1. Introduction

The present invention relates to the use of proteins, peptides and organic molecules capable of modulating inositol 1, ,5-triphosphate (IP ₃ ) receptor signal transduction in order to inhibit or reverse inappropriate growth of cells associated with

10 abnormalities of signal transduction associated with tyrosine kinases. The present invention also relates to the use of IP ₃ receptor mutants in the treatment of proliferative disorders associated with abnormalities of signal transduction associated with tyrosine m - kinases, including cancer.

The present invention also relates to the use of IP ₃ receptor and genetically engineered host cells that express the IP ₃ receptor to evaluate and screen for substances and compounds that modulate IP ₃ receptor

20 activities.

2. Background

Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate

_2S diverse cellular processes are relayed to the interior of cells. These processes include, but are not limited to, cell proliferation, differentiation and survival. A central feature of signal transduction is the reversible phosphorylation of certain proteins.

₃₀ (for reviews, see Posada et al . , 1992, Mol. Biol. Cell 2:583-392; Hardie, D.G. , 1990, Svmp. Soc. Exp. Biol. 4_4.:241-255) . The phosphorylation state of a protein is modified through the reciprocal actions of tyrosine kinases (TKs) , which function to phosphorylate

_ _s proteins, and tyrosine phosphatases (TPs) , which function to dephosphorylate proteins. Normal cellular

function requires a delicate balance between the activities of these two types of enzyme.

Receptor tyrosine kinases comprise a large f mily of trans embrane receptors for polypeptide growth factors with diverse biological activities and are composed of at least three domains: an extracellular ligand binding domain, a transmembrane domain and a cytoplasmic catalytic domain that can phosphorylate tyrosine residues. Their intrinsic tyrosine kinase function is activated upon ligand binding, which results in phosphorylation of the receptor and multiple cellular substrates, and subsequently in a variety of cellular responses (Ullrich et al . , 1990, Cell 11:203-212) . The secondary signal transducer molecules generated by activated receptors result in a signal cascade that regulates cell functions. For example, phosphorylation of phospholipase c activates this target molecule to hydrolyze phosphatidylinositol 4,5- bisphosphate, generating two secondary signal transducing molecules: inositol 1,4,5-triphosphate, which causes release of stored intracellular calcium, and diacylglycerol, which is the endogenous activator of a serine/threonine kinase, protein kinase C. Reviews describing intracellular signal transduction include Aaronson, S.A., 1991, Science 254:1146-1153; Schlessinger, J., 1988, Trends Biocheπt. Sci. 13:443- 447; and Ullrich and Schlessinger, 1990, Cell 61:203- 212. Various, cell proliferative disorders, including cancer, atherosclerosis, immune deficiency, neurodegenerative disease and hyperproliferative disease, such as psoriasis, have been associated with defects in different signaling pathways mediated by receptor tyrosine kinases. Examples of specific

receptor tyrosine kinases associated with cell proliferative disorders include platelet derived growth factor receptor (PDGFR) , epidermal growth factor receptor (EGFR) , and HER2. ^* The gene encoding HER2 (her-2) is also referred to as neu, and c-erbB-2 (Sla on et al., 1987 Science 235:177-182) .

Platelet-derived growth factors (PDGFs) and PDGF- receptors are expressed in cells of a variety of neoplasms including gliomas, lung carcinomas, ovarian tumors, and melanomas. For example, co-expression of PDGF and PDGF receptors in several human glioma cell lines has been reported (Nister et al . , 1991, J. Biol. Chem. 266:16755-16763; Nister et al . , 1988, Cancer Res. 48.:3910-3918) . PDGF and PDGF-S-receptors have been identified in primary human lung carcinomas

(Antoniades et al . , 1992, Proc. Natl. Acad. Sci. USA j3£:3942-3946) . Henriksen et al . , 1993, Cancer Res. 5.2:4550-4554) reported detecting PDGF and PDGF-α-receptor expression in malignant epithelial ovarian tumor specimens. PDGF receptors have been detected in several other cancers of epithelial origin, including human thyroid carcinoma cells (Heldin et al . , 1991, Endocrinology 129:2187-2193: Heldin et al . , 1988, Proc. Natl. Acad. Sci. USA 85_:9302-9306) , human breast cancer cells (Ginsburg et al., 1991, Cancer Letters 58:137-144). and primary gastric carcinomas (Chung et al . , 1992, Cancer Res. 52.:3453-3459) .

HER2/neu gene amplification has been linked by some investigators to neoplastic transformation. For example, the HER2/neu gene has been shown to be amplified in human breast cancer cells (Slamon et al. , 1987, supra) . Amplification and/or overexpression of HER2/neu has been detected in gastrointestinal, non- small cell lung, and ovarian adenocarcinomas and

occurs in a significant fraction of primary human breast cancers where it correlates with regionally advanced disease, increased probability of tumor recurrence, and reduced patient survival (Slamon et al . , 1987, supra and Slamon, 1989, Science 244:707- 712) .

Zeillinger et al., 1993, (Clin. Biochem. 26:221- 227) reported that increased expression of EGF receptor (EGF-R) has been found in various malignancies, including breast cancer. Positive EGF-R status has been associated with the aggressiveness of carcinomas of the cervix, ovaries, esophagus, and stomach. See, for example, Karameris et al . , 1993, Path. Res. Pract. 189:133-137; Hale et al . _f 1993, J^ Clin. Pathol. 46:149-153: Caraglia et al. , 1993,

Cancer Immunol. Immunother 2Z:150-156; and Koenders et al . , 1993, Breast Cancer Research and Treatment 25:21- 27.

The control of Ca ² * homeostasis, which plays an important physiological role in muscle contraction, cardiac function, maintenance of the integrity of membranes and coagulation of the blood, is mediated by the receptor-generated second messenger, inositol 1,4 ,5-trisphosphate (IP ₃ ) , and its binding to, and activation of, a family of intracellular Ca ² *-permeable channels, the IP ₃ receptors (IP ₃ Rs) (Siidhof et al . , 1991, EMBO J. 10:3199-3206; Ross et al . , 1992, Proc. Natl. Acad. Sci. USA 89:4265-4269: Berridge, M.J. , 1993, Nature 361:315-321: Pozzan et al . , 1994, Phvsiol. Rev.) . These channels are established by the interaction of four high Mr (about 300 kDa) identical or highly homologous subunits inserted into the endoplasmic reticulum membrane by means of 6 spanning sequences localized in the C-terminal domain of the IP ₃ receptors. The bulk of each subunit protrudes into

the cytosol (Furuichi et al., 1989. Nature 342:32-38; Maeda et al . , 1990, EMBO J 9:61-67 and 1991; Mignery et al . , 1990, EMBO J. 9:3893-3898: Mignery et al . ,

1989, Nature 342:192-195 and Mignery et al . , 1990, J.Biol. Chem. 265:12679-12685) . Deletion studies have revealed that the N-terminal domain of IP ₃ receptors contains the second messenger binding site and the C- terminal domain, with the transmembrane sequences, is necessary for membrane insertion and assembly of the subunits to yield the tetrameric organization of the IP ₃ Rs (Mignery et al., 1990, supra ; Mignery et al . ,

1990, supra ; Miyawaki et al . , 1991, Proc. Natl. Acad. Sci. USA 88.:4911-4915) .

3. Summary of Invention

The present invention relates to the use of proteins, peptides and organic molecules capable of modulating inositol 1,4,5-triphosphate (IP ₃ ) receptor signal transduction in order to inhibit or reverse inappropriate growth of cells associated with abnormalities of signal transduction associated with tyrosine kinases. The present invention also relates to the use of IP ₃ receptor mutants in the treatment of proliferative disorders associated with abnormalities of signal transduction associated with tyrosine kinases, including cancer.

The present invention also relates to the use of IP ₃ receptor and genetically engineered host cells that express the IP ₃ receptor to evaluate and screen for substances or compounds that modulate IP ₃ receptor activities.

The present invention is based, in part, upon the discovery that IP ₃ receptors and Ca ² + stores play an important role in intracellular signalling pathways. The present invention is also based, in part, on the

discovery that modulation of IP ₃ receptor signal transduction affects cell growth and a transformed phenotype.

The present invention is based, in part, upon the unexpected discovery that introduction of signalling incompetent IP ₃ receptor mutants to transformed fibroblast cells suppresses the level of transformation in those cells and the discovery that the suppression of transforming activities is not oncogene specific. In addition, the inventors have discovered that introduction of signalling incompetent IP ₃ receptor mutants to normal cells does not have a negative effect on cell growth or survival. The present invention is therefore based, in part, on the unexpected discovery that by introducing signalling incompetent IP ₃ receptor mutant gene sequences to cells, gene therapy may be used to inhibit or reverse tyrosine kinase induced cell transformation without affecting signalling properties of normal cells. The present invention also relates, in part, to the use of genetically engineered host cells expressing IP ₃ receptor or receptor mutants to screen and identify IP ₃ receptor agonists and antagonists. Soluble IP ₃ receptor mutants which retain the capability to bind IP ₃ may be used to screen, for example, peptide libraries or organic molecules capable of modulating the IP ₃ receptor signal transduction.

Pharmaceutical reagents designed to inhibit the IP ₃ /IP ₃ receptor interaction or other molecular interactions which activate or are activated by the IP ₃ signalling pathway may be useful in inhibition of innapropriate cell growth associated with abnormalities in signal transduction associated with

receptor tyrosine kinases including cancer, atherosclerosis and psoriasis.

4. Brief Description of the Figures Figures 1A-1B. Biogenesis and expression levels of the IP receptor and mutants s-IP ₃ R and Δ-IP ₃ R, of the major lumenal Ca ² * binding protein, calreticulin (CR),and of the SERCA ATPases in stable NIH 3T3 cell transfectant clones. Figure 1A shows audoradiographs of immunoprecipitates obtained with the anti-type I IP ₃ R-specific T210 Ab. Figure IB shows Western blotting of the transfected clones. Size marker positions in kDa are indicated to the left.

Figures 2A, 2B and 2C. Ca ² * responses and IP ₃ production induced by ATP and EGF in stable NIH 3 3 transfectant clones challenged in Ca ² *-poor incubation medium. Figure 2A shows the concentration-dependence of the [Ca ² * i responses induced by EGF or ATP in stable cell clones infected with NTK-HERc virus (M0I:5) and expressing endogenous IP ₃ receptors.

Closed circles axe 3T3/T10-HERC, closed triangles are 3T3/Δ-IP ₃ R5-HERc and closed squares are Δ-IP ₃ R6-HERc. Figure 2B shows the [ATP]-dependence of the [Ca ² *] ₁ responses in 3T3/T10-HERC and Δ-IP ₃ R6-HERc clones (circles and squaxes, respectively) with or without subsequent infection with ¥2-TGFα virus (MOI:3; filled and empty symbols, respectively) . Figure 2C show the IP ₃ production in the control and Δ-IP ₃ R expressing clones, labeled as in Figure 2A. In the investigated cells, the resting average [Ca ² *]i was 130 nM and the average radioactivity of IP ₃ was 7800 cpm. Results shown are means +/- SD of five-ten separate experiments.

Figure 3. Release of Ca ^2* from NIH 3T3 stable transfectant clones per eabilized with saponin and

then exposed either to various concentrations of IP ₃ or to Tg (100 nM) . The release values shown are the peak [Ca2+] increases revealed by fura-2 free acid (0.5 μM) added to the incubation medium. Results shown are means +/- SD of four experiments.

Figure 4. EGF-sti ulated [ ³ H] thymidine incorporation. Cells were infected with the NTK-HERc virus (M0I:5), expanded, seeded in 12-well plates, grown to confluence, and starved for 18 hours before stimulation. Error bars indicate the range of two independent experiments.

Figures 5A, 5B, 5C and 5D. Differential effects of IP ₃ receptor mutant expression of long-term growth of NIH 3T3 cells stimulated by autocrine TGFα and EGF- R overexpression. Cells were infected with NTK-HERc virus (MOI:5), superinfected with Ψ2-TGFα virus (MOI:3), and seeded in 96-well plates. Growth curves determined with the MTT assay are shown for 3T3/T10, 3T3/IP ₃ R16, 3T3/sIP ₃ R and 3T3/Δ-IP ₃ R5 cells maintained under the conditions indicated for 6 days [DMEM with 2___M glutamine supplemented with 0.5% FCS (represented by open circles) , 4% FCS (represented by closed circles) , or 10% FCS (represented by closed squares) . Error bars indicate SEM from triplicates. Figures 6A and 6B. Substrate tyrosine phosphorylation in control 3T3/T10 and 3T3/Δ-IP ₃ R cells. Cells were infected with NTK-HERc virus (M0I:5) together with ¥2-N2 virus (M0I:5) (lanes 1, 2, 4, 5) or *2-TGFα virus (M0I:3) (lanes 3,6). Cells were grown to confluence, starved for 24 hours in DMEM containing 0.5% FCS, and stimulated for 10 min with lOOng/ml EGF or by the autocrine loop with TGFα. The cells were solubilized, and for each lane 30 μg total protein separated by SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with

the antiphosphotyrosine antibody 5E2 (Fendly et al. Cancer Research .50:1550-1558, 1990), followed by the ECL detection system (Figure 6A) . Subsequently the nitrocellulose was stripped and reprobed with the anti-EGF-R Ab RK2 (Kris et al. Cell 40:619-625, 1985) for EGF-R control (Figure 6B) . Size marker positions are indicated in kDa.

Figures 7A-7F. The nucleic acid (SEQ ID N0:1) and deduced amino acid sequence (SEQ ID NO: 2) of rat IP3 receptor.

Figures 8A-8E. The shared amino acid sequence homology of rat IP3 receptor and human IP3 receptor.

5. Detailed Description The present invention relates to the use of proteins, peptides and organic molecules capable of modulating inositol 1,4,5-triphosphate (IP ₃ ) receptor signal transduction in order to inhibit or prevent inappropriate growth of cells associated with abnormalities in signal transduction associated with tyrosine kinases. Such modulators of IP ₃ may be used therapeutically. For example, antagonists of IP ₃ receptor signal transduction may find application in the treatment of proliferative disorders associated with abnormalities in signal transduction associated with tyrosine kinase, such as deregulation of tyrosine kinase function, including for example, cancer, atherosclerosis and psoriasis.

The present invention also relates to the use of IP ₃ receptor_mutants in the treatment of proliferative disorders associated with abnormalities of signal transduction associated with tyrosine kinases.

The present invention also relates to the use of IP ₃ receptor and genetically engineered host cells that express the IP ₃ receptor to evaluate and screen for

substances and compounds that modulate IP ₃ receptor activities.

The invention is based, in part, on the unexpected discovery that introduction of signalling incompetent IP ₃ receptor mutants to transformed fibroblast cells suppresses the level of transformation in those cells without corresponding adverse effects in normal cells, and the discovery that the suppression of transforming activities is not oncogene specific. Experiments are described herein, in which NIH 3T3 cells were transformed by coinfection of TGFα-expressing virus and the human EGF receptor, NTK-HERc. In transformed NIH 3T3 cells also expressing signalling-incompetent IP ₃ receptor mutants, the level of transformation was suppressed to a level of approximately 10%. In transformed NIH 3T3 cells also expressing soluble receptor mutants that retain the IP ₃ binding cite, the level of transformation was reduced significantly by about 35% in comparison to control cells.

The present invention is also based, in part, on the discovery that while the introduction of signalling incompetent IP ₃ receptor mutants to normal cells may modulate signal transduction and cellular Ca ² * homeostasis, other cellular mechanisms act to keep near normal cellular signalling properties.

As used herein, the phrase, "inappropriate growth of cells" includes abnormal, uncontrolled or deregulated cellular proliferation or differentiation, e .g. , under or over production of mature differentiated cells and inappropriate proliferation of immature cells, growth of abnormal cells and untimely cell death.

5.1. The IPj Receptor/Receptor Mutant Coding Sequences

Members of the IP ₃ receptor family have been described in Danoff et al . (Proσ. Neuropsvcho-

₅ Pharmacol. Biol. Psvchiatrv 18.:1-16, 1994) and Ross et al . (PNAS 89H01:4265-4269, 1992). Figures 7A-7F are the nucleic acid and deduced amino acid sequence of rat IP3 receptor. Figures 8A-8E demonstrate the shared amino acid sequence homology of rat IP3

₁₀ receptor and human IP3 receptor.

In accordance with the invention, any nucleotide sequence which encodes the amino acid sequence of an IP ₃ receptor gene product can be used to generate recombinant molecules which direct the expression of

- ₅ an IP ₃ receptor. The invention contemplates, in addition to the DNA sequences disclosed herein, 1) any DNA sequence that encodes the same amino acid sequence as encoded by the DNA sequences shown in Figures 7A- 7F; 2) any DNA sequence that hybridizes to the

₂₀ complement of the coding sequences disclosed herein

(see Figures 7A-7F) under highly stringent conditions, e . g. , washing in O.lxSSC/0.1% SDS at 68_>c (Ausubel F.M. et al . , eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing

₂₅ Associates, Inc., and John Wiley " Sons, Inc., New York, at p. 2.10.3) and encodes a functionally equivalent gene product; and/or 3) any DNA sequence that hybridizes to the complement of the coding sequences disclosed herein (see Figures 7A-7F) under

₃₀ less stringent conditions, such as moderately stringent conditions, e .g. , washing in 0.2xSSC/0.1% SDS at 42oC (Ausubel et al . , 1989, supra) , yet which still encodes a functionally equivalent gene product. The invention also encompasses 1) DNA vectors

__ that contain any of the coding sequences disclosed herein; and/or their complements (i.e., antisense) ; 2)

DNA expression vectors that contain any of the coding sequences disclosed herein, and/or their complements (i.e., antisense) , operatively associated with a regulatory element that directs the expression of the coding and/or antisense sequences; and 3) genetically engineered host cells that contain any of the coding sequences disclosed herein, and/or their complements (i.e., antisense), operatively associated with a regulatory element that directs the expression of the coding and/or antisense sequences in the host cell. Regulatory element includes but is not limited to inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression. The invention includes fragments of any of the DNA sequences disclosed herein.

As used herein, IP ₃ receptor is a term which refers to any member of the IP ₃ receptor family from any species, including, bovine, ovine, porcine, equine, and preferably human, in naturally occurring- sequence or in variant form, or from any source, whether natural, synthetic, or recombinant. As used herein, IP ₃ receptor mutant refers to a non-naturally occurring IP ₃ receptor. Preferred IP ₃ receptor mutants are those which lack the N-terminal IP ₃ binding domain and/or those which lack the C-terminal transmembrane domain. As used herein, the phrase "signalling incompetent IP ₃ receptor mutant", refers to an IP ₃ receptor mutant that is not capable of transducing a signal or that transduces a signal to a lesser extent than the naturally occurring IP ₃ receptor.

A particularly preferred IP ₃ receptor mutant of the present invention is Δ-IP ₃ R lacking 418 amino acids in the IP ₃ receptor N-terminal sequence, resulting in an IP ₃ receptor which lacks the IP ₃ binding domain and

which is expected to be signalling incompetent, i.e., unable to transduce a signal (Mignery and Sϋdhof et al . , 1990, supra ; Mignery et al., 1990, supra) . Δ-IP ₃ R is expected to retain the ability to assemble with naturally occurring IP ₃ receptor subunits to form a tetrameric Ca ² * channel across the endoplasmic reticulum membrane.

In another particularly preferred IP ₃ receptor mutant, s-IP ₃ R, a deletion of 379 amino acids is made in the C-terminal IP ₃ receptor sequence resulting in IP ₃ receptors which are expected to retain the IP ₃ binding domain but which lack the transmembrane domain necessary for transmembrane insertion (Mignery and Sϋdhof et al., 1990, supra ; Mignery et al . , 1990, supra) . s-IP ₃ R is expected to remain as a soluble monomer in the cytosol.

5.2. Expression of IP ₃ Receptor

In accordance with the invention, IP ₃ receptor polynucleotide sequences which encode naturally occurring IP ₃ receptors, peptide fragments of IP ₃ receptors, IP ₃ receptor fusion proteins or functional equivalents thereof, or IP ₃ receptor mutants, for example, signalling incompetent IP ₃ receptor mutants, may be used to generate recombinant DNA molecules that direct the expression of IP ₃ receptor protein, IP ₃ receptor peptide fragment, fusion proteins or a functional equivalent thereof or IP ₃ receptor mutants, in appropriate host cells. Such IP ₃ receptor polynucleotide sequences, as well as other polynucleotides which selectively hybridize to at least a part of such IP ₃ receptor polynucleotides or their complements, may also be used in nucleic acid hybridization assays, Southern and Northern blot analyses, etc.

Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence, may be used in the practice of the invention for the cloning and expression of IP ₃ receptor protein. Such DNA sequences include those which are capable of hybridizing to the human IP ₃ receptor sequence under stringent conditions. The phrase "stringent conditions" as used herein refers to those hybridizing conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50 ^C C; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M Sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 X SSC and 0.1% SDS.

Altered DNA sequences which may be used in accor¬ dance with the invention include deletions, additions or substitutions of different nucleotide residues resulting in a sequence that encodes the same or a functionally equivalent gene product. The gene product itself may contain deletions, additions or substitutions of amino acid residues within an IP ₃ receptor sequence, which result in a silent change thus producing a functionally equivalent IP ₃ receptor. Such amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipatic nature of the residues involved. For example,

negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include the following: leucine, isoleucine, valine; glycine, alanine; asparagine, gluta ine; serine, threonine; phenylalanine , tyrosine.

The DNA sequences of the invention may be engi¬ neered in order to alter an IP ₃ receptor coding sequence for a variety of ends including but not limited to alterations which modify processing and expression of the gene product. For example, mutations may be introduced using techniques which are well known in the art, e .g. , site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, phosphorylation, etc.

In another embodiment of the invention, an IP ₃ receptor or receptor mutant may be ligated to a heterologous sequence to encode a fusion protein. For example, for screening of peptide libraries for inhibitors of IP ₃ signal transduction, it may be useful to encode a chimeric IP ₃ receptor or receptor mutant protein expressing a heterologous epitope that is recognized by a commercially available antibody. A fusion protein may also be engineered to contain a cleavage site located between an IP ₃ receptor or receptor mutant sequence and the heterologous protein sequence, so that the IP ₃ receptor or receptor mutant may be cleaved away from the heterologous moiety. In an alternate embodiment of the invention, the coding sequence of an IP ₃ receptor or receptor mutant could be synthesized in whole or in part, using chemical methods well known in the art. See, for example, Caruthers et al . , 1980, Nuc. Acids Res. Sv p. Ser. 2:215-233; Crea et al . , 1980, Nuc. Acids Res.

