WO1992021701A1

WO1992021701A1 - Transfected mammalian cell lines expressing the a1 adenosine receptor

Info

Publication number: WO1992021701A1
Application number: PCT/US1992/004626
Authority: WO
Inventors: David R. Sibley; Lawrence C. Mahan; Elizabeth M. Smyk-Randall; Frederick J. Monsma, Jr.
Original assignee: The United States Of America Represented By The Secretary, Department Of Health And Human Services
Priority date: 1991-06-05
Filing date: 1992-06-05
Publication date: 1992-12-10
Also published as: AU2179192A

Abstract

The subject invention relates to mammalian cell lines which express an adenosine receptor referred to as A1. The cell lines may be utilized to screen for drugs which affect the activity of the A1 receptor. The cell lines are created by initially isolating a cDNA encoding the A1 adenosine receptor subtype, inserting the cDNA into a eukaryotic expression vector and transfecting a host cell with the vector.

Description

TRANSFECTED MAMMALIAN CELL LINES EXPRESSING THE A. ADENOSINE RECEPTOR

BACKGROUND OF THE INVENTION Technical Field

The subject invention relates to mammalian cell lines which express the A,, adenosine receptor and methods of use thereof. In particular, the cell lines may be utilized in the screening of drugs which affect the activity of the A, receptor.

Background Information Adenosine is a ubiquitous modulator of numerous physiological activities, particularly within the cardiovascular and nervous systems. The effects of adenosine appear to be mediated by specific membrane-bound receptor proteins. Adenosine receptors belong to a large class of neurotransmitter and hormone receptors which are linked to their signal transduction pathways via guanine nucleotide binding regulatory (G) proteins. Biochemical and pharmacological criteria have been used to divide adenosine receptors into two major subtypes referred to as A, and A₂ which either inhibit or stimulate, respectively, the enzyme adenylyl cyclase (Stiles, G.L., Trends Pharmacol. 7:486-49 (1986) and Williams, M. , Ann Rev. Pharmacol. Toxicol. 27:315- 45 (1987)). These receptors have been directly labeled using radioligand binding methods in various tissues and a great deal of information has been generated concerning their regulation and ligand binding characteristics (Stiles, supra (1986), Williams, supra (1987) and Stiles, G.L., Chemical Research 38:10-18 (1990)). Recently, substantial progress has been made in delineating the biochemical properties of adenosine receptors, particularly of the A, subtype. The ligand binding subunit of this receptor has been directly visualized utilizing photoaf inity labeling techniques and its glycoprotein characteristics investigated (Choca et al., Biochem. Biophvs. res. Corom. 131:115-21 (1985) Klotz et al., Klotz et al., J. Biol Chem. 260:1465a-64(1985) , Stiles et al..J. Neuro chem. 47:1020-25 (1986) Stiles, G.L., J. Biol. Chem. 261:10839-43 (1986)). The A, receptor has also recently been purified to homogeneity from rat (Nakata, H. , J. Biol. Chem. 264:16545-51 (1989)) and bovine (Olah et a. , Arch. Biochem.

Biophys.283:440-46 (1990)) brains as well as from rat testes ((Nakata, H. , J.Biol.Chem. 285:671-77 (1990)) . These studies have indicated that the A, receptor protein from various tissues is a single subunit glycoprotein with a molecular mass of approximately 36,00 Da. Interestingly, using some purification procedures, the A, receptor appears to co-purify with a guanine nucleotide regulatory (G) protein ((Stiles, G.L.,J. Biol.Chem., 260:6728-32 (1985) & Mushi et al. , J. Biol.Chem. 264:14853-59 (1989)), which may couple this receptor to its signal transduction pathway.

Adenosine receptors are important from a clinical therapeutic viewpoint as drugs which interact with these receptors may be used to treat Alzheimer's disease, epilepsy and disorders of memory, cognition and affect. One problem with currently available adenosine agonist and antagonist drugs is that they have many side effects, like many other drugs which work through interacting with receptors, despite their clinical utility. These side effects are predominantly due to a lack of receptor specificity in that the drug in use interacts not only with adenosine receptors but other neurotrans itter receptors as well. A major goal of clinical neuropharmacology and the pharmaceutical industry is the development of more highly selective drugs with even greater efficacy that those currently in use. An impediment to this process is the low abundance of adenosine receptor protein available to study in neural tissue and the lack of suitable homogeneous model systems of the receptor with which to screen drugs against. A novel approach to the solution of this problem is to clone cDNAs encoding adenosine receptors, construct eukaryotic expression vectors containing these cDNAs and create a series of stably transfected mammalian cell lines which express functional adenosine receptors in high abundance. These cell lines, which express a homogeneous population of adenosine receptors, can be used by the pharmaceutical industry or others to screen drugs, and study adenosine receptors, using a variety of biochemical, physiological, and pharmacological techiniques.

