCTGG GCTCACCAGCCGTTTCA
    TGAGC (121) CCCGCGCCCC (122)
    hCHN10 L231K CCCCTTGAAAAGCCTAAGAACTT GATGACCAAGTTCTTAG
    GGTCATC (123) GCTTTTCAAGGGG (124)
    • Example 3
  • Receptor Expression [0191]
  • Although a variety of cells are available to the art for the expression of proteins, it is most preferred that mammalian cells be utilized. The primary reason for this is predicated upon practicalities, i.e., utilization of, e.g., yeast cells for the expression of a GPCR, while possible, introduces into the protocol a non-mammalian cell which may not (indeed, in the case of yeast, does not) include the receptor-coupling, genetic-mechanism and secretary pathways that have evolved for mammalian systems—thus, results obtained in non-mammalian cells, while of potential use, are not as preferred as that obtained from mammalian cells. Of the mammalian cells, COS-7, 293 and 293T cells are particularly preferred, although the specific mammalian cell utilized can be predicated upon the particular needs of the artisan. [0192]
  • On day one, 1×10[0193] 7 293T cells per 150 mm plate were plated out. On day two, two reaction tubes were prepared (the proportions to follow for each tube are per plate): tube A was prepared by mixing 20 μg DNA (e.g., pCMV vector; pCMV vector with receptor cDNA, etc.) in 1.2 ml serum free DMEM (Irvine Scientific, Irvine, Calif.); tube B was prepared by mixing 120 μl lipofectamine (Gibco BRL) in 1.2 ml serum free DMEM. Tubes A and B were admixed by inversions (several times), followed by incubation at room temperature for 30-45 min. The admixture is referred to as the “transfection mixture”. Plated 293T cells were washed with 1×PBS, followed by addition of 10 ml serum free DMEM. 2.4 ml of the transfection mixture were added to the cells, followed by incubation for 4 hrs at 37° C./5% CO2. The transfection mixture was removed by aspiration, followed by the addition of 25 ml of DMEM/10% Fetal Bovine Serum. Cells were incubated at 37° C./5% CO2. After 72 hr incubation, cells were harvested and utilized for analysis.
    • Example 4
  • Assays for Determination of Constitutive Activity of Non-Endogenous GPCRs [0194]
  • A variety of approaches are available for assessment of constitutive activity of the non-endogenous human GPCRs. The following are illustrative; those of ordinary skill in the art are credited with the ability to determine those techniques that are preferentially beneficial for the needs of the artisan. [0195]
  • 1. Membrane Binding Assays: [[0196] 35S]GTPγS Assay
  • When a G protein-coupled receptor is in its active state, either as a result of ligand binding or constitutive activation, the receptor couples to a G protein and stimulates the release of GDP and subsequent binding of GTP to the G protein. The alpha subunit of the G protein-receptor complex acts as a GTPase and slowly hydrolyzes the GTP to GDP, at which point the receptor normally is deactivated. Constitutively activated receptors continue to exchange GDP for GTP. The non-hydrolyzable GTP analog, [[0197] 35S]GTPγS, can be utilized to demonstrate enhanced binding of [35S]GTPγS to membranes expressing constitutively activated receptors. The advantage of using [35S]GTPγS binding to measure constitutive activation is that: (a) it is generically applicable to all G protein-coupled receptors; (b) it is proximal at the membrane surface making it less likely to pick-up molecules which affect the intracellular cascade.
  • The assay utilizes the ability of G protein coupled receptors to stimulate [[0198] 35S]GTPγS binding to membranes expressing the relevant receptors. The assay can, therefore, be used in the direct identification method to screen candidate compounds to known, orphan and constitutively activated G protein-coupled receptors. The assay is generic and has application to drug discovery at all G protein-coupled receptors.
  • The [[0199] 35S]GTPγS assay can be incubated in 20 mM HEPES and between 1 and about 20 mM MgCl2 (this amount can be adjusted for optimization of results, although 20 mM is preferred) pH 7.4, binding buffer with between about 0.3 and about 1.2 nM [35S]GTPγS (this amount can be adjusted for optimization of results, although 1.2 is preferred) and 12.5 to 75 μg membrane protein (e.g, COS-7 cells expressing the receptor; this amount can be adjusted for optimization, although 75 μg is preferred) and 1 μM GDP (this amount can be changed for optimization) for 1 hour. Wheatgerm agglutinin beads (25 μl; Amersham) should then be added and the mixture incubated for another 30 minutes at room temperature. The tubes are then centrifuged at 1500×g for 5 minutes at room temperature and then counted in a scintillation counter.
  • A less costly but equally applicable alternative has been identified which also meets the needs of large scale screening. Flash plates™ and Wallac™ scintistrips may be utilized to format a high throughput [[0200] 35S]GTPγS binding assay. Furthermore, using this technique, the assay can be utilized for known GPCRs to simultaneously monitor tritiated ligand binding to the receptor at the same time as monitoring the efficacy via [35S]GTPγS binding. This is possible because the Wallac beta counter can switch energy windows to look at both tritium and 35S-labeled probes. This assay may also be used to detect other types of membrane activation events resulting in receptor activation. For example, the assay may be used to monitor 32P phosphorylation of a variety of receptors (both G protein coupled and tyrosine kinase receptors). When the membranes are centrifuged to the bottom of the well, the bound [35S]GTPγS or the 32P-phosphorylated receptor will activate the scintillant which is coated of the wells. Scinti® strips (Wallac) have been used to demonstrate this principle. In addition, the assay also has utility for measuring ligand binding to receptors using radioactively labeled ligands. In a similar manner, when the radiolabeled bound ligand is centrifuged to the bottom of the well, the scintistrip label comes into proximity with the radiolabeled ligand resulting in activation and detection.
  • 2. Adenylyl Cyclase [0201]
  • A Flash Plate™ Adenylyl Cyclase kit (New England Nuclear; Cat. No. SMP004A) designed for cell-based assays can be modified for use with crude plasma membranes. The Flash Plate wells contain a scintillant coating which also contains a specific antibody recognizing cAMP. The cAMP generated in the wells was quantitated by a direct competition for binding of radioactive cAMP tracer to the cAMP antibody. The following serves as a brief protocol for the measurement of changes in cAMP levels in membranes that express the receptors. [0202]
  • Transfected cells are harvested approximately three days after transfection. Membranes were prepared by homogenization of suspended cells in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCl[0203] 2. Homogenization is performed on ice using a Brinkman Polytron™ for approximately 10 seconds. The resulting homogenate is centrifuged at 49,000×g for 15 minutes at 4° C. The resulting pellet is then resuspended in buffer containing 20 mM HEPES, pH 7.4 and 0.1 mM EDTA, homogenized for 10 seconds, followed by centrifugation at 49,000×g for 15 minutes at 4° C. The resulting pellet can be stored at −80° C. until utilized. On the day of measurement, the membrane pellet is slowly thawed at room temperature, resuspended in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCL2 (these amounts can be optimized, although the values listed herein are preferred), to yield a final protein concentration of 0.60 mg/ml (the resuspended membranes were placed on ice until use).
  • cAMP standards and Detection Buffer (comprising 2 μCi of tracer [[0204] 125I cAMP (100 μl] to 11 ml Detection Buffer) are prepared and maintained in accordance with the manufacturer's instructions. Assay Buffer is prepared fresh for screening and contained 20 mM HEPES, pH 7.4, 10 mM MgCl2, 20 mM (Sigma), 0.1 units/ml creatine phosphokinase (Sigma), 50 μM GTP (Sigma), and 0.2 mM ATP (Sigma); Assay Buffer can be stored on ice until utilized. The assay is initiated by addition of 50 ul of assay buffer followed by addition of 50 ul of membrane suspension to the NEN Flash Plate. The resultant assay mixture is incubated for 60 minutes at room temperature followed by addition of 100 ul of detection buffer. Plates are then incubated an additional 2-4 hours followed by counting in a Wallac MicroBeta™ scintillation counter. Values of cAMP/well are extrapolated from a standard cAMP curve that is contained within each assay plate.
  • C. Reporter-Based Assays [0205]
  • 1. CREB Reporter Assay (Gs-Associated Receptors) [0206]
  • A method to detect Gs stimulation depends on the known property of the transcription factor CREB, which is activated in a cAMP-dependent manner. A PathDetect™ CREB trans-Reporting System (Stratagene, Catalogue #219010) can utilized to assay for Gs coupled activity in 293 or 293T cells. Cells are transfected with the plasmids components of this above system and the indicated expression plasmid encoding endogenous or mutant receptor using a Mammalian Transfection Kit (Stratagene, Catalogue #200285) according to the manufacturer's instructions. Briefly, 400 ng pFR-Luc (luciferase reporter plasmid containing Gal4 recognition sequences), 40 ng pFA2-CREB (Gal4-CREB fusion protein containing the Gal4 DNA-binding domain), 80 ng pCMV-receptor expression plasmid (comprising the receptor) and 20 ng CMV-SEAP (secreted alkaline phosphatase expression plasmid; alkaline phosphatase activity is measured in the media of transfected cells to control for variations in transfection efficiency between samples) are combined in a calcium phosphate precipitate as per the Kit's instructions. Half of the precipitate is equally distributed over 3 wells in a 96-well plate, kept on the cells overnight, and replaced with fresh medium the following morning. Forty-eight (48) hr after the start of the transfection, cells are treated and assayed for, e.g., luciferase activity [0207]
  • 2. AP1 Reporter Assay (Gq-Associated Receptors) [0208]
  • A method to detect Gq stimulation depends on the known property of Gq-dependent phospholipase C to cause the activation of genes containing AP1 elements in their promoter. A Pathdetect™ AP-1 cis-Reporting System (Stratagene, Catalogue #219073) can be utilized following the protocol set forth above with respect to the CREB reporter assay, except that the components of the calcium phosphate precipitate were 410 ng pAP1-Luc, 80 ng pCMV-receptor expression plasmid, and 20 ng CMV-SEAP. [0209]
  • 3. CRE-Luc Reporter Assay [0210]
  • 293 and 293T cells are plated-out on 96 well plates at a density of 2×10[0211] 4 cells per well and were transfected using Lipofectamine Reagent (BRL) the following day according to manufacturer instructions. A DNA/lipid mixture is prepared for each 6-well transfection as follows: 260 ng of plasmid DNA in 100 μl of DMEM were gently mixed with 2 μl of lipid in 100 μl of DMEM (the 260 ng of plasmid DNA consisted of 200 ng of a 8×CRE-Luc reporter plasmid (see below and FIG. 1 for a representation of a portion of the plasmid), 50 ng of pCMV comprising endogenous receptor or non-endogenous receptor or pCMV alone, and 10 ng of a GPRS expression plasmid (GPRS in pcDNA3 (Invitrogen)). The 8×CRE-Luc reporter plasmid was prepared as follows: vector SRIF-β-gal was obtained by cloning the rat somatostatin promoter (−71/+51) at BglV-HindIII site in the pβgal-Basic Vector (Clontech). Eight (8) copies of cAMP response element were obtained by PCR from an adenovirus template AdpCF126CCRE8 (see, 7 Human Gene Therapy 1883 (1996)) and cloned into the SRIF-β-gal vector at the Kpn-BglV site, resulting in the 8×CRE-β-gal reporter vector. The 8×CRE-Luc reporter plasmid was generated by replacing the beta-galactosidase gene in the 8×CRE-β-gal reporter vector with the luciferase gene obtained from the pGL3-basic vector (Promega) at the HindIII-BamHI site. Following 30 min. incubation at room temperature, the DNA/lipid mixture was diluted with 400 μl of DMEM and 100 μl of the diluted mixture was added to each well. 100 μl of DMEM with 10% FCS were added to each well after a 4 hr incubation in a cell culture incubator. The following day the transfected cells were changed with 200 μl/well of DMEM with 10% FCS. Eight (8) hours later, the wells were changed to 100 μl/well of DMEM without phenol red, after one wash with PBS. Luciferase activity were measured the next day using the LucLite™ reporter gene assay kit (Packard) following manufacturer instructions and read on a 1450 MicroBeta™ scintillation and luminescence counter (Wallac).
  • 4. SRF-LUC Reporter Assay [0212]
  • One method to detect Gq stimulation depends on the known property of Gq-dependent phospholipase C to cause the activation of genes containing serum response factors in their promoter. A Pathdetect™ SRF-Luc-Reporting System (Stratagene) can be utilized to assay for Gq coupled activity in, e.g., COS7 cells. Cells are transfected with the plasmid components of the system and the indicated expression plasmid encoding endogenous or non-endogenous GPCR using a Mammalian Transfection™ Kit (Stratagene, Catalogue #200285) according to the manufacturer's instructions. Briefly, 410 ng SRF-Luc, 80 ng pCMV-receptor expression plasmid and 20 ng CMV-SEAP (secreted alkaline phosphatase expression plasmid; alkaline phosphatase activity is measured in the media of transfected cells to control for variations in transfection efficiency between samples) are combined in a calcium phosphate precipitate as per the manufacturer's instructions. Half of the precipitate is equally distributed over 3 wells in a 96-well plate, kept on the cells in a serum free media for 24 hours. The last 5 hours the cells are incubated with 1 μM Angiotensin, where indicated. Cells are then lysed and assayed for luciferase activity using a Luclite™ Kit (Packard, Cat. #6016911) and “Trilux 1450 Microbeta” liquid scintillation and luminescence counter (Wallac) as per the manufacturer's instructions. The data can be analyzed using GraphPad Prism™ 2.0a (GraphPad Software Inc.). [0213]
  • 5. Intracellular IP[0214] 3 Accumulation Assay
  • On [0215] day 1, cells comprising the receptors (endogenous and/or non-endogenous) can be plated onto 24 well plates, usually 1×105 cells/well (although his umber can be optimized. On day 2 cells can be transfected by firstly mixing 0.25 ug DNA in 50 ul serum free DMEM/well and 2 ul lipofectamine in 50 μl serumfree DMEM/well. The solutions are gently mixed and incubated for 15-30 min at room temperature. Cells are washed with 0.5 ml PBS and 400 μl of serum free media is mixed with the transfection media and added to the cells. The cells are then incubated for 3-4 hrs at 37° C./5% CO2 and then the transfection media is removed and replaced with 1 ml/well of regular growth media. On day 3 the cells are labeled with 3H-myo-inositol. Briefly, the media is removed and the cells are washed with 0.5 ml PBS. Then 0.5 ml inositol-free/serum free media (GIBCO BRL) is added/well with 0.25 μCi of 3H-myo-inositol/well and the cells are incubated for 16-18 hrs o/n at 37° C./5% CO2. On Day 4 the cells are washed with 0.5 ml PBS and 0.45 ml of assay medium is added containing inositol-free/serum free media 10 μM pargyline 10 mM lithium chloride or 0.4 ml of assay medium and 50 ul of 10× ketanserin (ket) to final concentration of 10 μM. The cells are then incubated for 30 min at 37° C. The cells are then washed with 0.5 ml PBSand 200 ul of fresh/icecold stop solution (1M KOH; 18 mM Na-borate; 3.8 mM EDTA) is added/well. The solution is kept on ice for 5-10 min or until cells were lysed and then neutralized by 200 μl of fresh/ice cold neutralization sol. (7.5% HCL). The lysate is then transferred into 1.5 ml eppendorf tubes and 1 ml of chloroform/methanol (1:2) is added/tube. The solution is vortexed for 15 sec and the upper phase is applied to a Biorad AG1-X8™ anion exchange resin (100-200 mesh). Firstly, the resin is washed with water at 1:1.25 W/V and 0.9 ml of upper phase is loaded onto the column. The column is washed with 10 mls of 5 mM myo-inositol and 10 ml of 5 mM Na-borate/60 mM Na-formate. The inositol tris phosphates are eluted into scintillation vials containing 10 ml of scintillation cocktail with 2 ml of 0.1 M formic acid/1 M ammonium formate. The columns are regenerated by washing with 10 ml of 0.1 M formic acid/3M ammonium formate and rinsed twice with dd H2O and stored at 4° C. in water.
  • Exemplary results are presented below in Table I: [0216]
    TABLE I
    Signal Signal
    Generated: Generated:
    Endogenous Non-Endogenous
    Version Version
    Assay (Relative (Relative Percent
    Receptor Mutation Utilized Light Units) Light Units) Difference
    hAT1 F239K SRF-LUC 34 137 75%↑
    AT2K255IC3 SRF-LUC 34 127 73%↑
    hTDAG8 I225K CRE-LUC 2,715 14,440 81%↑
    (293 cells)
    I225K CRE-LUC 65,681 185,636 65%↑
    (293T cells)
    hH9 F236K CRE-LUC 1,887 6,096 69%↑
    hCCKB V332K CRE-LUC 785 3,223 76%↑
  • C. Cell-Based Detection Assay (Example—T[0217] DAG8)
  • 293 cells were plated-out on 150 mm plates at a density of 1.3×10[0218] 7 cells per plate, and were transfected using 12 ug of the respective DNA and 60 ul of Lipofectamine Reagent (BRL) per plate. The transfected cells were grown in media containing serum for an assay performed 24 hours post-transfection. For detection assay performed 48 hours post-transfection (assay comparing serum and serum-free media; see FIG. 3), the initial media was changed to either serum or serum-free media. The serum-free media was comprised solely of Dulbecco's Modified Eagle's (DME) High Glucose Medium (Irvine Scientific #9024). In addition to the above DME Medium, the media with serum contained the following: 10% Fetal Bovine Serum (Hyclone #SH30071.03), 1% of 100 mM Sodium Pyruvate (Irvine Scientific #9334),1% of20 mM L-Glutamine (Irvine Scientific #9317), and 1% of Penicillin-Streptomycin solution (Irvine Scientific #9366).
  • A 96-well Adenylyl Cyclase Activation Flashplate™ was used (NEN: #SMP004A). First, 50 ul of the standards for the assay were added to the plate, in duplicate, ranging from concentrations of 50 pmol to zero pmol cAMP per well. The standard cAMP (NEN: #SMP004A) was reconstituted in water, and serial dilutions were made using 1×PBS (Irvine Scientific: #9240). Next, 50 ul of the stimulation buffer (NEN: #SMP004A) was added to all wells. In the case of using compounds to measure activation or inactivation of cAMP, 10 ul of each compound, diluted in water, was added to its respective well, in triplicate. Various final concentrations used range from 1 uM up to 1 mM. [0219] Adenosine 5′-triphosphate, ATP, (Research Biochemicals International: #A-141) and Adenosine 5′-diphosphate, ADP, (Sigma: #A2754) were used in the assay. Next, the 293 cells transfected with the respective cDNA (CMV or TDAG8) were harvested 24 (assay detection in serum media) or 48 hours post-transfection (assay detection comparing serum and serum-free media). The media was aspirated and the cells washed once with 1×PBS. Then 5 ml of 1×PBS was added to the cells along with 3 ml of cell dissociation buffer (Sigma: #C-1544). The detached cells were transferred to a centrifuge tube and centrifuged at room temperature for five minutes. The supernatant was removed and the cell pellet was resuspended in an appropriate amount of 1×PBS to obtain a final concentration of 2×106 cells per milliliter. To the wells containing the compound, 50 ul of the cells in 1×PBS (1×105 cells/well) were added. The plate was incubated on a shaker for 15 minutes at room temperature. The detection buffer containing the tracer cAMP was prepared. In 11 ml of detection buffer (NEN: #SMP004A), 50 ul (equal to 1 uCi) of [125I]cAMP (NEN: #SMP004A) was added. Following incubation, 50 ul of this detection buffer containing tracer cAMP was added to each well. The plate was placed on a shaker and incubated at room temperature for two hours. Finally, the solution from the wells of the plate were aspirated and the flashplate was counted using the Wallac MicroBeta™ scintillation counter.
  • In FIG. 2A, ATP and ADP bind to endogenous TDAG8 resulting in an increase of cAMP of about 59% and about 55% respectively. FIG. 2B evidences ATP and ADP binding to endogenous TDAG8 where endogenous TDAG8 was transfected and grown in serum and serum-free medium. ATP binding to endogenous TDAG8 grown in serum media evidences an increase in cAMP of about 65%, compared to the endogenous TDAG8 with no compounds; in serum-free media there was an increase of about 68%. ADP binding to endogenous TDAG8 in serum evidences about a 61% increase, while in serum-free ADP binding evidences an increase of about 62% increase. ATP and ADP bind to endogenous TDAG8 with an EC50 value of 139.8 uM and 120.5 uM, respectively (data not shown). [0220]
  • Although the results presented in FIG. 2B indicate substantially the same results when serum and serum-free media were compared, our choice is to use a serum based media, although a serum-free media can also be utilized. [0221]
    • Example 6
  • GPCR Fusion Protein Preparation [0222]
  • The design of the constitutively activated GPCR-G protein fusion construct was accomplished as follows: both the 5′ and 3′ ends of the rat G protein Gsα (long form; Itoh, H. et al., 83 [0223] PNAS 3776 (1986)) were engineered to include a HindIII (5′-AAGCTT-3′) sequence thereon. Following confirmation of the correct sequence (including the flanking HindIII sequences), the entire sequence was shuttled into pcDNA3.1(−) (Invitrogen, cat. no. V795-20) by subcloning using the HindIII restriction site of that vector. The correct orientation for the Gsα sequence was determined after subcloning into pcDNA3.1(−). The modified pcDNA3.1(−) containing the rat Gsα gene at HindIII sequence was then verified; this vector was now available as a “universal” Gsα protein vector. The pcDNA3.1(−) vector contains a variety of well-known restriction sites upstream of the HindIII site, thus beneficially providing the ability to insert, upstream of the Gs protein, the coding sequence of an endogenous, constitutively active GPCR. This same approach can be utilized to create other “universal” G protein vectors, and, of course, other commercially available or proprietary vectors known to the artisan can be utilized—the important criteria is that the sequence for the GPCR be upstream and in-frame with that of the G protein.
  • TDAG8 couples via Gs, while H9 couples via Gz. For the following exemplary GPCR Fusion Proteins, fusion to Gsα was accomplished. [0224]
  • A TDAG8(1225K)-Gsα Fusion Protein construct was made as follows: primers were designed as follows: [0225]
    5′-gatcTCTAGAATGAACAGCACATGTATTGAAG-3′ (SEQ.ID.NO.: 125; sense)
    5′-ctagGGTACCCGCTCAAGGACCTCTAATTCCATAG-3′ (SEQ.ID.NO.: 126; antisense)
  • Nucleotides in lower caps are included as spacers in the restriction sites between the G protein and TDAG8. The sense and anti-sense primers included the restriction sites for XbaI and KpnI, respectively. [0226]
  • PCR was then utilized to secure the respective receptor sequences for fusion within the Gsα universal vector disclosed above, using the following protocol for each: 100 ng cDNA for TDAG8 was added to separate tubes containing 2 ul of each primer (sense and anti-sense), 3 uL of 10 mM dNTPs, 10 uL of 10×TaqPlus™ Precision buffer, 1 uL of TaqPlus™ Precision polymerase (Stratagene: #600211), and 80 uL of water. Reaction temperatures and cycle times for TDAG8 were as follows: the initial denaturing step was done it 94° C. for five minutes, and a cycle of 94° C. for 30 seconds; 55° C. for 30 seconds; 72° C. for two minutes. A final extension time was done at 72° C. for ten minutes. PCR product for was run on a 1% agarose gel and then purified (data not shown). The purified product was digested with XbaI and KpnI (New England Biolabs) and the desired inserts purified and ligated into the Gs universal vector at the respective restriction site. The positive clones was isolated following transformation and determined by restriction enzyme digest; expression using 293 cells was accomplished following the protocol set forth infra. Each positive clone for TDAG8:Gs-Fusion Protein was sequenced to verify correctness. [0227]
  • GPCR Fusion Proteins comprising non-endogenous, constitutively activated TDAG8(1225K) were analyzed as above and verified for constitutive activation. [0228]
  • An H9(F236K)-Gsα Fusion Protein construct was made as follows: primers were designed as follows: [0229]
    5′-TTAgatatcGGGGCCCACCCTAGCGGT-3′ (SEQ.ID.NO.: 145; sense)
    5′-ggtaccCCCACAGCCATTTCATCAGGATC-3′. (SEQ.ID.NO.: 146; antisense)
  • Nucleotides in lower caps are included as spacers in the restriction sites between the G protein and H9. The sense and anti-sense primers included the restriction sites for EcoRV and KpnI, respectively such that spacers (attributed to the restriction sites) exists between the G protein and H9. [0230]
  • PCR was then utilized to secure the respective receptor sequences for fusion within the Gsα universal vector disclosed above, using the following protocol for each: 80 ng cDNA for H9 was added to separate tubes containing 100 ng of each primer (sense and anti-sense), and 45 uL of PCR Supermix™ (Gibco-Brl, LifeTech) (50 ul total reaction volume). Reaction temperatures and cycle times for H9 were as follows: the initial denaturing step was done it 94° C. for one, and a cycle of 94° C. for 30 seconds; 55° C. for 30 seconds; 72° C. for two minutes. A final extension time was done at 72° C. for seven minutes. PCR product for was run on a 1% agarose gel and then purified (data not shown). The purified product was cloned into pCRII-TOPO™ System followed by identification of positive clones. Positive clones were isolated, digested with EcoRV and KpnI (New England Biolabs) and the desired inserts were isolated, purified and ligated into the Gs universal vector at the respective restriction site. The positive clones was isolated following transformation and determined by restriction enzyme digest; expression using 293 cells was accomplished following the protocol set forth infra. Each positive clone for H9(F236K):Gs—Fusion Protein was sequenced to verify correctness. Membranes were frozen (−80° C.) until utilized. [0231]
  • To ascertain the ability of measuring a cAMP response mediated by the Gs protein (even though H9 couples with Gz), the following cAMP membrane assay was utilized, based upon an NEN Adenyl Cyclase Activation Flahplate™ Assay kit (96 well format). “Binding Buffer” consisted of 10 mM HEPES, 100 mM NaCl and 10 mM MgCl (ph 7.4). “Regeneration Buffer” was prepared in Binding Buffer and consisted of 20 mM phosphocreatine, 20U creatine phosphokinase, 20 uM GTP, 0.2 mM ATP, and 0.6 mM IBMX. “cAMP Standards” were prepared in Binding Buffer as follows: [0232]
    cAMP Stock Added to indicted Final Assay Concentration
    (5,000 pmol/ml in amount of Binding (50 ul into 100 ul)
    2 ml H2O) in ul Buffer to achieve indicated pmol/well
    A 250 1 ml 50
    B 500 of A 500 ul 25
    C 500 of B 500 ul 12.5
    D 500 of C 750 ul 5.0
    E 500 of D 500 ul 2.5
    F 500 of E 500 ul 1.25
    G 500 of F 750 ul 0.5
  • Frozen membranes (both pCMV as control and the non-endogenous H(-Gs Fusion Protein) were thawed (on ice at room temperature until in solution). Membranes were homogenized with a polytron until in suspension (2×15 seconds). Membrane protein concentration was determined using the Bradford Assay Protocol (see infra). Membrane concentration was diluted to 0.5 mg/ml in Regeneration Buffer (final assay concentration—25 ug/well). Thereafter, 50 ul of Binding Buffer was added to each well. For control, 50 ul/well of cAMP standard was added to [0233] wells 11 and 12 A-G, with Binding Buffer alone to 12H (on the 96-well format). Thereafter, 50 ul/well of protein was added to the wells and incubated at room temperature (on shaker) for 60 min. 100 ul[125I]cAMP in Detection Buffer (see infra) was added to each well (final −50 ul[125I]cAMP into 11 ml Detection Buffer). These were incubated for 2 hrs at room temperature. Plates were aspirated with an 8 channel manifold and sealed with plate covers. Results (pmoles cAMP bound) were read in a Wallac™ 1450 on “prot #15). Results are presented in FIG. 3.
  • The results presented in FIG. 3 indicate that the Gs coupled fusion was able to “drive” the cyclase reaction such that measurement of the consitutive activation of H9(F236K) was viable. Based upon these results, the direct identification of candidate compounds that are inverse agonists, agonists and partial agonists is possible using a cyclase-based assay. [0234]
    • Example 6
  • Protocol: Direct Identification of Inverse Agonists and Agonists Using [[0235] 35S]GTPγS
  • Although we have utilized endogenous, constitutively active GPCRs for the direct identification of candidate compounds as, e.g., inverse agonists, for reasons that are not altogether understood, intra-assay variation can become exacerbated. Preferably, then, a GPCR Fusion Protein, as disclosed above, is also utilized with a non-endogenous, constitutively activated GPCR. We have determined that when such a protein is used, intra-assay variation appears to be substantially stabilized, whereby an effective signal-to-noise ratio is obtained. This has the beneficial result of allowing for a more robust identification of candidate compounds. Thus, it is preferred that for direct identification, a GPCR Fusion Protein be used and that when utilized, the following assay protocols be utilized. [0236]
  • Membrane Preparation [0237]
  • Membranes comprising the non-endogenous, constitutively active orphan GPCR Fusion Protein of interest and for use in the direct identification of candidate compounds as inverse agonists, agonists or partial agonists are preferably prepared as follows: [0238]
  • a. Materials [0239]
  • “Membrane Scrape Buffer” is comprised of 20 mM HEPES and 10 mM EDTA, pH 7.4; “Membrane Wash Buffer” is comprised of 20 mM HEPES and 0.1 mM EDTA, pH 7.4; “Binding Buffer” is comprised of 20 mM HEPES, 100 mM NaCl, and 10 mM MgCl[0240] 2, pH 7.4
  • b. Procedure [0241]
  • All materials are kept on ice throughout the procedure. Firstly, the media is aspirated from a confluent monolayer of cells, followed by rinse with 10 ml cold PBS, followed by aspiration. Thereafter, 5 ml of Membrane Scrape Buffer is added to scrape cells; this is followed by transfer of cellular extract into 50 ml centrifuge tubes (centrifuged at 20,000 rpm for 17 minutes at 4° C.). Thereafter, the supernatant is aspirated and the pellet is resuspended in 30 ml Membrane Wash Buffer followed by centrifuge at 20,000 rpm for 17 minutes at 4° C. The supernatant is then aspirated and the pellet resuspended in Binding Buffer. This is then homogenized using a Brinkman polytron™ homogenizer (15-20 second bursts until the all material is in suspension). This is referred to herein as “Membrane Protein”. [0242]
  • Bradford Protein Assay [0243]
  • Following the homogenization, protein concentration of the membranes is determined using the Bradford Protein Assay (protein can be diluted to about 1.5 mg/ml, aliquoted and frozen (−80° C.) for later use; when frozen, protocol for use is as follows: on the day of the assay, frozen Membrane Protein is thawed at room temperature, followed by vortex and then homogenized with a polytron at about 12×1,000 rpm for about 5-10 seconds; it is noted that for multiple preparations, the homogenizer should be thoroughly cleaned between homoginezation of different preparations). [0244]
  • a. Materials [0245]
  • Binding Buffer (as per above); Bradford Dye Reagent; Bradford Protein Standard are utilized, following manufacturer instructions (Biorad, cat. no. 500-0006). [0246]
  • b. Procedure [0247]
  • Duplicate tubes are prepared, one including the membrane, and one as a control “blank”. Each contained 800 ul Binding Buffer. Thereafter, 10 ul of Bradford Protein Standard (1 mg/ml) is added to each tube, and 10 ul of membrane Protein is then added to just one tube (not the blank). Thereafter, 200 ul of Bradford Dye Reagent is added to each tube, followed by vortex of each. After five (5) minutes, the tubes were re-vortexed and the material therein is transferred to cuvettes. The cuvettes are then read using a CECIL 3041 spectrophotometer, at wavelength 595. [0248]
  • Direct Identification Assay [0249]
  • a. Materials [0250]
  • GDP Buffer consists of 37.5 ml Binding Buffer and 2 mg GDP (Sigma, cat. no. G-7127), followed by a series of dilutions in Binding Buffer to obtain 0.2 uM GDP (final concentration of GDP in each well was 0.1 uM GDP); each well comprising a candidate compound, has a final volume of 200 ul consisting of 100 ul GDP Buffer (final concentration, 0.1 uM GDP), 50 ul Membrane Protein in Binding Buffer, and 50 ul [[0251] 35S]GTPγS (0.6 nM) in Binding Buffer (2.5 ul [35S]GTPγS per 10 ml Binding Buffer).
