CASPR/pl90, A FUNCTIONAL LlβAND FOR RPTP-BETA AND THE AXONAL CELL RECOGNITION MOLECULE CONTACTIN

The present application claims priority under 35 U.S.C.

§ 119(e) to provisional application serial No. 60/014,199,

5 filed March 27, 1996, the entire contents of which is incorporated herein by reference in its entirety.

1. INTRODUCTION

The present invention relates to the 190 Kd neuronal protein (hereinafter ^M pl90", "CASPR" or "CASPR/pl90") that interacts with contactin, and with the carbonic anhydrase ("CAH") domain of the receptor-type tyrosine phosphatase RPTP-3, specific peptides thereof and nucleic acid molecules encoding such pl90 proteins and peptides. The protein is

,- also referred to as CASPR, for Contactin Associated PRotein. The CAH domain of RPTPjS has previously been identified as a ligand for contactin, and the binding of the CAH domain of RPTP3 to the contactin on neural cells results in neurite growth, differentiation and survival. CASPR/pl90 has been

₂₀ identified as a potential bridge that couples contactin, a GPI-linked protein, with intracellular second messenger systems. The invention also relates to compounds that mimic, enhance, or suppress the effects of pl90, including those molecules which act downstream in the signal transduction

₂ _ pathway that results from the binding of the ligand to contactin. In addition, the invention also relates to the use of such compounds to treat neurologic diseases including those characterized by insufficient, aberrant, or excessive neurite growth, differentiation or survival.

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2. BACKGROUND OF THE INVENTION The ability of cells to respond to signals from their microenvironment is a fundamental feature of development. In the developing nervous system, neurons migrate and extend axons to establish their intricate network of synaptic connections (Goodman and Shatz, 1993, Cell/Neuron (Suppl.),

72/10:77-98) . During migration and axonal pathfinding, cells are guided by both attractive and repulsive signals (Hynes and Lander, 1992, Cell, 68:303-322; Keynes and Cook, 1992, Lurr. Opin. Neurobiol., 2:55-59). The ability of the neuron to respond to these signals requires cell surface molecules that are able to receive the signal and to transmit it to the cell interior resulting in specific biological responses.

It is well established that protein tyrosine phosphorylation is responsible for the regulation of many cellular responses to external stimuli crucial for cell growth, proliferation and differentiation (Schlessinger and Ullrich, 1992, Neuron, 9:383-391). Tyrosine phosphorylation has been implicated in several developmental processes in the nervous system. For example, receptor tyrosine kinases were shown to effect neuronal survival (Chao, 1992, Neuron, 9:583- 593), and cell fate determination (Zipursky and Rubin, 1994, Annu. Rev. Neurosci., 17:373-397). Non-receptor tyrosine kinases have been shown to be downstream elements in signaling via cell recognition molecules that play a role in cell guidance and migration (Ignelzi et al., 1994, Neuron, 12:873-884; Umemori et al., 1994, Nature, 367-572-586).

The transient nature of signaling by phosphorylation requires specific phosphatases for control and regulation (Hunter, 1995, Cell, 80:225-236). Indeed, many protein tyrosine phosphatases have been shown to be expressed in specific regions of the developing brain, including the olfactory neuroepithelium (Walton et al., 1993, Neuron, 11:387-400), the cortex (Sahin et al., 1995, J. Comp. Neurol., 351:617-631), and in retinal Muller glia (Shock et al., 1995, Mol. Brain Res., 28:110-116). Furthermore, expression of several tyrosine phosphatases, such as PTPα (den Hertog et al., 1993, EMBO J. , 12:3789-3798), PC12-PTP1 (Sharama and Lombroso, 1995, J. Biol. Chem., 270:49-53) and several forms of LAR (Zhang and Longo, 1995, J. Cell. Biol., 128:415-431) have been found to be regulated during neural differentiation of P19 or PC12 cells.