A canine cDNA has been cloned (i.e., clone RDC7) which may represent a species homolog of the rat receptor A, cDNA of the present invention; however, the scientists carrying out this work did not disclose the identity of the protein encoded by their cDNA (Libert et al., Science 244:569-72 (1989) & Libert et al. , Nuc. Acids Res. 18:1916 (1990)).

All U.S. patents and publications referred to herein are hereby incorporated by reference. SUMMARY OF THE INVENTION

The subject invention relates to mammalian cell lines which express an adenosine receptor referred to as A,. The cell lines may be utilized to screen for drugs which affect the activity of the A, receptor. The cell lines are created by initially isolating a cDNA encoding the A, adenosine receptor subtype, inserting the cDNA into a eukaryotic expression vector and transfecting a host cell with the vector.

More specifically, the present invention relates to a substantially pure form of an A, adenosine receptor wherein the receptor has an amino acid sequence as shown in Figure 1. The invention also relates to antibodies produced against this receptor.

Furthermore, the present invention also relates to a DNA segment encoding the receptor described-above.

The DNA segment has a nucleotide sequence as shown in Figure 7. The present invention also encompasses a portion of the segment, as well as a DNA segment which encodes an amino acid sequence having the adenylyl cyclase inhibition properties, pharmacological properties, and G regulatory protein coupling properties of a receptor having the amino acid sequence shown in Figure l.

The invention further relates to a recombinant DNA molecule comprising: a) the DNA segment discussed above and b) a vector for introducing this DNA molecule into eukaryotic or prokaryotic host cells. The vector may be a eukaryotic expression vector such as pCD-SRα. The DNA segment which is part of the recombinant molecule may encode the amino acid sequence shown in Figure 1. In particular, the present invention relates to a host cell stably transformed or transfected with the recombinant DNA molecule discussed above in a manner allowing expression of the receptor encoded by said recombinant DNA molecule. The host cell may be a prokaryotic or eukaryotic cell. Mammalian cells are particularly useful for transformation. Suitable mammalian host cells include, for example, Chinese hamster ovary cells or a A9-L cells.

The present invention further relates to a method of producing an adenosine receptor protein comprising culture the above-transformed host cell, under conditions such that the DNA segment is expressed and the receptor thereby produced. The receptor is isolated in the last step of the method. The invention also includes a method of screening drugs that interact with the A, receptor comprising contacting the transformed host cell with a drug under conditions such that the function of the receptor is either activated or blocked, and detecting the presence or absence of A, receptor activity.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the deduced amino acid sequence of the rat A, receptor protein. Transmembrane spanning domains are defined on the basis of hydropathy analysis. A potential N- 1inked glycosylation site is indicated by CHO. The cDNA encoding the A, receptor has been inserted into a eukaryotic expression vector and used in the construction of various mammalian cell lines expressing this functional protein for the purpose of, for example. A, receptor drug development. These A, receptor-expressing cell lines can be used to investigate the affinities and efficacies of agonist and antagonist drugs with the A, receptor using various techniques such as radioligland binding and second messenger assays.

Figure 2 shows a [ ]DPCPX saturation binding experiment in transfected A9-L cell membranes.

A: Transfected A9-L cell membranes were prepared and incubated with the indicated concentrations of [Η]DPCPX as described in Example IV. Nonspecific binding was determined by including 1 μM labelled DPCPX in the assays and was subtracted from the total binding values to yield the specific binding data.

B: Scatchard transformation of the specific binding data from A. Average binding parameters from 3 separate experiments are given in Example IV.

Figure 3 represents [ ]DPCPX competition binding assays in transfected A9-L cell membranes. Membranes were prepared and incubated with 0.5 nM [ ]DPCPX and the indicated concentrations of drugs as described in Example IV. The data points respresent the mean +/- SD from two independent experiments. The computer-derived affinity constants are given in the text. A, Antagonist competition curves: DPCPX (■) , XAC ( ) , and theophylline (Δ) . B, Agonist competition curves: CCPA ( ) , R-PIA (Δ),NECA (■) , and S-PIA (Δ) . c, R-PIA was incubated in the absence (■) or presence p) of 100 μM Gpp(NH)p. Figure 4 represents adenylyl cyclase assays in transfected CHO cell membranes. Membranes were prepared and adenylyl cyclase assays performed as described in Example V. A, 10^"* M concentrations of each of the indicated agonists were examined for their ability to inhibit forskolin-stimulated adenylyl cyclase activity. B, R-PIA was tested alone (10^"6) or in