  • b. Procedure [0252]
  • Candidate compounds are preferably screened using a 96-well plate format (these can be frozen at −80° C.). Membrane Protein (or membranes with expression vector excluding the GPCR Fusion Protein, as control), are homogenized briefly until in suspension. Protein concentration is then determined using the Bradford Protein Assay set forth above. Membrane Protein (and control) is then diluted to 0.25 mg/ml in Binding Buffer (final assay concentration, 12.5 ug/well). Thereafter, 100 ul GDP Buffer is added to each well of a Wallac Scintistrip™ (Wallac). A 5 ul pin-tool is then used to transfer 5 ul of a candidate compound into such well (i.e., 5 ul in total assay volume of 200 ul is a 1:40 ratio such that the final screening concentration of the candidate compound is 10 uM). Again, to avoid contamination, after each transfer step the pin tool should be rinsed in three reservoirs comprising water (1×), ethanol (1×) and water (2×)—excess liquid should be shaken from the tool after each rinse and dried with paper and kimwipes. Thereafter, 50 ul of Membrane Protein is added to each well (a control well comprising membranes without the GPCR Fusion Protein is also utilized), and pre-incubated for 5-10 minutes at room temperature. Thereafter, 50 ul of [[0253] 35S]GTPγS (0.6 nM) in Binding Buffer is added to each well, followed by incubation on a shaker for 60 minutes at room temperature (again, in this example, plates were covered with foil). The assay is then stopped by spinning of the plates at 4000 RPM for 15 minutes at 22° C. The plates are then aspirated with an 8 channel manifold and sealed with plate covers. The plates are then read on a Wallacc 1450 using setting “Prot. #37” (as per manufacturer instructions).
    • Example 7
  • Protocol: Confirmation Assay [0254]
  • Using an independent assay approach to provide confirmation of a directly identified candidate compound as set forth above, it is preferred that a confirmation assay then be utilized. In this case, the preferred confirmation assay is a cyclase-based assay. [0255]
  • A modified Flash Plate™ Adenylyl Cyclase kit (New England Nuclear; Cat. No. SMP004A) is preferably utilized for confirmation of candidate compounds directly identified as inverse agonists and agonists to non-endogenous, constitutively activated orphan GPCRs in accordance with the following protocol. [0256]
  • Transfected cells are harvested approximately three days after transfection. Membranes are prepared by homogenization of suspended cells in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCl[0257] 2. Homogenization is performed on ice using a Brinkman Polytron™ for approximately 10 seconds. The resulting homogenate is centrifuged at 49,000×g for 15 minutes at 4° C. The resulting pellet is then resuspended in buffer containing 20 mM HEPES, pH 7.4 and 0.1 mM EDTA, homogenized for 10 seconds, followed by centrifugation at 49,000×g for 15 minutes at 4° C. The resulting pellet can be stored at −80° C. until utilized. On the day of direct identification screening, the membrane pellet is slowly thawed at room temperature, resuspended in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCL2, to yield a final protein concentration of 0.60 mg/ml (the resuspended membranes are placed on ice until use).
  • cAMP standards and Detection Buffer (comprising 2 μCi of tracer [[0258] 125I ]cAMP (100 μl] to 11 ml Detection Buffer) are prepared and maintained in accordance with the manufacturer's instructions. Assay Buffer is prepared fresh for screening and contained 20 mM HEPES, pH 7.4, 10 mM MgCl2, 20 mM phospocreatine (Sigma), 0.1 units/ml creatine phosphokinase (Sigma), 50 μM GTP (Sigma), and 0.2 mM ATP (Sigma); Assay Buffer can be stored on ice until utilized.
  • Candidate compounds identified as per above (if frozen, thawed at room temperature) are added, preferably, to 96-well plate wells (3 μl/well; 12 μM final assay concentration), together with 40 μl Membrane Protein (30 μg/well) and 50 μl of Assay Buffer. This admixture is then incubated for 30 minutes at room temperature, with gentle shaking. [0259]
  • Following the incubation, 100 μl of Detection Buffer is added to each well, followed by incubation for 2-24 hours. Plates are then counted in a Wallac MicroBeta™ plate reader using “Prot. #31” (as per manufacturer instructions). [0260]
  • It is intended that each of the patents, applications, and printed publications mentioned in this patent document be hereby incorporated by reference in their entirety. [0261]
  • As those skilled in the art will appreciate, numerous changes and modifications may be made to the preferred embodiments of the invention without departing from the spirit of the invention. It is intended that all such variations fall within the scope of the invention. [0262]
  • Although a variety of expression vectors are available to those in the art, for purposes of utilization for both the endogenous and non-endogenous human GPCRs, it is most preferred that the vector utilized be pCMV. This vector was deposited with the American Type Culture Collection (ATCC) on Oct. 13, 1998 (10801 University Blvd., Manassas, Va. 20110-2209 USA) under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure. The DNA was tested by the ATCC and determined to be. The ATCC has assigned the following deposit number to pCMV: ATCC #20335 1. [0263]
  • 1 155 1 1260 DNA Homo sapiens 1 atggtcttct cggcagtgtt gactgcgttc cataccggga catccaacac aacatttgtc 60 gtgtatgaaa acacctacat gaatattaca ctccctccac cattccagca tcctgacctc 120 agtccattgc ttagatatag ttttgaaacc atggctccca ctggtttgag ttccttgacc 180 gtgaatagta cagctgtgcc cacaacacca gcagcattta agagcctaaa cttgcctctt 240 cagatcaccc tttctgctat aatgatattc attctgtttg tgtcttttct tgggaacttg 300 gttgtttgcc tcatggttta ccaaaaagct gccatgaggt ctgcaattaa catcctcctt 360 gccagcctag cttttgcaga catgttgctt gcagtgctga acatgccctt tgccctggta 420 actattctta ctacccgatg gatttttggg aaattcttct gtagggtatc tgctatgttt 480 ttctggttat ttgtgataga aggagtagcc atcctgctca tcattagcat agataggttc 540 cttattatag tccagaggca ggataagcta aacccatata gagctaaggt tctgattgca 600 gtttcttggg caacttcctt ttgtgtagct tttcctttag ccgtaggaaa ccccgacctg 660 cagatacctt cccgagctcc ccagtgtgtg tttgggtaca caaccaatcc aggctaccag 720 gcttatgtga ttttgatttc tctcatttct ttcttcatac ccttcctggt aatactgtac 780 tcatttatgg gcatactcaa cacccttcgg cacaatgcct tgaggatcca tagctaccct 840 gaaggtatat gcctcagcca ggccagcaaa ctgggtctca tgagtctgca gagacctttc 900 cagatgagca ttgacatggg ctttaaaaca cgtgccttca ccactatttt gattctcttt 960 gctgtcttca ttgtctgctg ggccccattc accacttaca gccttgtggc aacattcagt 1020 aagcactttt actatcagca caactttttt gagattagca cctggctact gtggctctgc 1080 tacctcaagt ctgcattgaa tccgctgatc tactactgga ggattaagaa attccatgat 1140 gcttgcctgg acatgatgcc taagtccttc aagtttttgc cgcagctccc tggtcacaca 1200 aagcgacgga tacgtcctag tgctgtctat gtgtgtgggg aacatcggac ggtggtgtga 1260 2 419 PRT Homo sapiens 2 Met Val Phe Ser Ala Val Leu Thr Ala Phe His Thr Gly Thr Ser Asn 1 5 10 15 Thr Thr Phe Val Val Tyr Glu Asn Thr Tyr Met Asn Ile Thr Leu Pro 20 25 30 Pro Pro Phe Gln His Pro Asp Leu Ser Pro Leu Leu Arg Tyr Ser Phe 35 40 45 Glu Thr Met Ala Pro Thr Gly Leu Ser Ser Leu Thr Val Asn Ser Thr 50 55 60 Ala Val Pro Thr Thr Pro Ala Ala Phe Lys Ser Leu Asn Leu Pro Leu 65 70 75 80 Gln Ile Thr Leu Ser Ala Ile Met Ile Phe Ile Leu Phe Val Ser Phe 85 90 95 Leu Gly Asn Leu Val Val Cys Leu Met Val Tyr Gln Lys Ala Ala Met 100 105 110 Arg Ser Ala Ile Asn Ile Leu Leu Ala Ser Leu Ala Phe Ala Asp Met 115 120 125 Leu Leu Ala Val Leu Asn Met Pro Phe Ala Leu Val Thr Ile Leu Thr 130 135 140 Thr Arg Trp Ile Phe Gly Lys Phe Phe Cys Arg Val Ser Ala Met Phe 145 150 155 160 Phe Trp Leu Phe Val Ile Glu Gly Val Ala Ile Leu Leu Ile Ile Ser 165 170 175 Ile Asp Arg Phe Leu Ile Ile Val Gln Arg Gln Asp Lys Leu Asn Pro 180 185 190 Tyr Arg Ala Lys Val Leu Ile Ala Val Ser Trp Ala Thr Ser Phe Cys 195 200 205 Val Ala Phe Pro Leu Ala Val Gly Asn Pro Asp Leu Gln Ile Pro Ser 210 215 220 Arg Ala Pro Gln Cys Val Phe Gly Tyr Thr Thr Asn Pro Gly Tyr Gln 225 230 235 240 Ala Tyr Val Ile Leu Ile Ser Leu Ile Ser Phe Phe Ile Pro Phe Leu 245 250 255 Val Ile Leu Tyr Ser Phe Met Gly Ile Leu Asn Thr Leu Arg His Asn 260 265 270 Ala Leu Arg Ile His Ser Tyr Pro Glu Gly Ile Cys Leu Ser Gln Ala 275 280 285 Ser Lys Leu Gly Leu Met Ser Leu Gln Arg Pro Phe Gln Met Ser Ile 290 295 300 Asp Met Gly Phe Lys Thr Arg Ala Phe Thr Thr Ile Leu Ile Leu Phe 305 310 315 320 Ala Val Phe Ile Val Cys Trp Ala Pro Phe Thr Thr Tyr Ser Leu Val 325 330 335 Ala Thr Phe Ser Lys His Phe Tyr Tyr Gln His Asn Phe Phe Glu Ile 340 345 350 Ser Thr Trp Leu Leu Trp Leu Cys Tyr Leu Lys Ser Ala Leu Asn Pro 355 360 365 Leu Ile Tyr Tyr Trp Arg Ile Lys Lys Phe His Asp Ala Cys Leu Asp 370 375 380 Met Met Pro Lys Ser Phe Lys Phe Leu Pro Gln Leu Pro Gly His Thr 385 390 395 400 Lys Arg Arg Ile Arg Pro Ser Ala Val Tyr Val Cys Gly Glu His Arg 405 410 415 Thr Val Val 3 1119 DNA Homo sapiens 3 atgttagcca acagctcctc aaccaacagt tctgttctcc cgtgtcctga ctaccgacct 60 acccaccgcc tgcacttggt ggtctacagc ttggtgctgg ctgccgggct ccccctcaac 120 gcgctagccc tctgggtctt cctgcgcgcg ctgcgcgtgc actcggtggt gagcgtgtac 180 atgtgtaacc tggcggccag cgacctgctc ttcaccctct cgctgcccgt tcgtctctcc 240 tactacgcac tgcaccactg gcccttcccc gacctcctgt gccagacgac gggcgccatc 300 ttccagatga acatgtacgg cagctgcatc ttcctgatgc tcatcaacgt ggaccgctac 360 gccgccatcg tgcacccgct gcgactgcgc cacctgcggc ggccccgcgt ggcgcggctg 420 ctctgcctgg gcgtgtgggc gctcatcctg gtgtttgccg tgcccgccgc ccgcgtgcac 480 aggccctcgc gttgccgcta ccgggacctc gaggtgcgcc tatgcttcga gagcttcagc 540 gacgagctgt ggaaaggcag gctgctgccc ctcgtgctgc tggccgaggc gctgggcttc 600 ctgctgcccc tggcggcggt ggtctactcg tcgggccgag tcttctggac gctggcgcgc 660 cccgacgcca cgcagagcca gcggcggcgg aagaccgtgc gcctcctgct ggctaacctc 720 gtcatcttcc tgctgtgctt cgtgccctac aacagcacgc tggcggtcta cgggctgctg 780 cggagcaagc tggtggcggc cagcgtgcct gcccgcgatc gcgtgcgcgg ggtgctgatg 840 gtgatggtgc tgctggccgg cgccaactgc gtgctggacc cgctggtgta ctactttagc 900 gccgagggct tccgcaacac cctgcgcggc ctgggcactc cgcaccgggc caggacctcg 960 gccaccaacg ggacgcgggc ggcgctcgcg caatccgaaa ggtccgccgt caccaccgac 1020 gccaccaggc cggatgccgc cagtcagggg ctgctccgac cctccgactc ccactctctg 1080 tcttccttca cacagtgtcc ccaggattcc gccctctga 1119 4 372 PRT Homo sapiens 4 Met Leu Ala Asn Ser Ser Ser Thr Asn Ser Ser Val Leu Pro Cys Pro 1 5 10 15 Asp Tyr Arg Pro Thr His Arg Leu His Leu Val Val Tyr Ser Leu Val 20 25 30 Leu Ala Ala Gly Leu Pro Leu Asn Ala Leu Ala Leu Trp Val Phe Leu 35 40 45 Arg Ala Leu Arg Val His Ser Val Val Ser Val Tyr Met Cys Asn Leu 50 55 60 Ala Ala Ser Asp Leu Leu Phe Thr Leu Ser Leu Pro Val Arg Leu Ser 65 70 75 80 Tyr Tyr Ala Leu His His Trp Pro Phe Pro Asp Leu Leu Cys Gln Thr 85 90 95 Thr Gly Ala Ile Phe Gln Met Asn Met Tyr Gly Ser Cys Ile Phe Leu 100 105 110 Met Leu Ile Asn Val Asp Arg Tyr Ala Ala Ile Val His Pro Leu Arg 115 120 125 Leu Arg His Leu Arg Arg Pro Arg Val Ala Arg Leu Leu Cys Leu Gly 130 135 140 Val Trp Ala Leu Ile Leu Val Phe Ala Val Pro Ala Ala Arg Val His 145 150 155 160 Arg Pro Ser Arg Cys Arg Tyr Arg Asp Leu Glu Val Arg Leu Cys Phe 165 170 175 Glu Ser Phe Ser Asp Glu Leu Trp Lys Gly Arg Leu Leu Pro Leu Val 180 185 190 Leu Leu Ala Glu Ala Leu Gly Phe Leu Leu Pro Leu Ala Ala Val Val 195 200 205 Tyr Ser Ser Gly Arg Val Phe Trp Thr Leu Ala Arg Pro Asp Ala Thr 210 215 220 Gln Ser Gln Arg Arg Arg Lys Thr Val Arg Leu Leu Leu Ala Asn Leu 225 230 235 240 Val Ile Phe Leu Leu Cys Phe Val Pro Tyr Asn Ser Thr Leu Ala Val 245 250 255 Tyr Gly Leu Leu Arg Ser Lys Leu Val Ala Ala Ser Val Pro Ala Arg 260 265 270 Asp Arg Val Arg Gly Val Leu Met Val Met Val Leu Leu Ala Gly Ala 275 280 285 Asn Cys Val Leu Asp Pro Leu Val Tyr Tyr Phe Ser Ala Glu Gly Phe 290 295 300 Arg Asn Thr Leu Arg Gly Leu Gly Thr Pro His Arg Ala Arg Thr Ser 305 310 315 320 Ala Thr Asn Gly Thr Arg Ala Ala Leu Ala Gln Ser Glu Arg Ser Ala 325 330 335 Val Thr Thr Asp Ala Thr Arg Pro Asp Ala Ala Ser Gln Gly Leu Leu 340 345 350 Arg Pro Ser Asp Ser His Ser Leu Ser Ser Phe Thr Gln Cys Pro Gln 355 360 365 Asp Ser Ala Leu 370 5 1107 DNA Homo sapiens 5 atggccaact ccacagggct gaacgcctca gaagtcgcag gctcgttggg gttgatcctg 60 gcagctgtcg tggaggtggg ggcactgctg ggcaacggcg cgctgctggt cgtggtgctg 120 cgcacgccgg gactgcgcga cgcgctctac ctggcgcacc tgtgcgtcgt ggacctgctg 180 gcggccgcct ccatcatgcc gctgggcctg ctggccgcac cgccgcccgg gctgggccgc 240 gtgcgcctgg gccccgcgcc atgccgcgcc gctcgcttcc tctccgccgc tctgctgccg 300 gcctgcacgc tcggggtggc cgcacttggc ctggcacgct accgcctcat cgtgcacccg 360 ctgcggccag gctcgcggcc gccgcctgtg ctcgtgctca ccgccgtgtg ggccgcggcg 420 ggactgctgg gcgcgctctc cctgctcggc ccgccgcccg caccgccccc tgctcctgct 480 cgctgctcgg tcctggctgg gggcctcggg cccttccggc cgctctgggc cctgctggcc 540 ttcgcgctgc ccgccctcct gctgctcggc gcctacggcg gcatcttcgt ggtggcgcgt 600 cgcgctgccc tgaggccccc acggccggcg cgcgggtccc gactccgctc ggactctctg 660 gatagccgcc tttccatctt gccgccgctc cggcctcgcc tgcccggggg caaggcggcc 720 ctggccccag cgctggccgt gggccaattt gcagcctgct ggctgcctta tggctgcgcg 780 tgcctggcgc ccgcagcgcg ggccgcggaa gccgaagcgg ctgtcacctg ggtcgcctac 840 tcggccttcg cggctcaccc cttcctgtac gggctgctgc agcgccccgt gcgcttggca 900 ctgggccgcc tctctcgccg tgcactgcct ggacctgtgc gggcctgcac tccgcaagcc 960 tggcacccgc gggcactctt gcaatgcctc cagagacccc cagagggccc tgccgtaggc 1020 ccttctgagg ctccagaaca gacccccgag ttggcaggag ggcggagccc cgcataccag 1080 gggccacctg agagttctct ctcctga 1107 6 368 PRT Homo sapiens 6 Met Ala Asn Ser Thr Gly Leu Asn Ala Ser Glu Val Ala Gly Ser Leu 1 5 10 15 Gly Leu Ile Leu Ala Ala Val Val Glu Val Gly Ala Leu Leu Gly Asn 20 25 30 Gly Ala Leu Leu Val Val Val Leu Arg Thr Pro Gly Leu Arg Asp Ala 35 40 45 Leu Tyr Leu Ala His Leu Cys Val Val Asp Leu Leu Ala Ala Ala Ser 50 55 60 Ile Met Pro Leu Gly Leu Leu Ala Ala Pro Pro Pro Gly Leu Gly Arg 65 70 75 80 Val Arg Leu Gly Pro Ala Pro Cys Arg Ala Ala Arg Phe Leu Ser Ala 85 90 95 Ala Leu Leu Pro Ala Cys Thr Leu Gly Val Ala Ala Leu Gly Leu Ala 100 105 110 Arg Tyr Arg Leu Ile Val His Pro Leu Arg Pro Gly Ser Arg Pro Pro 115 120 125 Pro Val Leu Val Leu Thr Ala Val Trp Ala Ala Ala Gly Leu Leu Gly 130 135 140 Ala Leu Ser Leu Leu Gly Pro Pro Pro Ala Pro Pro Pro Ala Pro Ala 145 150 155 160 Arg Cys Ser Val Leu Ala Gly Gly Leu Gly Pro Phe Arg Pro Leu Trp 165 170 175 Ala Leu Leu Ala Phe Ala Leu Pro Ala Leu Leu Leu Leu Gly Ala Tyr 180 185 190 Gly Gly Ile Phe Val Val Ala Arg Arg Ala Ala Leu Arg Pro Pro Arg 195 200 205 Pro Ala Arg Gly Ser Arg Leu Arg Ser Asp Ser Leu Asp Ser Arg Leu 210 215 220 Ser Ile Leu Pro Pro Leu Arg Pro Arg Leu Pro Gly Gly Lys Ala Ala 225 230 235 240 Leu Ala Pro Ala Leu Ala Val Gly Gln Phe Ala Ala Cys Trp Leu Pro 245 250 255 Tyr Gly Cys Ala Cys Leu Ala Pro Ala Ala Arg Ala Ala Glu Ala Glu 260 265 270 Ala Ala Val Thr Trp Val Ala Tyr Ser Ala Phe Ala Ala His Pro Phe 275 280 285 Leu Tyr Gly Leu Leu Gln Arg Pro Val Arg Leu Ala Leu Gly Arg Leu 290 295 300 Ser Arg Arg Ala Leu Pro Gly Pro Val Arg Ala Cys Thr Pro Gln Ala 305 310 315 320 Trp His Pro Arg Ala Leu Leu Gln Cys Leu Gln Arg Pro Pro Glu Gly 325 330 335 Pro Ala Val Gly Pro Ser Glu Ala Pro Glu Gln Thr Pro Glu Leu Ala 340 345 350 Gly Gly Arg Ser Pro Ala Tyr Gln Gly Pro Pro Glu Ser Ser Leu Ser 355 360 365 7 1008 DNA Homo sapiens 7 atggaatcat ctttctcatt tggagtgatc cttgctgtcc tggcctccct catcattgct 60 actaacacac tagtggctgt ggctgtgctg ctgttgatcc acaagaatga tggtgtcagt 120 ctctgcttca ccttgaatct ggctgtggct gacaccttga ttggtgtggc catctctggc 180 ctactcacag accagctctc cagcccttct cggcccacac agaagaccct gtgcagcctg 240 cggatggcat ttgtcacttc ctccgcagct gcctctgtcc tcacggtcat gctgatcacc 300 tttgacaggt accttgccat caagcagccc ttccgctact tgaagatcat gagtgggttc 360 gtggccgggg cctgcattgc cgggctgtgg ttagtgtctt acctcattgg cttcctccca 420 ctcggaatcc ccatgttcca gcagactgcc tacaaagggc agtgcagctt ctttgctgta 480 tttcaccctc acttcgtgct gaccctctcc tgcgttggct tcttcccagc catgctcctc 540 tttgtcttct tctactgcga catgctcaag attgcctcca tgcacagcca gcagattcga 600 aagatggaac atgcaggagc catggctgga ggttatcgat ccccacggac tcccagcgac 660 ttcaaagctc tccgtactgt gtctgttctc attgggagct ttgctctatc ctggaccccc 720 ttccttatca ctggcattgt gcaggtggcc tgccaggagt gtcacctcta cctagtgctg 780 gaacggtacc tgtggctgct cggcgtgggc aactccctgc tcaacccact catctatgcc 840 tattggcaga aggaggtgcg actgcagctc taccacatgg ccctaggagt gaagaaggtg 900 ctcacctcat tcctcctctt tctctcggcc aggaattgtg gcccagagag gcccagggaa 960 agttcctgtc acatcgtcac tatctccagc tcagagtttg atggctaa 1008 8 335 PRT Homo sapiens 8 Met Glu Ser Ser Phe Ser Phe Gly Val Ile Leu Ala Val Leu Ala Ser 1 5 10 15 Leu Ile Ile Ala Thr Asn Thr Leu Val Ala Val Ala Val Leu Leu Leu 20 25 30 Ile His Lys Asn Asp Gly Val Ser Leu Cys Phe Thr Leu Asn Leu Ala 35 40 45 Val Ala Asp Thr Leu Ile Gly Val Ala Ile Ser Gly Leu Leu Thr Asp 50 55 60 Gln Leu Ser Ser Pro Ser Arg Pro Thr Gln Lys Thr Leu Cys Ser Leu 65 70 75 80 Arg Met Ala Phe Val Thr Ser Ser Ala Ala Ala Ser Val Leu Thr Val 85 90 95 Met Leu Ile Thr Phe Asp Arg Tyr Leu Ala Ile Lys Gln Pro Phe Arg 100 105 110 Tyr Leu Lys Ile Met Ser Gly Phe Val Ala Gly Ala Cys Ile Ala Gly 115 120 125 Leu Trp Leu Val Ser Tyr Leu Ile Gly Phe Leu Pro Leu Gly Ile Pro 130 135 140 Met Phe Gln Gln Thr Ala Tyr Lys Gly Gln Cys Ser Phe Phe Ala Val 145 150 155 160 Phe His Pro His Phe Val Leu Thr Leu Ser Cys Val Gly Phe Phe Pro 165 170 175 Ala Met Leu Leu Phe Val Phe Phe Tyr Cys Asp Met Leu Lys Ile Ala 180 185 190 Ser Met His Ser Gln Gln Ile Arg Lys Met Glu His Ala Gly Ala Met 195 200 205 Ala Gly Gly Tyr Arg Ser Pro Arg Thr Pro Ser Asp Phe Lys Ala Leu 210 215 220 Arg Thr Val Ser Val Leu Ile Gly Ser Phe Ala Leu Ser Trp Thr Pro 225 230 235 240 Phe Leu Ile Thr Gly Ile Val Gln Val Ala Cys Gln Glu Cys His Leu 245 250 255 Tyr Leu Val Leu Glu Arg Tyr Leu Trp Leu Leu Gly Val Gly Asn Ser 260 265 270 Leu Leu Asn Pro Leu Ile Tyr Ala Tyr Trp Gln Lys Glu Val Arg Leu 275 280 285 Gln Leu Tyr His Met Ala Leu Gly Val Lys Lys Val Leu Thr Ser Phe 290 295 300 Leu Leu Phe Leu Ser Ala Arg Asn Cys Gly Pro Glu Arg Pro Arg Glu 305 310 315 320 Ser Ser Cys His Ile Val Thr Ile Ser Ser Ser Glu Phe Asp Gly 325 330 335 9 1413 DNA Homo sapiens 9 atggacacta ccatggaagc tgacctgggt gccactggcc acaggccccg cacagagctt 60 gatgatgagg actcctaccc ccaaggtggc tgggacacgg tcttcctggt ggccctgctg 120 ctccttgggc tgccagccaa tgggttgatg gcgtggctgg ccggctccca ggcccggcat 180 ggagctggca cgcgtctggc gctgctcctg ctcagcctgg ccctctctga cttcttgttc 240 ctggcagcag cggccttcca gatcctagag atccggcatg ggggacactg gccgctgggg 300 acagctgcct gccgcttcta ctacttccta tggggcgtgt cctactcctc cggcctcttc 360 ctgctggccg ccctcagcct cgaccgctgc ctgctggcgc tgtgcccaca ctggtaccct 420 gggcaccgcc cagtccgcct gcccctctgg gtctgcgccg gtgtctgggt gctggccaca 480 ctcttcagcg tgccctggct ggtcttcccc gaggctgccg tctggtggta cgacctggtc 540 atctgcctgg acttctggga cagcgaggag ctgtcgctga ggatgctgga ggtcctgggg 600 ggcttcctgc ctttcctcct gctgctcgtc tgccacgtgc tcacccaggc cacagcctgt 660 cgcacctgcc accgccaaca gcagcccgca gcctgccggg gcttcgcccg tgtggccagg 720 accattctgt cagcctatgt ggtcctgagg ctgccctacc agctggccca gctgctctac 780 ctggccttcc tgtgggacgt ctactctggc tacctgctct gggaggccct ggtctactcc 840 gactacctga tcctactcaa cagctgcctc agccccttcc tctgcctcat ggccagtgcc 900 gacctccgga ccctgctgcg ctccgtgctc tcgtccttcg cggcagctct ctgcgaggag 960 cggccgggca gcttcacgcc cactgagcca cagacccagc tagattctga gggtccaact 1020 ctgccagagc cgatggcaga ggcccagtca cagatggatc ctgtggccca gcctcaggtg 1080 aaccccacac tccagccacg atcggatccc acagctcagc cacagctgaa ccctacggcc 1140 cagccacagt cggatcccac agcccagcca cagctgaacc tcatggccca gccacagtca 1200 gattctgtgg cccagccaca ggcagacact aacgtccaga cccctgcacc tgctgccagt 1260 tctgtgccca gtccctgtga tgaagcttcc ccaaccccat cctcgcatcc taccccaggg 1320 gcccttgagg acccagccac acctcctgcc tctgaaggag aaagccccag cagcaccccg 1380 ccagaggcgg ccccgggcgc aggccccacg tga 1413 10 468 PRT Homo sapiens 10 Met Asp Thr Thr Met Glu Ala Asp Leu Gly Ala Thr Gly His Arg Pro 1 5 10 15 Arg Thr Glu Leu Asp Asp Glu Asp Ser Tyr Pro Gln Gly Gly Trp Asp 20 25 30 Thr Val Phe Leu Val Ala Leu Leu Leu Leu Gly Leu Pro Ala Asn Gly 35 40 45 Leu Met Ala Trp Leu Ala Gly Ser Gln Ala Arg His Gly Ala Gly Thr 50 55 60 Arg Leu Ala Leu Leu Leu Leu Ser Leu Ala Leu Ser Asp Phe Leu Phe 65 70 75 80 Leu Ala Ala Ala Ala Phe Gln Ile Leu Glu Ile Arg His Gly Gly His 85 90 95 Trp Pro Leu Gly Thr Ala Ala Cys Arg Phe Tyr Tyr Phe Leu Trp Gly 100 105 110 Val Ser Tyr Ser Ser Gly Leu Phe Leu Leu Ala Ala Leu Ser Leu Asp 115 120 125 Arg Cys Leu Leu Ala Leu Cys Pro His Trp Tyr Pro Gly His Arg Pro 130 135 140 Val Arg Leu Pro Leu Trp Val Cys Ala Gly Val Trp Val Leu Ala Thr 145 150 155 160 Leu Phe Ser Val Pro Trp Leu Val Phe Pro Glu Ala Ala Val Trp Trp 165 170 175 Tyr Asp Leu Val Ile Cys Leu Asp Phe Trp Asp Ser Glu Glu Leu Ser 180 185 190 Leu Arg Met Leu Glu Val Leu Gly Gly Phe Leu Pro Phe Leu Leu Leu 195 200 205 Leu Val Cys His Val Leu Thr Gln Ala Thr Arg Thr Cys His Arg Gln 210 215 220 Gln Gln Pro Ala Ala Cys Arg Gly Phe Ala Arg Val Ala Arg Thr Ile 225 230 235 240 Leu Ser Ala Tyr Val Val Leu Arg Leu Pro Tyr Gln Leu Ala Gln Leu 245 250 255 Leu Tyr Leu Ala Phe Leu Trp Asp Val Tyr Ser Gly Tyr Leu Leu Trp 260 265 270 Glu Ala Leu