9X101:2331; Matteucci et al . , 1980, Tetrahedron Letters 2_1:719; and Chow et al . , 1981, Nuc. Acids Res. 9(12) :2807-2817. Alternatively, the protein itself could be produced using chemical methods to synthesize an IP ₃ receptor or receptor mutant amino acid sequence in whole or in part. For example, peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high perform¬ ance liquid chromatography (e.g., see Creighton, 1983, Proteins Structures And Molecular Principles. W.H.

Freeman and Co., N.Y. pp. 50-60). The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Ed an degradation procedure; see Creighton, 1983, Proteins . Structures and Molecular Principles. W.H. Freeman and Co. , N.Y. , pp. 34-49).

In order to express a biologically active IP ₃ receptor or IP ₃ receptor mutant, the nucleotide sequence coding for IP ₃ receptor, receptor mutant or a functional equivalent, is inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. The IP ₃ receptor gene products as well as host cells or cell lines transfected or transformed with recombinant IP ₃ receptor expression vectors can be used for a variety of purposes. These include but are not limited to generating antibodies (i.e., monoclonal or polyclonal) that competitively inhibit activity of an IP ₃ receptor and neutralize its activity. Anti-IP ₃ receptor antibodies may be used in detecting and quantifying expression of an IP ₃ receptor in cells and tissues.

5.3. Expression Systems

Methods which are well known to those skilled in the art can be used to construct expression vectors containing an IP ₃ receptor or receptor mutant coding sequence and appropriate transcriptional/translational control signals. These methods include In vitro reσombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, for example, the techniques described in Maniatis et al . , 1989, Molecular Cloning A Laboratory Manual. Cold Spring Harbor Laboratory, N.Y. and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y.

A variety of host-expression vector systems may be utilized to express an IP ₃ receptor or receptor mutant coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing an IP ₃ receptor or receptor mutant coding sequence; yeast transformed with recombinant yeast expression vectors containing an IP ₃ receptor or receptor mutant coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g. , baculovirus) containing an IP ₃ receptor or receptor mutant coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expres- sion vectors (e.g., Ti plasmid) containing an IP ₃ receptor or receptor mutant coding sequence; or animal cell systems.

The expression elements of these systems vary in their strength and specificities. Depending on the host/vector system utilized, any of a number of

suitable transcription and translation elements, including constitutive and inducible promoters, may be used in the expression vector. For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used; when cloning in insect cell systems, promoters such as the baculovirus polyhedrin promoter may be used; when cloning in plant cell systems, promoters derived from the genome of plant cells (e.g., heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll a/b binding protein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; the coat protein promoter of TMV) may be used; when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5 K promoter) may be used; when generating cell lines that contain multiple copies of an IP ₃ receptor or receptor mutant DNA, SV40-, BPV- and EBV- based vectors may be used with an appropriate selectable marker.

In bacterial systems a number of expression vectors may be advantageously selected depending upon the use intended for the IP ₃ receptor or receptor mutant expressed _* For example, when large quantities of IP ₃ "receptor are to be produced for the generation of antibodies, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include but are not limited to the E . coli expression vector pUR278 (Ruther et al . , 1983, EMBO J. 2:1791), in which the IP ₃ receptor coding sequence may be ligated into the vector in frame with the lac Z coding

region so that a hybrid lac Z protein is produced; pIN vectors (Inouye et al . , 1985, Nucleic acids Res. 13:3101-3109; Van Heeke et al . , 1989, J. Biol. Chem. 264:5503-5509) ; and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST) . In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety. In yeast, a number of vectors containing constitutive or inducible promoters may be used. For a review see , Current Protocols in Molecular Biology. Vol. 2, 1988, Ed. Ausubel et al . , Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant et al . , 1987, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 1987, Acad. Press, N.Y. 153:516-544: Glover, 1986, DNA Cloning. Vol. II. IRL Press, Wash., D.C., Ch. 3; and Bitter, 1987, Heterologous Gene Expression in Yeast, Methods in Enzymology. Eds. Berger & Kimmel, Acad. Press, N.Y. 152:673-684: and The Molecular Biology of the Yeast Saccharomvces. 1982, Eds. Strathern et al.. Cold Spring Harbor Press, Vols. I and II.

In cases where plant expression vectors are used, the expression of an IP ₃ receptor or receptor mutant coding sequence may be driven by any of a number of promoters. For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al . , 1984, Nature 310:511-5141. or the coat protein promoter of TMV (Taka atsu et al . , 1987, EMBO J. 1:307-311) may be used; alternatively, plant promoters

such as the small subunit of RUBISCO (Coruzzi et al . , 1984, EMBO J. 2:1671-1680; Broglie et al . , 1984, Science 224:838-843) : or heat shock promoters, e.g., soybean hspl7.5-E or hspl7.3-B (Gurley et al . , 1986, Mol. Cell. Biol. 1:559-565) may be used. These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, etc. For reviews of such techniques see, for example, Weissbach & Weissbach, 1988, Methods for Plant

Molecular Biology. Academic Press, NY, Section VIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular Biology. 2d Ed., Blackie, London, Ch. 7-9. An alternative expression system which could be used to express an IP ₃ receptor or receptor mutant is an insect system. In one such system, Autographa californlca nuclear polyhidrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. An IP ₃ receptor or receptor mutant coding sequence may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example, the polyhedrin promoter) . Successful insertion of an IP ₃ receptor or receptor mutant coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene) . These recombinant viruses are then used to infect Spodoptera fpigiperda cells in which the inserted gene is expressed. (e.g., see Smith et al . , 1983, J. Viol. 4_6:584; Smith, U.S. Patent No. 4,215,051).

In mammalian host cells, a number of viral based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, an IP ₃

receptor or receptor mutant coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or In vivo recombination. Insertion in a non- essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing an IP ₃ receptor or receptor mutant in infected hosts (e.g.. See Logan et al . , 1984, Proc. Natl. Acad. Sci. (USA. 81:3655-3659). Alternatively, the vaccinia 7.5 K promoter may be used. (See, e .g. , Mackett et al . , 1982, Proc. Natl. Acad. Sci. (USA) 79:7415-7419; Mackett et al . , 1984, J. Virol. 49.:857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci. 79:4927-4931) .

Specific initiation signals may also be required for efficient translation of inserted IP ₃ receptor or receptor mutant coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire IP ₃ receptor or receptor mutant gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of an IP ₃ receptor or receptor mutant coding sequence is inserted, exogenous translational control signals, including the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of an IP ₃ receptor or receptor mutant coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression

may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al . , 1987, Methods in Enzv ol. 153:516-544). In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have charac¬ teristic and specific mechanisms for the post-transla- tional processing and modification of proteins. Appropriate cells lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, WI38, etc.

For long-term, high-yield production of recombi¬ nant proteins, stable expression is preferred. For example, cell lines which stably express an IP ₃ receptor or receptor mutant may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with IP ₃ receptor or receptor mutant DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription termina¬ tors, polyadenylation sites, etc.), and a selectable marker. Following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a

selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may be used advantageously to engineer cell lines which express an IP ₃ receptor or receptor mutant.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al . , 1977, Cell 11:223) . hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al . , 1962, Proc. Natl. Acad. Sci. USA 48.2026), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 2 _. 2:817) genes can be employed in tk, hgprt" or aprt" cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to ethotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 27:3567; O'Hare et al . , 1981, Proc. Natl. Acad. Sci. USA 2§.1527); gpt, which confers resistance to mycophenolic acid (Mulligan et al., 1981, Proc. Natl. Acad. Sci. USA 78.:2072) ; neo, which confers resistance to the a inoglycoside G-418 (Colberre-Garapin et al . , 1981, J. Mol. Biol. 150:1) ; and hygro, which confers resistance to hygromycin (Santerre et al . , 1984, Gene 3_0:147). Recently, additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman et al., 1988, Proc. Natl. Acad. Sci. USA 8j5:8047); and ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L. , 1987, In: Current Communications in

Molecular Biology. Cold Spring Harbor Laboratory, Ed.) .

5.4. Identification of Transfectants or

Transformants That Express IP ₃ Receptor or Receptor Mutant

The host cells which contain the coding sequence and which express the biologically active gene product may be identified by at least four general approaches; (a) DNA-DNA or DNA-RNA hybridization; (b) the presence or absence of "marker" gene functions; (c) assessing the level of transcription as measured by the expres¬ sion of IP ₃ receptor or receptor mutant mRNA transcripts in the host cell; and (d) detection of the gene product as measured by immunoassay or by its biological activity.

In the first approach, the presence of the IP ₃ receptor or receptor mutant coding sequence inserted in the expression vector can be detected by DNA-DNA or DNA-RNA hybridization using probes comprising nucleotide sequences that are homologous to the IP ₃ receptor or receptor mutant coding sequence, respectively, or portions or derivatives thereof.

In the second approach, the recombinant expres¬ sion vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.). For example, if the IP ₃ receptor or receptor mutant coding sequence is inserted within a marker gene sequence of the vector, recombinant cells containing the IP ₃ receptor or receptor mutant coding sequence can be identified by the absence of the marker gene function. Alternatively, a marker gene can be placed in tandem with an IP ₃ receptor or

receptor mutant sequence under the control of the same or different promoter used to control the expression of the IP ₃ receptor or receptor mutant coding sequence. Expression of the marker in response to induction or selection indicates expression of the IP ₃ receptor or receptor mutant coding sequence.

In the third approach, transcriptional activity for an IP ₃ receptor or receptor mutant coding region can be assessed by hybridization assays. For example, RNA can be isolated and analyzed by Northern blot using a probe homologous to an IP ₃ receptor or receptor coding sequence or particular portions thereof. Alternatively, total nucleic acids of the host cell may be extracted and assayed for hybridization to such probes.

In the fourth approach, the expression of an IP ₃ receptor or receptor mutant protein product can be assessed immunologically, for example by Western blots, immunoassays such as radioimmuno-precipitation, enzyme-linked immunoassays and the like.

5.5. Uses of IP ₃ Receptor and Engineered Cell Lines

Various cell proliferative disorders have been associated with defects in different signal transduction pathways mediated by receptor tyrosine kinases, including gliomas, lung carcinomas, ovarian tumors, thyroid carcinomas, human breast cancer, gastric carcinomas and melanomas; gastrointestinal, non-small cell lung and ovarian adenocarcinomas; cervical, ovarian, esophagal and stomach carcinomas; and atherosclerosis and psoriasis.

Modulation of receptor-generated signal transduction events can be mediated through secondary signal transducer molecules, such as IP ₃ . The term "secondary signal transduction molecules" as used

herein refers to any component or product found in the cascade of signal transduction events. IP ₃ and its binding to and activation of IP ₃ receptor, which in tetrameric organization forms intracellular Ca ² *- permeable channels, plays a role in Ca ² * homeostasis and intracellular signalling events. It has been observed that IP ₃ signal transduction affects cell growth and oncogenesis. Therefore, modulators of IP ₃ receptor signal transduction may be used therapeutically for the treatment of disorders and diseases states resulting from defects in different signal transduction pathways associated with receptor tyrosine kinases.

In an embodiment of the invention, an IP ₃ receptor or receptor mutant and/or cell line that expresses an IP ₃ receptor or receptor mutant may be used to screen for antibodies, peptides, or other molecules that act as agonists or antagonists of IP ₃ receptor through modulation of signal transduction pathways. For example, anti-IP ₃ receptor antibodies capable of neutralizing the activity of IP ₃ receptor may be used to inhibit an IP ₃ receptor associated signal transduction pathway. Such antibodies can act intracellularly utilizing the techniques described in Marasco et al., 1993 fPNAS 90:7889-7893. for example or through delivery by liposomes. Alternatively, screening of organic or peptide libraries with recombinantly expressed IP ₃ receptor or receptor mutant protein or cell lines expressing IP ₃ receptor or receptor mutant protein may be useful for identification of therapeutic molecules that function by modulating IP ₃ receptor signal transduction or Ca ² * homeostasis. Synthetic compounds, natural products, and other sources of potentially biologically active

materials can be screened in a number of ways deemed to be routine to those of skill in the art.

The ability of antibodies, peptides, or other molecules to prevent or mimic, the effect of IP ₃ 5 receptor signal transduction responses on IP ₃ receptor expressing cells may be measured. For example, responses such as modulation of Ca ^2* may be monitored. These assays may be performed using conventional techniques developed for these purposes, described 10 infra.

5.5.1. Antibody Production and Screening

Various procedures known in the art may be used

_ ₅ for the production of antibodies to epitopes of the recombinantly produced IP ₃ receptor or receptor mutants. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression

₂₀ library. Neutralizing antibodies, i.e., those which inhibit the signal transducing activity of an IP ₃ receptor are especially preferred for diagnostics and therapeutics.

For the production of antibodies, various host

_2S animals may be immunized by injection with an IP ₃ receptor protein including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's

₃₀ (complete and incomplete) , mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants

_3S such as BCG (bacilli Calmette-Guerin) and Corynebacterium parvum .

Monoclonal antibodies to an IP ₃ receptor or receptor mutant may be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler et al., 1975 (Nature 256:495-497) . the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today. 1:72; and Cote et al., 1983, Proc. Natl. Acad. Sci.. 80:2026-2030) and the EBV-hybridoma technique (Cole et al . , 1985, Monoclonal Antibodies and Cancer Therapy. Alan R. Liss, Inc., pp. 77-96). In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al . , 1984, Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger et al., 1984, Nature, 312:604- 608; and Takeda et al . , 1985, Nature 211:452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. Alternatively, techniques described for the production of single chain antibodies (U.S. Patent No. 4,946,778) can be adapted to produce IP ₃ receptor-specific single chain antibodies. Antibody fragments which contain specific binding sites of an IP ₃ receptor may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab') ₂ fragments which can be produced by pepsin digestion of the antibody molecule and the Fab .fragments which can be generated by reducing the disulfide bridges of the F(ab') ₂ fragments. Alternatively, Fab expression libraries may be constructed (Huse et al. , 1989, Science 246:1275-1281) to allow rapid and easy identification

of monoclonal Fab fragments with the desired specificity o the IP ₃ receptor.

5.5.2. Screening of Peptide Library with IPj Receptor or IP ₃ Receptor Engineered Cell Lines

Random peptide libraries consisting of all possible combinations of amino acids attached to a solid phase support may be used to identify peptides that are able to bind to the ligand binding site of a given receptor or other functional domains of a receptor such as kinase domains (Lam, K.S. et al . , 1991, Nature 354: 82-84) . The screening of peptide libraries may have therapeutic value in the discovery of pharmaceutical agents that act to modulate the biological activity of receptors through their interactions with the given receptor.

Identification of molecules that are able to bind to an IP ₃ receptor may be accomplished by screening a peptide library with a recombinant soluble IP ₃ receptor mutant. Soluble IP ₃ receptor mutant is described in Mignery and Sϋdhof et al . , 1990, supra and Mignery et al., 1990, supra . Methods for expression of IP ₃ receptor or IP ₃ receptor mutants are described in Section 5.2 and may be used to express recombinant IP ₃ receptor or receptor mutants or fragments of IP ₃ receptors depending on the functional domains of interest. For example, the extracellular ligand binding domains of IP ₃ receptor may be separately expressed and used to screen peptide libraries.

To identify and isolate the peptide/solid phase support that interacts and forms a complex with IP ₃ receptor or receptor mutant, it is necessary to label or "tag" the receptor or receptor mutant molecule. The IP ₃ receptor or receptor mutant protein may be conjugated to enzymes such as alkaline phosphatase or

horseradish peroxidase or to other reagents such as fluorescent labels which may include fluorescein isothyiocynate (FITC) , phycoerythrin (PE) or rhoda ine. conjugation of any given label to an IP ₃ receptor or receptor mutant may be performed using techniques that are routine in the art..

Alternatively, IP ₃ receptor or receptor mutant expression vectors may be engineered to express a chimeric IP ₃ receptor or receptor mutant protein containing an epitope for which a commercially available antibody exist. The epitope-specific antibody may be tagged using methods well known in the art including labeling with enzymes, fluorescent dyes or colored or magnetic beads. The "tagged" IP ₃ receptor or receptor mutant conjugate is incubated with the random peptide library for 30 minutes to one hour at 22°C to allow complex formation between IP ₃ receptor or receptor mutant and peptide species within the library. The library is then washed to remove any unbound IP ₃ receptor or receptor mutant protein. If the IP ₃ receptor or receptor mutant has been conjugated to alkaline phosphatase or horseradish peroxidase the whole library is poured into a petri dish containing a substrates for either alkaline phosphatase or peroxidase, for example, 5-bromo-4-chloro-3-indoyl phosphate (BCIP) or 3,3' ,4,4"-diamnobenzidine (DAB) , respectively. After incubating for several minutes, the peptide/solid phase-IP ₃ receptor complex changes color, and can be easily identified and isolated physically under a dissecting microscope with a micromanipulator. If a fluorescent tagged IP ₃ receptor molecule has been used, complexes may be isolated by fluorescent activated sorting. If a chimeric IP ₃ receptor protein expressing a heterologous epitope

has been used, detection of the peptide/IP ₃ receptor complex may be accomplished by using a labeled epitope specific antibody. Once isolated, the identity of the peptide attached to the solid phase support may be determined by peptide sequencing.

In another embodiment, it is possible to detect peptides that bind to cell surface receptors using intact cells. The use of intact cells is preferred for use with receptors that are multi-subunits or labile or with receptors that require the lipid domain of the cell membrane to be functional. Methods for generating cell lines expressing IP ₃ receptor or receptor mutants are described in Sections 5.3 and 5.4. The cells used in this technique may be either live or fixed cells. The cells will be incubated with the random peptide library and will bind to certain peptides in the library to form a "rosette" between the target cells and the relevant solid phase support/peptide. The rosette can thereafter be isolated by differential centrifugation or removed physically under a dissecting microscope.

As an alternative to whole cell assays for membrane bound receptors or receptors that require the lipid domain of the cell membrane to be functional, the receptor molecules can be reconstituted into liposomes where label or "tag" can be attached.

5.5.3. Screening of Organic Compounds with I , Receptor/Receptor Mutant rotein or Engineered Cell Lines

Cell lines that express IP ₃ receptor or IP ₃ receptor mutants may be used to screen for molecules that modulate IP ₃ signal transduction or affect Ca ^2* homeostasis. Such molecules may include small organic or inorganic compounds, or extracts of biological

materials such as plants, fungi, etc., or other molecules that modulate IP ₃ receptor signal transduction or that promote or prevent the formation of IP ₃ /IP ₃ receptor complex. Synthetic compounds, natural products, and other sources of potentially biologically active materials can be screened i a number of ways.

The ability of a test molecule to interfere with IP ₃ binding to the IP ₃ receptor and/or IP ₃ receptor signal transduction may be measured using standard biochemical techniques. Other responses such as phosphorylation or dephosphorylation of other proteins, activation or modulation of other molecules in the signal cascade, changes in cellular ion levels, association, dissociation or translocation of signalling molecules, or transcription or translation of specific genes may also be monitored. These assays may be performed using conventional techniques developed for these purposes in the course of screening.

Ligand binding to its cellular receptor may, via signal transduction pathways, affect a variety of cellular processes. Cellular processes under the control of the IP ₃ receptor signalling pathway may include, but are not limited to, normal cellular functions, Ca ² * homeostasis, proliferation, differentiation, in addition to abnormal or potentially deleterious processes such as unregulated cell proliferation, loss of contact inhibition, blocking of differentiation or untimely cell death. IP ₃ receptor, or functional derivatives thereof, useful in identifying compounds capable of modulating signal transduction may have, for example, amino acid deletions and/or insertions and/or substitutions as long as they retain significant ability to interact

with some or all relevant components of an IP ₃ receptor mediated signal transduction pathway. A functional derivative of an IP ₃ receptor may be prepared from a naturally occurring or recombinantly expressed IP ₃ receptor by proteolytic cleavage followed by conventional purification procedures known to those skilled in the art. Alternatively, the functional derivative may be produced by recombinant DNA technology by expressing parts of an IP ₃ receptor which include the functional domain, for example the ligand binding domain, in suitable cells. Functional derivatives may also be chemically synthesized. Cells expressing IP ₃ receptor may be used as a source of IP ₃ receptor, crude or purified for testing in these assays.

The qualitative or quantitative observation and measurement of any of the described cellular processes by techniques known in the art may be advantageously used as a means of scoring for signal transduction in the course of screening.

Various embodiments are described below for screening, identification and evaluation of compounds that interact with the IP ₃ receptor, which compounds may affect various cellular processes under the control of the IP ₃ receptor signalling pathway.

The present invention includes a method for identifying a compound which is capable of modulating signal transduction, comprising:

(a) contacting the compound with IP ₃ receptor, or a .functional derivative thereof, in pure or semi-pure form, in a membrane preparation, or in a whole live or fixed cell;

(b) incubating the mixture of step (a) in the presence of IP ₃ , for an interval sufficient

for the compound to stimulate or inhibit the signal transduction;

(c) measuring the signal transduction;

(d) comparing the signal transduction activity to that of IP ₃ receptor, or a functional derivative thereof, incubated without the compound, thereby determining whether the compound stimulates or inhibits signal transduction. IP ₃ receptor, or functional derivatives or IP ₃ receptor mutant thereof, useful in identifying compounds capable of modulating signal transduction may have, for example, amino acid deletions and/or insertions and/or substitutions as long as they retain significant signal transducing capacity. A preferred IP ₃ mutant is one lacking the C-terminal transmembrane sequences necessary for membrane insertion, i.e., a soluble IP ₃ receptor, while retaining the N-terminal IP ₃ binding domain. A functional derivative of IP ₃ receptor may be prepared from a naturally occurring or recombinantly expressed IP ₃ receptor by proteolytic cleavage followed by conventional purification procedures known to those skilled in the art. Alternatively, the functional derivative may be produced by recombinant DNA technology by expressing parts of IP ₃ receptor which include the functional domain in suitable cells. Functional derivatives may also be chemically synthesized. Cells expressing IP ₃ receptor or receptor mutants may be used as a source of IP ₃ recep.tor or receptor mutants, crude or purified, or in a membrane preparation, for testing in these assays. Alternatively, whole live or fixed cells may be used directly in those assays.

IP ₃ receptor signal transduction activity may be measured by standard biochemical techniques or by

monitoring the cellular processes controlled by the signal, such as Ca ² * levels.

