WO1999019488A1 - Novel human egf receptors and use thereof - Google Patents

Abstract

The present invention relates generally to epidermal growth factor receptors. More specifically, the present invention relates to novel isoforms of human epidermal growth factor receptor, particularly HER4/ErbB4, and uses thereof.

Description

NOVEL HUMAN EGF RECEPTORS AND USE THEREOF

The present invention was funded in part by grants from the United States Government and it has certain rights to the inventions described herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to generally to epidermal growth factor receptors. More specifically, the present invention relates to novel isoforms of human epidermal growth factor receptor, particularly HER4/ErbB4, and uses thereof.

2. Background

Numerous growth factors exert their biological effects by interacting with receptor tyrosine kinases (RTK) at the surface of the target cell. RTKs constitute a gene family of integral cell surface molecules that consist of functionally distinct domains ( 1). The N- terminal extracellular domain of a RTK interacts with specific ligands. A single transmembrane domain anchors the RTK to the cell membrane. An intracellular tyrosine kinase domain that is highly conserved among RTKs mediates ligand- dependent phosphorylation of tyrosine residues creating binding sites for SH2- and PTB-domain containing intracellular signaling molecules (2,3). Carboxy-terminal to the tyrosine kinase domain is a C-terminal tail whose length varies between different RTK subfamilies but that usually contains tyrosine residues that can be phosphorylated by the tyrosine kinase. Functional diversity is created by modifications of the RTK domain structure, for example, by alternative splicing of the RNA precursor molecules encoding the protein or by proteolytic processing of the mature protein product. Well characterized examples of alternative splicing of exons encoding RTKs are the isoforms of fibroblast growth factor receptor-2 (FGFR-2) in which sequences encoding the second half of the third immunoglobulin-like domain located at the proximal extracellular domain are responsible for a specific affinity for either FGF-2 or FGF-7 (4). Alternative splicing also results in the production of soluble FGFR- 1 or EGFR extracellular domains (5,6). There are numerous examples of release of RTK ectodomains by proteolytic processing including the colony stimulating factor- 1 receptor, Fms (7); the hepatocyte growth factor receptor, Met (8); the Kit ligand receptor, (9); an orphan receptor, Axl (10); and FGFR- 1 (11).

Human epidermal growth factor receptor 4 (HER4) is the most recently described member of the epidermal growth factor receptor (EGFR)-like subfamily of RTKs that consists of EGFR (HER1, ErbB l), HER2 (ErbB2, Neu), HER3 (ErbB3) and HER4 (ErbB4) (12-17). HER4 is a receptor for the neuregulins (NRG) (18), a group of alternatively spliced products of a single growth factor gene that include acetylcholine receptor inducing activity (ARIA), glial growth factor (GGF), heregulin (HRG) and Neu differentiation factor (NDF) ( 19-22). Two EGF-like growth factors, betacellulin (BTC) and heparin-binding EGF-like growth factor (HB-EGF) which, unlike NRGs, are ligands for EGFR (23,24), can also activate HER4 (25-28). Recently, a novel NRG-like gene, NRG-2, was identified and demonstrated to be a ligand for HER4 (29,30). Activation of HER4 in vitro leads to cellular proliferation, che o taxis or differentiation via activation of specific signal transduction cascades (27, 31-33). Expression of HER4 mRNA in several adult tissues, such as heart, kidney, brain and skeletal muscle (17) suggests that HER4 is involved in signaling necessary for the maintenance of a variety of mature organs. High HER4 expression levels in human breast cancer cell lines have further implicated HER4 as having a role in tumorigenesis (17). The biological significance for HER4 has been demonstrated in targeted null mice lacking HER4 (34). These homozygous HER4 knockout mice die at embryonic day 10- 1 1 and have cardiac and neural defects.

SUMMARY OF THE INVENTION

In the course of studying the interactions of HER4 with HB-EGF (27), and the role of HER4 signaling in the developing cerebellum (35), we sequenced two full length coding sequences of HER4 cDNA fragments isolated from cDNA libraries made independently from either a human MDA-MB-453 breast cancer cell line (17) or from human fetal brain tissue (33) and discovered structural differences in the two cDNAs. We have discovered that these two cDNA clones represent alternatively spliced isoforms that differ by insertion of either 23 (JM-a) or 13 (JM-b) alternative amino acids in the proximal extracellular domain just N- terminal to the transmembrane domain (juxtamembrane domain) . To date no alternatively spliced isoforms of HER4 have been described. The two isoforms are differentially expressed in mouse tissues, in particular in neural tissues, heart and kidney. When expressed in NIH 3T3 clone 7 cells, both isoforms could be activated by HB-EGF, NRG-αl , NRG-βl and BTC. On the other hand, a functional difference was observed in that pretreating the transfectants with a phorbol ester, phorbol 12-myristate 13-acetate (PMA), resulted in the loss of NRG-βl binding and a reduction in total cell-associated HER4 protein levels in HER4 JM-a, but not in HER4 JM-b-transfected cells. These results suggest that the JM-a, but not the JM-b, isoform can be cleaved in the juxtamembrane domain.

We have further identified another novel naturally occurring ErbB4 isoform, one that lacks amino acids 1046- 1061 when compared to the human ErbB4 sequence described originally (17). These 16 amino acids reside in the cytoplasmic tail of ErbB4 and include a Tyr-X-X-Met consensus sequence (residues 1056- 1059 in human ErbB4) that constitutes a consensus binding site for the p85 subunit of PI3-K (Songyang et al, 1993). Furthermore, we have demonstrated in cell culture, that the isoform lacking the p85 consensus binding site is functional in that it binds NRG- 1, is tyrosine phosphorylated in response to NRG- 1 , but has lost its capacity to bind p85 or to stimulate PI3-K activity. We refer to the novel isoform lacking the 16 amino acids as ErbB4 CYT-2 or HER4 CYT-2, and the isoform corresponding to the original ErbB4 sequence (17) as ErbB4 CYT- 1 or HER4 CYT-2. ErbB4 CYT-2 is expressed predominantly in neural tissues and kidney, while ErbB4 CYT- 1 is expressed predominantly in heart and breast. Taken together, these findings demonstrate the existence in vivo of two isoforms of ErbB4, which differ in their ability to activate specifically the PI3-K signal transduction pathway. We have also found that HER4 CYT- 1 and HER4 CYT-2 expression is regulated in a tissue-specific manner. Heart, breast and abdominal aorta expressed predominantly HER4 CYT- 1 whereas neural tissues and kidney expressed predominantly HER4 CYT- 2.

The present invention provides DNA segments encoding receptor proteins related to HER4 which previously has not been know or even suspected to exist.

The present invention also provides an isolated DNA encoding a protein comprising the amino acids of SEQ ID NO: l, as well as an isolated protein comprising the amino acids of SEQ ID NO: 1. Preferred proteins are novel HER4 isoforms including a HER4 isoform represented by the amino acid sequence of SEQ ID NO:3. Antibodies directed to SEQ ID NO: 1, or a protein comprising SEQ ID NO: 1, are also included. A DNA segment comprising the nucleotides of SEQ ID NOS: 2 is further provided.

The invention further provides an isolated DNA encoding a HER receptor having the amino acids of SEQ ID NO:4 deleted. Preferably, the HER receptor having amino acids of SEQ ID NO:4 deleted is represented by the amino acid sequence of SEQ ID NO: 6. A HER4 receptor is preferred.

As used herein the terms "ErbB4 receptor" "ErbB4 isoform" or "receptor" refers to a protein comprising the amino acids of SEQ ID NO: 1 or 3, or a HER receptor having the amino acids of SEQ ID NO:4 deleted (e.g., a receptor represented by SEQ ID NO:6).

The present invention further provides assays for expression of the RNA and protein products of the DNA of the present invention to enable determining whether abnormal expression of such DNA is involved with a particular disease, e.g., cancer, neural, cardiac or neuromuscular diseases.

The present invention also provides antibodies, either polyclonal or monoclonal, specific to a unique portion of the receptor protein; a method for detecting the presence of a receptor ligand that is capable of either activating or down- regulating, i.e., modulating, the receptor protein; a method of screen potential ligand analogs for their ability to modulate the receptor protein; and procedures for targeting a theraputic drug to cells having a high level of the receptor protein.

The present invention also provides binding assays that permit the ready screening for molecules that affect the binding of the receptors and their ligands.

The present invention further provides use of the receptors for intracellular or extracellular targets to affect binding. Intracellular targeting can be accomplished through the use of intracellularly expressed antibodies referred to as intrabodies. Extracellular targeting can be accomplished through the use of receptor specific antibodies. Additionally, the soluble form of the antibody can be used as a receptor decoy to inhibit binding. The present invention also provides an assay to determine the presence or absence of the receptors that can be used as a diagnostic /prognostic tool to identify the presence or stage of differentiation of tissue, e.g., tumor tissue.

Other aspects of the invention are disclosed infra.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows alternative HER4 juxtamembrane isoforms. A schematic diagram of HER4 with the deduced amino acid sequences of two alternative juxtamembrane domains (JM-a (SEQ ID NO:7) and JM-b (SEQ ID NO: l)) is shown. The alternative sequences are in boldface. The two contiguous amino acids that are at either end of the alternating juxtamembrane sequences are shown to help localize the juxtamembrane domains within the published sequence ( 17). Cys, cysteine rich domains; TM, transmembrane domain; TK, tyrosine kinase domain.

Figure 2 shows RT-PCR analysis of the distribution of HER4 juxtamembrane isoforms in mouse tissues. Total RNA was isolated from several mouse tissues, subjected to reverse transcription in the presence of random primers and amplified with primers flanking the mouse HER4 juxtamembrane domain (upper panel) or primers specific for mouse β- actin (lower panel) . PCR products separated on 2% agarose gels are shown. The expected size for the amplified HER4 JM-a isoform is 273 bp and for the amplified HER4 JM-b isoform 243 bp. A negative control (lane 22) shows the PCR product in the absence of a cDNA template.

Figure 3 illustrates a comparison of human and mouse HER4 juxtamembrane isoform sequences. Both nucleotide sequences and the deduced amino acid sequences ((JM-a SEQ ID NO:9; JM-b SEQ ID NO: l 1) are shown. A dot in the mouse sequences demonstrates an identical nucleotide or amino acid when compared to the respective human sequence. Sequences specific for the alternative juxtamembrane forms are shown in boldface. Number 624 is the position of the Gly residue in the published human HER4 sequence ( 17) and 33 is the position of the Gly residue in the partial mouse HER4 sequence determined in this report. The mouse HER4 JM-a and JM-b domains were sequenced from RT-PCR products obtained from kidney and heart RNA, respectively. The human HER4 JM-a and JM-b domains were sequenced from cH4M2 and pEV7-HER4 expression plasmids, respectively. An identical human HER4 JM-b sequence was also obtained from a heart tissue RT-PCR product.

Figures 4A-4C show oligo in situ hybridization of mouse cerebellum with HER4 JM-a and HER JM-b specific probes. Adjacent coronal sections of adult mouse cerebellum were hybridized with specific antisense oligonucleotide probes directed against HER4 JM-a (4A) or

HER4 JM-b (4B). The autoradiographic grains were visualized with dark field illumination. Figure 4C shows the same section as in Figure 4B stained with hematoxylin and visualized with bright field illumination. WM, white matter; GCL, granule cell layer; ML, molecular layer. Bar = 300 μm.

Figures 5A and 5B show tyrosine phosphorylation of HER4 juxtamembrane isoforms. (5A) The HER4 protein levels of an NIH 3T3 clone transfected with pMAMneo resistance gene plasmid alone (lane 1 ; ctl), or together with plasmids encoding HER4 JM-a (lane 2; clone #2) or HER4 JM-b (lane 3; clone #42), were determined by a combination of immunoprecipitation and Western blotting using HER4-specific antibodies. (5B) NIH 3T3 cells expressing no HER4 (top panel) or similar amounts of HER4 JM-a (middle panel; JM-a clone #2) or HER4 JM-b (bottom panel; JM-b clone #42) were starved in serum-free medium for 24 h and stimulated without (lane 1) or with 100 ng/ml of HB-EGF (lane 2), NRG-αl (lane 3), NRG-βl (lane 4) or BTC (lane 5). HER4-specific phosphorylation was measured by anti-phosphotyrosine Western blotting after immunoprecipitation with an anti-HER4 antibody. Arrows point to 180 kD HER4 bands.