Val Tyr Ser Asp Tyr Leu Ile Leu Leu Asn Ser Cys Leu 275 280 285 Ser Pro Phe Leu Cys Leu Met Ala Ser Ala Asp Leu Arg Thr Leu Leu 290 295 300 Arg Ser Val Leu Ser Ser Phe Ala Ala Ala Leu Cys Glu Glu Arg Pro 305 310 315 320 Gly Ser Phe Thr Pro Thr Glu Pro Gln Thr Gln Leu Asp Ser Glu Gly 325 330 335 Pro Thr Leu Pro Glu Pro Met Ala Glu Ala Gln Ser Gln Met Asp Pro 340 345 350 Val Ala Gln Pro Gln Val Asn Pro Thr Leu Gln Pro Arg Ser Asp Pro 355 360 365 Thr Ala Gln Pro Gln Leu Asn Pro Thr Ala Gln Pro Gln Ser Asp Pro 370 375 380 Thr Ala Gln Pro Gln Leu Asn Leu Met Ala Gln Pro Gln Ser Asp Ser 385 390 395 400 Val Ala Gln Pro Gln Ala Asp Thr Asn Val Gln Thr Pro Ala Pro Ala 405 410 415 Ala Ser Ser Val Pro Ser Pro Cys Asp Glu Ala Ser Pro Thr Pro Ser 420 425 430 Ser His Pro Thr Pro Gly Ala Leu Glu Asp Pro Ala Thr Pro Pro Ala 435 440 445 Ser Glu Gly Glu Ser Pro Ser Ser Thr Pro Pro Glu Ala Ala Pro Gly 450 455 460 Ala Gly Pro Thr 465 11 1248 DNA Homo sapiens 11 atgtcaggga tggaaaaact tcagaatgct tcctggatct accagcagaa actagaagat 60 ccattccaga aacacctgaa cagcaccgag gagtatctgg ccttcctctg cggacctcgg 120 cgcagccact tcttcctccc cgtgtctgtg gtgtatgtgc caatttttgt ggtgggggtc 180 attggcaatg tcctggtgtg cctggtgatt ctgcagcacc aggctatgaa gacgcccacc 240 aactactacc tcttcagcct ggcggtctct gacctcctgg tcctgctcct tggaatgccc 300 ctggaggtct atgagatgtg gcgcaactac cctttcttgt tcgggcccgt gggctgctac 360 ttcaagacgg ccctctttga gaccgtgtgc ttcgcctcca tcctcagcat caccaccgtc 420 agcgtggagc gctacgtggc catcctacac ccgttccgcg ccaaactgca gagcacccgg 480 cgccgggccc tcaggatcct cggcatcgtc tggggcttct ccgtgctctt ctccctgccc 540 aacaccagca tccatggcat caagttccac tacttcccca atgggtccct ggtcccaggt 600 tcggccacct gtacggtcat caagcccatg tggatctaca atttcatcat ccaggtcacc 660 tccttcctat tctacctcct ccccatgact gtcatcagtg tcctctacta cctcatggca 720 ctcagactaa agaaagacaa atctcttgag gcagatgaag ggaatgcaaa tattcaaaga 780 ccctgcagaa aatcagtcaa caagatgctg tttgtcttgg tcttagtgtt tgctatctgt 840 tgggccccgt tccacattga ccgactcttc ttcagctttg tggaggagtg gagtgaatcc 900 ctggctgctg tgttcaacct cgtccatgtg gtgtcaggtg tcttcttcta cctgagctca 960 gctgtcaacc ccattatcta taacctactg tctcgccgct tccaggcagc attccagaat 1020 gtgatctctt ctttccacaa acagtggcac tcccagcatg acccacagtt gccacctgcc 1080 cagcggaaca tcttcctgac agaatgccac tttgtggagc tgaccgaaga tataggtccc 1140 caattcccat gtcagtcatc catgcacaac tctcacctcc caacagccct ctctagtgaa 1200 cagatgtcaa gaacaaacta tcaaagcttc cactttaaca aaacctga 1248 12 415 PRT Homo sapiens 12 Met Ser Gly Met Glu Lys Leu Gln Asn Ala Ser Trp Ile Tyr Gln Gln 1 5 10 15 Lys Leu Glu Asp Pro Phe Gln Lys His Leu Asn Ser Thr Glu Glu Tyr 20 25 30 Leu Ala Phe Leu Cys Gly Pro Arg Arg Ser His Phe Phe Leu Pro Val 35 40 45 Ser Val Val Tyr Val Pro Ile Phe Val Val Gly Val Ile Gly Asn Val 50 55 60 Leu Val Cys Leu Val Ile Leu Gln His Gln Ala Met Lys Thr Pro Thr 65 70 75 80 Asn Tyr Tyr Leu Phe Ser Leu Ala Val Ser Asp Leu Leu Val Leu Leu 85 90 95 Leu Gly Met Pro Leu Glu Val Tyr Glu Met Trp Arg Asn Tyr Pro Phe 100 105 110 Leu Phe Gly Pro Val Gly Cys Tyr Phe Lys Thr Ala Leu Phe Glu Thr 115 120 125 Val Cys Phe Ala Ser Ile Leu Ser Ile Thr Thr Val Ser Val Glu Arg 130 135 140 Tyr Val Ala Ile Leu His Pro Phe Arg Ala Lys Leu Gln Ser Thr Arg 145 150 155 160 Arg Arg Ala Leu Arg Ile Leu Gly Ile Val Trp Gly Phe Ser Val Leu 165 170 175 Phe Ser Leu Pro Asn Thr Ser Ile His Gly Ile Lys Phe His Tyr Phe 180 185 190 Pro Asn Gly Ser Leu Val Pro Gly Ser Ala Thr Cys Thr Val Ile Lys 195 200 205 Pro Met Trp Ile Tyr Asn Phe Ile Ile Gln Val Thr Ser Phe Leu Phe 210 215 220 Tyr Leu Leu Pro Met Thr Val Ile Ser Val Leu Tyr Tyr Leu Met Ala 225 230 235 240 Leu Arg Leu Lys Lys Asp Lys Ser Leu Glu Ala Asp Glu Gly Asn Ala 245 250 255 Asn Ile Gln Arg Pro Cys Arg Lys Ser Val Asn Lys Met Leu Phe Val 260 265 270 Leu Val Leu Val Phe Ala Ile Cys Trp Ala Pro Phe His Ile Asp Arg 275 280 285 Leu Phe Phe Ser Phe Val Glu Glu Trp Ser Glu Ser Leu Ala Ala Val 290 295 300 Phe Asn Leu Val His Val Val Ser Gly Val Phe Phe Tyr Leu Ser Ser 305 310 315 320 Ala Val Asn Pro Ile Ile Tyr Asn Leu Leu Ser Arg Arg Phe Gln Ala 325 330 335 Ala Phe Gln Asn Val Ile Ser Ser Phe His Lys Gln Trp His Ser Gln 340 345 350 His Asp Pro Gln Leu Pro Pro Ala Gln Arg Asn Ile Phe Leu Thr Glu 355 360 365 Cys His Phe Val Glu Leu Thr Glu Asp Ile Gly Pro Gln Phe Pro Cys 370 375 380 Gln Ser Ser Met His Asn Ser His Leu Pro Thr Ala Leu Ser Ser Glu 385 390 395 400 Gln Met Ser Arg Thr Asn Tyr Gln Ser Phe His Phe Asn Lys Thr 405 410 415 13 1173 DNA Homo sapiens 13 atgccagata ctaatagcac aatcaattta tcactaagca ctcgtgttac tttagcattt 60 tttatgtcct tagtagcttt tgctataatg ctaggaaatg ctttggtcat tttagctttt 120 gtggtggaca aaaaccttag acatcgaagt agttattttt ttcttaactt ggccatctct 180 gacttctttg tgggtgtgat ctccattcct ttgtacatcc ctcacacgct gttcgaatgg 240 gattttggaa aggaaatctg tgtattttgg ctcactactg actatctgtt atgtacagca 300 tctgtatata acattgtcct catcagctat gatcgatacc tgtcagtctc aaatgctgtg 360 tcttatagaa ctcaacatac tggggtcttg aagattgtta ctctgatggt ggccgtttgg 420 gtgctggcct tcttagtgaa tgggccaatg attctagttt cagagtcttg gaaggatgaa 480 ggtagtgaat gtgaacctgg atttttttcg gaatggtaca tccttgccat cacatcattc 540 ttggaattcg tgatcccagt catcttagtc gcttatttca acatgaatat ttattggagc 600 ctgtggaagc gtgatcatct cagtaggtgc caaagccatc ctggactgac tgctgtctct 660 tccaacatct gtggacactc attcagaggt agactatctt caaggagatc tctttctgca 720 tcgacagaag ttcctgcatc ctttcattca gagagacaga ggagaaagag tagtctcatg 780 ttttcctcaa gaaccaagat gaatagcaat acaattgctt ccaaaatggg ttccttctcc 840 caatcagatt ctgtagctct tcaccaaagg gaacatgttg aactgcttag agccaggaga 900 ttagccaagt cactggccat tctcttaggg gtttttgctg tttgctgggc tccatattct 960 ctgttcacaa ttgtcctttc attttattcc tcagcaacag gtcctaaatc agtttggtat 1020 agaattgcat tttggcttca gtggttcaat tcctttgtca atcctctttt gtatccattg 1080 tgtcacaagc gctttcaaaa ggctttcttg aaaatatttt gtataaaaaa gcaacctcta 1140 ccatcacaac acagtcggtc agtatcttct taa 1173 14 390 PRT Homo sapiens 14 Met Pro Asp Thr Asn Ser Thr Ile Asn Leu Ser Leu Ser Thr Arg Val 1 5 10 15 Thr Leu Ala Phe Phe Met Ser Leu Val Ala Phe Ala Ile Met Leu Gly 20 25 30 Asn Ala Leu Val Ile Leu Ala Phe Val Val Asp Lys Asn Leu Arg His 35 40 45 Arg Ser Ser Tyr Phe Phe Leu Asn Leu Ala Ile Ser Asp Phe Phe Val 50 55 60 Gly Val Ile Ser Ile Pro Leu Tyr Ile Pro His Thr Leu Phe Glu Trp 65 70 75 80 Asp Phe Gly Lys Glu Ile Cys Val Phe Trp Leu Thr Thr Asp Tyr Leu 85 90 95 Leu Cys Thr Ala Ser Val Tyr Asn Ile Val Leu Ile Ser Tyr Asp Arg 100 105 110 Tyr Leu Ser Val Ser Asn Ala Val Ser Tyr Arg Thr Gln His Thr Gly 115 120 125 Val Leu Lys Ile Val Thr Leu Met Val Ala Val Trp Val Leu Ala Phe 130 135 140 Leu Val Asn Gly Pro Met Ile Leu Val Ser Glu Ser Trp Lys Asp Glu 145 150 155 160 Gly Ser Glu Cys Glu Pro Gly Phe Phe Ser Glu Trp Tyr Ile Leu Ala 165 170 175 Ile Thr Ser Phe Leu Glu Phe Val Ile Pro Val Ile Leu Val Ala Tyr 180 185 190 Phe Asn Met Asn Ile Tyr Trp Ser Leu Trp Lys Arg Asp His Leu Ser 195 200 205 Arg Cys Gln Ser His Pro Gly Leu Thr Ala Val Ser Ser Asn Ile Cys 210 215 220 Gly His Ser Phe Arg Gly Arg Leu Ser Ser Arg Arg Ser Leu Ser Ala 225 230 235 240 Ser Thr Glu Val Pro Ala Ser Phe His Ser Glu Arg Gln Arg Arg Lys 245 250 255 Ser Ser Leu Met Phe Ser Ser Arg Thr Lys Met Asn Ser Asn Thr Ile 260 265 270 Ala Ser Lys Met Gly Ser Phe Ser Gln Ser Asp Ser Val Ala Leu His 275 280 285 Gln Arg Glu His Val Glu Leu Leu Arg Ala Arg Arg Leu Ala Lys Ser 290 295 300 Leu Ala Ile Leu Leu Gly Val Phe Ala Val Cys Trp Ala Pro Tyr Ser 305 310 315 320 Leu Phe Thr Ile Val Leu Ser Phe Tyr Ser Ser Ala Thr Gly Pro Lys 325 330 335 Ser Val Trp Tyr Arg Ile Ala Phe Trp Leu Gln Trp Phe Asn Ser Phe 340 345 350 Val Asn Pro Leu Leu Tyr Pro Leu Cys His Lys Arg Phe Gln Lys Ala 355 360 365 Phe Leu Lys Ile Phe Cys Ile Lys Lys Gln Pro Leu Pro Ser Gln His 370 375 380 Ser Arg Ser Val Ser Ser 385 390 15 30 DNA Artificial Sequence Novel Sequence 15 ggaaagctta acgatcccca ggagcaacat 30 16 31 DNA Artificial Sequence Novel Sequence 16 ctgggatcct acgagagcat ttttcacaca g 31 17 1128 DNA Homo sapiens 17 atggcgaacg cgagcgagcc gggtggcagc ggcggcggcg aggcggccgc cctgggcctc 60 aagctggcca cgctcagcct gctgctgtgc gtgagcctag cgggcaacgt gctgttcgcg 120 ctgctgatcg tgcgggagcg cagcctgcac cgcgccccgt actacctgct gctcgacctg 180 tgcctggccg acgggctgcg cgcgctcgcc tgcctcccgg ccgtcatgct ggcggcgcgg 240 cgtgcggcgg ccgcggcggg ggcgccgccg ggcgcgctgg gctgcaagct gctcgccttc 300 ctggccgcgc tcttctgctt ccacgccgcc ttcctgctgc tgggcgtggg cgtcacccgc 360 tacctggcca tcgcgcacca ccgcttctat gcagagcgcc tggccggctg gccgtgcgcc 420 gccatgctgg tgtgcgccgc ctgggcgctg gcgctggccg cggccttccc gccagtgctg 480 gacggcggtg gcgacgacga ggacgcgccg tgcgccctgg agcagcggcc cgacggcgcc 540 cccggcgcgc tgggcttcct gctgctgctg gccgtggtgg tgggcgccac gcacctcgtc 600 tacctccgcc tgctcttctt catccacgac cgccgcaaga tgcggcccgc gcgcctggtg 660 cccgccgtca gccacgactg gaccttccac ggcccgggcg ccaccggcca ggcggccgcc 720 aactggacgg cgggcttcgg ccgcgggccc acgccgcccg cgcttgtggg catccggccc 780 gcagggccgg gccgcggcgc gcgccgcctc ctcgtgctgg aagaattcaa gacggagaag 840 aggctgtgca agatgttcta cgccgtcacg ctgctcttcc tgctcctctg ggggccctac 900 gtcgtggcca gctacctgcg ggtcctggtg cggcccggcg ccgtccccca ggcctacctg 960 acggcctccg tgtggctgac cttcgcgcag gccggcatca accccgtcgt gtgcttcctc 1020 ttcaacaggg agctgaggga ctgcttcagg gcccagttcc cctgctgcca gagcccccgg 1080 accacccagg cgacccatcc ctgcgacctg aaaggcattg gtttatga 1128 18 375 PRT Homo sapiens 18 Met Ala Asn Ala Ser Glu Pro Gly Gly Ser Gly Gly Gly Glu Ala Ala 1 5 10 15 Ala Leu Gly Leu Lys Leu Ala Thr Leu Ser Leu Leu Leu Cys Val Ser 20 25 30 Leu Ala Gly Asn Val Leu Phe Ala Leu Leu Ile Val Arg Glu Arg Ser 35 40 45 Leu His Arg Ala Pro Tyr Tyr Leu Leu Leu Asp Leu Cys Leu Ala Asp 50 55 60 Gly Leu Arg Ala Leu Ala Cys Leu Pro Ala Val Met Leu Ala Ala Arg 65 70 75 80 Arg Ala Ala Ala Ala Ala Gly Ala Pro Pro Gly Ala Leu Gly Cys Lys 85 90 95 Leu Leu Ala Phe Leu Ala Ala Leu Phe Cys Phe His Ala Ala Phe Leu 100 105 110 Leu Leu Gly Val Gly Val Thr Arg Tyr Leu Ala Ile Ala His His Arg 115 120 125 Phe Tyr Ala Glu Arg Leu Ala Gly Trp Pro Cys Ala Ala Met Leu Val 130 135 140 Cys Ala Ala Trp Ala Leu Ala Leu Ala Ala Ala Phe Pro Pro Val Leu 145 150 155 160 Asp Gly Gly Gly Asp Asp Glu Asp Ala Pro Cys Ala Leu Glu Gln Arg 165 170 175 Pro Asp Gly Ala Pro Gly Ala Leu Gly Phe Leu Leu Leu Leu Ala Val 180 185 190 Val Val Gly Ala Thr His Leu Val Tyr Leu Arg Leu Leu Phe Phe Ile 195 200 205 His Asp Arg Arg Lys Met Arg Pro Ala Arg Leu Val Pro Ala Val Ser 210 215 220 His Asp Trp Thr Phe His Gly Pro Gly Ala Thr Gly Gln Ala Ala Ala 225 230 235 240 Asn Trp Thr Ala Gly Phe Gly Arg Gly Pro Thr Pro Pro Ala Leu Val 245 250 255 Gly Ile Arg Pro Ala Gly Pro Gly Arg Gly Ala Arg Arg Leu Leu Val 260 265 270 Leu Glu Glu Phe Lys Thr Glu Lys Arg Leu Cys Lys Met Phe Tyr Ala 275 280 285 Val Thr Leu Leu Phe Leu Leu Leu Trp Gly Pro Tyr Val Val Ala Ser 290 295 300 Tyr Leu Arg Val Leu Val Arg Pro Gly Ala Val Pro Gln Ala Tyr Leu 305 310 315 320 Thr Ala Ser Val Trp Leu Thr Phe Ala Gln Ala Gly Ile Asn Pro Val 325 330 335 Val Cys Phe Leu Phe Asn Arg Glu Leu Arg Asp Cys Phe Arg Ala Gln 340 345 350 Phe Pro Cys Cys Gln Ser Pro Arg Thr Thr Gln Ala Thr His Pro Cys 355 360 365 Asp Leu Lys Gly Ile Gly Leu 370 375 19 1002 DNA Homo sapiens 19 atgaacacca cagtgatgca aggcttcaac agatctgagc ggtgccccag agacactcgg 60 atagtacagc tggtattccc agccctctac acagtggttt tcttgaccgg catcctgctg 120 aatactttgg ctctgtgggt gtttgttcac atccccagct cctccacctt catcatctac 180 ctcaaaaaca ctttggtggc cgacttgata atgacactca tgcttccttt caaaatcctc 240 tctgactcac acctggcacc ctggcagctc agagcttttg tgtgtcgttt ttcttcggtg 300 atattttatg agaccatgta tgtgggcatc gtgctgttag ggctcatagc ctttgacaga 360 ttcctcaaga tcatcagacc tttgagaaat atttttctaa aaaaacctgt ttttgcaaaa 420 acggtctcaa tcttcatctg gttctttttg ttcttcatct ccctgccaaa tacgatcttg 480 agcaacaagg aagcaacacc atcgtctgtg aaaaagtgtg cttccttaaa ggggcctctg 540 gggctgaaat ggcatcaaat ggtaaataac atatgccagt ttattttctg gactgttttt 600 atcctaatgc ttgtgtttta tgtggttatt gcaaaaaaag tatatgattc ttatagaaag 660 tccaaaagta aggacagaaa aaacaacaaa aagctggaag gcaaagtatt tgttgtcgtg 720 gctgtcttct ttgtgtgttt tgctccattt cattttgcca gagttccata tactcacagt 780 caaaccaaca ataagactga ctgtagactg caaaatcaac tgtttattgc taaagaaaca 840 actctctttt tggcagcaac taacatttgt atggatccct taatatacat attcttatgt 900 aaaaaattca cagaaaagct accatgtatg caagggagaa agaccacagc atcaagccaa 960 gaaaatcata gcagtcagac agacaacata accttaggct ga 1002 20 333 PRT Homo sapiens 20 Met Asn Thr Thr Val Met Gln Gly Phe Asn Arg Ser Glu Arg Cys Pro 1 5 10 15 Arg Asp Thr Arg Ile Val Gln Leu Val Phe Pro Ala Leu Tyr Thr Val 20 25 30 Val Phe Leu Thr Gly Ile Leu Leu Asn Thr Leu Ala Leu Trp Val Phe 35 40 45 Val His Ile Pro Ser Ser Ser Thr Phe Ile Ile Tyr Leu Lys Asn Thr 50 55 60 Leu Val Ala Asp Leu Ile Met Thr Leu Met Leu Pro Phe Lys Ile Leu 65 70 75 80 Ser Asp Ser His Leu Ala Pro Trp Gln Leu Arg Ala Phe Val Cys Arg 85 90 95 Phe Ser Ser Val Ile Phe Tyr Glu Thr Met Tyr Val Gly Ile Val Leu 100 105 110 Leu Gly Leu Ile Ala Phe Asp Arg Phe Leu Lys Ile Ile Arg Pro Leu 115 120 125 Arg Asn Ile Phe Leu Lys Lys Pro Val Phe Ala Lys Thr Val Ser Ile 130 135 140 Phe Ile Trp Phe Phe Leu Phe Phe Ile Ser Leu Pro Asn Thr Ile Leu 145 150 155 160 Ser Asn Lys Glu Ala Thr Pro Ser Ser Val Lys Lys Cys Ala Ser Leu 165 170 175 Lys Gly Pro Leu Gly Leu Lys Trp His Gln Met Val Asn Asn Ile Cys 180 185 190 Gln Phe Ile Phe Trp Thr Val Phe Ile Leu Met Leu Val Phe Tyr Val 195 200 205 Val Ile Ala Lys Lys Val Tyr Asp Ser Tyr Arg Lys Ser Lys Ser Lys 210 215 220 Asp Arg Lys Asn Asn Lys Lys Leu Glu Gly Lys Val Phe Val Val Val 225 230 235 240 Ala Val Phe Phe Val Cys Phe Ala Pro Phe His Phe Ala Arg Val Pro 245 250 255 Tyr Thr His Ser Gln Thr Asn Asn Lys Thr Asp Cys Arg Leu Gln Asn 260 265 270 Gln Leu Phe Ile Ala Lys Glu Thr Thr Leu Phe Leu Ala Ala Thr Asn 275 280 285 Ile Cys Met Asp Pro Leu Ile Tyr Ile Phe Leu Cys Lys Lys Phe Thr 290 295 300 Glu Lys Leu Pro Cys Met Gln Gly Arg Lys Thr Thr Ala Ser Ser Gln 305 310 315 320 Glu Asn His Ser Ser Gln Thr Asp Asn Ile Thr Leu Gly 325 330 21 1122 DNA Homo sapiens 21 atggccaaca ctaccggaga gcctgaggag gtgagcggcg ctctgtcccc accgtccgca 60 tcagcttatg tgaagctggt actgctggga ctgattatgt gcgtgagcct ggcgggtaac 120 gccatcttgt ccctgctggt gctcaaggag cgtgccctgc acaaggctcc ttactacttc 180 ctgctggacc tgtgcctggc cgatggcata cgctctgccg tctgcttccc ctttgtgctg 240 gcttctgtgc gccacggctc ttcatggacc ttcagtgcac tcagctgcaa gattgtggcc 300 tttatggccg tgctcttttg cttccatgcg gccttcatgc tgttctgcat cagcgtcacc 360 cgctacatgg ccatcgccca ccaccgcttc tacgccaagc gcatgacact ctggacatgc 420 gcggctgtca tctgcatggc ctggaccctg tctgtggcca tggccttccc acctgtcttt 480 gacgtgggca cctacaagtt tattcgggag gaggaccagt gcatctttga gcatcgctac 540 ttcaaggcca atgacacgct gggcttcatg cttatgttgg ctgtgctcat ggcagctacc 600 catgctgtct acggcaagct gctcctcttc gagtatcgtc accgcaagat gaagccagtg 660 cagatggtgc cagccatcag ccagaactgg acattccatg gtcccggggc caccggccag 720 gctgctgcca actggatcgc cggctttggc cgtgggccca tgccaccaac cctgctgggt 780 atccggcaga atgggcatgc agccagccgg cggctactgg gcatggacga ggtcaagggt 840 gaaaagcagc tgggccgcat gttctacgcg atcacactgc tctttctgct cctctggtca 900 ccctacatcg tggcctgcta ctggcgagtg tttgtgaaag cctgtgctgt gccccaccgc 960 tacctggcca ctgctgtttg gatgagcttc gcccaggctg ccgtcaaccc aattgtctgc 1020 ttcctgctca acaaggacct caagaagtgc ctgaccactc acgccccctg ctggggcaca 1080 ggaggtgccc cggctcccag agaaccctac tgtgtcatgt ga 1122 22 373 PRT Homo sapiens 22 Met Ala Asn Thr Thr Gly Glu Pro Glu Glu Val Ser Gly Ala Leu Ser 1 5 10 15 Pro Pro Ser Ala Ser Ala Tyr Val Lys Leu Val Leu Leu Gly Leu Ile 20 25 30 Met Cys Val Ser Leu Ala Gly Asn Ala Ile Leu Ser Leu Leu Val Leu 35 40 45 Lys Glu Arg Ala Leu His Lys Ala Pro Tyr Tyr Phe Leu Leu Asp Leu 50 55 60 Cys Leu Ala Asp Gly Ile Arg Ser Ala Val Cys Phe Pro Phe Val Leu 65 70 75 80 Ala Ser Val Arg His Gly Ser Ser Trp Thr Phe Ser Ala Leu Ser Cys 85 90 95 Lys Ile Val Ala Phe Met Ala Val Leu Phe Cys Phe His Ala Ala Phe 100 105 110 Met Leu Phe Cys Ile Ser Val Thr Arg Tyr Met Ala Ile Ala His His 115 120 125 Arg Phe Tyr Ala Lys Arg Met Thr Leu Trp Thr Cys Ala Ala Val Ile 130 135 140 Cys Met Ala Trp Thr Leu Ser Val Ala Met Ala Phe Pro Pro Val Phe 145 150 155 160 Asp Val Gly Thr Tyr Lys Phe Ile Arg Glu Glu Asp Gln Cys Ile Phe 165 170 175 Glu His Arg Tyr Phe Lys Ala Asn Asp Thr Leu Gly Phe Met Leu Met 180 185 190 Leu Ala Val Leu Met Ala Ala Thr His Ala Val Tyr Gly Lys Leu Leu 195 200 205 Leu Phe Glu Tyr Arg His Arg Lys Met Lys Pro Val Gln Met Val Pro 210 215 220 Ala Ile Ser Gln Asn Trp Thr Phe His Gly Pro Gly Ala Thr Gly Gln 225 230 235 240 Ala Ala Ala Asn Trp Ile Ala Gly Phe Gly Arg Gly Pro Met Pro Pro 245 250 255 Thr Leu Leu Gly Ile Arg Gln Asn Gly His Ala Ala Ser Arg Arg Leu 260 265 270 Leu Gly Met Asp Glu Val Lys Gly Glu Lys Gln Leu Gly Arg Met Phe 275 280 285 Tyr Ala Ile Thr Leu Leu Phe Leu Leu Leu Trp Ser Pro Tyr Ile Val 290 295 300 Ala Cys Tyr Trp Arg Val Phe Val Lys Ala Cys Ala Val Pro His Arg 305 310 315 320 Tyr Leu Ala Thr Ala Val Trp Met Ser Phe Ala Gln Ala Ala Val Asn 325 330 335 Pro Ile Val Cys Phe Leu Leu Asn Lys Asp Leu Lys Lys Cys Leu Thr 340 345 350 Thr His Ala Pro Cys Trp Gly Thr Gly Gly Ala Pro Ala Pro Arg Glu 355 360 365 Pro Tyr Cys Val Met 370 23 1053 DNA Homo sapiens 23 atggctttgg aacagaacca gtcaacagat tattattatg aggaaaatga aatgaatggc 60 acttatgact acagtcaata tgaattgatc tgtatcaaag aagatgtcag agaatttgca 120 aaagttttcc tccctgtatt cctcacaata gctttcgtca ttggacttgc aggcaattcc 180 atggtagtgg caatttatgc ctattacaag aaacagagaa ccaaaacaga tgtgtacatc 240 ctgaatttgg ctgtagcaga tttactcctt ctattcactc tgcctttttg ggctgttaat 300 gcagttcatg ggtgggtttt agggaaaata atgtgcaaaa taacttcagc cttgtacaca 360 ctaaactttg tctctggaat gcagtttctg gcttgcatca gcatagacag atatgtggca 420 gtaactaatg tccccagcca atcaggagtg ggaaaaccat gctggatcat ctgtttctgt 480 gtctggatgg ctgccatctt gctgagcata ccccagctgg ttttttatac agtaaatgac 540 aatgctaggt gcattcccat tttcccccgc tacctaggaa catcaatgaa agcattgatt 600 caaatgctag agatctgcat tggatttgta gtaccctttc ttattatggg ggtgtgctac 660 tttatcacgg caaggacact catgaagatg ccaaacatta aaatatctcg acccctaaaa 720 gttctgctca cagtcgttat agttttcatt gtcactcaac tgccttataa cattgtcaag 780 ttctgccgag ccatagacat catctactcc ctgatcacca gctgcaacat gagcaaacgc 840 atggacatcg ccatccaagt cacagaaagc attgcactct ttcacagctg cctcaaccca 900 atcctttatg tttttatggg agcatctttc aaaaactacg ttatgaaagt ggccaagaaa 960 tatgggtcct ggagaagaca gagacaaagt gtggaggagt ttccttttga ttctgagggt 1020 cctacagagc caaccagtac ttttagcatt taa 1053 24 350 PRT Homo sapiens 24 Met Ala Leu Glu Gln Asn Gln Ser Thr Asp Tyr Tyr Tyr Glu Glu Asn 1 5 10 15 Glu Met Asn Gly Thr Tyr Asp Tyr Ser Gln Tyr Glu Leu Ile Cys Ile 20 25 30 Lys Glu Asp Val Arg Glu Phe Ala Lys Val Phe Leu Pro Val Phe Leu 35 40 45 Thr Ile Ala Phe Val Ile Gly Leu Ala Gly Asn Ser Met Val Val Ala 50 55 60 Ile Tyr Ala Tyr Tyr Lys Lys Gln Arg Thr Lys Thr Asp Val Tyr Ile 65 70 75 80 Leu Asn Leu Ala Val Ala Asp Leu Leu Leu Leu Phe Thr Leu Pro Phe 85 90 95 Trp Ala Val Asn Ala Val His Gly Trp Val Leu Gly Lys Ile Met Cys 100 105 110 Lys Ile Thr Ser Ala Leu Tyr Thr Leu Asn Phe Val Ser Gly Met Gln 115 120 125 Phe Leu Ala Cys Ile Ser Ile Asp Arg Tyr Val Ala Val Thr Asn Val 130 135 140 Pro Ser Gln Ser Gly Val Gly Lys Pro Cys Trp Ile Ile Cys Phe Cys 145 150 155 160 Val Trp Met Ala Ala Ile Leu Leu Ser Ile Pro Gln Leu Val Phe Tyr 165 170 175 Thr Val Asn Asp Asn Ala Arg Cys Ile Pro Ile Phe Pro Arg Tyr Leu 180 185 190 Gly Thr Ser Met Lys Ala Leu Ile Gln Met Leu Glu Ile Cys Ile Gly 195 200 205 Phe Val Val Pro Phe Leu Ile Met Gly Val Cys Tyr Phe Ile Thr Ala 210 215 220 Arg Thr Leu Met Lys Met Pro Asn Ile Lys Ile Ser Arg Pro Leu Lys 225 230 235 240 Val Leu Leu Thr Val Val Ile Val Phe Ile Val Thr Gln Leu Pro Tyr 245 250 255 Asn Ile Val Lys Phe Cys Arg Ala Ile Asp Ile Ile Tyr Ser Leu Ile 260 265 270 Thr Ser Cys Asn Met Ser Lys Arg Met Asp Ile Ala Ile Gln Val Thr 275 280 285 Glu Ser Ile Ala Leu Phe His Ser Cys Leu Asn Pro Ile Leu Tyr Val 290 295 300 Phe Met Gly Ala Ser Phe Lys Asn Tyr Val Met Lys Val Ala Lys Lys 305 310 315 320 Tyr Gly Ser Trp Arg Arg Gln Arg Gln Ser Val Glu Glu Phe Pro Phe 325 330 335 Asp Ser Glu Gly Pro Thr Glu Pro Thr Ser Thr Phe Ser Ile 340 345 350 25 1116 DNA Homo sapiens 25 atgccaggaa acgccacccc agtgaccacc actgccccgt gggcctccct gggcctctcc 60 gccaagacct gcaacaacgt gtccttcgaa gagagcagga tagtcctggt cgtggtgtac 120 agcgcggtgt gcacgctggg ggtgccggcc aactgcctga ctgcgtggct ggcgctgctg 180 caggtactgc agggcaacgt gctggccgtc tacctgctct gcctggcact ctgcgaactg 240 ctgtacacag gcacgctgcc actctgggtc atctatatcc gcaaccagca ccgctggacc 300 ctaggcctgc tggcctcgaa ggtgaccgcc tacatcttct tctgcaacat ctacgtcagc 360 atcctcttcc tgtgctgcat ctcctgcgac cgcttcgtgg ccgtggtgta cgcgctggag 420 agtcggggcc gccgccgccg gaggaccgcc atcctcatct ccgcctgcat cttcatcctc 480 gtcgggatcg ttcactaccc ggtgttccag acggaagaca aggagacctg ctttgacatg 540 ctgcagatgg acagcaggat tgccgggtac tactacgcca ggttcaccgt tggctttgcc 600 atccctctct ccatcatcgc cttcaccaac caccggattt tcaggagcat caagcagagc 660 atgggcttaa gcgctgccca gaaggccaag gtgaagcact cggccatcgc ggtggttgtc 720 atcttcctag tctgcttcgc cccgtaccac ctggttctcc tcgtcaaagc cgctgccttt 780 tcctactaca gaggagacag gaacgccatg tgcggcttgg aggaaaggct gtacacagcc 840 tctgtggtgt ttctgtgcct gtccacggtg aacggcgtgg