The invention further provides for a method of screening compounds that, upon interacting with IP ₃ receptor, elicit or trigger a signal mimicking the action of IP ₃ binding to the IP ₃ receptor. Signal transduction is mimicked if the cellular processes under the control of the signalling pathway are affected in a way similar to that caused by ligand binding. Such compounds may be naturally occurring or synthetically produced molecules that activate the IP ₃ receptor.

The invention also includes a method for identifying a molecule in a chemical or biological preparation capable of binding to IP ₃ receptor, comprising:

(a) contacting the chemical or biological preparation with an IP ₃ receptor or a functional fragment thereof immobilized on a solid phase matrix for an interval and under conditions sufficient to allow binding to occur;

(b) removing unbound material from the solid phase matrix; and (c) detecting the presence of the compound bound to the solid phase, thereby identifying the compound. The above method may further include the step of:

(e) eluting the bound compound from the solid phase matrix, thereby isolating the compound. Compounds capable of binding to IP ₃ receptor may directly or indirectly modulate IP ₃ signal transduction and may include molecules that are naturally associated with the intracellular domain of IP,.

"Compounds capable of binding to IP ₃ receptor" refers to a naturally occurring or synthetically produced molecule which interacts with IP ₃ . Examples of such compounds are (i) a natural substrate of IP ₃ receptor; (ii) a naturally occurring molecule which is part of the signalling complex; and/or a naturally occurring si nalling molecule produced by other cell types.

5.6. Administration and Formulation of Therapeutic Molecules

In one embodiment of the invention, proteins, peptides or moleucles which bind IP ₃ receptor, IP ₃ receptor mutants, or a fragment containing the IP ₃ binding site, or an IP ₃ receptor anti-sense molecule containing a sequence complementary to at least a part of the coding sequence of IP ₃ receptor and which inhibits translation of IP ₃ receptor mRNA could be administered in vivo to modulate IP ₃ receptor signal transduction. For example, administration of an IP ₃ receptor mutant or a fragment containing the IP ₃ receptor binding domain or an organic molecule capable of binding to the IP ₃ receptor binding domain could competitively bind to IP ₃ and inhibit its interaction with the naturally occurring IP ₃ receptor in vivo to inhibit or reverse inappropriate growth of cells associated with abnormalities in signal transduction associated with tyrosine kinases. Alternatively, ligands for IP ₃ receptor, including anti-IP ₃ receptor antibodies or fragments thereof, may be used to ₀ modulate inappropriate cell growth associated with abnormalities in signal transduction associated with tyrosine kinases. Antagonists of IP ₃ activity may be used to inhibit tumor growth.

The particular peptides, proteins, organic _ compounds, antibodies, anti-sense molecules or IP ₃ receptor mutants that modulate IP, receptor signal

transduction can be administered to a patient either alone, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) .

Depending on the specific conditions being treated, these agents may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition. Suitable routes may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, or, in the case of solid tumors, directly injected into a solid tumor. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks ^, s solution. Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.. The compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. Determination

of the effective eimounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.

The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture,

and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP) . If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.

Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may be prepared, placed in an appropriate container, and labelled for treatment of an indicated condition. Suitable conditions indicated on the label may include treatment of a tumor, such as a glioma or breast cancer.

The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. Many of the IP ₃ receptor modulating compounds of the invention may be provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.

For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the PTP activity) . Such information can be used to more accurately determine useful doses in humans.

A therapeutically effective dose refers to that amount of the compound that results in amelioration of

symptoms or a prolongation of survival in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e .g. , for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) . The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with. little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e . g. , Fingl et al . , 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 pi). Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the IP ₃ receptor-inhibitory effects. Usual patient dosages for systemic administration range from 1 - 2000 mg/day, commonly from 1 - 250 mg/day, and typically from 10 -150 mg/day. Stated in terms of patient body weight, usual dosages range from 0.02 - 25 mg/kg/day, commonly from 0.02 - 3 mg/kg/day, typically from 0.2 - 1.5 mg/kg/day. Stated in terms of patient body surface areas, usual dosages range from 0.5 - 1200

mg/m ² /day, commonly from 0.5 - 150 mg/m ² /day, typically from 5 - 100 mg/m ² /day.

Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which axe sufficient to maintain the IP ₃ receptor-inhibitory effects. Usual average plasma levels should be maintained within 50 - 5000 μg/ l, commonly 50 - 1000 μg/ml, and typically 100 - 500 μg/ml. Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a tumor, often in a depot or sustained release formulation.

Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with tumor-specific antibody. The liposomes will be targeted to and taken up selectively by the tumor.

In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

5.7. Uses of IP _j Receptor-Mutant Polynucleotides An IP ₃ receptor coding sequence may be used for diagnostic purposes for detection of IP ₃ receptor expression. Included in the scope of the invention are oligoribonucleotide sequences, that include antisense RNA and DNA molecules and ribozymes that function to inhibit translation of IP ₃ receptor. Polynucleotides encoding an IP ₃ receptor mutant, for example a soluble or a signalling incompetent mutant, having the ability to compete with the endogenous IP ₃ receptor protein for access to molecules in the IP ₃ signalling pathway thereby exhibiting a dominant negative effect, may be expressed in targeted

cell populations to modulate the activity of naturally occurring IP ₃ receptor.

5.7.1. Therapeutic Uses of I ₃

Receptor Mutant Polynucleotide

The scope of the present invention encompasses oligoribonucleotide sequences, that include anti-sense RNA and DNA molecules and ribozymes that function to inhibit the translation of IP ₃ receptor mRNA. Anti¬ sense RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation. In regard to antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site, e.g., between -10 and +10 regions of the IP ₃ receptor nucleotide sequence, are preferred.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mecha¬ nism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complemen¬ tary target RNA, followed by a endonucleolytic cleav¬ age. Within the scope of the invention are engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of IP ₃ receptor RNA sequences.

Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligo- nucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing

their accessibility to hybridization with complemen¬ tary oligonucleotides, using ribonuclease protection assays.

Both anti-sense RNA and DNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively,

RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines. Various modifications to the DNA molecules may be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribo- or deoxy- nucleotides to the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phospho- diesterase linkages within the oligodeoxyribonucleo- tide backbone.

5.7.2. Use of Signalling Incompetent

IP ₃ Receptor Mutants in Gene Therapy

The IP ₃ receptor is known to assemble in tetrameric organization of identical or highly homologous subunits forming Ca ^2* -permeable channels.

IP ₃ receptor mutants can function as dominant negative mutations by suppressing the activation and response of naturally occurring IP ₃ receptors through formation of tetramers with naturally occurring receptors, or other mutant receptors, wherein such tetramers are signalling incompetent. IP ₃ receptor mutants lacking a transmembrane domain while retaining the IP ₃ binding domain may lack the ability to form tetramers with naturally occurring receptors and may also be signalling incompetent.

Signalling incompetent mutant IP ₃ receptors can be engineered into recombinant viral vectors and used in gene therapy in individuals that inappropriately express receptor tyrosine kinase or that exhibit inappropriate growth of cells associated with abnormalities in signal transduction associated with tyrosine kinases.

The capability of IP ₃ mutant receptors to suppress transformation of growth factor induced transformed NIH 3T3 cells is demonstrated in Section 7. The therapeutic potential of an IP ₃ receptor mutant used in gene therapy may be measured by examining suppression of receptor tyrosine kinase induced transforming activity. In an embodiment of the invention, mutant forms of the IP ₃ receptor having a therapeutic effect may be identified by expression in selected cells. Deletion or missense mutants of IP ₃ receptor that retain the ability to form tetramers with naturally occurring IP ₃ receptor protein or other mutants of IP ₃ receptors but which cannot function in signal transduction may be used to inhibit the biological activity of the naturally occurring IP ₃ receptor. For example, the IP ₃ binding domain of an IP ₃ receptor may be deleted resulting in a mutant IP ₃ receptor molecule that is

still able to undergo formation of tetramers with naturally occurring receptors but unable to transduce a signal. Accordingly, the invention provides a method of inhibiting the effects of IP ₃ receptor mediated signal transduction by an endogenous IP ₃ protein in a cell comprising delivering a DNA molecule encoding a signalling incompetent form of the IP ₃ receptor protein to the cell so that the signalling incompetent IP ₃ receptor protein is produced in the cell and competes with the endogenous IP ₃ receptor protein for access to molecules in the IP ₃ receptor protein signalling pathway which activate or are activated by the endogenous IP ₃ receptor protein.

Inappropriate growth of cells associated with abnormalities in signal transduction associated with tyrosine kinases is an important component of a variety of disorders and disease states such as cancer, atherosclerosis and psoriasis. Recombinant vixuses may be engineered to express signalling incompetent forms of the IP ₃ receptor which are capable of inhibiting the activity of naturally occurring IP ₃ receptor. These viruses may be used therapeutically for treatment of diseases resulting from aberrant expression or activity of receptor tyrosine kinases or activity of the IP ₃ receptor.

Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine pa illoma virus, may be used for delivery of recombinant IP ₃ receptor mutants into the targeted, cell population. Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors containing IP ₃ receptor mutant coding sequence. See, for example, the techniques described in Maniatis et al . , 1989, Molecular Cloning A Laboratory Manual. Cold Spring

Harbor Laboratory, N.Y. and Ausubel et al . , 1989, Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley Interscience, N.Y. Alternatively, recombinant IP ₃ receptor molecules can 5 be reconstituted into liposomes for delivery to target cells.

In a specific embodiment of the present invention, two IP ₃ receptor mutants Δ-IP ₃ R, lacking the N-terminal IP ₃ binding domain and s-IP ₃ R, lacking the 0 C-terminal transmembrane sequences necessary for membrane insertion, were shown to suppress the level of transformation in NIH T3 cells induced to transform by receptor tyrosine kinase.

5 6. Example; Characterization of IP ₃ Receptor and Ca ³ * Storage Systems

This example describes characterization of the expression and functioning of the IP ₃ receptor and the regulation of IP ₃ generation and Ca ² * storage systems - in transfected NIH 3T3 clones.

6.1. Materials and Methods

6.1.1. Generation of Stable Cell Lines NIH 3T3 cells were grown in Dulbecco's modified ₅ Eagle's medium (DMEM, GIIBCO) containing 10% fetal calf serum (FCS, GIBCO) and 2 mM glutamine. The expression vector under cytomegalovirus promoter control in which the full length wild type (wt) , or the mutant IP ₃ R cDNAs (Mignery and Sϋdhof et al., 1990, ₀ supra ; Mignery et al . , 1990, supra) were inserted, was previously described (Mignery et al . , 1990, supra) . Purified expression plasmid cDNAs were introduced into subconfluent cells by cotransfeetion with a plasmid conferring resistance to the antibiotic geneticin _S (G418), at a molar ratio of 10:1, using the calcium phosphate technique (Graham, F.L. and van der Eb,

A.J., 1973, Virology 12:456-467). Control cells were transfected in parallel with the vector and the resistance plasmid. One day after transfection, cells were split into DMEM/10% FCS/2 mM glutamine supplemented with 1 mg/ml G418. Expression of the wt and mutated IP ₃ R was established at day 11-14. G418 ^r colonies were picked and tested by in vivo labeling with [ ^3S S] methionine (Amersham, U.K.) and subsequent immunoprecipitation, and by Western blotting.

6.1.2. In Vivo Labelling of Cells and Immunoprecipitation

Cells were grown on 6 cm dishes to 80% confluence, washed with phosphate-buffered saline

(PBS) , and grown for 16 hrs in methionine-free DMEM containing 10% dialyzed FCS/2 mM glutamine and 40 μCi of [ ³⁵ S] methionine/ml. Cells were lysed in 0.6 ml lysis buffer [50 mM N-2-hydroxy-ethylpiperazine-N'-2- ethanesulfonic acid (HEPES; pH 7.5) , 150 mM NaCl, 1.5 mM MgCl ₂ 1 mM EGTA, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 5 μg of aprotinin/ml] at 4°C. Lysates were centrifuged for 10 min at 4 ^β C in an Eppendorf centrifuge (13000 x g) . The supernatants were incubated overnight at 4°C with an excess of the anti-IP ₃ receptor polyclonal antibody (Ab) T _210/ (raised against the 19°C terminus amino acids of the receptor, as described by Mignery et al., 1990, supra) and protein A-Sepharose. The immunoprecipitates were washed three times with washing buffer (20 mM HEPES pH

7.5, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol) and then resuspended in sample buffer, boiled for 3 min, and analyzed by SDS-PAGE. After treatment with 1 M sodium salicylate/30% methanol to amplify [ ^3S S] methionine radioactivity by fluorography (Chamberlain,

1979) , the gels were dxied and the [ ^3S S] methionine- labeled proteins were detected by autoradiography.

6.1.3. Western Blotting Nitrocellulose sheets, blotted from SDS gels (a 4-10% gradient for IP ₃ R and 10% for the other antigens) loaded with 250 μg lysate/lane, were reacted with the specific Abs and then decorated with " ⁵ I protein A as described elsewhere (Villa et al., 1993, J. Cell Biol. 121:1041-1051) . The Abs employed were the following: anti-IP ₃ R, the polyclonal T ₂₁₀ Ab (Mignery et al . , 1990, supra) ; anticalreticulin, the polyclonal rabbit Ab developed by Perrin et al . , 1991, FEBS Lett. 294:47- 50; anti-SERCA (sarcoplasmicendoplasmic reticulu Ca ^2* ATPase) , a mouse monoclonal Ab developed by Colyer et al . , 1989, Biochem. J. 262:439-446. Anti- phospholipase C (PLC) β and γ Abs were purchased from UBI Co., Frankfurt, Germany. Microdensitometry of the positive bands was carried out in a LKB chromatoscanner CS-380 microdensitometer.

6.1.4. Retrovirus-Mediated Gene Transfer

In order to obtain expression of the human EGF receptor (EGF-R) , HERc, subconfluent cells (1 x 10 ^s cells per 6-cm dish) were incubated with supernatants of GP+E-86 cells releasing high-titer NTK-HERc virus

(5 x 10 ^s G418 ^r CFU/ml) [multiplicity of infection

(MOI) : 5] . Stimulation of overexpressed EGF-R in infected cells was achieved by superinfection with supernatants of ¥2 cells releasing ¥2-TGFα virus

(Blasband et al . , 1990, Oncogene 5-1213-1221) (1 x 10 ^s

G418 ^r CFU/ml; kindly provided by David Lee) for 4-16 hrs in the presence of polybrene (8 μg/ml; Aldrich) at a MOI of 3. As a control, parallel cell monolayers were superinfected with *2 cell supernatants of 2

cells releasing ¥2-N2 control virus (5xlO ^s G418 ² CFU/ml) in the same experimental conditions. The retroviral expression vectors pN2 and pNTK-HERc have been described previously (Keller et al., 1985, Nature 318:149-154: von Ruden et al., 1988, EMBO J. 2:2749- 2756) . Virus titers were determined by infecting NIH 3T3 cells with serial dilutions of retrovirus containing cell-free conditioned media of the individual virus-producer lines and determining the number of the G418 resistant colonies.

6.1.5. [Ca ² *] Measurements and ⁴⁵ Ca Release from Intact and Permeabilized Cells

For intact cell sample preparation, monolayers were washed with PBS without Ca ² * and Mg ² *, then detached from culture dishes by gentle trypsinization (0.05% trypsin in the same buffer), washed and resuspended in Krebs-Ringer-HEPES (KRH) medium (25 mM HEPES pH 7.4; 125 mM NaCl; 5 mM KC1; 1.2 mM KH ₂ P0 _« ; 1.2 mM MgS0 ₄ ; 2 mM CaCl ₂ ; 6 mM glucose) . Cells were loaded for 30 min at 25°C with the Ca ² * sensitive fluorescent dye, fura-2 (Calbiochem) , added as the acetoxymethylester at the final concentration of 3 μM. Subsequently the cells were supplemented with 250 μM sulfinpyrazone (Sigma) , to prevent dye leakage, and left to sediment for 20 min at 25°C. Samples were then centrifuged for 20 min (48 x g) , washed with KRH supplemented with 3% bovine serum albumin, resuspended in the same medium, divided into aliquots of 1-2 x 10 ^β cells, and kept at room temperature until use.

To measure [Ca ² *]i, cell aliquots were resuspended in KRH medium containing 0.2 mM CaCl ₂ supplemented with 250 μM sulfinpyrazone, then transferred to a thermostatted cuvette (37°C) , maintained under stirring and analyzed in a Perkin Elmer LS-5B

fluorimeter as described by Grynkiewicz et al . , 1985, J. Biol. Chem. 260:3440-3450. For the experiments measuring [Ca ² *] in permeabilized cell-free systems, aliquots of 3xl0 ⁷ cells were washed three times with an intracellular-like solution supplemented with the ATP regeneration system containing: 120 mM, KC1; 1 mM, MgS0 ₄ ; 1.2 mM KH ₂ P0 ₄ ; 25 mM HEPES; 5 mM NaCl; 6 mM glucose; 10 mM phosphocreatine; 10 units/ml creatine phosphokinase; pH 7.2. After resuspension in the intracellular-like solution, 30 μg/ml saponin was added and the cells were stirred at room temperature for 10 min. This treatment yielded 90 + 5% cells permeable to trypan blue. Cells were then transferred to the ther ostatted cuvette, fura-2 free acid, 0.5 μM, and ATP, 1 mM, were added 5 min before addition of either various concentrations of IP ₃ or 100 nM of the SERCA blocker, thapsigargin (Tg, Sigma) , and [Ca ^2* ^ measurements were carried out as described above.

6.1.6. IP ₃ Measurements

Cells were labelled for 48 hours with 2 μCi/ml of myo-[2- ³ H]inositol (Amersham) in basal Eagle diploid modified medium (BME, GIBCO) containing 3% inositol- free FCS, and subsequently detached. Aliquots of 1 x io ^β cells, preincubated for 10 min with 10 mM LiCl, were stimulated by addition of ATP or other nucleotides and then stopped after 20 sec by the addition of trichloroacetic acid to a final 7.5% concentration. Samples were centrifuged, and the supernatants _^ extracted three times with diethylether. Radioactive inositol phosphates were separated by either stepwise elution ion-exchange chromatography or HPLC, and radioactivity counted in a Beckman β counter. Note that all IP ₃ , measurements were performed together with [C ^2* ^ measurements, employing

parallel aliquots of the same batches of cells processed concomitantly.

In the experiments in which IP ₃ generation by A1F ₄ - was investigated, cell aliquots were resuspended in KRH supplemented with 10 μM A1C1 ₃ and 10 mM NaF, in the presence of 10 mM LiCl, and incubated at 37° for 1 hr, after which the supernatants were processed as described for IP ₃ release.

6.1.7. IP _j Binding to Microsomes

NIH-3T3 microsomal fractions, prepared as described by Alderson et al., 1989, (Arch. Biochem. Biophvs. 272:162-174) were resuspended in a medium containing: 100 mM KC1; 10 mM NaCl; 10 mM Tris-HCl buffer; pH 8.4. Binding to the fractions (1 mg protein/ml) was measured in the same buffer, supplemented with 0.1 mM EGTA and 5 nM [ ³ H]IP ₃ (44 Ci/mmol, Amersham) mixed with non-radioactive IP ₃ in the 0-150 nM range. To measure unspecific binding, parallel samples were prepared with excess IP ₃ (5 μM) added. After 10 min incubation at 4 ^β C, bound and free counts were separated by filtration through glass fiber filters (GF/C, Whatman) , which were washed twice with cold buffer.

6.1.8. [ ^3S S] ATPγS Binding to P _2u Receptors of Intact Cells

Cells detached from Petri dishes were washed as described in Section 6.1.7 and resuspended in a modified KRH medium free of Ca ² * and Mg ² * and supplemented with 1 mM EDTA. After equilibration at

37 ^β C for 30 min, 1 ml aliquots of the suspensions containing 10 ⁷ cells were preincubated for 10 min at

4°C and then labeled for 5 min at the same temperature with 1 μM [ ³⁵ S] ATPγS (50 Ci/mmol, Amersham) , with or without 10 mM ATP or UTP. Specific binding vas

estimated as the radioactivity displaceable by the unlabeled nucleotides. Bound and free counts were separated by filtration through glass fiber filters which were washed twice with cold buffer.

6.1.9. [*H] Thymidine Incorporation Cells were seeded on 12-well Costax plates at a density of 5x10* cells per well in DMEM containing 10% FCS/2 mM glutamine and grown for three days. After washing with PBS, the confluent cell layers were starved for 24 hrs in DMEM containing 0.5% FCS/2 mM glutamine, stimulated with EGF for 18 hrs in DMEM containing 0.5% bovine serum albumin (BSA) , 2 mM glutamine, and pulsed for 4 hrs with 0.5 μCi of [methyl- ³ H]thymidine (Amersham, U.K.). The incorporation of radioactive thymidine was stopped on ice, the cells were washed twice with PBS, and soluble radioactivity was extracted with 0.5 ml of 10% trichloroacetic acid for 20 min on ice, followed by another wash with the same solution. Precipitates were then lysed in 0.2 ml 0.2 N NaOH/0.2% SDS and later neutralized with 0.2 N HC1. The incorporated radioactivity was quantitated by scintillation counting.

6.1.10. Colorimetrie Growth Assay Cells were seeded on 96-well plates (1 x 10 ³ cells/well) in DMEM with 2 mM glutamine containing 10%, 4% or 0.5% FCS in a total volume of 0.1 ml. At day 4, cells- were fed with the appropriate medium. The colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]- 2,5-diphenyltetrazolium bromide; Sigma) assay was performed essentially as described by Mosman, 1983 (J. Immunol. Meth. 6_5:55-63). At the time points indicated, MTT stock solution (2.5 mg/ml in PBS,

filtered) was added, and plates were incubated for another 2 hrs at 37 ^β C. Cells were subsequently lysed and the dye-crystals solubilized in acid/isopropanol (0.04 N HCI in isopropanol) . The plates were read within one hour on a Dynatech MR5000 Microelisa Reader, using a test wavelength of 570 nm and a reference wavelength of 690 nm.

6.1.11. Colony Formation in Soft Agar To examine the ability of stable NIH 3T3 clones to form colonies in soft agar after infection with the EGF receptor, NTK-HERc and *2-TGFα virus, cells were plated in 6-cm dishes with Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) , 2 mM glutamine and 0.2% agar. The plates had been previously coated with a bottom layer of DMEM containing 10% FCS/2 mM glutamine and 0.5% agar. Colonies were visible within 4 weeks, and were stained before counting with MTT stock solution (see Section 6.1.10) for two hours at 37°c.