Figures 6A-6C show the effect of PMA on 125I-NRG-βl binding to cells transfected with HER4 JM isoforms. (6A) Confluent 6-well plate wells of NIH 3T3 cells expressing HER4 JM-a (clones #2 and #102) or HER4 JM-b (clones # 15 and #42) were pretreated for 45 min with 100 ng/ml PMA and then incubated with 20 ng/ml ¹²5I-NRG-βl . After washing, the amount of bound ¹²⁵I-NRG-βl was measured with a γ- counter. The amount of radioactivity bound to control transfected cells not expressing HER4 was subtracted. (6B) Confluent 12-well plate wells of NIH 3T3 cells expressing HER4 JM-a (clone # 102) or HER4 JM-b (clone # 15) were pretreated for the time periods indicated with 100 ng/ml PMA and ¹²⁵I-NRG-βl binding was measured as in panel A. (6C) Control transfectants (lanes 1 and 2), cells expressing HER4 JM-a (lanes 3 and 4; clone #2) and cells expressing HER4 JM-b (lanes 5 and 6; clone #15) were treated with or without 100 ng/ml PMA for 45 min. Total cellular HER4 protein levels were determined by immunoprecipitation and Western blotting with HER4-specific antibodies. The arrow points to a 180 kD HER4 band.

Figure 7 shows the effect of PMA on HER4 cell surface immunoreactivity. NIH 3T3 cells transfected with an antibiotic resistance gene encoding plasmid alone (Control), or together with plasmids encoding HER4 JM-a (clone #2; JM-a) or HER4 JM-b (clone #15; JM-b) were treated with 0 ng/ml (top panels) or 100 ng/ml (lower panels) PMA for 45 min. The cells were fixed and the expression of cell surface HER4 was detected using a monoclonal antibody directed against the extracellular domain. Bar = 50 μm.

Figure 8 shows RT-PCR analysis with ErbB4-specific primers designed corresponding to sequences flanking the PI3-K binding site. Total RNA was isolated from human heart (lane 1), human kidney (lane 2), mouse heart (lane 3) and mouse kidney (lane 4) and subjected to RT- PCR analysis. The PCR products were separated on a 2% agarose gel and visualized under ultraviolet light after staining with ethidium bromide. A 1 Kb ladder was used as a size marker. The arrows point to the 2 bands that were cloned, solid arrows for the middle bands and open arrows for the lower bands.

Figures 9A and 9B show the nucleotide (SEQ ID NO: 13, mouse SEQ ID NO: 14) and deduced amino acid sequences (SEQ ID NO: 12) of the ErbB4 cytoplasmic isoforms. (9A) The RT-PCR products obtained using primers flanking the PI3-K binding site derived from the human kidney and mouse heart samples in Figure 8 (two lower bands in each species) were cloned into a pCR3.1 vector and the inserts were sequenced. The 48 nucleotide (16 amino acid) insert including the PI3-K binding site present in ErbB4 CYT- 1 , but not in ErbB4 CYT-2, is shown in boldface, and two amino acids (NR and NQ) flanking either end are shown in plain font to put the deletion into context. The numbers 1046 and 1061 refer to the positions within the published human ErbB4 sequence (Plowman et al, 1993) of the first (S) and last amino acid residues (G) which are missing in CYT-2. The dots within the mouse sequences indicate nucleotides identical to the human sequences. (9B) A schematic diagram of ErbB4 CYT- 1 and ErbB4 CYT-2 isoforms. The amino acid sequence present in the CYT- 1 isoform, but not in the CYT-2 isoform, is shown in boldface. The horizontal dashes within the CYT-2 sequence indicate the sites of the missing amino acids when compared to the CYT- 1 sequence. The binding sequence for the p85 subunit of PI3-K, Tyr-Thr-Pro-Met, is underlined. Cys, cysteine rich domain; TM, transmembrane domain; TK, tyrosine kinase domain.

Figure 10 shows RT-PCR analysis of the distribution of ErbB4 CYT isoforms in mouse tissues. Total RNA was isolated and subjected to RT- PCR analysis with mouse ErbB4-specific primers designed corresponding to cDNA sequences flanking the PI3-K binding site. As an internal standard, all the templates were also analyzed by PCR using primers specific for mouse β-actin (bottom panel). Lane 22 (negative control) shows a PCR reactions in the absence of templates.

Figure 1 1 illustrates ErbB4 protein levels in cells expressing ErbB4 cytoplasmic isoforms. Lysates of a control clone transfected with a plasmid encoding a neomycin resistance gene alone (lane 1), of two independent clones (bl .42 and b l .amg) co-transfected with a neomycin resistance gene plasmid and an expression plasmid for ErbB4 CYT- 1 (lanes 2 and 3), and of two independent clones (B2.1 1 and b2.15) cotransfected a neomycin resistance gene plasmid and an expression plasmid for ErbB4 CYT-2 (lanes 4 and 5) were prepared. Protein levels in cell lysates were analyzed by a combination of immunoprecipitation and Western blotting, using anti-ErbB4-specific antibodies. The samples were separated on a 6% SDS-PAGE and visualized by ECL. An arrow points to the position of ErbB4.

Figure 12 shows cross-linking of 125^T_NRQ_ I to cells expressing ErbB4 cytoplasmic isoforms. Control cells (lane 1), cells expressing

ErbB4 CYT-1 (lanes 2 and 3) and cells expressing ErbB4 CYT-2 (lanes 4 and 5) were cross-linked with DSS in the presence of 125^T_NRG- 1. The cells were lysed and the cross-linked complexes were separated on a 6% SDS-PAGE and visualized by autoradiography. An arrow points to the 190 kD 125ι_NRG- l/ErbB4 complex.

Figure 13 shows tyrosine phosphorylation of ErbB4 cytoplasmic isoforms. Control cells (lanes 1 and 2), cells expressing ErbB4 CYT- 1 (lanes 3-6) or cells expressing ErbB4 CYT-2 (lanes 7- 10) were stimulated without (lanes 1, 3, 5, 7 and 9) or with (lanes 2, 4, 6, 8 and 10) 100 ng/ml NRG- 1. The cells were lysed and the lysates were immunoprecipitated with an anti-ErbB4 antibody. The precipitated material was separated on a 6% SDS-PAGE and the tyrosine phosphorylated proteins were visualized by Western blotting with an antiphosphotyrosine antibody followed by ECL. An arrow points to the position of ErbB4.

Figures 14A and 14B show the association of PI3-K with ErbB4 cytoplasmic isoforms. ( 14A) Co-precipitation of ErbB4 with p85. Control cells (lanes 1 and 2), cells expressing ErbB4 CYT- 1 (bl . amg, lanes 3 and

4) or cells expressing ErbB4 CYT-2 (b2.15, lanes 5 and 6) were stimulated without (lanes 1 , 3 and 5) or with (lanes 2, 4 and 6) 100 ng/ml NRG- 1. Cells were lysed and the lysates were immunoprecipitated with an anti-p85 antibody. The precipitated material was separated on a 6% SDS-PAGE and analyzed by Western blotting with an anti-ErbB4 antibody followed by ECL. An arrow points to the position of ErbB4. (14B) Association of PI3-K activity with ErbB4. Control cells (lanes 1 and 2), cells expressing ErbB4 CYT- 1 (lanes 3, 4 and 7) or cells expressing ErbB4 CYT-2 (lanes 5, 6 and 8) were stimulated without (lanes 1, 3 and

5) or with (lanes 2, 4 and 6-8) NRG-1. Cells were lysed and lysates were imunoprecipitated with an anti-ErbB4 antibody (lanes 1-6) or anti-p85 antibody (lanes 7 and 8). The precipitated material was analyzed for PI3- K activity in vitro by visualizing the incorporation of 32p _mt_o Pl32p wi h TLC and autoradiography. The intensity of Pl32p signals was quantitated by densitometry using the NIH Image program. Both the TLC signals and densitometric results are shown. Similar results were obtained when the independent clones bl.42 and b2.1 1 were analyzed.

Figures 15A and 15B show activation of PI3-K by ErbB4 cytoplasmic isoforms. (15A) Tyrosine phosphorylation of p85. Control cells (lanes 1 and 2), cells expressing ErbB4 CYT- 1 (bl . amg, lanes 3 and 4) or cells expressing ErbB4 CYT-2 (b2.15, lanes 5 and 6) were stimulated without (lanes 1 , 3 and 5) or with (lanes 2, 4 and 6) 100 ng/ml NRG- 1. The cells were lysed and the lysates were immunoprecipitated with an anti-p85 antibody. The precipitated material was separated on a 6% SDS-PAGE and the tyrosine phosphorylated proteins were visualized by Western blotting with an antiphosphotyrosine antibody followed by ECL. Arrows points to positions of the 85 kD p85 band and the 180 kD ErbB4 band. (15B) Association of PI3-K activity with phosphotyrosine. Control cells (lanes 1 and 2), cells expressing ErbB4 CYT- 1 (lanes 3, 4 and 7) or cells expressing ErbB4 CYT-2 (lanes 5, 6 and 8) were stimulated without (lanes 1 , 3 and 5) or with (lanes 2, 4 and 6-8) NRG- 1. Cells were lysed and lysates were imunoprecipitated with an antiphosphotyrosine antibody (lanes 1-6) or anti-p85 antibody (lanes 7 and 8). The precipitated material was then analyzed for PI3-K activity in vitro by visualizing the incorporation of 32p in o Pl32p with TLC and autoradiography. The intensity of Pl32p signals was quantitated by densitometry using the NIH Image program. Both the TLC signals and densitometric results are shown. Similar results were obtained when the independent clones bl .42 and b2.1 1 were analyzed.

DETAILED DESCRIPTION OF THE INVENTION

HER4 is a major receptor for a class of molecules called "neuregulins." These molecules are involved in motor function and neuromuscular diseases. We have discovered isoforms of ErbB4 (HER4) that differ by alternatively spliced exons in the extracellular juxtamembrane region (ErbB4 JM-a and ErbB4 JM-b) or by the presence or absense of a binding site for an intracellular signal transduction molecule, phosphatidyl inositol 3-kinase (ErbB4 CYT- 1 and ErB4 CYT-2). While not wishing to be bound by theory, it is believed that the presence of the receptors of the present invention is associated with neural, neuromuscular or cardiac disease.

As noted above, the receptor CYT-2 lacks the binding site for PI3 kinase (SEQ ID NO:4), and it is believed that cell possessing such a receptor are more susceptable to apoptosis and have less motility than normal cells. Thus, transfer of DNA encoding CYT-2 to tumor cell can be used to inhibit cell motility and increase apotosis of such cells. The receptors of the present invention may also be used diagnostically. The ligand for HER4 is made in the uterus at the time of blastula implantation. Binding of HPGF to HER4 is an important adhesion step in getting the blastula implanted into the uterine wall. It is believed that the presence of the receptor of the present invention can lead to failure of implantation of the blastula. In the event of spontaneous abortion, the blastula can be analyzed for the presence or absence of the receptors of the present invention.

Determining the level of these receptors in individuals can be an important tool in determining whether an individual is at a greater risk for diseases such as neural and neuromuscular diseases. This knowledge can be used in determining the type of treatment for that individual.

The determination of the number of receptors present on the cells of an individual can readily be accomplished by standard means, for example, using FACS analysis or analysis of RNA levels. The level can be compared to a reference level, which can be determined by standard means. These assays are further discussed below.

Another preferred embodiment of this invention is in the diagnosis of diseases associated with these receptors. The receptors, nucleotide sequences encoding receptors and antibodies that bind to receptors can be particularly useful for diagnosis of cardiac, neural or neuromuscular diseases.

Using any suitable technique known in the art, such as Northern blotting, quantitative PCR, reverse transcriptase PCR, etc. the nucleotide sequences of the receptors or fragments thereof can be used to measure levels of receptor RNA expression. Alternatively, the antibodies of the invention can be used in standard techniques such as Western blotting to detect the presence of cells expressing receptors and using standard techniques, e.g. FACS or ELISA, to quantify the level of expression.

One can treat diseases associated with the expression of the receptors of the present invention by blocking receptor /ligand interaction. This can be accomplished by a range of different approaches. For example, antibodies, decoys, small molecules, antagonists, etc. One preferred approach is the use of antibodies to these receptors. Antibodies specifically binding to SEQ ID NO: l are preferred. Antibodies to these receptors can be prepared by standard means. For example, one can use single chain antibodies to target these receptors.

An alternative strategy is to use receptor decoys. For example, one could prepare a decoy comprising the portion of these receptors present on the exterior of the cell membrane. Another strategy is to prepare soluble forms of these receptors. This can be done by standard means including using PCR to clone a gene, site- directed mutagenesis to make changes in the structure, deletions to make fragments, etc. as discussed below.

