Abstract:
The present invention discloses a system and method using isoforms of the human ErbB3 receptor as negative regulators of heregulin-stimulated ErbB2, ErbB3, and ErbB4 activation. The present invention also discloses a system and method of one such isoform, p85-sErbB3 binding to heregulin with an affinity comparable to that of full-length ErbB3, and competitively inhibiting high affinity heregulin binding to ErbB2/3 heterodimers on the cell surface of breast carcinoma cells. The present invention also uses p85-sErbB3 to inhibit heregulin-induced phosphorylation of ErbB2, ErbB3, and ErbB4 in cells and uses p85-sErbB3 as a negative regulator of heregulin-stimulated signal transduction and as a block for cell growth. The present invention is also directed to nucleic acids and expression vectors encoding p85-sErbB3, host cells harboring such expression vectors, and methods of preparing the protein. The present invention discloses a system and method of therapeutic applications in human malignancies associated with heregulin-mediated cell growth such as breast and prostate cancer.

Description:
[0001]    This application claims priority from U.S. Application No. 60/157,144, filed Sep. 30, 1999, entitled “Soluble Epidermal Growth Factor Receptor-Like Proteins and Their Uses,” and U.S. application Ser. No. 09/676,380, filed Sep. 29, 2000, entitled, “Soluble Epidermal Growth Factor Receptor-Like Proteins and Their Uses in Cancer Detection Methods,” and U.S. Application No. 60/294,824 filed May 31, 2001 entitled, “Use of Secreted Human ErbB3 Receptor Isoform to Inhibit Heregulin Simulated Activation of ErbB2, ErbB3 and ErbB4, all of which are included herein by reference. This application is a continuation in part of U.S. Applications 60/157,144 and Ser. No. 09/676,380. 
     
    
     
         [0002]    The disclosed invention was made with the support of a grant from the National Cancer Institute (CA85133). The United States Government has certain rights in the invention.  
         FIELD OF INVENTION  
         [0003]    The present invention is directed to a system and method that uses soluble ErbB3 proteins, including p85-sErbB3, p45-sErbB3 and other isoforms of ErbB3, wherein said sErbB3 protein binds to heregulins and antagonizes heregulin-stimulated activation of the ErbB receptors and blocks the cell proliferative activity thereof. The present invention is also directed to expression vectors encoding a sErbB3 protein, including p85-sErbB3, p45-sErbB3 and other isoforms of ErbB3, host cells harboring such expression vectors, methods of preparing such proteins, and methods and systems utilizing such proteins for the treatment of conditions associated with undesired heregulin stimulation.  
         BACKGROUND OF THE INVENTION  
         [0004]    The heregulins (also called neuregulins, neu differentiation factor (NDF), acetylcholin receptor inducing activity (ARIA), glial growth factors (GGFs)) are a family of epidermal growth factor-like growth factors that activate members of the ErbB/EGF receptor family (Holmes, Sliwkowski et al. 1992; Peles, Bacus et al. 1992; Wen, Peles et al. 1992; Falls, Rosen et al. 1993; Marchionni, Goodearl et al. 1993). Isoforms of heregulins, all of which arise from splice variants of a single gene, NRG-1 (neuregulin-1), have been cloned and classified into the α and β subgroups based on structural differences in their EGF binding domains (Holmes, Sliwkowski et al. 1992).  
           [0005]    ErbB3-mediated signal transduction exerted by heregulins has been implicated in the regulation of diverse biological events including Schwann cell differentiation, neural regulation of skeletal muscle differentiation, heart development, and proliferation and differentiation of normal and malignant breast epithelial cells (Alroy and Yarden 1997; Sundaresan, Penuel et al. 1999). Researches have shown that breast carcinoma cells respond to heregulin through proliferation, differentiation, as well as morphogenesis. Carcinoma cells expressing heregulin are hormone-independent and correlated to the ability for metastasis in experimental studies.  
           [0006]    ErbB3 is a transmembrane glycoprotein encoded by the c-erbB3 gene (Kraus, Issing et al. 1989; Plowman, Whitney et al. 1990). The ErbB3 receptor belongs to the ErbB family which is composed of four growth factor receptor tyrosine kinases, known as ErbB1/EGFR, ErbB2/Neu, ErbB4, as well as ErbB3. ErbB3 and ErbB4 are receptors for heregulins and ErbB2 is a coreceptor (Carraway and Burden 1995). These receptors are structurally related and include three functional domains: an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic tyrosine kinase domain (Plowman, Culouscou et al. 1993). The extracellular domain can be further divided into four subdomains (I-IV), including two cystein-rich regions (II and IV) and two flanking regions (I and III). The ErbB3 is unusual among receptor tyrosine kinases in that its catalytic domain is defective. Despite its lack of intrinsic catalytic activity, ErbB3 is an important mediator of heregulin responsiveness. heregulin binding induces ErbB3 to associate with other members of the ErbB family to form heterodimeric receptor complexes. ErbB3 then transactivates the kinase of its partner receptor which initiates a variety of cytoplasmic signaling cascades.  
           [0007]    The ErbB3 receptor, together with ErbB2, is an important receptor involved in cellular growth and differentiation. Particular attention has focused on the role of ErbB3 as a coreceptor of ErbB2 in the area of cancer research. Transgenic mice that have been engineered to overexpress heregulin in mammary glands have been reported to exhibit persistent terminal end buds and, over time, to develop mammary adenocarcinomas (Krane and Leder 1996). ErbB3 expression studies on tumor tissues and on cell lines show frequent co-expression of ErbB2 and ErbB3 receptors (Alimandi, Heidaran et al. 1995; Meyer and Birchmeier 1995; Robinson, He et al. 1996; Siegel, Ryan et al. 1999). In addition, both ErbB2 and ErbB3 are activated in mammary tumors formed in transgenic mice harboring only the activated form of ErbB2 (Siegel, Ryan et al. 1999). A lot of cell lines used for experimental tumor formation studies are either estrogen-dependent (MCF-7 and T47D, the low ErbB2 expressers) or estrogen-independent (SKBR3, high ErbB2 expressers). However, these cell lines do not exhibit metastatic phenotypes. When MCF-7 cells are transfected to overexpress ErbB2, MCF-7 cells gain estrogen-independent phenotype, however, they never metastasize. On the other hand, the MCF-7 cells overexpressing heregulin gains metastatic phenotype, suggesting heregulin&#39;s active role in metastasis (Hijazi, Thompson et al. 2000; Tsai, Homby et al. 2000).  
           [0008]    Five alternate ErbB3 transcripts arise from read-through of an intron and the use of alternative polyadenylation signals (Lee and Maihle 1998; Katoh, Yazaki et al. 1993). Using 3′-RACE the inventors have isolated four novel c-erbB3 cDNA clones of 1.6, 1.7, 2.1, and 2.3 kb from a human ovarian carcinoma-derived cell line (Lee and Maihle 1998). p85-sErbB3 of 543 aa, encoded by a 2.1 kb alternate c-erbB3 transcript (cDNA clone R31F), is composed of subdomains I through III and the first third of subdomain IV, and has a unique 24 amino acid carboxy-terminal sequence. p45-sErbB3 of 312 aa, encoded by a 1.7 kb alternate c-erbB3 transcript (cDNA clone R2F) contains subdomains I, II, and a portion of subdomain III of the extracellular domain of ErbB-3 followed by two unique glycine residues. p50-sErbB3 of 381 aa, encoded by a 1.6 kb alternate c-erbB3 transcript (cDNA clone R1F) contains subdomains I, II, and a portion of subdomain III of the extracellular domain of ErbB-3 followed by 30 unique amino acids. p75-sErbB3 of 515 aa, encoded by a 2.3 kb alternate c-erbB3 transcript (cDNA clone R35F), is composed of subdomains I through III, and has a unique 41 amino acid carboxy-terminal sequence (FIG. 1) (Lee and Maihle 1998).  
           [0009]    Using various recombinant truncated forms of EGFR, it has been shown that efficient inhibition of full-length EGFR activation by dominant-negative heterodimerization occurs only when these deletion mutants retain the transmembrane domain in addition to the extracellular domain (Redemann, Holzmann et al. 1992). Similarly, a recombinant dominant-negative ErbB3 mutant with a deleted cytoplasmic domain but which retains its transmembrane domain can inhibit full-length ErbB2 and ErbB3 activation (Ram, Schelling et al. 2000). In contrast, in avian tissues, expression of a naturally occurring sEGFR/ErbB1 inhibits TGFα dependent transformation (Flickinger, Maihle et al. 1992). Soluble EGFR secreted by the A431 human carcinoma cell line also has been reported to inhibit the kinase activity of purified fill-length EGFR in a ligand-independent manner (Basu, Raghunath et al. 1989). In no case do these soluble EGF/ErbB1 receptors function as antagonists through high affinity ligand-binding. Similarly, herstatin, a naturally occurring soluble ErbB2 protein which inhibits ErbB2 activation appears to function by blocking ErbB2 dimerization (Doherty, Bond et al. 1999).  
           [0010]    The ErbB3 protein, specifically the p85-sErbB3 and p45 sErbB3 isoforms, is unique among other naturally occurring or recombinant soluble ErbB receptors in that it binds specifically to heregulin with high affinity and inhibits its binding to cell surface receptors and consequently inhibit heregulin-induced activation of the receptors and their downstream effectors. Thus sErbB3, specifically p85-sErbB3 and p45-sErbB3, can be used as a therapeutic reagent for heregulin-induced malignancy such as mammary and prostate tumors.  
           [0011]    Heretofore, production and purification methods for, therapeutic uses of, and useful compositions containing, this protein, referred to herein as p85-sErbB3 have not been available.  
         SUMMARY OF THE INVENTION  
         [0012]    The present invention provides several novel isolated and purified nucleic acids which encode soluble isoforms of ErbB3. Preferred embodiments of this aspect of the invention are nucleic acid sequences which specifically encode a soluble form of ErbB3 whose amino acid sequence comprises the sequence of SEQ ID NO: 1. The nucleic acid embodiments include DNA SEQ ID NO:2  
           [0013]    The present invention discloses a system and process that uses isoforms of sErbB3 to bind to HRG with high affinity and effectively block HRG binding to cell surface receptors. More specifically, the present invention discloses a system and process that uses p85-sErbB3 to bind to HRG with high affininity and substantially block HRB binding to cell surface receptors. The present invention also discloses the diagnosis and treatment of carcinoma cells with p85-sErbB3 and other ErbB3 isoforms.  
           [0014]    A preferred embodiment of the present invention uses an expression vector, such as a plasmid or virus, containing the isolated cDNA encoding p85-sErbB3 and other ErbB3 isoforms, as well as a cell, either eukaryotic or prokaryotic, containing the expression vector.  
           [0015]    The present invention also discloses a process for producing the p85-sErbB3 molecule and other ErbB3 isoforms, which includes the steps of ligating the isolated DNA into an expression vector capable of expressing the isolated DNA in a suitable host; transforming the host with the expression vector; culturing the host under conditions suitable for expression of the isolated DNA and production of the p85-sErbB3 protein and other ErbB3 isoforms, and isolating the protein from the host. The host cell may be a prokaryote, or a eukaryote.  
           [0016]    The invention further discloses a method and system for the production of polyclonal or monoclonal antibodies directed against unique p85-sErbB3 and other ErbB isoform epitopes. The inventors have generated polyclonal antibodies specific to p85-sErbB3 using a C-terminal unique sequence of the p85-sErbB3 as an antigen. The affinity-purified antibody can be used to detectp85-sErbB3 using immunoblot analysis and other detection methods.  
           [0017]    Another embodiment of the invention discloses a system and method of detecting p85-sErbB3 and other ErbB3 isoforms in a mammalian biological specimen which is selected from the group consisting of fluids (including blood, serum, plasma, urine and ascites), tissues, and their derivatives. Further the inventors disclose an immunoprecipitation followed by immunoblot analysis to detect p85-sErbB3 using anti-ErbB3 antibodies.  
           [0018]    Yet another embodiment of this invention provides a vector for gene therapy, comprising a nucleic acid molecule having i) a transcription regulatory segment; and ii) a second segment coding for p85-sErbB3 or other ErbB3 isoforms under transcriptional control of the transcription regulatory sequence; and a delivery vehicle for delivering the nucleic acid molecule.  
           [0019]    Other aspects, embodiments, features and advantages of the present invention will be apparent from a reading of the description of the following preferred embodiments. 
       
    
    