6.1.12. Substrate Tyrosine

Phosphorylation Analysis

Cells were seeded on 6-cm dishes and, after reaching confluence, starved for 24 hrs with DMEM/2 M glutamine/0.5% FCS. They were then stimulated for 10 min at 37°C with EGF or TGFα (100 ng/ml) , obtained from Toyoba, stopped on ice, washed twice with PBS, and lysed in 100 μl lysis buffer described in Section

6.1.2, supplemented with 1 mM sodium orthovanadate and

0.3 mM 4-nitrophenylphosphate (Sigma) at 4 ^β C. The lysates were centrifuged for 10 min at 4°C in an

Eppendorf centrifuge (13 000 x g) . The total amount of protein in the lysates was determined using the BCA assay (Mikro BCA Protein Assay reagent; Pierce) .

Thirty μg of total protein for each lane was adjusted

in volume, mixed with double-concentrated sample buffer, boiled for 3 min, and analyzed by SDS-PAGE. Proteins were transferred electrophoretically to nitrocellulose and subsequently incubated with the mouse antiphosphotyrosine monoclonal Ab 5E2 (Fendly et al., 1990, Cancer Research 50:1550-1558). For detection, the nitrocellulose filter was incubated with peroxidase-coupled goat anti-mouse Abs, followed by the enhanced chemiluminescence (ECL) substrate reaction (Amersham) . After detection of the ECL substrate reaction on Kodak X-Omat film, nitrocellulose filters were stripped with stripping buffer (100 mM 0-mercaptoethanol, 62.5 mM Tris- (hydroxymethyl)aminoethane, 2% SDS) for 30 min at 50 ^β C, and reprobed with the anti-EGF-R Ab RK2 (Kris et al . , 1985, Cell 4^:619-625) as an EGF-R control. The second antibody was a peroxidase-coupled goat anti- rabbit Ab followed by the ECL substrate reaction.

6.2. Expression of IP ₃ Receptors, SERCAs and Calreticulin

Figures 1A and IB illustrates the data obtained with isolated 3T3 clones transfected with expression plasmids under cytomegalovirus promoter control, either alone (3T3/T10) or containing; rat type I IP ₃ R cDNA, both wild type (wt) and deletion mutants, s-IP ₃ R and Δ-IP ₃ R. Two independently isolated clones for each receptor construct were used in the transfections. Figure 1A shows immunoprecipitation results of lysates obtained from cells prelabeled with [ ^3S S] methionine for 16 hours ^* . As shown in Figure 1A, the signal obtained from the endogenous IP ₃ R of control cells transfected with the vector alone was too low to induce a visible band, whereas in the cell clone transfected with the wt receptor the band was prominent, ~ 20 fold higher than in the controls.

Unexpectedly, in the preparations obtained from the deletion mutant clones, the endogenous band appeared, accompanied by even more evident, lower Mr bands corresponding to the deleted s- and Δ-IP ₃ R, respectively. Taken together, the labeling of the intact + deleted receptor bands of the s and Δ clones was, on the average, » 20 fold higher than the controls. When the transfected clones were investigated by Western blotting (Figure IB) , the large quantitive differences revealed by the [ ³⁵ S] methionine labeling experiments did not appear. Only in the clones transfected with the rat wild type (wt) receptor cDNA (IP ₃ R clones) , and expressing therefore the endogenous together with the exogenous receptor, the cognate band was consistently increased by approximately 30%. The lower Mr bands, corresponding to the deleted receptor forms, were not stronger but considerably weaker than that of the endogenous wt receptor. In one Δ-IP ₃ R clone, Δ-IP ₃ R5, shown in Figure IB and in the other clone investigated, Δ-IP ₃ R6 the truncated forms accounted for 30 and 21.7% of the wt values, respectively; in the sIP ₃ R clone shown in IB, and in two other sIP ₃ R clones, the corresponding values ranged from 15 to 23%. Two additional proteins of intracellular Ca ^2* stores were investigated by Western blotting, the intracellular Ca2+ ATPases, the SERCAs, and calreticulin (CR) .

With respect to the SERCAs, differences among the various IP ₃ receptor clones were revealed. In particular, some clones exhibited two immunolabeled bands around 100 kDa probably corresponding to the SERCAs 2a and 2b isoforms (Pozzan et al. , 1994, Phvsiol. Rev.) , and others the f ster running band only (Figure IB) . In the analyzed clone population.

these features were observed apparently at random, with no correlation with any of the transfected IP ₃ Rs. In contrast, the levels of calreticulin (CR) , the major Ca ² * binding protein of the ER lumen, were shown to be yery similar among the clones (variability <20%, Figure IB) .

6.3. Ca ² * and IP ₃ Responses in Intact Cells These experiments were designed to distinguish between the effects triggered by the discharge of intracellular Ca ² * stores and those induced by the activation of plasma membrane channels. The incubation medium employed contained 1/10 the usual [Ca ² *] ₀ , i.e., 0.2 instead of 2 mM. The first [Ca ^* li responses investigated were those induced by the SERCA blocker, thapsigargin (Tg) (Sigma) , that induces leakage of the Ca ² * segregated within the rapidly exchanging Ca ² * stores, which are believed to be proportional to the overall Ca ² * capacity of these stores and are, therefore, commonly used to assess their size. The results obtained were not appreciably different among all the investigated 3T3 clones, whether transfected with IP ₃ R or not.

The responsiveness of our clones to high concentrations of agonists known in other cells to activate receptors coupled to polyphosphoinositide (PI) hydrolysis was investigated. Bradykinin, bombesin, vasopressin, acetylcholine analogues, angiotensin, histamine, serotonin and thrombin caused no [Ca ² *^ responses, whereas the growth factor EGF and the nucleotide ATP did. The [Ca*] effects of EGF were most probably mediated by the EGF receptor, HERc, expressed by our cells as a consequence of their infection with the NTK-HERc virus, while those of ATP, because of their pharmacological properties (in

particular, low and high sensitivity to ADP and UTP, respectively, and insensitivity to pertussis toxin) were attributed to a receptor of the P _2u type (Lustig et al., 1993, PNAS USA 90:5113-5117). Figure 2A shows the concentration dependence of the EGF and ATP- induced [Ca ² *]i responses in some of the transfected clones. The effects of EGF were small, whereas those of ATP were considerable.

Among the clones, those expressing the s-IP ₃ R and those overexpressing the wt IP ₃ R did not differ significantly from the controls, whereas those with the Δ-IP ₃ Rs exhibited a small, but reproducible, shift to the right. Figure 2B compares the ATP concentration-dependence curves of controls and Δ-IP ₃ R clones with and without activation of an autocrine loop sustained by infection of TGFα. In the control clones expressing the growth factor the curve was significantly shifted to the left, whereas that of the Δ-IP ₃ R clones was not. The intracellular Ca ² * release responses induced by ATP are mediated by IP ₃ . Receptor-induced generation of the second messenger, IP ₃ , was investigated. Figure 2C shows the ATP concentration dependence of IP ₃ accumulation estimated at the peak, i.e., 20 sec after addition of the stimulant. The

ATP-induced increase of IP ₃ was considerable in control fibroblasts and in those overexpressing the wt IP ₃ R whereas it was markedly smaller in the Δ-IP ₃ R clones, especially in Δ-IP ₃ R5 (on the average -65%, Figure 2C) . Taking together the results of Figures 2A and 2C, it is concluded that at least two signalling changes are operative in the clones expressing the Δ-IP ₃ R: a markedly decreased generation of IP ₃ in response to ATP stimulation; and an increased sensitivity of the IP ₃ R with respect to its ligand, which compensates in large

part for the defect of IP ₃ generation and thus keeps moderate the shift to the right of the [Ca ² *]i release response curves with respect to controls. These differences were not due to changes in either the 5 level of expression or affinity of the IP ₃ R because [ ³ H]IP ₃ binding experiments, carried out in the microsomal fractions isolated from two clones, the control 3T3/TIO and the 3T3/Δ-IP ₃ R5 clones, yielded similar values: Kd= 12 and 17 nM; Bmax 0.52 and 0.60 10 pmoles/mg protein, respectively

6.4. IP ₃ Induced Ca ² * Release From Permeabilized Cells

The increased IP ₃ R sensitivity of the Δ-IP ₃ R

-_ clones was further confirmed using cells permeabilized with saponin (90% positivity to the vital dye, trypan blue) that were exposed to various concentrations of IP ₃ while bathed in an intracellular-type medium supplemented with fuxa-2 free acid.- As can be seen in ₀ Figure 3, only the Δ-IP ₃ R-expressing cells showed appreciable Ca ^2* release responses after application of 1 μM IP ₃ , whereas controls did not. With increased IP ₃ concentrations (5, 10 and 30 μM) release appeared also from control cells, however those from the Δ clone

_2S cells were still much greater. In parallel aliquots of intact and permeabilized cells, administration of Tg yielded analogous release responses from all the clones investigated. Thus, the different results obtained with IP ₃ do not seem to be due to variability

₃ _ among the clones in intracellular Ca ² * store capacity but rather to a different sensitivity to IP ₃ of their receptors.

6.5. ATP Binding and IP ₃ Generating Systems

₃₅ The results of Figures 2A-2C and Figure 3 suggest that expression of Δ-IP ₃ R subunits induces effects at

the level of not only the internal stores but also the receptor-triggered generation of the second messenger,

In order to identify the level of this latter effect, we investigated both the binding of ATPγS, an analogue of the surface receptor ligand, ATP, and the functioning of the IP ₃ generation systems. As can be seen in Table 1, both the Δ-IP ₃ R clones exhibited decreased ATPγS binding, more distinct in the clone designated Δ-IP ₃ R5 than in the clone designated Δ- IP ₃ RR6.

In contrast, IP ₃ generation induced by AIF ₄ " (a compound that bypasses the receptor and acts directly on the G protein) was not decreased but was moderately increased in the Δ-IP ₃ R5 clone. Likewise, both the Δ- IP ₃ R5 and the Δ-IP ₃ R6 clones exhibited levels of phospholipase C, of both the β and γ types (presumably activated by the ATP and the EGF receptor, respectively) , not much different from those of the controls (see Table I) . These results suggest that the cell surface effects of the Δ-IP ₃ R expression occur at the level of the P _2u receptor, which is down regulated, with no effect on its coupling and effector mechanisms, which are apparently unaffected.

6.6. Results

6.6.1. I _j Receptor Biogenesis and Expression Levels

In the control cells, cultured for 16 hrs with [ ^3S S] methionine, incorporation into the receptor subunits was very low, as it is the case with most ER, slowly turning over, membrane proteins. In contrast, in the IP ₃ R-transfected cells, incorporation was increased ~ 20 fold, and the increased labeling was not limited to the transfected receptor forms but

involved, to some extent, the endogenous wt molecules. In spite of these large labeling changes, in the cells transfected with the wt receptor, the IP ₃ R levels (as shown by Western blotting) were increased by only 30%, while the IP ₃ R mutant clones were unchanged and the deleted forms accounted for only a fraction (15- 30%) of these level. The large discrepancy between labeling and Western blot results reveals a considerable acceleration of the IP ₃ R turnover in the transfected cells. The transfected cells are able to activate a sort of a defense program by which the overall IP ₃ R level is kept near the controls.

The changes discussed above could have involved other components of the ER which participate with the IP ₃ R in the rapidly exchanging Ca ² * stores. Some changes were observed in the levels of the SERCAs, the Ca ² * ATPases that accumulate Ca ² * within the ER lumen (Pozzan et al., Phvsiol. Rev. 1994). The study of numerous clones, however, revealed that these changes did not correlate with the expression of any IP ₃ R forms, and had apparently no functional consequence. In contrast, the levels of CR, the major Ca ² * binding protein, were changed little among the clones.

The results suggest that any changes of Ca ² * homeostasis observed in the IP ₃ R transfected cells cannot be due to these other components but should be safely attributed to the molecular changes of the IP ₃ R itself.

6.6.2. PI Hydrolysis and

Intracellular Ca ² * Release

The physiological role of the IP ₃ R is to mediate release of Ca ² * from specific, rapidly exchanging Ca ^2* stores. The wt-overexpressing and s form-expressing subunits did not induced any appreciable changes of the Ca ^2* release activity.

In contrast to the s form, at least two effects of the Δ form expression were visible. First, the cells of the Δ clones generated less IP ₃ when exposed to ATP and secondly, their IP ₃ Rs were found to be distinctly more sensitive to IP ₃ , in both intact and permeabilized cells. These two changes tend to compensate for each other in terms of Ca ² * release, and the concentration dependence of the [Ca ² *^ responses induced by ATP in intact cells of the Δ clones was only moderately shifted to the right with respect to the controls.

The increased IP ₃ R sensitivity of the Δ clone cells may be a direct consequence of the Δ subunit expression. Based on the expression data in the analyzed clones, and assuming the Δ to resemble the wt subunits in their assembly properties, a 1/3 combination of the two subunits in the receptor tetramers might be frequently expected. Since the IP ₃ binding experiments failed to reveal any significant differences between Δ and control clones, the increased sensitivity appears to concern not binding but activation of IP ₃ Rs. The latter process is still debated (see Meyer et al.. Science 240:653-656 1988; Finch et al. , Science 252:443-446. 1991; Berridge, Nature 361:315-321. 1993) .

In case it does require binding of IP ₃ to each of the- four subunits of the tetramer, the Δ subunit may be permanently activated, independently of IP ₃ binding, thus explaining the increased sensitivity observed. As far as the decreased generation of IP ₃ , the results suggest it to be due to reduced expression (down regulation) of the surface ATP receptors (of the P _2u type) and not to a defect of the G protein function or PLC expression. Experiments seem to exclude another possible mechanism for inhibition of IP ₃ generation in

the Δ clones, i.e., the drop of the cellular phosphatidylinositol 4,5-bisphosphate concentration. Down regulation of PI hydrolysis-coupled surface receptors may be another component by which the cell defends itself from excessive sensitivity of IP ₃ R to its ligand.

7. Example: Expression of IP ₃ Receptor Mutants in Normal and Transformed Fibroblast Cells This Example describes the ability of IP ₃ receptor mutants to suppress growth and transformation of NIH

3T3 cell induced to transform by overexpression of growth factor receptor tyrosine kinases.

7.1. Materials and Methods

Materials and methods are those described in Section 6.

7.2. EGF and Serum-Induced Cell Growth Confluent, quiescent cell monolayers were stimulated with EGF (whose receptor is overexpressed in the clones) , and [ ³ H]thymidine incorporation was measured. As shown in Figure 4, no appreciable difference was observed between the 3T3/T10 control and the N terminal truncated clone Δ-IP ₃ R6. Analogous results were obtained with the Δ-IP ₃ R5 clone and with the transfectants expressing wt IP ₃ Rs and s-IP ₃ Rs.

Somewhat different results were obtained in the analysis of long-term growth properties of the NIH 3T3 transfectants stimulated with FCS. As shown in Figure 5A-5D, the NIH 3T3-HERc cells transfected with the wt rat IP ₃ R displayed similar growth rates under all FCS concentrations and reached the same cell density, whereas the two mutant IP ₃ R-expressing cell lines displayed a reduction in their rate of growth (15-20 and 30-40% for s- and Δ-IP ₃ R-HERc, respectively) and

failed to reach cell densities comparable to control cells. Thus, long-term growth behavior of fibroblasts was partly impaired by expression of either IP ₃ R mutant.

7.3. EGF-Receptor Mediated Transformation of IP ₃ Receptor Mutant Cell Lines

Since overexpression of the EGF-R with stimulation by its ligand is known to cause 0 transformation of NIH 3T3 cells (Di Fiore et al .,

1987, Cell 51:1063-1070; Riedel et al., 1989, PNAS USA

11:1477-1481; Velu et al., 1987, Science 237:1408-

1410) , an autocrine stimulation loop was developed

(Redemann et al . , 1992, Mol. Cell. Biol. 12:491-498) 5 by superinfection of our cells with the TGFα- expression, virus (*2-TGFα; Blasband et al . , 1990,

Oncogene 1:1213-1221), and the ability of the various clones to form colonies in soft agar was compared.

Controls for each of the cell clones infected 0 with a single virus containing either HERc or TGFα alone did not form colonies in soft agar, while the control 3T3/T10 cells were efficiently transformed by coinfection with NTK-HERc and *2-TGFα viruses and formed approximately 300 colonies per 10 ^s infectious _S units of the EGF-R virus. In IP ₃ R 3T3 cells, reproducible and consistent changes of the EGF-R/TGFα transforming potency were observed. The transfected wt IP ₃ R enhanced this potency by about 40% and the s- IP ₃ R reduced it significantly by about 35% in ₀ comparison to control cells. In the Δ-IP ₃ R clones, transformation was suppressed to a level of approximately 10%, see Table II. Table II shows colony formation in soft agar. Two independent stable NIH 3T3 colonies each expressing subunits of the ₅ transfected wild-type IP ₃ -receptor (IP ₃ R) , the soluble IP ₃ receptor (s-IP ₃ R) , or the IP ₃ -receptor lacking its

ligand binding domain (Δ-IP ₃ R) , were compared for their ability to form colonies in soft agar after infection with NTK-HERc virus (MOI:5) and *2-TGFα virus (MOI:3). Colonies were stained after 5 weeks. 3T3=3T3/T10-HERc 5 controls. The number of colonies indicated on Table II represents an average of four independent experiments.

Transforming activities of other tyrosine kinases such as PDGF/PDGF-R?, HER2/neu, and EGF-R carrying v- 0 erbB mutations (Massoglia et al . , 1990, Mol. Cell. Biol. 11:3048-3055) were suppressed to a similar extent (5-10% of controls) by Δ-IP ₃ R expression, suggesting that the observed inhibitory effect is not oncogene-specific. 5

7.4 Ligand-Stimulated Receptor and Total Substrate Phosphorylation

Ligand binding to the EGF-R initiates dimerization and activation of its intrinsic tyrosine ₀ kinase activity, in turn rapidly phosphorylating carboxy-terminal tyrosine residues of the receptor itself and tyrosine residues of substrates downstream of the receptor (Downward et al . , 1984, Nature 311:483-485: Margolis et al . , 1989, J. Biol. Chem. s 211:10667-10671; Canals, 1992, Biochemistry 31:4493- 4501) . To investigate whether the changes of the EGF- induced growth and transformation of the IP ₃ R transfectants were due to changes of transmembrane signalling at the EGF receptor, all clones were ₀ analyzed by SDS gel electrophoresis after stimulation with either EGF or by infection with the *2-TGFα virus. Tyrosine phosphorylation of proteins was detected using Western blotting with the antiphosphotyrosine-specific antibody 5E2 (Fendly et ₅ al . , 1990 , Cancer Research 50:1550-1558) (Figure 6A) , and the amount of EGF-R by subsequent detection with

the anti-EGF-R Ab RK2 (Kris et al . , 1985, Cell 10:619- 625; Figure 6B) .

EGF addition to the cell lines infected with NTK- HERc virus induced tyrosine phosphorylation of the 170 kDa EGF-R and of a number of polypeptides in the range of 50-200 kDa (Figure 6A, lane 2 and 5) . The same protein tyrosine phosphorylation pattern was obtained with TGFα, consistent with the knowledge that the same signaling pathways are activated by both ligands (Figure 6A, lanes 3 and 6) . Moreover, no consistent major differences were observed in the tyrosine phosphorylation pattern of EGF- or TGFα-stimulated Δ- IP ₃ R cell clones (lanes 2 and 5 with 3 and 6, respectively) and all other cell clones characterized, indicating that the observed inhibition of EGF-R/TGFα- induced transformation was not due to a major direct interference with signalling, through tyrosine phosphorylation.

7.5 Results: Cell Growth and Transformation

To investigate the significance of IP ₃ and [Ca ² *] regulation on cell growth and transformation, the stable NIH-3T3 clones expressing IP ₃ Rs, wt and mutants, were employed after infection with the NTK-HERc virus, alone or together with the γ2-TGFα viruses. In these cells, the results could not be influenced by the possible down regulation of the endogenous EGF-R. In spite of the unaffected expression of the EGF/EGF-R system, a change still appeared in our mutated IP ₃ R clones.

In control cells, the expression of the TGFα-HERc loop resulted in a shift to the left of the ATP concentration dependent [Ca ² *^ increase curve, whereas in the Δ-IP ₃ R clones such a shift was lost (Figure 2B) .

Results obtained in our cell clones concerned their growth characteristics in monolayers and soft agar. The mitogenic response to exogenous EGF, measured by [ ³ H] thymidine incorporation into DNA, was not significantly different in cells expressing transfected, wt or mutant IP ₃ R, confirming previous conclusions that PI hydrolysis and Ca ² * have no major role in the short-term events induced by growth factors. Differences became apparent in long-term experiments that analyzed growth rate, in particular, growth of the Δ and the s clones was repressed by about 30-40 and 15-20% at all serum concentrations tested, respectively. Differences were found when the transformation parameters of the NTK-HERc and 2-TGFα-infected clones were experimentally addressed. Growth of cells expressing the IP ₃ R mutants was markedly suppressed, especially that of the Δ clone cells, which essentially failed to grow in soft agar. These results were duplicated when the EGF-R/TGFα growth stimulation system was replaced by others (PDGF/PDGF- Rβ; HER2/neu and v-erb B) . IP ₃ Rs that include Δ subunits appear therefore to modify profoundly the fibroblasts and to make them incompetent for tumoral growth, even under conditions in which the receptors and the ligands that in the controls induce the response are certainly operative, and the [Ca ² *^ responses are largely maintained. The results reveal that expression in intact cells of mutated IP ₃ Rs induces a complex array of signal transduction changes developed by the cell to defend its molecular and functional identity. These changes appear to modify profoundly a basic function of the cell, its growth.

Unexpectedly, the processes leading to cell transformation and oncogenesis were particularly affected, especially in the Δ clones, revealing a differential dependence of normal and cancer cells on Ca ² * and IP ₃ second messenger systems. These results identify the IP ₃ Rs and the intracellular Ca ² * stores as important crossroads of signalling pathways involved in growth regulation.

Various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description, such modifications are intended to fall within the scope of the appended claims. It is also to be understood that all base pair sizes given for nucleotides are approximate and are used for purposes of description.

All references cited herein are hereby incorporated by reference in their entirety.

TABLE 1

ATPγS binding, AIFI ₄ "-induced IP ₃ generation and phospholipase β and γ expression in 3T3 fibroblasts expressing endogenous and mutated, Δ type IP ₃ receptors.