ctgaccccat tatctacgtg 900 ctggccacgg accattcccg ccaagaagtg tccagaatcc ataaggggtg gaaagagtgg 960 tccatgaaga cagacgtcac caggctcacc cacagcaggg acaccgagga gctgcagtcg 1020 cccgtggccc ttgcagacca ctacaccttc tccaggcccg tgcacccacc agggtcacca 1080 tgccctgcaa agaggctgat tgaggagtcc tgctga 1116 26 371 PRT Homo sapiens 26 Met Pro Gly Asn Ala Thr Pro Val Thr Thr Thr Ala Pro Trp Ala Ser 1 5 10 15 Leu Gly Leu Ser Ala Lys Thr Cys Asn Asn Val Ser Phe Glu Glu Ser 20 25 30 Arg Ile Val Leu Val Val Val Tyr Ser Ala Val Cys Thr Leu Gly Val 35 40 45 Pro Ala Asn Cys Leu Thr Ala Trp Leu Ala Leu Leu Gln Val Leu Gln 50 55 60 Gly Asn Val Leu Ala Val Tyr Leu Leu Cys Leu Ala Leu Cys Glu Leu 65 70 75 80 Leu Tyr Thr Gly Thr Leu Pro Leu Trp Val Ile Tyr Ile Arg Asn Gln 85 90 95 His Arg Trp Thr Leu Gly Leu Leu Ala Ser Lys Val Thr Ala Tyr Ile 100 105 110 Phe Phe Cys Asn Ile Tyr Val Ser Ile Leu Phe Leu Cys Cys Ile Ser 115 120 125 Cys Asp Arg Phe Val Ala Val Val Tyr Ala Leu Glu Ser Arg Gly Arg 130 135 140 Arg Arg Arg Arg Thr Ala Ile Leu Ile Ser Ala Cys Ile Phe Ile Leu 145 150 155 160 Val Gly Ile Val His Tyr Pro Val Phe Gln Thr Glu Asp Lys Glu Thr 165 170 175 Cys Phe Asp Met Leu Gln Met Asp Ser Arg Ile Ala Gly Tyr Tyr Tyr 180 185 190 Ala Arg Phe Thr Val Gly Phe Ala Ile Pro Leu Ser Ile Ile Ala Phe 195 200 205 Thr Asn His Arg Ile Phe Arg Ser Ile Lys Gln Ser Met Gly Leu Ser 210 215 220 Ala Ala Gln Lys Ala Lys Val Lys His Ser Ala Ile Ala Val Val Val 225 230 235 240 Ile Phe Leu Val Cys Phe Ala Pro Tyr His Leu Val Leu Leu Val Lys 245 250 255 Ala Ala Ala Phe Ser Tyr Tyr Arg Gly Asp Arg Asn Ala Met Cys Gly 260 265 270 Leu Glu Glu Arg Leu Tyr Thr Ala Ser Val Val Phe Leu Cys Leu Ser 275 280 285 Thr Val Asn Gly Val Ala Asp Pro Ile Ile Tyr Val Leu Ala Thr Asp 290 295 300 His Ser Arg Gln Glu Val Ser Arg Ile His Lys Gly Trp Lys Glu Trp 305 310 315 320 Ser Met Lys Thr Asp Val Thr Arg Leu Thr His Ser Arg Asp Thr Glu 325 330 335 Glu Leu Gln Ser Pro Val Ala Leu Ala Asp His Tyr Thr Phe Ser Arg 340 345 350 Pro Val His Pro Pro Gly Ser Pro Cys Pro Ala Lys Arg Leu Ile Glu 355 360 365 Glu Ser Cys 370 27 1113 DNA Homo sapiens 27 atggcgaact atagccatgc agctgacaac attttgcaaa atctctcgcc tctaacagcc 60 tttctgaaac tgacttcctt gggtttcata ataggagtca gcgtggtggg caacctcctg 120 atctccattt tgctagtgaa agataagacc ttgcatagag caccttacta cttcctgttg 180 gatctttgct gttcagatat cctcagatct gcaatttgtt tcccatttgt gttcaactct 240 gtcaaaaatg gctctacctg gacttatggg actctgactt gcaaagtgat tgcctttctg 300 ggggttttgt cctgtttcca cactgctttc atgctcttct gcatcagtgt caccagatac 360 ttagctatcg cccatcaccg cttctataca aagaggctga ccttttggac gtgtctggct 420 gtgatctgta tggtgtggac tctgtctgtg gccatggcat ttcccccggt tttagacgtg 480 ggcacttact cattcattag ggaggaagat caatgcacct tccaacaccg ctccttcagg 540 gctaatgatt ccttaggatt tatgctgctt cttgctctca tcctcctagc cacacagctt 600 gtctacctca agctgatatt tttcgtccac gatcgaagaa aaatgaagcc agtccagttt 660 gtagcagcag tcagccagaa ctggactttt catggtcctg gagccagtgg ccaggcagct 720 gccaattggc tagcaggatt tggaaggggt cccacaccac ccaccttgct gggcatcagg 780 caaaatgcaa acaccacagg cagaagaagg ctattggtct tagacgagtt caaaatggag 840 aaaagaatca gcagaatgtt ctatataatg acttttctgt ttctaacctt gtggggcccc 900 tacctggtgg cctgttattg gagagttttt gcaagagggc ctgtagtacc agggggattt 960 ctaacagctg ctgtctggat gagttttgcc caagcaggaa tcaatccttt tgtctgcatt 1020 ttctcaaaca gggagctgag gcgctgtttc agcacaaccc ttctttactg cagaaaatcc 1080 aggttaccaa gggaacctta ctgtgttata tga 1113 28 370 PRT Homo sapiens 28 Met Ala Asn Tyr Ser His Ala Ala Asp Asn Ile Leu Gln Asn Leu Ser 1 5 10 15 Pro Leu Thr Ala Phe Leu Lys Leu Thr Ser Leu Gly Phe Ile Ile Gly 20 25 30 Val Ser Val Val Gly Asn Leu Leu Ile Ser Ile Leu Leu Val Lys Asp 35 40 45 Lys Thr Leu His Arg Ala Pro Tyr Tyr Phe Leu Leu Asp Leu Cys Cys 50 55 60 Ser Asp Ile Leu Arg Ser Ala Ile Cys Phe Pro Phe Val Phe Asn Ser 65 70 75 80 Val Lys Asn Gly Ser Thr Trp Thr Tyr Gly Thr Leu Thr Cys Lys Val 85 90 95 Ile Ala Phe Leu Gly Val Leu Ser Cys Phe His Thr Ala Phe Met Leu 100 105 110 Phe Cys Ile Ser Val Thr Arg Tyr Leu Ala Ile Ala His His Arg Phe 115 120 125 Tyr Thr Lys Arg Leu Thr Phe Trp Thr Cys Leu Ala Val Ile Cys Met 130 135 140 Val Trp Thr Leu Ser Val Ala Met Ala Phe Pro Pro Val Leu Asp Val 145 150 155 160 Gly Thr Tyr Ser Phe Ile Arg Glu Glu Asp Gln Cys Thr Phe Gln His 165 170 175 Arg Ser Phe Arg Ala Asn Asp Ser Leu Gly Phe Met Leu Leu Leu Ala 180 185 190 Leu Ile Leu Leu Ala Thr Gln Leu Val Tyr Leu Lys Leu Ile Phe Phe 195 200 205 Val His Asp Arg Arg Lys Met Lys Pro Val Gln Phe Val Ala Ala Val 210 215 220 Ser Gln Asn Trp Thr Phe His Gly Pro Gly Ala Ser Gly Gln Ala Ala 225 230 235 240 Ala Asn Trp Leu Ala Gly Phe Gly Arg Gly Pro Thr Pro Pro Thr Leu 245 250 255 Leu Gly Ile Arg Gln Asn Ala Asn Thr Thr Gly Arg Arg Arg Leu Leu 260 265 270 Val Leu Asp Glu Phe Lys Met Glu Lys Arg Ile Ser Arg Met Phe Tyr 275 280 285 Ile Met Thr Phe Leu Phe Leu Thr Leu Trp Gly Pro Tyr Leu Val Ala 290 295 300 Cys Tyr Trp Arg Val Phe Ala Arg Gly Pro Val Val Pro Gly Gly Phe 305 310 315 320 Leu Thr Ala Ala Val Trp Met Ser Phe Ala Gln Ala Gly Ile Asn Pro 325 330 335 Phe Val Cys Ile Phe Ser Asn Arg Glu Leu Arg Arg Cys Phe Ser Thr 340 345 350 Thr Leu Leu Tyr Cys Arg Lys Ser Arg Leu Pro Arg Glu Pro Tyr Cys 355 360 365 Val Ile 370 29 1080 DNA Homo sapiens 29 atgcaggtcc cgaacagcac cggcccggac aacgcgacgc tgcagatgct gcggaacccg 60 gcgatcgcgg tggccctgcc cgtggtgtac tcgctggtgg cggcggtcag catcccgggc 120 aacctcttct ctctgtgggt gctgtgccgg cgcatggggc ccagatcccc gtcggtcatc 180 ttcatgatca acctgagcgt cacggacctg atgctggcca gcgtgttgcc tttccaaatc 240 tactaccatt gcaaccgcca ccactgggta ttcggggtgc tgctttgcaa cgtggtgacc 300 gtggcctttt acgcaaacat gtattccagc atcctcacca tgacctgtat cagcgtggag 360 cgcttcctgg gggtcctgta cccgctcagc tccaagcgct ggcgccgccg tcgttacgcg 420 gtggccgcgt gtgcagggac ctggctgctg ctcctgaccg ccctgtgccc gctggcgcgc 480 accgatctca cctacccggt gcacgccctg ggcatcatca cctgcttcga cgtcctcaag 540 tggacgatgc tccccagcgt ggccatgtgg gccgtgttcc tcttcaccat cttcatcctg 600 ctgttcctca tcccgttcgt gatcaccgtg gcttgttaca cggccaccat cctcaagctg 660 ttgcgcacgg aggaggcgca cggccgggag cagcggaggc gcgcggtggg cctggccgcg 720 gtggtcttgc tggcctttgt cacctgcttc gcccccaaca acttcgtgct cctggcgcac 780 atcgtgagcc gcctgttcta cggcaagagc tactaccacg tgtacaagct cacgctgtgt 840 ctcagctgcc tcaacaactg tctggacccg tttgtttatt actttgcgtc ccgggaattc 900 cagctgcgcc tgcgggaata tttgggctgc cgccgggtgc ccagagacac cctggacacg 960 cgccgcgaga gcctcttctc cgccaggacc acgtccgtgc gctccgaggc cggtgcgcac 1020 cctgaaggga tggagggagc caccaggccc ggcctccaga ggcaggagag tgtgttctga 1080 30 359 PRT Homo sapiens 30 Met Gln Val Pro Asn Ser Thr Gly Pro Asp Asn Ala Thr Leu Gln Met 1 5 10 15 Leu Arg Asn Pro Ala Ile Ala Val Ala Leu Pro Val Val Tyr Ser Leu 20 25 30 Val Ala Ala Val Ser Ile Pro Gly Asn Leu Phe Ser Leu Trp Val Leu 35 40 45 Cys Arg Arg Met Gly Pro Arg Ser Pro Ser Val Ile Phe Met Ile Asn 50 55 60 Leu Ser Val Thr Asp Leu Met Leu Ala Ser Val Leu Pro Phe Gln Ile 65 70 75 80 Tyr Tyr His Cys Asn Arg His His Trp Val Phe Gly Val Leu Leu Cys 85 90 95 Asn Val Val Thr Val Ala Phe Tyr Ala Asn Met Tyr Ser Ser Ile Leu 100 105 110 Thr Met Thr Cys Ile Ser Val Glu Arg Phe Leu Gly Val Leu Tyr Pro 115 120 125 Leu Ser Ser Lys Arg Trp Arg Arg Arg Arg Tyr Ala Val Ala Ala Cys 130 135 140 Ala Gly Thr Trp Leu Leu Leu Leu Thr Ala Leu Cys Pro Leu Ala Arg 145 150 155 160 Thr Asp Leu Thr Tyr Pro Val His Ala Leu Gly Ile Ile Thr Cys Phe 165 170 175 Asp Val Leu Lys Trp Thr Met Leu Pro Ser Val Ala Met Trp Ala Val 180 185 190 Phe Leu Phe Thr Ile Phe Ile Leu Leu Phe Leu Ile Pro Phe Val Ile 195 200 205 Thr Val Ala Cys Tyr Thr Ala Thr Ile Leu Lys Leu Leu Arg Thr Glu 210 215 220 Glu Ala His Gly Arg Glu Gln Arg Arg Arg Ala Val Gly Leu Ala Ala 225 230 235 240 Val Val Leu Leu Ala Phe Val Thr Cys Phe Ala Pro Asn Asn Phe Val 245 250 255 Leu Leu Ala His Ile Val Ser Arg Leu Phe Tyr Gly Lys Ser Tyr Tyr 260 265 270 His Val Tyr Lys Leu Thr Leu Cys Leu Ser Cys Leu Asn Asn Cys Leu 275 280 285 Asp Pro Phe Val Tyr Tyr Phe Ala Ser Arg Glu Phe Gln Leu Arg Leu 290 295 300 Arg Glu Tyr Leu Gly Cys Arg Arg Val Pro Arg Asp Thr Leu Asp Thr 305 310 315 320 Arg Arg Glu Ser Leu Phe Ser Ala Arg Thr Thr Ser Val Arg Ser Glu 325 330 335 Ala Gly Ala His Pro Glu Gly Met Glu Gly Ala Thr Arg Pro Gly Leu 340 345 350 Gln Arg Gln Glu Ser Val Phe 355 31 1503 DNA Homo sapiens 31 atggagcgtc cctgggagga cagcccaggc ccggaggggg cagctgaggg ctcgcctgtg 60 ccagtcgccg ccggggcgcg ctccggtgcc gcggcgagtg gcacaggctg gcagccatgg 120 gctgagtgcc cgggacccaa ggggaggggg caactgctgg cgaccgccgg ccctttgcgt 180 cgctggcccg ccccctcgcc tgccagctcc agccccgccc ccggagcggc gtccgctcac 240 tcggttcaag gcagcgcgac tgcgggtggc gcacgaccag ggcgcagacc ttggggcgcg 300 cggcccatgg agtcggggct gctgcggccg gcgccggtga gcgaggtcat cgtcctgcat 360 tacaactaca ccggcaagct ccgcggtgcg agctaccagc cgggtgccgg cctgcgcgcc 420 gacgccgtgg tgtgcctggc ggtgtgcgcc ttcatcgtgc tagagaatct agccgtgttg 480 ttggtgctcg gacgccaccc gcgcttccac gctcccatgt tcctgctcct gggcagcctc 540 acgttgtcgg atctgctggc aggcgccgcc tacgccgcca acatcctact gtcggggccg 600 ctcacgctga aactgtcccc cgcgctctgg ttcgcacggg agggaggcgt cttcgtggca 660 ctcactgcgt ccgtgctgag cctcctggcc atcgcgctgg agcgcagcct caccatggcg 720 cgcagggggc ccgcgcccgt ctccagtcgg gggcgcacgc tggcgatggc agccgcggcc 780 tggggcgtgt cgctgctcct cgggctcctg ccagcgctgg gctggaattg cctgggtcgc 840 ctggacgctt gctccactgt cttgccgctc tacgccaagg cctacgtgct cttctgcgtg 900 ctcgccttcg tgggcatcct ggccgcgatc tgtgcactct acgcgcgcat ctactgccag 960 gtacgcgcca acgcgcggcg cctgccggca cggcccggga ctgcggggac cacctcgacc 1020 cgggcgcgtc gcaagccgcg ctctctggcc ttgctgcgca cgctcagcgt ggtgctcctg 1080 gcctttgtgg catgttgggg ccccctcttc ctgctgctgt tgctcgacgt ggcgtgcccg 1140 gcgcgcacct gtcctgtact cctgcaggcc gatcccttcc tgggactggc catggccaac 1200 tcacttctga accccatcat ctacacgctc accaaccgcg acctgcgcca cgcgctcctg 1260 cgcctggtct gctgcggacg ccactcctgc ggcagagacc cgagtggctc ccagcagtcg 1320 gcgagcgcgg ctgaggcttc cgggggcctg cgccgctgcc tgcccccggg ccttgatggg 1380 agcttcagcg gctcggagcg ctcatcgccc cagcgcgacg ggctggacac cagcggctcc 1440 acaggcagcc ccggtgcacc cacagccgcc cggactctgg tatcagaacc ggctgcagac 1500 tga 1503 32 500 PRT Homo sapiens 32 Met Glu Arg Pro Trp Glu Asp Ser Pro Gly Pro Glu Gly Ala Ala Glu 1 5 10 15 Gly Ser Pro Val Pro Val Ala Ala Gly Ala Arg Ser Gly Ala Ala Ala 20 25 30 Ser Gly Thr Gly Trp Gln Pro Trp Ala Glu Cys Pro Gly Pro Lys Gly 35 40 45 Arg Gly Gln Leu Leu Ala Thr Ala Gly Pro Leu Arg Arg Trp Pro Ala 50 55 60 Pro Ser Pro Ala Ser Ser Ser Pro Ala Pro Gly Ala Ala Ser Ala His 65 70 75 80 Ser Val Gln Gly Ser Ala Thr Ala Gly Gly Ala Arg Pro Gly Arg Arg 85 90 95 Pro Trp Gly Ala Arg Pro Met Glu Ser Gly Leu Leu Arg Pro Ala Pro 100 105 110 Val Ser Glu Val Ile Val Leu His Tyr Asn Tyr Thr Gly Lys Leu Arg 115 120 125 Gly Ala Ser Tyr Gln Pro Gly Ala Gly Leu Arg Ala Asp Ala Val Val 130 135 140 Cys Leu Ala Val Cys Ala Phe Ile Val Leu Glu Asn Leu Ala Val Leu 145 150 155 160 Leu Val Leu Gly Arg His Pro Arg Phe His Ala Pro Met Phe Leu Leu 165 170 175 Leu Gly Ser Leu Thr Leu Ser Asp Leu Leu Ala Gly Ala Ala Tyr Ala 180 185 190 Ala Asn Ile Leu Leu Ser Gly Pro Leu Thr Leu Lys Leu Ser Pro Ala 195 200 205 Leu Trp Phe Ala Arg Glu Gly Gly Val Phe Val Ala Leu Thr Ala Ser 210 215 220 Val Leu Ser Leu Leu Ala Ile Ala Leu Glu Arg Ser Leu Thr Met Ala 225 230 235 240 Arg Arg Gly Pro Ala Pro Val Ser Ser Arg Gly Arg Thr Leu Ala Met 245 250 255 Ala Ala Ala Ala Trp Gly Val Ser Leu Leu Leu Gly Leu Leu Pro Ala 260 265 270 Leu Gly Trp Asn Cys Leu Gly Arg Leu Asp Ala Cys Ser Thr Val Leu 275 280 285 Pro Leu Tyr Ala Lys Ala Tyr Val Leu Phe Cys Val Leu Ala Phe Val 290 295 300 Gly Ile Leu Ala Ala Ile Cys Ala Leu Tyr Ala Arg Ile Tyr Cys Gln 305 310 315 320 Val Arg Ala Asn Ala Arg Arg Leu Pro Ala Arg Pro Gly Thr Ala Gly 325 330 335 Thr Thr Ser Thr Arg Ala Arg Arg Lys Pro Arg Ser Leu Ala Leu Leu 340 345 350 Arg Thr Leu Ser Val Val Leu Leu Ala Phe Val Ala Cys Trp Gly Pro 355 360 365 Leu Phe Leu Leu Leu Leu Leu Asp Val Ala Cys Pro Ala Arg Thr Cys 370 375 380 Pro Val Leu Leu Gln Ala Asp Pro Phe Leu Gly Leu Ala Met Ala Asn 385 390 395 400 Ser Leu Leu Asn Pro Ile Ile Tyr Thr Leu Thr Asn Arg Asp Leu Arg 405 410 415 His Ala Leu Leu Arg Leu Val Cys Cys Gly Arg His Ser Cys Gly Arg 420 425 430 Asp Pro Ser Gly Ser Gln Gln Ser Ala Ser Ala Ala Glu Ala Ser Gly 435 440 445 Gly Leu Arg Arg Cys Leu Pro Pro Gly Leu Asp Gly Ser Phe Ser Gly 450 455 460 Ser Glu Arg Ser Ser Pro Gln Arg Asp Gly Leu Asp Thr Ser Gly Ser 465 470 475 480 Thr Gly Ser Pro Gly Ala Pro Thr Ala Ala Arg Thr Leu Val Ser Glu 485 490 495 Pro Ala Ala Asp 500 33 1029 DNA Homo sapiens 33 atgcaagccg tcgacaatct cacctctgcg cctgggaaca ccagtctgtg caccagagac 60 tacaaaatca cccaggtcct cttcccactg ctctacactg tcctgttttt tgttggactt 120 atcacaaatg gcctggcgat gaggattttc tttcaaatcc ggagtaaatc aaactttatt 180 atttttctta agaacacagt catttctgat cttctcatga ttctgacttt tccattcaaa 240 attcttagtg atgccaaact gggaacagga ccactgagaa cttttgtgtg tcaagttacc 300 tccgtcatat tttatttcac aatgtatatc agtatttcat tcctgggact gataactatc 360 gatcgctacc agaagaccac caggccattt aaaacatcca accccaaaaa tctcttgggg 420 gctaagattc tctctgttgt catctgggca ttcatgttct tactctcttt gcctaacatg 480 attctgacca acaggcagcc gagagacaag aatgtgaaga aatgctcttt ccttaaatca 540 gagttcggtc tagtctggca tgaaatagta aattacatct gtcaagtcat tttctggatt 600 aatttcttaa ttgttattgt atgttataca ctcattacaa aagaactgta ccggtcatac 660 gtaagaacga ggggtgtagg taaagtcccc aggaaaaagg tgaacgtcaa agttttcatt 720 atcattgctg tattctttat ttgttttgtt cctttccatt ttgcccgaat tccttacacc 780 ctgagccaaa cccgggatgt ctttgactgc actgctgaaa atactctgtt ctatgtgaaa 840 gagagcactc tgtggttaac ttccttaaat gcatgcctgg atccgttcat ctattttttc 900 ctttgcaagt ccttcagaaa ttccttgata agtatgctga agtgccccaa ttctgcaaca 960 tctctgtccc aggacaatag gaaaaaagaa caggatggtg gtgacccaaa tgaagagact 1020 ccaatgtaa 1029 34 342 PRT Homo sapiens 34 Met Gln Ala Val Asp Asn Leu Thr Ser Ala Pro Gly Asn Thr Ser Leu 1 5 10 15 Cys Thr Arg Asp Tyr Lys Ile Thr Gln Val Leu Phe Pro Leu Leu Tyr 20 25 30 Thr Val Leu Phe Phe Val Gly Leu Ile Thr Asn Gly Leu Ala Met Arg 35 40 45 Ile Phe Phe Gln Ile Arg Ser Lys Ser Asn Phe Ile Ile Phe Leu Lys 50 55 60 Asn Thr Val Ile Ser Asp Leu Leu Met Ile Leu Thr Phe Pro Phe Lys 65 70 75 80 Ile Leu Ser Asp Ala Lys Leu Gly Thr Gly Pro Leu Arg Thr Phe Val 85 90 95 Cys Gln Val Thr Ser Val Ile Phe Tyr Phe Thr Met Tyr Ile Ser Ile 100 105 110 Ser Phe Leu Gly Leu Ile Thr Ile Asp Arg Tyr Gln Lys Thr Thr Arg 115 120 125 Pro Phe Lys Thr Ser Asn Pro Lys Asn Leu Leu Gly Ala Lys Ile Leu 130 135 140 Ser Val Val Ile Trp Ala Phe Met Phe Leu Leu Ser Leu Pro Asn Met 145 150 155 160 Ile Leu Thr Asn Arg Gln Pro Arg Asp Lys Asn Val Lys Lys Cys Ser 165 170 175 Phe Leu Lys Ser Glu Phe Gly Leu Val Trp His Glu Ile Val Asn Tyr 180 185 190 Ile Cys Gln Val Ile Phe Trp Ile Asn Phe Leu Ile Val Ile Val Cys 195 200 205 Tyr Thr Leu Ile Thr Lys Glu Leu Tyr Arg Ser Tyr Val Arg Thr Arg 210 215 220 Gly Val Gly Lys Val Pro Arg Lys Lys Val Asn Val Lys Val Phe Ile 225 230 235 240 Ile Ile Ala Val Phe Phe Ile Cys Phe Val Pro Phe His Phe Ala Arg 245 250 255 Ile Pro Tyr Thr Leu Ser Gln Thr Arg Asp Val Phe Asp Cys Thr Ala 260 265 270 Glu Asn Thr Leu Phe Tyr Val Lys Glu Ser Thr Leu Trp Leu Thr Ser 275 280 285 Leu Asn Ala Cys Leu Asp Pro Phe Ile Tyr Phe Phe Leu Cys Lys Ser 290 295 300 Phe Arg Asn Ser Leu Ile Ser Met Leu Lys Cys Pro Asn Ser Ala Thr 305 310 315 320 Ser Leu Ser Gln Asp Asn Arg Lys Lys Glu Gln Asp Gly Gly Asp Pro 325 330 335 Asn Glu Glu Thr Pro Met 340 35 1077 DNA Homo sapiens 35 atgtcggtct gctaccgtcc cccagggaac gagacactgc tgagctggaa gacttcgcgg 60 gccacaggca cagccttcct gctgctggcg gcgctgctgg ggctgcctgg caacggcttc 120 gtggtgtgga gcttggcggg ctggcggcct gcacgggggc gaccgctggc ggccacgctt 180 gtgctgcacc tggcgctggc cgacggcgcg gtgctgctgc tcacgccgct ctttgtggcc 240 ttcctgaccc ggcaggcctg gccgctgggc caggcgggct gcaaggcggt gtactacgtg 300 tgcgcgctca gcatgtacgc cagcgtgctg ctcaccggcc tgctcagcct gcagcgctgc 360 ctcgcagtca cccgcccctt cctggcgcct cggctgcgca gcccggccct ggcccgccgc 420 ctgctgctgg cggtctggct ggccgccctg ttgctcgccg tcccggccgc cgtctaccgc 480 cacctgtgga gggaccgcgt atgccagctg tgccacccgt cgccggtcca cgccgccgcc 540 cacctgagcc tggagactct gaccgctttc gtgcttcctt tcgggctgat gctcggctgc 600 tacagcgtga cgctggcacg gctgcggggc gcccgctggg gctccgggcg gcacggggcg 660 cgggtgggcc ggctggtgag cgccatcgtg cttgccttcg gcttgctctg ggccccctac 720 cacgcagtca accttctgca ggcggtcgca gcgctggctc caccggaagg ggccttggcg 780 aagctgggcg gagccggcca ggcggcgcga gcgggaacta cggccttggc cttcttcagt 840 tctagcgtca acccggtgct ctacgtcttc accgctggag atctgctgcc ccgggcaggt 900 ccccgtttcc tcacgcggct cttcgaaggc tctggggagg cccgaggggg cggccgctct 960 agggaaggga ccatggagct ccgaactacc cctcagctga aagtggtggg gcagggccgc 1020 ggcaatggag acccgggggg tgggatggag aaggacggtc cggaatggga cctttga 1077 36 358 PRT Homo sapiens 36 Met Ser Val Cys Tyr Arg Pro Pro Gly Asn Glu Thr Leu Leu Ser Trp 1 5 10 15 Lys Thr Ser Arg Ala Thr Gly Thr Ala Phe Leu Leu Leu Ala Ala Leu 20 25 30 Leu Gly Leu Pro Gly Asn Gly Phe Val Val Trp Ser Leu Ala Gly Trp 35 40 45 Arg Pro Ala Arg Gly Arg Pro Leu Ala Ala Thr Leu Val Leu His Leu 50 55 60 Ala Leu Ala Asp Gly Ala Val Leu Leu Leu Thr Pro Leu Phe Val Ala 65 70 75 80 Phe Leu Thr Arg Gln Ala Trp Pro Leu Gly Gln Ala Gly Cys Lys Ala 85 90 95 Val Tyr Tyr Val Cys Ala Leu Ser Met Tyr Ala Ser Val Leu Leu Thr 100 105 110 Gly Leu Leu Ser Leu Gln Arg Cys Leu Ala Val Thr Arg Pro Phe Leu 115 120 125 Ala Pro Arg Leu Arg Ser Pro Ala Leu Ala Arg Arg Leu Leu Leu Ala 130 135 140 Val Trp Leu Ala Ala Leu Leu Leu Ala Val Pro Ala Ala Val Tyr Arg 145 150 155 160 His Leu Trp Arg Asp Arg Val Cys Gln Leu Cys His Pro Ser Pro Val 165 170 175 His Ala Ala Ala His Leu Ser Leu Glu Thr Leu Thr Ala Phe Val Leu 180 185 190 Pro Phe Gly Leu Met Leu Gly Cys Tyr Ser Val Thr Leu Ala Arg Leu 195 200 205 Arg Gly Ala Arg Trp Gly Ser Gly Arg His Gly Ala Arg Val Gly Arg 210 215 220 Leu Val Ser Ala Ile Val Leu Ala Phe Gly Leu Leu Trp Ala Pro Tyr 225 230 235 240 His Ala Val Asn Leu Leu Gln Ala Val Ala Ala Leu Ala Pro Pro Glu 245 250 255 Gly Ala Leu Ala Lys Leu Gly Gly Ala Gly Gln Ala Ala Arg Ala Gly 260 265 270 Thr Thr Ala Leu Ala Phe Phe Ser Ser Ser Val Asn Pro Val Leu Tyr 275 280 285 Val Phe Thr Ala Gly Asp Leu Leu Pro Arg Ala Gly Pro Arg Phe Leu 290 295 300 Thr Arg Leu Phe Glu Gly Ser Gly Glu Ala Arg Gly Gly Gly Arg Ser 305 310 315 320 Arg Glu Gly Thr Met Glu Leu Arg Thr Thr Pro Gln Leu Lys Val Val 325 330 335 Gly Gln Gly Arg Gly Asn Gly Asp Pro Gly Gly Gly Met Glu Lys Asp 340 345 350 Gly Pro Glu Trp Asp Leu 355 37 1005 DNA Homo sapiens 37 atgctgggga tcatggcatg gaatgcaact tgcaaaaact ggctggcagc agaggctgcc 60 ctggaaaagt actacctttc cattttttat gggattgagt tcgttgtggg agtccttgga 120 aataccattg ttgtttacgg ctacatcttc tctctgaaga actggaacag cagtaatatt 180 tatctcttta acctctctgt ctctgactta gcttttctgt gcaccctccc catgctgata 240 aggagttatg ccaatggaaa ctggatatat ggagacgtgc tctgcataag caaccgatat 300 gtgcttcatg ccaacctcta taccagcatt ctctttctca cttttatcag catagatcga 360 tacttgataa ttaagtatcc tttccgagaa caccttctgc aaaagaaaga gtttgctatt 420 ttaatctcct tggccatttg ggttttagta accttagagt tactacccat acttcccctt 480 ataaatcctg ttataactga caatggcacc acctgtaatg attttgcaag ttctggagac 540 cccaactaca acctcattta cagcatgtgt ctaacactgt tggggttcct tattcctctt 600 tttgtgatgt gtttctttta ttacaagatt gctctcttcc taaagcagag gaataggcag 660 gttgctactg ctctgcccct tgaaaagcct ctcaacttgg tcatcatggc agtggtaatc 720 ttctctgtgc tttttacacc ctatcacgtc atgcggaatg tgaggatcgc ttcacgcctg 780 gggagttgga agcagtatca gtgcactcag gtcgtcatca actcctttta cattgtgaca 840 cggcctttgg cctttctgaa cagtgtcatc aaccctgtct tctattttct tttgggagat 900 cacttcaggg acatgctgat gaatcaactg agacacaact tcaaatccct tacatccttt 960 agcagatggg ctcatgaact cctactttca ttcagagaaa agtga 1005 38 334 PRT Homo sapiens 38 Met Leu Gly Ile Met Ala Trp Asn Ala Thr Cys Lys Asn Trp Leu Ala 1 5 10 15 Ala Glu Ala Ala Leu Glu Lys Tyr Tyr Leu Ser Ile Phe Tyr Gly Ile 20 25 30 Glu Phe Val Val Gly Val Leu Gly Asn Thr Ile Val Val Tyr Gly Tyr 35 40 45 Ile Phe Ser Leu Lys Asn Trp Asn Ser Ser Asn Ile Tyr Leu Phe Asn 50 55 60 Leu Ser Val Ser Asp Leu Ala Phe Leu Cys Thr Leu Pro Met Leu Ile 65 70 75 80 Arg Ser Tyr Ala Asn Gly Asn Trp Ile Tyr Gly Asp Val Leu Cys Ile 85 90 95 Ser Asn Arg Tyr Val Leu His Ala Asn Leu Tyr Thr Ser Ile Leu Phe 100 105 110 Leu Thr Phe Ile Ser Ile Asp Arg Tyr Leu Ile Ile Lys Tyr Pro Phe 115 120 125 Arg Glu His Leu Leu Gln Lys Lys Glu Phe Ala Ile Leu Ile Ser Leu 130 135 140 Ala Ile Trp Val Leu Val Thr Leu Glu Leu Leu Pro Ile Leu Pro Leu 145 150 155 160 Ile Asn Pro Val Ile Thr Asp Asn Gly Thr Thr Cys Asn Asp Phe Ala 165 170 175 Ser Ser Gly Asp Pro Asn Tyr Asn Leu Ile Tyr Ser Met Cys Leu Thr 180 185 190 Leu Leu Gly Phe Leu Ile Pro Leu Phe Val Met Cys Phe Phe Tyr Tyr 195 200 205 Lys Ile Ala Leu Phe Leu Lys Gln Arg Asn Arg Gln Val Ala Thr Ala 210 215 220 Leu Pro Leu Glu Lys Pro Leu Asn Leu Val Ile Met Ala Val Val Ile 225 230 235 240 Phe Ser Val Leu Phe Thr Pro Tyr His Val Met Arg Asn Val Arg Ile 245 250 255 Ala Ser Arg Leu Gly Ser Trp Lys Gln Tyr Gln Cys Thr Gln Val Val 260 265 270 Ile Asn Ser Phe Tyr Ile Val Thr Arg Pro Leu Ala Phe Leu Asn Ser 275 280 285 Val Ile Asn Pro Val Phe Tyr Phe Leu Leu Gly Asp His Phe Arg Asp 290 