SUBSTITUTE SHEET combination with 10^"5 M concentrations of either DPCPX or XAC. The data represent the mean +/- SEM replicate values (n=4) from a single experiment which was replicated twice with similar results. Typical basal and forskolin-sti ulated adenylyl cyclase activities were 5.5 and 30 pmol cAMP/min/mg, respectively. None of the adenosine agonists affected the basal enzyme activity (data not shown) . Figure 5 represents northern blot analyses of the A, adenosine receptor mRNA in various tissues. 2 μg Poly(A)^* RNA samples were run on denaturing 1% formaldehyde-agarose gels, electrophoretically transferred to GeneScreen (NEN) nitrocellulose, hybridized with a cDNA- specific, [^MP]-labeled oligodeoxynucleotide probe, washed under high stringency and exposed to Kodak X-AR film as described in the Examples. RNA molecular size markers are indicated on the left. A, CNS tissues: mRNA of approximately 3.1 and 5.6 kb (arrows) in length were detected and located predominantly in the cortex (CTX) , cerebellum (CB) , and hippocampus (HIP) . Other tissues shown are total brain (TB) , olfactory bulb (OFB) , mesencephalon (MES) , retina (RET) , striatum (STR) and pituitary (PIT). B, Peripheral tissues: tissues indicated are heart (HT) , lung (LNG) , liver (LIV) , kidney (KID) , spleen (SPL) , stomach (STM) , intestine (INT) , uterus (UT) and testis (TST) .

Figure 6 represents in situ hybridization histochemical analysis of the distribution of mRNA for the A, adenosine receptor. Low-magnification autoradiographic analysis of the distribution of mRNA for this receptor in coronal (A,B) and sagittal (C) sections of the adult rat brain. Areas with specific mRNA expression are indicated: CB, cerebellum; CTX, cortex; HIP, hippocampus; MGN; medial geniculate nuclei; STR, striatu , TH, thalamus; and VT, ventral tegmental area. D, High magnification of the CA4 region of the hippocampus indicating specific localization to neuronal (N) nuclei as opposed to glial (G) nuclei.

Fi_gur_{es 1A}-1_J s_how the genetic sequence of the cDNA of the present invention. This cDNA encodes an A, receptor linked to the inhibition of adenylyl cyclase activity.

DETAILED DESCRIPTION OF THE INVENTION As noted above, the present invention relates to mammalian cell lines which express the A, receptor. These cell lines are homogeneous, can be grown in large quantities and are easily manipulated. Most importantly, such cells result in the production of large quantities of adenosine receptor A,. Thus, using the cell lines, one may readily screen drugs that activate or block the receptor and thus affect the production of adenylyl cyclase.

The A, receptor of the present invention is linked to the inhibition of adenylyl cyclase activity and is coupled with the guanine nucleotide binding regulatory protein. The receptor can have an amino acid sequence corresponding to the sequence shown in Figure 1 or can, of course, have the sequence of a molecule having substantially the same adenylyl cyclase activation properties, pharmacological properties and G regulatory protein coupling properties of the molecule corresponding to Figure l. The receptor protein may also have an amino acid sequence corresponding to any portion of the protein shown in Figure 1, allelic variations of

SUBSTITUTE SHEET this portion or allelic variations of the entire protein.

The receptor may be present in a substantially pure form. In other words, the receptor may be present in a form that is substantially free of proteins and nucleic acids with which it is normally associated. The receptor may be purified using methods commonly known in the receptor. Furthermore, the A, receptor may be used as an antigen, according to methods known in the art, in order to produce antibodies thereto. Such antibodies may be monoclonal as well as polyclonal.

As noted above, the present invention relates to cell lines which express the A, receptor. However, the invention also relates to genetic sequences, in particular, cDNA sequences, which encode the amino acid sequence given in Figure 1. More specifically. Figure 7 shows the sequence of a cDNA clone which encodes the A, receptor of the present invention. Thus, the present invention encompasses the genetic sequence shown in Figure 7 as well as allelic variations thereof or portions thereof. The present invention also relates to a recombinant construct or recombinant DNA molecule comprising the DNA segment or sequence shown in Figure 7 (or a portion thereof) and a vector for introducing the sequence into a host cell. The DNA sequence utilized may encode either the receptor shown in Figure 1 or a receptor having the adenylyl cyclase inhibiting properties, pharmacological, and G regulatory protein coupling properties of the A, receptor. The cDNA of the present invention has been inserted into a bacterial cell and deposited with the American Type Culture Collection in Rockville, Maryland. The accession number of the deposit is .

The vector which is used in creating the recombinant construct may either be a prokaryotic or eukaryotic expression vector. For example, a plasmid, bacteriophage, or virus (such as pCD- SRα) be used. The DNA sequence can be present in the vector operably linked to regulatory elements including, but not limited to, a promoter. The recombinant construct may be utilized to transform or transfect either prokaryotic or eukaryotic host cells.