Values shown are averages of the results obtained in 2-4 experiments, expressed as % of the control clone 3T3/T10-HERC.

(1) At the [ATPγS] employed (1 μM) 100% specific binding was 9500 cpm, corresponding to 4.7 pmoles/mg protein.

(2) The 100% IP ₃ radioactivity was 19,230 cpm.

(3) Measured by microdensitometry of the corresponding Western-blotted bands.

TABLE II

COLONY FORMATION IN SOFT AGAR

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Fischer, Gabriela A. Ullrich, Axel

(ii) TITLE OF INVENTION: Methods For Treating Cell Proliferative Disorders By Modulating Signal Transduction

(iii) NUMBER OF SEQUENCES: 3

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Pennie _A 6 Edmonds

(B) STREET: 1155 Avenue of the Americas

(C) CITY: New York

(D) STATE: New York

(E) COUNTRY: U.S.A.

(F) ZIP: 10036-2711

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/268,390

(B) FILING DATE: 30-JUN-1994

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Coruzzi, Laura A.

(B) REGISTRATION NUMBER: 30,742

(C) REFERENCE/DOCKET NUMBER: 7683-057

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (212)790-9090

(B) TELEFAX: (212)869-9741

(C) TELEX: 66141 PENNIE

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8806 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 135..8144

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: CCGGATCGTC CAAGTCGCCG GGCTGCGAGG TGGCACTGCG CTCCTTTCCG CCCAGCGCGC 60

CTCAGTCCTC TGCACCGAGC CTGGCAGCTC GCTTCTGGGC GACTTTGCCG CCTCGAGCCC 120

CGGAGGCCAA AGCC ATG AAT GAA ATG TCC AGC TTT CTT CAC ATC GGG GAC 170 Met Asn Glu Met Ser Ser Phe Leu His lie Gly Asp 1 5 10

ATT GTG TCC CTG TAC GCC GAG GGC TCT GTC AAC GGC TTC ATC AGC ACT 218 lie Val Ser Leu Tyr Ala Glu Gly Ser Val Asn Gly Phe lie Ser Thr 15 20 25

TTG GGG CTG GTG GAT GAC CGT TGT GTG GTG GAG CCT GCA GCT GGG GAC 266 Leu Gly Leu Val Asp Asp Arg Cys Val Val Glu Pro Ala Ala Gly Asp 30 35 40

CTG GAC AAC CCC CCC AAG AAG TTC CGA GAT TGC CTC TTC AAA GTG TGC 314 Leu Asp Asn Pro Pro Lys Lys Phe Arg Asp Cys Leu Phe Lys Val Cys 45 50 55 60

CCC ATG AAC CGC TAC TCC GCA CAG AAG CAG TAC TGG AAA GCC AAG CAG 362 Pro Met Asn Arg Tyr Ser Ala Gin Lys Gin Tyr Trp Lys Ala Lys Gin 65 70 75

ACT AAG CAG GAC AAA GAG AAG ATC GCC GAT GTG GTG TTG CTG CAG AAG 410 Thr Lys Gin Asp Lys Glu Lys lie Ala Asp Val Val Leu Leu Gin Lys 80 85 90

CTG CAG CAT GCA GCC CAG ATG GAG CAG AAG CAA AAC GAC ACA GAG AAC 458 Leu Gin His Ala Ala Gin Met Glu Gin Lys Gin Asn Asp Thr Glu Asn 95 100 105

AAG AAG GTG CAC GGG GAC GTG GTG AAG TAC GGC AGC GTG ATT CAG CTT 506 Lys Lys Val His Gly Asp Val Val Lys Tyr Gly Ser Val He Gin Leu 110 115 120

CTG CAC ATG AAG AGC AAC AAA TAC TTA ACG GTG AAC AAG CGG CTG CCG 554 Leu His Met Lys Ser Asn Lys Tyr Leu Thr Val Asn Lys Arg Leu Pro 125 130 135 140

GCC CTG CTG GAG AAG AAC GCC ATG CGG GTG ACG CTG GAC GCG ACG GGC 602 Ala Leu Leu Glu Lys Asn Ala Met Arg Val Thr Leu Asp Ala Thr Gly 145 ISO 155

AAC GAG GGC TCC TGG CTC TTC ATC CAG CCC TTC TGG AAG CTT CGG AGC 650 Asn Glu Gly Ser Trp Leu Phe He G n Pro Phe Trp Lys Leu Arg Ser 160 165 170

AAT GGG GAT AAC GTG GTT GTG GGG GAC AAG GTG ATC CTG AAC CCT GTC 698 Asn Gly Asp Asn Val Val Val Gly Asp Lys Val He Leu Asn Pro Val 175 180 185

AAC GCA GGG CAG CCT CTA CAC GCC AGC AAC TAT GAG CTT AGT GAT AAC 7 6 Asn Ala Gly Gin Pro Leu His Ala Ser Asn Tyr Glu Leu Ser Asp Asn 190 195 200

GTT GGC TGC AAG GAG GΪC AAC TCT GTC AAC TGC AAC ACC AGC TGG AAG 794 Val Gly Cys Lys Glu Val Asn Ser Val Asn Cys Asn Thr Ser Trp Lys 205 210 215 220

ATC AAC TTA TTC ATG CAG TTC CGG GAC CAT CTG GAG GAG GTG TTG AAG 842 He Asn Leu Phe Met Gin Phe Arg Asp His Leu Glu Glu Val Leu Lys 225 230 235

GGG GGA GAC GTG GTG CGG CTG TTC CAT GCA GAG CAG GAG AAG TTC CTG 890

Gly Gly Asp Val Val Arg Leu Phe His Ala Glu Gin Glu Lys Phe Leu 240 245 250

ACC TGT GAC GAG TAC CGG GGC AAG CTT CAG GTG TTC CTG AGG ACC ACG 9 8 Thr Cys Asp Glu Tyr Arg Gly Lys Leu Gin Val Phe Leu Arg Thr Thr

255 260 265

CTG CGT CAG TCC GCC ACC TCG GCC ACC AGC TCC AAC GCG CTC TGG GAG 986 Leu Arg Gin Ser Ala Thr Ser Ala Thr Ser Ser Asn Ala Leu Trp Glu 270 275 280

GTG GAG GTG GTC CAC CAT GAC CCC TGC CGC GGC GGA GCG GGA CAC TGG 1034

Val Glu Val Val His His Asp Pro Cys Arg Gly Gly Ala Gly His Trp 285 290 295 300

AAT GGT CTG TAC AGG TTC AAG CAC CTG GCC ACG GGC AAC TAT CTG GCG 1082

Asn Gly Leu Tyr Arg Phe Lys His Leu Ala Thr Gly Asn Tyr Leu Ala 305 310 315

GCT GAG GAA AAC CCC AGC TAC AAG GGT GAT GTC TCA GAT CCT AAG GCA 1130

Ala Glu Glu Asn Pro Ser Tyr Lys Gly Asp Val Ser Asp Pro Lys Ala 320 325 330

GCA GGA CCA GGG GCC CAG AGC CGC ACA GGC CGC AGA AAC GCC GGC GAG 1178

Ala Gly Pro Gly Ala Gin Ser Arg Thr Gly Arg Arg Asn Ala Gly Glu

335 340 345

AAG ATC AAG TAC CGT CTG GTG GCT GTG CCA CAT GGC AAC GAC ATC GCG 1226

Lys He Lys Tyr Arg Leu Val Ala Val Pro His Gly Asn Asp He Ala 350 355 360

TCT CTC TTC GAG CTG GAC CCC ACC ACG CTG CAG AAA ACC GAC TCC TTC 1274

Ser Leu Phe Glu Leu Asp Pro Thr Thr Leu Gin Lys Thr Asp Ser Phe 365 370 375 380

GTG CCC CGG AAC TCC TAC GTG CGG CTG CGG CAC CTC TGT ACC AAC ACC 1322

Val Pro Arg Asn Ser Tyr Val Arg Leu Arg His Leu Cys Thr Asn Thr 385 390 395

TGG ATC CAG AGC ACG AAC GCG CCC ATC GAC GTG GAG GAG GAA CGG CCT 1370

Trp He Gin Ser Thr Asn Ala Pro He Asp Val Glu Glu Glu Arg Pro 400 405 410

ATC CGG CTC ATG TTG GGC ACG TGC CCC ACC AAG GAG GAC AAG GAG GCC 1418

He Arg Leu Met Leu Gly Thr Cys Pro Thr Lys Glu Asp Lys Glu Ala

415 420 425

TTT GCC ATT GTG TCG GTT CCC GTG TCT GAG ATC CGC GAC CTG GAC TTT 1466

Phe Ala He Val Ser Val Pro Val Ser Glu He Arg Asp Leu Asp Phe 430 435 440

GCC AAC GAT GCA AGC TCC ATG CTG GCC AGC GCT GTG GAG AAA CTC AAC 1514

Ala Asn Asp Ala Ser Ser Met Leu Ala Ser Ala Val Glu Lys Leu Asn 445 450 455 460

GAG GGC TTC ATC AGC CAG AAC GAC CGC AGG TTT GTC ATC CAG CTG TTG 1562

Glu Gly Phe .lie Ser Gin Asn Asp Arg Arg Phe Val He Gin Leu Leu 465 470 475

GAG GAC TTG GTG TTT TTT GTC AGC GAT GTT CCC AAC AAT GGG CAG AAT 1610

Glu Asp Leu Val Phe Phe Val Ser Asp Val Pro Asn Asn Gly Gin Asn 480 485 490

GTC CTG GAC ATC ATG GTC ACC AAA CCC AAC CGA GAG CGG CAG AAG CTG 1658 Val Leu Asp He Met Val Thr Lys Pro Asn Arg Glu Arg Gin Lys Leu 495 500 505

ATG CGG GAC GAG AAC ATA CTT AAG CAG ATC TTT GGC ATC CTG AAG GCT 1706 Met Arg Asp Glu Asn He Leu Lys Gin He Phe Gly He Leu Lys Ala 510 515 520'

CCA TTC CGT GAC AAG GGG GGT GAA GGG CCA TTG GTG CGT CTG GAG GAA 1754 Pro Phe Arg Asp Lys Gly Gly Glu Gly Pro Leu Val Arg Leu Glu Glu 525 530 535 540

CTG TCT GAC CAG AAG AAC GCC CCC TAC CAG TAC ATG TTC CGC CTG TGT 1802 Leu Ser Asp Gin Lys Asn Ala Pro Tyr Gin Tyr Met Phe Arg Leu Cys 545 SSO 555

TAC CGG GTG CTC CGG CAC TCC CAG GAG GAC TAC CGT AAG AAC CAG GAG 1850 Tyr Arg Val Leu Arg His Ser Gin Glu Asp Tyr Arg Lys Asn Gin Glu 560 565 570

CAC ATA GCC AAG CAG TTC GGG ATG ATG CAG TCA CAG ATT GGC TAT GAC 1898 His He Ala Lys Gin Phe Gly Met Met Gin Ser Gin He Gly Tyr Asp 575 580 585

ATC CTG GCC GAA GAC ACC ATC ACG GCC CTG CTG CAC AAC AAC CGG AAG 1946 He Leu Ala Glu Asp Thr He Thr Ala Leu Leu His Asn Asn Arg Lys 590 595 600

CTG CTG GAG AAG CAC ATC ACC AAG ACC GAG GTG GAA ACC TTC GTG AGC 1994 Leu Leu Glu Lys His He Thr Lys Thr Glu Val Glu Thr Phe Val Ser 605 610 615 620

CTC GTG CGC AAG AAC AGG GAG CCC AGG TTC CTG GAC TAC CTC TCC GAC 2042 Leu Val Arg Lys Asn Arg Glu Pro Arg Phe Leu Asp Tyr Leu Ser Asp 625 630 635

CTG TGC GTG TCC AAC CGC ATT GCC ATC CCC GTC ACC CAG GAG CTG ATT 2090 Leu Cys Val Ser Asn Arg He Ala He Pro Val Thr Gin Glu Leu He 640 645 650

TGC AAG TGT GTG CTG GAC CCC AAG AAC AGT GAC ATC CTT ATC CAG ACT 2138 Cys Lys Cys Val Leu Asp Pro Lys Asn Ser Asp He Leu He Gin Thr 655 660 665

GAG CTT CGG CCC GTG AAG GAG ATG GCT CAG TCT CAC GAG TAC CTC AGC 2186 Glu Leu Arg Pro Val Lys Glu Met Ala Gin Ser His Glu Tyr Leu Ser 670 675 680

ATC GAG TAC TCG GAG GAG GAG GTG TGG CTC ACA TGG ACG GAC AGG AAC 2234 He Glu Tyr Ser Glu Glu Glu Val Trp Leu Thr Trp Thr Asp Arg Asn 685 690 695 700

AAC GAG CAT CAC GAG AAG AGT GTG AGG CAG CTG GCC CAG GAG GCA AGG 2282 Asn Glu His His Glu Lys Ser Val Arg Gin Leu Ala Gin Glu Ala Arg 705 ^* 710 715

GCC GGC AAC GCA CAC GAC GAG AAC GTG CTC AGC TAC TAC AGG TAC CAG 2330 Ala Gly Asn Ala His Asp Glu Asn Val Leu Ser Tyr Tyr Arg Tyr Gin 720 725 730

TTG AAG CTG TTT GCC CGC ATG TGC CTA GAC CGC CAG TAC CTG GCC ATC 2378 Leu Lys Leu Phe Ala Arg Met Cys Leu Asp Arg Gin Tyr Leu Ala He 735 740 745

GAC GAG ATC TCC AAG CAA CTG GGT GTG GAG CTG CTT TTC CTA TGC ATG 2426 Asp Glu He Ser Lys Gin Leu Gly Val Glu Leu Leu Phe Leu Cys Met 750 755 760

GCG GAC GAG ATG CTG CCC TTT GAC CTC CGC GCC TCA TTC TGC CAC CTG 2474 Ala Asp Glu Met Leu Pro Phe Asp Leu Arg Ala Ser Phe Cys His Leu 765 770 775 780

ATG CTA CAT GTG CAC GTG GAC CGT GAC CCC CAG GAG CTG GTG ACC CCT 2S22 Met Leu His Val His Val Asp Arg Asp Pro Gin Glu Leu Val Thr Pro 785 790 795

GTC AAG TTT GCC CGC CTC TGG ACA GAG ATC CCC ACG GCC ATC ACC ATC 2S70 Val Lys Phe Ala Arg Leu Trp Thr Glu He Pro Thr Ala He Thr He 800 805 810

AAA GAC TAT GAT TCC AAT CTC AAC GCC TCC CGA GAT GAC AAG AAA AAC 2618 Lys Asp Tyr Asp Ser Asn Leu Asn Ala Ser Arg Asp Asp Lys Lys Asn 815 820 825

AAG TTT GCC AGC ACC ATG GAG TTC GTG GAG GAT TAC CTT AAC AAC GTG 2666 Lys Phe Ala Ser Thr Met Glu Phe Val Glu Asp Tyr Leu Asn Asn Val 830 835 840

GTC GGC GAG GCC GTG CCC TTT GCC AAC GAT GAG AAG AAC ATA CTG ACC 2714 Val Gly Glu Ala Val Pro Phe Ala Asn Asp Glu Lys Asn He Leu Thr B45 850 855 860

TTT GAG GTG GTC AGC CTG GCA CAC AAC CTC ATC TAC TTC GGG TTC TAC 2762 Phe Glu Val Val Ser Leu Ala His Asn Leu He Tyr Phe Gly Phe Tyr 865 870 875

AGC TTC AGC GAG CTG CTG CGA CTC ACA CGC ACG TTG CTG GGC ATC ATC 2810 Ser Phe Ser Glu Leu Leu Arg Leu Thr Arg Thr Leu Leu Gly He He 880 885 890

GAC TGC ATT CAG GCA CCA GCC GCT GTG CTG CAG GCC TAT GAG GAG CCT 2858 Asp Cys He Gin Ala Pro Ala Ala Val Leu Gin Ala Tyr Glu Glu Pro 895 900 905

GGC GGC AAG AAC GTG CGG AGG TCC ATC CAG GGG GTG GGA CAC ATG ATG 2906 Gly Gly Lys Asn Val Arg Arg Ser He Gin Gly Val Gly His Met Met 910 915 920

TCC ACC ATG GTG CTA AGC CGC AAG CAG TCT GTA TTT GGT GCC TCC AGC 2954 Ser Thr Met Val Leu Ser Arg Lys Gin Ser Val Phe Gly Ala Ser Ser 925 930 935 940

CTG CCT ACT GGA GTG GGT GTC CCC GAG CAG CTG GAC AGA AGC AAA TTT 3002 Leu Pro Thr Gly Val Gly Val Pro Glu Gin Leu Asp Arg Ser Lys Phe 945 950 955

GAG GAC AAT GAA CAC ACC GTG GTG ATG GAG ACC AAG CTG AAG ATC CTG 3050 Glu Asp Asn Glu His Thr Val Val Met Glu Thr Lys Leu Lys He Leu 960 ^' 965 970

GAG ATC CTG CAG TTC ATC CTC AAC GTG CGC CTA GAC TAC CGC ATC TCC 098 Glu He Leu Gin Phe He Leu Asn Val Arg Leu Asp Tyr Arg He Ser 975 980 985

TAC CTG CTG TCG GTC TTC AAG AAG GAG TTT GTG GAG GTC TTT CCC ATG 3146 Tyr Leu Leu Ser Val Phe Lys Lys Glu Phe Val Glu Val Phe Pro Met 990 995 1000

CAG GAC AGC GGG GCA GAC GGC ACA GCC CCC GCC TTC GAT TCC TCT ACT 3194 Gin Asp Ser Gly Ala Asp Gly Thr Ala Pro Ala Phe Asp Ser Ser Thr 1005 1010 1015 1020

GCC AAC ATG AAT CTG GAC CGC ATC GGG GAG CAG GCA GAG GCC ATG TTT 3242 Ala Asn Met Asn Leu Asp Arg He Gly Glu Gin Ala Glu Ala Met Phe 1025 1030 1035

GGA GTA GGG AAG ACC AGC AGC ATG CTA GAG GTG GAT GAC GAA GGG GGT 3290 Gly Val Gly Lys Thr Ser Ser Met Leu Glu Val Asp Asp Glu Gly Gly 1040 1045 1050

CGC ATG TTC CTG CGA GTG CTG CTG CAC CTC ACC ATG CAC GAC TAC CCA 3338 Arg Met Phe Leu Arg Val Leu Leu His Leu Thr Met His Asp Tyr Pro 1055 1060 1065

CCT CTG GTC TCC GGC GCC CTG CAG CTG CTC TTC AAA CAT TTC AGC CAG 3386 Pro Leu Val Ser Gly Ala Leu Gin Leu Leu Phe Lys His Phe Ser Gin 1070 1075 1080

CGC CAG GAG GCC ATG CAC ACC TTC AAG CAG GTG CAG CTG CTC ATC TCA 3434 Arg Gin Glu Ala Met His Thr Phe Lys Gin Val Gin Leu Leu He Ser 108S 1090 1095 1100

GCC CAG GAT GTG GAG AAC TAC AAA GTG ATC AAG TCG GAA CTG GAT CGA 3482 Ala Gin Asp Val Glu Asn Tyr Lys Val He Lys Ser Glu Leu Asp Arg 1105 1110 1115

CTG CGG ACG ATG GTG GAG AAG TCG GAG CTG TGG GTG GAC AAG AAA GGC 3530 Leu Arg Thr Met Val Glu Lys Ser Glu Leu Trp Val Asp Lys Lys Gly 1120 1125 1130

AGC GTC AAG GGC GAG GAG GGG GAG GCG GGC GCC AGC AAG GAT AAG AAG 3578 Ser Val Lys Gly Glu Glu Gly Glu Ala Gly Ala Ser Lys Asp Lys Lys 1135 1140 1145

GAG CGG CCC TCG GAT GAG GAG GGA TTT CTG CAG CCA CAC GGG GAG AAG 3626 Glu Arg Pro Ser Asp Glu Glu Gly Phe Leu Gin Pro His Gly Glu Lys 1150 1155 1160

AGC AGT GAG AAC TAC CAG ATT GTC AAA GGC ATC CTG GAG AGG CTG AAC 3674 Ser Ser Glu Asn Tyr Gin He Val Lys Gly He Leu Glu Arg Leu Asn 1165 1170 1175 1180

AAG ATG TGT GGG GTC GGG GAG CAG ATG CGA AAG AAG CAA CAG AGA CTG 3722 Lys Met Cys Gly Val Gly Glu Gin Met Arg Lys Lys Gin Gin Arg Leu 1185 1190 1195

CTG AAG AAC ATG GAC GCC CAC AAG GTC ATG CTG GAC CTG CTG CAG ATC 3770 Leu Lys Asn Met Asp Ala His Lys Val Met Leu Asp Leu Leu Gin He 1200 1205 1210

CCT TAT GAC AAG AAC GAC AAC AAG ATG ATG GAG ATC CTG CGC TAC ACA 3818 Pro Tyr Asp Lys Asn Asp Asn Lys Met Met Glu He Leu Arg Tyr Thr 1215 " 1220 1225

CAC CAG TTC CTA CAG AAG TTC TGT GCC GGG AAC CCT GGC AAC CAG GCC 3866 His Gin Phe Leu Gin Lys Phe Cys Ala Gly Asn Pro Gly Asn Gin Ala 1230 1235 1240

CTT CTG CAC AAG CAC TTG CAG CTC TTC CTC ACG CCC GGG CTC CTG GAG 3914 Leu Leu His Lys His Leu Gin Leu Phe Leu Thr Pro Gly Leu Leu Glu 1245 1250 1255 1260

GCT GAG ACC ATG CAG CAC ATT TTC CTC AAC AAC TAT CAG CTG TGC TCT 3962 Ala Glu Thr Met Gin His He Phe Leu Asn Asn Tyr Gin Leu Cys Ser 1265 1270 1275

GAG ATC AGC GAG CCG GTG CTG CAA CAC TTC GTG CAC TGC TGG CCG ACA 4010 Glu He Ser Glu Pro Val Leu Gin His Phe Val His Cys Trp Pro Thr 1280 1285 1290

CAC GGG CGC CAC GTG CAG TAC CTG GAC TTC CTG CAT ACC GTC ATC AAG 4058 His Gly Arg His Val Gin Tyr Leu Asp Phe Leu His Thr Val He Lys 1295 1300 1305