     BRIEF DESCRIPTION OF THE FIGURES AND DEFINITIONS  
       [0020]    [0020]FIG. 1. Diagram of soluble ErbB3 (sErbB3) proteins. ErbB3 is composed of a 19 aa signal peptide sequence that is cleaved (gray box), an extracellular ligand-binding domain (aa 1-620), a transmembrane domain (aa 621-646; indicated as TM), and an intracellular domain (aa 647-1323). The extracellular domain of the receptor can be further divided into four subdomains (I-IV), as noted in the text. The alternate c-erbB3 transcripts arise from read-through of an intron and the use of alternative polyadenylation signals. p45-sErbB3 contains the amino-terminal 310 amino acids of ErbB3 and two unique carboxy-terminal amino acid residues. p50-sErbB3 contains the amino-terminal 351 amino acids of ErbB3 and 30 unique carboxy-terminal amino acid residues. p75-sErbB3 contains the amino-terminal 474 amino acids of ErbB3 and 41 unique carboxy-terminal amino acid residues. p85-sErbB3 contains the amino-terminal 519 amino acids of ErbB3 and 24 unique carboxy-terminal amino acid residues. The carboxy-terminal unique sequences are denoted as black boxes.  
         [0021]    [0021]FIG. 2. p45-sErbB3 and p85-sErbB3 in conditioned media can block HRG-induced activation of ErbB3. (A) p45-sErbB3 and p85-sErbB3 in the concentrated conditioned media were detected by Western blotting using an anti-ErbB3 antibody recognizing the extracellular region of ErbB3. Increasing volumes (5, 10, 20 μl; left to right) of the concentrated conditioned media (×15) were loaded on an SDS-PAGE gel. (B) and (C) The Ba/F3 (ErbB2+ErbB3) cells were stimulated with HRGα (panel B) and HRGβ (panel C) with or without the concentrated conditioned media for 10 min at room temperature prior to lysis. ErbB3 was immunoprecipitated with an anti-ErbB3 antibody from equal amounts of total protein, subjected to SDS-PAGE, and analyzed by Western blotting using an anti-phosphotyrosine antibody (αPY). Filters were stripped and reprobed with anti-ErbB3 antibody recognizing the intracellular region of ErbB3.  
         [0022]    [0022]FIG. 3. p85-sErbB3 binds to HRG. (A) HRGβ was crosslinked to p85-sErbB3 (25 nM) with BS 3  after incubating in the presence of 50 nM  125 I-HRGβ without or with increasing concentrations (0.16, 0.32, 0.64, 1.25 μM) of unlabeled HRGβ. Insulin (1.25 μM) was used as a negative control. The arrowhead indicates a 90 kDa complex of  125 I-HRGβ and p85-sErbB3. (B) and (C) Binding analysis of  125 1-HRG to p85-sErbB3 and ErbB3-IgG fusion protein. Binding assays were performed in a 96-well plate format as described in Materials and Methods. Binding results were analyzed by using Scatchard method and by plotting the displacement of  125 I-HRGβ 177-244  binding by unlabeled HRGβ 177-244  (Inset).  
         [0023]    [0023]FIG. 4. Inhibition of HRGβ binding by p85-sErbB3 and by 2C4, a monoclonal antibody specific for ErbB2. T47D cells were incubated with the indicated concentrations of p85-sErbB3 and 2C4 at room temperature for 30 min.  125 I-HRGβ 177-244  (0.1 nM) was then added and binding reactions were performed as described in Materials and Methods.  125 I-HRGβ 177-244  bound to the cell surface was measured using a gamma counter.  
         [0024]    [0024]FIG. 5. p85-sErbB3 blocks HRG-induced activation of ErbB2 and ErbB3 in the Ba/F3 (ErbB2+ErbB3) cells. Cells were untreated or stimulated with HRGβ 1-241  alone or HRGβ 1-241  plus purified p85-sErbB3 for 10 min at room temperature. Receptor phosphorylation levels and ErbB2 and ErbB3 receptor levels were determined by anti-ErbB2 (A) and anti-ErbB3 (B) immunoprecipitation followed by Western blotting as described in FIG. 2.  
         [0025]    [0025]FIG. 6. p85-sErbB3 blocks HRG-induced activation of ErbB proteins and their downstream activators MAPK, PI3K (p85), and Akt. (A) p85-sErbB3 blocks HRG-induced activation of ErbB2, ErbB3, and ErbB4 in T47D and MCF7 breast carcinoma cells. Serum-starved cells were stimulated with no HRGβ, HRGβ alone, or 6 nM HRGβ plus 36 nM p85-sErbB3 for 10 min at room temperature. Receptor phosphorylation levels and ErbB2, ErbB3, and ErbB4 receptor levels were determined by immunoprecipitation followed by Western blotting. (B) p85-sErbB3 inhibits HRG-induced association of PI3K (p85) with ErbB3 and activation of MAPK and Akt in T47D cells. Cells were treated with 1 nM HRGβ and 10 nM p85-sErbB3 for 10 min or 30 min and analyzed for activation of ErbB3. Association of PI3K (p85) with ErbB3 was analyzed by immunoprecipitation of cell lysates using an anti-ErbB3 antibody followed by Western blotting of anti-PI3K (p85) antibody. Activation of MAPK and Akt was examined by Western blotting of cell lysates using antibodies specific to phospho-MAPK and phospho-Akt.  
         [0026]    [0026]FIG. 7. p85-sErbB3 inhibits cell growth stimulation by HRG. MCF7 cells were trypsinized, washed, and plated at a density of 5,000 (squares) or 8,000 cells/well (triangles) in 96-well plates with increasing concentrations of HRGβ in serum-free medium and growth was measured after 3 days (inset). MCF7 cells were trypsinized, washed, incubated with p85-sErbB3 for 30 min, and plated with or without 0.4 nM HRGβ in serum-free medium. At 40 nM (a 100-fold molar excess to HRGβ) in the presence of HRGβ, p85-sErbB3 inhibited cell growth by 75% and 90%, at densities of 5,000 and 8,000 cells/well, respectively, whereas the same concentration of p85-sErbB3 did not affect cell growth in the absence of HRGβ. The data presented are the mean±standard deviation of six replicates. This experiment was repeated three times and the results shown represent all three trials. 
     