Receptor-type tyrosine phosphatases (RPTPs) have been subdivided into several groups based on structural characteristics of their extracellular domains (Charbonneau and Tonkε, 1992, Annu. Rev. Cell Biol., 8:463-493; Barnea et al., 1993, Mol. Cell. Biol., 13:1497-1506). RPTPø/f and RPTP7 are members of a distinct group of phosphatases, characterized by the presence of a carbonic anhydrase-like domains (CAH) , fibronectin type III repeats (FNIII) , and a long cysteine free region (spacer domain) in their extracellular domain (Barnea et al. , 1993, Mol. Cell. Biol., 13:1497-1506; Krueger et al., 1992, Proc. Natl. Acad. Sci. USA, 89:7417-7421; Levy et al., 1993, J. Biol. Chem., 268:10573-10581). The expression of RPTPjS is restricted to the central and peripheral nervous system, while RPTPγ is expressed both in the developing nervous system, as well as, in a variety of other tissues in adult rat (Canoll et al.,

1993, Dev. Brain Res., 75:293-298; Barnea et al., 1993, Mol. Cell. Biol., 13:1497-1506). RPTP3 exists in three forms, one secreted form and two membrane bound forms, that differ by the absence of 860 residues from the spacer domain (Levy et al. , 1993, J. Biol. Chem., 268:1053-10582; Maurel et al.,

1994, Proc. Natl. Acad. Sci. USA, 91:2512-2516). The secreted form has been identified as a chondroitin sulfate proteoglycan from rat brain called phosphocan (3F8 proteoglycan) (Barnea et al., 1994, Cell, 76:205; Maurel et al., 1994, Proc. Natl. Acad. Sci. USA, 91:2512-2516; Shitara et al., 1994, J. Biol. Chem. 269:20189-20193). The transmembrane form has also been shown to be expressed in a form of a chondroitin sulfate proteoglycan (Barnea et al., 1994, J. Biol. Chem., 269:14349-14352). Purified phosphocan can interact in vitro with the extracellular matrix protein tenascin, and with the adhesion molecules, N-CAM and Ng-CAM (Barnea et al., 1994, J. Biol. Chem., 269:14349-14352; Grumet et al., 1993, J. Cell. Biol., 120:815-824; Grumet et al., 1994, J. Biol. Chem., 269:12142-12146; Milev et al., 1994, J. Cell. Biol., 127:2512-2516).

3. SUMMARY OF THE INVENTION The present invention relates to the 190 Kd neuronal protein (hereinafter "pl90 ^M , "CASPR" or "CASPR/pl90") that interacts with contactin, and with the carbonic anhydrase ("CAH") domain of the receptor-type tyrosine phosphatase RPTP-/3, specific peptides thereof and nucleic acid molecules encoding such pl90 proteins and peptides.

The invention further relates to the use of pl90 and related compounds to treat neurologic diseases including those characterized by insufficient, aberrant, or excessive neurite growth, differentiation or survival. More specifically, the invention relates to the use of compounds that mimic, enhance or suppress the effects of pl90 on neurite growth, differentiation and survival. The invention is based, in part, on the discovery that the CAH domain of RPTP0 (RPTP0-CAH) is the ligand for contactin and that its binding results in neurite growth, differentiation and survival, and on the further discovery that pl90 acts as the bridge between contactin and intracellular second messenger systems.

In the examples described infra , it is shown that receptor phosphatase RPTP(8 specifically interacts with two ligands, one on the surface of glial cells, and the other on the surface of neuronal cells. Using expression cloning in COS7 cells and bioaffinity purification, the neuronal ligand was identified to be the rat homologue of the cell recognition molecule contactin (F11/F3) . Using combinations of soluble and membrane bound forms of RPTPS and contactin it is demonstrated that the reciprocal interaction between the two molecules is mediated by the CAH domain of the phosphatase. Moreover, it is found that when used as a substrate, the CAH domain of RPTP3 induced neurite growth, differentiation and survival of primary neurons and IMR-32 neuroblastoma cells. Using antibody perturbation experiments, the contactin ligand was found to be a neuronal receptor for the CAH domain of RPTP/S. The data indicate that

the interactions between contactin, a cell recognition molecule, and RPTBjS, a transmembrane protein tyrosine phosphatase, plays an important role in neuronal development and differentiation. As explained more fully in Section 5.2, the further experiments of the examples were conducted to elucidate the interaction between contactin and intracellular second messenger systems. Binding experiments revealed that the interaction between pl90 and contactin is important for the role of contactin and RPTP/3-CAH in neuronal growth, development and differentiation.