Compounds that affect this receptor/ ligand interaction can be directly screened for example using a direct binding assay. For example, the compound of interest can be added before or after the addition of the labeled ligand and the effect of the compound on binding can be determined by comparing the degree of binding in that situation against a base line standard with that ligand, not in the presence of the compound. The binding assay can be adapted depending upon precisely what is being tested. The DNA segments according to this invention are useful for detection of expression of the receptors in tissues, as described in the Examples below. Therefore, in yet another aspect, the present invention relates to a bioassay for determining the amount of receptor mRNA in a biological sample comprising the steps of: i) contacting that biological sample with a nucleic acid isolate consisting essentially of a nucleotide sequence that encodes the receptor or a unique portion thereof, e.g., SEQ ID. NO:2 or a DNA encoding HER4 lacking the SEQ ID NO:5 under conditions such that a nucleic acid:RNA hybrid molecule, such as a DNA: RNA hybrid molecule, can be formed; and ii) determining the amount of hybrid molecule present, the amount of hybrid molecule indicating the amount of receptor mRNA in the sample. Findings described in the Examples, below, indicate that increased expression of the receptors of the present invention, as detected by this method of this invention, may play a role in some human malignancies, as is the case for the EGFR (erbB) and erbB-2 genes, as well as cardiac, neural or neuromuscular diseases.

Of course, it will be understood by one skilled in the art of genetic engineering that in relation to production of polypeptide products, the present invention also includes DNA segments having DNA sequences other than those in the present examples that also encode the amino acid sequence of the polypeptide product of the receptor gene. For example, it is known that by reference to the universal genetic code, standard genetic engineering methods can be used to produce synthetic DNA segments having various sequences that encode any given amino acid sequence. Such synthetic DNA segments encoding at least a portion of the amino acid sequence of the polypeptide product of the receptor gene also fall within the scope of the present invention. Further, it is known that different individuals may have slightly different DNA sequences for any given human gene and, in some cases, such mutant or variant genes encode polypeptide products having amino acid sequences which differ among individuals without affecting the essential function of the polypeptide product. Still further, it is also known that many amino acid substitutions can be made in a polypeptide product by genetic engineering methods without affecting the essential function of that polypeptide. Accordingly, the present invention further relates to a DNA segment having a nucleotide sequence that encodes an amino acid sequence differing in at least one amino acid from the amino acid sequence of receptor, or a unique portion thereof, and having greater overall similarity to the amino acid sequence of the receptor than to that of any other polypeptide. The amino acid sequence of this DNA segment includes at least about 4 to 6 amino acids which are sufficient to provide a binding site for an antibody specific for the portion of a polypeptide containing this sequence.

The present invention further relates to a recombinant DNA molecule comprising a DNA segment of this invention and a vector. In yet another aspect, the present invention relates to a culture of cells transformed with a DNA segment according to this invention. These host cells transformed with DNAs of the invention include both higher eukaryotes, including animal, plant and insect cells, and lower eukaryotes, such as yeast cells, as well as prokaryotic hosts including bacterial cells such as those of E. coli and Bacillus subtills.

Various standard recombinant systems, such as those cited above as well as others known in the art, are suitable as well for production of large amounts of the novel receptor proteins using methods of isolation for receptor proteins that are well known in the art. Therefore, the present invention also encompasses an isolated polypeptide having at least a portion of the amino acid sequence of SEQ ID NO: 1, 3 or 6.

The isolated nucleotide sequences and isolated polypeptides of the invention encoding receptors can be mutagenized by any of several standard methods including treatment with hydroxylamine, passage through mutagenic bacterial strains, etc. The mutagenized sequences can then be classified "wild type" or "non-wild type" depending whether it will still facilitate infectivity or not.

Mutagenized sequences can contain point mutations, deletions, substitutions, rearrangements etc. Mutagenized sequences can be used to define the cellular function of different regions of the receptors they encode. This information can be used to assist in the design of small molecules or peptides mimicking the interactive part of the receptor.

Another approach is to use small molecules that will selectively bind to one of the receptors. Such molecules and peptides can be synthesized by known techniques.

Another strategy is to express antibodies to these receptors in individuals intracellularly. This can be done by the method of Marasco and Haseltine set forth in WO94-02610 (PCT/US93/06735 filed July 16, 1993) published February 3, 1994.

In addition, additional compounds that bind to these receptors can readily be screened for. For example, one can select cells expressing high numbers of these receptors, plate them; e.g. add labeled ligand and screen for compounds or combinations of compounds that will interact with, e.g. binding of, these receptors by standard techniques. Alternatively, one can use known techniques to prepare cells that will express these receptors and use those cells in drug screens.

One can also prepare cell lines stably expressing the receptors. Such cells can be used for a variety of purposes including an excellent source of antigen for preparing a range of antibodies using techniques well known in the art. Therapeutic and Pharmaceutic Compositions

An exemplary pharmaceutical composition is a therapeutically effective amount of a decoy, antibody etc. that affects the ability of the receptor to bind ligand optionally included in a pharmaceutically- acceptable and compatible carrier. The term "ph,armaceutically- acceptable and compatible carrier" as used herein, and described more fully below, includes (i) one or more compatible solid or liquid filler diluents or encapsulating substances that are suitable for administration to a human or other animal, and/ or (ii) a system, such as a retroviral vector, capable of delivering the molecule to a target cell. In the present invention, the term "carrier" thus denotes an organic or inorganic ingredient, natural or synthetic, with which the molecules of the invention are combined to facilitate application. The term "therapeutically-effective amount" is that amount of the present pharmaceutical compositions which produces a desired result or exerts a desired influence on the particular condition being treated. Various concentrations may be used in preparing compositions incorporating the same ingredient to provide for variations in the age of the patient to be treated, the severity of the condition, the duration of the treatment and the mode of administration.

The term "compatible", as used herein, means that the components of the pharmaceutical compositions are capable of being commingled with a small molecule, nucleic acid and/ or polypeptides of the present invention, and with each other, in a manner such that does not substantially impair the desired pharmaceutical efficacy.

Dose of the pharmaceutical compositions of the invention will vary depending on the subject and upon particular route of administration used. Dosages can range from 0.1 to 100,000 μg/kg per day, more preferably 1 to 10,000 μg/kg. By way of an example only, an overall dose range of from about, for example, 1 microgram to about 300 micrograms might be used for human use. This dose can be delivered at periodic intervals based upon the composition. For example on at least two separate occasions, preferably spaced apart by about 4 weeks. Other compounds might be administered daily. Pharmaceutical compositions of the present invention can also be administered to a subject according to a variety of other, well- characterized protocols. For example, certain currently accepted immunization regimens can include the following: (i) administration times are a first dose at elected date; a second dose at 1 month after first dose; and a third dose at 5 months after second dose. See Product Information, Physician's Desk Reference, Merck Sharp & Dohme ( 1990), at 1442-43. (e.g., Hepatitis B Vaccine-type protocol); (ii) Recommended administration for children is first dose at elected date (at age 6 weeks old or older); a second dose at 4-8 weeks after first dose; a third dose at 4-8 weeks after second dose; a fourth dose at 6- 12 months after third dose; a fifth dose at age 4-6 years old; and additional boosters every 10 years after last dose. See Product Information, Physician's Desk Reference, Merck Sharp 86 Dohme (1990), at 879 (e.g., Diptheria, Tetanus and Pertussis-type vaccine protocols). Desired time intervals for delivery of multiple doses of a particular composition can be determined by one of ordinary skill in the art employing no more than routine experimentation.

The small molecules and polypeptides of the invention may also be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of this invention. Such pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene-sulfonic, tartaric, citric, meth.anesulphonic, formic, malonic, succinic, naphthalene-2- sulfonic, and benzenesulphonic. Also, pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group. Thus, the present invention also provides pharmaceutical compositions, for medical use, which comprise nucleic acid and/or polypeptides of the invention together with one or more pharmaceutically acceptable carriers thereof and optionally any other therapeutic ingredients.

The compositions include those suitable for oral, rectal, intravaginal, topical, nasal, ophthalmic or parenteral administration, all of which may be used as routes of administration using the materials of the present invention. Other suitable routes of administration include intrathecal administration directly into spinal fluid (CSF), direct injection onto an arterial surface and intraparenchymal injection directly into targeted areas of an organ. Compositions suitable for parenteral administration are preferred. The term "parenteral" includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.

The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Methods typically include the step of bringing the active ingredients of the invention into association with a carrier which constitutes one or more accessory ingredients.

Compositions of the present invention suitable for oral aα ninistration may be presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the nucleic acid and/ or polypeptide of the invention in liposomes or as a suspension in an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, or an emulsion.

Preferred compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the molecule of the invention which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenteraUy- acceptable diluent or solvent, for example as a solution in 1,3 -butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectibles.

Antibodies

The term "antibodies" is meant to include monoclonal antibodies, polyclonal antibodies and antibodies prepared by recombinant nucleic acid techniques that are selectively reactive with polypeptides encoded by eukaryotic nucleotide sequences of the present invention. The term "selectively reactive" refers to those antibodies that react with one or more antigenic determinants of the receptors and do not react with other polypeptides. Antigenic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics. Antibodies can be used for diagnostic applications or for research purposes.

For example, antibodies may be raised against ammo-terminal (N- terminal) or carboxyl-terminal (C-terminal) peptides of a polypeptide encoded by the receptors. A preferred region is that of SEQ ID NO: 1.

One approach is to isolate a peptide sequence that contains an antigenic determinant for use as an immunogen. This peptide immunogen can be attached to a carrier to enhance the immunogenic response. Although the peptide immunogen can correspond to any portion of a polypeptide encoded by a eukaryotic nucleotide sequence of the invention, certain amino acid sequences are more likely than others to provoke an immediate response, for example, an amino acid sequence including the N- or C-terminus of a polypeptide encoded by a gene that contains nucleotide sequences of the invention. Preferably one can use a cell line expressing only the receptor, select those cells with the highest levels of expression and use the whole cell as an antigen.

For example, cDNA clone encoding a receptor or a fragment thereof may be expressed in a host using standard techniques (see above; see Sambrook et al., Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York: 1989) such that 5-20% of the total protein that can be recovered from the host is the desired protein. Recovered proteins can be electrophoresed using PAGE and the appropriate protein band can be cut out of the gel. The desired protein sample can then be eluted from the gel slice and prepared for immunization. Alternatively, a protein of interest can be purified by using conventional methods such as, for example, ion exchange hydrophobic, size exclusion, or affinity chromatography.

Once the protein immunogen is prepared, mice can be immunized twice intraperitoneally with approximately 50 micrograms of protein immunogen per mouse. Sera from such immunized mice can be tested for antibody activity by immunohistology or i munocytology on any host system expressing such polypeptide and by ELISA with the expressed polypeptide. For immunohistology, active antibodies of the present invention can be identified using a biotin-conjugated anti-mouse immunoglobulin followed by avidin-peroxidase and a chromogenic peroxidase substrate. Preparations of such reagents are commercially available; for example, from Zymad Corp., San Francisco, California. Mice whose sera contain detectable active antibodies according to the invention can be sacrificed three days later and their spleens removed for fusion and hybridoma production. Positive supernatants of such hybridomas can be identified using the assays described above and by, for example, Western blot analysis.

To further improve the likelihood of producing an antibody as provided by the invention, the amino acid sequence of polypeptides encoded by a eukaryotic nucleotide sequence of the present invention may be analyzed in order to identify portions of amino acid sequence which may be associated with increased immunogenicity. For example, polypeptide sequences may be subjected to computer analysis to identify potentially immunogenic surface epitopes. Such computer analysis can include generating plots of antigenic index, hydrophilicity, structural features such as amphophilic helices or amphophilic sheets and the like.

For preparation of monoclonal antibodies directed toward polypeptides encoded by a eukaryotic nucleotide sequence of the invention, any technique that provides for the production of antibody molecules by continuous cell lines may be used. For example, the hybridoma technique originally developed by Kohler and Milstein

(Nature, 256: 495-497, 1973), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today, 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies, and the like, are within the scope of the present invention. See, generally Larrick et al., U.S. Patent 5,001,065 and references cited therein. Further, single-chain antibody (SCA) methods are also available to produce antibodies against polypeptides encoded by a eukaryotic nucleotide sequence of the invention (Ladner et al. U.S. patents 4,704,694 and 4,976,778).

The monoclonal antibodies may be human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies. The present invention provides for antibody molecules as well as fragments of such antibody molecules.

Those of ordinary skill in the art will recognize that a large variety of possible moieties can be coupled to the resultant antibodies or to other molecules of the invention. See, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr (eds), Carger Press, New York, ( 1989), the entire contents of which are incorporated herein by reference.