    
     DEFINITIONS  
       [0027]    As used herein, the term “soluble” ErbB3 (sErbB3) means that the ErbB3 polypeptide is found in a form that is not anchored to the membrane of a cell, i.e., a portion of the sErbB3 is not found physically embedded in the lipid bilayer which comprises the cell membrane in the organism of its origin. As used herein, the term “biological activity” of a peptide of the invention is defined to mean a polypeptide comprising a subunit of a peptide having SEQ ID NO: 1, or a variant thereof, which has at least about 10%, preferably at least about 50%, and more preferably at least about 90%, of the activity of a peptide having SEQ ID NO: 1. The activity of a peptide of the invention can be measured by methods well known in the art including, but not limited to, the ability to bind heregulins, or the ability of the peptide to elicit a sequence-specific immune response when the peptide is administered to an organism, e.g., goat, sheep or mice.  
         [0028]    The terms “recombinant nucleic acid” or “preselected nucleic acid,” e.g., “recombinant DNA sequence or segment” or “preselected DNA sequence or segment” refer to a nucleic acid that has been derived or isolated from any appropriate tissue source and that may be subsequently chemically altered, typically in vitro, so that its sequence is not naturally occurring, or corresponds to naturally occurring sequences that are not positioned as they would be positioned in a genome which has not been transformed with exogenous DNA. An example of preselected DNA “derived” from a source, would be a DNA sequence that is identified as a useful fragment within a given organism, and which is then chemically synthesized in essentially pure form. An example of such DNA “isolated” from a source would be a useful DNA sequence that is excised or removed from said source by chemical means, e.g., by the use of restriction endonucleases, so that it can be further manipulated, e.g., amplified, for use in the invention, by the methodology of genetic engineering.  
         [0029]    “Regulatory sequences” is defined to mean RNA or DNA sequences necessary for the expression, post-transcriptional modification, translation, and post-translational modification of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotic cells, for example, include a promoter, and optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, stop sequences, enhancers, splicing, and polyadenylation signal sequences, as well as glycosylation and secretory signal sequences.  
         [0030]    As used herein, the term “cell line” or “host cell” is intended to refer to well-characterized homogenous, biologically pure populations of cells. These cells may be eukaryotic cells that are neoplastic or which have been “immortalized” in vitro by methods known in the art, as well as primary cells, or prokaryotic cells. The cell line or host cell is preferably of mammalian origin, but cell lines or host cells of non-mammalian origin may be employed, including avian, plant, insect, yeast, fungal or bacterial sources. Generally, the preselected DNA sequence is related to a DNA sequence that is resident in the genome of the host cell but is not expressed, or not highly expressed, or, alternatively, over-expressed.  
         [0031]    The terms “transfected” or “transformed” are used herein to include any host cell or cell line, the genome of which has been altered or augmented by the presence of at least one preselected DNA sequence, which DNA is also referred to in the art of genetic engineering as “heterologous DNA,” “recombinant DNA,” “exogenous DNA,” “genetically engineered DNA,” “non-native DNA,” or “foreign DNA,” wherein said DNA was isolated and introduced into the genome of the host cell or cell line by the process of genetic engineering. The host cells of the present invention are typically produced by transfection with a DNA sequence in a plasmid expression vector, a viral expression vector, or as an isolated linear DNA sequence. Preferably, the transfected DNA is a chromosomally integrated recombinant DNA sequence, which comprises a gene encoding an sErbB3 isoform, which host cell may or may not express significant levels of autologous or “native” ErbB3 isoforms.  
       DESCRIPTION OF THE INVENTION  
       [0032]    Using various recombinant truncated forms of EGFR, it has been shown that efficient inhibition of full-length EGFR activation by dominant-negative heterodimerization occurs only when these deletion mutants retain the transmembrane domain and the extracellular domain. Similarly, a recombinant dominant-negative ErbB3 mutant with a deleted cytoplasmic domain but which retains its transmembrane domain can inhibit full-length ErbB2 and ErbB3 activation. In contrast, in avian tissues, expression of a naturally occurring sEGFR/ErbB1 inhibits TGFA dependent transformation. Soluble EGFR secreted by the A431 human carcinoma cell line also has been reported to inhibit the kinase activity of purified full-length EGFR in a ligand-independent manner. These soluble EGF/ErbB1 receptors do not function as antagonists through high affinity ligand-binding. Similarly, herstatin, a naturally occurring soluble ErbB2 protein which inhibits ErbB2 activation appears to function by blocking ErbB2 dimerization; this inhibition is thought to be mediated via ligand-independent binding of herstatin to ErbB2. In contrast, sErbB3, including p85-sErbB3 and p45-sErbB3, inhibits HRG-induced stimulation of ErbB2, ErbB3, and ErbB4, at least in part, by neutralizing ligand activity through competitive binding. The present invention discloses that p85-sErbB3 is capable of binding to the cell surface.  
         [0033]    The physiological role of p85-sErbB3 in normal tissues also has not been understood. The present invention indicates that although a much higher concentration (100-fold) was required to inhibit cell growth, a 10-fold molar excess of p85-sErbB3 was sufficient for inhibition of phosphorylation of ErbB receptors. At this ratio, a small fraction of receptors are still activated are enough for growth stimulation. It is known that the 2.1 kb transcript encoding p85-sErbB3 is expressed at low levels compared to the full-length c-erbB3 transcript in all cell lines and tissues examined to date, however, local expression of this transcript has been studied in detail. It is, therefore, plausible that p85-sErbB3 acts as an HRG antagonist locally in a tissue-specific and/or stage-specific manner, and related studies to examine the distribution of p85-sErbB3 in selected tissues are currently underway. Evidence suggests that local concentrations of autocrine growth factors such as EGF are exquisitely regulated and do not travel far from the cell surface from which they are released. In this context, tightly regulated levels of local p85-sErbB3 expression have important consequences for HRG-mediated cell growth. These consequences are even more dramatic in cancer cells where cell polarity is typically lost, resulting in deregulation of normal spatial and temporal control of growth factor:receptor interactions.  
         [0034]    The present invention provides several novel isolated and purified nucleic acids which encode isoforms of ErbB3 and nucleic acids encoding engineered variants of these proteins. Preferred embodiments are nucleic acids which specifically encode isoforms of ErbB3 whose amino acid sequence comprises the sequence of SEQ ID NO: 1 and SEQ ID NO: 2. How about NO3 and 4?The present invention also defines the biochemical and biological characteristics of a novel sErbB3 isoform designated p85-sErbB3. The present invention discloses the use of p85-sErbB3 as a unique HRG inhibitor because it can block HRG binding to cell surface receptors via binding to HRG with high affinity, thereby, inhibiting HRG-induced stimulation of ErbB2, ErbB3, and ErbB4. This inhibition is sufficient to effectively block HRG-stimulated cell growth. This novel ErbB3 receptor isoform, therefore, is disclosed as a potent modulator of HRG regulated cell proliferation and differentiation in normal human tissues, and an ideal candidate for the development of novel cancer therapeutics.  
       EXAMPLES AND PREFERRED EMBODIMENTS  
       [0035]    Conditioned Media from Cells Expressing p45-sErbB3 and p85-sErbB3 Inhibit HRG Activation of ErbB3. p45-sErbB3 and p85-sErbB3 are naturally occurring secreted products of the ErbB3 gene (Lee and Maihle 1998). p45-sErbB3 contains the amino-terminal 310 amino acids of ErbB3 and two unique carboxy-terminal amino acid residues. p85-sErbB3 contains the amino-terminal 519 amino acids of ErbB3 and 24 unique carboxy-terminal amino acid residues (See FIG. 1). To examine whether p45-sErbB3 and p85-sErbB3 can modulate HRG receptor activation cells stably transfected with these corresponding cDNA clones were isolated. These cells secrete p45-sErbB3 and p85-sErbB3 into the culture medium (See FIG. 2A). The conditioned medium from these cells was used as the source of p45-sErbB3 or p85-sErbB3 in a series of preliminary experiments described below.  
         [0036]    To test the ability of p45-sErbB3 and p85-sErbB3 to modulate aspects of HRG-mediated ErbB receptor activation a clonal derivative of the Ba/F3 cell line expressing exogenous ErbB2 and ErbB3 was stimulated with HRGβ EGF domain 177-241  (HRGα) and HRGβ1 1176 -246 (HRGβ) in the absence or presence of concentrated conditioned media containing p45-sErbB3 and p85-sErbB3. As shown in FIG. 2, HRGβ was at least 20-fold more effective than HRGα in stimulating ErbB3 tyrosine phosphorylation. Conditioned media containing sErbB3 inhibited HRGα-stimulated ErbB3 activation by 40% (p45-sErbB3) and 80% (p85-sErbB3) at 1 μg/ml HRGA, as determined by densitometric analysis. However, at a higher concentration (2 μg/ml), conditioned media containing p85-sErbB3 decreased activation by 30%, although inhibition by conditioned media containing p45-sErbB3 was negligible (See FIG. 2A). In the presence of conditioned medium containing either p45-sErbB3 or p85-sErbB3, ligand stimulation of ErbB3 tyrosine phosphorylation was decreased by 60% and 90%, respectively, at both 50 and 100 ng/ml HRGβ (See FIG. 2C). These data indicate that p85-sErbB3 inhibited ErbB3 phosphorylation in response to both HRGα and HRGβ more effectively than p45-sErbB3, although the concentration of p85-sErbB3 used in these studies was lower than that of p45-sErbB3 (FIG. 2A).  
         [0037]    Purification of p85-sErbB3. p85-sErbB3 was isolated from a concentrated conditioned medium of cells stably transfected with a cDNA clone R31F encoding p85-sErbB3 (ref) and was purified in two steps. The first step was lectin affinity chromatography with a Con A column (Sigma). The bound p85-sErbB3 was washed with column buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM MnCl 2 , and 1 mM CaCl 2 ) and eluted using column buffer containing 1 M α-methyl D-mannoside, then dialyzed against 20 mM Tris-HCl, pH 7.5 overnight. The second step of purification was accomplished using a Mono Q ion exchange FPLC column (Pharmacia). The bound p85-sErbB3 was eluted from the column with 0-500 mM NaCl gradient containing 20 mM Tris-HCl, pH 7.5. Samples taken from each step were subjected to SDS-PAGE in duplicate and analyzed by Coomassie staining and by Western blot using anti-ErbB3 236 antibody recognizing the extracellular domain of the ErbB3 (Lee and Maihle 1998). The final p85-sErbB3 pool was homogeneous on SDS-PAGE, and the identity of the purified protein was confirmed by Western blot analysis. Purified preparations of p85-sErbB3 were used in all subsequent experiments.  
         [0038]    p85-sErbB3 Binds to HRG with High Affinity. Previous reports based the assignation of the subdomain boundaries of the ErbB3 extracellular domain on the subdomain boundaries of EGFR (Lee and Maihle 1998) as defined by the genomic structure of avian ErbB1(Callaghan, Antczak et al. 1993). Accordingly, p85-sErbB3 is composed of subdomains I through III and includes the first 45 amino acids of subdomain IV (aa 1-519), and a unique twenty-four amino acid sequence at the carboxy-terminus. Binding studies using heregulins indicate that subdomains I and II are required for heregulin binding (Singer, Landgraf et al. 2001). On the other hand, for EGF binding to EGFR subdomains I and III are low and high affinity binding sites, respectively (Lax, Bellot et al. 1989). Because p85-sErbB3 contains both subdomains I through III the present invention determined that p85-sErbB3 would bind to heregulins.  
         [0039]    Direct binding between p85-sErbB3 and radiolabeled HRGβ was examined using the chemical crosslinker BS 3 . As shown in FIG. 3A, a protein complex of 90 kDa was formed between p85-sErbB3 and  125 I-HRGβ. Formation of this complex could be inhibited by addition of excess cold HRGβ but not by addition of excess insulin, indicating that p85-sErbB3 binding to HRGβ is specific and that purified preparations of p85-sErbB3 are biologically active. An analysis of  125 I-HRGβ 177-244  binding to immobilized p85-sErbB3 was then performed using an ErbB3-IgG homodimer as a positive control. As shown in FIG. 3, p85-sErbB3 binds to HRGβ 177-244  with a K D  of 3.0±0.2 nM. In comparison, ErbB3-IgG binds to HRGβ 177-244  with a K D  of 4.7±0.2 nM. These results demonstrate that p85-sErbB3 binds to HRGβ 177-244  with an affinity similar to that of the extracellular domain of ErbB3. Based on the results of these two complementary experimental approaches, the present invention establishes the use of p85-sErbB3 to bind to HRG with an affinity equivalent to the affinity of HRG for the full-length extracellular domain of ErbB3.  
         [0040]    p85-sErbB3 Inhibits Binding of HRG to Receptors on the Cell Surface. The present invention also discloses the use of p85-sErbB3 to effectively limit binding of heregulin to cell surface receptors in the breast carcinoma cell line T47D. This cell line expresses all four ErbB receptors at moderate levels. Cells were incubated with varying concentrations of p85-sErbB3 in the presence of  125 I-labeled HRGβ 177-244 . Simultaneously, a separate group of cells was incubated with  125 I-HRGβ 177-244  in the presence of varying concentrations of 2C4, a monoclonal antibody specific for the ErbB2 extracellular domain (Lewis, Lofgren et al. 1996). As shown by the inhibition curves (See FIG. 4), p85-sErbB3 and 2C4 inhibit HRGβ 177-244  binding to cell surface receptors with similar IC 50  values (0.45±0.03 nM and 0.55±0.03 nM, respectively) although the mechanism of inhibition by these two molecules is distinct. Although 2C4 inhibits heregulin binding to cell surface receptors by blocking ErbB2-ErbB3 heterodimerization via binding to the ErbB2 extracellular domain (Fitzpatrick, Pisacane et al. 1998), p85-sErbB3 inhibited receptor activation, at least in part, by competing for heregulin binding to the cell surface.  
         [0041]    p85-sErbB3 Blocks HRG-Induced Activation of ErbB2, ErbB3, and ErbB4. The present invention also examined the ability of p85-sErbB3 to modulate HRG-stimulated receptor activation in the Ba/F3 (ErbB2+ErbB3) cell line using purified p85-sErbB3. This allowed an analysis of the mechanism of p85-sErbB3 mediated inhibition in a quantitative manner. As shown in FIG. 5, when Ba/F3 (ErbB2+ErbB3) cells were treated with p85-sErbB3 at a 10-fold molar excess over HRGβ 1-241 , ErbB3 phosphorylation levels were reduced to basal levels. A similar level of receptor inhibition also was apparent when either a 2.5- or 5-fold molar excess of p85-sErbB3 was used in these experiments. Exogenous addition of p85-sErbB3 also inhibited HRG-induced ErbB2 activation. p85-sErbB3 blocked HRG stimulation whether the cells were treated with the EGF-like domain of HRG (HRGα or HRGβ), as shown in FIG. 2, or with HRGβ 1-241  (See FIG. 5), suggesting that inhibition by p85-sErbB3 occurs, at least in part, through a direct interaction between p85-sErbB3 and the EGF-like domain of HRG. Cells treated with the same concentration of p85-sErbB3 but not stimulated with HRG did not exhibit altered ErbB2 or ErbB3 tyrosine phosphorylation, or show any change in the level of either ErbB2 or ErbB3 expression, suggesting that p85-sErbB3 does not function as a “ligand” for these receptors.  
         [0042]    To examine whether exogenous addition of p85-sErbB3 exerts the same inhibitory effect on endogenously expressed ErbB receptors, and to determine whether p85-sErbB3 could modulate other members of the EGF receptor family, the activity of p85-sErbB3 in two breast carcinoma cell lines, i.e., T47D and MCF7, was tested. As shown in FIG. 6A, addition of p85-sErbB3 (at a 6-fold molar excess relative to HRGβ) inhibited HRG-induced activation of ErbB2, ErbB3, and ErbB4 in both the T47D and MCF7 cell lines. In contrast, at least in these two cell lines which express low EGFR levels, EGFR phosphorylation remained at basal level in cells treated with HRGβ regardless of whether p85-sErbB3 was present or not. Similarly, EGF-induced phosphorylation of EGFR or ErbB2, or, to a lesser degree, phosphorylation of ErbB3, was not decreased by p85-sErbB3. These results demonstrate that inhibition by p85-sErbB3 is specific for HRG-induced activation of ErbB2, ErbB3, and ErbB4.  
         [0043]    It is notable that in the T47D cells, a decrease in ErbB2, ErbB3, and ErbB4 protein levels following HRG stimulation was observed. In MCF7 cells a decrease in ErbB3 levels also was apparent when HRG was added to the culture medium (See FIG. 6A). It has been reported that the polyclonal ErbB3 antibody specific to the carboxy-terminal 17 aa used in this study preferentially recognizes non-phosphorylated ErbB3 on Western blots (Vartanian, Goodearl et al. 1997). Thus, when T47D or MCF7 cells are stimulated with HRG, a significant fraction of ErbB3 is phosphorylated, and, therefore, undetectable with this particular ErbB3 antibody. The anti-ErbB antibodies used in these experiments recognize the carboxy-terminal 17 aa (ErbB3) and 18 aa (ErbB2 and ErbB4) sequences of these receptors. Each of these sequences contains one tyrosine residue. Immunoblot detection by the anti-ErbB2 and ErbB4 antibodies used in this study, therefore, may reflect either the level of receptor expression or the unphosphorylated fraction of these receptors.  
         [0044]    p85-sErbB3 Inhibits Activation of Downstream Effectors of HRG. HRG-stimulated activation of ErbB2, ErbB3, and ErbB4 leads to activation of two major signal transduction pathways: the PI3K pathway and the MAPK pathway (Wallasch, Weiss et al. 1995). The present invention tested whether p85-sErbB3 could inhibit activation of these two downstream effector pathways in T47D cells. Specifically, the present invention examined activation of MAPK and Akt by analyzing the phosphorylation levels of these proteins, and examined the ability of p85 phosphatidylinositide 3-kinase (“PI3K”) to interact with ErbB3 following HRGβ treatment. In the presence of p85-sErbB3 (10-fold molar excess relative to HRGβ), tyrosine phosphorylation of ErbB3 was reduced to basal levels. In the same cell population, addition of exogenous p85-sErbB3 abrogated the phosphorylation of both MAPK and Akt as determined by Western blot analysis, and inhibited ErbB3&#39;s association with p85 PI3K (See FIG. 6B). These results further demonstrate that p85-sErbB3 can inhibit the activation of ErbB2, ErbB3, and ErbB4, and this inhibition affects the activation of downstream signaling molecules such as MAPK, Akt, and PI3K.  
         [0045]    p85-sErbB3 Inhibits HRG-stimulated Cell Growth. The present invention also discloses the inhibition of HRG-induced phosphorylation of ErbB receptors by p85-sErbB3 as correlated with the modulation of HRG&#39;s biological effects. Specifically, a cell growth assay using MCF7 cells stimulated with HRGβ was performed and showed that, within the concentration range  
       REFERENCES  
       [0046]    Alimandi, M., M. Heidaran, et al. (1995). “Cooperative signaling of ErbB3 and ErbB2 in neoplastic transformation and human mammary carcinomas.”  Oncogene  10: 1813-1821.  
         [0047]    Alroy, I. and Y. Yarden (1997). “The ErbB signaling network in embryogenesis and oncogenesis: signal diversification through combinatorial ligand-receptor interactions.”  FEBS Lett.  410(1): 83-86.  
         [0048]    Basu, A., M. Raghunath, et al. (1989). “Inhibition of tyrosine kinase activity of the epidermal growth factor (EGF) receptor by a truncated receptor form that binds to EGF: role for interreceptor interaction in kinase regulation.”  Mol. Cell. Biol.  9(2): 671-677.  
         [0049]    Callaghan, T., M. Antczak, et al. (1993). “A complete description of the EGF-receptor exon structure: implication in oncogenic activation and domain evolution.”  Oncogene  8: 2939-2948.  
         [0050]    Carraway, K. L. I. and S. J. Burden (1995). “Neuregulins and their receptors.”  Current Opinion in Neurobiology  5: 606-612.  
         [0051]    Doherty, J. K., C. Bond, et al. (1999). “The HER-2/neu receptor tyrosine kinase gene encodes a secreted autoinhibitor.”  Proc. Natl. Acad. Sci. USA  96(19): 10869-10874.  
         [0052]    Falls, D. L., K. M. Rosen, et al. (1993). “ARIA, a protein that stimulates acetylcholine receptor synthesis, is a member of the neu ligand family.”  Cell  72(5): 801-15.  
         [0053]    Fitzpatrick, V. D., P. I. Pisacane, et al. (1998). “Formation of a high affinity heregulin binding site using the soluble extracellular domains of ErbB2 with ErbB3 or ErbB4 .” FEBS Lett.  431(1): 102-106.  
         [0054]    Flickinger, T. W., N. J. Maihle, et al. (1992). “An alternatively processed mRNA from the avian c-erbB gene encodes a soluble, truncated form of the receptor that can block ligand-dependent transformation.”  Mol. Cell. Biol.  12(2): 883-893.  
         [0055]    Hijazi, M. M., E. W. Thompson, et al. (2000). “Heregulin regulates the actin cytoskeleton and promotes invasive properties in breast cancer cell lines.”  International Journal of Oncology  17(4): 629-41.  
         [0056]    Holmes, W. E., M. X. Sliwkowski, et al. (1992). “Identification of heregulin, a specific activator of p185erbB2 .” Science  256(5060): 1205-1210.  
         [0057]    Katoh, M., Y. Yazaki, et al. (1993). “c-erbB3 gene encodes secreted as well as transmembrane receptor tyrosine kinase.”  Biochem. Biophys. Res. Commun.  192(3): 1189-1197.  
         [0058]    Krane, I. M. and P. Leder (1996). “NDF/heregulin induces persistence of terminal end buds and adenocarcinomas in the mammary glands of transgenic mice.”  Oncogene  12(8): 1781-1788.  
         [0059]    Kraus, M. H., W. Issing, et al. (1989). “Isolation and characterization of ERBB3, a third member of the ERBB/epidermal growth factor receptor family: evidence for overexpression in a subset of human mammary tumors.”  Proc. Natl. Acad. Sci. USA  86: 9193-9197.  
         [0060]    Lax, I., F. Bellot, et al. (1989). “Functional analysis of the ligand binding site of EGF-receptor utilizing chimeric chicken/human receptor molecules.”  EMBO J.  8(2): 421-427.  
         [0061]    Lee, H. and N. J. Maihle (1998). “Isolation and characterization of four alternate c-erbB3 transcripts expressed in ovarian carcinoma-derived cell lines and normal human tissues.”  Oncogene  16(25): 3243-3252.  
         [0062]    Lewis, G. D., J. A. Lofgren, et al. (1996). “Growth regulation of human breast and ovarian tumor cells by heregulin: Evidence for the requirement of ErbB2 as a critical component in mediating heregulin responsiveness.”  Cancer Res.  56(6): 1457-1465.  
         [0063]    Marchionni, M. A., A. D. Goodearl, et al. (1993). “Glial growth factors are alternatively spliced erbB2 ligands expressed in the nervous system.”  Nature  362(6418): 312-8.  
         [0064]    Meyer, D. and C. Birchmeier (1995). “Multiple essential functions of neuregulin in development [see comments] [published erratum appears in Nature Dec. 14, 1995;378(6558):753 ].” Nature  378(6555): 386-390.  
         [0065]    Peles, E., S. S. Bacus, et al. (1992). “Isolation of the neu/HER-2 stimulatory ligand: a 44 kd glycoprotein that induces differentiation of mammary tumor cells.”  Cell  69(1): 205-16.  
         [0066]    Plowman, G. D., J. M. Culouscou, et al. (1993). “Ligand-specific activation of HER4/p180erbB4, a fourth member of the epidermal growth factor receptor family.”  Proc. Natl. Acad. Sci. USA  90(5): 1746-1750.  
         [0067]    Plowman, G. D., G. S. Whitney, et al. (1990). “Molecular cloning and expression of an additional epidermal growth factor receptor-related gene.”  Proc. Natl. Acad. Sci. USA  87(13): 4905-4909.  
         [0068]    Ram, T. G., M. E. Schelling, et al. (2000). “Blocking HER-2/HER-3 function with a dominant negative form of HER-3 in cells stimulated by heregulin and in breast cancer cells with HER-2 gene amplification.”  Cell Growth Differ.  11(3): 173-183.  
         [0069]    Redemann, N., B. Holzmann, et al. (1992). “Anti-oncogenic activity of signalling-defective epidermal growth factor receptor mutants.”  Mol. Cell. Biol.  12(2): 491-498.  
         [0070]    Robinson, D., F. He, et al. (1996). “A tyrosine kinase profile of prostate carcinoma.”  Proc. Natl. Acad. Sci. USA  93(12): 5958-5962.  
         [0071]    Siegel, P. M., E. D. Ryan, et al. (1999). “Elevated expression of activated forms of Neu/ErbB-2 and ErbB-3 are involved in the induction of mammary tumors in transgenic mice: implications for human breast cancer.”  EMBO Journal  18(8): 2149-2164.  
         [0072]    Singer, E., R. Landgraf, et al. (2001). “Identification of a heregulin binding site in HER3 extracellular domain.”  Journal of Biological Chemistry  276(47): 44266-74.  
         [0073]    Sundaresan, S., E. Penuel, et al. (1999). “The biology of human epidermal growth factor receptor 2 .” Curr. Oncol. Report  1: 16-22.  
         [0074]    Tsai, M. S., A. E. Hornby, et al. (2000). “Expression and function of CYR61, an angiogenic factor, in breast cancer cell lines and tumor biopsies.”  Cancer Research  60(20): 5603-7.  
         [0075]    Vartanian, T., A. Goodearl, et al. (1997). “Axonal Neuregulin Signals Cells of the Oligodendrocyte Lineage through Activation of HER4 and Schwann Cells through HER2 and HER3 .” J. Cell Biol.  137: 211.  
         [0076]    Wallasch, C., F. U. Weiss, et al. (1995). “Heregulin-dependent regulation of HER2/neu oncogenic signaling by heterodimerization with HER3 .” EMBO J.  14(17): 4267-4275.  
         [0077]    Wen, D., E. Peles, et al. (1992). “Neu differentiation factor: a transmembrane glycoprotein containing an EGF domain and an immunoglobulin homology unit.”  Cell  69(3): 559-72.  
         [0078]    p85-sErbB3 protein  
                           AA 1-538: The N-terminal ErbB3 sequence           AA 1-19: signal peptide       AA 539-562: p85-sErbB3 unique sequence                                   10                             20       MetArgAlaAsnAspAlaLeuGlnValLeu GlyLeuLeuPheSerLeuAlaArgGlySer&gt;                                           30                             40       GluValGlyAsnSerGlnAlaValCysPro GlyThrLeuAsnGlyLeuSerValThrGly&gt;                                           50                             60       AspAlaGluAsnGlnTyrGlnThrLeuTyr LysLeuTyrGluArgCysGluValValMet&gt;                                           70                             80       GlyAsnLeuGluIleValLeuThrGlyHis AsnAlaAspLeuSerPheLeuGlnTrpIle&gt;                                           90                            100       ArgGluValThrGlyTyrValLeuValAla MetAsnGluPheSerThrLeuProbeuPro&gt;                                          110                            120       AsnLeuArgValValArgGlyThrGlnVal TyrAspGlyLysPheAlaIlePheValMet&gt;                                          130                            140       LeuAsnTyrAsnThrAsnSerSerHisAla LeuArgGlnLeuArgLeuThrGlnLeuThr&gt;                                          150                            160       GluIleLeuSerGlyGlyValTyrIleGlu LysAsnAspLysLeuCysHisMetAspThr&gt;                                          170                            180       IleAspTrpArgAspIleValArgAspArg AspAlaGluIleValValLysAspAsnGly&gt;                                          190                            200       ArgSerCysProProCysHisGluValCys LysGlyArgCysTrpGlyProGlySerGlu&gt;                                          210                            220       AspCysGlnThrLeuThrLysThrIleCys AlaProGlnCysAsnGlyHisCysPheGly&gt;                                          230                            240       ProAsnProAsnGlnCysCysHisAspGlu CysAlaGlyGlyCysSerGlyProGlnAsp&gt;                                          250                            260       ThrAspCysPheAlaCysArgHisPheAsn AspSerGlyAlaCysValProArgCysPro&gt;                                          270                            280       GlnProLeuValTyrAsnLysLeuThrPhe GlnLeuGluProAsnProHisThrLysTyr&gt;                                          290                            300       GlnTyrGlyGlyValCysValAlaSerCys ProHisAsnPheValValAspGlnThrSer&gt;                                          310                            320       CyaValArgAlaCysProProAspLysMet GluValAspLysAsnGlyLeuLysMetCys&gt;                                          330                            340       GluProCysGlyGlyLeuCysProLysAla CysGluGlyThrGlySerGlySerArgPhe&gt;                                          350                            360       GlnThrValAspSerSerAsnIleAspGly PheValAsnCysThrLysIleLeuGlyAsn&gt;                                          370                            380       LeuAspPheLeuIleThrGlyLeuAsnGly AspProTrpHisLysIleProAlaLeuAsp&gt;                                          390                            400       ProGluLysLeuAsnValPheArgThrVal ArgGluIleThrGlyTyrLeuAsnIleGln&gt;                                          410                            420       SerTrpProProHisMetHisAsnPheSer ValPheSerAsnLeuThrThrIleGlyGly&gt;                                          430                            440       ArgSerLeuTyrAsnArgGlyPheSerLeu LeuIleMetLysAsnLeuAsnValThrSer&gt;                                          450                            460       LeuGlyPheArgSerLeuLysGluIleSer AlaGlyArgIleTyrIleSerAlaAsnArg&gt;                                          470                            480       GlnLeuCysTyrHisHisSerLeuAsnTrp ThrLysValLeuArgGlyProThrGluGlu&gt;                                          490                            500       ArgLeuAspIleLysHisAsnArgProArg ArgAspCysValAlaGluGlyLysValCys&gt;                                          510                            520       AspProLeuCysSerSerGlyGlyCysTrp GlyProGlyProGlyGlnCysLeuSerCys&gt;                                          530                            540       ArgAsnTyrSerArgGlyGlyValCysVal ThrHisCysAsnPheLeuAsnGlyTyrSer&gt;                                          550                            560       LysGlySerGlnSerArgMetGlyGlyGly GlyAlaLeuGlnTrpAsnCysSerGlyGly&gt;                  562       IleGln               p85-sErbB3 cDNA sequence (R31F)       nt 1-1673: The N-terminal ErbB3 sequence       nt 1674-1761: unique to p85-sErbB3               10        20         30        40         50        60       cgggccccccctcgaggtcg ggccggacttggctgggctc ccttcaccctctgcggagtc                       70        80         90       100        110       120       ATGAGGGCGAACGACGCTCT GCAGGTGCTGGGCTTGCTTT TCAGCCTGGCCCGGGGCTCC                      130       140        150       160        170       180       GAGGTGGGCAACTCTCAGGC AGTGTGTCCTGGGACTCTGA ATGGCCTGAGTGTGACCGGC                      190       200        210       220        230       240       GATGCTGAGAACCAATACCA GACACTGTACAAGCTCTACG AGAGGTGTGAGGTGGTGATG                      250       260        270       280        290       300       GGGAACCTTGAGATTGTGCT CACGGGACACAATGCCGACC TCTCCTTCCTGCAGTGGATT                      310       320        330       340        350       360       CGAGAAGTGACAGGCTATGT CCTCGTGGCCATGAATGAAT TCTCTACTCTACCATTGCCC                      370       380        390       400        410       420       AACCTCCGCGTGGTGCGAGG GACCCAGGTCTACGATGGGA AGTTTGCCATCTTCGTCATG                      430       440        450       460        470       480       TTGAACTATAACACCAACTC CAGCCACGCTCTGCGCCAGC TCCGCTTGACTCAGCTCACC                      490       500        510       520        530       540       GAGATTCTGTCAGGGGGTGT TTATATTGAGAAGAACGATA AGCTTTGTCACATGGACACA                      550       560        570       580        590       600       ATTGACTGGAGGGACATCGT GAGGGACCGAGATGCTGAGA TAGTGGTGAAGGACAATGGC                      610       620        630       640        650       660       AGAAGCTGTCCCCCCTGTCA TGAGGTTTGCAAGGGGCGAT GCTGGGGTCCTGGATCAGAA                      670       680        690       700        710       720       GACTGCCAGACATTGACCAA GACCATCTGTGCTCCTCAGT GTAATGGTCACTGCTTTGGG                      730       740        750       760        770       780       CCCAACCCCAACCAGTGCTG CCATGATGAGTGTGCCGGGG GCTGCTCAGGCCCTCAGGAC                      790       800        810       820        830       840       ACAGACTGCTTTGCCTGCCG GCACTTCAATGACAGTGGAG CCTGTGTACCTCGCTGTCCA                      850       860        870       880        890       900       CAGCCTCTTGTCTACAACAA GCTAACTTTCCAGCTGGAAC CCAATCCCCACACCAAGTAT                      910       920        930       940        950       960       CAGTATGGAGGAGTTTGTGT AGCCAGCTGTCCCCATAACT TTGTGGTGGATCAAACATCC                      970       980        990      1000       1010      1020       TGTGTCAGGGCCTGTCCTCC TGACAAGATGGAAGTAGATA AAAATGGGCTCAAGATGTGT                     1030      1040       1050      1060       1070      1080       GAGCCTTGTGGGGGACTATG TCCCAAAGCCTGTGAGGGAA CAGGCTCTGGGAGCCGCTTC                     1090      1100       1110      1120       1130      1140       CAGACTGTGGACTCGAGCAA CATTGATGGATTTGTGAACT GCACCAAGATCCTGGGCAAC                     1150      1160       1170      1180       1190      1200       CTGGACTTTCTGATCACCGG CCTCAATGGAGACCCCTGGC ACAAGATCCCTGCCCTGGAC                     1210      1220       1230      1240       1250      1260       CCAGAGAAGCTCAATGTCTT CCGGACAGTACGGGAGATCA CAGGTTACCTGAACATCCAG                     1270      1280       1290      1300       1310      1320       TCCTGGCCGCCCCACATGCA CAACTTCAGTGTTTTTTCCA ATTTGACAACCATTGGAGGC                     1330      1340       1350      1360       1370      1380       AGAAGCCTCTACAACCGGGG CTTCTCATTGTTGATCATGA AGAACTTGAATGTCACATCT                     1390      1400       1410      1420       1430      1440       CTGGGCTTCCGATCCCTGAA GGAAATTAGTGCTGGGCGTA TCTATATAAGTGCCAATAGG                     1450      1460       1470      1480       1490      1500       CAGCTCTGCTACCACCACTC TTTGAACTGGACCAAGGTGC TTCGGGGGCCTACGGAAGAG                     1510      1520       1530      1540       1550      1560       CGACTAGACATCAAGCATAA TCGGCCGCGCAGAGACTGCG TGGCAGAGGGCAAAGTGTGT                     1570      1580       1590      1600       1610      1620       GACCCACTGTGCTCCTCTGG GGGATGCTGGGGCCCAGGCC CTGGTCAGTGCTTGTCCTGT                     1630      1640       1650      1660       1670      1680       CGAAATTATAGCCGAGGAGG TGTCTGTGTGACCCACTGCA ACTTTTTGAATGGGTACAGT                     1690      1700       1710      1720       1730      1740       AAGGGGAGCCAGTCAAGGAT GGGTGGGGGTGGGGCCCTGC AATGGAACTGTTCAGGTGGC                     1750      1760       1770      1780       1790      1800       ATACAATAAaagtctttaga caaaaaaaaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaa          
 