295 300 Met Leu Met Asn Gln Leu Arg His Asn Phe Lys Ser Leu Thr Ser Phe 305 310 315 320 Ser Arg Trp Ala His Glu Leu Leu Leu Ser Phe Arg Glu Lys 325 330 39 1296 DNA Homo sapiens 39 atgcaggcgc ttaacattac cccggagcag ttctctcggc tgctgcggga ccacaacctg 60 acgcgggagc agttcatcgc tctgtaccgg ctgcgaccgc tcgtctacac cccagagctg 120 ccgggacgcg ccaagctggc cctcgtgctc accggcgtgc tcatcttcgc cctggcgctc 180 tttggcaatg ctctggtgtt ctacgtggtg acccgcagca aggccatgcg caccgtcacc 240 aacatcttta tctgctcctt ggcgctcagt gacctgctca tcaccttctt ctgcattccc 300 gtcaccatgc tccagaacat ttccgacaac tggctggggg gtgctttcat ttgcaagatg 360 gtgccatttg tccagtctac cgctgttgtg acagaaatgc tcactatgac ctgcattgct 420 gtggaaaggc accagggact tgtgcatcct tttaaaatga agtggcaata caccaaccga 480 agggctttca caatgctagg tgtggtctgg ctggtggcag tcatcgtagg atcacccatg 540 tggcacgtgc aacaacttga gatcaaatat gacttcctat atgaaaagga acacatctgc 600 tgcttagaag agtggaccag ccctgtgcac cagaagatct acaccacctt catccttgtc 660 atcctcttcc tcctgcctct tatggtgatg cttattctgt acagtaaaat tggttatgaa 720 ctttggataa agaaaagagt tggggatggt tcagtgcttc gaactattca tggaaaagaa 780 atgtccaaaa tagccaggaa gaagaaacga gctgtcatta tgatggtgac agtggtggct 840 ctctttgctg tgtgctgggc accattccat gttgtccata tgatgattga atacagtaat 900 tttgaaaagg aatatgatga tgtcacaatc aagatgattt ttgctatcgt gcaaattatt 960 ggattttcca actccatctg taatcccatt gtctatgcat ttatgaatga aaacttcaaa 1020 aaaaatgttt tgtctgcagt ttgttattgc atagtaaata aaaccttctc tccagcacaa 1080 aggcatggaa attcaggaat tacaatgatg cggaagaaag caaagttttc cctcagagag 1140 aatccagtgg aggaaaccaa aggagaagca ttcagtgatg gcaacattga agtcaaattg 1200 tgtgaacaga cagaggagaa gaaaaagctc aaacgacatc ttgctctctt taggtctgaa 1260 ctggctgaga attctccttt agacagtggg cattaa 1296 40 431 PRT Homo sapiens 40 Met Gln Ala Leu Asn Ile Thr Pro Glu Gln Phe Ser Arg Leu Leu Arg 1 5 10 15 Asp His Asn Leu Thr Arg Glu Gln Phe Ile Ala Leu Tyr Arg Leu Arg 20 25 30 Pro Leu Val Tyr Thr Pro Glu Leu Pro Gly Arg Ala Lys Leu Ala Leu 35 40 45 Val Leu Thr Gly Val Leu Ile Phe Ala Leu Ala Leu Phe Gly Asn Ala 50 55 60 Leu Val Phe Tyr Val Val Thr Arg Ser Lys Ala Met Arg Thr Val Thr 65 70 75 80 Asn Ile Phe Ile Cys Ser Leu Ala Leu Ser Asp Leu Leu Ile Thr Phe 85 90 95 Phe Cys Ile Pro Val Thr Met Leu Gln Asn Ile Ser Asp Asn Trp Leu 100 105 110 Gly Gly Ala Phe Ile Cys Lys Met Val Pro Phe Val Gln Ser Thr Ala 115 120 125 Val Val Thr Glu Met Leu Thr Met Thr Cys Ile Ala Val Glu Arg His 130 135 140 Gln Gly Leu Val His Pro Phe Lys Met Lys Trp Gln Tyr Thr Asn Arg 145 150 155 160 Arg Ala Phe Thr Met Leu Gly Val Val Trp Leu Val Ala Val Ile Val 165 170 175 Gly Ser Pro Met Trp His Val Gln Gln Leu Glu Ile Lys Tyr Asp Phe 180 185 190 Leu Tyr Glu Lys Glu His Ile Cys Cys Leu Glu Glu Trp Thr Ser Pro 195 200 205 Val His Gln Lys Ile Tyr Thr Thr Phe Ile Leu Val Ile Leu Phe Leu 210 215 220 Leu Pro Leu Met Val Met Leu Ile Leu Tyr Ser Lys Ile Gly Tyr Glu 225 230 235 240 Leu Trp Ile Lys Lys Arg Val Gly Asp Gly Ser Val Leu Arg Thr Ile 245 250 255 His Gly Lys Glu Met Ser Lys Ile Ala Arg Lys Lys Lys Arg Ala Val 260 265 270 Ile Met Met Val Thr Val Val Ala Leu Phe Ala Val Cys Trp Ala Pro 275 280 285 Phe His Val Val His Met Met Ile Glu Tyr Ser Asn Phe Glu Lys Glu 290 295 300 Tyr Asp Asp Val Thr Ile Lys Met Ile Phe Ala Ile Val Gln Ile Ile 305 310 315 320 Gly Phe Ser Asn Ser Ile Cys Asn Pro Ile Val Tyr Ala Phe Met Asn 325 330 335 Glu Asn Phe Lys Lys Asn Val Leu Ser Ala Val Cys Tyr Cys Ile Val 340 345 350 Asn Lys Thr Phe Ser Pro Ala Gln Arg His Gly Asn Ser Gly Ile Thr 355 360 365 Met Met Arg Lys Lys Ala Lys Phe Ser Leu Arg Glu Asn Pro Val Glu 370 375 380 Glu Thr Lys Gly Glu Ala Phe Ser Asp Gly Asn Ile Glu Val Lys Leu 385 390 395 400 Cys Glu Gln Thr Glu Glu Lys Lys Lys Leu Lys Arg His Leu Ala Leu 405 410 415 Phe Arg Ser Glu Leu Ala Glu Asn Ser Pro Leu Asp Ser Gly His 420 425 430 41 24 DNA Artificial Sequence Novel Sequence 41 ctgtgtacag cagttcgcag agtg 24 42 24 DNA Artificial Sequence Novel Sequence 42 gagtgccagg cagagcaggt agac 24 43 31 DNA Artificial Sequence Novel Sequence 43 cccgaattcc tgcttgctcc cagcttggcc c 31 44 32 DNA Artificial Sequence Novel Sequence 44 tgtggatcct gctgtcaaag gtcccattcc gg 32 45 20 DNA Artificial Sequence Novel Sequence 45 tcacaatgct aggtgtggtc 20 46 22 DNA Artificial Sequence Novel Sequence 46 tgcatagaca atgggattac ag 22 47 511 DNA Homo sapiens 47 tcacaatgct aggtgtggtc tggctggtgg cagtcatcgt aggatcaccc atgtggcacg 60 tgcaacaact tgagatcaaa tatgacttcc tatatgaaaa ggaacacatc tgctgcttag 120 aagagtggac cagccctgtg caccagaaga tctacaccac cttcatcctt gtcatcctct 180 tcctcctgcc tcttatggtg atgcttattc tgtacgtaaa attggttatg aactttggat 240 aaagaaaaga gttggggatg gttcagtgct tcgaactatt catggaaaag aaatgtccaa 300 aatagccagg aagaagaaac gagctgtcat tatgatggtg acagtggtgg ctctctttgc 360 tgtgtgctgg gcaccattcc atgttgtcca tatgatgatt gaatacagta attttgaaaa 420 ggaatatgat gatgtcacaa tcaagatgat ttttgctatc gtgcaaatta ttggattttc 480 caactccatc tgtaatccca ttgtctatgc a 511 48 21 DNA Artificial Sequence Novel Sequence 48 ctgcttagaa gagtggacca g 21 49 22 DNA Artificial Sequence Novel Sequence 49 ctgtgcacca gaagatctac ac 22 50 21 DNA Artificial Sequence Novel Sequence 50 caaggatgaa ggtggtgtag a 21 51 23 DNA Artificial Sequence Novel Sequence 51 gtgtagatct tctggtgcac agg 23 52 21 DNA Artificial Sequence Novel Sequence 52 gcaatgcagg tcatagtgag c 21 53 27 DNA Artificial Sequence Novel Sequence 53 tggagcatgg tgacgggaat gcagaag 27 54 27 DNA Artificial Sequence Novel Sequence 54 gtgatgagca ggtcactgag cgccaag 27 55 23 DNA Artificial Sequence Novel Sequence 55 gcaatgcagg cgcttaacat tac 23 56 22 DNA Artificial Sequence Novel Sequence 56 ttgggttaca atctgaaggg ca 22 57 23 DNA Artificial Sequence Novel Sequence 57 actccgtgtc cagcaggact ctg 23 58 24 DNA Artificial Sequence Novel Sequence 58 tgcgtgttcc tggaccctca cgtg 24 59 29 DNA Artificial Sequence Novel Sequence 59 caggccttgg attttaatgt cagggatgg 29 60 27 DNA Artificial Sequence Novel Sequence 60 ggagagtcag ctctgaaaga attcagg 27 61 27 DNA Artificial Sequence Novel Sequence 61 tgatgtgatg ccagatacta atagcac 27 62 27 DNA Artificial Sequence Novel Sequence 62 cctgattcat ttaggtgaga ttgagac 27 63 26 DNA Artificial Sequence Novel Sequence 63 cccaagcttc cccaggtgta tttgat 26 64 26 DNA Artificial Sequence Novel Sequence 64 gttggatcca cataatgcat tttctc 26 65 1080 DNA Homo sapiens 65 atgattctca actcttctac tgaagatggt attaaaagaa tccaagatga ttgtcccaaa 60 gctggaaggc ataattacat atttgtcatg attcctactt tatacagtat catctttgtg 120 gtgggaatat ttggaaacag cttggtggtg atagtcattt acttttatat gaagctgaag 180 actgtggcca gtgtttttct tttgaattta gcactggctg acttatgctt tttactgact 240 ttgccactat gggctgtcta cacagctatg gaataccgct ggccctttgg caattaccta 300 tgtaagattg cttcagccag cgtcagtttc aacctgtacg ctagtgtgtt tctactcacg 360 tgtctcagca ttgatcgata cctggctatt gttcacccaa tgaagtcccg ccttcgacgc 420 acaatgcttg tagccaaagt cacctgcatc atcatttggc tgctggcagg cttggccagt 480 ttgccagcta taatccatcg aaatgtattt ttcattgaga acaccaatat tacagtttgt 540 gctttccatt atgagtccca aaattcaacc cttccgatag ggctgggcct gaccaaaaat 600 atactgggtt tcctgtttcc ttttctgatc attcttacaa gttatactct tatttggaag 660 gccctaaaga aggcttatga aattcagaag aacaaaccaa gaaatgatga tatttttaag 720 ataattatgg caattgtgct tttctttttc ttttcctgga ttccccacca aatattcact 780 tttctggatg tattgattca actaggcatc atacgtgact gtagaattgc agatattgtg 840 gacacggcca tgcctatcac catttgtata gcttatttta acaattgcct gaatcctctt 900 ttttatggct ttctggggaa aaaatttaaa agatattttc tccagcttct aaaatatatt 960 cccccaaaag ccaaatccca ctcaaacctt tcaacaaaaa tgagcacgct ttcctaccgc 1020 ccctcagata atgtaagctc atccaccaag aagcctgcac catgttttga ggttgagtga 1080 66 359 PRT Homo sapiens 66 Met Ile Leu Asn Ser Ser Thr Glu Asp Gly Ile Lys Arg Ile Gln Asp 1 5 10 15 Asp Cys Pro Lys Ala Gly Arg His Asn Tyr Ile Phe Val Met Ile Pro 20 25 30 Thr Leu Tyr Ser Ile Ile Phe Val Val Gly Ile Phe Gly Asn Ser Leu 35 40 45 Val Val Ile Val Ile Tyr Phe Tyr Met Lys Leu Lys Thr Val Ala Ser 50 55 60 Val Phe Leu Leu Asn Leu Ala Leu Ala Asp Leu Cys Phe Leu Leu Thr 65 70 75 80 Leu Pro Leu Trp Ala Val Tyr Thr Ala Met Glu Tyr Arg Trp Pro Phe 85 90 95 Gly Asn Tyr Leu Cys Lys Ile Ala Ser Ala Ser Val Ser Phe Asn Leu 100 105 110 Tyr Ala Ser Val Phe Leu Leu Thr Cys Leu Ser Ile Asp Arg Tyr Leu 115 120 125 Ala Ile Val His Pro Met Lys Ser Arg Leu Arg Arg Thr Met Leu Val 130 135 140 Ala Lys Val Thr Cys Ile Ile Ile Trp Leu Leu Ala Gly Leu Ala Ser 145 150 155 160 Leu Pro Ala Ile Ile His Arg Asn Val Phe Phe Ile Glu Asn Thr Asn 165 170 175 Ile Thr Val Cys Ala Phe His Tyr Glu Ser Gln Asn Ser Thr Leu Pro 180 185 190 Ile Gly Leu Gly Leu Thr Lys Asn Ile Leu Gly Phe Leu Phe Pro Phe 195 200 205 Leu Ile Ile Leu Thr Ser Tyr Thr Leu Ile Trp Lys Ala Leu Lys Lys 210 215 220 Ala Tyr Glu Ile Gln Lys Asn Lys Pro Arg Asn Asp Asp Ile Phe Lys 225 230 235 240 Ile Ile Met Ala Ile Val Leu Phe Phe Phe Phe Ser Trp Ile Pro His 245 250 255 Gln Ile Phe Thr Phe Leu Asp Val Leu Ile Gln Leu Gly Ile Ile Arg 260 265 270 Asp Cys Arg Ile Ala Asp Ile Val Asp Thr Ala Met Pro Ile Thr Ile 275 280 285 Cys Ile Ala Tyr Phe Asn Asn Cys Leu Asn Pro Leu Phe Tyr Gly Phe 290 295 300 Leu Gly Lys Lys Phe Lys Arg Tyr Phe Leu Gln Leu Leu Lys Tyr Ile 305 310 315 320 Pro Pro Lys Ala Lys Ser His Ser Asn Leu Ser Thr Lys Met Ser Thr 325 330 335 Leu Ser Tyr Arg Pro Ser Asp Asn Val Ser Ser Ser Thr Lys Lys Pro 340 345 350 Ala Pro Cys Phe Glu Val Glu 355 67 27 DNA Artificial Sequence Novel Sequence 67 accatgggca gcccctggaa cggcagc 27 68 39 DNA Artificial Sequence Novel Sequence 68 agaaccacca ccagcaggac gcggacggtc tgccggtgg 39 69 39 DNA Artificial Sequence Novel Sequence 69 gtccgcgtcc tgctggtggt ggttctggca tttataatt 39 70 33 DNA Artificial Sequence Novel Sequence 70 cctggatcct tatcccatcg tcttcacgtt agc 33 71 26 DNA Artificial Sequence Novel Sequence 71 ctggaattct cctgccagca tggtga 26 72 30 DNA Artificial Sequence Novel Sequence 72 gcaggatcct atattgcgtg ctctgtcccc 30 73 999 DNA Homo sapiens 73 atggtgaact ccacccaccg tgggatgcac acttctctgc acctctggaa ccgcagcagt 60 tacagactgc acagcaatgc cagtgagtcc cttggaaaag gctactctga tggagggtgc 120 tacgagcaac tttttgtctc tcctgaggtg tttgtgactc tgggtgtcat cagcttgttg 180 gagaatatct tagtgattgt ggcaatagcc aagaacaaga atctgcattc acccatgtac 240 tttttcatct gcagcttggc tgtggctgat atgctggtga gcgtttcaaa tggatcagaa 300 accattatca tcaccctatt aaacagtaca gatacggatg cacagagttt cacagtgaat 360 attgataatg tcattgactc ggtgatctgt agctccttgc ttgcatccat ttgcagcctg 420 ctttcaattg cagtggacag gtactttact atcttctatg ctctccagta ccataacatt 480 atgacagtta agcgggttgg gatcagcata agttgtatct gggcagcttg cacggtttca 540 ggcattttgt tcatcattta ctcagatagt agtgctgtca tcatctgcct catcaccatg 600 ttcttcacca tgctggctct catggcttct ctctatgtcc acatgttcct gatggccagg 660 cttcacatta agaggattgc tgtcctcccc ggcactggtg ccatccgcca aggtgccaat 720 atgaagggag cgattacctt gaccatcctg attggcgtct ttgttgtctg ctgggcccca 780 ttcttcctcc acttaatatt ctacatctct tgtcctcaga atccatattg tgtgtgcttc 840 atgtctcact ttaacttgta tctcatactg atcatgtgta attcaatcat cgatcctctg 900 atttatgcac tccggagtca agaactgagg aaaaccttca aagagatcat ctgttgctat 960 cccctgggag gcctttgtga cttgtctagc agatattaa 999 74 332 PRT Homo sapiens 74 Met Val Asn Ser Thr His Arg Gly Met His Thr Ser Leu His Leu Trp 1 5 10 15 Asn Arg Ser Ser Tyr Arg Leu His Ser Asn Ala Ser Glu Ser Leu Gly 20 25 30 Lys Gly Tyr Ser Asp Gly Gly Cys Tyr Glu Gln Leu Phe Val Ser Pro 35 40 45 Glu Val Phe Val Thr Leu Gly Val Ile Ser Leu Leu Glu Asn Ile Leu 50 55 60 Val Ile Val Ala Ile Ala Lys Asn Lys Asn Leu His Ser Pro Met Tyr 65 70 75 80 Phe Phe Ile Cys Ser Leu Ala Val Ala Asp Met Leu Val Ser Val Ser 85 90 95 Asn Gly Ser Glu Thr Ile Ile Ile Thr Leu Leu Asn Ser Thr Asp Thr 100 105 110 Asp Ala Gln Ser Phe Thr Val Asn Ile Asp Asn Val Ile Asp Ser Val 115 120 125 Ile Cys Ser Ser Leu Leu Ala Ser Ile Cys Ser Leu Leu Ser Ile Ala 130 135 140 Val Asp Arg Tyr Phe Thr Ile Phe Tyr Ala Leu Gln Tyr His Asn Ile 145 150 155 160 Met Thr Val Lys Arg Val Gly Ile Ser Ile Ser Cys Ile Trp Ala Ala 165 170 175 Cys Thr Val Ser Gly Ile Leu Phe Ile Ile Tyr Ser Asp Ser Ser Ala 180 185 190 Val Ile Ile Cys Leu Ile Thr Met Phe Phe Thr Met Leu Ala Leu Met 195 200 205 Ala Ser Leu Tyr Val His Met Phe Leu Met Ala Arg Leu His Ile Lys 210 215 220 Arg Ile Ala Val Leu Pro Gly Thr Gly Ala Ile Arg Gln Gly Ala Asn 225 230 235 240 Met Lys Gly Ala Ile Thr Leu Thr Ile Leu Ile Gly Val Phe Val Val 245 250 255 Cys Trp Ala Pro Phe Phe Leu His Leu Ile Phe Tyr Ile Ser Cys Pro 260 265 270 Gln Asn Pro Tyr Cys Val Cys Phe Met Ser His Phe Asn Leu Tyr Leu 275 280 285 Ile Leu Ile Met Cys Asn Ser Ile Ile Asp Pro Leu Ile Tyr Ala Leu 290 295 300 Arg Ser Gln Glu Leu Arg Lys Thr Phe Lys Glu Ile Ile Cys Cys Tyr 305 310 315 320 Pro Leu Gly Gly Leu Cys Asp Leu Ser Ser Arg Tyr 325 330 75 32 DNA Artificial Sequence Novel Sequence 75 ccgaagcttc gagctgagta aggcggcggg ct 32 76 31 DNA Artificial Sequence Novel Sequence 76 gtggaattca tttgccctgc ctcaaccccc a 31 77 1344 DNA Homo sapiens 77 atggagctgc taaagctgaa ccggagcgtg cagggaaccg gacccgggcc gggggcttcc 60 ctgtgccgcc cgggggcgcc tctcctcaac agcagcagtg tgggcaacct cagctgcgag 120 ccccctcgca ttcgcggagc cgggacacga gaattggagc tggccattag aatcactctt 180 tacgcagtga tcttcctgat gagcgttgga ggaaatatgc tcatcatcgt ggtcctggga 240 ctgagccgcc gcctgaggac tgtcaccaat gccttcctcc tctcactggc agtcagcgac 300 ctcctgctgg ctgtggcttg catgcccttc accctcctgc ccaatctcat gggcacattc 360 atctttggca ccgtcatctg caaggcggtt tcctacctca tgggggtgtc tgtgagtgtg 420 tccacgctaa gcctcgtggc catcgcactg gagcgatata gcgccatctg ccgaccactg 480 caggcacgag tgtggcagac gcgctcccac gcggctcgcg tgattgtagc cacgtggctg 540 ctgtccggac tactcatggt gccctacccc gtgtacactg tcgtgcaacc agtggggcct 600 cgtgtgctgc agtgcgtgca tcgctggccc agtgcgcggg tccgccagac ctggtccgta 660 ctgctgcttc tgctcttgtt cttcatccca ggtgtggtta tggccgtggc ctacgggctt 720 atctctcgcg agctctactt agggcttcgc tttgacggcg acagtgacag cgacagccaa 780 agcagggtcc gaaaccaagg cgggctgcca ggggctgttc accagaacgg gcgttgccgg 840 cctgagactg gcgcggttgg caaagacagc gatggctgct acgtgcaact tccacgttcc 900 cggcctgccc tggagctgac ggcgctgacg gctcctgggc cgggatccgg ctcccggccc 960 acccaggcca agctgctggc taagaagcgc gtggtgcgaa tgttgctggt gatcgttgtg 1020 cttttttttc tgtgttggtt gccagtttat agtgccaaca cgtggcgcgc ctttgatggc 1080 ccgggtgcac accgagcact ctcgggtgct cctatctcct tcattcactt gctgagctac 1140 gcctcggcct gtgtcaaccc cctggtctac tgcttcatgc accgtcgctt tcgccaggcc 1200 tgcctggaaa cttgcgctcg ctgctgcccc cggcctccac gagctcgccc cagggctctt 1260 cccgatgagg accctcccac tccctccatt gcttcgctgt ccaggcttag ctacaccacc 1320 atcagcacac tgggccctgg ctga 1344 78 447 PRT Homo sapiens 78 Met Glu Leu Leu Lys Leu Asn Arg Ser Val Gln Gly Thr Gly Pro Gly 1 5 10 15 Pro Gly Ala Ser Leu Cys Arg Pro Gly Ala Pro Leu Leu Asn Ser Ser 20 25 30 Ser Val Gly Asn Leu Ser Cys Glu Pro Pro Arg Ile Arg Gly Ala Gly 35 40 45 Thr Arg Glu Leu Glu Leu Ala Ile Arg Ile Thr Leu Tyr Ala Val Ile 50 55 60 Phe Leu Met Ser Val Gly Gly Asn Met Leu Ile Ile Val Val Leu Gly 65 70 75 80 Leu Ser Arg Arg Leu Arg Thr Val Thr Asn Ala Phe Leu Leu Ser Leu 85 90 95 Ala Val Ser Asp Leu Leu Leu Ala Val Ala Cys Met Pro Phe Thr Leu 100 105 110 Leu Pro Asn Leu Met Gly Thr Phe Ile Phe Gly Thr Val Ile Cys Lys 115 120 125 Ala Val Ser Tyr Leu Met Gly Val Ser Val Ser Val Ser Thr Leu Ser 130 135 140 Leu Val Ala Ile Ala Leu Glu Arg Tyr Ser Ala Ile Cys Arg Pro Leu 145 150 155 160 Gln Ala Arg Val Trp Gln Thr Arg Ser His Ala Ala Arg Val Ile Val 165 170 175 Ala Thr Trp Leu Leu Ser Gly Leu Leu Met Val Pro Tyr Pro Val Tyr 180 185 190 Thr Val Val Gln Pro Val Gly Pro Arg Val Leu Gln Cys Val His Arg 195 200 205 Trp Pro Ser Ala Arg Val Arg Gln Thr Trp Ser Val Leu Leu Leu Leu 210 215 220 Leu Leu Phe Phe Ile Pro Gly Val Val Met Ala Val Ala Tyr Gly Leu 225 230 235 240 Ile Ser Arg Glu Leu Tyr Leu Gly Leu Arg Phe Asp Gly Asp Ser Asp 245 250 255 Ser Asp Ser Gln Ser Arg Val Arg Asn Gln Gly Gly Leu Pro Gly Ala 260 265 270 Val His Gln Asn Gly Arg Cys Arg Pro Glu Thr Gly Ala Val Gly Lys 275 280 285 Asp Ser Asp Gly Cys Tyr Val Gln Leu Pro Arg Ser Arg Pro Ala Leu 290 295 300 Glu Leu Thr Ala Leu Thr Ala Pro Gly Pro Gly Ser Gly Ser Arg Pro 305 310 315 320 Thr Gln Ala Lys Leu Leu Ala Lys Lys Arg Val Val Arg Met Leu Leu 325 330 335 Val Ile Val Val Leu Phe Phe Leu Cys Trp Leu Pro Val Tyr Ser Ala 340 345 350 Asn Thr Trp Arg Ala Phe Asp Gly Pro Gly Ala His Arg Ala Leu Ser 355 360 365 Val Ala Pro Ile Ser Phe Ile His Leu Leu Ser Tyr Ala Ser Ala Cys 370 375 380 Val Asn Pro Leu Val Tyr Cys Phe Met His Arg Arg Phe Arg Gln Ala 385 390 395 400 Cys Leu Glu Thr Cys Ala Arg Cys Cys Pro Arg Pro Pro Arg Ala Arg 405 410 415 Pro Arg Ala Leu Pro Asp Glu Asp Pro Pro Thr Pro Ser Ile Ala Ser 420 425 430 Leu Ser Arg Leu Ser Tyr Thr Thr Ile Ser Thr Leu Gly Pro Gly 435 440 445 79 30 DNA Artificial Sequence Novel Sequence 79 tgcaagctta aaaaggaaaa aatgaacagc 30 80 30 DNA Artificial Sequence Novel Sequence 80 taaggatccc ttcccttcaa aacatccttg 30 81 1014 DNA Homo sapiens 81 atgaacagca catgtattga agaacagcat gacctggatc actatttgtt tcccattgtt 60 tacatctttg tgattatagt cagcattcca gccaatattg gatctctgtg tgtgtctttc 120 ctgcaaccca agaaggaaag tgaactagga atttacctct tcagtttgtc actatcagat 180 ttactctatg cattaactct ccctttatgg attgattata cttggaataa agacaactgg 240 actttctctc ctgccttgtg caaagggagt gcttttctca tgtacatgaa gttttacagc 300 agcacagcat tcctcacctg cattgccgtt gatcggtatt tggctgttgt ctaccctttg 360 aagttttttt tcctaaggac aagaagaatt gcactcatgg tcagcctgtc catctggata 420 ttggaaacca tcttcaatgc tgtcatgttg tgggaagatg aaacagttgt tgaatattgc 480 gatgccgaaa agtctaattt tactttatgc tatgacaaat accctttaga gaaatggcaa 540 atcaacctca acttgttcag gacgtgtaca ggctatgcaa tacctttggt caccatcctg 600 atctgtaacc ggaaagtcta ccaagctgtg cggcacaata aagccacgga aaacaaggaa 660 aagaagagaa tcataaaact acttgtcagc atcacagtta cttttgtctt atgctttact 720 ccctttcatg tgatgttgct gattcgctgc attttagagc atgctgtgaa cttcgaagac 780 cacagcaatt ctgggaagcg aacttacaca atgtatagaa tcacggttgc attaacaagt 840 ttaaattgtg ttgctgatcc aattctgtac tgttttgtta ccgaaacagg aagatatgat 900 atgtggaata tattaaaatt ctgcactggg aggtgtaata catcacaaag acaaagaaaa 960 cgcatacttt ctgtgtctac aaaagatact atggaattag aggtccttga gtag 1014 82 337 PRT Homo sapiens 82 Met Asn Ser Thr Cys Ile Glu Glu Gln His Asp Leu Asp His Tyr Leu 1 5 10 15 Phe Pro Ile Val Tyr Ile Phe Val Ile Ile Val Ser Ile Pro Ala Asn 20 25 30 Ile Gly Ser Leu Cys Val Ser Phe Leu Gln Pro Lys Lys Glu Ser Glu 35 40 45 Leu Gly Ile Tyr Leu Phe Ser Leu Ser Leu Ser Asp Leu Leu Tyr Ala 50 55 60 Leu Thr Leu Pro Leu Trp Ile Asp Tyr Thr Trp Asn Lys Asp Asn Trp 65 70 75 80 Thr Phe Ser Pro Ala Leu Cys Lys Gly Ser Ala Phe Leu Met Tyr Met 85 90 95 Lys Phe Tyr Ser Ser Thr Ala Phe Leu Thr Cys Ile Ala Val Asp Arg 100 105 110 Tyr Leu Ala Val Val Tyr Pro Leu Lys Phe Phe Phe Leu Arg Thr Arg 115 120 125 Arg Ile Ala Leu Met Val Ser Leu Ser Ile Trp Ile Leu Glu Thr Ile 130 135 140 Phe Asn Ala Val Met Leu Trp Glu Asp Glu Thr Val Val Glu Tyr Cys 145 150 155 160 Asp Ala Glu Lys Ser Asn Phe Thr Leu Cys Tyr Asp Lys Tyr Pro Leu 165 170 175 Glu Lys Trp Gln Ile Asn Leu Asn Leu Phe Arg Thr Cys Thr Gly Tyr 180 185 190 Ala Ile Pro Leu Val Thr Ile Leu Ile Cys Asn Arg Lys Val Tyr Gln 195 200 205 Ala Val Arg His Asn Lys Ala Thr Glu Asn Lys Glu Lys Lys Arg Ile 210 215 220 Ile Lys Leu Leu Val Ser Ile Thr Val Thr Phe Val Leu Cys Phe Thr 225 230 235 240 Pro Phe His Val Met Leu Leu Ile Arg Cys Ile Leu Glu His Ala Val 245 250 255 Asn Phe Glu Asp His Ser Asn Ser Gly Lys Arg Thr Tyr Thr Met Tyr 260 265 270 Arg Ile Thr Val Ala Leu Thr Ser Leu Asn Cys Val Ala Asp Pro Ile 275 280 285 Leu Tyr Cys Phe Val Thr Glu Thr Gly Arg Tyr Asp Met Trp Asn Ile 290 295 300 Leu Lys Phe Cys Thr Gly Arg Cys Asn Thr Ser Gln Arg Gln Arg Lys 305 310 315 320 Arg Ile Leu Ser Val Ser Thr Lys Asp Thr Met Glu Leu Glu Val Leu 325 330 335 Glu 83 40 DNA Artificial Sequence Novel Sequence 83 caggaagaag aaacgagctg tcattatgat ggtgacagtg 40 84 40 DNA Artificial Sequence Novel Sequence 84 cactgtcacc atcataatga cagctcgttt cttcttcctg 40 85 30 DNA Artificial Sequence Novel Sequence 85 ggccaccggc agaccaaacg cgtcctgctg 30 86 31 DNA Artificial Sequence Novel Sequence 86 ctccttcggt cctcctatcg ttgtcagaag t 31 87 37 DNA Artificial Sequence Novel Sequence 87 ggaaaagaag agaatcaaaa aactacttgt cagcatc 37 88 31 DNA Artificial Sequence Novel Sequence 88 ctccttcggt cctcctatcg ttgtcagaag t 31 89 1080 DNA Homo sapiens 89 atgattctca actcttctac tgaagatggt attaaaagaa tccaagatga ttgtcccaaa 60 gctggaaggc ataattacat atttgtcatg attcctactt tatacagtat catctttgtg 120 gtgggaatat ttggaaacag cttggtggtg atagtcattt acttttatat gaagctgaag 180 actgtggcca gtgtttttct tttgaattta gcactggctg acttatgctt tttactgact 240 ttgccactat gggctgtcta cacagctatg gaataccgct ggccctttgg caattaccta 300 tgtaagattg cttcagccag cgtcagtttc aacctgtacg ctagtgtgtt tctactcacg 360 tgtctcagca ttgatcgata cctggctatt gttcacccaa tgaagtcccg ccttcgacgc 420 acaatgcttg tagccaaagt cacctgcatc atcatttggc tgctggcagg cttggccagt 480 ttgccagcta taatccatcg aaatgtattt ttcattgaga acaccaatat tacagtttgt 540 gctttccatt atgagtccca aaattcaacc cttccgatag ggctgggcct gaccaaaaat 600 atactgggtt tcctgtttcc ttttctgatc attcttacaa gttatactct tatttggaag 660 gccctaaaga aggcttatga aattcagaag aacaaaccaa gaaatgatga tattaaaaag 720 ataattatgg caattgtgct tttctttttc ttttcctgga ttccccacca aatattcact 780 tttctggatg