3.1. DEFINITIONS As used herein, the following terms and abbreviations shall have the meanings indicated below:

Table 1

base pair(s) bp carbonic anhydrase CAH carbonic anhydrase domain of RPTP/3 RPTPS complementary DNA cDNA counts per minute cpm deoxyribonucleic acid DNA fibronectin type III FNIII glycosyl-phosphatedylinositol GPI kilobase pairs kb kilodation kDa micrograms μg micrometer μm nanograms ng nanometer nm nucleotide nt phospholipase C PI-PLC polyacrylamide gel electrophoresis PAGE polymerase chain reaction PCR receptor type tyrosine phosphatase beta RPTP/8 ribonucleic acid RNA sodium dodecyl sulfate SDS units u

As used herein, the word "modulate" shall have its usual meaning, but shall also encompaβs the meanings of the words enhance, inhibit, and mimic. In addition, as used herein, the word "expression", when used in connection with a gene such as pl90, shall have its usual meaning, but shall also encompass the transcription of the gene, the longevity of the functional mRNA transcribed from the gene, the translation of that mRNA, and the activity of the gene product.

4. DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the homology between human and rat CASPR/p190 proteins. Also shown are the important domains of the proteins as more fully described infra at Section 5.3.

5. DETAILED DESCRIPTION OF THE INVENTION

A large group of protein tyrosine phosphatases have structural characteristics suggesting that they function as cell surface receptors. Receptor type tyrosine phosphatase β (RPTP/S) is expressed in the developing nervous system and it contains a carbonic anhydrase (CAH) domain as well as a fibronectin type III (FNIII) repeat in its extracellular domain. A variety of experiments were conducted to search for ligands of RPTP/3. These experiments led to the surprising recognition that the CAH domain of RPTP/8 is a functional ligand for contactin, a GPI-membrane anchored neuronal cell recognition molecule that functions as a receptor on neurons. The CAH domain of RPTP/S (RPTP/8-CAH) induces cell adhesion and neurite growth of primary tectal neurons, and differentiation of neuroblastoma cells. Further experiments led to the recognition that the interaction between pl90 and contactin is important in mediating the effects of contactin and RPTP0-CAH. The assays of the invention identify compounds that mimic, enhance, or inhibit the pl90 mediated effects of contactin/RPTP/3-CAH on neural cells including, but not limited to, agonists and antagonists of contactin/RPTP/3-CAH. Therapeutic uses of compounds so identified are also provided. The invention is described in

detail in the following subsections and examples for purposes of clarity and not by way of limitation.

5.1. BIOLOGY OF THE INTERACTION BETWEEN

CONTACTIN AND THE CAH DOMAIN OF RPTPθ

During development of the nervous systems, neurons are guided by secreted and cell bound molecules that provide both negative and positive cues. The experiments described in the examples of Sections 6.1 and 6.2 show that RPTP/3, a receptor type protein tyrosine phosphatase, may provide such a signal by interacting with the axonal recognition molecule contactin. RPTP/S is a developmentally regulated protein that exists in three forms, one secreted and two membrane bound.

The extracellular region of RPTP/S has a multidomain structure consisting of a CAH-like domain, a single FNIII repeat, and a long cysteine free spacer region. The complex structural nature of its extracellular region may result in a multifunctional protein that is able to interact with different proteins. As documented by the data shown herein, the CAH and the FNIII domains bind to at least two potential ligands present on neurons or glial cells. Functional expression cloning in COS7 cells and affinity purification with a specific affinity matrix followed by microsequencing enabled unequivocal identification of the cell recognition molecule contactin (F3/F11) as a neuronal ligand of RPTP/S.

The interaction between contactin and RPTP/3 is mediated via the CAH domain of the phosphatase, while the FNIII domain appears to bind to another molecule expressed on the surface of glial cells. It was previously shown that the secreted proteoglycan form of RPTP _/ S interacts with tenascin, N-CAM and

Ng-CAM (Grumet et al. , 1994, J. Biol. Chem., 269:12142-12146;

Barnea et al., 1994, J. Biol. Chem., 269:14349-14352; Grumet et al., 1993, J. Cell. Biol., 120:815-724; Milev et al.,