         [0079]    (1) Information for SEQ ID NO:2:  
         [0080]    (i) Sequence characteristics:  
         [0081]    (ii) Length  
         [0082]    (iii) Type  
         [0083]    (iv) Molecule type  
         [0084]    (v) Original source:  
         [0085]    (vi) Organism: homo sapience  
                           p45-sErbB3 protein           AA 1-329: The N-terminal ErbB3 sequence       AA 1-19: signal peptide       AA 330-331: p45-sErbB3 unique sequence       10                             20       MetArgAlaAsnAspAlaLeuGlnValLeu GlyLeuLeuPheserLeuAlaArgGlySer&gt;                                           30                             40       GluValGlyAsnSerGlnAlaValCysPro GlyThrLeuAsnGlyLeuSerValThrGly&gt;                                           50                             60       AspAlaGluAsnGlnTyrGlnThrLeuTyr LysLeuTyrGluArgCysGluValValMet&gt;                                           70                             80       GlyAsnLeuGluIleValLeuThrGlyHis AsnAlaAspLeuSerPheLeuGlnTrpIle&gt;                                           90                            100       ArgGluValThrGlyTyrValLeuValAla MetAsnGluPheSerThrLeuProLeuPro&gt;                                          110                            120       AsnLeuArgValValArgGlyThrGlnVal TyrAspGlyLysPheAlaIlePheValMet&gt;                                          130                            140       LeuAsnTyrAsnThrAsnSerserHisAla LeuArgGlnLeuArgLeuThrGlnLeuThr&gt;                                          150                            160       GluIleLeuSerGlyGlyValTyrIleGlu LysAsnAspLysLeuCysHisMetAspThr&gt;                                          170                            180       IleAspTrpArgAspIleValArgAspArg AspAlaGluIleValValLysAspAsnGly&gt;                                          190                            200       ArgSerCysProProCysHisGluValCys LysGlyArgCysTrpGlyProGlySerGlu&gt;                                          210                            220       AspCysGlnThrLeuThrLysThrIleCys AlaProGlnCysAsnGlyHisCysPheGly&gt;                                          230                            240       ProAsnProAsnGlnCysCysHisAspGlu CysAlaGlyGlyCysSerGlyProGlnAsp&gt;                                          250                            260       ThrAspCysPheAlaCysArgHisPheAsn AspSerGlyAlaCysValProArgCysPro&gt;                                          270                            280       GlnProLeuValTyrAsnLysLeuThrPhe GlnLeuGluProAsnProHisThrLysTyr&gt;                                          290                            300       GlnTyrGlyGlyValCysValAlaSerCys ProHisAsnPheValValAspGlnThrSer&gt;                                          310                            320       CysValArgAlaCysProProAspLysMet GluValAspLysASnGlyLeuLysMetCys&gt;                                          330       GluProCysGlyGlyLeuCysProLysGly Gly               P45-sErbB3 cDNA sequence (R2F)       nt 1-1048: The N-terminal ErbB3 sequence       nt 1049-1056: unique to p45-sErbB3       cgggccccccctcgaggtcg ggccggacttggctgggctc ccttcaccctctgcggagtc               70        80         90       100        110       120       ATGAGGGCGAACGACGCTCT GCAGGTGCTGGGCTTGCTTT TCAGCCTGGCCCGGGGCTCC                      130       140        150       160        170       180       GAGGTGGGCAACTCTCAGGC AGTGTGTCCTGGGACTCTGA ATGGCCTGAGTGTGACCGGC                      190       200        210       220        230       240       GATGCTGAGAACCAATACCA GACACTGTACAAGCTCTACG AGAGGTGTGAGGTGGTGATG                      250       260        270       280        290       300       GGGAACCTTGAGATTGTGCT CACGGGACACAATGCCGACC TCTCCTTCCTGCAGTGGATT                      310       320        330       340        350       360       CGAGAAGTGACAGGCTATGT CCTCGTGGCCATGAATGAAT TCTCTACTCTACCATTGCCC                      370       380        390       400        410       420       AACCTCCGCGTGGTGCGAGG GACCCAGGTCTACGATGGGA AGTTTGCCATCTTCGTCATG                      430       440        450       460        470       480       TTGAACTATAACACCAACTC CAGCCACGCTCTGCGCCAGC TCCGCTTGACTCAGCTCACC                      490       500        510       520        530       540       GAGATTCTGTCAGGGGGTGT TTATATTGAGAAGAACGATA AGCTTTGTCACATGGACACA                      550       560        570       580        590       600       ATTGACTGGAGGGACATCGT GAGGGACCGAGATGCTGAGA TAGTGGTGAAGGACAATGGC                      610       620        630       640        650       660       AGAAGCTGTCCCCCCTGTCA TGAGGTTTGCAAGGGGCGAT GCTGGGGTCCTGGATCAGAA                      670       680        690       700        710       720       GACTGCCAGACATTGACCAA GACCATCTGTGCTCCTCAGT GTAATGGTCACTGCTTTGGG                      730       740        750       760        770       780       CCCAACCCCAACCAGTGCTG CCATGATGAGTGTGCCGGGG GCTGCTCAGGCCCTCAGGAC                      790       800        810       820        830       840       ACAGACTGCTTTGCCTGCCG GCACTTCAATGACAGTGGAG CCTGTGTACCTCGCTGTCCA                      850       860        870       880        890       900       CAGCCTCTTGTCTACAACAA GCTAACTTTCCAGCTGGAAC CCAATCCCCACACCAAGTAT                      910       920        930       940       950       960       CAGTATGGAGGAGTTTGTGT AGCCAGCTGTCCCCATAACT TTGTGGTGGATCAAACATCC                      970       980        990      1000       1010      1020       TGTGTCAGGGCCTGTCCTCC TGACAAGATGGAAGTAGATA AAAATGGGCTCAAGATGTGT                     1030      1040       1050      1060       1070      1080       GAGCCTTGTGGGGGACTATG TCCCAAAGGTGGGTAGgaga tggtaagaagttgtaaagag                     1090     1100        1110      1120       1130      1140       acagcctttcctctgagcct gcgcagaccacccccactga acctctcttacatttgcagc                     1150      1160       1170      1180       1190      1200       ctgtgagggaacaggctctg ggagccgcttccagactgtg gactcgagcaacattgatgg                     1210      1220       1230      1240       1250      1260       atttgtgaactgcaccaaga tcctgggcaacctggacttt ctgatcaccggcctcaatgg                     1270      1280       1290      1300       1310      1320       gttagagatcctgccttccc tccttagaccccagcccacg cacccctcacagttcatttc                     1330      1340       1350      1360       1370      1380       attggccaaaactttcctat gtggagctgactaggaatca aagtcataaaattctagcct                     1390      1400       1410      1420       gttacaaaggaaaaaaaaaa aaaaaaaaaaaaaaaaaaaa          
 