Coupling may be accomplished by any chemical reaction that will bind the two molecules so long as the antibody and the other moiety retain their respective activities. This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation. The preferred binding is, however, covalent binding. Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of the present invention, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehydes, diazobenzenes and hexamethylene diamines. This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents. (See Killen and Lindstrom 1984, "Specific killing of lymphocytes that cause experimental Autoimmune Myasthenia Gravis by toxin- acetylcholine receptor conjugates." Jour. Immun. 133: 1335-2549; Jansen, F.K., H.E. Blythman, D. Carriere, P. Casella, O. Gros, P. Gros, J.C. Laurent, F. Paolucci, B. Pau, P. Poncelet, G. Richer, H. Vidal, and G.A. Voisin. 1982. "Immunotoxins: Hybrid molecules combining high specificity and potent cytotoxicity". Immunological Reviews 62: 185-216; and Vitetta et al., supra). Preferred linkers are described in the literature. See, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 ( 1984) describing use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, Umemoto et al. U.S. Patent 5,030,719, describing use of halogenated acetyl hydrazide derivative coupled to an antibody by way of an oligopeptide linker. Particularly preferred linkers include: (i) EDC (1- ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)- toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 [3- (2-pyridyldithio) propionamido] hexanoate (Pierce Chem. Co., Cat #21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6 [3-(2-pyridyldithio)- propianamide] hexanoate (Pierce Chem. Co. Cat. #2165-G); and (v) sulfo- NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. #24510) conjugated to EDC.

The linkers described above contain components that have different attributes, thus leading to conjugates with differing physiochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS- ester containing linkers are less soluble than sulfo-NHS esters. Further, the linker SMPT contains a sterically hindered disulfide bond, and can form conjugates with increased stability. Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available. Sulfo-NHS, in particular, can enhance the stability of carbodimide couplings. Carbodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone.

These antibodies may also be used as carriers to form immunotoxins. As such, they may be used to deliver a desired chemical or cytotoxic moiety to cell expressing the receptor. The cytotoxic moiety of the immunotoxin may be a cytotoxic drug or an enzymatically active toxin of bacterial, fungal or plant origin, or an enzymatically active polypeptide chain or fragment ("A chain") of such a toxin. Enzymatically active toxins and fragments thereof are preferred and are exemplified by diphtheria toxin A fragment, non-binding active fragments of diphtheria toxin, exotoxin A (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alphasarcin, certain Aleurites fordii proteins, certain Dianthin proteins, Phytolacca americana proteins (PAP, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin, Ricin A chain, Pseudomonas aeruginosa exotoxin A and PAP are preferred.

Conjugates of the monoclonal antibody and such cytotoxic moieties may be made using a variety of bifunctional protein coupling agents. Examples of such reagents are N-succinimidyl-3- (2- pyridyldithio) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters such as dimethyl adeipimidate HCI, active esters such as disuccinimidyl suberate, aldehydes such as glutaradehyde, bis-azido compounds such as bis(p-diazoniumbenzoyl)- ethylenediamine, diisocyanates such as tolylene 2,6-diisocyante, and bis- active fluorine compounds such as l ,5-difluoro-2,4-dinitrobenzene.

The enzymatically active polypeptide of the immunotoxins according to the invention may be recombinantly produced.

Recombinantly produced ricin toxin A chain (rRTA) may be produced in accordance with the methods disclosed in PCT W085/03508 published August 15, 1985. Recombinantly produced diphtheria toxin A chain and non-binding active fragments thereof are also described in PCT W085/03508 published August 15, 1985.

Antibodies of the present invention can be detected by appropriate assays, e.g., conventional types of immunoassays. For example, a sandwich assay can be performed in which the receptor or fragment thereof is affixed to a solid phase. Incubation is maintained for a sufficient period of time to allow the antibody in the sample to bind to the immobilized polypeptide on the solid phase. After this first incubation, the solid phase is separated from the sample. The solid phase is washed to remove unbound materials and interfering substances such as nonspecific proteins which may also be present in the sample. The solid phase containing the antibody of interest bound to the immobilized polypeptide of the present invention is subsequently incubated with labeled antibody or antibody bound to a coupling agent such as biotin or avidin. Labels for antibodies are well-known in the art and include radionu elides, enzymes (e.g. maleate dehydrogenase, horseradish peroxidase, glucose oxidase, catalase), fluors (fluorescein isothiocyanate, rhodamine, phycocyanin, fluorescamine), biotin, and the like. The labeled antibodies are incubated with the solid and the label bound to the solid phase is measured, the amount of the label detected serving as a measure of the amount of anti-urea transporter antibody present in the sample. These and other immunoassays can be easily performed by those of ordinary skill in the art.

The following Examples serve to illustrate the present invention, and are not intended to limit the invention in any manner.

EXAMPLES Example 1

Sequencing of HER4 expression vectors

Full length HER4 coding region inserts were cloned into pCDM8 (Invitrogen) and pEV7 (33) mammalian expression vectors to generate cH4M2 and pEV7-HER4 plasmids, respectively. The origin of the HER4 insert in cH4M2 was a cDNA library produced from an MDA-MB-453 breast cancer cell line (17) and, that of the HER4 insert in pEV7-HER4, a cDNA library produced from human fetal brain tissue (33). The coding regions of both HER4 inserts were sequenced from one strand by chain termination sequencing using a Sequenase Version 2.0 DNA sequencing kit (USB). The primers used were designed according to the one published HER4 sequence (17) (GenBank accession number L07868). When compared to this sequence, there were two changes in the HER4 coding sequence of pEV7-HER4. These were: i) a major alteration in the external juxtamembrane region (Fig. 1 and Fig. 3) in which 69 nucleotides (23 amino acids) are replaced by 39 different nucleotides (13 arnino acids), and ii) a minor one base replacement of G1751 by A1751 in pEV7-HER4 which would result in a Gly to Asp change in the middle of the C-terminal Cys-rich domain. This change may reflect polymorphism or might be a cloning artifact. There were also two differences in the cH4M2 HER4 insert compared to the published sequence, both in the very 5' end. These were i) a change of AAG37-39 in the published sequence to CGA37-39 resulting in a conserved Lys to Arg change, and ii) a change of G45 in the published sequence to T45, which would not result in an amino acid change. These two changes had been generated to optimize the Kozak consensus for an initiating Met and to optimize PCR-mediated amplification, respectively, and are not present in vivo.

RT-PCR amplification and cloning of HER4 isoform sequences

Total RNA was prepared from various tissues obtained from 19-21 g Swiss Webster mice (Charles River Laboratories) and from the myocardium of the left ventricle of a human heart (obtained from a valve operation) by using the RNAzol B reagent according to the manufacturer's instructions (Tel-Test, Inc.). Total RNA (2.5 μg) was subsequently reverse transcribed to cDNA with Superscript II enzyme according to the manufacturer's instructions (GIBCO BRL) using random oligonucleotide primers (GIBCO BRL). Specific fragments of this cDNA were amplified with 30 (mouse samples) or 40 (human heart sample) cycles of PCR amplification. All PCR reactions were carried out in a total volume of 30 μl including 2 μl of template (10% vol/vol of reverse transcriptase reaction) or 2 μl of H20 (negative control), 20 pmol of specific 5' and 3' primers (see below), 2 U of Taq DNA polymerase (Boehringer Mannheim), 5 nmol of each dNTP (Boehringer Mannheim) and 3 μl of lOx Taq DNA polymerase buffer (Boehringer Mannheim). Mouse HER4 juxtamembrane domain cDNA was amplified with mouse HER4 JM 5' primer (5'-GAA ATG TCC AGA TGG CCT ACA GGG-3' SEQ ID NO: 16) and mouse HER4 JM 3' primer (5'-CTT TTT GAT GCT CTT TCT TCT GAC-3' SEQ ID NO: 17) (sequences kindly provided by Dr. Cary Lai, Scripps Research Institute, La Jolla, CA). Human HER4 juxtamembrane domain cDNA was amplified with human HER4 JM 5' primer (5'-CAG

TGT GAG AAG ATG GAA GAT G-3' SEQ ID NO: 18) and human HER4 JM 3' primer (5'-CTT TTT GAT GAT CTT CCT TCT AAC-3' SEQ ID NO: 19) (17). Mouse β-actin cDNA was amplified with mouse β-actin 5' primer (5'- CTA CAA TGA GCT GCG TGT GG-3' (SEQ ID NO:20) and mouse β-actin 3'primer (5'-TAG CTC TTC TCC AGG GAG GA-3' SEQ ID NO:21) (36). PCR samples were denatured at 94°C for 3 min and subsequently cycled through 30 sec steps of annealing (at 63°C for mouse HER4 juxtamembrane, at 60°C for human HER4 juxtamembrane and at 55°C for mouse β-actin PCR, respectively), extension (at 72°C) and denaturation (at 94°C) steps. The duration of the extension step (72°C) of the last cycle was increased to 20 min. PCR products were separated by electrophoresis using 2% agarose gels, stained with ethidium bromide and visualized with UV light. A 1 Kb DNA ladder (GIBCO BRL) was used as a size marker.

For sequencing, juxtamembrane HER4 RT-PCR amplicons from mouse heart and kidney tissues were cloned into pBluescript vector (Stratagene) and sequenced in both orientations with T3 and T7 primers. The human juxtamembrane HER4 cDNA amplified from heart tissue was purified with a Qiaquick PCR Purification Kit (QIAGEN) and sequenced with human HER4 JM 3' primer. In situ hybridization

Adult Swiss Webster mice were sacrificed and fixed with 4% paraformaldehyde in PBS by intracardial perfusion. After dissection, the cerebella were fixed in 4% paraformaldehyde in PBS overnight at 4°C, equilibrated with 30% sucrose, embedded in Tissue Tek (Miles, Inc.) and frozen. Coronal sections (15 μm) were sectioned on glass microscope slides (Superfrost Plus, Fisher) with a cryostat. Two 46-mer antisense oligonucleotides, one directed against HER JM-a (5'- GGGGTGTAACGGTCCCACTAGTCATGACTGCATTTACTACCCATGG- 3ΗSEQ ID NO:22) and the other against HER4 JM-b (5'-

GTGCATAGGTTCAAGCATTGAAGACTGCATCGGCCTGACGGAATAG-3') (SEQ ID NO:23) (Fig. 3; mouse sequences), were synthesized and purified by gel electrophoresis (IDT). The oligonucleotides were end-labeled with 35S-α-dATP (3000 Ci/mmol; New England Nuclear) using terminal deoxynucleotidyl transferase (GIBCO BRL), and separated from unincorporated nucleotides using a Nuctrap column (Stratagene) .

Tissue sections were prehybridized in 0.25% acetic anhydride in 0.1 M triethanolamine for 10 min at room temperature and incubated in 2x SSC for 1 h at 42°C. The hybridization was carried out in 50% deionized formamide, 0.2 M sodium phosphate, 4x SSC, 10% dextran sulfate, lx Denhardt's solution, 0.1% SDS, 250 μg/ml total yeast RNA, 200 mM DTT, 500 μg/ml salmon sperm DNA and 3000 cpm/μl 35S- labeled antisense oligonucleotide probes for 18 h at 42°C. During the hybridization, the sections were covered with Parafilm and stored in a humid chamber. Following the hybridization, the slides were rinsed once in lx SSC at 55°C, washed four times for 15 min at 55°C and once for one h at room temperature in lx SSC, and rinsed in H20. Sections were dehydrated, processed for autoradiography using NTB-2 Kodak emulsion (Kodak), exposed for 4 weeks at 4°C and examined using both light- and dark-field illumination (Darklite; MVI) under a compound microscope (Microphot FX; Nikon). Expression vectors and transfection

To generate stable cell lines expressing HER4 JM-a or HER4 JM-b under direction of the same CMV promoter, the JM-b sequence in pEV7- HER4 was switched into cH4M2. To do this, a 2.3 kb BstE II-Nsi I fragment which included the juxtamembrane domain was digested and purified from the pEV7-HER4 (HER4 JM-b isoform) and ligated into cH4M2 (HER4 JM-a isoform) from which the corresponding BstE II-Nsi I fragment had first been removed. This procedure generated a cH4M2 JM- b expression plasmid that differed from the original cH4M2 JM-a expression plasmid only in the alternative juxtamembrane sequences within the 2.3 kb BstE II-Nsi I fragment. Both cH4M2 JM-a and a cH4M2 JM-b were separately co-transfected with an antibiotic resistance gene encoding plasmid (pMAMneo; Clontech) into NIH 3T3 clone 7 cells (33) using Lipofectin (GIBCO BRL) according to the manufacturer's recommendations. Clones transfected with pMAMneo plasmid alone were generated to be used as a transfection control. Cells were subsequently cultured in DMEM supplemented with 10% fetal bovine serum, 1% glutamme/penici n/ streptomycin supplement (GPS; Irvine Scientific), 4.5 g/1 glucose and 500 μg/ml G418 (Geneticin; GIBCO BRL). G418- resistant clones were screened for their HER4 expression levels by immunoprecipitation and Western blotting as described below.