tattgattca actaggcatc atacgtgact gtagaattgc agatattgtg 840 gacacggcca tgcctatcac catttgtata gcttatttta acaattgcct gaatcctctt 900 ttttatggct ttctggggaa aaaatttaaa agatattttc tccagcttct aaaatatatt 960 cccccaaaag ccaaatccca ctcaaacctt tcaacaaaaa tgagcacgct ttcctaccgc 1020 ccctcagata atgtaagctc atccaccaag aagcctgcac catgttttga ggttgagtga 1080 90 359 PRT Homo sapiens 90 Met Ile Leu Asn Ser Ser Thr Glu Asp Gly Ile Lys Arg Ile Gln Asp 1 5 10 15 Asp Cys Pro Lys Ala Gly Arg His Asn Tyr Ile Phe Val Met Ile Pro 20 25 30 Thr Leu Tyr Ser Ile Ile Phe Val Val Gly Ile Phe Gly Asn Ser Leu 35 40 45 Val Val Ile Val Ile Tyr Phe Tyr Met Lys Leu Lys Thr Val Ala Ser 50 55 60 Val Phe Leu Leu Asn Leu Ala Leu Ala Asp Leu Cys Phe Leu Leu Thr 65 70 75 80 Leu Pro Leu Trp Ala Val Tyr Thr Ala Met Glu Tyr Arg Trp Pro Phe 85 90 95 Gly Asn Tyr Leu Cys Lys Ile Ala Ser Ala Ser Val Ser Phe Asn Leu 100 105 110 Tyr Ala Ser Val Phe Leu Leu Thr Cys Leu Ser Ile Asp Arg Tyr Leu 115 120 125 Ala Ile Val His Pro Met Lys Ser Arg Leu Arg Arg Thr Met Leu Val 130 135 140 Ala Lys Val Thr Cys Ile Ile Ile Trp Leu Leu Ala Gly Leu Ala Ser 145 150 155 160 Leu Pro Ala Ile Ile His Arg Asn Val Phe Phe Ile Glu Asn Thr Asn 165 170 175 Ile Thr Val Cys Ala Phe His Tyr Glu Ser Gln Asn Ser Thr Leu Pro 180 185 190 Ile Gly Leu Gly Leu Thr Lys Asn Ile Leu Gly Phe Leu Phe Pro Phe 195 200 205 Leu Ile Ile Leu Thr Ser Tyr Thr Leu Ile Trp Lys Ala Leu Lys Lys 210 215 220 Ala Tyr Glu Ile Gln Lys Asn Lys Pro Arg Asn Asp Asp Ile Lys Lys 225 230 235 240 Ile Ile Met Ala Ile Val Leu Phe Phe Phe Phe Ser Trp Ile Pro His 245 250 255 Gln Ile Phe Thr Phe Leu Asp Val Leu Ile Gln Leu Gly Ile Ile Arg 260 265 270 Asp Cys Arg Ile Ala Asp Ile Val Asp Thr Ala Met Pro Ile Thr Ile 275 280 285 Cys Ile Ala Tyr Phe Asn Asn Cys Leu Asn Pro Leu Phe Tyr Gly Phe 290 295 300 Leu Gly Lys Lys Phe Lys Arg Tyr Phe Leu Gln Leu Leu Lys Tyr Ile 305 310 315 320 Pro Pro Lys Ala Lys Ser His Ser Asn Leu Ser Thr Lys Met Ser Thr 325 330 335 Leu Ser Tyr Arg Pro Ser Asp Asn Val Ser Ser Ser Thr Lys Lys Pro 340 345 350 Ala Pro Cys Phe Glu Val Glu 355 91 35 DNA Artificial Sequence Novel Sequence 91 ccaagaaatg atgatattaa aaagataatt atggc 35 92 31 DNA Artificial Sequence Novel Sequence 92 ctccttcggt cctcctatcg ttgtcagaag t 31 93 1080 DNA Homo sapiens 93 atgattctca actcttctac tgaagatggt attaaaagaa tccaagatga ttgtcccaaa 60 gctggaaggc ataattacat atttgtcatg attcctactt tatacagtat catctttgtg 120 gtgggaatat ttggaaacag cttggtggtg atagtcattt acttttatat gaagctgaag 180 actgtggcca gtgtttttct tttgaattta gcactggctg acttatgctt tttactgact 240 ttgccactat gggctgtcta cacagctatg gaataccgct ggccctttgg caattaccta 300 tgtaagattg cttcagccag cgtcagtttc gccctgtacg ctagtgtgtt tctactcacg 360 tgtctcagca ttgatcgata cctggctatt gttcacccaa tgaagtcccg ccttcgacgc 420 acaatgcttg tagccaaagt cacctgcatc atcatttggc tgctggcagg cttggccagt 480 ttgccagcta taatccatcg aaatgtattt ttcattgaga acaccaatat tacagtttgt 540 gctttccatt atgagtccca aaattcaacc cttccgatag ggctgggcct gaccaaaaat 600 atactgggtt tcctgtttcc ttttctgatc attcttacaa gttatactct tatttggaag 660 gccctaaaga aggcttatga aattcagaag aacaaaccaa gaaatgatga tatttttaag 720 ataattatgg caattgtgct tttctttttc ttttcctgga ttccccacca aatattcact 780 tttctggatg tattgattca actaggcatc atacgtgact gtagaattgc agatattgtg 840 gacacggcca tgcctatcac catttgtata gcttatttta acaattgcct gaatcctctt 900 ttttatggct ttctggggaa aaaatttaaa agatattttc tccagcttct aaaatatatt 960 cccccaaaag ccaaatccca ctcaaacctt tcaacaaaaa tgagcacgct ttcctaccgc 1020 ccctcagata atgtaagctc atccaccaag aagcctgcac catgttttga ggttgagtga 1080 94 359 PRT Homo sapiens 94 Met Ile Leu Asn Ser Ser Thr Glu Asp Gly Ile Lys Arg Ile Gln Asp 1 5 10 15 Asp Cys Pro Lys Ala Gly Arg His Asn Tyr Ile Phe Val Met Ile Pro 20 25 30 Thr Leu Tyr Ser Ile Ile Phe Val Val Gly Ile Phe Gly Asn Ser Leu 35 40 45 Val Val Ile Val Ile Tyr Phe Tyr Met Lys Leu Lys Thr Val Ala Ser 50 55 60 Val Phe Leu Leu Asn Leu Ala Leu Ala Asp Leu Cys Phe Leu Leu Thr 65 70 75 80 Leu Pro Leu Trp Ala Val Tyr Thr Ala Met Glu Tyr Arg Trp Pro Phe 85 90 95 Gly Asn Tyr Leu Cys Lys Ile Ala Ser Ala Ser Val Ser Phe Ala Leu 100 105 110 Tyr Ala Ser Val Phe Leu Leu Thr Cys Leu Ser Ile Asp Arg Tyr Leu 115 120 125 Ala Ile Val His Pro Met Lys Ser Arg Leu Arg Arg Thr Met Leu Val 130 135 140 Ala Lys Val Thr Cys Ile Ile Ile Trp Leu Leu Ala Gly Leu Ala Ser 145 150 155 160 Leu Pro Ala Ile Ile His Arg Asn Val Phe Phe Ile Glu Asn Thr Asn 165 170 175 Ile Thr Val Cys Ala Phe His Tyr Glu Ser Gln Asn Ser Thr Leu Pro 180 185 190 Ile Gly Leu Gly Leu Thr Lys Asn Ile Leu Gly Phe Leu Phe Pro Phe 195 200 205 Leu Ile Ile Leu Thr Ser Tyr Thr Leu Ile Trp Lys Ala Leu Lys Lys 210 215 220 Ala Tyr Glu Ile Gln Lys Asn Lys Pro Arg Asn Asp Asp Ile Phe Lys 225 230 235 240 Ile Ile Met Ala Ile Val Leu Phe Phe Phe Phe Ser Trp Ile Pro His 245 250 255 Gln Ile Phe Thr Phe Leu Asp Val Leu Ile Gln Leu Gly Ile Ile Arg 260 265 270 Asp Cys Arg Ile Ala Asp Ile Val Asp Thr Ala Met Pro Ile Thr Ile 275 280 285 Cys Ile Ala Tyr Phe Asn Asn Cys Leu Asn Pro Leu Phe Tyr Gly Phe 290 295 300 Leu Gly Lys Lys Phe Lys Arg Tyr Phe Leu Gln Leu Leu Lys Tyr Ile 305 310 315 320 Pro Pro Lys Ala Lys Ser His Ser Asn Leu Ser Thr Lys Met Ser Thr 325 330 335 Leu Ser Tyr Arg Pro Ser Asp Asn Val Ser Ser Ser Thr Lys Lys Pro 340 345 350 Ala Pro Cys Phe Glu Val Glu 355 95 26 DNA Artificial Sequence Novel Sequence 95 cccaagcttc cccaggtgta tttgat 26 96 29 DNA Artificial Sequence Novel Sequence 96 cctgcaggcg aaactgactc tggctgaag 29 97 42 DNA Artificial Sequence Novel Sequence 97 ctgtacgcta gtgtgtttct actcacgtgt ctcagcattg at 42 98 26 DNA Artificial Sequence Novel Sequence 98 gttggatcca cataatgcat tttctc 26 99 1080 DNA Homo sapiens 99 atgattctca actcttctac tgaagatggt attaaaagaa tccaagatga ttgtcccaaa 60 gctggaaggc ataattacat atttgtcatg attcctactt tatacagtat catctttgtg 120 gtgggaatat ttggaaacag cttggtggtg atagtcattt acttttatat gaagctgaag 180 actgtggcca gtgtttttct tttgaattta gcactggctg acttatgctt tttactgact 240 ttgccactat gggctgtcta cacagctatg gaataccgct ggccctttgg caattaccta 300 tgtaagattg cttcagccag cgtcagtttc aacctgtacg ctagtgtgtt tctactcacg 360 tgtctcagca ttgatcgata cctggctatt gttcacccaa tgaagtcccg ccttcgacgc 420 acaatgcttg tagccaaagt cacctgcatc atcatttggc tgctggcagg cttggccagt 480 ttgccagcta taatccatcg aaatgtattt ttcattgaga acaccaatat tacagtttgt 540 gctttccatt atgagtccca aaattcaacc cttccgatag ggctgggcct gaccaaaaat 600 atactgggtt tcctgtttcc ttttctgatc attcttacaa gttattttgg aattcgaaaa 660 cacttactga agacgaatag ctatgggaag aacaggataa cccgtgacca agttaagaag 720 ataattatgg caattgtgct tttctttttc ttttcctgga ttccccacca aatattcact 780 tttctggatg tattgattca actaggcatc atacgtgact gtagaattgc agatattgtg 840 gacacggcca tgcctatcac catttgtata gcttatttta acaattgcct gaatcctctt 900 ttttatggct ttctggggaa aaaatttaaa agatattttc tccagcttct aaaatatatt 960 cccccaaaag ccaaatccca ctcaaacctt tcaacaaaaa tgagcacgct ttcctaccgc 1020 ccctcagata atgtaagctc atccaccaag aagcctgcac catgttttga ggttgagtga 1080 100 359 PRT Homo sapiens 100 Met Ile Leu Asn Ser Ser Thr Glu Asp Gly Ile Lys Arg Ile Gln Asp 1 5 10 15 Asp Cys Pro Lys Ala Gly Arg His Asn Tyr Ile Phe Val Met Ile Pro 20 25 30 Thr Leu Tyr Ser Ile Ile Phe Val Val Gly Ile Phe Gly Asn Ser Leu 35 40 45 Val Val Ile Val Ile Tyr Phe Tyr Met Lys Leu Lys Thr Val Ala Ser 50 55 60 Val Phe Leu Leu Asn Leu Ala Leu Ala Asp Leu Cys Phe Leu Leu Thr 65 70 75 80 Leu Pro Leu Trp Ala Val Tyr Thr Ala Met Glu Tyr Arg Trp Pro Phe 85 90 95 Gly Asn Tyr Leu Cys Lys Ile Ala Ser Ala Ser Val Ser Phe Asn Leu 100 105 110 Tyr Ala Ser Val Phe Leu Leu Thr Cys Leu Ser Ile Asp Arg Tyr Leu 115 120 125 Ala Ile Val His Pro Met Lys Ser Arg Leu Arg Arg Thr Met Leu Val 130 135 140 Ala Lys Val Thr Cys Ile Ile Ile Trp Leu Leu Ala Gly Leu Ala Ser 145 150 155 160 Leu Pro Ala Ile Ile His Arg Asn Val Phe Phe Ile Glu Asn Thr Asn 165 170 175 Ile Thr Val Cys Ala Phe His Tyr Glu Ser Gln Asn Ser Thr Leu Pro 180 185 190 Ile Gly Leu Gly Leu Thr Lys Asn Ile Leu Gly Phe Leu Phe Pro Phe 195 200 205 Leu Ile Ile Leu Thr Ser Tyr Phe Gly Ile Arg Lys His Leu Leu Lys 210 215 220 Thr Asn Ser Tyr Gly Lys Asn Arg Ile Thr Arg Asp Gln Val Lys Lys 225 230 235 240 Ile Ile Met Ala Ile Val Leu Phe Phe Phe Phe Ser Trp Ile Pro His 245 250 255 Gln Ile Phe Thr Phe Leu Asp Val Leu Ile Gln Leu Gly Ile Ile Arg 260 265 270 Asp Cys Arg Ile Ala Asp Ile Val Asp Thr Ala Met Pro Ile Thr Ile 275 280 285 Cys Ile Ala Tyr Phe Asn Asn Cys Leu Asn Pro Leu Phe Tyr Gly Phe 290 295 300 Leu Gly Lys Lys Phe Lys Arg Tyr Phe Leu Gln Leu Leu Lys Tyr Ile 305 310 315 320 Pro Pro Lys Ala Lys Ser His Ser Asn Leu Ser Thr Lys Met Ser Thr 325 330 335 Leu Ser Tyr Arg Pro Ser Asp Asn Val Ser Ser Ser Thr Lys Lys Pro 340 345 350 Ala Pro Cys Phe Glu Val Glu 355 101 37 DNA Artificial Sequence Novel Sequence 101 tccgaattcc aaaataactt gtaagaatga tcagaaa 37 102 33 DNA Artificial Sequence Novel Sequence 102 agatcttaag aagataatta tggcaattgt gct 33 103 62 DNA Artificial Sequence Novel Sequence 103 aattcgaaaa cacttactga agacgaatag ctatgggaag aacaggataa cccgtgacca 60 ag 62 104 62 DNA Artificial Sequence Novel Sequence 104 ttaacttggt cacgggttat cctgttcttc ccatagctat tcgtcttcag taagtgtttt 60 cg 62 105 1083 DNA Homo sapiens 105 atgattctca actcttctac tgaagatggt attaaaagaa tccaagatga ttgtcccaaa 60 gctggaaggc ataattacat atttgtcatg attcctactt tatacagtat catctttgtg 120 gtgggaatat ttggaaacag cttggtggtg atagtcattt acttttatat gaagctgaag 180 actgtggcca gtgtttttct tttgaattta gcactggctg acttatgctt tttactgact 240 ttgccactat gggctgtcta cacagctatg gaataccgct ggccctttgg caattaccta 300 tgtaagattg cttcagccag cgtcagtttc aacctgtacg ctagtgtgtt tctactcacg 360 tgtctcagca ttgatcgata cctggctatt gttcacccaa tgaagtcccg ccttcgacgc 420 acaatgcttg tagccaaagt cacctgcatc atcatttggc tgctggcagg cttggccagt 480 ttgccagcta taatccatcg aaatgtattt ttcattgaga acaccaatat tacagtttgt 540 gctttccatt atgagtccca aaattcaacc cttccgatag ggctgggcct gaccaaaaat 600 atactgggtt tcctgtttcc ttttctgatc attcttacaa gttatactct tatttggaag 660 gccctaaaga aggcttatga aattcagaag aacaaaccaa gaaatgatga tatttttaag 720 ataattatgg cagcaattgt gcttttcttt ttcttttcct ggattcccca ccaaatattc 780 acttttctgg atgtattgat tcaactaggc atcatacgtg actgtagaat tgcagatatt 840 gtggacacgg ccatgcctat caccatttgt atagcttatt ttaacaattg cctgaatcct 900 cttttttatg gctttctggg gaaaaaattt aaaagatatt ttctccagct tctaaaatat 960 attcccccaa aagccaaatc ccactcaaac ctttcaacaa aaatgagcac gctttcctac 1020 cgcccctcag ataatgtaag ctcatccacc aagaagcctg caccatgttt tgaggttgag 1080 tga 1083 106 360 PRT Homo sapiens 106 Met Ile Leu Asn Ser Ser Thr Glu Asp Gly Ile Lys Arg Ile Gln Asp 1 5 10 15 Asp Cys Pro Lys Ala Gly Arg His Asn Tyr Ile Phe Val Met Ile Pro 20 25 30 Thr Leu Tyr Ser Ile Ile Phe Val Val Gly Ile Phe Gly Asn Ser Leu 35 40 45 Val Val Ile Val Ile Tyr Phe Tyr Met Lys Leu Lys Thr Val Ala Ser 50 55 60 Val Phe Leu Leu Asn Leu Ala Leu Ala Asp Leu Cys Phe Leu Leu Thr 65 70 75 80 Leu Pro Leu Trp Ala Val Tyr Thr Ala Met Glu Tyr Arg Trp Pro Phe 85 90 95 Gly Asn Tyr Leu Cys Lys Ile Ala Ser Ala Ser Val Ser Phe Asn Leu 100 105 110 Tyr Ala Ser Val Phe Leu Leu Thr Cys Leu Ser Ile Asp Arg Tyr Leu 115 120 125 Ala Ile Val His Pro Met Lys Ser Arg Leu Arg Arg Thr Met Leu Val 130 135 140 Ala Lys Val Thr Cys Ile Ile Ile Trp Leu Leu Ala Gly Leu Ala Ser 145 150 155 160 Leu Pro Ala Ile Ile His Arg Asn Val Phe Phe Ile Glu Asn Thr Asn 165 170 175 Ile Thr Val Cys Ala Phe His Tyr Glu Ser Gln Asn Ser Thr Leu Pro 180 185 190 Ile Gly Leu Gly Leu Thr Lys Asn Ile Leu Gly Phe Leu Phe Pro Phe 195 200 205 Leu Ile Ile Leu Thr Ser Tyr Thr Leu Ile Trp Lys Ala Leu Lys Lys 210 215 220 Ala Tyr Glu Ile Gln Lys Asn Lys Pro Arg Asn Asp Asp Ile Phe Lys 225 230 235 240 Ile Ile Met Ala Ala Ile Val Leu Phe Phe Phe Phe Ser Trp Ile Pro 245 250 255 His Gln Ile Phe Thr Phe Leu Asp Val Leu Ile Gln Leu Gly Ile Ile 260 265 270 Arg Asp Cys Arg Ile Ala Asp Ile Val Asp Thr Ala Met Pro Ile Thr 275 280 285 Ile Cys Ile Ala Tyr Phe Asn Asn Cys Leu Asn Pro Leu Phe Tyr Gly 290 295 300 Phe Leu Gly Lys Lys Phe Lys Arg Tyr Phe Leu Gln Leu Leu Lys Tyr 305 310 315 320 Ile Pro Pro Lys Ala Lys Ser His Ser Asn Leu Ser Thr Lys Met Ser 325 330 335 Thr Leu Ser Tyr Arg Pro Ser Asp Asn Val Ser Ser Ser Thr Lys Lys 340 345 350 Pro Ala Pro Cys Phe Glu Val Glu 355 360 107 26 DNA Artificial Sequence Novel Sequence 107 cccaagcttc cccaggtgta tttgat 26 108 38 DNA Artificial Sequence Novel Sequence 108 aagcacaatt gctgcataat tatcttaaaa atatcatc 38 109 39 DNA Artificial Sequence Novel Sequence 109 aagataatta tggcagcaat tgtgcttttc tttttcttt 39 110 26 DNA Artificial Sequence Novel Sequence 110 gttggatcca cataatgcat tttctc 26 111 1344 DNA Homo sapiens 111 atggagctgc taaagctgaa ccggagcgtg cagggaaccg gacccgggcc gggggcttcc 60 ctgtgccgcc cgggggcgcc tctcctcaac agcagcagtg tgggcaacct cagctgcgag 120 ccccctcgca ttcgcggagc cgggacacga gaattggagc tggccattag aatcactctt 180 tacgcagtga tcttcctgat gagcgttgga ggaaatatgc tcatcatcgt ggtcctggga 240 ctgagccgcc gcctgaggac tgtcaccaat gccttcctcc tctcactggc agtcagcgac 300 ctcctgctgg ctgtggcttg catgcccttc accctcctgc ccaatctcat gggcacattc 360 atctttggca ccgtcatctg caaggcggtt tcctacctca tgggggtgtc tgtgagtgtg 420 tccacgctaa gcctcgtggc catcgcactg gagcgatata gcgccatctg ccgaccactg 480 caggcacgag tgtggcagac gcgctcccac gcggctcgcg tgattgtagc cacgtggctg 540 ctgtccggac tactcatggt gccctacccc gtgtacactg tcgtgcaacc agtggggcct 600 cgtgtgctgc agtgcgtgca tcgctggccc agtgcgcggg tccgccagac ctggtccgta 660 ctgctgcttc tgctcttgtt cttcatccca ggtgtggtta tggccgtggc ctacgggctt 720 atctctcgcg agctctactt agggcttcgc tttgacggcg acagtgacag cgacagccaa 780 agcagggtcc gaaaccaagg cgggctgcca ggggctgttc accagaacgg gcgttgccgg 840 cctgagactg gcgcggttgg caaagacagc gatggctgct acgtgcaact tccacgttcc 900 cggcctgccc tggagctgac ggcgctgacg gctcctgggc cgggatccgg ctcccggccc 960 acccaggcca agctgctggc taagaagcgc gtgaaacgaa tgttgctggt gatcgttgtg 1020 cttttttttc tgtgttggtt gccagtttat agtgccaaca cgtggcgcgc ctttgatggc 1080 ccgggtgcac accgagcact ctcgggtgct cctatctcct tcattcactt gctgagctac 1140 gcctcggcct gtgtcaaccc cctggtctac tgcttcatgc accgtcgctt tcgccaggcc 1200 tgcctggaaa cttgcgctcg ctgctgcccc cggcctccac gagctcgccc cagggctctt 1260 cccgatgagg accctcccac tccctccatt gcttcgctgt ccaggcttag ctacaccacc 1320 atcagcacac tgggccctgg ctga 1344 112 447 PRT Homo sapiens 112 Met Glu Leu Leu Lys Leu Asn Arg Ser Val Gln Gly Thr Gly Pro Gly 1 5 10 15 Pro Gly Ala Ser Leu Cys Arg Pro Gly Ala Pro Leu Leu Asn Ser Ser 20 25 30 Ser Val Gly Asn Leu Ser Cys Glu Pro Pro Arg Ile Arg Gly Ala Gly 35 40 45 Thr Arg Glu Leu Glu Leu Ala Ile Arg Ile Thr Leu Tyr Ala Val Ile 50 55 60 Phe Leu Met Ser Val Gly Gly Asn Met Leu Ile Ile Val Val Leu Gly 65 70 75 80 Leu Ser Arg Arg Leu Arg Thr Val Thr Asn Ala Phe Leu Leu Ser Leu 85 90 95 Ala Val Ser Asp Leu Leu Leu Ala Val Ala Cys Met Pro Phe Thr Leu 100 105 110 Leu Pro Asn Leu Met Gly Thr Phe Ile Phe Gly Thr Val Ile Cys Lys 115 120 125 Ala Val Ser Tyr Leu Met Gly Val Ser Val Ser Val Ser Thr Leu Ser 130 135 140 Leu Val Ala Ile Ala Leu Glu Arg Tyr Ser Ala Ile Cys Arg Pro Leu 145 150 155 160 Gln Ala Arg Val Trp Gln Thr Arg Ser His Ala Ala Arg Val Ile Val 165 170 175 Ala Thr Trp Leu Leu Ser Gly Leu Leu Met Val Pro Tyr Pro Val Tyr 180 185 190 Thr Val Val Gln Pro Val Gly Pro Arg Val Leu Gln Cys Val His Arg 195 200 205 Trp Pro Ser Ala Arg Val Arg Gln Thr Trp Ser Val Leu Leu Leu Leu 210 215 220 Leu Leu Phe Phe Ile Pro Gly Val Val Met Ala Val Ala Tyr Gly Leu 225 230 235 240 Ile Ser Arg Glu Leu Tyr Leu Gly Leu Arg Phe Asp Gly Asp Ser Asp 245 250 255 Ser Asp Ser Gln Ser Arg Val Arg Asn Gln Gly Gly Leu Pro Gly Ala 260 265 270 Val His Gln Asn Gly Arg Cys Arg Pro Glu Thr Gly Ala Val Gly Lys 275 280 285 Asp Ser Asp Gly Cys Tyr Val Gln Leu Pro Arg Ser Arg Pro Ala Leu 290 295 300 Glu Leu Thr Ala Leu Thr Ala Pro Gly Pro Gly Ser Gly Ser Arg Pro 305 310 315 320 Thr Gln Ala Lys Leu Leu Ala Lys Lys Arg Val Lys Arg Met Leu Leu 325 330 335 Val Ile Val Val Leu Phe Phe Leu Cys Trp Leu Pro Val Tyr Ser Ala 340 345 350 Asn Thr Trp Arg Ala Phe Asp Gly Pro Gly Ala His Arg Ala Leu Ser 355 360 365 Val Ala Pro Ile Ser Phe Ile His Leu Leu Ser Tyr Ala Ser Ala Cys 370 375 380 Val Asn Pro Leu Val Tyr Cys Phe Met His Arg Arg Phe Arg Gln Ala 385 390 395 400 Cys Leu Glu Thr Cys Ala Arg Cys Cys Pro Arg Pro Pro Arg Ala Arg 405 410 415 Pro Arg Ala Leu Pro Asp Glu Asp Pro Pro Thr Pro Ser Ile Ala Ser 420 425 430 Leu Ser Arg Leu Ser Tyr Thr Thr Ile Ser Thr Leu Gly Pro Gly 435 440 445 113 34 DNA Artificial Sequence Novel Sequence 113 cagcagcatg cgcttcacgc gcttcttagc ccag 34 114 35 DNA Artificial Sequence Novel Sequence 114 agaagcgcgt gaagcgcatg ctgctggtga tcgtt 35 115 33 DNA Artificial Sequence Novel Sequence 115 atggagaaaa gaatcaaaag aatgttctat ata 33 116 33 DNA Artificial Sequence Novel Sequence 116 tatatagaac attcttttga ttcttttctc cat 33 117 30 DNA Artificial Sequence Novel Sequence 117 cgctctctgg ccttgaagcg cacgctcagc 30 118 30 DNA Artificial Sequence Novel Sequence 118 gctgagcgtg cgcttcaagg ccagagagcg 30 119 30 DNA Artificial Sequence Novel Sequence 119 cccaggaaaa aggtgaaagt caaagttttc 30 120 30 DNA Artificial Sequence Novel Sequence 120 gaaaactttg actttcacct ttttcctggg 30 121 27 DNA Artificial Sequence Novel Sequence 121 ggggcgcggg tgaaacggct ggtgagc 27 122 27 DNA Artificial Sequence Novel Sequence 122 gctcaccagc cgtttcaccc gcgcccc 27 123 30 DNA Artificial Sequence Novel Sequence 123 ccccttgaaa agcctaagaa cttggtcatc 30 124 30 DNA Artificial Sequence Novel Sequence 124 gatgaccaag ttcttaggct tttcaagggg 30 125 32 DNA Artificial Sequence Novel Sequence 125 gatctctaga atgaacagca catgtattga ag 32 126 35 DNA Artificial Sequence Novel Sequence 126 ctagggtacc cgctcaagga cctctaattc catag 35 127 1296 DNA Homo sapiens 127 atgcaggcgc ttaacattac cccggagcag ttctctcggc tgctgcggga ccacaacctg 60 acgcgggagc agttcatcgc tctgtaccgg ctgcgaccgc tcgtctacac cccagagctg 120 ccgggacgcg ccaagctggc cctcgtgctc accggcgtgc tcatcttcgc cctggcgctc 180 tttggcaatg ctctggtgtt ctacgtggtg acccgcagca aggccatgcg caccgtcacc 240 aacatcttta tctgctcctt ggcgctcagt gacctgctca tcaccttctt ctgcattccc 300 gtcaccatgc tccagaacat ttccgacaac tggctggggg gtgctttcat ttgcaagatg 360 gtgccatttg tccagtctac cgctgttgtg acagaaatgc tcactatgac ctgcattgct 420 gtggaaaggc accagggact tgtgcatcct tttaaaatga agtggcaata caccaaccga 480 agggctttca caatgctagg tgtggtctgg ctggtggcag tcatcgtagg atcacccatg 540 tggcacgtgc aacaacttga gatcaaatat gacttcctat atgaaaagga acacatctgc 600 tgcttagaag agtggaccag ccctgtgcac cagaagatct acaccacctt catccttgtc 660 atcctcttcc tcctgcctct tatggtgatg cttattctgt acagtaaaat tggttatgaa 720 ctttggataa agaaaagagt tggggatggt tcagtgcttc gaactattca tggaaaagaa 780 atgtccaaaa tagccaggaa gaagaaacga gctaagatta tgatggtgac agtggtggct 840 ctctttgctg tgtgctgggc accattccat gttgtccata tgatgattga atacagtaat 900 tttgaaaagg aatatgatga tgtcacaatc aagatgattt ttgctatcgt gcaaattatt 960 ggattttcca actccatctg taatcccatt gtctatgcat ttatgaatga aaacttcaaa 1020 aaaaatgttt tgtctgcagt ttgttattgc atagtaaata aaaccttctc tccagcacaa 1080 aggcatggaa attcaggaat tacaatgatg cggaagaaag caaagttttc cctcagagag 1140 aatccagtgg aggaaaccaa aggagaagca ttcagtgatg gcaacattga agtcaaattg 1200 tgtgaacaga cagaggagaa gaaaaagctc aaacgacatc ttgctctctt taggtctgaa 1260 ctggctgaga attctccttt agacagtggg cattaa 1296 128 431 PRT Homo sapiens 128 Met Gln Ala Leu Asn Ile Thr Pro Glu Gln Phe Ser Arg Leu Leu Arg 1 5 10 15 Asp His Asn Leu Thr Arg Glu Gln Phe Ile Ala Leu Tyr Arg Leu Arg 20 25 30 Pro Leu Val Tyr Thr Pro Glu Leu Pro Gly Arg Ala Lys Leu Ala Leu 35 40 45 Val Leu Thr Gly Val Leu Ile Phe Ala Leu Ala Leu Phe Gly Asn Ala 50 55 60 Leu Val Phe Tyr Val Val Thr Arg Ser Lys Ala Met Arg Thr Val Thr 65 70 75 80 Asn Ile Phe Ile Cys Ser Leu Ala Leu Ser Asp Leu Leu Ile Thr Phe 85 90 95 Phe Cys Ile Pro Val Thr Met Leu Gln Asn Ile Ser Asp Asn Trp Leu 100 105 110 Gly Gly Ala Phe Ile Cys Lys Met Val Pro Phe Val Gln Ser Thr Ala 115 120 125 Val Val Thr Glu Met Leu Thr Met Thr Cys Ile Ala Val Glu Arg His 130 135 140 Gln Gly Leu Val His Pro Phe Lys Met Lys Trp Gln Tyr Thr Asn Arg 145 150 155 160 Arg Ala Phe Thr Met Leu Gly Val Val Trp Leu Val Ala Val Ile Val 165 170 175 Gly Ser Pro Met Trp His Val Gln Gln Leu Glu Ile Lys Tyr Asp Phe 180 185 190 Leu Tyr Glu Lys Glu His Ile Cys Cys Leu Glu Glu Trp Thr Ser Pro 195 200 205 Val His Gln Lys Ile Tyr Thr Thr Phe Ile Leu Val Ile Leu Phe Leu 210 215 220 Leu Pro Leu Met Val Met Leu Ile Leu Tyr Ser Lys Ile Gly Tyr Glu 225 230 235 240 Leu Trp Ile Lys Lys Arg Val Gly Asp Gly Ser Val Leu Arg Thr Ile 245 250 255 His Gly Lys Glu Met Ser Lys Ile Ala Arg Lys Lys Lys Arg Ala Lys 260 265 270 Ile Met Met Val Thr Val Val Ala Leu Phe Ala Val Cys Trp Ala Pro 275 280 285 Phe His Val Val His Met Met Ile Glu Tyr Ser Asn Phe Glu Lys Glu 290 295 300 Tyr Asp Asp Val Thr Ile Lys Met Ile Phe Ala Ile Val Gln Ile Ile 305 310 315 320 Gly Phe Ser Asn Ser Ile Cys Asn Pro Ile Val Tyr Ala Phe Met Asn 325 330 335 Glu Asn Phe Lys Lys Asn Val Leu Ser Ala Val Cys Tyr Cys Ile Val 340 345 350 Asn Lys Thr Phe Ser Pro Ala Gln Arg His Gly Asn Ser Gly Ile Thr 355 360 365 Met Met Arg Lys Lys Ala Lys Phe Ser Leu Arg Glu Asn Pro Val Glu 370 375 380 Glu Thr Lys Gly Glu Ala Phe Ser Asp Gly Asn Ile Glu Val Lys Leu 385 390 395 400 Cys Glu Gln Thr Glu Glu Lys Lys Lys Leu Lys Arg