         [0086]    (2) Information for SEQ ID NO:3:  
         [0087]    (i) Sequence characteristics:  
         [0088]    (ii) Length  
         [0089]    (iii) Type  
         [0090]    (iv) Molecule type  
         [0091]    (v) Original source:  
         [0092]    (vi) Organism: homo sapience  
                           p75-sErbB3 protein (eDNA clone R35F)           AA 1-493: The N-terminal ErbB3 sequence       AA 1-19: signal peptide       AA 494-534: p75-sErbB3 unique sequence       ````````````````````````````10`````````````````````````````20       MetArgAlaAsnAspAlaLeuGlnValLeu GlyLeuLeuPheSerLeuAlaArgGlySer&gt;               ````````````````````````````30`````````````````````````````40       GluValGlyAsnSerGlnAlaValCysPro GlyThrLeuAsnGlyLeuSerValThrGly&gt;               ````````````````````````````50`````````````````````````````60       AspAlaGluASnGlnTyrGlnThrLeuTyr LysLeuTyrGluArgCysGluValValMet&gt;               ````````````````````````````70`````````````````````````````80       GlyAsnLeuGluIleValLeuThrGlyHis AsnAlaAspLeuSerPheLeuGlnTrpIle&gt;               ````````````````````````````90````````````````````````````100       ArgGluValThrGlyTyrValLeuValAla MetAsnGluPheSerThrLeuProLeuPro&gt;               ```````````````````````````110````````````````````````````120       AsnLeuArgValValArgGlyThrGlnVal TyrAspGlyLysPheAlaIlePheValMet&gt;               ```````````````````````````130````````````````````````````140       LeuAsnTyrAsnThrAsnSerSerHisAla LeuArgGlnLeuArgLeuThrGlnLeuThr&gt;               ```````````````````````````150````````````````````````````160       GluIleLeuSerGlyGlyValTyrIleGlu LysAsnAspLysLeuCysHisMetAspThr&gt;               ```````````````````````````170````````````````````````````180       IleAspTrpArgAspIleValArgAspArg AspAlaGluIleValValLysAspAnsGly&gt;               ```````````````````````````190````````````````````````````200       ArgSerCysProProCysHisGluValCys LysGlyArgCysTrpGlyProGlySerGlu&gt;               ```````````````````````````210````````````````````````````220       AspCysGlnThrLeuThrLysThrIleCys AlaProGlnCysAsnGlyHisCysPheGly&gt;               ```````````````````````````230````````````````````````````240       ProAsnProAsnGlnCysCysHisAspGlu CysAlaGlyGlyCysSerGlyProGlnAsp&gt;               ```````````````````````````250````````````````````````````260       ThrAspCysPheAlaCysArgHisPheAsn AspSerGlyAlaCysValProArgCysPro&gt;               ```````````````````````````270````````````````````````````280       GlnProLeuValTyrAsnLySLeuThrPhe GlnLeuGluProAsnProHisThrLysTyr&gt;               ```````````````````````````290````````````````````````````300       GlnTyrGlyGlyValCysValAlaSerCys ProHisAsnPheValValAspGlnThrSer&gt;               ```````````````````````````310````````````````````````````320       CysValArqAlaCysProProAspLysMet GluValAspLysAsnGlyLeuLysMetCys&gt;               ```````````````````````````330````````````````````````````340       GluProCysGlyGlyLeuCysProLysAla CysGluGlyThrGlySerGlySerArgPhe&gt;               ```````````````````````````350````````````````````````````360       GlnThrValAspSerSerAsnIleAspGly PheValAsnCysThrLysIleLeuGlyAsn&gt;               ```````````````````````````370````````````````````````````380       LeuAspPheLeuIleThrGlyLeuAsnGly AspProTrpHisLysIleProAlaLesAsp&gt;               ```````````````````````````390````````````````````````````400       ProGluLysLeuAsnValPheArgThrVal ArgGluIleThrGlyTyrLeuAsnIleGln&gt;               ```````````````````````````410````````````````````````````420       SerTrpProProHisMetHisAsnPheSer ValPheSerAsnLeuThrThrIleGlyGly&gt;               ```````````````````````````430````````````````````````````440       ArgSerLeuTyrAsnArgGlyPheSerLeu LeuIleMetLysAsnLeuAsnValThrSer&gt;               ```````````````````````````450````````````````````````````460       LeuGlyPheArgSerLeuLysGluIleSer AlaGlyArgIleTyrIleSerAlaAsnArg&gt;               ```````````````````````````470````````````````````````````480       GlnLeuCysTyrHisHisSerLeuAsnTrp ThrLysValLeuArgGlyProThrGluGlu&gt;               ```````````````````````````490````````````````````````````500       ArgLeuAspIleLysHisAsnArgProArg ArgAspCysGlyGluGlyLysGlyLeuLeu&gt;               ```````````````````````````510````````````````````````````520       GlyGlyGluAsnArgGluSerGlyArgArg GlyLeuLysGlyLeuPheCysProArgArg&gt;               ```````````````````````````530``````````534       GlySerArgValGluGlyTrpAsnGlnGly GluGlyGlyCys               P75-sErbB3 cDNA sequence (clone R35F)       nt 1-1540: The N-terminal ErbB3 sequence       nt 1541-1665: unique to p75-sErbB3       ````````10````````20`````````30````````40`````````50````````60       cgggccccccctcgaggtcg ggccggacttggctgggctc ccttcaccctctgcggagtc               ````````70````````80`````````90```````100````````110```````120       ATGAGGGCGAACGACGCTCT GCAGGTGCTGGGCTTGCTTT TCAGCCTGGCCCGGGGCTCC               ```````130```````140````````150```````160````````170```````180       GAGGTGGGCAACTCTCAGGC AGTGTGTCCTGGGACTCTGA ATGGCCTGAGTGTGACCGGC               ```````190```````200````````210```````220````````230```````240       GATGCTGAGAACCAATACCA GACACTGTACAAGCTCTACG AGAGGTGTGAGGTGGTGATG               ```````250```````260````````270```````280````````290```````300       GGGAACCTTGAGATTGTGCT CACGGGACACAATGCCGACC TCTCCTTCCTGCAGTGGATT               ```````310```````320````````330```````340````````350```````360       CGAGAAGTGACAGGCTATGT CCTCGTGGCCATGAATGAAT TCTCTACTCTACCATTGCCC               ```````370```````380````````390```````400````````410```````420       AACCTCCGCGTGGTGCGAGG GACCCAGGTCTACGATGGGA AGTTTGCCATCTTCGTCATG               ```````430```````440````````450```````460````````470```````480       TTGAACTATAACACCAACTC CAGCCACGCTCTGCGCCAGC TCCGCTTGACTCAGCTCACC               ```````490```````500````````510```````520````````530```````540       GAGATTCTGTCAGGGGGTGT TTATATTGAGAAGAACGATA AGCTTTGTCACATGGACACA               ``````550````````560````````570```````580````````590```````600       ATTGACTGGAGGGACATCGT GAGGGACCGAGATGCTGAGA TAGTGGTGAAGGACAATGGC               ``````610````````620````````630```````640````````650```````660       AGAAGCTGTCCCCCCTGTCA TGAGGTTTGCAAGGGGCGAT GCTGGGGTCCTGGATCAGAA               ``````670````````680````````690```````700````````710```````720       GACTGCCAGACATTGACCAA GACCATCTGTGCTCCTCAGT GTAATGGTCACTGCTTTGGG               ``````730````````740````````750```````760````````770```````780       CCCAACCCCAACCAGTGCTG CCATGATGAGTGTGCCGGGG GCTGCTCAGGCCCTCAGGAC               ``````790````````800````````810```````820````````830```````840       ACAGACTGCTTTGCCTGCCG GCACTTCAATGACAGTGGAG CCTGTGTACCTCGCTGTCCA               ``````850````````860````````870```````880````````890```````900       CAGCCTCTTGTCTACAACAA GCTAACTTTCCAGCTGGAAC CCAATCCCCACACCAAGTAT               ``````910````````920````````930```````940````````950```````960       CAGTATGGAGGAGTTTGTGT AGCCAGCTGTCCCCATAACT TTGTGGTGGATCAAACATCC               ``````970````````980````````990``````1000```````1010``````1020       TGTGTCAGGGCCTGTCCTCC TGACAAGATGGAAGTAGATA AAAATGGGCTCAAGATGTGT               `````1030```````1040```````1050``````1060```````1070``````1080       GAGCCTTGTGGGGGACTATG TCCCAAAGCCTGTGAGGGAA CAGGCTCTGGGAGCCGCTTC               `````1090```````1100```````1110``````1120```````1130``````1140       CAGACTGTGGACTCGAGCAA CATTGATGGATTTGTGAACT GCACCAAGATCCTGGGCAAC               `````1150```````1160```````1170``````1180```````1190``````1200       CTGGACTTTCTGATCACCGG CCTCAATGGAGACCCCTGGC ACAAGATCCCTGCCCTGGAC               `````1210```````1220```````1230``````1240```````1250``````1260       CCAGAGAAGCTCAATGTCTT CCGGACAGTACGGGAGATCA CAGGTTACCTGAACATCCAG               `````1270```````1280```````1290``````1300```````1310``````1320       TCCTGGCCGCCCCACATGCA CAACTTCAGTGTTTTTTCCA ATTTGACAACCATTGGAGGC               `````1330```````1340```````1350``````1360```````1370``````1380       AGAAGCCTCTACAACCGGGG CTTCTCATTGTTGATCATGA AGAACTTGAATGTCACATCT               `````1390```````1400```````1410``````1420```````1430``````1440       CTGGGCTTCCGATCCCTGAA GGAAATTAGTGCTGGGCGTA TCTATATAAGTGCCAATAGG               `````1450```````1460```````1470``````1480```````1490``````1500       CAGCTCTGCTACCACCACTC TTTGAACTGGACCAAGGTGC TTCGGGGGCCTACGGAAGAG               `````1510```````1520```````1530``````1540```````1550``````1560       CGACTAGACATCAAGCATAA TCGGCCGCGCAGAGACTGCG GTGAGGGAAAGGGTCTGCTA               `````1570```````1580```````1590``````1600```````1610``````1620       GGTGGTGAGAATAGGGAGTC AGGGAGGAGAGGGCTGAAAG GACTATTCTGCCCTAGACGT               `````1630```````1640```````1650``````1660```````1670``````1680       GGGAGTAGGGTTGAGGGATG GAACCAAGGAGAAGGGGGCT GTTAGgctggaagcagtaac               `````1690```````1700```````1710``````1720```````1730``````1740       gaggaagaataatgaagaga gggcttgctgggagtcctca gactcctctcctaacccacc               `````1750```````1760```````1770``````1780```````1790``````1800       ccttcctttccagtggcaga gggcaaagtgtgtgacccac tgtgctcctctgggggatgc               `````1810```````1820```````1830`````1840````````1850``````1860       tggggcccaggccctggtca gtgcttgtcctgtcgaaatt atagccgaggaggtgtctgt               `````1870```````1880```````1890``````1900```````1910``````1920       gtgacccactgcaactttct gaatgggtacagtaagggga gccagtcaaggatgggtggg               `````1930```````1940```````1950``````1960```````1970``````1980       ggtggggccctgcaatggaa ctgttcaggtggcatacaat aaaagtctttagacagcaaa               `````1990```````2000       aaaaaaaaaaaaaaaaaaaaaa          
 