Phorbol ester treatment of cells

The media were aspirated and replaced with DMEM containing 0 or 100 ng/ml of a phorbol ester, phorbol 12-myristate 13-acetate (PMA; Sigma). One hundred ng/ml has been suggested to be the optimal concentration for stimulating HER4 processing in NIH 3T3 cells (37). PMA treatments were carried out at 37° C for time periods ranging from 0 to 60 min for the ¹²⁵I-NRG-βl binding assay and for 45 min for the irrirnunoprecipitation and Western blot analysis of HER4 protein amounts and for the anti-HER4 immunocytochemistry. Immunoprecipitation and Western blot analysis of HER4 levels

To screen for HER4 expression levels, individual HER4 transfected clones were grown to confluence in 6-well dishes, lysed and immunoprecipitated with a 1 : 150 dilution of a mouse monoclonal antibody that recognizes an epitope within the extracellular domain of human HER4 (clone H4.77.16; Neomarkers) as described previously (27). The immunoprecipitates were separated on 6% SDS-PAGE gels and transferred to 0.1 μm nitrocellulose membranes (Schleicher and Schuell). The filters were incubated in the presence of a 1:35 dilution of a rabbit polyclonal antibody raised against a peptide corresponding to a sequence in the cytoplasmic domain of human HER4 (C- 18; Santa Cruz Biotechnology, Inc.) and the bound antibody was visualized using a peroxidase conjugated donkey anti-rabbit IgG secondary antibody (1 : 10,000 dilution; Amersham) and enhanced chemiluminescence (ECL) (27).

To analyze total cellular HER4 protein levels after PMA treatment, confluent 100 mm dish cultures were lysed in a buffer containing 1% NP- 40, 150 mM NaCl, 5 mM EDTA, 1 mM benzamidine, 1 μg/ml leupeptin, 100 μM phenylmethylsulfonyl fluoride and 1 mM sodium ortho-vanadate. Insoluble components were removed by centrifugation and the supernatants were subjected to immunoprecipitation with a polyclonal anti-HER4 antibody recognizing the cytoplasmic domain of human HER4 (0618; kindly provided by Dr. Cary Lai, Scripps Research Institute, La Jolla, CA). Immunoprecipitated samples were separated in 7.5% SDS- PAGE gels and transferred to polyvinylidene difluoride membranes (Millipore Inc.). The HER4 proteins on the membranes were subsequently detected by Western blotting using a HER4-specific antibody as described above.

HER4 tyrosine phosphorylation analysis

Tyrosine phosphorylation of HER4 in response to growth factor stimulation was analyzed in cultures (6-well dishes) of NIH 3T3 clone 7 transfectants similar to those used for screening of HER4 protein levels. Serum-starved cells were incubated with or without 100 ng/ml of HB- EGF, NRG-αl , NRG-βl and BTC in DMEM and the levels of HER4- specific tyrosine phosphorylation was measured as described (27). Recombinant human HB-EGF was kindly provided by Dr. J.Abraham

(Scios Nova, Mountain View, CA), recombinant human NRG-βl (residues 177-241 corresponding to the EGF-like domain of HRG-βl) by Dr. M.Sliwkowski (Genetech, Inc., South San Francisco, CA) and recombinant human BTC by Drs. Y.Shing and J.Folkman (Children's Hospital, Boston, MA). Recombinant human NRG-αl (residues 177-241 corresponding to the EGF-like domain of heregulin-αl) was purchased from R&D.

125I-NRG-βl binding assay NRG-βl was radio-iodinated using IODO-BEADS (Pierce) as described (27). Specific activities of 46,000 cpm/ng and 70,000 cpm/ng were achieved in two independent iodinations. The 125I-NRG-βl binding assay was performed following a published protocol (37) with minor modifications. NIH-3T3 clone 7 transfectants were grown to confluence in 6- or 12-well dishes. After PMA stimulation, cell layers were washed twice with ice-cold PBS and incubated for one h on ice with 20 ng/ml of 1251- NRG-βl in DMEM, 20 mM HEPES, pH 7.4, 0.1 mg/ml gelatin. Cells were washed again three times with ice cold PBS and lysed in 0.2 N NaOH. The amount of bound 125I-NRG-βl was measured with a γ-counter. Non- specific binding was estimated as the amount of binding to control- (pMAMneo-) transfected clones and was subtracted from the results.

Immunocytochemistry

NIH 3T3 clone 7 transfectants were grown to 50% confluence in 24-well dishes. The cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature, rinsed with PBS, blocked with 6% BSA in PBS for one h at room temperature and incubated with a monoclonal anti-HER4 antibody (clone H4.77.16; Neomarkers) overnight at 4°C. The cells were washed with PBS and the primary antibody was detected using a Cy3- conj gated anti-mouse secondary antibody (Jackson Immunoresearch Laboratories). Photographs were obtained with an Olympus 1X70 inverted microscope.

RESULTS

Identification of HER4 juxtamembrane isoforms — Two plasmids containing full length human HER4 cDNA inserts were obtained, cH4M2 which contained an insert originally cloned from a human MDA-MB-453 breast cancer cell line (17), and pEV7-HER4 which contained an insert originally cloned from fetal human brain tissue (33). The HER4 coding regions of the two plasmids were sequenced and just one major difference was observed. The HER4 coding region of pEV7-HER4 differed from the published MDA-MB-453-derived HER4 sequence ( 17) (GenBank accession no. L07868) by an in-frame alteration in which 69 nucleotides in the extracellular juxtamembrane domain of the published sequence were replaced with an unrelated sequence of 39 nucleotides. The deduced difference in amino acid sequence resulting from this replacement was a switch from 23 juxtamembrane amino acids in cH4M2 to 13 different amino acids in pEV7-HER4 (Fig. 1). The alteration in nucleotide sequence is shown in Fig. 3. The 23 amino acid form was named HER4 JM-a, and the alternative 13 amino acid form was named HER4 JM-b, based on the location of the alternative sequences within the juxtamembrane (JM) domain and the chronology of their description. The virtual identity of the 3924 bp and 3894 bp full length coding sequences of cH4M2 and pEV7-HER4, respectively, with the exception of the juxtamembrane regions, suggested that the differential juxt4amembrane sequences were generated as a result of alternative splicing of RNA derived from a single gene as opposed to originating from two genes.

RT-PCR analysis of HER4 JM isoforms in mouse and human tissues — To demonstrate that both the HER4 JM-a and JM-b isoforms existed in vivo, total RNA was isolated from several mouse tissues, subjected to reverse transcription and analyzed by PCR using primers flanking the variable juxtamembrane region of mouse HER4. The expected size for a PCR product amplified from HER4 JM-a-derived cDNA was 273 bp and from HER4 JM-b-derived cDNA 243 bp, respectively. After separating the PCR products in a 2% agarose gel, bands corresponding to the expected sizes of either one or both isoforms were found but with different tissue distributions (Fig. 2, lanes 1-21, top panel). For example, cerebellum expressed both isoforms, kidney expressed solely the JM-a isoform and heart expressed solely the JM-b isoform (Fig. 2, lanes 11, 5 and 1 , respectively). As a control to ensure that all samples analyzed by PCR contained cDNA templates, the same reverse transcriptase products that were used to amplify HER4 JM fragments were used in PCR analysis with mouse β-actin specific primers. A single β-actin PCR product of expected size (450 bp) was detected in all tissues (Fig. 2, lanes 1-21 , bottom panel). When the PCR reaction was carried out in the absence of cDNA template, no PCR product was detected with either set of primers (Fig. 2, lane 22). RT-PCR was also carried out with selected human tissues. Human cerebellum was found to express both HER4 isoforms but human heart only the JM- b isoform (data not shown), consistent with the mouse RT-PCR distribution results.

The identities of the amplified mouse RT-PCR products were confirmed by cloning the single HER4 juxtamembrane RT-PCR products derived from mouse kidney and heart, into a pBluescript vector and sequencing the inserts. Mouse and human HER4 juxtamembrane domains were found to be highly homologous. The RT-PCR product from mouse kidney (Fig. 2, lane 5, JM-a) contained a sequence differing in only one nucleotide out of 69 from the sequence of the expression plasmid cH4M2 encoding human HER4 JM-a (Fig. 3). The deduced JM-a amino acid sequence was identical between human and mouse (Fig. 3) . In addition, the RT-PCR product from mouse heart (Fig. 2, lane 1 , JM-b) contained a sequence differing in only 3 nucleotides out of 39 from the sequence of the expression plasmid pEV7-HER4 encoding human HER4 JM-b (Fig. 3). One of these nucleotide differences resulted in a difference (Met vs. Thr) in the deduced JM-b amino acid sequence between human and mouse (Fig. 3). The presence of the novel JM-b sequence in human tissues was further confirmed by showing that a sequence identical to that obtained from the pEV7-HER4 expression vector could be obtained from the single HER4 juxtamembrane RT-PCR product derived from human heart tissue (Fig. 3). Taken together, these data indicate that both HER4 isoforms exist in vivo, that they exist in both mouse and human tissues and that the respective juxtamembrane domains are highly conserved between mouse and human.

In situ hybridization analysis of HER4 JM isoforms — The RT- PCR analysis of HER4 isoform expression in mouse tissues (Fig. 2) suggested that in neural- derived tissues both HER4 JM-a and HER4 JM- b mRNAs are produced simultaneously. To determine if the two HER4 isoforms are expressed in the same or distinct anatomical regions, we studied the distribution of mRNAs encoding each isoform in the mouse cerebellum, a tissue that apparently expresses relatively high levels of each HER4 isoform. Using ³⁵S-labelled antisense oligonucleotide probes corresponding to the different juxtamembrane sequences, and therefore specific for each isoform, both HER4 JM-a (Fig. 4A) and HER4 JM-b (Fig. 4B) were found to be expressed in the granule cell layer (GCL) and in the cerebellar white matter (WM) but not in the molecular layer (ML). The anatomical regions are visualized with hematoxylin staining in Fig. 4C. Qualitatively similar results were obtained using a riboprobe directed against the intracellular domain of HER4 which recognizes both isoforms (not shown) . The signals detected in the granule cell layer reflect the expression of both HER4 isoforms in the neuronal population within this layer. The signals in the white matter represent most probably the expression of both HER4 isoforms in oligodendrocytes, which are the main cell type in this region (38). In agreement with the results obtained with RT-PCR, a weak in situ hybridization signal was detected with the HER4 JM-a probe, but not with the HER4 JM-b probe, in mouse kidney sections (not shown) demonstrating the specificity of the probes.