1994, J. Cell. Biol., 127:1703-1715). Since N-CAM and Ng-CAM do not bind directly to the CAH or the FNIII domain of RPTP/S, they may interact with the large spacer domain of the phosphatase. Alternatively, they could interact with RPTPS

through a third component. Contactin may fulfill this function since it has been shown to interact with Ng-CAM, Nr- CAM, and the matrix proteins tenascin and restriction (Brtimmendorf et al., 1993, Neuron, 10:711-727; Morales et al., 1993, Neuron, 11:1113-1122; Zisch et al., 1992, J. Cell. Biol., 119:203-213). The various subdomains of the extracellular region of RPTP/3 are able to interact with several distinct proteins that are expressed on diverse cell types in the central nervous system. In contrast to other cell recognition molecules that are widely expressed in the nervous system, members of the contactin subgroup appear to be expressed in a restricted manner on specific axons during development (Dodd et al., 1988, Neuron, 1:105-116; Faivre-Sarrailh et al., 1992, J. Neurosci., 12:257-267). The spatial and temporal expression pattern of these proteins indicates they play an important role during development of the nervous system. Contactin was found to be exclusively expressed on neurons during development in fiber-rich areas of the retina, tectum, spinal cord and cerebellum (Ranscht, 1988, J. Cell. Biol, 107:1561- 1573) . It was found to be localized in the postnatal and adult mouse cerebellum in axonal extensions of the granule cells in the parallel layer (Faivre-Sarrailh et al., 1992, J. Neurosci., 12:257-267). This pattern of expression is overlapping with the expression pattern of RPTP/3 in the rat. RPTP/3 was shown to be expressed in fiber-rich regions such as the parallel fibers of the cerebellum and the spinal cord (Canoll et al., 1993, Dev. Brain Res., 75:293-298; Milev et al., 1994, J. Cell. Biol., 127:1703-1715). RPTP/3 is also expressed on glial and radial glial cells, and its secreted form is produced by astrocytes. Therefore, both forms of RPTP/3 may modulate neuronal function via interactions with contactin.

The contactin subgroup of glycoproteins all share structural similarity in that they are, glycosyl¬ phosphatidylinositol (GPI)-anchored proteins. They also exist in soluble forms generated aε a result of membrane

release or by expression of alternative spliced forms (Briimmendorf and Rathjen, 1993, J. Neurochem., 61:1207-1219). Differential expression of the membrane-bound and soluble forms of contactin was found in the hypothalamus-hypophyseal system (Rougon et al., 1994, Braz. J. Med. Biol. Res., 2:409- 414) . RPTP/S also exists in either membrane bound or secreted forms that are developmentally regulated. Therefore, both RPTP/S and contactin may act as either a ligand or a receptor for each other. Hence, the classical notion of ligand receptor interaction does not fully explain this system since both components might switch roles at different stages of development. For example, the soluble form of RPTP/S produced by glial cells may act as a ligand for the membrane bound form of contactin expressed on the surface of neuronal cells. Conversely, the soluble form of contactin may act aε ligand for the membrane bound form of RPTP/3 expressed on the surface of glial cells. Moreover, interaction between the membrane bound forms of contactin expressed on the surface of neurons with the membrane form of RPTP/3 expressed on the surface of glial cells may lead to bidirectional signals between these two cell types. Such complex interactions between the various forms of RPTP/3 and contactin may generate developmentally regulated unidirectional and bidirectional signals. While not being limited to any theory or explanation of how the invention works, the following is hypothesized to explain how the CAH domain of RPTB/3 binds to contactin. Carbonic anhydrases are highly efficient enzymes that catalyze the hydration of C0 ₂ . Yet, the CAH domain of PTPases were not thought to be endowed with enzymatic activity due to substitution of two of the three key histidine residues that are esεential for enzymatic activity (Barnea et al., 1993, Mol. Cell. Biol., 13:1497-1506). In contradistinction, the highly packed hydrophobic core as well as the hydrophobic residues that are exposed on the surface of carbonic anhydrase structure and which are conserved in the CAH domains of RPTP7 and /S may be involved in protein-protein