In particular, the present invention relates to host cells which may be transformed with the recombinant construct discussed directly above. The host cell may be prokaryotic (e.g., a bacterial cell), lower eukaryotic (e.g., fungus, such as yeast cells), or higher eukaryotic (e.g., all mammalian cells, for example, rat or human cells) . Mammalian cells are preferred. For example, both Chinese hamster ovary cells (CHO) as well as A9-L cells may be stably transformed with the recombinant construct of the present invention. Transformation may be accomplished by any method commonly utilized in the art. The transformed cells may be used as a source for the DNA sequence shown in Figure 7 (or an allelic variation or portion thereof) . Of course, if the host cell is to be used for expression purposes, the transformed cell is used as a source for the A, receptor.

The A, receptor protein produced by the transformed cells may be detected in a sample, for example, a cell or tissue culture, by contacting the sample with an antibody which binds to the receptor. The detection of the presence or the absence of the resulting antibody-receptor complex may be achieved by methods known in the art. Moreover, the presence or absence of a DNA segment encoding the A, receptor protein may be detected in a sample, such as a cell or tissue culture, by contacting the sample with a DNA probe that is comprised of the DNA segment of interest. Utilizing methods which are known in the art and under conditions such that hybridization will occur, a complex can form which consists of the probe and the DNA segment from the sample. Again, detection of the presence or absence of the complex can be carried out using conventional methods.

The cell lines of the present invention can be used for many purposes related to the expression of the protein. In particular, such cell lines can be used in the study and elucidation of receptor proteins and the production thereof. The cell lines may also be used in a clinical setting in order to determine which drugs enhance or interfere with the activity of the receptor and thus affect the regulation of adenylyl cyclase. As noted above, activation of the receptor by an agonist will inhibit the production of cAMP whereas blockage of the receptor will prevent this effect. Thus, activation of the receptor by an agonist will inhibit adenylyl cyclase activity whereas inhibition or blockage of the receptor by an antagonist will increase adenylyl cyclase activity. (Activation of the receptor will also inhibit phosphatidylinositol turnover, inhibit Ca^2* mobilization and increase K^* channel activity.) Thus, the cell line of the present invention can be utilized to investigate the affinities and efficacies of A, agonist and antagonist drugs using several techniques, for example, radioligand binding (see Example IV) and second messenger assays. The activity of the drug-treated cell can be compared to a control cell in order to evaluate the activation or blockage of the receptor. It should also be noted that expression of the A, receptor cDNA can be measured for diagnostic purposes using conventional methods. This is carried out by utilizing antibodies to the receptor protein. Such antibodies are prepared by injecting all (or a portion) of the receptor protein into a mammal in order to elicit an immune response.

The present invention can be illustrated by the use of the following non-limiting examples: Example I

Amplification of cDNA Fragments and Characterization Thereof

PCR Amplification:

Poly (A)^* RNA was prepared from rat striatum and fractionated according to size by sucrose gradient centrifugation as previously described (Mahan et al., Proc. Natl. Acad. Sci. USA 87:2196-2200 (1990)). RNA of -3 kb was used as a template for first strand synthesis of cDNA. 1-2 μg of RNA was denatured at 65°C for 5 minutes and reverse transcription was performed at 39- 40°C in PCR buffer (GeneAmp, Perkin Elmer-Cetus) with AMV reverse transcriptase (Promega) and 1.2 μM of a 64-fold degenerate consensus primer to the sixth transmembrane region of G protein-coupled receptors described previously (Mahan et al. , Proc. Natl. Acad. Sci. USA 87: 2196-220 (1990)). Second strand synthesis and subsequent amplification was performed by the addition of 1.2 μM of a 256-fold degenerate consensus primer to the third transmembrane region (Libert et al.. 13 Supra 1989) and TAQ polymerase (Perkin Elmer- Cetus) . Primers contained Sal 1 and Hind III linker sites to facilitate subcloning. Thirty cycles of 93°C, 1.5 minutes (denaturation) ; 55°C, 2 minutes (annealing) and 72°C, 4 minutes (extension) were carried out with a final extension for 15 minutes at 72°C in 100 μl final volume. Products were analyzed and purified by electrophoresis in 1% LMP agarose (FMC Bioproducts) . Individual bands were excised, phenol extracted, ethanol precipitated, digested with Sal 1 and Hind III (New England Biolabs) , and subcloned into pGEM HZf(+) (Promega) followed by transformation into E. Coli strain EJM109. Plas id preparations were prepared for double-stranded DNA sequencing of individual inserts using SP6 and T7 primers as described below. cDNA Library Screening and DNA Seguencing:

1 x 10⁶ recombinants from a rat striatal cDNA library, constructed in the λZAP II vector (Stratagene) , were screened with a PCR fragment which was ³P-labeled via nick translation. Duplicate nitrocellulose filters were hybridized in 50% formamide, 0.75 M NaCl/0.075 M sodium citrate (5X SSC) , 5X

Denhardt's solution, 0.02 M NajHP0_, 0.25% SDS, 0.15 mg/ml salmon sperm DNA, and 4 x 10⁶ dpm/ml of ³²P-labelled probe for 24 hr at 37°C. High stringency washing of the filters was performed with IX SSC and 0.1% SDS at 65°C prior to autoradiography. A phage found to hybridize to the probe were subsequently plaque purified. In vivo excision and rescue of the nested pBluescript plasmids from the Λ ZAP II clones were performed using helper phage according to the Stratagene protocol. Nucleotide sequence analysis was performed using the Sanger dideoxy nucleotide chain termination method with Sequenase (US Biochemical Corp.) on denatured doubled-stranded plasmid templates. Primers were synthetic oligonucleotides which were either vector-specific or derived from prior sequence information.

Sequence analysis and comparisons were performed on GCG Sequence Analysis Software (Univ. of Wisconsin) and Genbank.

As can be observed from the above discussion, the polymerase chain reaction was used to amplify several DNA fragments which were then preliminarily characterized by DNA sequence analysis. One of these fragments was found to exhibit considerable (> 90%) sequence homology to the RDC7 cDNA clone which was recently isolated from a drug thyroid library and apparently encodes a G protein-linked receptor of unknown function (Libert et al., Science 244:569-72 (1989) and Libert et al., Nuc. Acids Res. 18:1916 (1990)). This PCR-generated cDNA fragment was subsequently used to screen a rat striatal cDNA library in order to isolate a full-length clone. One positive clone containing a cDNA insert of - 2.2kb was isolated and the complete nucleotide sequence determined. (See Figure 1.) The longest open reading frame in this cDNA codes for a 326 residue protein with a theoretical molecular weight of 36,692 Da.

Example II mRNA Analysis

Northern blot and in situ hybridization histochemical analyses were performed as previously described (Monsma et al. , Nature 342:926-29 (1989)). A 48-base oligodeoxynucleotide probe, 5'-

CCCGTAGTACTTCrTGGGGGT<_ΛCCGGAGGAGGCrGACACCTTTTTGTT- 15 3' , was synthesized from sequence specific to the putative third intracellular loop. This probe was radiolabelled using terminal deoxynucleotidlytransferase (Boehringer Mannheim) with either [α-"P]ATP (Northern blots) or [α- ^3SS]ATP (in situ hybridization) . Hybridization reactions were carried out on 2 μg samples of poly (A)^* RNA or serial 12 μm sagittal and coronal sections of adult rat brain. RNA markers (0.24- 9.5kb) were purchased from Bethesda Research Labs.

Example III Transfection of the Vector. Transfection of Eukaryotic Cells and Expression of the Receptor Protect! A full-length cDNA insert was subcloned into the pCD-SRα expression vector (Takebe et al., Molec. Cell. Biol. 8:466-72 (1988)) containing a modified polylinker. Competent DH5 cells were transformed and clones containing the appropriate cDNA insert were used for large-scale plasmid preparations via the CsCl₂ gradient purification method. DNA from the resulting plasmid construct (30 μg) , along with 3 μg of pMAMneo (Clontech) for a selectable marker, was used to transfect CHO and A9-L cells by the

CaPθ₄ precipitation technique (Chen et al., Malec. Cell Biol. 7:2745-52 (1987)). Cells were cultured in Dulbeccos Modified Eagle's Media containing high glucose (4,500 mg/1) , sodium pyruvate (lmM) , and 10% fetal bovine serum, in a humidified atmosphere of 5% C0₂ in air at 37^βC. Selection with the neomycin analog G-418 (500 μg/ml) was started 72 hr after transfection and continued for up to 2 weeks. Cell colonies exhibiting stable transfection of the pCD-SRα-cDNA construct were identi ied via dot-blot hybridization of cellular RNA as described above for Northern blot analysis. Cell culture media, reagents and fetal bovine serum were obtained from Gibco Laboratories (Grand Island, NY) . EXAMPLE IV

Radioligand Binding Assay Host cell membranes were prepared as follows:

Cells were detached from 150 cm² flasks with 1 mM EDTA in Ca^*/Mg^2* free Earle's balanced salt solution (EBSS) and were washed by centrifugation at 300 x g and resuspension with cold EBSS (complete) . The cells were then suspended in ice-cold lysis buffer (5 mM Tris HC1, pH 7.4, 5 mM MgClj and transferred to a Dounce homogenizer on ice and homogenized using 10 strokes with an A pestle. The homogenate was suspended in 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 5 mM KC1, 1.5 mM CaCl₂, 4 mM MgCl₂, 120 mM NaCl and centrifuged for 10 min at 43,500 x g. The crude membrane pellet was resuspended in 50 mM Tris- HCl, pH 7.2, lmM EDTA, 10 mM MgCl₂ containing 2 U/ml of adenosine deaminase (Sigma, St. Louis, MO) and incubated for 30 min at 30°C. The membrane homogenate was subsequently centrifuged for 10 min at 43,500 g and the resulting membrane pellet resuspended in the appropriate assay buffer. Protein concentrations were determined using the bicinchoninic acid (BCA) protein reagent (Pierce; Rockford, IL) as described in Smith et al, Anal Biochem. 150:76-85 (1985).

A, receptor binding assays using [³H]DPCPX (96 Ci/mmol, Amersham Corp.) were then performed as previously described ((Nakata, H., J. Biol. Chem. 264:16545-51 (1989) and Nakata, H., J. Biol. Chem. 265: 671-77 (1990)). Briefly, the membrane preparation was suspended with the appropriate ligands in 40 mM Tris-acetate, pH 7.5, 0.8 mM EDTA, and 4 mM MgCI₂ for a final assay concentration of 150 μM DPCPX. Incubations were carried out at 30°C for 1 hr, then terminated by rapid filtration under vacuum through Whatman GF/B filters pretreated with 0.3% polyethyleneimine. The filters were washed with 5 x 4 ml of ice-cold 50 mM Tris-HCl (pH 7.4) and the retained radioactivity was quantitated by liquid scintillation spectroscopy in 5 ml Aquasol

(National Diagnostics, Palmetto, FL) at a counting efficiency of 47%. All adenosine agonists and antagonists were purchased from Research Bioche icals (Natick, MA) . The data were analyzed with the program "LIGAND" ((Munson et al.. Anal Biochem. 107:220-23 (1980)) which performs weighted non-linear least squares curve fitting to the general model of Feldman ((Feldman, H.A. , Anal. Biochem. 48:317-38 (1972)) involving the interaction of several ligands with several independent classes of sites according to the law of mass action. Deviation of the observed points from the predicted values were weighted according to the reciprocal of the predicted variance. Competition curves were analyzed using models for competition of radioligand and competitor to one or two independent sites. Results from fits using a two site model were retained only when the two site model fit the data significantly better than a one site model as determined by the partial F test at a significance level of p<0.05.

The above radioligand binding competition assay, using adenosine antagonist (Figure 3A) and agonist (Figure 3B) ligands, allowed for the further identification and characterization of the receptor express in the transfected A9-L cells. Unlabeled DPCPX was the most potent (K,=0.85 nM) antagonist of [^H]DPCPX binding followed by XAC (K,=2.0 nM) , a potetn A,-selective antagonist. In contrast, the non-selective adenosine antagonist, theophylline, was more than three orders of magnitude less potent (K,=7.3 μM) . Such high affinity displacement observed with both DPCPX and XAC is suggestive of an A, receptor subtype. The Hill coefficients for all antagonist/[3H]DPCX competition curves exhibited Hill coefficients less than unity (ranging from 0.48 to 0.69) and were best described by the existence of one high (K„) and one low (K affinity agonist-receptor binding state (Fig. 3B) . The high affinity agonist state (RH) constituted the majority of receptor sites (67-78%) in all cases (Fig. 3B) . The two A,-selective agonists, CCPA (1^=0.71 nM, Iζ_= 97.5 nM;%R„=78) and R-PIA (1^=0.77 nM, K_L=61.2 nM;%R„=69), exhibited the highest affinity (Fig. 3B) . The [ ]DPCPX binding sites expressed in A9- L cells demonstrated approximately 15-20 fold stereoselectivity for the R-PIA entantiomer versus the S-PIA enantiomer (K„=0.77 nM, KL= 1.4 μM; %R„=67) . The adenosine agonist, NECA, was intermediate in potency (K_H=4.3ΠM, K,_=0.66 μM;%R„=74) relative to the PIA enantiomers (Fig. 3B) . This rank order of agonist potency:CCPA=R- PIA>NECA>S-PIA is characteristic of the A, adenosine receptor subtype in rat brain ((Nakata H., J. Biol. Chem. 264:1645-551 (1989)) and other tissues ((Stiles, G.L. Trends Pharmacol. 7:486-90 (1986) ) . Taken together, the radioligand binding data in Figs. 2 and 3 indicate that this rat striatal cDNA encodes an A, adenosine receptor protein. Linkage of the cloned A, adenosine receptor endogenous G proteins in the A9-L cells was initially investigated using the non- 19 hydrolyzable GTP analogue, Gpp(NH)p. Addition of