Ligand activation of the HER4 JM isoforms — Tissue specific expression of HER4 JM-a or JM-b isoforms suggests that the isoforms may have different functions. To address this question, stable transfectants expressing either HER4 JM-a or HER4 JM-b were produced. To ensure that identical expression vectors were used, a 2.3 kb BstE II-Nsil fragment including the sequence coding for the juxtamembrane domain was isolated from HER4-pEV7 (HER4 JM-b) and ligated into cH4M2 (HER4 JM-a) to replace the corresponding BstE II- Nsi I fragment. Both the original cH4M2 plasmid (cH4M2 JM-a) and the cH4M2 plasmid with a swapped juxtamembrane domain (cH4M2 JM-b) were subsequently transfected into NIH 3T3 clone 7 cells. Cell lines expressing similar levels of HER4 protein, as determined by immunoprecipitation and Western blotting, namely clones JM-a #2 and JM-b #42 (Fig. 5A, lanes 2 and 3), were incubated in the presence of HER4 ligands. The cells were lysed and tyrosine phosphorylation of HER4 was assessed by immunoprecipitation with an anti-HER4 antibody followed by Western blotting with an anti-phosphotyrosine antibody. Each of the ligands examined, HB-EGF, NRG-αl , NRG-βl and BTC, stimulated tyrosine phosphorylation of both HER4 JM-a and HER4 JM-b well above control and for a given ligand each of the receptor isoforms was activated to the same extent (Fig. 5B). The relative levels of HER4 activity induced by HB-EGF and NRG-αl (Fig. 5B, lanes 2 and 3) were clearly lower than the levels achieved with NRG-βl and BTC (Fig. 5B, lanes 4 and 5) consistent with previous reports (25,27,39). Control cells transfected with pMAMneo alone did not express detectable levels of endogenous HER4 protein (Fig. 5A, lane 1) nor were they activated by the HER4 ligands (Fig. 5B, control panel). Phorbol ester treatment of cells expressing HER4 JM isoforms HER4 has been reported to be proteolytically cleaved in response to stimulation with a phorbol ester, PMA (37). Since cleavage of many transmembrane proteins occurs in the external juxtamernbrane region, we wanted to determine whether the two different HER4 juxtamembrane isoforms responded differently or similarly to PMA treatment. In a previous study (37), cleavage of the transmembrane form of HER4 was maximal after a 30-60 min treatment with a concentration of 100 ng/ml of PMA as ascertained by a reduction in the binding of radio-iodinated NRG-βl to the surface of the HER4 transfected cells. Therefore, we incubated cells expressing HER4 JM-a (clones JM-a #2 and JM-a # 102) or HER4 JM-b (clones JM-b # 15 and JM-b #42) with or without 100 ng/ml of PMA for 45 min at 37°C and then with 20 ng/ml 125I-NRG- 1 for 1 h on ice and the amount of radioactivity associated with the cells was measured (Fig. 6A). PMA treatment resulted in a reduction in the amount of 125I-NRG- 1 binding to two independent HER4 JM-a clones by about 80-85% but had no significant effect on 125I-NRG-βl binding to two independent HER4 JM-b clones (Fig. 6A). To determine whether the differences observed were a result of different kinetics of processing of the two receptor isoforms, the time course of PMA effects on the level of 125I-NRG-βl binding was measured (Fig. 6B). A reduction in the amount of 125I-NRG-βl binding to HER4 JM-a cells was detected by 20 min and was maximal by 40 min after PMA treatment but no reduction of 1251- NRG-βl binding to HER4 JM-b cells was observed even at 60 min. When the total cellular HER4 protein levels in these cells were analyzed by a combination of immunoprecipitation and Western blotting, 100 ng/ml PMA treatment for 45 min resulted in a loss of cell-associated HER4 JM- a (Fig. 6C, clone JM-a #2, lane 4 compared to lane 3) but not of cell- associated HER4 JM-b (Fig. 6C, clone JM-b # 15, lane 6 compared to lane 5). PMA treatment actually increased the 125I-NRG-βl binding to cells expressing HER4 JM-b (Fig. 6B) and the relative amount of cell surface HER4 JM-b (Fig. 6C), consistent with possible PMA stimulation of HER4 JM-b translocation to the cell surface and lack of HER4 JM-b cleavage. In order to further confirm these differential effects of PMA, cells expressing HER4 JM-a or HER4 JM-b were stimulated for 45 min with 100 ng/ml PMA, fixed without permeabilization and immunostained with an antibody recognizing the extracellular domain of HER4 (Fig. 7). PMA treatment of cells expressing HER4 JM-a led to a virtual total disappearance of HER4 immunoreactivity from the cell surface. In contrast, PMA did not reduce at all the HER4 immunoreactivity on the surface of cells expressing HER4 JM-b. Consistent with the results of 125I-NRG- 1 binding (Fig. 6B) and HER4 immunoprecipitation and Western blotting (Fig. 6C), there was a slight increase in HER4 immunostaining after PMA treatment of cells expressing HER4 JM-b (Fig. 7).

Taken together, these results suggest that the two HER4 isoforms differ in their susceptibility to proteolytic cleavage in response to PMA. While PMA treatment leads to a virtually total reduction in the amount of cell surface HER4 JM-a protein it does not reduce the amount of cell surface HER4 JM-b protein.

DISCUSSION

We have demonstrated that HER4 exists in vivo in two alternatively spliced isoforms that differ in having either 23 or 13 alternative amino acids in the extracellular juxtamembrane domain immediately N-terminal to the transmembrane domain. The 23 amino acid isoform has been designated as HER4 JM-a and the 13 amino acid isoform as HER4 JM-b. The two isoforms appear to differ functionally in their response to phorbol ester in that HER4 JM-a but not HER4 JM-b is processed. The juxtamembrane alterations are the only significant differences in the full length coding sequences of cDNAs originating from two independent sources, human breast cancer cells and human fetal brain tissue, suggesting that they represent alternatively spliced forms derived from a single HER4 gene. This is consistent with the finding that a single HER4 gene is localized to q33.3-34 of human chromosome 2 (40). The two juxtamembrane sequences differ substantially and the only conserved sequence in the two forms is Asp-Cys-Ile. Conservation of the only Cys residue in the juxtamembrane domain is consistent with the suggestion that the Cys residues in the proximal Cys-rich domain are critical for correct disulfide bonding (41).

The finding that there are alternative exons that encode alternative HER4 amino acid sequences is novel since these types of isoforms have not been described to date for members of the EGF receptor subfamily. So far, the only example of an alternatively spliced isoform of a receptor of the EGF receptor subfamily in normal mammalian tissues is the truncation of EGFR (ErbBl) that generates a soluble ectodomain (6). Alternative transcripts coding for truncated extracellular domains of EGFR, HER2 and HER3 are also generated as a result of a read-through of the splice donor site and the presence of a stop codon and a polyadenylation sites within an intron (42-45). In addition, numerous aberrant forms of EGFR are expressed in tumor tissues in association with gene amplification and chromosomal rearrangements (12,46-48). Alternative splicing of exons encoding the juxtamembrane domain may be a specific characteristic of HER4 since our RT-PCR analysis of different mouse tissues did not reveal the existence of alternative EGFR or HER2 juxtamembrane isoforms (our unpublished data). As set forth in the below example, we have also detected alternative sequences in the HER4 cytoplasmic tail that either contain or do not contain the binding site for p85, the regulatory subunit of phosphatidyl inositol 3-kinase. Taken together, HER4 resembles more the members of the FGF receptor subfamily where the existence of alternatively spliced isoforms is a common phenomenon (49) rather than other members of the EGF receptor subfamily of RTKs.

While not wishing to be bound by theory, it is believed that alternative splicing of the juxtamembrane domain leads to differential binding of HER4 ligands and activation of this receptor. The precedents are the FGF receptors in which alternative splicing of exons encoding 47- 50 amino acids of the C-terminal half of the third immunoglobulin-like domain in the juxtamembrane region of FGFR- 1, FGFR-2 and FGFR-3 determines the binding affinities of these receptors for various FGF ligands (4,50-52). Similarly, the presence or absence of an exon encoding 1 1 amino acids of the juxtamembrane domain of TrkB regulates its affinity to different neurotrophins (53). However, when HER4 JM-a and HER4 JM-b were expressed at equal protein levels in NIH 3T3 clone 7 cells, no qualitative differences were found in the ability of HB-EGF,

NRG-αl, NRG-βl or BTC to activate tyrosine phosphorylation in either of the two receptor isoforms. Whether there may be differential downstream effects on the two receptor isoforms is not known.

Another possibility is that the juxtamembrane domain of HER4 contains the cleavage site for protease-induced release of a soluble ectodomain as has been shown for the juxtamembrane domains of other RTKs (7,9- 1 1,54) and that differences between HER4 JM-a and HER4 JM-b are reflected in differential transmembrane receptor processing. PMA has previously been demonstrated to induce proteolytic cleavage of HER4 in transfected NIH 3T3 cells (37). When NIH 3T3 clone 7 cells expressing either of the HER4 juxtamembrane isoforms were exposed to a phorbol ester, PMA, a differential response was noted, as follows, i) PMA treatment prevented the binding of 125I-NRG-βl to cells expressing HER4 JM-a but not to those expressing HER4 JM-b. ii)

Immunoprecipitation and Western blot analysis revealed a diminution of cell-associated 180 kD HER4 protein after PMA treatment of cells expressing HER4 JM-a but not after treatment of cells expressing HER4 JM-b. iii) Cell surface immunostaining with an antibody directed against the HER4 extracellular domain revealed loss of HER4 immunoreactivity in response to PMA in cells expressing HER4 JM-a but not in cells expressing HER4 JM-b. An alternative possibility is that phorbol ester treatment results in internalization of HER4 JM-a leading to diminished binding of 125I-NRG- βl. However, our immunoprecipitation and Western blot experiments using whole cell lysates show a diminution of HER4 JM-a levels rather than the constant levels which might be expected if the HER4 was being translocated into the cell. Furthermore, unlike EGFR that is readily internalized after ligand binding, HER4 has been demonstrated not to be endocytosed effectively (55). Taken together, the most plausible explanation for the differential effects of phorbol ester on the JM-a and JM-b isoforms is that the HER4 JM-a isoform contains a specific protease binding site for ectodomain release that is not present in the HER4 JM-b isoform. The ability of cells to express cleavable and/or non- cleavable foms of HER4 might result in another level of regulating the activities of the four known ligands for HER4.

The HER4 JM isoforms are expressed in vivo in a differential manner suggesting that transcription of the HER4 gene and the splicing of its RNA precursor are regulated in a tissue specific manner and that a level of specificity in isoform-specific function might exist. RT-PCR analysis of mouse tissues demonstrated that some tissues (lung, placenta, bladder, liver, stomach) express little if any of the two isoforms, that some tissues (cerebellum, cerebral cortex, spinal cord, medulla oblongata, eye) express both simultaneously, that some tissues (e.g. kidney) express solely JM-a while other tissues (e.g. heart) express solely JM-b. Besides RT-PCR, the expression of both JM-a and JM-b isoforms could be demonstrated in cerebellum by in situ hybridization using oligonucleotide probes that could hybridize with one but not the other isoform. This analysis suggested that both isoforms are transcribed by neurons in the granule cell layer and by oligodendrocytes in the white matter. Both of these cell types have been shown to express HER4 in vitro (our unpublished data) (56). The signal for HER4 JM-a was stronger in the granule cell layer, while the signal for HER4 JM-b was stronger in the white matter. While these results are not quantitative, they suggest that the level of expression of the each isoform may be different in these two cell populations. Although the HER4 JM isoforms are expressed in a tissue specific manner, preliminary results using RT-PCR indicate that there is no specific correlation in the expression pattern of either HER4 JM-a or HER4 JM-b with the expression pattern of either the NRG or HB- EGF ligands (our unpublished data).

EXAMPLE 2 Identification of ErbB4/HER4 cytoplasmic isoforms. Materials and Methods RT-PCR and sequencing

Total RNA was isolated from the myocardium of the left ventricle of human heart, from human kidney and from various tissues obtained from 19-21 g Swiss Webster mice (Charles River Laboratories) by using RNAzol B reagent according to the manufacturer's instructions (Tel-Test, Inc.). Total RNA (2.5 μg) was subsequently reverse transcribed to cDNA with Superscript II enzyme according to the manufacturer's instructions (GIBCO BRL) using random oligonucleotide primers (GIBCO BRL). The cDNA was subjected to PCR analysis with primer pairs flanking the sequence encoding the p85 binding site in the cytoplasmic tail of ErbB4 or with primers specific for β-actin. Human ErbB4 cDNA was amplified with primers S'-GAAGAGGATTTGGAAGATATGATG-S' (SEQ ID NO:24) and 5'-ACAGCAGGAGTCATCAAAAATCTC-3' (SEQ ID NO:25) (17), mouse ErbB4 cDNA with primers S'-GCTGAGGAATATTTGGTCCCCCAG-S' (SEQ ID NO:26)and 5'-AAACATCTCAGCCGTTGCACCCTG-3' (SEQ ID NO:27) (73), and mouse β-actin cDNA with primers 5'- CTACAATGAGCTGCGTGTGG-3' (SEQ ID NO:28) and 5'- TAGCTCTTCTCCAGGGAGGA-3' (SEQ ID NO:29) (36), as described above. The PCR reactions were carried out for 40 cycles with the annealing steps at 60°C. The PCR products were separated on a 2% agarose g el and visualized under ultraviolet light after staining with ethidium bromide. A 1 Kb DNA ladder (GIBCO BRL) was used as a size marker. For sequencing, cytoplasmic ErbB4 RT-PCR amplicons from human kidney and mouse heart were cloned into a pCR3.1 vector using a TA Cloning Kit (Invitrogen) . The inserts were sequenced using T7 or pCR3.1 reverse primers (Invitrogen) by chain termination sequencing using a Sequenase Version 2.0 DNA sequencing kit (USB).