His Leu Ala Leu 405 410 415 Phe Arg Ser Glu Leu Ala Glu Asn Ser Pro Leu Asp Ser Gly His 420 425 430 129 2040 DNA Homo sapiens 129 atgggcagcc cctggaacgg cagcgacggc cccgaggggg cgcgggagcc gccgtggccc 60 gcgctgccgc cttgcgacga gcgccgctgc tcgccctttc ccctgggggc gctggtgccg 120 gtgaccgctg tgtgcctgtg cctgttcgtc gtcggggtga gcggcaacgt ggtgaccgtg 180 atgctgatcg ggcgctaccg ggacatgcgg accaccacca acttgtacct gggcagcatg 240 gccgtgtccg acctactcat cctgctcggg ctgccgttcg acctgtaccg cctctggcgc 300 tcgcggccct gggtgttcgg gccgctgctc tgccgcctgt ccctctacgt gggcgagggc 360 tgcacctacg ccacgctgct gcacatgacc gcgctcagcg tcgagcgcta cctggccatc 420 tgccgcccgc tccgcgcccg cgtcttggtc acccggcgcc gcgtccgcgc gctcatcgct 480 gtgctctggg ccgtggcgct gctctctgcc ggtcccttct tgttcctggt gggcgtcgag 540 caggaccccg gcatctccgt agtcccgggc ctcaatggca ccgcgcggat cgcctcctcg 600 cctctcgcct cgtcgccgcc tctctggctc tcgcgggcgc caccgccgtc cccgccgtcg 660 gggcccgaga ccgcggaggc cgcggcgctg ttcagccgcg aatgccggcc gagccccgcg 720 cagctgggcg cgctgcgtgt catgctgtgg gtcaccaccg cctacttctt cctgcccttt 780 ctgtgcctca gcatcctcta cgggctcatc gggcgggagc tgtggagcag ccggcggccg 840 ctgcgaggcc cggccgcctc ggggcgggag agaggccacc ggcagaccaa acgcgtcctg 900 cgtaagtgga gccgccgtgg ttccaaagac gcctgcctgc agtccgcccc gccggggacc 960 gcgcaaacgc tgggtcccct tcccctgctc gcccagctct gggcgccgct tccagctccc 1020 tttcctattt cgattccagc ctccacccgc cggtacttcc catcccccga gaaaaccatg 1080 tcctgtcccc caggagctct gggggacccc agggcgcttt gagggtggga tccccggatc 1140 cgattcagta accagcagtg cttttccaga gcctctgaga ccagaaagga gagttggtaa 1200 ttcttaatcc aaccacctgt tagatgccac aaatgaggag tcctcacagt gctcttgaga 1260 agacgaggga gatttcatta agctaaaatt ttttatttaa tgttaagtga tgctgaaggc 1320 taaagtaaac cttgctcgta tcaaaaagta aagattgtgc agacctgttg tagaattctt 1380 ttcaacagag aacagaaaac ttgtctccga agtgggtttg tggaaggaag cctgccaagg 1440 cggcttgttc agagaaattg ctccttctgg tttatgtcca gccttgataa cacatatggg 1500 agcctactat gcagttttaa agcaagtatc catgcagcct gcagcctggt cattttttct 1560 ggggtgagga tctgcctagg tagaagtttt ctctaattta ttttgctgtt acttgttatt 1620 gcagatggtt ccttgtcggg gtggggggtt tatttgcttc ccaatgcttt tgttaatccc 1680 ggtgctgtgt cttatgttgc agtggtggtg gttctggcat ttataatttg ctggttgccc 1740 ttccacgttg gcagaatcat ttacataaac acggaagatt cgcggatgat gtacttctct 1800 cagtacttta acatcgtcgc tctgcaactt ttctatctga gcgcatctat caacccaatc 1860 ctctacaacc tcatttcaaa gaagtacaga gcggcggcct ttaaactgct gctcgcaagg 1920 aagtccaggc cgagaggctt ccacagaagc agggacactg cgggggaagt tgcaggggac 1980 actggaggag acacggtggg ctacaccgag acaagcgcta acgtgaagac gatgggataa 2040 130 412 PRT Homo sapiens 130 Met Gly Ser Pro Trp Asn Gly Ser Asp Gly Pro Glu Gly Ala Arg Glu 1 5 10 15 Pro Pro Trp Pro Ala Leu Pro Pro Cys Asp Glu Arg Arg Cys Ser Pro 20 25 30 Phe Pro Leu Gly Ala Leu Val Pro Val Thr Ala Val Cys Leu Cys Leu 35 40 45 Phe Val Val Gly Val Ser Gly Asn Val Val Thr Val Met Leu Ile Gly 50 55 60 Arg Tyr Arg Asp Met Arg Thr Thr Thr Asn Leu Tyr Leu Gly Ser Met 65 70 75 80 Ala Val Ser Asp Leu Leu Ile Leu Leu Gly Leu Pro Phe Asp Leu Tyr 85 90 95 Arg Leu Trp Arg Ser Arg Pro Trp Val Phe Gly Pro Leu Leu Cys Arg 100 105 110 Leu Ser Leu Tyr Val Gly Glu Gly Cys Thr Tyr Ala Thr Leu Leu His 115 120 125 Met Thr Ala Leu Ser Val Glu Arg Tyr Leu Ala Ile Cys Arg Pro Leu 130 135 140 Arg Ala Arg Val Leu Val Thr Arg Arg Arg Val Arg Ala Leu Ile Ala 145 150 155 160 Val Leu Trp Ala Val Ala Leu Leu Ser Ala Gly Pro Phe Leu Phe Leu 165 170 175 Val Gly Val Glu Gln Asp Pro Gly Ile Ser Val Val Pro Gly Leu Asn 180 185 190 Gly Thr Ala Arg Ile Ala Ser Ser Pro Leu Ala Ser Ser Pro Pro Leu 195 200 205 Trp Leu Ser Arg Ala Pro Pro Pro Ser Pro Pro Ser Gly Pro Glu Thr 210 215 220 Ala Glu Ala Ala Ala Leu Phe Ser Arg Glu Cys Arg Pro Ser Pro Ala 225 230 235 240 Gln Leu Gly Ala Leu Arg Val Met Leu Trp Val Thr Thr Ala Tyr Phe 245 250 255 Phe Leu Pro Phe Leu Cys Leu Ser Ile Leu Tyr Gly Leu Ile Gly Arg 260 265 270 Glu Leu Trp Ser Ser Arg Arg Pro Leu Arg Gly Pro Ala Ala Ser Gly 275 280 285 Arg Glu Arg Gly His Arg Gln Thr Lys Arg Val Leu Leu Val Val Val 290 295 300 Leu Ala Phe Ile Ile Cys Trp Leu Pro Phe His Val Gly Arg Ile Ile 305 310 315 320 Tyr Ile Asn Thr Glu Asp Ser Arg Met Met Tyr Phe Ser Gln Tyr Phe 325 330 335 Asn Ile Val Ala Leu Gln Leu Phe Tyr Leu Ser Ala Ser Ile Asn Pro 340 345 350 Ile Leu Tyr Asn Leu Ile Ser Lys Lys Tyr Arg Ala Ala Ala Phe Lys 355 360 365 Leu Leu Leu Ala Arg Lys Ser Arg Pro Arg Gly Phe His Arg Ser Arg 370 375 380 Asp Thr Ala Gly Glu Val Ala Gly Asp Thr Gly Gly Asp Thr Val Gly 385 390 395 400 Tyr Thr Glu Thr Ser Ala Asn Val Lys Thr Met Gly 405 410 131 1344 DNA Homo sapiens 131 atggagctgc taaagctgaa ccggagcgtg cagggaaccg gacccgggcc gggggcttcc 60 ctgtgccgcc cgggggcgcc tctcctcaac agcagcagtg tgggcaacct cagctgcgag 120 ccccctcgca ttcgcggagc cgggacacga gaattggagc tggccattag aatcactctt 180 tacgcagtga tcttcctgat gagcgttgga ggaaatatgc tcatcatcgt ggtcctggga 240 ctgagccgcc gcctgaggac tgtcaccaat gccttcctcc tctcactggc agtcagcgac 300 ctcctgctgg ctgtggcttg catgcccttc accctcctgc ccaatctcat gggcacattc 360 atctttggca ccgtcatctg caaggcggtt tcctacctca tgggggtgtc tgtgagtgtg 420 tccacgctaa gcctcgtggc catcgcactg gagcgatata gcgccatctg ccgaccactg 480 caggcacgag tgtggcagac gcgctcccac gcggctcgcg tgattgtagc cacgtggctg 540 ctgtccggac tactcatggt gccctacccc gtgtacactg tcgtgcaacc agtggggcct 600 cgtgtgctgc agtgcgtgca tcgctggccc agtgcgcggg tccgccagac ctggtccgta 660 ctgctgcttc tgctcttgtt cttcatccca ggtgtggtta tggccgtggc ctacgggctt 720 atctctcgcg agctctactt agggcttcgc tttgacggcg acagtgacag cgacagccaa 780 agcagggtcc gaaaccaagg cgggctgcca ggggctgttc accagaacgg gcgttgccgg 840 cctgagactg gcgcggttgg caaagacagc gatggctgct acgtgcaact tccacgttcc 900 cggcctgccc tggagctgac ggcgctgacg gctcctgggc cgggatccgg ctcccggccc 960 acccaggcca agctgctggc taagaagcgc gtgaaacgaa tgttgctggt gatcgttgtg 1020 cttttttttc tgtgttggtt gccagtttat agtgccaaca cgtggcgcgc ctttgatggc 1080 ccgggtgcac accgagcact ctcgggtgct cctatctcct tcattcactt gctgagctac 1140 gcctcggcct gtgtcaaccc cctggtctac tgcttcatgc accgtcgctt tcgccaggcc 1200 tgcctggaaa cttgcgctcg ctgctgcccc cggcctccac gagctcgccc cagggctctt 1260 cccgatgagg accctcccac tccctccatt gcttcgctgt ccaggcttag ctacaccacc 1320 atcagcacac tgggccctgg ctga 1344 132 447 PRT Homo sapiens 132 Met Glu Leu Leu Lys Leu Asn Arg Ser Val Gln Gly Thr Gly Pro Gly 1 5 10 15 Pro Gly Ala Ser Leu Cys Arg Pro Gly Ala Pro Leu Leu Asn Ser Ser 20 25 30 Ser Val Gly Asn Leu Ser Cys Glu Pro Pro Arg Ile Arg Gly Ala Gly 35 40 45 Thr Arg Glu Leu Glu Leu Ala Ile Arg Ile Thr Leu Tyr Ala Val Ile 50 55 60 Phe Leu Met Ser Val Gly Gly Asn Met Leu Ile Ile Val Val Leu Gly 65 70 75 80 Leu Ser Arg Arg Leu Arg Thr Val Thr Asn Ala Phe Leu Leu Ser Leu 85 90 95 Ala Val Ser Asp Leu Leu Leu Ala Val Ala Cys Met Pro Phe Thr Leu 100 105 110 Leu Pro Asn Leu Met Gly Thr Phe Ile Phe Gly Thr Val Ile Cys Lys 115 120 125 Ala Val Ser Tyr Leu Met Gly Val Ser Val Ser Val Ser Thr Leu Ser 130 135 140 Leu Val Ala Ile Ala Leu Glu Arg Tyr Ser Ala Ile Cys Arg Pro Leu 145 150 155 160 Gln Ala Arg Val Trp Gln Thr Arg Ser His Ala Ala Arg Val Ile Val 165 170 175 Ala Thr Trp Leu Leu Ser Gly Leu Leu Met Val Pro Tyr Pro Val Tyr 180 185 190 Thr Val Val Gln Pro Val Gly Pro Arg Val Leu Gln Cys Val His Arg 195 200 205 Trp Pro Ser Ala Arg Val Arg Gln Thr Trp Ser Val Leu Leu Leu Leu 210 215 220 Leu Leu Phe Phe Ile Pro Gly Val Val Met Ala Val Ala Tyr Gly Leu 225 230 235 240 Ile Ser Arg Glu Leu Tyr Leu Gly Leu Arg Phe Asp Gly Asp Ser Asp 245 250 255 Ser Asp Ser Gln Ser Arg Val Arg Asn Gln Gly Gly Leu Pro Gly Ala 260 265 270 Val His Gln Asn Gly Arg Cys Arg Pro Glu Thr Gly Ala Val Gly Lys 275 280 285 Asp Ser Asp Gly Cys Tyr Val Gln Leu Pro Arg Ser Arg Pro Ala Leu 290 295 300 Glu Leu Thr Ala Leu Thr Ala Pro Gly Pro Gly Ser Gly Ser Arg Pro 305 310 315 320 Thr Gln Ala Lys Leu Leu Ala Lys Lys Arg Val Lys Arg Met Leu Leu 325 330 335 Val Ile Val Val Leu Phe Phe Leu Cys Trp Leu Pro Val Tyr Ser Ala 340 345 350 Asn Thr Trp Arg Ala Phe Asp Gly Pro Gly Ala His Arg Ala Leu Ser 355 360 365 Val Ala Pro Ile Ser Phe Ile His Leu Leu Ser Tyr Ala Ser Ala Cys 370 375 380 Val Asn Pro Leu Val Tyr Cys Phe Met His Arg Arg Phe Arg Gln Ala 385 390 395 400 Cys Leu Glu Thr Cys Ala Arg Cys Cys Pro Arg Pro Pro Arg Ala Arg 405 410 415 Pro Arg Ala Leu Pro Asp Glu Asp Pro Pro Thr Pro Ser Ile Ala Ser 420 425 430 Leu Ser Arg Leu Ser Tyr Thr Thr Ile Ser Thr Leu Gly Pro Gly 435 440 445 133 1014 DNA Homo sapiens 133 atgaacagca catgtattga agaacagcat gacctggatc actatttgtt tcccattgtt 60 tacatctttg tgattatagt cagcattcca gccaatattg gatctctgtg tgtgtctttc 120 ctgcaagcaa agaaggaaag tgaactagga atttacctct tcagtttgtc actatcagat 180 ttactctatg cattaactct ccctttatgg attgattata cttggaataa agacaactgg 240 actttctctc ctgccttgtg caaagggagt gcttttctca tgtacatgaa tttttacagc 300 agcacagcat tcctcacctg cattgccgtt gatcggtatt tggctgttgt ctaccctttg 360 aagttttttt tcctaaggac aagaagattt gcactcatgg tcagcctgtc catctggata 420 ttggaaacca tcttcaatgc tgtcatgttg tgggaagatg aaacagttgt tgaatattgc 480 gatgccgaaa agtctaattt tactttatgc tatgacaaat accctttaga gaaatggcaa 540 atcaacctca acttgttcag gacgtgtaca ggctatgcaa tacctttggt caccatcctg 600 atctgtaacc ggaaagtcta ccaagctgtg cggcacaata aagccacgga aaacaaggaa 660 aagaagagaa tcaaaaaact acttgtcagc atcacagtta cttttgtctt atgctttact 720 ccctttcatg tgatgttgct gattcgctgc attttagagc atgctgtgaa cttcgaagac 780 cacagcaatt ctgggaagcg aacttacaca atgtatagaa tcacggttgc attaacaagt 840 ttaaattgtg ttgctgatcc aattctgtac tgttttgtta ccgaaacagg aagatatgat 900 atgtggaata tattaaaatt ctgcactggg aggtgtaata catcacaaag acaaagaaaa 960 cgcatacttt ctgtgtctac aaaagatact atggaattag aggtccttga gtag 1014 134 337 PRT Homo sapiens 134 Met Asn Ser Thr Cys Ile Glu Glu Gln His Asp Leu Asp His Tyr Leu 1 5 10 15 Phe Pro Ile Val Tyr Ile Phe Val Ile Ile Val Ser Ile Pro Ala Asn 20 25 30 Ile Gly Ser Leu Cys Val Ser Phe Leu Gln Ala Lys Lys Glu Ser Glu 35 40 45 Leu Gly Ile Tyr Leu Phe Ser Leu Ser Leu Ser Asp Leu Leu Tyr Ala 50 55 60 Leu Thr Leu Pro Leu Trp Ile Asp Tyr Thr Trp Asn Lys Asp Asn Trp 65 70 75 80 Thr Phe Ser Pro Ala Leu Cys Lys Gly Ser Ala Phe Leu Met Tyr Met 85 90 95 Asn Phe Tyr Ser Ser Thr Ala Phe Leu Thr Cys Ile Ala Val Asp Arg 100 105 110 Tyr Leu Ala Val Val Tyr Pro Leu Lys Phe Phe Phe Leu Arg Thr Arg 115 120 125 Arg Phe Ala Leu Met Val Ser Leu Ser Ile Trp Ile Leu Glu Thr Ile 130 135 140 Phe Asn Ala Val Met Leu Trp Glu Asp Glu Thr Val Val Glu Tyr Cys 145 150 155 160 Asp Ala Glu Lys Ser Asn Phe Thr Leu Cys Tyr Asp Lys Tyr Pro Leu 165 170 175 Glu Lys Trp Gln Ile Asn Leu Asn Leu Phe Arg Thr Cys Thr Gly Tyr 180 185 190 Ala Ile Pro Leu Val Thr Ile Leu Ile Cys Asn Arg Lys Val Tyr Gln 195 200 205 Ala Val Arg His Asn Lys Ala Thr Glu Asn Lys Glu Lys Lys Arg Ile 210 215 220 Lys Lys Leu Leu Val Ser Ile Thr Val Thr Phe Val Leu Cys Phe Thr 225 230 235 240 Pro Phe His Val Met Leu Leu Ile Arg Cys Ile Leu Glu His Ala Val 245 250 255 Asn Phe Glu Asp His Ser Asn Ser Gly Lys Arg Thr Tyr Thr Met Tyr 260 265 270 Arg Ile Thr Val Ala Leu Thr Ser Leu Asn Cys Val Ala Asp Pro Ile 275 280 285 Leu Tyr Cys Phe Val Thr Glu Thr Gly Arg Tyr Asp Met Trp Asn Ile 290 295 300 Leu Lys Phe Cys Thr Gly Arg Cys Asn Thr Ser Gln Arg Gln Arg Lys 305 310 315 320 Arg Ile Leu Ser Val Ser Thr Lys Asp Thr Met Glu Leu Glu Val Leu 325 330 335 Glu 135 999 DNA Homo sapiens 135 atggtgaact ccacccaccg tgggatgcac acttctctgc acctctggaa ccgcagcagt 60 tacagactgc acagcaatgc cagtgagtcc cttggaaaag gctactctga tggagggtgc 120 tacgagcaac tttttgtctc tcctgaggtg tttgtgactc tgggtgtcat cagcttgttg 180 gagaatatct tagtgattgt ggcaatagcc aagaacaaga atctgcattc acccatgtac 240 tttttcatct gcagcttggc tgtggctgat atgctggtga gcgtttcaaa tggatcagaa 300 accattatca tcaccctatt aaacagtaca gatacggatg cacagagttt cacagtgaat 360 attgataatg tcattgactc ggtgatctgt agctccttgc ttgcatccat ttgcagcctg 420 ctttcaattg cagtggacag gtactttact atcttctatg ctctccagta ccataacatt 480 atgacagtta agcgggttgg gatcagcata agttgtatct gggcagcttg cacggtttca 540 ggcattttgt tcatcattta ctcagatagt agtgctgtca tcatctgcct catcaccatg 600 ttcttcacca tgctggctct catggcttct ctctatgtcc acatgttcct gatggccagg 660 cttcacatta agaggattgc tgtcctcccc ggcactggtg ccatccgcca aggtgccaat 720 atgaagggaa aaattacctt gaccatcctg attggcgtct ttgttgtctg ctgggcccca 780 ttcttcctcc acttaatatt ctacatctct tgtcctcaga atccatattg tgtgtgcttc 840 atgtctcact ttaacttgta tctcatactg atcatgtgta attcaatcat cgatcctctg 900 atttatgcac tccggagtca agaactgagg aaaaccttca aagagatcat ctgttgctat 960 cccctgggag gcctttgtga cttgtctagc agatattaa 999 136 332 PRT Homo sapiens 136 Met Val Asn Ser Thr His Arg Gly Met His Thr Ser Leu His Leu Trp 1 5 10 15 Asn Arg Ser Ser Tyr Arg Leu His Ser Asn Ala Ser Glu Ser Leu Gly 20 25 30 Lys Gly Tyr Ser Asp Gly Gly Cys Tyr Glu Gln Leu Phe Val Ser Pro 35 40 45 Glu Val Phe Val Thr Leu Gly Val Ile Ser Leu Leu Glu Asn Ile Leu 50 55 60 Val Ile Val Ala Ile Ala Lys Asn Lys Asn Leu His Ser Pro Met Tyr 65 70 75 80 Phe Phe Ile Cys Ser Leu Ala Val Ala Asp Met Leu Val Ser Val Ser 85 90 95 Asn Gly Ser Glu Thr Ile Ile Ile Thr Leu Leu Asn Ser Thr Asp Thr 100 105 110 Asp Ala Gln Ser Phe Thr Val Asn Ile Asp Asn Val Ile Asp Ser Val 115 120 125 Ile Cys Ser Ser Leu Leu Ala Ser Ile Cys Ser Leu Leu Ser Ile Ala 130 135 140 Val Asp Arg Tyr Phe Thr Ile Phe Tyr Ala Leu Gln Tyr His Asn Ile 145 150 155 160 Met Thr Val Lys Arg Val Gly Ile Ser Ile Ser Cys Ile Trp Ala Ala 165 170 175 Cys Thr Val Ser Gly Ile Leu Phe Ile Ile Tyr Ser Asp Ser Ser Ala 180 185 190 Val Ile Ile Cys Leu Ile Thr Met Phe Phe Thr Met Leu Ala Leu Met 195 200 205 Ala Ser Leu Tyr Val His Met Phe Leu Met Ala Arg Leu His Ile Lys 210 215 220 Arg Ile Ala Val Leu Pro Gly Thr Gly Ala Ile Arg Gln Gly Ala Asn 225 230 235 240 Met Lys Gly Lys Ile Thr Leu Thr Ile Leu Ile Gly Val Phe Val Val 245 250 255 Cys Trp Ala Pro Phe Phe Leu His Leu Ile Phe Tyr Ile Ser Cys Pro 260 265 270 Gln Asn Pro Tyr Cys Val Cys Phe Met Ser His Phe Asn Leu Tyr Leu 275 280 285 Ile Leu Ile Met Cys Asn Ser Ile Ile Asp Pro Leu Ile Tyr Ala Leu 290 295 300 Arg Ser Gln Glu Leu Arg Lys Thr Phe Lys Glu Ile Ile Cys Cys Tyr 305 310 315 320 Pro Leu Gly Gly Leu Cys Asp Leu Ser Ser Arg Tyr 325 330 137 33 DNA Artificial Sequence Novel Sequence 137 gccaatatga agggaaaaat taccttgacc atc 33 138 31 DNA Artificial Sequence Novel Sequence 138 ctccttcggt cctcctatcg ttgtcagaag t 31 139 1842 DNA Homo sapiens 139 atggggccca ccctagcggt tcccaccccc tatggctgta ttggctgtaa gctaccccag 60 ccagaatacc caccggctct aatcatcttt atgttctgcg cgatggttat caccatcgtt 120 gtagacctaa tcggcaactc catggtcatt ttggctgtga cgaagaacaa gaagctccgg 180 aattctggca acatcttcgt ggtcagtctc tctgtggccg atatgctggt ggccatctac 240 ccataccctt tgatgctgca tgccatgtcc attgggggct gggatctgag ccagttacag 300 tgccagatgg tcgggttcat cacagggctg agtgtggtcg gctccatctt caacatcgtg 360 gcaatcgcta tcaaccgtta ctgctacatc tgccacagcc tccagtacga acggatcttc 420 agtgtgcgca atacctgcat ctacctggtc atcacctgga tcatgaccgt cctggctgtc 480 ctgcccaaca tgtacattgg caccatcgag tacgatcctc gcacctacac ctgcatcttc 540 aactatctga acaaccctgt cttcactgtt accatcgtct gcatccactt cgtcctccct 600 ctcctcatcg tgggtttctg ctacgtgagg atctggacca aagtgctggc ggcccgtgac 660 cctgcagggc agaatcctga caaccaactt gctgaggttc gcaattttct aaccatgttt 720 gtgatcttcc tcctctttgc agtgtgctgg tgccctatca acgtgctcac tgtcttggtg 780 gctgtcagtc cgaaggagat ggcaggcaag atccccaact ggctttatct tgcagcctac 840 ttcatagcct acttcaacag ctgcctcaac gctgtgatct acgggctcct caatgagaat 900 ttccgaagag aatactggac catcttccat gctatgcggc accctatcat attcttccct 960 ggcctcatca gtgatattcg tgagatgcag gaggcccgta ccctggcccg cgcccgtgcc 1020 catgctcgcg accaagctcg tgaacaagac cgtgcccatg cctgtcctgc tgtggaggaa 1080 accccgatga atgtccggaa tgttccatta cctggtgatg ctgcagctgg ccaccccgac 1140 cgtgcctctg gccaccctaa gccccattcc agatcctcct ctgcctatcg caaatctgcc 1200 tctacccacc acaagtctgt ctttagccac tccaaggctg cctctggtca cctcaagcct 1260 gtctctggcc actccaagcc tgcctctggt caccccaagt ctgccactgt ctaccctaag 1320 cctgcctctg tccatttcaa gggtgactct gtccatttca agggtgactc tgtccatttc 1380 aagcctgact ctgttcattt caagcctgct tccagcaacc ccaagcccat cactggccac 1440 catgtctctg ctggcagcca ctccaagtct gccttcagtg ctgccaccag ccaccctaaa 1500 cccatcaagc cagctaccag ccatgctgag cccaccactg ctgactatcc caagcctgcc 1560 actaccagcc accctaagcc cgctgctgct gacaaccctg agctctctgc ctcccattgc 1620 cccgagatcc ctgccattgc ccaccctgtg tctgacgaca gtgacctccc tgagtcggcc 1680 tctagccctg ccgctgggcc caccaagcct gctgccagcc agctggagtc tgacaccatc 1740 gctgaccttc ctgaccctac tgtagtcact accagtacca atgattacca tgatgtcgtg 1800 gttgttgatg ttgaagatga tcctgatgaa atggctgtgt ga 1842 140 613 PRT Homo sapiens 140 Met Gly Pro Thr Leu Ala Val Pro Thr Pro Tyr Gly Cys Ile Gly Cys 1 5 10 15 Lys Leu Pro Gln Pro Glu Tyr Pro Pro Ala Leu Ile Ile Phe Met Phe 20 25 30 Cys Ala Met Val Ile Thr Ile Val Val Asp Leu Ile Gly Asn Ser Met 35 40 45 Val Ile Leu Ala Val Thr Lys Asn Lys Lys Leu Arg Asn Ser Gly Asn 50 55 60 Ile Phe Val Val Ser Leu Ser Val Ala Asp Met Leu Val Ala Ile Tyr 65 70 75 80 Pro Tyr Pro Leu Met Leu His Ala Met Ser Ile Gly Gly Trp Asp Leu 85 90 95 Ser Gln Leu Gln Cys Gln Met Val Gly Phe Ile Thr Gly Leu Ser Val 100 105 110 Val Gly Ser Ile Phe Asn Ile Val Ala Ile Ala Ile Asn Arg Tyr Cys 115 120 125 Tyr Ile Cys His Ser Leu Gln Tyr Glu Arg Ile Phe Ser Val Arg Asn 130 135 140 Thr Cys Ile Tyr Leu Val Ile Thr Trp Ile Met Thr Val Leu Ala Val 145 150 155 160 Leu Pro Asn Met Tyr Ile Gly Thr Ile Glu Tyr Asp Pro Arg Thr Tyr 165 170 175 Thr Cys Ile Phe Asn Tyr Leu Asn Asn Pro Val Phe Thr Val Thr Ile 180 185 190 Val Cys Ile His Phe Val Leu Pro Leu Leu Ile Val Gly Phe Cys Tyr 195 200 205 Val Arg Ile Trp Thr Lys Val Leu Ala Ala Arg Asp Pro Ala Gly Gln 210 215 220 Asn Pro Asp Asn Gln Leu Ala Glu Val Arg Asn Phe Leu Thr Met Phe 225 230 235 240 Val Ile Phe Leu Leu Phe Ala Val Cys Trp Cys Pro Ile Asn Val Leu 245 250 255 Thr Val Leu Val Ala Val Ser Pro Lys Glu Met Ala Gly Lys Ile Pro 260 265 270 Asn Trp Leu Tyr Leu Ala Ala Tyr Phe Ile Ala Tyr Phe Asn Ser Cys 275 280 285 Leu Asn Ala Val Ile Tyr Gly Leu Leu Asn Glu Asn Phe Arg Arg Glu 290 295 300 Tyr Trp Thr Ile Phe His Ala Met Arg His Pro Ile Ile Phe Phe Pro 305 310 315 320 Gly Leu Ile Ser Asp Ile Arg Glu Met Gln Glu Ala Arg Thr Leu Ala 325 330 335 Arg Ala Arg Ala His Ala Arg Asp Gln Ala Arg Glu Gln Asp Arg Ala 340 345 350 His Ala Cys Pro Ala Val Glu Glu Thr Pro Met Asn Val Arg Asn Val 355 360 365 Pro Leu Pro Gly Asp Ala Ala Ala Gly His Pro Asp Arg Ala Ser Gly 370 375 380 His Pro Lys Pro His Ser Arg Ser Ser Ser Ala Tyr Arg Lys Ser Ala 385 390 395 400 Ser Thr His His Lys Ser Val Phe Ser His Ser Lys Ala Ala Ser Gly 405 410 415 His Leu Lys Pro Val Ser Gly His Ser Lys Pro Ala Ser Gly His Pro 420 425 430 Lys Ser Ala Thr Val Tyr Pro Lys Pro Ala Ser Val His Phe Lys Gly 435 440 445 Asp Ser Val His Phe Lys Gly Asp Ser Val His Phe Lys Pro Asp Ser 450 455 460 Val His Phe Lys Pro Ala Ser Ser Asn Pro Lys Pro Ile Thr Gly His 465 470 475 480 His Val Ser Ala Gly Ser His Ser Lys Ser Ala Phe Ser Ala Ala Thr 485 490 495 Ser His Pro Lys Pro Ile Lys Pro Ala Thr Ser His Ala Glu Pro Thr 500 505 510 Thr Ala Asp Tyr Pro Lys Pro Ala Thr Thr Ser His Pro Lys Pro Ala 515 520 525 Ala Ala Asp Asn Pro Glu Leu Ser Ala Ser His Cys Pro Glu Ile Pro 530 535 540 Ala Ile Ala His Pro Val Ser Asp Asp Ser Asp Leu Pro Glu Ser Ala 545 550 555 560 Ser Ser Pro Ala Ala Gly Pro Thr Lys Pro Ala Ala Ser Gln Leu Glu 565 570 575 Ser Asp Thr Ile Ala Asp Leu Pro Asp Pro Thr Val Val Thr Thr Ser 580 585 590 Thr Asn Asp Tyr His Asp Val Val Val Val Asp Val Glu Asp Asp Pro 595 600 605 Asp Glu Met Ala Val 610 141 1842 DNA Homo sapiens 141 atggggccca ccctagcggt tcccaccccc tatggctgta ttggctgtaa gctaccccag 60 ccagaatacc caccggctct aatcatcttt atgttctgcg cgatggttat caccatcgtt 120 gtagacctaa tcggcaactc catggtcatt ttggctgtga cgaagaacaa gaagctccgg 180 aattctggca acatcttcgt ggtcagtctc tctgtggccg atatgctggt ggccatctac 240 ccataccctt tgatgctgca tgccatgtcc attgggggct gggatctgag ccagttacag 300 tgccagatgg tcgggttcat cacagggctg agtgtggtcg gctccatctt caacatcgtg 360 gcaatcgcta tcaaccgtta ctgctacatc tgccacagcc tccagtacga acggatcttc 420 agtgtgcgca atacctgcat ctacctggtc atcacctgga tcatgaccgt cctggctgtc 480 ctgcccaaca tgtacattgg caccatcgag tacgatcctc gcacctacac ctgcatcttc 540 aactatctga acaaccctgt cttcactgtt accatcgtct gcatccactt cgtcctccct 600 ctcctcatcg tgggtttctg ctacgtgagg atctggacca aagtgctggc ggcccgtgac 660 cctgcagggc agaatcctga caaccaactt gctgaggttc gcaataaact aaccatgttt 720 gtgatcttcc tcctctttgc agtgtgctgg tgccctatca acgtgctcac tgtcttggtg 780 gctgtcagtc cgaaggagat ggcaggcaag atccccaact ggctttatct tgcagcctac 840 ttcatagcct acttcaacag ctgcctcaac gctgtgatct acgggctcct caatgagaat 900 ttccgaagag aatactggac catcttccat gctatgcggc accctatcat attcttctct 960 ggcctcatca gtgatattcg tgagatgcag gaggcccgta ccctggcccg cgcccgtgcc 1020 catgctcgcg accaagctcg tgaacaagac cgtgcccatg cctgtcctgc tgtggaggaa 1080 accccgatga atgtccggaa tgttccatta cctggtgatg ctgcagctgg ccaccccgac 1140 cgtgcctctg gccaccctaa gccccattcc agatcctcct ctgcctatcg caaatctgcc 1200 tctacccacc acaagtctgt ctttagccac tccaaggctg cctctggtca cctcaagcct 1260 gtctctggcc actccaagcc tgcctctggt caccccaagt ctgccactgt ctaccctaag 1320 cctgcctctg tccatttcaa ggctgactct gtccatttca agggtgactc tgtccatttc 1380 aagcctgact ctgttcattt caagcctgct tccagcaacc ccaagcccat cactggccac 1440 catgtctctg