GCC GAG GGC AAG TAC GTG AAG AAG TGT CAG GAC ATG ATC ATG ACC GAG 4106 Ala Glu Gly Lys Tyr Val Lys Lys Cys Gin Asp Met He Met Thr Glu 1310 1315 1320

CTG ACC AAC GCA GGC GAC GAC GTG GTG GTG TTC TAC AAT GAC AAG GCC 4154 Leu Thr Asn Ala Gly Asp Asp Val Val Val Phe Tyr Asn Asp Lys Ala 1325 1330 1335 1340

TCT CTG GCC CAT CTG CTG GAC ATG ATG AAG GCA GCC CGA GAT GGC GTG 4202 Ser Leu Ala His Leu Leu Asp Met Met Lys Ala Ala Arg Asp Gly Val 1345 1350 1355

GAG GAC CAC AGC CCC CTC ATG TAC CAC ATC TCC CTG GTG GAC TTG CTG 4250 Glu Asp His Ser Pro Leu Met Tyr His He Ser Leu Val Asp Leu Leu 1360 1365 1370

GCT GCC TGT GCA GAA GGC AAA AAT GTC TAC ACA GAG ATC AAG TGC ACT 4298 Ala Ala Cys Ala Glu Gly Lys Asn Val Tyr Thr Glu He Lys Cys Thr 1375 1380 1385

TCC CTG CTG CCT CTA GAG GAC GTG GTG TCG GTG GTG ACC CAC GAG GAC 43 6 Ser Leu Leu Pro Leu Glu Asp Val Val Ser Val Val Thr His Glu Asp 1390 1395 1400

TGC ATC ACG GAG GTT AAA ATG GCG TAT GTG AAC TTC GTG AAC CAC TGC 4394 Cys He Thr Glu Val Lys Met Ala Tyr Val Asn Phe Val Asn His Cys 1405 1410 1415 1420

TAT GTA GAC ACG GAG GTG GAG ATG AAG GAG ATC TAT ACC AGC AAC CAC 4442 Tyr Val Asp Thr Glu Val Glu Met Lys Glu He Tyr Thr Ser Asn His 1425 1430 1435

ATC TGG ACG CTC TTC GAG AAC TTC ACC CTG GAC ATG GCT CTG GTC TGT 4490 He Trp Thr Leu Phe Glu Asn Phe Thr Leu Asp Met Ala Leu Val Cys 1440 1445 14S0

AAC AAG CGG GAG AAG CGC CTG TCA GAC CCC ACC CTT GAG AAG TAC GTG 4538 Asn Lys Arg Glu Lys Arg Leu Ser Asp Pro Thr Leu Glu Lys Tyr Val 1455 1460 1465

CTC ACC GTG GTG CTG GAC ACC ATC AGC GCC TTC TTC AGT TCC CCA TTT 4586 Leu Thr Val Val Leu Asp Thr He Ser Ala Phe Phe Ser Ser Pro Phe 1470 .1475 1480

TCA GAG AAC AGC ACA TCC CTA CAG ACG CAT CAG ACA ATT GTG GTT CAG 4634 Ser Glu Asn Ser Thr Ser Leu Gin Thr His Gin Thr He Val Val Gin 1485 1490 1495 1500

CTG CTG CAG TCC ACC ACC CGG CTG CTC GAG TGT CCT TGG CTG CAG CAG 6B2 Leu Leu Gin Ser Thr Thr Arg Leu Leu Glu Cys Pro Trp Leu Gin Gin 1505 1510 1515

CAG CAC AAG GGC TCG GTG GAG GCC TGT GTC CGC ACC CTC GCC ATG GTG 4730 Gin His Lys Gly Ser Val Glu Ala Cys Val Arg Thr Leu Ala Met Val 1520 1S25 1530

GCC AAG AGT AGA GCC ATC TTG CTG CCG ATG GAT CTG GAT GCC CAC ATG 477B Ala Lys Ser Arg Ala He Leu Leu Pro Met Asp Leu Asp Ala His Met 1535 1540 1545

AGC GCT CTG CTC AGC AGT GGG GGC AGC TGC TCA GCC GCG GCC CAA CGC 4826 Ser Ala Leu Leu Ser Ser Gly Gly Ser Cys Ser Ala Ala Ala Gin Arg 1550 1555 1560

AGC GCT GCC AAC TAC AAG ACG GCC ACG AGG ACC TTC CCT CGG GTC ATC 4874 Ser Ala Ala Asn Tyr Lys Thr Ala Thr Arg Thr Phe Pro Arg Val He 1565 1S70 1575 1580

CCC ACC GCC AAC CAG TGG GAC TAC AAG AAC ATC ATT GAG AAG TTA CAG 4922 Pro Thr Ala Asn Gin Trp Asp Tyr Lys Asn He He Glu Lys Leu Gin 1585 1590 1595

GAC ATC ATC ACG GCC CTA GAA GAG CGG CTG AAG CCT CTG GTG CAG GCC 4970 Asp He He Thr Ala Leu Glu Glu Arg Leu Lys Pro Leu Val Gin Ala 1600 1605 1610

GAG CTC TCG GTG CTG GTG GAC ATG CTG CAC TGG CCC GAG CTG CTC TTT 5018 Glu Leu Ser Val Leu Val Asp Met Leu His Trp Pro Glu Leu Leu Phe 1615 1620 1625

CTA GAG GGC AGT GAG GCC TAC CAG CGC TGC GAG AGT GGC GGC TTC CTG 5066 Leu Glu Gly Ser Glu Ala Tyr Gin Arg Cys Glu Ser Gly Gly Phe Leu 1630 1635 1640

TCC AAG CTC ATC CGT CAC ACC AAG GGT CTC ATG GAG TCG GAG GAG AAG 5114 S~z Lys Leu He Arg His Thr Lys Gly Leu Met Glu Ser Glu Glu Lys 1645 1650 1655 1660

CTG TGC GTG AAG GTG CTG CGG ACG CTG CAG CAG ATG CTG CAG AAG AAG 5162 Leu Cys Val Lys Val Leu Arg Thr Leu Gin Gin Met Leu Gin Lys Lys 1665 1670 1675

AGC AAG TAT GGG GAC CGG GGC AAC CAG CTA AGG AAG ATG CTG CTG CAG 5210 Ser Lys Tyr Gly Asp Arg Gly Asn Gin Leu Arg Lys Met Leu Leu Gin 1680 1685 1690

AAT TAC CTC CAG AAC CGG AAG TCC GGT CCC CGG GGC GAG CTC ACT GAC 5258 Asn Tyr Leu Gin Asn Arg Lys Ser Gly Pro Arg Gly Glu Leu Thr Asp 1695 1700 1705

CCC ACA GGC TCT GGC GTG GAT CAG GAC TGG TCC GCC ATC GCA GCC ACC 5306 Pro Thr Gly Ser Gly Val Asp Gin Asp Trp Ser Ala He Ala Ala Thr 1710 1715 1720

CAG TGC CGG CTG GAC AAG GAG GGG GCC ACC AAG TTG GTG TGT GAT CTC 5354 Gin Cys Arg Leu Asp Lys Glu Gly Ala Thr Lys Leu Val Cys Asp Leu 172S 1730 1735 1740

ATC ACC AGC ACC AAG AAC GAG AAG ATC TTC CAG GAG AGC ATC GGC CTG 5402 He Thr Ser Thr Lys Asn Glu Lys He Phe Gin Glu Ser He Gly Leu 1745 1750 1755

GCC ATC CGC CTG CTG GAC GGG GGC AAC ACT GAG ATC CAG AAG TCT TTC 5450 Ala He Arg Leu Leu Asp Gly Gly Asn Thr Glu He Gin Lys Ser Phe 1760 1765 1770

TAC AAC CTG ATG ACA AGT GAC AAG AAG TCA GAG CGC TTC TTC AAA GTG 5498 Tyr Asn Leu Met Thr Ser Asp Lys Lys Ser Glu Arg Phe Phe Lys Val 1775 1780 1785

CTG CAT GAC CGC ATG AAG CGT GCC CAG CAG GAG ACC AAG TCC ACA GTT 5546 Leu His Asp Arg Met Lys Arg Ala Gin Gin Glu Thr Lys Ser Thr Val 1790 1795 1800 -

GCC GTC AAC ATG AGT GAC CTG GGC AGC CAG CCT CGT GAG GAC CGT GAG 5594 Ala Val Asn Met Ser Asp Leu Gly Ser Gin Pro Arg Glu Asp Arg Glu 1805 1810 1815 1820

CCA GCA GAC CCT ACC ACC AAA GGT CGC GTG TCC TCC TTC TCC ATG CCC 5642 Pro Ala Asp Pro Thr Thr Lys Gly Arg Val Ser Ser Phe Ser Met Pro 1825 1830 1835

AGC TCC TCC CGA TAT TCG CTG GGC CCT GGC TTG CAC CGG GGG CAC GAC 5690 Ser Ser Ser Arg Tyr Ser Leu Gly Pro Gly Leu His Arg Gly His Asp 1840 1845 1850

GTG AGC GAG CGT GCG CAG AAC AAC GAG ATG GGG ACC TCG GTG CTC ATC 5738 Val Ser Glu Arg Ala Gin Asn Asn Glu Met Gly Thr Ser Val Leu He 1855 1860 1865

ATG CGG CCC ATC CTG CGC TTC CTG CAG CTG CTG TGT GAG AAC CAT AAC 5786 Met Arg Pro He Leu Arg Phe Leu Gin Leu Leu Cys Glu Asn His Asn 1870 1875 1880

CGG GAC CTG CAG AAC TTC CTG CGC TGT CAG AAC AAC AAG ACC AAC TAC 5834 Arg Asp Leu Gin Asn Phe Leu Arg Cys Gin Asn Asn Lys Thr Asn Tyr 1885 1890 1895 1900

AAC CTC GTG TGT GAG ACC CTG CAG TTC CTG GAC ATC ATG TGT GGC AGC 5882 Asn Leu Val Cys Glu Thr Leu Gin Phe Leu Asp He Met Cys Gly Ser 1905 1910 1915

ACC ACA GGG GGC CTG GGG CTG CTG GGG CTC TAC ATC AAC GAG GAC AAT 59 0 Thr Thr Gly Gly Leu Gly Leu Leu Gly Leu Tyr He Asn Glu Asp Asn 1920 1925 1930

GTG GGC CTG GTC ATC CAG ACC TTG GAG ACC CTC ACA GAG TAC TGC CAG 5978 Val Gly Leu Val He Gin Thr Leu Glu Thr Leu Thr Glu Tyr Cys Gin 1935 1940 1945

GGC CCG TGC CAT GAG AAC CAG ACC TGC ATC GTG ACT CAC GAG TCC AAC 6026 Gly Pro Cys His Glu Asn Gin Thr Cys He Val Thr His Glu Ser Asn 1950 19S5 1960

GGC ATT GAC ATC ATC ACG GCG CTG ATC CTC AAT GAC ATC AGC CCG CTC 6074 Gly He Asp He He Thr Ala Leu He Leu Asn Asp He Ser Pro Leu 1965 1970 1975 1980

TGC AAG TAC CGC ATG GAC CTG GTG CTG CAG CTC AAG GAC AAT GCC TCC 6122 Cys Lys Tyr Arg Met Asp Leu Val Leu Gin Leu Lys Asp Asn Ala Ser 1985 - 1990 1995

AAG CTG CTC CTG GCT CTG ATG GAG AGT CGA CAT GAC AGC GAG AAT GCA 6170 Lys Leu Leu Leu Ala Leu Met Glu Ser Arg His Asp Ser Glu Asn Ala 2000 2005 2010

GAA CGC ATC CTC ATC AGC CTT CGG CCT CAG GAG CTG GTG GAC GTG ATC 6218 Glu Arg He Leu He Ser Leu Arg Pro Gin Glu Leu Val Asp Val He 2015 2020 2025

AAG AAG GCC TAC CTG CAG GAG GAG GAG CGG GAA AAT TCC GAA GTG AGC 626 Lys Lys Ala Tyr Leu Gin Glu Glu Glu Arg Glu Asn Ser Glu Val Ser 2030 2035 2040

CCA CGT GAA GTG GGC CAC AAC ATC TAC ATC CTG GCA CTG CAG CTT TCC 631 Pro Arg Glu Val Gly His Asn He Tyr He Leu Ala Leu Gin Leu Ser 2045 2050 20S5 2060

AGG CAC AAC AAG CAG CTA CAG CAC CTG CTG AAG CCG GTG AAG CGC ATC 636 Arg His Asn Lys Gin Leu Gin His Leu Leu Lys Pro Val Lys Arg He 2065 2070 2075

CAG GAG GAG GAG GCC GAG GGC ATC TCT TCC ATG CTC AGC CTC AAC AAC 641 Gin Glu Glu Glu Ala Glu Gly He Ser Ser Met Leu Ser Leu Asn Asn 2080 2085 2090

AAG CAG CTC TCT CAG ATG CTC AAG TCC TCA GCC CCT GCC CAA GAG GAG 645 Lys Gin Leu Ser Gin Met Leu Lys Ser Ser Ala Pro Ala Gin Glu Glu 2095 2100 2105

GAG GAA GAC CCG CTG GCC TAC TAT GAG AAC CAC ACC TCG CAG ATT GAG 650 Glu Glu Asp Pro Leu Ala Tyr Tyr Glu Asn His Thr Ser Gin He Glu 2110 2115 2120

ATT GTG CGG CAA GAC CGA AGC ATG GAG CAG ATC GTG TTC CCG GTA CCT 655 He Val Arg Gin Asp Arg Ser Met Glu Gin He Val Phe Pro Val Pro 2125 2130 2135 2140

GCC ATC TGC CAG TTC CTG ACA GAG GAG ACC AAG CAC CGG CTC TTC ACC 660 Ala He Cys Gin Phe Leu Thr Glu Glu Thr Lys His Arg Leu Phe Thr 2145 2150 2155

ACC ACG GAG CAG GAC GAG CAG GGC AGC AAA GTG AGC GAC TTC TTC GAC 665 Thr Thr Glu Gin Asp Glu Gin Gly Ser Lys Val Ser Asp Phe Phe Asp 2160 2165 2170

CAG TCC TCC TTC CTG CAC AAC GAG ATG GAG TGG CAG CGC CGG CTT CGC 669 Gin Ser Ser Phe Leu His Asn Glu Met Glu Trp Gin Arg Arg Leu Arg 2175 2180 2185

AGC ATG CCG CTC ATC TAC TGG TTC TCC CGC CGC ATG ACG CTG TGG GGT 674 Ser Met Pro Leu He Tyr Trp Phe Ser Arg Arg Met Thr Leu Trp Gly 2190 2195 2200

AGC ATC TCC TTC AAC CTG GCA GTG TTC ATC AAC ATC ATC ATT GCC TTC 679 Ser He Ser Phe Asn Leu Ala Val Phe He Asn He He He Ala Phe 2205 2210 2215 2220

TTC TAC CCC TAC GTG GAA GGC GCG TCC ACG GGC GTG CTG GGC TCC CCA 684 Phe Tyr Pro Tyr Val Glu Gly Ala Ser Thr Gly Val Leu Gly Ser Pro 2225 2230 2235

CTC ATC TCG CTC CTC TTC TGG ATC CTC ATC TGC TTC TCC ATT GCC GCG 689 Leu He Ser Leu Leu Phe Trp He Leu He Cys Phe Ser He Ala Ala 2240 ' 2245 2250

CTG TTC ACC AAG CAC TAC AGC GTC CGT CCC CTC ATT GTG GCA CTG GTC 693 Leu Phe Thr Lys His Tyr Ser Val Arg Pro Leu He Val Ala Leu Val 2255 2260 2265

CTT CGC TCC ATC TAC TAC CTG GGC ATT GGG CCC ACG CTC AAC ATC CTG 698 Leu Arg Ser He Tyr Tyr Leu Gly He Gly Pro Thr Leu Asn He Leu 2270 2275 2280

GGT GCT CTC AAT CTG ACC AAC AAG ATC GTG TTT GTG GTG AGC TTT GTG 7034 Gly Ala Leu Asn Leu Thr Asn Lys He Val Phe Val Val Ser Phe Val 2285 2290 2295 2300

GGC AAC CGC GGC ACC TTC ATC CGT GGC TAT AAA GCA ATG GTC ATG GAC 7082 Gly Asn Arg Gly Thr Phe He Arg Gly Tyr Lys Ala Met Val Met Asp 2305 2310 2315

ATG GAG TTC CTG TAT CAC GTG GGC TAC ATC CTG ACG AGT GTC CTG GGC 7130 Met Glu Phe Leu Tyr His Val Gly Tyr He Leu Thr Ser Val Leu Gly 2320 2325 2330

CTC TTT GCT CAT GAG CTC TTC TAC AGT ATC CTG CTC TTT GAC CTC ATC 7178 Leu Phe Ala His Glu Leu Phe Tyr Ser He Leu Leu Phe Asp Leu He 2335 2340 2345

TAC CGA GAG GAG ACG CTG TTT AAT GTC ATC AAG AGC GTG ACC CGC AAT 7226 Tyr Arg Glu Glu Thr Leu Phe Asn Val He Lys Ser Val Thr Arg Asn 2350 2355 2360

GGC CGC TCC ATC CTG CTA ACT GCC CTG CTG GCC CTC ATC CTT GTC TAC 7274 Gly Arg Ser He Leu Leu Thr Ala Leu Leu Ala Leu He Leu Val Tyr 2365 2370 2375 2380

CTC TTC TCC ATC GTG GGC TTC CTC TTC CTC AAG GAT GAC TTC ATC CTG 7322 Leu Phe Ser He Val Gly Phe Leu Phe Leu Lys Asp Asp Phe He Leu 2385 2390 2395

GAG GTG GAC CGG CTG CCT GGG AAC CAC TCC AGA GCC AGC ACC CTG GGG 7370 Glu Val Asp Arg Leu Pro Gly Asn His Ser Arg Ala Ser Thr Leu Gly 2400 2405 2410

ATG CCA CAC GGA GCT GCC ACA TTT ATG GGC ACA TGT AGT GGG GAC AAG 7418 Met Pro His Gly Ala Ala Thr Phe Met Gly Thr Cys Ser Gly Asp Lys 2415 2420 2425

ATG GAC TGT GTC TCT GAG GTC TCG GTG CCT GAG ATC CTA GAA GAG GAC 7466 Met Asp Cys Val Ser Glu Val Ser Val Pro Glu He Leu Glu Glu Asp 2430 2435 2440

GAG GAG CTG GAC AGC ACA GAG CGG GCC TGT GAT ACT CTG CTT ATG TGT 7514 Glu Glu Leu Asp Ser Thr Glu Arg Ala Cys Asp Thr Leu Leu Met Cys 2445 2450 2455 2460

ATT GTC ACC GTC ATG AAC CAC GGA CTG AGG AAC GGT GGC GGC GTG GGC 7562 He Val Thr Val Met Asn His Gly Leu Arg Asn Gly Gly Gly Val Gly 2465 2470 2475

GAC ATC CTC CGC AAG CCC TCC AAA GAC GAG TCG CTC TTC CCA GCC AGG 7610 Asp He Leu Arg Lys Pro Ser Lys Asp Glu Ser Leu Phe Pro Ala Arg 2480 2485 2490

GTG GTG TAC GAC CTC CTG TTC TTC TTC ATT GTC ATC ATT ATT GTC CTG 7658 Val Val Tyr Asp Leu Leu Phe Phe Phe He Val He He He Val Leu 2495 2500 2505

AAC CTC ATC TTT GGG GTG ATC ATC GAC ACC TTC GCC GAC CTG CGC AGC 7706 Asn Leu He Phe Gly Val He He Asp Thr Phe Ala Asp Leu Arg Ser 2510 2S15 2520

GAG AAG CAG AAG AAA GAG GAA ATC CTC AAG ACC ACG TGC TTC ATC TGT 7754 Glu Lys Gin Lys Lys Glu Glu He Leu Lys Thr Thr Cys Phe He Cys ■2525 2530 2535 2540

GGT CTG GAG AGG GAC AAG TTT GAC AAC AAG ACA GTG TCC TTT GAA GAG 7B02 Gly Leu Glu Arg Asp Lys Phe Asp Asn Lys Thr Val Ser Phe Glu Glu 2545 2550 2555

CAC ATC AAA CTG GAG CAC AAC ATG TGG AAC TAC CTG TAC TTC ATT GTG 7850 His He Lys Leu Glu His Asn Met Trp Asn Tyr Leu Tyr Phe He Val 2560 2565 2570

CTG GTC CGT GTC AAG AAC AAG ACA GAC TAC ACG GGC CCC GAG AGC TAC 7898 Leu Val Arg Val Lys Asn Lys Thr Asp Tyr Thr Gly Pro Glu Ser Tyr 2575 2580 2585

GTG GCT CAG ATG ATC AAG AAC AAG AAC CTG GAC TGG TTC CCA CGG ATG 7946 Val Ala Gin Met He Lys Asn Lys Asn Leu Asp Trp Phe Pro Arg Met 2590 2595 2600

CGC GCC ATG TCC TTG GTG AGC GGC GAG GGC GAG GGC GAG CAG AAC GAG 7994 Arg Ala Met Ser Leu Val Ser Gly Glu Gly Glu Gly Glu Gin Asn Glu 2605 2610 2615 2620

ATC CGC ATC CTG CAG GAG AAG CTT GGC TCC ACC ATG AAG CTG GTC TCG 8042 He Arg He Leu Gin Glu Lys Leu Gly Ser Thr Met Lys Leu Val Ser 2625 2630 2635

CAC CTC ACC GCC CAG CTC AAT GAG CTC AAG GAA CAG ATG ACG GAG CAG 8090 His Leu Thr Ala Gin Leu Asn Glu Leu Lys Glu Gin Met Thr Glu Gin 2640 2645 2650

CGG AAG CGG AGG CAA CGT CTG GGC TTC GTG GAC GTG CAG AAC TGC ATG 8138 Arg Lys Arg Arg Gin Arg Leu Gly Phe Val Asp Val Gin Asn Cys Met 2655 2660 2665