Expression vectors and transfection

To generate stable cell lines expressing ErbB4 CYT- 1 and ErbB4 CYT-2, the CYT-2 sequence was amplified by RT-PCR from human kidney and introduced into an ErbB4 CYT- 1 expression vector, cH4M2 JM-b (62). To achieve this, ErbB4 cDNA was amplified with a primer pair 5'-AGTTTTCAAGGATGGCTCGAGACC-3' (SEQ ID NO:30) and 5'- ACCATTGGATGCATTGTGATATTC-3' (SEQ ID NO:31 )specifιc for sequences flanking the alternative cytoplasmic region ( 17). The RT-PCR products that were generated (665 bp and 617 bp) were cloned into the pCR3.1 vector using a TA Cloning Kit (Invitrogen). A 592 bp Xhol/Nsύ fragment of the smaller 617 bp insert (matching the size of the CYT-2 form) was then cloned into the cH4M2 JM-b expression vector to replace the corresponding 640 bp Xhol/Nsi fragment. This procedure generated a cH4M2 JM-b CYT-2 expression plasmid that differed from the original cH4M2 JM-b CYT- 1 expression plasmid only in the alternative cytoplasmic sequences. The sequences of the alternative domains of both expression vectors were confirmed by sequencing. Both cH4M2 JM-b CYT- 1 and a cH4M2 JM-b CYT-2 were separately cotransfected with a neomycin resistance gene encoding plasmid (pMAMneo; Clontech) into NIH 3T3 clone 7 cells (33) using Lipofectin (GIBCO BRL) according to the manufacturer's recommendations. Clones transfected with pMAMneo plasmid alone were generated and used as negative controls. Cells were subsequently cultured in DMEM supplemented with 10% fetal bovine serum, 1% glutamine/penicillin/ streptomycin supplement (GPS; Irvine Scientific), 4.5 g/1 glucose and 500 μg/ml G418 (Geneticin; GIBCO BRL). G418-resistant clones were screened for their ErbB4 expression levels by immunoprecipitation and Western blotting as described below. The clone bl.amg has been described previously (33). Immunoprecipitation and Western blot analysis of ErbB4 levels To screen for ErbB4 expression levels, individual ErbB4 transfected clones were grown to confluence, lysed and immunoprecipitated with a 1: 150 dilution of a mouse monoclonal antibody that recognizes an epitope within the extracellular domain of human ErbB4 (clone H4.77.16; Neomarkers) as described previously (27). The immunoprecipitates were separated on 6% SDS-PAGE gels and transferred to 0.1 μm nitrocellulose membranes (Schleicher and Schuell). The filters were incubated in the presence of a 1 :50 dilution of a rabbit polyclonal antibody raised against a peptide corresponding to a sequence in the cytoplasmic domain of human ErbB4 (C- 18; Santa Cruz Biotechnology, Inc.) and the bound antibody was visualized using a peroxidase conjugated anti-rabbit IgG secondary antibody (1: 10.000 dilution; Jackson Immunoresearch Laboratories, Inc.) combined with enhanced chen iluπiinescence (ECL; Amersham).

Tyrosine phosphorylation and coprecipitation analysis

Tyrosine phosphorylation and coprecipitation in response to growth factor stimulation was analyzed in confluent cultures of NIH 3T3 clone 7 transfectants. Cells starved without serum for 24 hours were incubated with or without 100 ng/ml of NRG-1 in DMEM and the levels of ErbB4-specific tyrosine phosphorylation were measured by immunoprecipitation with the monoclonal anti-ErbB4 antibody followed by Western blotting using 4G10 antiphosphotyrosine antibody (a kind gift from Dr. B. Drucker, Dana Farber Cancer Institute, Boston, MA), peroxidase conjugated anti-mouse IgG secondary antibody (1: 10.000 dilution; Cappel) and ECL. The PI3-K-associated tyrosine phosphorylation was measured by immunoprecipitation with a monoclonal antibody against the p85 subunit of PI3-K (UBI) followed by Western blotting with 4G10. Co-precipitation of ErbB4 and PI3-K was analyzed by immunoprecipitation with the anti-p85 antibody followed by Western blotting with the polyclonal anti-ErbB4 as described above. Recombinant human NRG- 1 (residues 177-241 corresponding to the EGF-like domain of heregulin- 1) was kindly provided by Dr. M.Sliwkowski (Genetech, Inc., South San Francisco, CA).

Radioiodination and cross-finking

NRG- 1 was radio-iodinated using IODO-BEADS (Pierce) as described (Elenius et al, 1997b). A specific activity of 1 17.000 cpm/ng was achieved. For cross-finking of 125I_NRG_ I to cell surface receptors, cells were plated on 20 cm2 dishes and grown to confluency. Cells were cross-linked with 200 μM disuccinimidyl suberate (DSS; Pierce) in the presence of 15-30 ng/ml of 125I_NRG- 1 and the cross-linked complexes were visualized by 6% SDS-PAGE and autoradiography (Elenius et al, 1997b).

PI3-K in vitro kinase assay

For PI3-K in vitro kinase assays, NIH 3T3 transfectants were grown to confluence in 177 cm2 dishes, starved without serum for 24 hours, stimulated with or without 100 ng/ml NRG- 1 and lysed. The lysates were then immunoprecipitated with polyclonal anti-ErbB4, monoclonal antiphosphotyrosine (4G10) or monoclonal anti-p85 antibodies as described (27). An in vitro kinase assay for PI3-K was used to measure PI3-K activity in the immunoprecipitates as previously described (89, 57). Briefly, the phosphorylation of phosphatidyl inositol

(PI; Avanti Polar Lipids) with [ - ²P]-ATP (DuPont) to form PI³ P was analyzed using thin-layer chromatography (TLC) and Pl32p was visualized by autoradiography and quantitated with densitometry.

Results

Identification and sequencing of ErbB4 cytoplasmic isoforms In order to analyze human and mouse ErbB4 mRNAs in various tissues, primers were designed corresponding to a cytoplasmic sequence of human ErbB4 cDNA (17, 18) and to a homologous region in the partial mouse ErbB4 cDNA sequence (73). These primers were used for RT-PCR analysis of total RNA isolated from human and mouse tissues. RT-PCR of both human and mouse heart and kidney tissues produced, however, three distinct bands each, rather than the one that had been expected (Fig. 8). In order to characterize the RT-PCR products more fully, they were cloned into a pCR3.1 vector. The identities of the bands derived from human kidney and mouse heart were determined by sequencing the inserts (Fig. 9A). The sequence of the 294 bp product (Fig. 8, lane 2, middle band) of human kidney was identical to a cytoplasmic one in the original human ErbB4 sequence (17, 18). The smaller 246 bp PCR product (Fig. 8, lane 2, lower band) differed from the original human ErbB4 sequence in having an in-frame deletion of 48 nucleotides encoding 16 amino acids at positions 1046- 1061 in the cytoplasmic domain. The 252 bp PCR product (Fig. 8, lane 3, middle band), cloned from mouse heart was homologous to the 294 bp human product. The 204 bp PCR product from mouse heart (Fig. 8, lane 3, lower band) was identical to a partial mouse ErbB4 sequence (73) and differed from the mouse 252 bp PCR product by having a deletion of 48 nucleotides encoding 16 amino acids corresponding to positions 1046- 1061 in the human ErbB4 sequence. The largest bands generated by RT-PCR (human approximately 360 bp and mouse approximately 310 bp) were eventually found to be artifacts produced as a result of annealing of the two smaller products to each other during the PCR cycling (data not shown).

These results demonstrated that two isoforms with or without a 16 amino acid deletion within the cytoplasmic tail of the ErbB4 receptor exist in both human and mouse tissues. The isoform identical to the published human sequence was named ErbB4 CYT- 1 , and the isoform with the 16 amino acid deletion was named ErbB4 CYT-2, based on the deletion being within the cytoplasmic domain (Fig. 9B) and the order of their discovery. The 16 amino acid sequence found in ErbB4 CYT- 1 and lacking in ErbB4 CYT-2 contained a Tyr-Thr-Pro-Met sequence, previously suggested to serve as the site for the binding of p85 to the activated ErbB4 (Fig. 9B) (60).

The expression of ErbB4 cytoplasmic isoforms in mouse tissues is regulated in a tissue-specific manner

A series of mouse tissues were analyzed for ErbB4 cytoplasmic isoform expression (Fig.10). RNA was purified from over 20 mouse tissues and analyzed by RT-PCR using primers flanking the differential cytoplasmic region. Whereas some tissues such as heart, breast and abdominal aorta (Fig. 10, top panel, lanes 1 , 2 and 20, respectively) expressed predominantly ErbB4 CYT- 1 , others, in particular neural tissues such as cerebellum, cerebral cortex, spinal cord and medulla oblongata (Fig. 10, top panel, lanes 1 1 , 12, 15 and 16, respectively) expressed predominantly ErbB4 CYT-2. Some tissues, including lung, bladder and liver (Fig. 10, top panel, lanes 3, 9 and 14) expressed very little, if any, of either one of the isoforms. To generate an internal standard for the RT-PCR reaction, the same reverse transcriptase products used for ErbB4-specific PCR were analyzed by PCR using primers specific for mouse β-actin. A single band of the expected size (450 bp) was detected in all the samples (Fig. 10, bottom panel, lanes 1- 21). No product was detected when PCR was performed with either set of primers in the absence of a cDNA template (Fig. 10, both panels, lane 22). These studies indicated that the expression of the two cytoplasmic isoforms was regulated in a tissue-specific manner.

Both ErbB4 cytoplasmic isoforms are functional receptors

ErbB4 CYT-2 lacks the Tyr-X-X-Met consensus sequence (residues 1056- 1059) that constitutes a consensus binding site for the p85 subunit of PI3-K (84). In order to demonstrate that this isoform is functional but has lost its ability to bind p85 and to stimulate PI3-K activity, stable NIH 3T3 cell transfectants expressing either ErbB4 CYT- 1 or ErbB4 CYT-2 were generated. A previously described expression vector (cH4M2 JM-b) was used to express the ErbB4 CYT-1 isoform (62). To construct an ErbB4 CYT-2 expression vector, RT-PCR products from human kidney were generated with primers flanking the alternative cytoplasmic region. A 592 bp Xhol/Nsύ fragment of a RT-PCR product matching the size of the CYT-2 form was cloned into the cH4M2 JM-b expression vector to replace the corresponding 640 bp Xhόl/Nsύ fragment. This vector was designated cH4M2 JM-b CYT-2 and its sequence was confirmed. The two expression vectors were introduced into NIH 3T3 clone 7 cells, which are devoid of detectable levels of any endogenous ErbB receptor expression (33), and which have been previously used to analyze the function of ErbB4 juxtamembrane isoforms (62). Several clones expressing one or the other of the ErbB4 cytoplasmic isoforms were obtained. The clones expressing the JM-b CYT- 1 isoform of ErbB4 were designated with the prefix b 1 , and the clones expressing the JM-b CYT-2 isoform with the prefix b2. Control clones transfected with a neomycin resistance gene were designated with the prefix neo. ErbB4 protein synthesis was analyzed by immunoprecipitation and Western blotting with anti-ErbB4 antibodies (Fig. 11). No ErbB4 protein was detected in clones transfected with the vector encoding the neomycin resistance gene alone (Fig. 1 1 , lane 1). Two clones (bl.42 and b l.amg) expressed ErbB4 CYT- 1 (Fig. 4, lanes 2 and 3) and two clones (b2.1 1 and b2.15) expressed ErbB4 CYT-2 (Fig. 1 1, lanes 4 and 5) . The expression levels of ErbB4 proteins by these cell lines were fairly equivalent.

To determine whether both types of cytoplasmic isoforms were expressed at the cell surfaces and were capable of binding soluble ErbB4 ligands, cross-linking analysis with radioiodinated NRG- 1 (125τ_ NRG- 1) was performed. Cross-linking of 1 5ι_NRG- 1 to both clones expressing ErbB4 CYT- 1 and to both clones expressing ErbB4 CYT-2 resulted in the formation of a 190 kDa complex, as expected for the covalent binding of a single 7 kD NRG-1 molecule to a single 180 kD ErbB4 molecule (Fig. 12, lanes 2-5). No cross-linked complex was observed when 125j._NRG- l was cross-linked to control cells transfected with the neomycin resistance gene alone and not expressing ErbB4 (Fig. 12, lane 1).

To examine whether the intrinsic tyrosine kinase activity of both ErbB4 isoforms were activated in response to ligand binding, two clones each of cells expressing ErbB4 CYT- 1 and ErbB4 CYT-2 were stimulated with or without NRG- 1 and analyzed for ErbB4 tyrosine phosphorylation. Both ErbB4 cytoplasmic isoforms were activated to a similar extent by NRG- 1 (Fig. 13, lanes 3-10). No ErbB4 phosphorylation was observed in the control cells transfected with the neomycin resistance gene alone (Fig. 13, lanes 1 and 2). Taken together, these two experiments indicate that both ErbB4 cytoplasmic isoforms are functional cell surface receptors capable of binding an ErbB ligand and activating tyrosine phosphorylation.