         [0093]    (3) Information for SEQ ID NO:4:  
         [0094]    (i) Sequence characteristics:  
         [0095]    (ii) Length  
         [0096]    (iii) Type  
         [0097]    (iv) Molecule type  
         [0098]    (v) Original source:  
         [0099]    (vi) Organism: homo sapience  
                           p50-sErbB3 protein (cDNA clone R1F)           AA 1-370: The N-terminal ErbB3 sequence       AA 1-19: signal peptide       AA 371-390: p50-sErbB3 unique sequence                                   10                             20       MetArgAlaAsnAspAlaLeuGlnValLeu GlyLeuLeuPheSerLeuAlaArgGlySer&gt;                                           30                             40       GluValGlyAsnSerGlnAlaValCysPro GlyThrLeuAsnGlyLeuSerValThrGly&gt;                                           50                             60       AspAlaGluAsnGlnTyrGlnThrLeuTyr LysLeuTyrGluArgCysGluValValMet&gt;                                           70                             80       GlyAsnLeuGluIleValLeuThrGlyHis AsnAlaAspLeuSerPheLeuGlnTrpIle&gt;                                           90                            100       ArgGluValThrGlyTyrValLeuValAla MetAsnGluPheSerThrLeuProLeuPro&gt;                                          110                            120       AsnLeuArgValValArgGlyThrGlnVal TyrAspGlyLysPheAlaIlePheValMet&gt;                                          130                            140       LeuAsnTyrAsnThrAsnSerSerHisAla LeuArgGlnLeuArgLeuThrGlnLeuThr&gt;                                          150                            160       GluIleLeuSerGlyGlyValTyrIleGlu LysAsnAspLysLeuCysHisMetAspThr&gt;                                          170                            180       IleAspTrpArgAspIleValArgAspArg AspAlaGluIleValValLysAspAsnGly&gt;                                          190                            200       ArgSerCysProProCysHisGluValCys LysGlyArgCysTrpGlyProGlySerGlu&gt;                                          210                            220       AspCysGlnThrLeuThrLysThrIleCys AlaProGlnCysAsnGlyHisCysPheGly&gt;                                          230                            240       ProAsnProAsnGlnCysCysHisAspGlu CysAlaGlyGlyCysSerGlyProGlnAsp&gt;                                          250                            260       ThrAspCysPheAlaCysArgHisPheAsn AspSerGlyAlaCysValProArgCysPro&gt;                                          270                            280       GlnProLeuValTyrAsnLysLeuThrPhe GlnLeuGluProAsnProHisThrLysTyr&gt;                                          290                            300       GlnTyrGlyGlyValCysValAlaSerCys ProHisAsnPheValValAspGlnThrSer&gt;                                          310                            320       CysValArgAlaCysProProAspLysMet GluValAspLysAsnGlyLeuLysMetCys&gt;                                          330                            340       GluProCysGlyGlyLeuCysProLysAla CysGluGlyThrGlySerGlySerArgPhe&gt;                                          350                            360       GlnThrValAspSerSerAsnhleAspGly PheValAsnCysThrLysIleLeuGlyAsn&gt;                                          370                            380       LeuAspPheLeuIleThrGlyLeuAsnGly LeuGluIleLeuProSerLeuLeuArgPro&gt;                                          380                            390       GlnProThrHisProSerGlnPheIleSer LeuAlaLysThrPheLeuCysGlyAlaAsp               p50-sErbB3 cDNA sequence (R1F)       nt 1-1169: The N-terminal ErbB3 sequence       nt 1170-1263: unique to p50-sErbB3               10        20         30        40         50        60       cgggccccccctcgaggtcg ggccggacttggctgggctc ccttcaccctctgcggagtc                       70        80         90       100        110       120       ATGAGGGCGAACGACGCTCT GCAGGTGCTGGGCTTGCTTT TCAGCCTGGCCCGGGGCTCC                      130       140        150       160        170       180       GAGGTGGGCAACTCTCAGGC AGTGTGTCCTGGGACTCTGA ATGGCCTGAGTGTGACCGGC                      190       200        210       220        230       240       GATGCTGAGAACCAATACCA GACACTGTACAAGCTCTACG AGAGGTGTGAGGTGGTGATG                      250       260        270       280        290       300       GGGAACCTTGAGATTGTGCT CACGGGACACAATGCCGACC TCTCCTTCCTGCAGTGGATT                      310       320        330       340       350       360       CGAGAAGTGACAGGCTATGT CCTCGTGGCCATGAATGAAT TCTCTACTCTACCATTGCCC                      370      380        390       400        410       420       AACCTCCGCGTGGTGCGAGG GACCCAGGTCTACGATGGGA AGTTTGCCATCTTCGTCATG                      430       440        450       460        470       480       TTGAACTATAACACCAACTC CAGCCACGCTCTGCGCCAGC TCCGCTTGACTCAGCTCACC                      490       500        510       520        530       540       GAGATTCTGTCAGGGGGTGT TTATATTGAGAAGAACGATA AGCTTTGTCACATGGACACA                      550       560        570       580        590       600       ATTGACTGGAGGGACATCGT GAGGGACCGAGATGCTGAGA TAGTGGTGAAGGACAATGGC                      610       620        630       640        650       660       AGAAGCTGTCCCCCCTGTCA TGAGGTTTGCAAGGGGCGAT GCTGGGGTCCTGGATCAGAA                      670       680        690       700        710       720       GACTGCCAGACATTGACCAA GACCATCTGTGCTCCTCAGT GTAATGGTCACTGCTTTGGG                      730       740        750       760        770       780       CCCAACCCCAACCAGTGCTG CCATGATGAGTGTGCCGGGG GCTGCTCAGGCCCTCAGGAC                      790       800        810       820        830       840       ACAGACTGCTTTGCCTGCCG GCACTTCAATGACAGTGGAG CCTGTGTACCTCGCTGTCCA                      850       860        870       880        890       900       CAGCCTCTTGTCTACAACAA GCTAACTTTCCAGCTGGAAC CCAATCCCCACACCAAGTAT                      910       920        930       940        950       960       CAGTATGGAGGAGTTTGTGT AGCCAGCTGTCCCCATAACT TTGTGGTGGATCAAACATCC                      970       980        990      1000       1010      1020       TGTGTCAGGGCCTGTCCTCC TGACAAGATGGAAGTAGATA AAAATGGGCTCAAGATGTGT                     1030      1040       1050      1060       1070      1080       GAGCCTTGTGGGGGACTATG TCCCAAAGCCTGTGAGGGAA CAGGCTCTGGGAGCCGCTTC                     1090      1100       1110      1120       1130      1140       CAGACTGTGGACTCGAGCAA CATTGATGGATTTGTGAACT GCACCAAGATCCTGGGCAAC                     1150      1160       1170      1180       1190      1200       CTGGACTTTCTGATCACCGG CCTCAATGGGTTAGAGATCC TGCCTTCCCTCCTTAGACCC                     1210      1220       1230      1240       1250      1260       CAGCCCACGCACCCCTCACA GTTCATTTCATTGGCCAAAA CTTTCCTATGTGGAGCTGAC                     1270      1280       1290      1300       1310      1320       TAGgaatcaaagtcataaaa ttctagcctgttaaaaaaaa aaaaaaaaaaaaaaaaaaaa          
 
         [0100]    tested, growth of this cell line was dose-dependent (See FIG. 7). It was observed that at a concentration of 0.4 nM HRGβ the cell growth rate was half of the rate observed at saturating levels of HRGβ. In cell cultures grown in the presence of 0.4 nM HRGβ and p85-sErbB3 (a 100-fold molar excess relative to HRGβ), p85-sErbB3 inhibited cell growth by 75% and 90%, at densities of 5,000 and 8,000 cells/well, respectively, whereas the same concentration of p85-sErbB3 did not affect cell growth in the absence of HRGβ (See FIG. 7). Thus, the present invention discloses the use of p85-sErbB3 as a potent inhibitor of HRG-dependent breast carcinoma cell growth in vitro.  
     
       
       
         1 
         
           
             8  
           
           
             1  
             1800  
             DNA  
             Homo sapiens  
             
               CDS  
               (61)..(1746)  
             
           
            1 

cgggcccccc ctcgaggtcg ggccggactt ggctgggctc ccttcaccct ctgcggagtc     60 

atg agg gcg aac gac gct ctg cag gtg ctg ggc ttg ctt ttc agc ctg      108 
Met Arg Ala Asn Asp Ala Leu Gln Val Leu Gly Leu Leu Phe Ser Leu 
1               5                   10                  15 

gcc cgg ggc tcc gag gtg ggc aac tct cag gca gtg tgt cct ggg act      156 
Ala Arg Gly Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr 
            20                  25                  30 

ctg aat ggc ctg agt gtg acc ggc gat gct gag aac caa tac cag aca      204 
Leu Asn Gly Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr 
        35                  40                  45 

ctg tac aag ctc tac gag agg tgt gag gtg gtg atg ggg aac ctt gag      252 
Leu Tyr Lys Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu 
    50                  55                  60 

att gtg ctc acg gga cac aat gcc gac ctc tcc ttc ctg cag tgg att      300 
Ile Val Leu Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile 
65                  70                  75                  80 

cga gaa gtg aca ggc tat gtc ctc gtg gcc atg aat gaa ttc tct act      348 
Arg Glu Val Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr 
                85                  90                  95 

cta cca ttg ccc aac ctc cgc gtg gtg cga ggg acc cag gtc tac gat      396 
Leu Pro Leu Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp 
            100                 105                 110 

ggg aag ttt gcc atc ttc gtc atg ttg aac tat aac acc aac tcc agc      444 
Gly Lys Phe Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser 
        115                 120                 125 

cac gct ctg cgc cag ctc cgc ttg act cag ctc acc gag att ctg tca      492 
His Ala Leu Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser 
    130                 135                 140 

ggg ggt gtt tat att gag aag aac gat aag ctt tgt cac atg gac aca      540 
Gly Gly Val Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr 
145                 150                 155                 160 

att gac tgg agg gac atc gtg agg gac cga gat gct gag ata gtg gtg      588 
Ile Asp Trp Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val Val 
                165                 170                 175 

aag gac aat ggc aga agc tgt ccc ccc tgt cat gag gtt tgc aag ggg      636 
Lys Asp Asn Gly Arg Ser Cys Pro Pro Cys His Glu Val Cys Lys Gly 
            180                 185                 190 

cga tgc tgg ggt cct gga tca gaa gac tgc cag aca ttg acc aag acc      684 
Arg Cys Trp Gly Pro Gly Ser Glu Asp Cys Gln Thr Leu Thr Lys Thr 
        195                 200                 205 

atc tgt gct cct cag tgt aat ggt cac tgc ttt ggg ccc aac ccc aac      732 
Ile Cys Ala Pro Gln Cys Asn Gly His Cys Phe Gly Pro Asn Pro Asn 
    210                 215                 220 

cag tgc tgc cat gat gag tgt gcc ggg ggc tgc tca ggc cct cag gac      780 
Gln Cys Cys His Asp Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp 
225                 230                 235                 240 

aca gac tgc ttt gcc tgc cgg cac ttc aat gac agt gga gcc tgt gta      828 
Thr Asp Cys Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val 
                245                 250                 255 

cct cgc tgt cca cag cct ctt gtc tac aac aag cta act ttc cag ctg      876 
Pro Arg Cys Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu 
            260                 265                 270 

gaa ccc aat ccc cac acc aag tat cag tat gga gga gtt tgt gta gcc      924 
Glu Pro Asn Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala 
        275                 280                 285 

agc tgt ccc cat aac ttt gtg gtg gat caa aca tcc tgt gtc agg gcc      972 
Ser Cys Pro His Asn Phe Val Val Asp Gln Thr Ser Cys Val Arg Ala 
    290                 295                 300 

tgt cct cct gac aag atg gaa gta gat aaa aat ggg ctc aag atg tgt     1020 
Cys Pro Pro Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys Met Cys 
305                 310                 315                 320 

gag cct tgt ggg gga cta tgt ccc aaa gcc tgt gag gga aca ggc tct     1068 
Glu Pro Cys Gly Gly Leu Cys Pro Lys Ala Cys Glu Gly Thr Gly Ser 
                325                 330                 335 

ggg agc cgc ttc cag act gtg gac tcg agc aac att gat gga ttt gtg     1116 
Gly Ser Arg Phe Gln Thr Val Asp Ser Ser Asn Ile Asp Gly Phe Val 
            340                 345                 350 

aac tgc acc aag atc ctg ggc aac ctg gac ttt ctg atc acc ggc ctc     1164 
Asn Cys Thr Lys Ile Leu Gly Asn Leu Asp Phe Leu Ile Thr Gly Leu 
        355                 360                 365 

aat gga gac ccc tgg cac aag atc cct gcc ctg gac cca gag aag ctc     1212 
Asn Gly Asp Pro Trp His Lys Ile Pro Ala Leu Asp Pro Glu Lys Leu 
    370                 375                 380 

aat gtc ttc cgg aca gta cgg gag atc aca ggt tac ctg aac atc cag     1260 
Asn Val Phe Arg Thr Val Arg Glu Ile Thr Gly Tyr Leu Asn Ile Gln 
385                 390                 395                 400 

tcc tgg ccg ccc cac atg cac aac ttc agt gtt ttt tcc aat ttg aca     1308 
Ser Trp Pro Pro His Met His Asn Phe Ser Val Phe Ser Asn Leu Thr 
                405                 410                 415 

acc att gga ggc aga agc ctc tac aac cgg ggc ttc tca ttg ttg atc     1356 
Thr Ile Gly Gly Arg Ser Leu Tyr Asn Arg Gly Phe Ser Leu Leu Ile 
            420                 425                 430 

atg aag aac ttg aat gtc aca tct ctg ggc ttc cga tcc ctg aag gaa     1404 
Met Lys Asn Leu Asn Val Thr Ser Leu Gly Phe Arg Ser Leu Lys Glu 
        435                 440                 445 

att agt gct ggg cgt atc tat ata agt gcc aat agg cag ctc tgc tac     1452 
Ile Ser Ala Gly Arg Ile Tyr Ile Ser Ala Asn Arg Gln Leu Cys Tyr 
    450                 455                 460 

cac cac tct ttg aac tgg acc aag gtg ctt cgg ggg cct acg gaa gag     1500 
His His Ser Leu Asn Trp Thr Lys Val Leu Arg Gly Pro Thr Glu Glu 
465                 470                 475                 480 

cga cta gac atc aag cat aat cgg ccg cgc aga gac tgc gtg gca gag     1548 
Arg Leu Asp Ile Lys His Asn Arg Pro Arg Arg Asp Cys Val Ala Glu 
                485                 490                 495 

ggc aaa gtg tgt gac cca ctg tgc tcc tct ggg gga tgc tgg ggc cca     1596 
Gly Lys Val Cys Asp Pro Leu Cys Ser Ser Gly Gly Cys Trp Gly Pro 
            500                 505                 510 

ggc cct ggt cag tgc ttg tcc tgt cga aat tat agc cga gga ggt gtc     1644 
Gly Pro Gly Gln Cys Leu Ser Cys Arg Asn Tyr Ser Arg Gly Gly Val 
        515                 520                 525 

tgt gtg acc cac tgc aac ttt ttg aat ggg tac agt aag ggg agc cag     1692 
Cys Val Thr His Cys Asn Phe Leu Asn Gly Tyr Ser Lys Gly Ser Gln 
    530                 535                 540 

tca agg atg ggt ggg ggt ggg gcc ctg caa tgg aac tgt tca ggt ggc     1740 
Ser Arg Met Gly Gly Gly Gly Ala Leu Gln Trp Asn Cys Ser Gly Gly 
545                 550                 555                 560 

ata caa taaaagtctt tagacaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa      1796 
Ile Gln 

aaaa                                                                1800 

 
           
             2  
             562  
             PRT  
             Homo sapiens  
           
            2 

Met Arg Ala Asn Asp Ala Leu Gln Val Leu Gly Leu Leu Phe Ser Leu 
1               5                   10                  15 

Ala Arg Gly Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr 
            20                  25                  30 