ctggcagcca ctccaagtct gccttcaatg ctgccaccag ccaccctaaa 1500 cccatcaagc cagctaccag ccatgctgag cccaccactg ctgactatcc caagcctgcc 1560 actaccagcc accctaagcc cgctgctgct gacaaccctg agctctctgc ctcccattgc 1620 cccgagatcc ctgccattgc ccaccctgtg tctgacgaca gtgacctccc tgagtcggcc 1680 tctagccctg ccgctgggcc caccaagcct gctgccagcc agctggagtc tgacaccatc 1740 gctgaccttc ctgaccctac tgtagtcact accagtacca atgattacca tgatgtcgtg 1800 gttgttgatg ttgaagatga tcctgatgaa atggctgtgt ga 1842 142 613 PRT Homo sapiens 142 Met Gly Pro Thr Leu Ala Val Pro Thr Pro Tyr Gly Cys Ile Gly Cys 1 5 10 15 Lys Leu Pro Gln Pro Glu Tyr Pro Pro Ala Leu Ile Ile Phe Met Phe 20 25 30 Cys Ala Met Val Ile Thr Ile Val Val Asp Leu Ile Gly Asn Ser Met 35 40 45 Val Ile Leu Ala Val Thr Lys Asn Lys Lys Leu Arg Asn Ser Gly Asn 50 55 60 Ile Phe Val Val Ser Leu Ser Val Ala Asp Met Leu Val Ala Ile Tyr 65 70 75 80 Pro Tyr Pro Leu Met Leu His Ala Met Ser Ile Gly Gly Trp Asp Leu 85 90 95 Ser Gln Leu Gln Cys Gln Met Val Gly Phe Ile Thr Gly Leu Ser Val 100 105 110 Val Gly Ser Ile Phe Asn Ile Val Ala Ile Ala Ile Asn Arg Tyr Cys 115 120 125 Tyr Ile Cys His Ser Leu Gln Tyr Glu Arg Ile Phe Ser Val Arg Asn 130 135 140 Thr Cys Ile Tyr Leu Val Ile Thr Trp Ile Met Thr Val Leu Ala Val 145 150 155 160 Leu Pro Asn Met Tyr Ile Gly Thr Ile Glu Tyr Asp Pro Arg Thr Tyr 165 170 175 Thr Cys Ile Phe Asn Tyr Leu Asn Asn Pro Val Phe Thr Val Thr Ile 180 185 190 Val Cys Ile His Phe Val Leu Pro Leu Leu Ile Val Gly Phe Cys Tyr 195 200 205 Val Arg Ile Trp Thr Lys Val Leu Ala Ala Arg Asp Pro Ala Gly Gln 210 215 220 Asn Pro Asp Asn Gln Leu Ala Glu Val Arg Asn Lys Leu Thr Met Phe 225 230 235 240 Val Ile Phe Leu Leu Phe Ala Val Cys Trp Cys Pro Ile Asn Val Leu 245 250 255 Thr Val Leu Val Ala Val Ser Pro Lys Glu Met Ala Gly Lys Ile Pro 260 265 270 Asn Trp Leu Tyr Leu Ala Ala Tyr Phe Ile Ala Tyr Phe Asn Ser Cys 275 280 285 Leu Asn Ala Val Ile Tyr Gly Leu Leu Asn Glu Asn Phe Arg Arg Glu 290 295 300 Tyr Trp Thr Ile Phe His Ala Met Arg His Pro Ile Ile Phe Phe Ser 305 310 315 320 Gly Leu Ile Ser Asp Ile Arg Glu Met Gln Glu Ala Arg Thr Leu Ala 325 330 335 Arg Ala Arg Ala His Ala Arg Asp Gln Ala Arg Glu Gln Asp Arg Ala 340 345 350 His Ala Cys Pro Ala Val Glu Glu Thr Pro Met Asn Val Arg Asn Val 355 360 365 Pro Leu Pro Gly Asp Ala Ala Ala Gly His Pro Asp Arg Ala Ser Gly 370 375 380 His Pro Lys Pro His Ser Arg Ser Ser Ser Ala Tyr Arg Lys Ser Ala 385 390 395 400 Ser Thr His His Lys Ser Val Phe Ser His Ser Lys Ala Ala Ser Gly 405 410 415 His Leu Lys Pro Val Ser Gly His Ser Lys Pro Ala Ser Gly His Pro 420 425 430 Lys Ser Ala Thr Val Tyr Pro Lys Pro Ala Ser Val His Phe Lys Ala 435 440 445 Asp Ser Val His Phe Lys Gly Asp Ser Val His Phe Lys Pro Asp Ser 450 455 460 Val His Phe Lys Pro Ala Ser Ser Asn Pro Lys Pro Ile Thr Gly His 465 470 475 480 His Val Ser Ala Gly Ser His Ser Lys Ser Ala Phe Asn Ala Ala Thr 485 490 495 Ser His Pro Lys Pro Ile Lys Pro Ala Thr Ser His Ala Glu Pro Thr 500 505 510 Thr Ala Asp Tyr Pro Lys Pro Ala Thr Thr Ser His Pro Lys Pro Ala 515 520 525 Ala Ala Asp Asn Pro Glu Leu Ser Ala Ser His Cys Pro Glu Ile Pro 530 535 540 Ala Ile Ala His Pro Val Ser Asp Asp Ser Asp Leu Pro Glu Ser Ala 545 550 555 560 Ser Ser Pro Ala Ala Gly Pro Thr Lys Pro Ala Ala Ser Gln Leu Glu 565 570 575 Ser Asp Thr Ile Ala Asp Leu Pro Asp Pro Thr Val Val Thr Thr Ser 580 585 590 Thr Asn Asp Tyr His Asp Val Val Val Val Asp Val Glu Asp Asp Pro 595 600 605 Asp Glu Met Ala Val 610 143 33 DNA Artificial Sequence Novel Sequence 143 gctgaggttc gcaataaact aaccatgttt gtg 33 144 31 DNA Artificial Sequence Novel Sequence 144 ctccttcggt cctcctatcg ttgtcagaag t 31 145 27 DNA Artificial Sequence Novel Sequence 145 ttagatatcg gggcccaccc tagcggt 27 146 29 DNA Artificial Sequence Novel Sequence 146 ggtaccccca cagccatttc atcaggatc 29 147 31 DNA Artificial Sequence Novel Sequence 147 gatctggagt accccattga cgtcaatggg g 31 148 31 DNA Artificial Sequence Novel Sequence 148 gatcccccat tgacgtcaat ggggtactcc a 31 149 372 PRT Homo sapiens 149 Met Leu Ala Asn Ser Ser Ser Thr Asn Ser Ser Val Leu Pro Cys Pro 1 5 10 15 Asp Tyr Arg Pro Thr His Arg Leu His Leu Val Val Tyr Ser Leu Val 20 25 30 Leu Ala Ala Gly Leu Pro Leu Asn Ala Leu Ala Leu Trp Val Phe Leu 35 40 45 Arg Ala Leu Arg Val His Ser Val Val Ser Val Tyr Met Cys Asn Leu 50 55 60 Ala Ala Ser Asp Leu Leu Phe Thr Leu Ser Leu Pro Val Arg Leu Ser 65 70 75 80 Tyr Tyr Ala Leu His His Trp Pro Phe Pro Asp Leu Leu Cys Gln Thr 85 90 95 Thr Gly Ala Ile Phe Gln Met Asn Met Tyr Gly Ser Cys Ile Phe Leu 100 105 110 Met Leu Ile Asn Val Asp Arg Tyr Ala Ala Ile Val His Pro Leu Arg 115 120 125 Leu Arg His Leu Arg Arg Pro Arg Val Ala Arg Leu Leu Cys Leu Gly 130 135 140 Val Trp Ala Leu Ile Leu Val Phe Ala Val Pro Ala Ala Arg Val His 145 150 155 160 Arg Pro Ser Arg Cys Arg Tyr Arg Asp Leu Glu Val Arg Leu Cys Phe 165 170 175 Glu Ser Phe Ser Asp Glu Leu Trp Lys Gly Arg Leu Leu Pro Leu Val 180 185 190 Leu Leu Ala Glu Ala Leu Gly Phe Leu Leu Pro Leu Ala Ala Val Val 195 200 205 Tyr Ser Ser Gly Arg Val Phe Trp Thr Leu Ala Arg Pro Asp Ala Thr 210 215 220 Gln Ser Gln Arg Arg Arg Lys Thr Lys Arg Leu Leu Leu Ala Asn Leu 225 230 235 240 Val Ile Phe Leu Leu Cys Phe Val Pro Tyr Asn Ser Thr Leu Ala Val 245 250 255 Tyr Gly Leu Leu Arg Ser Lys Leu Val Ala Ala Ser Val Pro Ala Arg 260 265 270 Asp Arg Val Arg Gly Val Leu Met Val Met Val Leu Leu Ala Gly Ala 275 280 285 Asn Cys Val Leu Asp Pro Leu Val Tyr Tyr Phe Ser Ala Glu Gly Phe 290 295 300 Arg Asn Thr Leu Arg Gly Leu Gly Thr Pro His Arg Ala Arg Thr Ser 305 310 315 320 Ala Thr Asn Gly Thr Arg Ala Ala Leu Ala Gln Ser Glu Arg Ser Ala 325 330 335 Val Thr Thr Asp Ala Thr Arg Pro Asp Ala Ala Ser Gln Gly Leu Leu 340 345 350 Arg Pro Ser Asp Ser His Ser Leu Ser Ser Phe Thr Gln Cys Pro Gln 355 360 365 Asp Ser Ala Leu 370 150 412 PRT Homo sapiens 150 Met Gly Ser Pro Trp Asn Gly Ser Asp Gly Pro Glu Gly Ala Arg Glu 1 5 10 15 Pro Pro Trp Pro Ala Leu Pro Pro Cys Asp Glu Arg Arg Cys Ser Pro 20 25 30 Phe Pro Leu Gly Ala Leu Val Pro Val Thr Ala Val Cys Leu Cys Leu 35 40 45 Phe Val Val Gly Val Ser Gly Asn Val Val Thr Val Met Leu Ile Gly 50 55 60 Arg Tyr Arg Asp Met Arg Thr Thr Thr Asn Leu Tyr Leu Gly Ser Met 65 70 75 80 Ala Val Ser Asp Leu Leu Ile Leu Leu Gly Leu Pro Phe Asp Leu Tyr 85 90 95 Arg Leu Trp Arg Ser Arg Pro Trp Val Phe Gly Pro Leu Leu Cys Arg 100 105 110 Leu Ser Leu Tyr Val Gly Glu Gly Cys Thr Tyr Ala Thr Leu Leu His 115 120 125 Met Thr Ala Leu Ser Val Glu Arg Tyr Leu Ala Ile Cys Arg Pro Leu 130 135 140 Arg Ala Arg Val Leu Val Thr Arg Arg Arg Val Arg Ala Leu Ile Ala 145 150 155 160 Val Leu Trp Ala Val Ala Leu Leu Ser Ala Gly Pro Phe Leu Phe Leu 165 170 175 Val Gly Val Glu Gln Asp Pro Gly Ile Ser Val Val Pro Gly Leu Asn 180 185 190 Gly Thr Ala Arg Ile Ala Ser Ser Pro Leu Ala Ser Ser Pro Pro Leu 195 200 205 Trp Leu Ser Arg Ala Pro Pro Pro Ser Pro Pro Ser Gly Pro Glu Thr 210 215 220 Ala Glu Ala Ala Ala Leu Phe Ser Arg Glu Cys Arg Pro Ser Pro Ala 225 230 235 240 Gln Leu Gly Ala Leu Arg Val Met Leu Trp Val Thr Thr Ala Tyr Phe 245 250 255 Phe Leu Pro Phe Leu Cys Leu Ser Ile Leu Tyr Gly Leu Ile Gly Arg 260 265 270 Glu Leu Trp Ser Ser Arg Arg Pro Leu Arg Gly Pro Ala Ala Ser Gly 275 280 285 Arg Glu Arg Gly His Arg Gln Thr Lys Arg Val Leu Leu Val Val Val 290 295 300 Leu Ala Phe Ile Ile Cys Trp Leu Pro Phe His Val Gly Arg Ile Ile 305 310 315 320 Tyr Ile Asn Thr Glu Asp Ser Arg Met Met Tyr Phe Ser Gln Tyr Phe 325 330 335 Asn Ile Val Ala Leu Gln Leu Phe Tyr Leu Ser Ala Ser Ile Asn Pro 340 345 350 Ile Leu Tyr Asn Leu Ile Ser Lys Lys Tyr Arg Ala Ala Ala Phe Lys 355 360 365 Leu Leu Leu Ala Arg Lys Ser Arg Pro Arg Gly Phe His Arg Ser Arg 370 375 380 Asp Thr Ala Gly Glu Val Ala Gly Asp Thr Gly Gly Asp Thr Val Gly 385 390 395 400 Tyr Thr Glu Thr Ser Ala Asn Val Lys Thr Met Gly 405 410 151 2040 DNA Homo sapiens 151 atgggcagcc cctggaacgg cagcgacggc cccgaggggg cgcgggagcc gccgtggccc 60 gcgctgccgc cttgcgacgc gcgccgctgc tcgccctttc ccctgggggc gctggtgccg 120 gtgaccgctg tgtgcctgtg cctgttcgtc gtcggggtga gcggcaacgt ggtgaccgtg 180 atgctgatcg ggcgctaccg ggacatgcgg accaccacca acttgtacct gggcagcatg 240 gccgtgtccg acctactcat aatgctcggg ctgccgttcg acctgtaccg cctctggcgc 300 tcgcggccct gggtgttcgg gccgctgctc tgccgcctgt ccctctacgt gggcgagggc 360 tgcacctacg ccacgctgct gcacatgacc gcgctcagcg tcgagcgcta cctggccatc 420 tgccgcccgc tccgcgcccg cgtcttggtc acccggcgcc gcgtccgcgc gctcatcgct 480 gtgctctggg ccgtggcgct gctctctgcc ggtcccttct tgttcctggt gggcgtcgag 540 caggaccccg gcatctccgt agtcccgggc ctcaatggca ccgcgcggat cgcctcctcg 600 cctctcgcct cgtcgccgcc tctctggctc tcgcgggcgc caccgccgtc cccgccgtcg 660 gggcccgaga ccgcggaggc cgcggcgctg ttcagccgcg aatgccggcc gagccccgcg 720 cagctgggcg cgctgcgtgt catgctgtgg gtcaccaccg cctacttctt cctgcccttt 780 ctgtgcctca gcatcctcta cgggctcatc gggcgggagc tgtggagcag ccggcggccg 840 ctgcgaggcc cggccgcctc ggggcgggag agaggccacc ggcagaccaa acgcgtcctg 900 cgtaagttga gccgccgtgg ttccaaagac gcctgcctgc agtccgcccc gccggggacc 960 gcgcaaacgc tgggtcccct tcccctgctc gcccagctct gggcgccgct tccagctccc 1020 tttcctattt cgattccagc ctccacccgc cggtacttcc catcccccga gaaaaccatg 1080 tcctgtcccc caggagctct gggggacccc agggcgcttt gagggtggga tccccggatc 1140 cgattcagta accagcagtg cttttccaga gcctctgaga ccagaaagga gagttggtaa 1200 ttcttaatcc aaccacctgt tagatgccac aaatgaggag tcctcacagt gctcttgaga 1260 agacgaggga gatttcatta agctaaaatt ttttatttaa tgttaagtga tgctgaaggc 1320 taaagtaaac cttgctcgta tcaaaaagta aagattgtgc agacctgttg tagaattctt 1380 ttcaacagag aacagaaaac ttgtctccga agtgggtttg tggaaggaag cctgccaagg 1440 cggcttgttc agagaaattg ctccttctgg tttatgtcca gccttgataa cacatatggg 1500 agcctactat gcagttttaa agcaagtatc catgcagcct ccagcctggt cattttttct 1560 ggggtgagga tctgcctagg tagaagtttt ctctaattta ttttgctgtt acttgttatt 1620 gcagatggtt ccttgtcggg gtggggggtt tatttgcttc ccaatgcttt tgttaatccc 1680 ggtgctgtgt cttatgttgc agtggtggtg gttctggcat ttataatttg ctggttgccc 1740 ttccacgttg gcagaatcat ttacataaac acggaagatt cgcggatgat gtacttctct 1800 cagtacttta acatcgtcgc tctgcaactt ttctatctga gcgcatctat caacccaatc 1860 ctctacaacc tcatttcaaa gaagtacaga gcggcggcct ttaaactgct gctcgcaagg 1920 aagtccaggc cgagaggctt ccacagaagc agggacactg cgggggaagt tgcaggggac 1980 actggaggag acacggtggg ctacaccgag acaagcgcta acgtgaagac gatgggataa 2040 152 30 DNA Artificial Sequence Novel Sequence 152 ggccaccggc agaccaaacg cgtcctgctg 30 153 31 DNA Artificial Sequence Novel Sequence 153 ctccttcggt cctcctatcg ttgtcagaag t 31 154 24 DNA Artificial Sequence Novel Sequence 154 gcactcatgg tcagcctgtc catc 24 155 23 DNA Artificial Sequence Novel Sequence 155 gtacagaatt ggatcagcaa cac 23

Claims (80)

What is claimed is:
1. A cDNA encoding a non-endogenous, constitutively activated version of a human G protein-coupled receptor comprising hARE-3(F313K).
2. A non-endogenous version of a human G protein-coupled receptor encoded by the cDNA of claim 1.
3. A Plasmid comprising a Vector and the cDNA of claim 1.
4. A Host Cell comprising the Plasmid of claim 3.
5. A cDNA encoding a non-endogenous, constitutively activated version of a human G protein-coupled receptor comprising hARE-4(V233K)
6. A non-endogenous version of a human G protein-coupled receptor encoded by the cDNA of claim 5.
7. A Plasmid comprising a Vector and the cDNA of claim 5.
8. A Host Cell comprising the Plasmid of claim 7.
9. A cDNA encoding a non-endogenous, constitutively activated version of a human G protein-coupled receptor comprising hARE-5(A240K).
10. A non-endogenous version of a human G protein-coupled receptor encoded by the cDNA of claim 9.
11. A Plasmid comprising a Vector and the cDNA of claim 5.
12. A Host Cell comprising the Plasmid of claim 11.
13. A cDNA encoding a non-endogenous, constitutively activated version of a human G protein-coupled receptor comprising hGPCR14(L257K).
14. A non-endogenous version of a human G protein-coupled receptor encoded by the cDNA of claim 13.
15. A Plasmid comprising a Vector and the cDNA of claim 13.
16. A Host Cell comprising the Plasmid of claim 15.
17. A cDNA encoding a non-endogenous, constitutively activated version of a human G protein-coupled receptor comprising hGPCR27(C283K).
18. A non-endogenous version of a human G protein-coupled receptor encoded by the cDNA of claim 17.
19. A Plasmid comprising a Vector and the cDNA of claim 17.
20. A Host Cell comprising the Plasmid of claim 19.
21. A cDNA encoding a non-endogenous, constitutively activated version of a human G protein-coupled receptor comprising hARE-1(E232K).
22. A non-endogenous version of a human G protein-coupled receptor encoded by the cDNA of claim 21.
23. A Plasmid comprising a Vector and the cDNA of claim 21.
24. A Host Cell comprising the Plasmid of claim 23.
25. A cDNA encoding a non-endogenous, constitutively activated version of a human G protein-coupled receptor comprising hARE-2(G285K).
26. A non-endogenous version of a human G protein-coupled receptor encoded by the cDNA of claim 25.
27. A Plasmid comprising a Vector and the cDNA of claim 25.
28. A Host Cell comprising the Plasmid of claim 27.
29. A cDNA encoding a non-endogenous, constitutively activated version of a human G protein-coupled receptor comprising hPPR1(L239K).
30. A non-endogenous version of a human G protein-coupled receptor encoded by the cDNA of claim 29.
31. A Plasmid comprising a Vector and the cDNA of claim 29.
32. A Host Cell comprising the Plasmid of claim 31.
33. A cDNA encoding a non-endogenous, constitutively activated version of a human G protein-coupled receptor comprising hG2A(K232A).
34. A non-endogenous version of a human G protein-coupled receptor encoded by the cDNA of claim 33.
35. A Plasmid comprising a Vector and the cDNA of claim 33.
36. A Host Cell comprising the Plasmid of claim 35.
37. A cDNA encoding a non-endogenous, constitutively activated version of a human G protein-coupled receptor comprising hRUP3(L224K).
38. A non-endogenous version of a human G protein-coupled receptor encoded by the cDNA of claim 37.
39. A Plasmid comprising a Vector and the cDNA of claim 37.
40. A Host Cell comprising the Plasmid of claim 39.
41. A cDNA encoding a non-endogenous, constitutively activated version of a human G protein-coupled receptor comprising hRUP5(A236K).
42. A non-endogenous version of a human G protein-coupled receptor encoded by the cDNA of claim 41.
43. A Plasmid comprising a Vector and the cDNA of claim 41.
44. A Host Cell comprising the Plasmid of claim 42.
45. A cDNA encoding a non-endogenous, constitutively activated version of a human G protein-coupled receptor comprising hRUP6(N267K)
46. A non-endogenous version of a human G protein-coupled receptor encoded by the cDNA of claim 45.
47. A Plasmid comprising a Vector and the cDNA of claim 45.
48. A Host Cell comprising the Plasmid of claim 47.
49. A cDNA encoding a non-endogenous, constitutively activated version of a human G protein-coupled receptor comprising hRUP7(A302K).
50. A non-endogenous version of a human G protein-coupled receptor encoded by the cDNA of claim 49.
51. A Plasmid comprising a Vector and the cDNA of claim 49.
52. A Host Cell comprising the Plasmid of claim 51.
53. A cDNA encoding a non-endogenous, constitutively activated version of a human G protein-coupled receptor comprising hCHN4(V236K).
54. A non-endogenous version of a human G protein-coupled receptor encoded by the cDNA of claim 53.
55. A Plasmid comprising a Vector and the cDNA of claim 53.
56. A Host Cell comprising the Plasmid of claim 55.
57. A cDNA encoding a non-endogenous, constitutively activated version of a human G protein-coupled receptor comprising hMC4(A244K).
58. A non-endogenous version of a human G protein-coupled receptor encoded by the cDNA of claim 57.
59. A Plasmid comprising a Vector and the cDNA of claim 57.
60. A Host Cell comprising the Plasmid of claim 60.
61. A cDNA encoding a non-endogenous, constitutively activated version of a human G protein-coupled receptor comprising hCHN3(S284K).
62. A non-endogenous version of a human G protein-coupled receptor encoded by the cDNA of claim 61.
63. A Plasmid comprising a Vector and the cDNA of claim 61.
64. A Host Cell comprising the Plasmid of claim 63.
65. A cDNA encoding a non-endogenous, constitutively activated version of a human G protein-coupled receptor comprising hCHN6(L352K).
66. A non-endogenous version of a human G protein-coupled receptor encoded by the cDNA of claim 65.
67. A Plasmid comprising a Vector and the cDNA of claim 65.
68. A Host Cell comprising the Plasmid of claim 67.
69. A cDNA encoding a non-endogenous, constitutively activated version of a human G protein-coupled receptor comprising hCHN8(N235K).
70. A non-endogenous version of a human G protein-coupled receptor encoded by the cDNA of claim 69.
71. A Plasmid comprising a Vector and the cDNA of claim 69.
72. A Host Cell comprising the Plasmid of claim 71.
73. A cDNA encoding a non-endogenous, constitutively activated version of a human G protein-coupled receptor comprising hH9(F236K).
74. A non-endogenous version of a human G protein-coupled receptor encoded by the cDNA of claim 73.
75. A Plasmid comprising a Vector and the cDNA of claim 73.
76. A Host Cell comprising the Plasmid of claim 74.
77. A cDNA encoding a non-endogenous, constitutively activated version of a human G protein-coupled AT1 receptor selected from the group consisting of: hAT1(F239K); hAT1(N111A); hAT1(AT2K255IC3); and hAT1(A243+).
78. A non-endogenous version of a human G protein-coupled receptor encoded by a cDNA of claim 77.
79. A Plasmid comprising a Vector and the cDNA of claim 77.
80. A Host Cell comprising the Plasmid of claim 79.
US10/417,820 1998-10-13 2003-04-16 Constitutively activated human G protein coupled receptors Abandoned US20030229216A1 (en)

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Publication number Priority date Publication date Assignee Title
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6084083A (en) * 1995-03-03 2000-07-04 Milennium Pharmaceuticals, Inc. Compositions and methods for the treatment and diagnosis of immune disorders
US6436703B1 (en) * 2000-03-31 2002-08-20 Hyseq, Inc. Nucleic acids and polypeptides
US6455685B1 (en) * 1995-03-03 2002-09-24 Millennium Pharmaceuticals, Inc. Compositions and methods for the treatment and diagnosis of immune disorders

Family Cites Families (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US146757A (en) * 1874-01-27 Improvement in loom-temples
US106741A (en) * 1870-08-23 Improvement in gauge for cutting bias pieces of cloth
US61567A (en) * 1867-01-29 William d
CA2076386C (en) 1990-02-26 2003-04-22 David S. Hogness Identification and expression of insect steroid receptor dna sequence
WO1993007294A1 (en) 1991-10-01 1993-04-15 The United States Of America, Represented By The Secretary, Department Of Health And Human Service A method of identifying ligands and antagonists of ligands
WO1995013373A1 (en) 1993-11-10 1995-05-18 Arch Development Corporation Ubiquitous nuclear receptor: compositions and methods
US5573944A (en) 1994-07-22 1996-11-12 President And Fellows Of Harvard College Yeast cell expressing heterologous receptor kinase
US20020106741A1 (en) 1995-06-06 2002-08-08 Human Genome Sciences, Inc. G protein receptor HTNAD29
US5750353A (en) 1995-12-11 1998-05-12 New England Medical Center Hospitals, Inc. Assay for non-peptide agonists to peptide hormone receptors
WO1998032858A2 (en) 1997-01-23 1998-07-30 Schering Corporation Mammalian chemokines; receptors; reagents; uses
EP0942271A1 (en) 1998-03-10 1999-09-15 Oxford Instruments (Uk) Limited Improvements in resistance thermometry
CA2330251A1 (en) 1998-06-12 1999-12-16 Merck & Co., Inc. Cloning and identification of the motilin receptor
IL155450A0 (en) 2000-10-20 2003-11-23 Expression Diagnostics Inc Leukocyte expression profiling
WO2002061087A2 (en) 2000-12-19 2002-08-08 Lifespan Biosciences, Inc. Antigenic peptides, such as for g protein-coupled receptors (gpcrs), antibodies thereto, and systems for identifying such antigenic peptides

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6084083A (en) * 1995-03-03 2000-07-04 Milennium Pharmaceuticals, Inc. Compositions and methods for the treatment and diagnosis of immune disorders
US6156887A (en) * 1995-03-03 2000-12-05 Millennium Pharmaceuticals, Inc. Compositions and methods for the treatment and diagnosis of immune disorders
US6204371B1 (en) * 1995-03-03 2001-03-20 Millennium Pharmaceuticals, Inc. Compositions and methods for the treatment and diagnosis of immune disorders
US6288218B1 (en) * 1995-03-03 2001-09-11 Douglas Adam Levinson Compositions and methods for the treatment and diagnosis of immune disorders
US6414117B1 (en) * 1995-03-03 2002-07-02 Millennium Pharmaceuticals, Inc. Compositions and methods for the treatment and diagnosis of immune disorders
US6455685B1 (en) * 1995-03-03 2002-09-24 Millennium Pharmaceuticals, Inc. Compositions and methods for the treatment and diagnosis of immune disorders
US6436703B1 (en) * 2000-03-31 2002-08-20 Hyseq, Inc. Nucleic acids and polypeptides

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