AGC CGC TGAGCAGAGC GGAGCCTCGG AGGCCCCCAC GGGAGGCCGG CTTGCCCACA 8194 Ser Arg 2670

GCTGCCGCCC TTCCTTCAGG TCAGGTAGAA GCCAGGTGCC TGCCCCATCT TCAGCTCAGC 8254

TGCCACCCTC CCTCCCCAAG CCAGGCTCTG GCCAGCGGGA GTGCCAGAGC AGAGCACGGT 8314

CCTGCTTAGT AAGTACCCTT GAAGAGACTC GGTTTACCTT AGTGCCTTAG GGGATGCAAG 8374

TTCTCCAGAC TGCTGTCCTT CGAAGCAGTG ACTGGCATTT CTCAGTGGCC CTGGCAGGTG 8434

GTTAAAAGTG GGCTGGCTCT CACCTCCAAG CACTACATTG TGGCTGTCCC CTGCCTGCCA 8494

CCCTGGCTAG GAAGGTGGTA GCCTGCTGTT ATTAGCTTCC TAGGGCTTTG ATATTTAACT 8554

TATTCTGGGG GCCATCCCAA CCTCCTGTGG CCTGCTGGGG ACAGTGTGGC CCCGTCCCAG 8614

GAGCCTCATG ACTCAGGTGG ACCTTGCTCC CAGGACTTTC TCTAGGCTTT GTAGGCAAGC 8674

CTCACAAGCC CAGCAACAGG CCTGGGCTGT GTGACAGTGG CTTCTCTCTT TTTGGTGTAA 8734

TGTTTTACAT GTCCTGTGTC CTGGAGACTA ATGTGTTAAT TGCCTTAAAT AAATTAATAG 8794

ACTCCAAAAA AA 8806

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2670 amino acids

(B) TYPE : amino acid (D) TOPOLOGY : linear

( ii) MOLECULE TYPE : protein

(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 2 :

Met Asn Glu Met Ser Ser Phe Leu His He Gly Asp He Val Ser Leu

1 5 10 15

Tyr Ala Glu Gly Ser Val Asn Gly Phe He Ser Thr Leu Gly Leu Val 20 25 30

Asp Asp Arg Cys Val Val Glu Pro Ala Ala Gly Asp Leu Asp Asn Pro 35 40 45

Pro Lys Lys Phe Arg Asp Cys Leu Phe Lys Val Cys Pro Met Asn Arg 50 55 60

Tyr Ser Ala Gin Lys Gin Tyr Trp Lys Ala Lys Gin Thr Lys Gin Asp .65 70 75 80

Lys Glu Lys He Ala Asp Val Val Leu Leu Gin Lys Leu Gin His Ala 85 90 95

Ala Gin Met Glu Gin Lys Gin Asn Asp Thr Glu Asn Lys Lys Val His 100 105 110

Gly Asp Val Val Lys Tyr Gly Ser Val He Gin Leu Leu His Met Lys 115 120 125

Ser Asn Lys Tyr Leu Thr Val Asn Lys Arg Leu Pro Ala Leu Leu Glu 130 135 140

Lys Asn Ala Met Arg Val Thr Leu Asp Ala Thr Gly Asn Glu Gly Ser 145 150 155 160

Trp Leu Phe He Gin Pro Phe Trp Lys Leu Arg Ser Asn Gly Asp Asn 165 170 175

Val Val Val Gly Asp Lys Val He Leu Asn Pro Val Asn Ala Gly Gin 180 185 190

Pro Leu His Ala Ser Asn Tyr Glu Leu Ser Asp Asn Val Gly Cys Lys 195 200 205

Glu Val Asn Ser Val Asn Cys Asn Thr Ser Trp Lys He Asn Leu Phe 210 215 220

Met Gin Phe Arg Asp His Leu Glu Glu Val Leu Lys Gly Gly Asp Val 225 230 235 240

Val Arg Leu Phe His Ala Glu Gin Glu Lys Phe Leu Thr Cys Asp Glu 245 250 255

Tyr Arg Gly Lys Leu Gin Val Phe Leu Arg Thr Thr Leu Arg Gin Ser 260 265 270

Ala Thr Ser Ala Thr Ser Ser Asn Ala Leu Trp Glu Val Glu Val Val 275 280 285

His His Asp Pro Cys Arg Gly Gly Ala Gly His Trp Asn Gly Leu Tyr 290 295 300

Arg Phe Lys His Leu Ala Thr Gly Asn Tyr Leu Ala Ala Glu Glu Asn 305 310 315 320

Pro Ser Tyr Lys Gly Asp Val Ser Asp Pro Lys Ala Ala Gly Pro Gly 325 330 335

Ala Gin Ser Arg Thr Gly Arg Arg Asn Ala Gly Glu Lys He Lys Tyr 340 345 -350

Arg Leu Val Ala Val Pro His Gly Asn Asp He Ala Ser Leu Phe Glu 355 360 365

Leu Asp Pro Thr Thr Leu Gin Lys Thr Asp Ser Phe Val Pro Arg Asn 370 375 380

Ser Tyr Val Arg Leu Arg His Leu Cys Thr Asn Thr Trp He Gin Ser 385 390 395 400

Thr Asn Ala Pro He Asp Val Glu Glu Glu Arg Pro He Arg Leu Met 405 410 415

Leu Gly Thr Cys Pro Thr Lys Glu Asp Lys Glu Ala Phe Ala He Val 420 425 430

Ser Val Pro Val Ser Glu He Arg Asp Leu Asp Phe Ala Asn Asp Ala 435 440 445

Ser Ser Met Leu Ala Ser Ala Val Glu Lys Leu Asn Glu Gly Phe He 450 455 460

Ser Gin Asn Asp Arg Arg Phe Val He Gin Leu Leu Glu Asp Leu Val 465 470 475 480

Phe Phe Val Ser Asp Val Pro Asn Asn Gly Gin Asn Val Leu Asp He 485 490 495

Met Val Thr Lys Pro Asn Arg Glu Arg Gin Lys Leu Met Arg Asp Glu 500 505 510

Asn He Leu Lys Gin He Phe Gly He Leu Lys Ala Pro Phe Arg Asp 515 520 525

Lys Gly Gly Glu Gly Pro Leu Val Arg Leu Glu Glu Leu Ser Asp Gin 530 535 540

Lys Asn Ala Pro Tyr Gin Tyr Met Phe Arg Leu Cys Tyr Arg Val Leu 545 550 555 560

Arg His Ser Gin Glu Asp Tyr Arg Lys Asn Gin Glu His He Ala Lys 565 570 575

Gin Phe Gly Met Met Gin Ser Gin He Gly Tyr Asp He Leu Ala Glu 580 585 590

Asp Thr He Thr Ala.Leu Leu His Asn Asn Arg Lys Leu Leu Glu Lys 595 600 605

His He Thr Lys Thr Glu Val Glu Thr Phe Val Ser Leu Val Arg Lys 610 615 620

Asn Arg Glu Pro Arg Phe Leu Asp Tyr Leu Ser Asp Leu Cys Val Ser 625 630 635 640

Asn Arg He Ala He Pro Val Thr Gin Glu Leu He Cys Lys Cys Val 645 650 655

Leu Asp Pro Lys Asn Ser Asp He Leu He Gin Thr Glu Leu Arg Pro 660 665 670

Val Lys Glu Met Ala Gin Ser His Glu Tyr Leu Ser He Glu Tyr Ser 675 680 685

Glu Glu Glu Val Trp Leu Thr Trp Thr Asp Arg Asn Aβn Glu His His 690 695 700

Glu Lys Ser Val Arg Gin Leu Ala Gin Glu Ala Arg Ala Gly Asn Ala 705 710 715 720

His Asp Glu Asn Val Leu Ser Tyr Tyr Arg Tyr Gin Leu Lys Leu Phe 72S 730 735

Ala Arg Met Cys Leu Asp Arg Gin Tyr Leu Ala He Asp Glu He Ser 740 745 750

Lys G n Leu Gly Val Glu Leu Leu Phe Leu Cys Met Ala Asp Glu Met 755 760 765

Leu Pro Phe Asp Leu Arg Ala Ser Phe Cys His Leu Met Leu His Val 770 775 780

His Val Asp Arg Asp Pro Gin Glu Leu Val Thr Pro Val Lys Phe Ala 785 790 795 800

Arg Leu Trp Thr Glu He Pro Thr Ala He Thr He Lys Asp Tyr Asp 805 810 615

Ser Asn Leu Asn Ala Ser Arg Asp Asp Lys Lys Asn Lys Phe Ala Ser 820 825 830

Thr Met Glu Phe Val Glu Asp Tyr Leu Asn Asn Val Val Gly Glu Ala 835 840 845

Val Pro Phe Ala Asn Asp Glu Lys Asn He Leu Thr Phe Glu Val Val 850 855 860

Ser Leu Ala His Asn Leu He Tyr Phe Gly Phe Tyr Ser Phe Ser Glu 865 870 875 880

Leu Leu Arg Leu Thr Arg Thr Leu Leu Gly He He Asp Cys He Gin 885 890 895

Ala Pro Ala Ala Val Leu Gin Ala Tyr Glu Glu Pro Gly Gly Lys Asn 900 90S 910

Val Arg Arg Ser He Gin Gly Val Gly His Met Met Ser Thr Met Val 915 920 925

Leu Ser Arg Lys Gin Ser Val Phe Gly Ala Ser Ser Leu Pro Thr Gly 930 935 940

Val Gly Val Pro Glu Gin Leu Asp Arg Ser Lys Phe Glu Asp Asn Glu 945 950 955 960

His Thr Val Val Met Glu Thr Lys Leu Lys He Leu Glu He Leu Gin 965 970 975

Phe He Leu Asn Val Arg Leu Asp Tyr Arg He Ser Tyr Leu Leu Ser 980 985 990

Val Phe Lys Lys Glu Phe Val Glu Val Phe Pro Met Gin Asp Ser Gly 995 1000 1005

Ala Asp Gly Thr Ala Pro Ala Phe Asp Ser Ser Thr Ala Asn Met Asn 1010 1015 1020

Leu Asp Arg He Gly Glu Gin Ala Glu Ala Met Phe Gly Val Gly Lys 1025 1030 1035 1040

Thr Ser Ser Met Leu Glu Val Asp Asp Glu Gly Gly Arg Met Phe Leu 1045 1050 1055

Arg Val Leu Leu His Leu Thr Met His Asp Tyr Pro Pro Leu Val Ser 1060 1065 1070

Gly Ala Leu Gin Leu Leu Phe Lys His Phe Ser Gin Arg Gin Glu Ala 1075 1080 1085

Met His Thr Phe Lys Gin Val Gin Leu Leu He Ser Ala Gin Asp Val 1090 1095 1100

Glu Asn Tyr Lys Val He Lys Ser Glu Leu Asp Arg Leu Arg Thr Met 1105 1110 1115 1120

Val Glu Lys Ser Glu Leu Trp Val Asp Lys Lys Gly Ser Val Lys Gly 1125 1130 1135

Glu Glu Gly Glu Ala Gly Ala Ser Lys Asp Lys Lys Glu Arg Pro Ser 1140 1145 1150

Asp Glu Glu Gly Phe Leu Gin Pro His Gly Glu Lys Ser Ser Glu Asn 1155 1160 1165

Tyr Gin He Val Lys Gly He Leu Glu Arg Leu Asn Lys Met Cys Gly 1170 1175 1180

Val Gly Glu Gin Met Arg Lys Lys Gin Gin Arg Leu Leu Lys Asn Met 1185 1190 1195 1200

Asp Ala His Lys Val Met Leu Asp Leu Leu Gin He Pro Tyr Asp Lys 1205 1210 1215

Asn Asp Asn Lys Met Met Glu He Leu Arg Tyr Thr His Gin Phe Leu 1220 1225 1230

Gin Lys Phe Cys Ala Gly Asn Pro Gly Asn Gin Ala Leu Leu His Lys 1235 1240 1245

His Leu Gin Leu Phe Leu Thr Pro Gly Leu Leu Glu Ala Glu Thr Met 1250 1255 1260

Gin His He Phe Leu- Asn Asn Tyr Gin Leu Cys Ser Glu He Ser Glu 1265 1270 1275 1280

Pro Val Leu Gin His Phe Val His Cys Trp Pro Thr His Gly Arg His 1285 1290 1295

Val Gin Tyr Leu Asp Phe Leu His Thr Val He Lys Ala Glu Gly Lys 1300 1305 1310

87 -

Tyr Val Lys Lys Cys Gin Asp Met He Met Thr Glu Leu Thr Asn Ala 1315 1320 1325

Gly Asp Asp Val Val Val Phe Tyr Asn Asp Lys Ala Ser Leu Ala His 1330 1335 1340

Leu Leu Asp Met Met Lys Ala Ala Arg Asp Gly Val.Glu Asp His Ser 1345 1350 1355 1360

Pro Leu Met Tyr His He Ser Leu Val Asp Leu Leu Ala Ala Cys Ala 1365 1370 1375

Glu Gly Lys Asn Val Tyr Thr Glu He Lys Cys Thr Ser Leu Leu Pro 1380 1385 1390

Leu Glu Asp Val Val Ser Val Val Thr His Glu Asp Cys He Thr Glu 1395 1400 1405

Val Lys Met Ala Tyr Val Asn Phe Val Asn His Cys Tyr Val Asp Thr 1410 1415 1420

Glu Val Glu Met Lys Glu He Tyr Thr Ser Asn His He Trp Thr Leu 1425 1430 1435 1440

Phe Glu Asn Phe Thr Leu Asp Met Ala Leu Val Cys Asn Lys Arg Glu 1445 1450 1455

Lys Arg Leu Ser Asp Pro Thr Leu Glu Lys Tyr Val Leu Thr Val Val 1460 1465 1470

Leu Asp Thr He Ser Ala Phe Phe Ser Ser Pro Phe Ser Glu Asn Ser 1475 1480 1485

Thr Ser Leu Gin Thr His Gin Thr He Val Val Gin Leu Leu Gin Ser 1490 1495 1500

Thr Thr Arg Leu Leu Glu Cys Pro Trp Leu Gin Gin Gin His Lys Gly 1505 1510 1515 1520

Ser Val Glu Ala Cys Val Arg Thr Leu Ala Met Val Ala Lys Ser Arg 1525 1530 1535

Ala He Leu Leu Pro Met Asp Leu Asp Ala His Met Ser Ala Leu Leu 1540 1545 1550

Ser Ser Gly Gly Ser Cys Ser Ala Ala Ala Gin Arg Ser Ala Ala Asn 1555 1560 1565

Tyr Lys Thr Ala Thr Arg Thr Phe Pro Arg Val He Pro Thr Ala Asn 1570 1575 1580

Gin Trp Asp Tyr Lys Asn He He Glu Lys Leu Gin Asp He He Thr 1585 1590 1595 1600

Ala Leu Glu Glu Arg-Leu Lys Pro Leu Val Gin Ala Glu Leu Ser Val 1605 1610 1615

Leu Val Asp Met Leu His Trp Pro Glu Leu Leu Phe Leu Glu Gly Ser 1620 1625 1630

Glu Ala Tyr Gin Arg Cys Glu Ser Gly Gly Phe Leu Ser Lys Leu He 1635 1640 1645

Arg His Thr Lys Gly Leu Met Glu Ser Glu Glu Lys Leu Cys Val Lys 1650 1655 1660

Val Leu Arg Thr Leu Gin Gin Met Leu Gin Lys Lys Ser Lys Tyr Gly 1665 1670 167S 1680

Asp Arg Gly Asn Gin Leu Arg Lys Met Leu Leu Gin Asn Tyr Leu Gin 1685 1690 1695

Asn Arg Lys Ser Gly Pro Arg Gly Glu Leu Thr Asp Pro Thr Gly Ser 1700 1705 1710

Gly Val Asp Gin Asp Trp Ser Ala He Ala Ala Thr Gin Cys Arg Leu 1715 1720 1725

Asp Lys Glu Gly Ala Thr Lys Leu Val Cys Asp Leu He Thr Ser Thr 1730 1735 1740

Lys Asn Glu Lys He Phe Gin Glu Ser He Gly Leu Ala He Arg Leu 1745 1750 1755 1760

Leu Asp Gly Gly Asn Thr Glu He Gin Lys Ser Phe Tyr Asn Leu Met 1765 1770 1775

Thr Ser Asp Lys Lys Ser Glu Arg Phe Phe Lys Val Leu His Asp Arg 1780 1785 1790

Met Lys Arg Ala Gin Gin Glu Thr Lys Ser Thr Val Ala Val Asn Met 1795 1800 1805

Ser Asp Leu Gly Ser Gin Pro Arg Glu Asp Arg Glu Pro Ala Asp Pro 1810 1815 1820

Thr Thr Lys Gly Arg Val Ser Ser Phe Ser Met Pro Ser Ser Ser Arg 1825 1830 1835 1840

Tyr Ser Leu Gly Pro Gly Leu His Arg Gly His Asp Val Ser Glu Arg 1845 1850 1855

Ala Gin Asn Asn Glu Met Gly Thr Ser Val Leu He Met Arg Pro He 1860 1865 1870

Leu Arg Phe Leu Gin Leu Leu Cys Glu Asn His Asn Arg Asp Leu Gin 1875 1880 1885

Asn Phe Leu Arg Cys Gin Asn Asn Lys Thr Asn Tyr Asn Leu Val Cys 1890 1895 1900

Glu Thr Leu Gin Phe Leu Asp He Met Cys Gly Ser Thr Thr Gly Gly 1905 1910 1915 1920

Leu Gly Leu Leu Gly Leu Tyr He Asn Glu Asp Asn Val Gly Leu Val 1925 1930 1935

He Gin Thr Leu Glu-Thr Leu Thr Glu Tyr Cys Gin Gly Pro Cys His 1940 1945 1950

Glu Asn Gin Thr Cys He Val Thr His Glu Ser Asn Gly He Asp He 1955 1960 1965

He Thr Ala Leu He Leu Asn Asp He Ser Pro Leu Cys Lys Tyr Arg 1970 1975 1980

Met Asp Leu Val Leu Gin Leu Lys Asp Asn Ala Ser Lys Leu Leu Leu 1985 1990 1995 2000

Ala Leu Met Glu Ser Arg His Asp Ser Glu Asn Ala Glu Arg He Leu 2005 2010 2015

He Ser Leu Arg Pro Gin Glu Leu Val Asp Val He Lys Lye Ala Tyr 2020 2025 2030

Leu Gin Glu Glu Glu Arg Glu Asn Ser Glu Val Ser Pro Arg Glu Val 2035 2040 2045

Gly His Asn He Tyr He Leu Ala Leu Gin Leu Ser Arg His Asn Lys 2050 2055 2060

Gin Leu Gin His Leu Leu Lys Pro Val Lys Arg He Gin Glu Glu Glu 2065 2070 2075 2080

Ala Glu Gly He Ser Ser Met Leu Ser Leu Asn Asn Lys Gin Leu Ser 2085 2090 2095

Gin Met Leu Lys Ser Ser Ala Pro Ala Gin Glu Glu Glu Glu Asp Pro 2100 2105 2110

Leu Ala Tyr Tyr Glu Asn His Thr Ser Gin He Glu He Val Arg Gin 2115 2120 2125

Asp Arg Ser Met Glu Gin He Val Phe Pro Val Pro Ala He Cys Gin 2130 2135 2140

Phe Leu Thr Glu Glu Thr Lys His Arg Leu Phe Thr Thr Thr Glu Gin 2145 2150 2155 2160

Asp Glu Gin Gly Ser Lys Val Ser Asp Phe Phe Asp Gin Ser Ser Phe 2165 2170 2175

Leu His Asn Glu Met Glu Trp Gin Arg Arg Leu Arg Ser Met Pro Leu 2180 2185 2190

He Tyr Trp Phe Ser Arg Arg Met Thr Leu Trp Gly Ser He Ser Phe 2195 2200 2205

Asn Leu Ala Val Phe He Asn He He He Ala Phe Phe Tyr Pro Tyr 2210 2215 2220

Val Glu Gly Ala Ser Thr Gly Val Leu Gly Ser Pro Leu He Ser Leu 2225 2230 2235 2240

Leu Phe Trp He Leu He Cys Phe Ser He Ala Ala Leu Phe Thr Lys 2245 2250 2255

His Tyr Ser Val Arg Pro Leu He Val Ala Leu Val Leu Arg Ser He 2260 2265 2270

Tyr Tyr Leu Gly He'Gly Pro Thr Leu Asn He Leu Gly Ala Leu Asn 2275 2280 2285

Leu Thr Asn Lys He Val Phe Val Val Ser Phe Val Gly Asn Arg Gly 2290 2295 2300

Thr Phe He Arg Gly Tyr Lys Ala Met Val Met Asp Met Glu Phe Leu 2305 2310 2315 2320

Tyr His Val Gly Tyr He Leu Thr Ser Val Leu Gly Leu Phe Ala His 2325 2330 2335

Glu Leu Phe Tyr Ser He Leu Leu Phe Asp Leu He Tyr Arg Glu Glu 2340 2345 2350

Thr Leu Phe Asn Val He Lys Ser Val Thr Arg Asn ^' Gly Arg Ser He 2355 2360 2365

Leu Leu Thr Ala Leu Leu Ala Leu He Leu Val Tyr Leu Phe Ser He 2370 2375 2380

Val Gly Phe Leu Phe Leu Lys Asp Asp Phe He Leu Glu Val Asp Arg 238S 2390 2395 2400

Leu Pro Gly Asn His Ser Arg Ala Ser Thr Leu Gly Met Pro His Gly 2405 2410 2415

Ala Ala Thr Phe Met Gly Thr Cys Ser Gly Asp Lys Met Asp Cys Val 2420 2425 2430

Ser Glu Val Ser Val Pro Glu He Leu Glu Glu Asp Glu Glu Leu Asp 2435 2440 2445

Ser Thr Glu Arg Ala Cys Asp Thr Leu Leu Met Cys He Val Thr Val 2450 2455 2460

Met Asn His Gly Leu Arg Asn Gly Gly Gly Val Gly Asp He Leu Arg 2465 2470 2475 2480

Lys Pro Ser Lys Asp Glu Ser Leu Phe Pro Ala Arg Val Val Tyr Asp 2485 2490 2495

Leu Leu Phe Phe Phe He Val He He He Val Leu Asn Leu He Phe 2500 2505 2510

Gly Val He He Asp Thr Phe Ala Asp Leu Arg Ser Glu Lys Gin Lys 2515 2S20 2S25

Lys Glu Glu He Leu Lys Thr Thr Cys Phe He Cys Gly Leu Glu Arg 2530 2535 2540

Asp Lys Phe Asp Asn Lys Thr Val Ser Phe Glu Glu His He Lys Leu 2545 2550 2555 2560