interaction and thus function as a ligand binding domain (Barnea et al., 1993, Mol. Cell. Biol., 13:1497-1506). It is of note that Vaccinia virus contains a transmembrane protein with a CAH-like domain in its extracellular domain, which waε thought to be involved in binding of the virion to host proteins (Maa et al., 1990, J. Biol. Chem., 265: 1669-1577). Therefore, in theory but not by way of limitation, compounds exhibiting effects which mimic, enhance, or inhibit the contactin mediated effects of RPTP/3-CAH on neuronal cells may do so via other members of the contactin family of glycoproteins, and may do so even if lacking in CAH activity. A number of models may be proposed for how contactin, a GPI-linked protein that is inserted into the outer leaflet of the plasma membrane, transmits a signal into the cells to promote neurite outgrowth. In theory, and not by way of limitation, one possibility is that contactin is able to interact with a transmembrane signaling component. The pl90 (also referred to as pl80) protein that was coprecipitated with contactin has been a candidate for such a signaling protein. pl90 may be membrane-associated since it may not be released by phospholipase C treatment. Another potential signal transducer may be Ll/Ng-CAM or a related molecule. This transmembrane CAM was shown to interact with contactin (Brummendorf et al., 1993, Neuron, 10:711-727), and to initiate second messenger cascade via its cytoplasmic domain (Doherty and Walsh, 1994, Curr. Opin. Neurobiol., 4:49-55). The best characterized GPI linked signaling protein is the ciliary neurotrophic factor receptor (CNTF receptor) . Following ligand binding, the CNTFR interacts with the signal transducer gpl30. The gpl30 protein that is shared by several lymphokines and cytokineε εuch aε IL-6, LIF and Oncostatin, undergoeε dimerization followed by recruitment of the cytoplasmic Jak protein tyrosine kinases. Stimulation of the Jak kinases leads to activation of both the Ras/MAP kinase and the Stat signaling pathways that relay signals from the cell surface to the nucleus. A contactin associated

protein such as pl90 may have a function εimilar to the function of gpl30.

Aε demonstrated by the examples infra , the binding of the CAH domain of RPTP/S to contactin leads to cell adheεion and neurite outgrowth. It seems unlikely that the induction of neurite growth iε a default reεponse resulting from cell adhesion per se . Neurons were found to adhere to extracellular matrix proteins such as tenascin and restriction in short term binding assays, but these substrates did not promote further neurite extension

(Schachner et al., 1994, Perβpect. Dev. Neurobiol., 1:33-41). It waε recently reported that the FNIII domain of contactin iε reεponsible for adhesion, while the neurite promoting activity was attributed to the Ig domains (Durbec et al., 1994, Eur. J. Neuro. , 6:461-472). Another εtudy demonεtrated that contactin can mediate the repulsion of neurons by restriction (Pesheva et al., 1993, Neuron, 10:69-82). Again, this effect was proposed to occur in a stepwise manner, first an adhesion step that was followed by a signal that was transduced to the cellε leading to retraction. Therefore, in light of the reεultε presented herein, it may be that in response to different stimuli, the εame molecule can tranεmit opposite signals depending on the context or milieu. Whatever the mechanism, the results presented here demonstrate that a receptor type tyrosine phosphataεe serves as a functional ligand for a GPI-anchored cell adhesion molecule.

Contactin may also serve as a functional ligand for RPTP/S. Modulation of phosphataεe activity by neuronal contactin may result in εignaling to glial cellε. If this does occur, this kind of bidirectional flow of information should allow the interacting cellε to respond quickly to local environmental changes during development. Two other receptor type tyrosine phosphatases RPTPμ and RPTPK were εhown to mediate cell-cell interaction in a hemophilic manner (Brady-Kalany et al., 1993, J. Cell. Biol., 122:961-972; Gebbink et al., 1993, J. Biol. Chem., 268:16101-16104; Sap et

al., 1994, Mol. Cell. Biol., 14:1-9). However, changes in catalytic activity as a result of these interactions could not be detected. These phosphatases are joining a growing family of proteins that are involved in cellular recognition that contain intrinsic enzymatic activities, including kinaεes (Dtrk; Pulido et al., 1992), EMBO J. , 11:391-404, β subunit of Na ⁺ , K ⁺ -ATPase (AMOG; Gloor et al. , 1990, J. Cell. Biol., 109:755-788), and β subunit of prolyl 4-hydroxylase (cognin; Rao and Hausman, 1993, Proc. Natl. Acad. Sci. USA, 90:2950-2954) .

In summary, the experiments and data described herein demonstrate that RPTP/8 is a functional ligand for the GPI- anchored cell recognition molecule contactin. The interactions between these two proteins is mediated by the CAH domain of the phosphatase. In addition, the FNIII of RPTP/S repeat is required for interaction with glia cells, demonstrating that the multidomain structure of RPTP/S enableε interactionε with different proteins, and indicates that other potential ligands may modulate these interactions.

5.2 BIOLOGY OF THE P190 INTERACTION

Applicants have discovered that contactin functionally interacts with pl90, a novel mammalian protein described herein. In light of thiε information, pl90 may play an important role as the link between contactin mediated neurite growth, differentiation and survival and the intracellular second messenger signalling responεible for this contactin mediated effect.