Leu Asn Gly Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr 
        35                  40                  45 

Leu Tyr Lys Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu 
    50                  55                  60 

Ile Val Leu Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile 
65                  70                  75                  80 

Arg Glu Val Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr 
                85                  90                  95 

Leu Pro Leu Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp 
            100                 105                 110 

Gly Lys Phe Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser 
        115                 120                 125 

His Ala Leu Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser 
    130                 135                 140 

Gly Gly Val Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr 
145                 150                 155                 160 

Ile Asp Trp Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val Val 
                165                 170                 175 

Lys Asp Asn Gly Arg Ser Cys Pro Pro Cys His Glu Val Cys Lys Gly 
            180                 185                 190 

Arg Cys Trp Gly Pro Gly Ser Glu Asp Cys Gln Thr Leu Thr Lys Thr 
        195                 200                 205 

Ile Cys Ala Pro Gln Cys Asn Gly His Cys Phe Gly Pro Asn Pro Asn 
    210                 215                 220 

Gln Cys Cys His Asp Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp 
225                 230                 235                 240 

Thr Asp Cys Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val 
                245                 250                 255 

Pro Arg Cys Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu 
            260                 265                 270 

Glu Pro Asn Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala 
        275                 280                 285 

Ser Cys Pro His Asn Phe Val Val Asp Gln Thr Ser Cys Val Arg Ala 
    290                 295                 300 

Cys Pro Pro Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys Met Cys 
305                 310                 315                 320 

Glu Pro Cys Gly Gly Leu Cys Pro Lys Ala Cys Glu Gly Thr Gly Ser 
                325                 330                 335 

Gly Ser Arg Phe Gln Thr Val Asp Ser Ser Asn Ile Asp Gly Phe Val 
            340                 345                 350 

Asn Cys Thr Lys Ile Leu Gly Asn Leu Asp Phe Leu Ile Thr Gly Leu 
        355                 360                 365 

Asn Gly Asp Pro Trp His Lys Ile Pro Ala Leu Asp Pro Glu Lys Leu 
    370                 375                 380 

Asn Val Phe Arg Thr Val Arg Glu Ile Thr Gly Tyr Leu Asn Ile Gln 
385                 390                 395                 400 

Ser Trp Pro Pro His Met His Asn Phe Ser Val Phe Ser Asn Leu Thr 
                405                 410                 415 

Thr Ile Gly Gly Arg Ser Leu Tyr Asn Arg Gly Phe Ser Leu Leu Ile 
            420                 425                 430 

Met Lys Asn Leu Asn Val Thr Ser Leu Gly Phe Arg Ser Leu Lys Glu 
        435                 440                 445 

Ile Ser Ala Gly Arg Ile Tyr Ile Ser Ala Asn Arg Gln Leu Cys Tyr 
    450                 455                 460 

His His Ser Leu Asn Trp Thr Lys Val Leu Arg Gly Pro Thr Glu Glu 
465                 470                 475                 480 

Arg Leu Asp Ile Lys His Asn Arg Pro Arg Arg Asp Cys Val Ala Glu 
                485                 490                 495 

Gly Lys Val Cys Asp Pro Leu Cys Ser Ser Gly Gly Cys Trp Gly Pro 
            500                 505                 510 

Gly Pro Gly Gln Cys Leu Ser Cys Arg Asn Tyr Ser Arg Gly Gly Val 
        515                 520                 525 

Cys Val Thr His Cys Asn Phe Leu Asn Gly Tyr Ser Lys Gly Ser Gln 
    530                 535                 540 

Ser Arg Met Gly Gly Gly Gly Ala Leu Gln Trp Asn Cys Ser Gly Gly 
545                 550                 555                 560 

Ile Gln 

 
           
             3  
             1420  
             DNA  
             Homo sapiens  
             
               CDS  
               (61)..(1053)  
             
           
            3 

cgggcccccc ctcgaggtcg ggccggactt ggctgggctc ccttcaccct ctgcggagtc     60 

atg agg gcg aac gac gct ctg cag gtg ctg ggc ttg ctt ttc agc ctg      108 
Met Arg Ala Asn Asp Ala Leu Gln Val Leu Gly Leu Leu Phe Ser Leu 
1               5                   10                  15 

gcc cgg ggc tcc gag gtg ggc aac tct cag gca gtg tgt cct ggg act      156 
Ala Arg Gly Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr 
            20                  25                  30 

ctg aat ggc ctg agt gtg acc ggc gat gct gag aac caa tac cag aca      204 
Leu Asn Gly Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr 
        35                  40                  45 

ctg tac aag ctc tac gag agg tgt gag gtg gtg atg ggg aac ctt gag      252 
Leu Tyr Lys Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu 
    50                  55                  60 

att gtg ctc acg gga cac aat gcc gac ctc tcc ttc ctg cag tgg att      300 
Ile Val Leu Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile 
65                  70                  75                  80 

cga gaa gtg aca ggc tat gtc ctc gtg gcc atg aat gaa ttc tct act      348 
Arg Glu Val Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr 
                85                  90                  95 

cta cca ttg ccc aac ctc cgc gtg gtg cga ggg acc cag gtc tac gat      396 
Leu Pro Leu Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp 
            100                 105                 110 

ggg aag ttt gcc atc ttc gtc atg ttg aac tat aac acc aac tcc agc      444 
Gly Lys Phe Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser 
        115                 120                 125 

cac gct ctg cgc cag ctc cgc ttg act cag ctc acc gag att ctg tca      492 
His Ala Leu Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser 
    130                 135                 140 

ggg ggt gtt tat att gag aag aac gat aag ctt tgt cac atg gac aca      540 
Gly Gly Val Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr 
145                 150                 155                 160 

att gac tgg agg gac atc gtg agg gac cga gat gct gag ata gtg gtg      588 
Ile Asp Trp Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val Val 
                165                 170                 175 

aag gac aat ggc aga agc tgt ccc ccc tgt cat gag gtt tgc aag ggg      636 
Lys Asp Asn Gly Arg Ser Cys Pro Pro Cys His Glu Val Cys Lys Gly 
            180                 185                 190 

cga tgc tgg ggt cct gga tca gaa gac tgc cag aca ttg acc aag acc      684 
Arg Cys Trp Gly Pro Gly Ser Glu Asp Cys Gln Thr Leu Thr Lys Thr 
        195                 200                 205 

atc tgt gct cct cag tgt aat ggt cac tgc ttt ggg ccc aac ccc aac      732 
Ile Cys Ala Pro Gln Cys Asn Gly His Cys Phe Gly Pro Asn Pro Asn 
    210                 215                 220 

cag tgc tgc cat gat gag tgt gcc ggg ggc tgc tca ggc cct cag gac      780 
Gln Cys Cys His Asp Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp 
225                 230                 235                 240 

aca gac tgc ttt gcc tgc cgg cac ttc aat gac agt gga gcc tgt gta      828 
Thr Asp Cys Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val 
                245                 250                 255 

cct cgc tgt cca cag cct ctt gtc tac aac aag cta act ttc cag ctg      876 
Pro Arg Cys Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu 
            260                 265                 270 

gaa ccc aat ccc cac acc aag tat cag tat gga gga gtt tgt gta gcc      924 
Glu Pro Asn Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala 
        275                 280                 285 

agc tgt ccc cat aac ttt gtg gtg gat caa aca tcc tgt gtc agg gcc      972 
Ser Cys Pro His Asn Phe Val Val Asp Gln Thr Ser Cys Val Arg Ala 
    290                 295                 300 

tgt cct cct gac aag atg gaa gta gat aaa aat ggg ctc aag atg tgt     1020 
Cys Pro Pro Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys Met Cys 
305                 310                 315                 320 

gag cct tgt ggg gga cta tgt ccc aaa ggt ggg taggagatgg taagaagttg   1073 
Glu Pro Cys Gly Gly Leu Cys Pro Lys Gly Gly 
                325                 330 

taaagagaca gcctttcctc tgagcctgcg cagaccaccc ccactgaacc tctcttacat   1133 

ttgcagcctg tgagggaaca ggctctggga gccgcttcca gactgtggac tcgagcaaca   1193 

ttgatggatt tgtgaactgc accaagatcc tgggcaacct ggactttctg atcaccggcc   1253 

tcaatgggtt agagatcctg ccttccctcc ttagacccca gcccacgcac ccctcacagt   1313 

tcatttcatt ggccaaaact ttcctatgtg gagctgacta ggaatcaaag tcataaaatt   1373 

ctagcctgtt acaaaggaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa                 1420 

 
           
             4  
             331  
             PRT  
             Homo sapiens  
           
            4 

Met Arg Ala Asn Asp Ala Leu Gln Val Leu Gly Leu Leu Phe Ser Leu 
1               5                   10                  15 

Ala Arg Gly Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr 
            20                  25                  30 

Leu Asn Gly Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr 
        35                  40                  45 

Leu Tyr Lys Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu 
    50                  55                  60 

Ile Val Leu Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile 
65                  70                  75                  80 

Arg Glu Val Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr 
                85                  90                  95 

Leu Pro Leu Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp 
            100                 105                 110 

Gly Lys Phe Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser 
        115                 120                 125 

His Ala Leu Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser 
    130                 135                 140 

Gly Gly Val Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr 
145                 150                 155                 160 

Ile Asp Trp Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val Val 
                165                 170                 175 

Lys Asp Asn Gly Arg Ser Cys Pro Pro Cys His Glu Val Cys Lys Gly 
            180                 185                 190 

Arg Cys Trp Gly Pro Gly Ser Glu Asp Cys Gln Thr Leu Thr Lys Thr 
        195                 200                 205 

Ile Cys Ala Pro Gln Cys Asn Gly His Cys Phe Gly Pro Asn Pro Asn 
    210                 215                 220 

Gln Cys Cys His Asp Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp 
225                 230                 235                 240 

Thr Asp Cys Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val 
                245                 250                 255 

Pro Arg Cys Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu 
            260                 265                 270 

Glu Pro Asn Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala 
        275                 280                 285 

Ser Cys Pro His Asn Phe Val Val Asp Gln Thr Ser Cys Val Arg Ala 
    290                 295                 300 

Cys Pro Pro Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys Met Cys 
305                 310                 315                 320 

Glu Pro Cys Gly Gly Leu Cys Pro Lys Gly Gly 
                325                 330 

 
           
             5  
             2005  
             DNA  
             Homo sapiens  
             
               CDS  
               (61)..(1662)  
             
           
            5 

cgggcccccc ctcgaggtcg ggccggactt ggctgggctc ccttcaccct ctgcggagtc     60 

atg agg gcg aac gac gct ctg cag gtg ctg ggc ttg ctt ttc agc ctg      108 
Met Arg Ala Asn Asp Ala Leu Gln Val Leu Gly Leu Leu Phe Ser Leu 
1               5                   10                  15 

gcc cgg ggc tcc gag gtg ggc aac tct cag gca gtg tgt cct ggg act      156 
Ala Arg Gly Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr 
            20                  25                  30 

ctg aat ggc ctg agt gtg acc ggc gat gct gag aac caa tac cag aca      204 
Leu Asn Gly Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr 
        35                  40                  45 

ctg tac aag ctc tac gag agg tgt gag gtg gtg atg ggg aac ctt gag      252 
Leu Tyr Lys Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu 
    50                  55                  60 

att gtg ctc acg gga cac aat gcc gac ctc tcc ttc ctg cag tgg att      300 
Ile Val Leu Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile 
65                  70                  75                  80 

cga gaa gtg aca ggc tat gtc ctc gtg gcc atg aat gaa ttc tct act      348 
Arg Glu Val Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr 
                85                  90                  95 

cta cca ttg ccc aac ctc cgc gtg gtg cga ggg acc cag gtc tac gat      396 
Leu Pro Leu Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp 
            100                 105                 110 

ggg aag ttt gcc atc ttc gtc atg ttg aac tat aac acc aac tcc agc      444 
Gly Lys Phe Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser 
        115                 120                 125 

cac gct ctg cgc cag ctc cgc ttg act cag ctc acc gag att ctg tca      492 
His Ala Leu Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser 
    130                 135                 140 

ggg ggt gtt tat att gag aag aac gat aag ctt tgt cac atg gac aca      540 
Gly Gly Val Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr 
145                 150                 155                 160 

att gac tgg agg gac atc gtg agg gac cga gat gct gag ata gtg gtg      588 
Ile Asp Trp Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val Val 
                165                 170                 175 

aag gac aat ggc aga agc tgt ccc ccc tgt cat gag gtt tgc aag ggg      636 
Lys Asp Asn Gly Arg Ser Cys Pro Pro Cys His Glu Val Cys Lys Gly 
            180                 185                 190 

cga tgc tgg ggt cct gga tca gaa gac tgc cag aca ttg acc aag acc      684 
Arg Cys Trp Gly Pro Gly Ser Glu Asp Cys Gln Thr Leu Thr Lys Thr 
        195                 200                 205 

atc tgt gct cct cag tgt aat ggt cac tgc ttt ggg ccc aac ccc aac      732 
Ile Cys Ala Pro Gln Cys Asn Gly His Cys Phe Gly Pro Asn Pro Asn 
    210                 215                 220 

cag tgc tgc cat gat gag tgt gcc ggg ggc tgc tca ggc cct cag gac      780 
Gln Cys Cys His Asp Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp 
225                 230                 235                 240 

aca gac tgc ttt gcc tgc cgg cac ttc aat gac agt gga gcc tgt gta      828 
Thr Asp Cys Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val 
                245                 250                 255 

cct cgc tgt cca cag cct ctt gtc tac aac aag cta act ttc cag ctg      876 
Pro Arg Cys Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu 
            260                 265                 270 

gaa ccc aat ccc cac acc aag tat cag tat gga gga gtt tgt gta gcc      924 
Glu Pro Asn Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala 
        275                 280                 285 

agc tgt ccc cat aac ttt gtg gtg gat caa aca tcc tgt gtc agg gcc      972 
Ser Cys Pro His Asn Phe Val Val Asp Gln Thr Ser Cys Val Arg Ala 
    290                 295                 300 

tgt cct cct gac aag atg gaa gta gat aaa aat ggg ctc aag atg tgt     1020 
Cys Pro Pro Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys Met Cys 
305                 310                 315                 320 

gag cct tgt ggg gga cta tgt ccc aaa gcc tgt gag gga aca ggc tct     1068 
Glu Pro Cys Gly Gly Leu Cys Pro Lys Ala Cys Glu Gly Thr Gly Ser 
                325                 330                 335 

ggg agc cgc ttc cag act gtg gac tcg agc aac att gat gga ttt gtg     1116 
Gly Ser Arg Phe Gln Thr Val Asp Ser Ser Asn Ile Asp Gly Phe Val 
            340                 345                 350 

aac tgc acc aag atc ctg ggc aac ctg gac ttt ctg atc acc ggc ctc     1164 
Asn Cys Thr Lys Ile Leu Gly Asn Leu Asp Phe Leu Ile Thr Gly Leu 
        355                 360                 365 