Glu His Asn Met Trp Asn Tyr Leu Tyr Phe He Val Leu Val Arg Val 2565 2570 2575

Lys Asn Lys Thr Asp Tyr Thr Gly Pro Glu Ser Tyr Val Ala Gin Met 2S80 2585 2590

He Lys Asn Lys Asn Leu Asp Trp Phe Pro Arg Met Arg Ala Met Ser 2595 2600 2605

Leu Val Ser Gly Glu'Gly Glu Gly Glu Gin Asn Glu He Arg He Leu 2610 2615 2620

Gin Glu Lys Leu Gly Ser Thr Met Lys Leu Val Ser His Leu Thr Ala 2625 2630 2635 2640

Gin Leu Asn Glu Leu Lys Glu Gin Met Thr Glu Gin Arg Lys Arg Arg 2645 2650 2655

in Arg Leu Gly Phe Val Asp Val Gin Asn Cys Met Ser Arg 2660 2665 2670

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2670 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Met Ser Glu Met Ser Ser Phe Leu His He Gly Asp He Val Ser Leu 1 5 10 15

Tyr Ala Glu Gly Ser Val Asn Gly Phe He Ser Thr Leu Gly Leu Val 20 25 30

Asp Asp Arg Cys Val Val Glu Pro Ala Ala Gly Asp Leu Asp Asn Pro 35 40 45

Pro Lys Lys Phe Arg Asp Cys Leu Phe Lys Val Cys Pro Met Asn Arg 50 55 60

Tyr Ser Ala Gin Lys Gin Tyr Trp Lys Ala Lys Gin Thr Lys Gin Asp 65 70 75 80

Lys Glu Lys He Ala Asp Val Val Leu Leu Gin Lys Leu Gin His Ala 85 90 95

Ala Gin Met Glu Gin Lys Gin Asn Asp Thr Glu Asn Lys Lys Val His 100 105 110

Gly Asp Val Val Lys Tyr Gly Ser Val He Gin Leu Leu His Met Lys 115 120 125

Ser Asn Lys Tyr Leu Thr Val Asn Lys Arg Leu Pro Ala Leu Leu Glu 130 135 140

Lys Asn Ala Met Arg Val Thr Leu Asp Ala Thr Gly Asn Glu Gly Ser 145 150 155 160

Trp Leu Phe He Gin Pro Phe Trp Lys Leu Arg Ser Asn Gly Asp Asn 165 170 17S

Val Val Val Gly Asp Lys Val He Leu Asn Pro Val Asn Ala Gly Gin 180 185 190

Pro Leu His Ala Ser Asn Tyr Glu Leu Ser Asp Asn Ala Gly Cys Lys 195 ^* 200 205

Glu Val Asn Ser Val Asn Cys Asn Thr Ser Trp Lys He Asn Leu Phe 210 215 220

Met Gin Phe Arg Asp His Leu Glu Glu Val Leu Lys Gly Gly Asp Val 225 230 235 240

Val Arg Leu Phe His Ala Glu Gin Glu Lys Phe Leu Thr Cys Asp Glu

245 250 255

Tyr Lys Gly Lys Leu Gin Val Phe Leu Arg Thr Thr Leu Arg Gin Ser 260 265 270

Ala Thr Ser Ala Thr Ser Ser Asn Ala Leu Trp Glu Val Glu Val Val 27S 280 285

His His Asp Pro Cys Arg Gly Gly Ala Gly His Trp Asn Gly Leu Tyr 290 295 300

Arg Phe Lys His Leu Ala Thr Gly Asn Tyr Leu Ala Ala Glu Glu Asn 305 310 315 320

Pro Ser Tyr Lys Gly Asp Ala Ser Asp Pro Lys Ala Ala Gly Met Gly 325 330 335

Ala Gin Gly Arg Thr Gly Arg Arg Asn Ala Gly Glu Lys He Lys Tyr 340 345 350

Cys Leu Val Ala Val Pro His Gly Asn Asp He Ala Ser Leu Phe Glu 355 360 365

Leu Asp Pro Thr Thr Leu Gin Lys Thr Asp Ser Phe Val Pro Arg Asn 370 375 380

Ser Tyr Val Arg Leu Arg His Leu Cys Thr Asn Thr Trp He Gin Ser 385 390 395 400

Thr Asn Val Pro He Asp He Glu Glu Glu Arg Pro He Arg Leu Met 405 410 415

Leu Gly Thr Cys Pro Thr Lys Glu Asp Lys Glu Ala Phe Ala He Val 420 425 430

Ser Val Pro Val Ser Glu He Arg Asp Leu Asp Phe Ala Asn Asp Ala 435 440 445

Ser Ser Met Leu Ala Ser Ala Val Glu Lys Leu Asn Glu Gly Phe He 450 455 460

Ser Gin Asn Asp Arg Arg Phe Val He Gin Leu Leu Glu Asp Leu Val 465 470 475 480

Phe Phe Val Ser Asp Val Pro Asn Asn Gly Gin Asn Val Leu Asp He 485 490 495

Met Val Thr Lys Pro Asn Arg Glu Arg Gin Lys Leu Met Arg Glu Gin 500 505 510

Asn He Leu Gin Val Phe Gly He Leu Lys Val Pro Phe Arg Glu Lys 515 520 525

Gly Gly Glu Gly Pro Leu Val Arg Leu Glu Glu Leu Ser Asp Gin Lys 530 535 540

Asn Ala Pro Tyr Gin His Met Phe Arg Leu Cys Tyr Arg Val Leu Arg 545 550 555 560

Tyr Ser Gin Glu Asp Tyr Arg Lys Asn Gin Glu His He Ala Lys Gin 565 570 575

Phe Gly Met Met Gin Ser Gin He Gly Tyr Asp He Leu Ala Glu Asp

580 585 590

Thr He Thr Ala Leu Leu His Asn Asn Arg Lys Leu Leu Glu Lys His 595 600 605

He Thr Lys Thr Glu Val Glu Thr Phe Val Ser Leu Val Arg Lys Asn 610 615 620

Arg Glu Pro Arg Phe Leu Asp Tyr Leu Ser Asp Leu Cys Val Ser Asn 625 630 635 640

His He Ala He Pro Val Thr Gin Glu Leu He Cys Lys Cys Val Leu 645 650 655

Asp Pro Lys Asn Ser Asp He Leu He Arg Thr Glu Leu Arg Pro Val 660 665 670

Lys Glu Met Ala Gin Ser His Glu Tyr Leu Ser He Glu Tyr Ser Glu 675 680 685

Glu Glu Val Trp Leu Thr Trp Thr Asp Lys Asn Asn Glu His His Glu 690 695 700

Lys Ser Val Arg Gin Leu Ala Gin Glu Ala Arg Ala Gly Asn Ala His 705 710 715 720

Asp Glu Asn Val Leu Ser Tyr Tyr Arg Tyr Gin Leu Lys Leu Phe Ala 725 730 735

Arg Met Cys Leu Asp Arg Gin Tyr Leu Ala He Asp Glu He Ser Gin 740 745 7S0

Gin Leu Gly Val Asp Leu He Phe Leu Cys Met Ala Asp Glu Met Leu 755 760 765

Pro Phe Asp Leu Arg Ala Ser Phe Cys His Leu Met Leu His Val His 770 775 780

Val Asp Arg Asp Pro Gin Glu Leu Val Thr Pro Val Lys Phe Ala Arg 785 790 795 800

Leu Trp Thr Glu He Pro Thr Ala He Thr He Lys Asp Tyr Asp Ser 805 810 815

Asn Leu Asn Ala Ser Arg Asp Asp Lys Lys Asn Lys Phe Ala Asn Thr 820 825 830

Met Glu Phe Val Glu Asp Tyr Leu Asn Asn Val Val Ser Glu Ala Val 835 840 845

Pro Phe Ala Asn Glu Glu Lys Asn Lys Leu Thr Phe Glu Val Val Ser 850 855 860

Leu Ala His Asn Leu He Tyr Phe Gly Phe Tyr Ser Phe Ser Glu Leu 865 870 875 880

Leu Arg Leu Thr Arg Thr Leu Leu Gly He He Asp Cys Val Gin Gly 885 890 895

Pro Pro Ala Met Leu Gin Ala Tyr Glu Asp Pro Gly Gly Lys Asn Val 900 905 910

Arg Arg Ser He Gin Gly Val Gly His Met Met Ser Thr Met Val Leu

915 920 925

Ser Arg Lys Gin Ser Val Phe Ser Ala Pro Ser Leu Ser Ala Gly Ala 930 935 940

Ser Ala Ala Glu Pro Leu Asp Arg Ser Lys Phe Glu Glu Asn Glu Asp 945 950 955 960

He Val Val Met Glu Thr Lys Leu Lys He Leu Glu He Leu Gin Phe 965 970 975

He Leu Asn Val Arg Leu Asp Tyr Arg He Ser Tyr Leu Leu Ser Val 980 985 990

Phe Lys Lys Glu Phe Val Glu Val Phe Pro Met Gin Asp Ser Gly Ala 995 1000 1005

Asp Gly Thr Ala Pro Ala Phe Asp Ser Thr Thr Ala Asn Met Asn Leu 1010 1015 1020

Asp Arg He Gly Glu Gin Ala Glu Ala Met Phe Gly Val Gly Lys Thr 1025 1030 1035 1040

Ser Ser Met Leu Glu Val Asp Asp Glu Gly Gly Arg Met Phe Leu Arg 1045 1050 1055

Val Leu He His Leu Thr Met His Asp Tyr Ala Pro Leu Val Ser Gly 1060 1065 1070

Ala Leu Gin Leu Leu Phe Lys His Phe Ser Gin Arg Gin Glu Ala Met 1075 1080 1085

His Thr Phe Lys Gin Val Gin Leu Leu He Ser Ala Gin Asp Val Glu 1090 1095 1100

Asn Tyr Lys Val He Lys Ser Glu Leu Asp Arg Leu Arg Thr Met Val 1105 1110 1115 1120

Glu Lys Ser Glu Leu Trp Val Asp Lys Lys Gly Ser Gly Lys Gly Glu 1125 1130 1135

Glu Val Glu Ala Gly Thr Ala Lys Asp Lys Lys Glu Arg Pro Thr Asp 1140 1145 1150

Glu Glu Gly Phe Leu His Pro Pro Gly Glu Lys Ser Ser Glu Asn Tyr 1155 1160 1165

Gin He Val Lys Gly He Leu Glu Arg Leu Asn Lys Met Cys Gly Val 1170 1175 1180

Gly Glu Gin Met Arg Lys Lys Gin Gin Arg Leu Leu Lys Asn Met Asp 1185 1190 1195 1200

Ala His Lys Val Met Leu Asp Leu Leu Gin He Pro Tyr Asp Lys Gly 1205 1210 1215

Asp Ala Lys Met Met Glu He Leu Arg Tyr Thr His Gin Phe Leu Gin 1220 1225 1230

Lys Phe Cys Ala Gly Asn Pro Gly Asn Gin Ala Leu Leu His Lys His 1235 1240 1245

Leu His Leu Phe Leu Thr Pro Gly Leu Leu Glu Ala Glu Thr Met Gin

1250 12S5 1260

His He Phe Leu Asn Asn Tyr Gin Leu Cys Ser Glu He Ser Glu Pro 1265 1270 1275 1280

Val Leu Gin His Phe Val His Leu Leu Ala Thr His Gly Arg His Val 1285 1290 1295

Gin Tyr Leu Asp Phe Leu His Thr Val He Lys Ala Glu Gly Lys Tyr 1300 1305 1310

Val Lys Lys Cys Gin Asp Met He Met Thr Glu Leu Thr Asn Ala Gly 1315 1320 1325

Asp Asp Val Val Val Phe Tyr Asn Asp Lys Ala Ser Leu Ala His Leu 1330 1335 1340

Leu Asp Met Met Lys Ala Ala Arg Asp Gly Val Glu Asp His Ser Pro 1345 1350 1355 1360

Leu Met Tyr His He Ser Leu Val Asp Leu Leu Ala Ala Cys Ala Glu 1365 1370 1375

Gly Lys Asn Val Tyr Thr Glu He Lys Cys Thr Ser Leu Val Pro Leu 1380 1385 1390

Glu Asp Val Val Ser Val Val Thr His Glu Asp Cys He Thr Glu Val 1395 1400 1405

Lys Met Ala Tyr Val Asn Phe Val Asn His Cys Tyr Val Asp Thr Glu 1410 1415 1420

Val Glu Met Lys Glu He Tyr Thr Ser Asn His He Trp Thr Leu Phe 1425 1430 1435 1440

Glu Asn Phe Thr Leu Asp Met Ala Arg Val Cys Ser Lys Arg Glu Lys 1445 1450 1455

Arg Val Ala Asp Pro Thr Leu Glu Lys Tyr Val Leu Ser Val Val Leu 1460 1465 1470

Asp Thr He Asn Ala Phe Phe Ser Ser Pro Phe Ser Glu Asn Ser Thr 1475 1480 1485

Ser Leu Gin Thr His Gin Pro Val Val Val Gin Leu Leu Gin Ser Thr 1490 1495 1500

Thr Arg Leu Leu Glu Cys Pro Trp Leu Gin Gin Gin His Lys Gly Ser 1505 1510 1515 1520

Val Glu Ala Cys He Arg Thr Leu Ala Met Val Ala Lys Gly Arg Ala 1525 1530. 1535

He Leu Leu Pro Met Asp Leu Asp Ala His He Ser Ser Met Leu Ser 1540 1545 1550

Ser Gly Ala Ser Cys Ala Ala Ala Ala Gin Arg Asn Ala Ser Ser Tyr 1555 1560 1565

Lys Ala Thr Thr Arg Ala Phe Pro Arg Val Thr Pro Thr Ala Asn Gin 1570 1575 1580

Trp Asp Tyr Lys Asn He He Glu Lys Leu Gin Asp He He Thr Ala

1585 1590 1595 1600

Leu Glu Glu Arg Leu Lys Pro Leu Val Gin Ala Glu Leu Ser Val Leu 1605 1610 1615

Val Asp Val Leu His Trp Pro Glu Leu Leu Phe Leu Glu Gly Ser Glu 1620 1625 1630

Ala Tyr Gin Arg Cys Glu Ser Gly Gly Phe Leu Ser Lys Leu He Gin 1635 1640 1645

His Thr Lys Asp Leu Met Glu Ser Glu Glu Lys Leu Cys He Lys Val 1650 1655 1660

Leu Arg Thr Leu Gin Gin Met Leu Val Lys Lys Thr Lys Tyr Gly Asp 1665 1670 1675 1680

Arg Gly Asn Gin Leu Arg Lys Met Leu Leu Gin Asn Tyr Leu Gin Asn 1685 1690 1695

Arg Lys Ser Thr Ser Arg Gly Asp Leu Pro Asp Pro He Gly Thr Gly 1700 1705 1710

Leu Asp Pro Asp Trp Ser Ala He Ala Ala Thr Gin Cys Arg Leu Asp 1715 1720 1725

Lys Glu Gly Ala Thr Lys Leu Val Cys Asp Leu He Thr Ser Thr Lys 1730 1735 1740

Asn Glu Lys He Phe Gin Glu Ser He Gly Leu Ala He His Leu Leu 1745 1750 1755 1760

Asp Gly Gly Asn Thr Glu He Gin Lys Ser Phe His Asn Leu Met Met 1765 1770 1775

Ser Asp Lys Lys Ser Glu Arg Phe Phe Lys Val Leu His Asp Arg Met 1780 1785 1790

Lys Arg Ala Gin Gin Glu Thr Lys Ser Thr Val Ala Val Asn Met Asn 1795 1800 1805

Asp Leu Gly Ser Gin Pro His Glu Asp Arg Glu Pro Val Asp Pro Thr 1810 1815 1820

Thr Lys Gly Arg Val Ala Ser Phe Ser He Pro Gly Ser Ser Ser Arg 1825 1830 1835 1840

Tyr Ser Leu Gly Pro Ser Leu Arg Arg Gly His Glu Val Ser Glu Arg 1845 1850 1855

Val Gin Ser Ser Glu Met Gly Thr Ser Val Leu He Met Gin Pro He I860 1B65 1870

Leu Arg Phe Leu Gin Leu Leu Cys Glu Asn His Asn Arg Asp Leu Gin 1875 1880 1885

Asn Phe Leu Arg Cys Gin Asn Asn Lys Thr Asn Tyr Asn Leu Val Cys 1890 1895 1900

Glu Thr Leu Gin Phe Leu Asp He Met Cys Gly Ser Thr Thr Gly Gly 1905 1910 1915 1920

Leu Gly Leu Leu Gly Leu Tyr He Asn Glu Asp Asn Val Gly Leu Val

1925 1930 1935

He Gin Thr Leu Glu Thr Leu Thr Glu Tyr Cys Gin Gly Pro Cys His 1940 1945 1950

Glu Asn Gin Thr Cys He Val Thr His Glu Ser Asn Gly He Asp He 1955 1960 1965

He Thr Ala Leu He Leu Asn Asp He Ser Pro Leu Cys Lys Tyr Arg 1970 1975 1980

Met Asp Leu Val Leu Gin Leu Lys Asp Asn Ala Ser Lys Leu Leu Leu 1985 1990 1995 2000

Ala Leu Met Glu Ser Arg His Asp Ser Glu Asn Ala Glu Arg He Leu 2005 2010 2015

He Ser Leu Arg Pro Gin Glu Leu Val Asp Val He Lys Lys Ala Tyr 2020 2025 2030

Leu Gin Glu Glu Glu Arg Glu Asn Ser Glu Val Ser Pro Arg Glu Val 2035 2040 2045

Gly His Asn He Tyr He Leu Ala Leu Gin Leu Ser Arg His Asn Lys 2050 2055 2060

Gin Leu Gin His Leu Leu Lys Pro Val Lys Arg He Gin Glu Glu Glu 2065 2070 2075 2080

Ala Glu Gly He Ser Ser Met Leu Ser Leu Asn Asn Lys Gin Leu Ser 2085 2090 2095

Gin Met Leu Lys Ser Ser Ala Pro Ala Gin Glu Glu Glu Glu Asp Pro 2100 2105 2110

Leu Ala Tyr Tyr Glu Asn His Thr Ser Gin He Glu He Val Arg Gin 2115 2120 2125

Asp Arg Ser Met Glu Gin He Val Phe Pro Val Pro Gly He Cys Gin 2130 2135 2140

Phe Leu Thr Glu Glu Thr Lys His Arg Leu Phe Thr Thr Thr Glu Gin 2145 2150 2155 2160

Asp Glu Gin Gly Ser Lys Val Ser Asp Phe Phe Asp Gin Ser Ser Phe 2165 2170 2175

Leu His Asn Glu Met Glu Trp Gin Arg Asn Val Arg Ser Met Pro Leu 2180 2185 2190

He Tyr Trp Phe Ser Arg Arg Met Thr Leu Trp Gly Ser He Ser Phe 2195 2200 2205

Asn Leu Ala Val Phe He Asn He He He Ala Phe Phe Tyr Pro Tyr 2210 2215 2220

Met Glu Gly Ala Ser Thr Gly Val Leu Asp Ser Pro Leu He Ser Leu 2225 2230 2235 2240

Leu Phe Trp He Leu He Cys Phe Ser He Ala Ala Leu Phe Thr Lys 2245 2250 2255

Arg Tyr Ser He Arg Pro Leu He Val Ala Leu He Leu Arg Ser He

2260 2265 2270

Tyr Tyr Leu Gly He Gly Pro Thr Leu Asn He Leu Gly Ala Leu Asn 2275 2280 2285

Leu Thr Asn Lys He Val Phe Val Val Ser Phe Val Gly Asn Arg Gly 2290 2295 2300

Thr Phe He Arg Gly Tyr Lys Ala Met Val Met Asp Met Glu Phe Leu 2305 2310 2315 2320

Tyr His Val Gly Tyr He Leu Thr Ser Val Leu Gly Leu Phe Ala His 2325 2330 2335

Glu Leu Phe Tyr Ser He Leu Leu Phe Asp Leu He Tyr Arg Glu Glu 2340 2345 2350

Thr Leu Phe Asn Val He Lys Ser Val Thr Arg Asn Gly Arg Ser He 2355 2360 2365

Leu Leu Thr Ala Leu Leu Ala Leu He Leu Val Tyr Leu Phe Ser He 2370 2375 2380

Val Gly Phe Leu Phe Leu Lys Asp Asp Phe He Leu Glu Val Asp Arg 2385 2390 2395 2400

Leu Pro Asn Asn His Ser Thr Ala Ser Pro Leu Gly Met Pro His Gly 2405 2410 2415

Ala Ala Ala Phe Val Asp Thr Cys Ser Gly Asp Lys Met Asp Cys Val 2420 2425 2430

Ser Gly Leu Ser Val Pro Glu Val Leu Glu Glu Asp Arg Glu Leu Asp 2435 2440 2445

Ser Thr Glu Arg Ala Cys Asp Thr Leu Leu Met Cys He Val Thr Val 2450 2455 2460

Met Asn His Gly Leu Arg Asn Gly Gly Gly Val Gly Asp He Leu Arg 2465 2470 2475 2480

Lys Pro Ser Lys Asp Glu Ser Leu Phe Pro Ala Arg Val Val Tyr Asp 2485 2490 2495

Leu Leu Phe Phe Phe He Val He He He Val Leu Asn Leu He Phe 2500 2505 2510

Gly Val He He Asp Thr Phe Ala Asp Leu Arg Ser Glu Lys Gin Lys 2515 2520 2525

Lys Glu Glu He Leu Lys Thr Thr Cys Phe He Cys Gly Leu Glu Arg 2530 2535 2540

Asp Lys Phe Asp Asn Lys Thr Val Ser Phe Glu Glu His He Lys Leu 2545 2550 2555 2S60

Glu His Asn Met Trp Asn Tyr Leu Tyr Phe He Val Leu Val Arg Val 2565 2570 2575

Lys Asn Lys Thr Asp Tyr Thr Gly Pro Glu Ser Tyr Val Ala Gin Met 2580 2585 2590

He Lys Asn Lys Asn Leu Asp Trp Phe Pro Arg Met Arg Ala Met Ser

2595 2600 2605

Leu Val Ser Asn Glu Gly Glu Gly Glu Gin Asn Glu He Arg He Leu 2610 2615 2620

Gin Asp Lys Leu Asn Ser Thr Met Lys Leu Val Ser His Leu Thr Ala 2625 2630 2635 2640

Gin Leu Asn Glu Leu Lys Glu Gin Met Thr Glu Gin Arg Lys Arg Arg 2645 2650 2655

Gin Arg Leu Gly Phe Val Asp Val Gin Asn Cys He Ser Arg 2660 2665 2670