Cell recognition molecules that contain immunoglobulin (Ig)-like domains and fibronectin type III repeats (FNIII) mediate the interaction of neurons with their local environment during development (Edel an et al., 1991, Annu. Rev. Biochem., 60:155-190; Rathjen et al., 1991, Semin. Neurosci., 3:297-307; Sonderegger et al., 1991, J. Cell. Biol., 119:1387-1394). Baεed on εtructural εimilarity they are εubdivided to three groupε. The first is represented by NCAM that exist in several alternatively spliced forms

(Cunningham et al., 1987, Science, 236:799-805). The εecond iε the Ll/Ng-CAM subgroup that alεo contains Nr-CAM and Neurofaεcin (Grumet, 1992, J. Neuroεci. Reε., 31:1-13). The third group containε contactin and its mouse and chicken ho ologueε F3 and Fll (Ranscht, 1988, J. Cell. Biol.,

107:1561-1573; Brum endorf et al., 1989, Neuron, 2:1351-1361; Gennarini et al. , 1989, J. Cell. Biol., 109:755-788; Reid et al., 1994, Brain Reε. Mol. Brain Reε., 21:1-8; Berglund et al., 1994, Genomicε, 21:571-582), TAG-1 and itε chick and human homologueε Axonin 1 and TAX-1 (Furley et al., 1990, Cell 61:157-170; Hasler et al., 1993, Eur. J. Biochem., 211:329-339; Zuellig et al., 1992, Eur. J. Biochem., 204:453- 463) and BIG-l (Yoshihara et al., 1994, Neuron, 13:415-426). The glycoproteinε from the contactin subgroup are all glycosylphosphatydylinositol (GPI)-anchored proteins composed of six C2 type Ig-like domains and four fibronectin type III repeats. They can also be found as secreted proteins as a result of membrane release and shedding or by alternative splicing that generate soluble forms (Brummendorf et al., 1993, J. Neurochem., 61:1207-1219). In contrast to other cell recognition molecules that are widely expressed in the nervous system, members of the contactin subgroup are expressed in a more restricted manner on specific axons during development (Dodd et al., 1985, Neuron, 1:105-116; Faivre-Sarrailh et al., 1992, J. Neurosci., 12:257-267;

Yoshihara et al., 1994, Neuron, 13:415-426). This spatial and temporal expresεion pattern suggests that they play a key role during axonal guidance and synapse formation.

Contactin interacts with other members of the Ig superfamily and with extracellular matrix components. Direct interaction was demonstrated between contactin and NgCAM and NrCAM, the extracellular matrix proteins tenascin and restrictin and with the carbonic anhydraεe domain of the receptor type tyrosine phosphataεe β (RPTP/S) (Briimmendorf et al, 1993, Neuron, 10:711-727; Ziεch et al., 1992, J. Cell. Biol., 119:203-213; Zisch et al., 1992, J. Mol. Neurosci.; Pesheva et al., 1993, Neuron, 10:69-82; Peles et al., 1995,

Cell, 82:251-260). These interactions are mediated by different Ig-like domains, the first and second domains bind to tenascin and Ng-CAM while the second and third mediate its interaction with restrictin (Zisch et al., 1992, J. cell. Biol., 119:203-213; Zisch et al., 1992, J. Mol. Neurosci.; Briimmendorf et al, 1993, Neuron, 10:711-727.). Moreover, contactin has been shown to be involved in both positive and negative responses of neurons to various stimuli (Peles et al., 1995, Cell, 82:251-260; Pesheva et al., 1993, Neuron, 10:69-82). When presented aε a ligand to neurons, either aε a membrane-bound or a εoluble form, contactin induceε axonal growth (Clarke et al., 1993, Eur. J. Cell. Biol., 61:108-115; Durbec et al., 1992, J. Cell. Biol., 117:877-887; Gennarini et al., 1989, J. Cell. Biol., 109:755-788). Itε neuronal receptor has been identified aε the recognition molecule Nr- CAM (Morales et al., 1993, Neuron, 11:1113-1122). Contactin itself functions as a receptor present on neurons. It mediates their repulsion by the extracellular matrix protein restrictin and neurite outgrowth induced by the CAH domain of RPTP/3 (Pesheva et al., 1993, Neuron, 10:69-82; Peles et al., 1995, Cell, 82:251-260). Thus, depending on the cellular context and ligand, contactin can mediate two opposite cellular responses (e.g. repulsion vs. adhesion and outgrowth) . The function of cell recognition molecules involves two stages, first an adhesion step and then a signal transduction step. Signaling by these molecules has been shown to utilize different second messenger systemε including GTP-binding proteinε, calcium influx and tyroεine kinases (Reviewed in Doherty et al., 1994, Curr. Opin. Neurobiol., 4:49-55). Non- receptor tyrosine kinases of the src family connect different external signals with intracellular signaling pathways. They are highly expresεed in developing neurons and are enriched in the nerve growth cones (Bare et al., 1993, Oncogene, 8:1429-1436; Maness et al., 1994, J. Biol. Chem., 193:5001- 5005; Sudol et al., 1988, Oncogene Res., 2:345-355.). There iε increasing evidence that links these kinases to signaling