aat gga gac ccc tgg cac aag atc cct gcc ctg gac cca gag aag ctc     1212 
Asn Gly Asp Pro Trp His Lys Ile Pro Ala Leu Asp Pro Glu Lys Leu 
    370                 375                 380 

aat gtc ttc cgg aca gta cgg gag atc aca ggt tac ctg aac atc cag     1260 
Asn Val Phe Arg Thr Val Arg Glu Ile Thr Gly Tyr Leu Asn Ile Gln 
385                 390                 395                 400 

tcc tgg ccg ccc cac atg cac aac ttc agt gtt ttt tcc aat ttg aca     1308 
Ser Trp Pro Pro His Met His Asn Phe Ser Val Phe Ser Asn Leu Thr 
                405                 410                 415 

acc att gga ggc aga agc ctc tac aac cgg ggc ttc tca ttg ttg atc     1356 
Thr Ile Gly Gly Arg Ser Leu Tyr Asn Arg Gly Phe Ser Leu Leu Ile 
            420                 425                 430 

atg aag aac ttg aat gtc aca tct ctg ggc ttc cga tcc ctg aag gaa     1404 
Met Lys Asn Leu Asn Val Thr Ser Leu Gly Phe Arg Ser Leu Lys Glu 
        435                 440                 445 

att agt gct ggg cgt atc tat ata agt gcc aat agg cag ctc tgc tac     1452 
Ile Ser Ala Gly Arg Ile Tyr Ile Ser Ala Asn Arg Gln Leu Cys Tyr 
    450                 455                 460 

cac cac tct ttg aac tgg acc aag gtg ctt cgg ggg cct acg gaa gag     1500 
His His Ser Leu Asn Trp Thr Lys Val Leu Arg Gly Pro Thr Glu Glu 
465                 470                 475                 480 

cga cta gac atc aag cat aat cgg ccg cgc aga gac tgc ggt gag gga     1548 
Arg Leu Asp Ile Lys His Asn Arg Pro Arg Arg Asp Cys Gly Glu Gly 
                485                 490                 495 

aag ggt ctg cta ggt ggt gag aat agg gag tca ggg agg aga ggg ctg     1596 
Lys Gly Leu Leu Gly Gly Glu Asn Arg Glu Ser Gly Arg Arg Gly Leu 
            500                 505                 510 

aaa gga cta ttc tgc cct aga cgt ggg agt agg gtt gag gga tgg aac     1644 
Lys Gly Leu Phe Cys Pro Arg Arg Gly Ser Arg Val Glu Gly Trp Asn 
        515                 520                 525 

caa gga gaa ggg ggc tgt taggctggaa gcagtaacga ggaagaataa            1692 
Gln Gly Glu Gly Gly Cys 
    530 

tgaagagagg gcttgctggg agtcctcaga ctcctctcct aacccacccc ttcctttcca   1752 

gtggcagagg gcaaagtgtg tgacccactg tgctcctctg ggggatgctg gggcccaggc   1812 

cctggtcagt gcttgtcctg tcgaaattat agccgaggag gtgtctgtgt gacccactgc   1872 

aactttctga atgggtacag taaggggagc cagtcaagga tgggtggggg tggggccctg   1932 

caatggaact gttcaggtgg catacaataa aagtctttag acagcaaaaa aaaaaaaaaa   1992 

aaaaaaaaaa aaa                                                      2005 

 
           
             6  
             534  
             PRT  
             Homo sapiens  
           
            6 

Met Arg Ala Asn Asp Ala Leu Gln Val Leu Gly Leu Leu Phe Ser Leu 
1               5                   10                  15 

Ala Arg Gly Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr 
            20                  25                  30 

Leu Asn Gly Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr 
        35                  40                  45 

Leu Tyr Lys Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu 
    50                  55                  60 

Ile Val Leu Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile 
65                  70                  75                  80 

Arg Glu Val Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr 
                85                  90                  95 

Leu Pro Leu Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp 
            100                 105                 110 

Gly Lys Phe Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser 
        115                 120                 125 

His Ala Leu Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser 
    130                 135                 140 

Gly Gly Val Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr 
145                 150                 155                 160 

Ile Asp Trp Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val Val 
                165                 170                 175 

Lys Asp Asn Gly Arg Ser Cys Pro Pro Cys His Glu Val Cys Lys Gly 
            180                 185                 190 

Arg Cys Trp Gly Pro Gly Ser Glu Asp Cys Gln Thr Leu Thr Lys Thr 
        195                 200                 205 

Ile Cys Ala Pro Gln Cys Asn Gly His Cys Phe Gly Pro Asn Pro Asn 
    210                 215                 220 

Gln Cys Cys His Asp Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp 
225                 230                 235                 240 

Thr Asp Cys Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val 
                245                 250                 255 

Pro Arg Cys Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu 
            260                 265                 270 

Glu Pro Asn Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala 
        275                 280                 285 

Ser Cys Pro His Asn Phe Val Val Asp Gln Thr Ser Cys Val Arg Ala 
    290                 295                 300 

Cys Pro Pro Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys Met Cys 
305                 310                 315                 320 

Glu Pro Cys Gly Gly Leu Cys Pro Lys Ala Cys Glu Gly Thr Gly Ser 
                325                 330                 335 

Gly Ser Arg Phe Gln Thr Val Asp Ser Ser Asn Ile Asp Gly Phe Val 
            340                 345                 350 

Asn Cys Thr Lys Ile Leu Gly Asn Leu Asp Phe Leu Ile Thr Gly Leu 
        355                 360                 365 

Asn Gly Asp Pro Trp His Lys Ile Pro Ala Leu Asp Pro Glu Lys Leu 
    370                 375                 380 

Asn Val Phe Arg Thr Val Arg Glu Ile Thr Gly Tyr Leu Asn Ile Gln 
385                 390                 395                 400 

Ser Trp Pro Pro His Met His Asn Phe Ser Val Phe Ser Asn Leu Thr 
                405                 410                 415 

Thr Ile Gly Gly Arg Ser Leu Tyr Asn Arg Gly Phe Ser Leu Leu Ile 
            420                 425                 430 

Met Lys Asn Leu Asn Val Thr Ser Leu Gly Phe Arg Ser Leu Lys Glu 
        435                 440                 445 

Ile Ser Ala Gly Arg Ile Tyr Ile Ser Ala Asn Arg Gln Leu Cys Tyr 
    450                 455                 460 

His His Ser Leu Asn Trp Thr Lys Val Leu Arg Gly Pro Thr Glu Glu 
465                 470                 475                 480 

Arg Leu Asp Ile Lys His Asn Arg Pro Arg Arg Asp Cys Gly Glu Gly 
                485                 490                 495 

Lys Gly Leu Leu Gly Gly Glu Asn Arg Glu Ser Gly Arg Arg Gly Leu 
            500                 505                 510 

Lys Gly Leu Phe Cys Pro Arg Arg Gly Ser Arg Val Glu Gly Trp Asn 
        515                 520                 525 

Gln Gly Glu Gly Gly Cys 
    530 

 
           
             7  
             1320  
             DNA  
             Homo sapiens  
             
               CDS  
               (61)..(1260)  
             
           
            7 

cgggcccccc ctcgaggtcg ggccggactt ggctgggctc ccttcaccct ctgcggagtc     60 

atg agg gcg aac gac gct ctg cag gtg ctg ggc ttg ctt ttc agc ctg      108 
Met Arg Ala Asn Asp Ala Leu Gln Val Leu Gly Leu Leu Phe Ser Leu 
1               5                   10                  15 

gcc cgg ggc tcc gag gtg ggc aac tct cag gca gtg tgt cct ggg act      156 
Ala Arg Gly Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr 
            20                  25                  30 

ctg aat ggc ctg agt gtg acc ggc gat gct gag aac caa tac cag aca      204 
Leu Asn Gly Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr 
        35                  40                  45 

ctg tac aag ctc tac gag agg tgt gag gtg gtg atg ggg aac ctt gag      252 
Leu Tyr Lys Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu 
    50                  55                  60 

att gtg ctc acg gga cac aat gcc gac ctc tcc ttc ctg cag tgg att      300 
Ile Val Leu Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile 
65                  70                  75                  80 

cga gaa gtg aca ggc tat gtc ctc gtg gcc atg aat gaa ttc tct act      348 
Arg Glu Val Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr 
                85                  90                  95 

cta cca ttg ccc aac ctc cgc gtg gtg cga ggg acc cag gtc tac gat      396 
Leu Pro Leu Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp 
            100                 105                 110 

ggg aag ttt gcc atc ttc gtc atg ttg aac tat aac acc aac tcc agc      444 
Gly Lys Phe Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser 
        115                 120                 125 

cac gct ctg cgc cag ctc cgc ttg act cag ctc acc gag att ctg tca      492 
His Ala Leu Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser 
    130                 135                 140 

ggg ggt gtt tat att gag aag aac gat aag ctt tgt cac atg gac aca      540 
Gly Gly Val Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr 
145                 150                 155                 160 

att gac tgg agg gac atc gtg agg gac cga gat gct gag ata gtg gtg      588 
Ile Asp Trp Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val Val 
                165                 170                 175 

aag gac aat ggc aga agc tgt ccc ccc tgt cat gag gtt tgc aag ggg      636 
Lys Asp Asn Gly Arg Ser Cys Pro Pro Cys His Glu Val Cys Lys Gly 
            180                 185                 190 

cga tgc tgg ggt cct gga tca gaa gac tgc cag aca ttg acc aag acc      684 
Arg Cys Trp Gly Pro Gly Ser Glu Asp Cys Gln Thr Leu Thr Lys Thr 
        195                 200                 205 

atc tgt gct cct cag tgt aat ggt cac tgc ttt ggg ccc aac ccc aac      732 
Ile Cys Ala Pro Gln Cys Asn Gly His Cys Phe Gly Pro Asn Pro Asn 
    210                 215                 220 

cag tgc tgc cat gat gag tgt gcc ggg ggc tgc tca ggc cct cag gac      780 
Gln Cys Cys His Asp Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp 
225                 230                 235                 240 

aca gac tgc ttt gcc tgc cgg cac ttc aat gac agt gga gcc tgt gta      828 
Thr Asp Cys Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val 
                245                 250                 255 

cct cgc tgt cca cag cct ctt gtc tac aac aag cta act ttc cag ctg      876 
Pro Arg Cys Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu 
            260                 265                 270 

gaa ccc aat ccc cac acc aag tat cag tat gga gga gtt tgt gta gcc      924 
Glu Pro Asn Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala 
        275                 280                 285 

agc tgt ccc cat aac ttt gtg gtg gat caa aca tcc tgt gtc agg gcc      972 
Ser Cys Pro His Asn Phe Val Val Asp Gln Thr Ser Cys Val Arg Ala 
    290                 295                 300 

tgt cct cct gac aag atg gaa gta gat aaa aat ggg ctc aag atg tgt     1020 
Cys Pro Pro Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys Met Cys 
305                 310                 315                 320 

gag cct tgt ggg gga cta tgt ccc aaa gcc tgt gag gga aca ggc tct     1068 
Glu Pro Cys Gly Gly Leu Cys Pro Lys Ala Cys Glu Gly Thr Gly Ser 
                325                 330                 335 

ggg agc cgc ttc cag act gtg gac tcg agc aac att gat gga ttt gtg     1116 
Gly Ser Arg Phe Gln Thr Val Asp Ser Ser Asn Ile Asp Gly Phe Val 
            340                 345                 350 

aac tgc acc aag atc ctg ggc aac ctg gac ttt ctg atc acc ggc ctc     1164 
Asn Cys Thr Lys Ile Leu Gly Asn Leu Asp Phe Leu Ile Thr Gly Leu 
        355                 360                 365 

aat ggg tta gag atc ctg cct tcc ctc ctt aga ccc cag ccc acg cac     1212 
Asn Gly Leu Glu Ile Leu Pro Ser Leu Leu Arg Pro Gln Pro Thr His 
    370                 375                 380 

ccc tca cag ttc att tca ttg gcc aaa act ttc cta tgt gga gct gac     1260 
Pro Ser Gln Phe Ile Ser Leu Ala Lys Thr Phe Leu Cys Gly Ala Asp 
385                 390                 395                 400 

taggaatcaa agtcataaaa ttctagcctg ttaaaaaaaa aaaaaaaaaa aaaaaaaaaa   1320 

 
           
             8  
             400  
             PRT  
             Homo sapiens  
           
            8 

Met Arg Ala Asn Asp Ala Leu Gln Val Leu Gly Leu Leu Phe Ser Leu 
1               5                   10                  15 

Ala Arg Gly Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr 
            20                  25                  30 

Leu Asn Gly Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr 
        35                  40                  45 

Leu Tyr Lys Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu 
    50                  55                  60 

Ile Val Leu Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile 
65                  70                  75                  80 

Arg Glu Val Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr 
                85                  90                  95 

Leu Pro Leu Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp 
            100                 105                 110 

Gly Lys Phe Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser 
        115                 120                 125 

His Ala Leu Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser 
    130                 135                 140 

Gly Gly Val Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr 
145                 150                 155                 160 

Ile Asp Trp Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val Val 
                165                 170                 175 

Lys Asp Asn Gly Arg Ser Cys Pro Pro Cys His Glu Val Cys Lys Gly 
            180                 185                 190 

Arg Cys Trp Gly Pro Gly Ser Glu Asp Cys Gln Thr Leu Thr Lys Thr 
        195                 200                 205 

Ile Cys Ala Pro Gln Cys Asn Gly His Cys Phe Gly Pro Asn Pro Asn 
    210                 215                 220 

Gln Cys Cys His Asp Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp 
225                 230                 235                 240 

Thr Asp Cys Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val 
                245                 250                 255 

Pro Arg Cys Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu 
            260                 265                 270 

Glu Pro Asn Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala 
        275                 280                 285 

Ser Cys Pro His Asn Phe Val Val Asp Gln Thr Ser Cys Val Arg Ala 
    290                 295                 300 

Cys Pro Pro Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys Met Cys 
305                 310                 315                 320 

Glu Pro Cys Gly Gly Leu Cys Pro Lys Ala Cys Glu Gly Thr Gly Ser 
                325                 330                 335 

Gly Ser Arg Phe Gln Thr Val Asp Ser Ser Asn Ile Asp Gly Phe Val 
            340                 345                 350 

Asn Cys Thr Lys Ile Leu Gly Asn Leu Asp Phe Leu Ile Thr Gly Leu 
        355                 360                 365 

Asn Gly Leu Glu Ile Leu Pro Ser Leu Leu Arg Pro Gln Pro Thr His 
    370                 375                 380 

Pro Ser Gln Phe Ile Ser Leu Ala Lys Thr Phe Leu Cys Gly Ala Asp 
385                 390                 395                 400