100 μM Gpp(NH)p to the R-PIA/[³H]DPCPX competition assay (Fig. 3B inset) caused a marked reduction in the number of high affinity agonist binding sites, 69% to 35%, with an approximate 10-fold shift in the IC₅₀ of the competition curve from -3 nM to -50 nM (n=2) . Furthermore, the presence of Gpp(NH)p increased the specific binding of [³H]DPCPX by about two-fold in these experiments (data not shown) , similar to that reported for rat brain membranes (Ramkumar et al., J. Pharmacol. EXP. Ther. 246:1194-1200 (1988)) and solubilized A, receptors from rat testis (Nakata, H. , J. Biol. Chem. 265:671-77(1990)). The cloned A, receptor expressed in A9-L cells thus appears to be functionally coupled in the membranes to an endogenous G protein.

EXAMPLE V Determination of cAMP production As A, receptors have been reported to be functionally linked to the inhibition of adenylyl cyclase activity ((Stiles et al., Trends Pharmacel. 7:486-90 (1986) & Williams, H. Ann. Rev. Pharmacol. Toxicol. 28:315-45 (1987)), the present inventors tested for this response in the transfected A9-L cell membranes. Preliminary experiments did indeed demonstrate some adenosine agonist inhibition of adenylyl cyclase activity, however, this was variable and confounded by the unexpected presence of an endogenous A₂ adenosine receptor linked to stimulation of cAMP production (data not shown) . Thus, the inventors turned to the CHO cells which had been transfected with the A, receptor cDNA and preliminarly characterized as expressing >1 pmol/mg protein specific [³H]DPCPX binding sites (data not shown) . The experiment was carried out as follows: 50μl of cell membranes (100 μg protein) suspended in AC buffer (75 mM Tris-HCl, pH 7.4, 250 mM sucrose, 12.5 mM MgCl₂, 1.5 mM EDTA) containing 1 mM DTT and 200 μM sodium metabisulfite and supplemented with 2.75 mM phosphoenolpyruvate, 53 μM GTP, 0.12 mM ATP, 1.0 U myokinase, 0.2 U pyruvate kinase and 100 μM RO- 20-1724 (a phosphodiesterase inhibitor, Biomol, Plymouth Meeting, PA) were added to tubes on ice containing 10 μl H₂0 or 10 μl of appropriate test compounds. The membranes were incubated for 5 min at 37°C to generate cAMP and the reaction was stopped by a 3 min incubation in boiling H₂0. The cAMP generated was assayed by the method of Brown et al. by incubation with cAMP binding protein, prepared from bovine adrenal gland, in the presence of [³H]cAMP (45 Ci/mmol, Amersham Corp., Arlington Heights, IL) at 4°C for 2-16 hr as previously described ((Brown et al., Biochem. J. 121:561-67 (1971) and Barton et al., Molec. Pharmacol. 38:581-41 (1990)). Following incubation with the cAMP binding protein, free [ jcA P was removed by treatment with charcoal/BSA solution, and the bound [^JcAMP remaining in the supernatant was quantitated by liquid scintillation spectroscopy. The cAMP concentrations produced in the assay were determined by comparison with a standard curve which was linear in the range of 1-30 pMoles cAMP per assay tube.

Fig. 4A shows the effect of various adenosine agonists on inhibiting adenylyl cyclase activity. When tested at 10 nM concentrations, the potent Al selective agonists, CCPA and R-PIA, both significantly inhibited forskolin cAMP production. NECA exhibited less of a response while S-PIA was relatively ineffective at 10 nM (Fig. 4A) . In contrast, 10 nM concentrations of the A₂-selective agonists, CGS21680 and CV1808, did not affect adenylyl cyclase activity (Fig. 4A) . No adenosine agonist inhibition of adenylyl cyclase activity (or the presence of specific [³H]DPCPX binding) was observed in untransfected CHO cells (data not shown) . Fig. 4B shows that the R-PIA inhibitory response could be completely blocked by the A,-selective anatagonists, DPCPX and XAC. This functional agonist and antagonist pharmacology (Fig. 4) agrees well with that observed for the radioligand binding analyses in Figs. 2 and 3. The cloned Al receptor thus appears to be capable of functional coupling to the inhibition of adenyl cyclase activity.