pathways that are utilized by neural cell recognition molecules. Recently, the potential role for Src and Fyn kinaseε aε a downstream component in Ll and N-CAM signaling was demonstrated using cerebellar neurons from src and fyn- knockout mice (Beggs et al. , 1993, J. Cell Biol. 127:825-833; Ignelzi et al., 1994, Neuron, 12:873-884). In addition, activation of Fyn by the cell adhesion molecule MAG in oligodendrocytes was implicated as a regulatory signaling event during myelination (Umemori et al. , 1994, Nature, 367:572-576). Finally, Fyn has been shown to associate with contactin in mouse cerebellum and in chick neurons in culture (Olive et al., 1995, J. Neurochem., 65; Zisch et al., 1992, J. Cell. Biol., 119:203-213; Zisch et al. , 1992, J. Mol. Neurosci. The method by which contactin, a GPI-linked protein, associates with a cytoplasmic kinases is unclear. One possibility is that contactin interacts with a transmembrane protein that acts as a "bridge" to the cell interior.

The experiments described herein by the Examples of Section 8 describe the cloning of such candidate molecules termed CASPR/pl90 (for Contactin Associated PRotein) . These 190 kDa proteins are found in a complex with contactin and the CAH domain of RPTP/S, but only when both pl90 and RPTP/S are present on the same surface of the membrane. The cytoplasmic tail of CASPR/pl90 contains a proline rich sequence that interacts with the SH3 domain of Src family kinaεeε. Therefore this molecule could be a potential bridge that couples contactin, a GPI-linked protein, with intracellular second mesεenger systems.

5.3 MAMMALIAN P190 GENES AND GENE PRODUCTS The present invention includes, but is not limited to CASPR/pl90 peptides, polypeptides, polypeptide fragments and fusion proteins as described herein. The present invention further includes CASPR/pl90 nucleic acid molecules are described herein.

In one embodiment, such CASPR/pl90 genes and gene products are mammalian, preferably human or rodent, genes and gene products. In another embodiment, such genes and gene products are naturally occurring genes and gene products. The purification and sequencing of pl90 protein and the cloning of mammalian pl90 cDNA may be conducted aε deεcribed for human and rat pl90 cDNA in the Examples of Section 8.

The human and rat CASPR/pl90 transcripts have open reading frames that encode for 1384 and 1381 amino acids, respectively, and share 93% identity at the amino acid level. CASPR/pl90 iε a putative type I transmembrane protein with a short proline-rich cytoplasmic domain. (The transmembrane domain iε marked as TMD in Figure 1) .

A description of the CASPR/pl90 gene product follows. Such CASPR/pl90 gene products include, but are not limited to gene products containing the amino acid sequence depicted in SEQ ID NOS:2 or 4, or the amino acid sequence of at least one of the domainε depicted in SEQ ID NOS:2 or 4 and/or aε depicted in Figure 1 and/or aε deεcribed below. The firεt CASPR/pl90 methionine iε followed by a stretch of 19-20 amino acid residues rich in hydrophobic residues, which probably acts as a εignal sequence. The extracellular domainε of rat and human CASPR/pl90 contain 1281 and 1282 amino acid residues, respectively. The extracellular region of CASPR/pl90 contains 16 potential N-linked glycosylation sites followed by a second hydrophobic stretch that is a typical transmembrane domain.

The CASPR/pl90 extracellular domain is a novel mosaic of several motifs that to mediate protein-protein interactions. Near the N-terminus of mature CASPR/pl90 (109 amino acid residueε) iε a domain with 31-33% amino acid identity to the Cl and C2 terminal domains of coagulation factors V and VIII, 26% identity with the neuronal adhesion molecule neurophilin (previously known as the neuronal A5 antigen) and 20% identity to a region of discoidin I, a lectin from the slime mold Dictyosteliu diεcoideum. The domain iε marked aε DISC in Figure l.