ErbB4 CYT-2 does not bind to or activate PI3-K

The lack of a PI3-K consensus binding site in the cytoplasmic domain of ErbB4 CYT-2 suggests that the interaction of this isoform with PI3-K would be impaired. Accordingly, the association of the two ErbB4 cytoplasmic isoforms with PI3-K was assessed by co-precipitation experiments. Control cells expressing neomycin resistance gene alone and cells expressing ErbB4 CYT- 1 or ErbB4 CYT-2 were stimulated with or without NRG- 1 , their lysates were immunoprecipitated with an antibody directed against the p85 subunit of PI3-K and the immunoprecipitated material was analyzed by Western blotting with an antibody directed against ErbB4 (Fig. 14A). When cells expressing ErbB4 CYT- 1 were analyzed, a 180 kD band immunoreactive with anti-ErbB4 was co-precipitated with anti-p85 (Fig. 14A, lane 3). The intensity of this band increased in response to stimulation of the cells with NRG- 1 (Fig. 14A, lane 4), consistent with previous reports that NRG- 1 up-regulates the association of p85 with ErbB4 (60;27). On the other hand, no association of p85 with ErbB4 was detected in cells expressing ErbB4 CYT-2 (Fig. 14A, lanes 5 and 6) or in neo control cells not expressing ErbB4 (Fig. 14A, lanes 1 and 2) either with or without NRG-1 stimulation. These results demonstrate that the 16 amino acids missing in the cytoplasmic domain of ErbB4 CYT-2 include the major binding site in ErbB4 for PI3-K and rule out the possibility that there may be other direct PI3-K binding sites elsewhere in the cytoplasmic domain.

To analyze PI3-K activity, control cells and cells expressing ErbB4 CYT- 1 or ErbB4 CYT-2 were analyzed by anti-ErbB4 immunoprecipitation followed by a PI3-K in vitro kinase assay and thin layer chromatography (TLC) (Fig. 14B). Cells expressing ErbB4 CYT- 1 demonstrated NRG- 1 -induced association of PI3-K activity with ErbB4 as measured by formation of PI³ P (Fig. 14B, lanes 3 and 4) whereas no ErbB4-associated PI3-K activity was detected in cells expressing ErbB4 CYT-2 (Fig. 14B, lanes 5 and 6) or in neo controls not expressing ErbB4 (Fig. 14B, lanes 1 and 2). These results are consistent with the immunoprecipitation data shown in Fig. 14A. To obtain standards for the migration of the phosphorylated phosphatidyl inositol substrate on the TLC plate, PI3-K activity was immunoprecipitated directly from NRG-1- stimulated ErbB4 CYT- 1 and ErbB4 CYT-2 cell clones respectively with an anti-p85 antibody (Fig. 14B, lanes 7 and 8).

We have demonstrated that ErbB4 CYT-2 cannot directly bind PI3-K. This isoform could, however, activate PI3-K indirectly, for example via an adapter molecule such as Grb-2-associated binder- 1 (Gab- 1) that links PI3-K to Grb-2 (66). To assess whether ErbB4 CYT-2 could activate PI3-K indirectly, tyrosine phosphorylation of the p85 subunit of PI3-K was analyzed by immunoprecipitation with anti-p85 antibody followed by Western blot with anti-phospotyrosine antibody (Fig. 15A). Lysates of cells expressing ErbB4 CYT- 1 contained specific tyrosine phosphorylated proteins of 85 kD and 180 kD, whose phosphorylation was induced by NRG-1 (Fig. 15A, lanes 3 and 4). Re-blotting the membrane with anti-p85 and anti-ErbB4 antibodies demonstrated that these proteins were p85 and the co-precipitated 180 kD ErbB4 receptor, respectively (not shown). In contrast, no significant tyrosine phosphorylation of 85 kD or 180 kD bands was detected in cells expressing ErbB4 CYT-2 (Fig. 15A, lanes 5 and 6) or neo control cells (Fig. 15A, lanes 1 and 2). These results demonstrating that p85 was tyrosine phosphorylated in cells expressing ErbB4 CYT- 1 but not in cells expressing ErbB4 CYT-2. Next a PI3-K in vitro kinase assay was performed after immunoprecipitation with an antiphosphotyrosine antibody. Phosphotyrosine-associated PI3-K activity was detected in cells expressing ErbB4 CYT- 1 after stimulation with NRG- 1 (Fig. 15B, lanes 3 and 4) but no such activity was detected in cells expressing ErbB4 CYT-2 (Fig. 15B, lanes 5 and 6) or in control cells (Fig. 15B, lanes 1 and 2). PI³²P standards were obtained by direct anti- p85 immunoprecipitation from NRG- 1 -stimulated ErbB4 CYT- 1 and ErbB4 CYT-2 cell clones (Fig. 15B, lanes 7 and 8). These results suggested that PI3-K was not activated even indirectly in cells expressing ErbB4 CYT-2.

Discussion

This example presents evidence that the ErbB4 receptor exists in vivo or isoforms, probably as a result of alternative splicing, that differ in whether or not they include a 16 amino acid stretch in the cytoplasmic domain that contains a consensus binding site for the p85 subunit of PI3-K. These isoforms, ErbB4 CYT- 1 , which includes the PI3-K binding domain, and ErbB4 CYT-2, which does not, were detected by RT-PCR analysis of human and mouse, heart and kidney tissue. Both isoforms were demonstrated to be functional NRG- 1 receptors. When expressed in NIH 3T3 cells, they bound NRG- 1 to the cell surface and were tyrosine phosphorylated in response to NRG- 1 stimulation. However, the isoforms differ in that cells expressing ErbB4 CYT-2 could not bind or activate PI3-K in a ligand-dependent manner. These results suggest strongly that Tyrιo56 of human ErbB4, which is located in the 16 amino acid sequence lacking in ErbB4 CYT-2, is the only tyrosine residue within the cytoplasmic tail of ErbB4 that can interact with the p85 subunit of PI3- K. This result is consistent with the report that a synthetic peptide corresponding to residues 1054- 1063 including a phosphorylated Tyriosβ inhibits the binding of PI3-K to ErbB4 (60). There is the possibility, however, that the ErbB4 isoform lacking the PI3-K binding domain could activate PI3-K signaling indirectly via an adapter molecule or by ErbB receptor heterodimerization. For example, ErbBl has been reported to activate PI3-K by indirect mechanisms, via the ErbB3 receptor (83, or the adapter molecules Cbl (82) and Gab- 1 (66). However, these considerations probably do not apply to the ErbB4 CYT-2 isoform since the cells lines used here do not express ErbB3 (33) and Cbl has been shown to selectively bind to ErbB l but not to ErbB4 (71). Furthermore, we were able to rule out experimentally indirect PI3-K activation in cells expressing ErbB4 CYT-2 by demonstrating that NRG- 1 stimulation could not induce tyrosine phosphorylation of p85, nor detectable phosphotyrosine-associated PI3-K activity in these cells. Taken together, our results suggest that ErbB4 CYT-2 is a functional receptor that responds to NRG- 1 but it is a receptor that is unable to stimulate PI3-K activity in NIH 3T3 cells either directly or indirectly due to lack of a p85 binding site.

An isoform that lacks the ability to activate PI3-K signaling might be functionally significant since signaling through the PI3-K pathway has been demonstrated to lead to numerous cellular responses, such as proliferation, survival, chemotaxis, actin rearrangements and changes in adhesion and protein trafficking (86). In a previous study of ErbB4- mediated chemotaxis, we demonstrated that NRG- 1 and HB-EGF stimulated chemotaxis of cells expressing ErbB4 in the absence of other EGFR-like receptors (27). Activation of ErbB4 by NRG- 1 and HB-EGF stimulated a direct association of PI3-K activity with ErbB4. Nanomolar concentrations of wortmannin, a known inhibitor of PI3-K activity, were effective in abolishing the chemotactic response after stimulation with ErbB4 ligands (27). These results suggested a role for PI3-K activity in the signal transduction pathway leading from activated ErbB4 to chemotaxis. However, to date we have not been able to determine definitively whether the ErbB4 CYT-2 isoform is impaired or not in mediating chemotaxis via PI3-K. Another functional possibility is that cells expressing ErbB4 CYT-2 do not survive as well as do cells expressing ErbB4 CYT- 1 in response to adverse conditions. PI3-K signaling has recently been implicated as having a central role in the pathway leading from an activated RTK to cellular survival (91 ; 63). NRG- 1 , a ligand that can activate ErbB4, promotes survival signaling in various cell types (74; 61 ; 58; 59; 93). Our preliminary data suggest that NRG-1 promotes survival of serum-starved NIH 3T3 cells expressing ErbB4 CYT- 1 but not of cells expressing ErbB4 CYT-2.

ErbB4 isoforms differing in the presence of a PI3-K binding domain are not the only ones that exist. In Example 1 we found that there were ErbB4 isoforms that differed in the structure of the extracellular juxtamembrane domain, whereby 23 amino acids in one isoform were replaced by 13 other amino acids in the other isoform, suggesting alternative splicing of exons. These juxtamembrane isoforms were named ErbB4 JM-a and ErbB4 JM-b for the 23 amino acid form and the 13 amino acid form, respectively. Both ErbB4 JM-a and ErbB4 JM-b were equally activated by known ErbB4 ligands. However, ErbB4 JM-a was found to be susceptible to proteolytic cleavage of the receptor extracellular domain in response to treatment with phorbol ester, whereas ErbB4 JM-b was not. Given the existence of juxtamembrane and cytoplasmic isoform pairs, it appears that there are at least four possible ErbB4 isoforms in existence and possibly other ones as yet undiscovered.

Only one human ErbB4 gene has been identified (40). Since the sequences of ErbB4 CYT- 1 and ErbB4 CYT-2 flanking the alternative 16 amino acid insert were identical, it is most probable that these two isoforms are generated by alternative splicing of a single ErbB4 RNA precursor molecule. Differential expression of ErbB4 CYT- 1 and ErbB4 CYT-2 by different mouse tissues suggested that this alternative splicing 56

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The references cited above are hereby incorporated herein by reference.

This invention has been described in detail including the preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of this disclosure may make modifications and improvements thereon without departing from the spirit and scope of the invention as set forth in the claims.

Claims

63We claim:

1. An isolated DNA segment encoding a protein comprising the amino acids of SEQ ID NO: 1 or 3.

2. A DNA segment encoding a human epidermal growth factor receptor having the amino acid of SEQ ID NO:5 deleted.

3. The DNA of claim 2, wherein the receptor is represented by the amino acid sequence of SEQ ID NO:6.

4. A protein encoded by the DNA of claim 1, 2 or 3.

5. An antibody directed to the protein of claim 4.

6. The DNA of claim 1, wherein the protein is a human epidermal growth factor receptor.

7. An isolated DNA segment comprising the nucleotide sequence of SEQ ID NO:2, or the complement thereof.

8. A host cell containing the DNA of claim 1 , 2 or 3.

9. A bioassay for detecting ErbB4 isoform mRNA in a biological sample comprising the steps of: i) contacting said biological sample with a DNA segment according to claims 1, 2 or 3 under conditions such that a DNA:RNA hybrid molecule containing said DNA segment and complementary RNA can be formed; and ii) determining the amount of said DNA segment present in said hybrid molecule. 64

10. A bioassay for testing potential analogs of ligands of ErbB4 receptors for the ability to affect an activity mediated by said ErbB4 receptors, comprising the steps of: i) contacting a molecule suspected of being a ligand with ErbB4 receptors produced by a cell according to claim 8; and ii) determining the amount of a biological activity mediated by said ErbB4 receptors in said cells.

11. An assay for detecting an ErbB4 antigen in a biological sample comprising the steps of: i) contacting said sample with an antibody according to claim 5, under conditions such that a specific complex of said antibody and said antigen can be formed ; and ii) determining the amount of said antibody present as said complexes.

12. A method for targeting a therapeutic drug to cells having high levels of ErbB4 receptors, comprising the steps of: i) conjugating an antibody according to claim 5, or an active fragment thereof, to said drug; and ii) administering the resulting conjugate to an individual with cells having high levels of ErbB4 receptors in an effective amount and by an effective route such that said antibody is able to bind to said receptor on said cells.

13. Use of the antibody of claim 5, or an active fragment thereof, conjugated to a therapeutic drug to target said therapeutic drug to cells having high levels of ErbB4 receptors.