EXAMPLE VI

Tissue Distribution of mRNA Corresponding to A. Receptor cDNA

As a further characterization of the A, receptor cDNA, the present inventors investigated the tissue distribution of its corresponding mRNA. Northern blot analysis of a variety of rat brain tissue revealed two species of mRNA for this A, adenosine receptor; one -3.1 kb and a less prominent, higher molecular weight species of -5.6 kb (Fig. 5A) . Highest expression was observed in the cortex, cerebellum, and hippocampus. Olfactory bulb, esencephalon, and striatum also exhibited moderate levels of both messages, while the retina appeared to predominantly express the 5.6 kb species. No expression was apparent in the pituitary. The 3.1 kb species of mRNA appeared to be the predominant form in all peripheral rat tissues examined (Fig. 5B) . These transcripts were detectable in the spleen and stomach with some expression also being observed in the heart and testis. These data contrast somewhat with Northern blots performed on RDC-7 mRNA where a single transcript of 2.5 kb was observed in dog tissues ((Libert et. al. , Science 244:569072

(1989) & Ramkumar et al., J. Pharmacol. Exp. Ther. 246:1194-1200 (1988)). The origin of the two mRNAs in rat and the relationship of these to the 2.5 kb dog transcript will require further investigation.

In situ hybridization histochemical studies (Fig. 6A-D) confirmed the tissue distribution of mRNA expression seen with the Northern blot analyses. In addition, these studies revealed marked expression of A, receptor mRNA in thalamic nuclei, the medial geniculate nucleus and the ventral tegmental area (Fig. 6A- B) . High levels of expression observed in the cerebellum were confined predominantly to the granule cell layer (Fig. 6C) . In most cases, autoradiographic labeling was restricted to neuronal, not glial, nuclei (Fig. 6D) . The expression of A, receptor mRNA and A, receptor protein co-localized in a number of brain tissues. Highest levels of A, adenosine receptor binding sites (-200-600 fmol/mg protein) were found in cortex, cerebellum, hippocampus, striatum and thalamus utilizing [ΗlCH , an A, adenosine agonist, or ['H]DPCPX for binding to membrane preparations or in autoradiographic analyses (Breens et al, Naunyn-Schmiedeberg's Arch. Pharmacol. 335:59-63 (1987), Breens et al., "Adenosine Receptor Subtypes:Binding Studies," In Topics and Perspectives in Adenosine Research. Gerlack et al. (eds.), Springer-Verlag, pp. 59-73 (1987)). In addition, these anatomical findings are in agreement with preliminary information on RDC7 mRNA distribution (Libert et al., Supra (1989) & Scheffmann et al., Brain Res. 519:333-37 (1990)) .

Claims

WHAT IS CLAIMED IS:

1. A substantially pure form of an A, adenosine receptor wherein said receptor has an amino acid sequence as shown in Figure 1.

2. An antibody to the receptor of claim

3. A DNA segment encoding the receptor of claim 1.

4. The DNA segment of claim 3 wherein said segment has a nucleotide sequence as shown in

Figure 7.

5. A unique portion of said DNA segment of claim 4.

6. The DNA segment of claim 4 wherein said DNA segment encodes an amino acid sequence having the adenylyl cyclase inhibition properties, pharmacological properties, and G regulatory protein coupling properties of a receptor having the amino acid sequence shown in Figure 1.

7. A recombinant DNA molecule comprising: a) said DNA segment according to claim 3; and b) a vector for introducing said DNA molecule into eukaryotic or prokaryotic host cells.

8. The recombinant DNA molecule according to claim 7 wherein said vector is a eukaryotic expression vector.

9. The recombinant DNA molecule according to claim 8 wherein said vector is pCD- SR .

10. The recombinant DNA molecule of claim 7 wherein said DNA segment encodes the amino acid sequence shown in Figure 1.

11. A host cell stably transformed with the recombinant DNA molecule of claim 7 in a manner allowing expression of said receptor encoded by said recombinant DNA molecule.

12. The host cell of claim 11 wherein said host cell is a prokaryotic or eukaryotic cell.

13. The host cell of claim 12 wherein said host cell is a mammalian cell.

14. The host cell of claim 13 wherein said host cell is a Chinese hamster ovary cell or an A9-L cell.

15. A method of producing an adenosine receptor protein comprising culture said host cell of claim 11, under conditions such that said DNA segment is expressed and said receptor thereby produced, and isolating said receptor.

16. A method of screening drugs that interact with an A, receptor comprising contacting said host cell of claim 11 with a drug under conditions such that function of said receptor is either activated or blocked, and detecting the presence or absence of A, receptor activity.

17. A method of detecting the presence of A, receptor in a sample comprising contacting said sample with said antibody of claim 2, and detecting the presence or absence of a complex formed between said receptor and said antibody.

18. A method of detecting the presence or absence of a DNA segment encoding A, receptor in a sample comprising contacting said sample with a DNA probe comprising said DNA segment of claim 3 under conditions such that hybridization between said probe and said DNA segment of said sample occurs, and detecting the presence or absence of a complex formed between said probe and said DNA segment.