The extracellular domain of CASPR/pl90 also contains four repeats, of approximately 140 amino acid residues each, with homology to neurexinε, a family of polymorphic neuronal cell εurface proteins. These domains are marked as NX1-NX4 in Figure 1.

CASPR/pl90 also contains two epidermal growth factor (EGF)-like modules (marked as EGF1-EGF2 in Figure 1).

A single domain related to the C-terminal region of fibrinogen beta/gamma (marked as FIB in Figure 1) is flanked by an EGF and neurexin motif.

CASPR/pl90 contains a stretch of 47 amino acids that is identical between human and rat CASPR/pl90, and contains seven copies of Pro-Gly-Tyr-X _j _ ₂ and three additional imperfect repeats of this sequence (marked as PGY in Figure 1) . The cytoplasmic domain of human and rat CASPR/pl90 contain 78 and 74 amino acids, respectively. These include a 38-42 amino acid proline-rich motif (38% proline) , the majority of which conεists of proline residues alternating with alanine, glycine, or threonine residueε (marked aε PRO in Figure 1) .

In addition to full length CASPR/pl90 gene products,

CASPR/pl90 polypeptide fragments are also included within the scope of the invention. In this εense, the term "CASPR/pl90 polypeptide fragments" encompasses polypeptides that comprise pl90 fragments, deletions, including internal deletions or any combination of pl90 fragments or deletions. In particular, pl90 polypeptides are those that specifically include or lack any of the domains listed in Table 2, below, or any combination thereof. TABLE 2

CASPR/pl90 AMINO ACID RESIDUES AS SHOWN IN FIGURE 1 AND IN SEQ DOMAIN NAME ID NOS: 1 or 3

DISC 40-168 (SEQ ID NO:l)

41-169 (SEQ ID NO:3)

NX1 199-330 (SEQ ID NO:l)

200-331 (SEQ ID NO:3)

NX2 362-486 (SEQ ID N0:1) 363-487 (SEQ ID NO:3) EGF1 544-576 (SEQ ID N0:1) 544-577 (SEQ ID NO:3) FIB 582-739 (SEQ ID N0:1) 583-740 (SEQ ID N0:3) NX3 809-938 (SEQ ID N0:1) 810-939 (SEQ ID NO:3) EGF2 961-985 (SEQ ID N0:1) 962-986 (SEQ ID NO:3) PGY 1031-1077 (SEQ ID NO:l) 1032-1078 (SEQ ID NO:3) NX4 1083-1218 (SEQ ID NO:l) 1084-1219 (SEQ ID NO:3) TMD 1282-1306 (SEQ ID NO:l) 1283-1307 (SEQ ID NO:3) PRO 1328-1369 (SEQ ID NO:l) 1329-1366 (SEQ ID NO:3)

In a further embodiment of the invention, the pl90 DNA or a modi ied εequence thereof may be ligated to a heterologous sequence to encode a CASPR/pl90 fusion protein. For example, for screening peptide libraries it may be useful to encode a chimeric pl90 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 the pl90 sequence and the heterologous protein sequence, so that the pl90 protein or protein fragment can be cleaved away from the heterologous moiety. In another embodiment, DNA sequences encoding a fusion protein comprising all or a portion of the pl90 protein fused to another protein with a desired activity are within the scope of the invention; e.g., enzymes εuch aε GUS (/3-glucuronidase) , /3-galactoεidase, luciferase, etc.

With respect to nucleic acid molecules, the invention contemplates nucleic acid molecules containing: 1) any DNA sequence that encodes the same amino acid εequence as encoded by the DNA sequenceε εhown in SEQ ID NO:l and SEQ ID NO:3; 2) any DNA εequence that hybridize to the complement of the

coding εequences disclosed herein under highly εtringent conditions, e.g., washing in O.lxSSC/0.1% SDS at 68°C (Ausubel, et al., eds., 1989, Current Protocolε in Molecular Biology, Vol. I, Green Publiεhing Associateε, Inc., and John Wiley & sons, Inc., New York, at p. 2.10.3; εee also

Sambrook, J. et al., (1989) Molecular cloning, Colo. Spring Harbor Press, USA, pp. 9.47-9.55), and which can encode a functionally equivalent gene product; and/or 3) any DNA sequence that hybridizes to the complement of the coding sequences disclosed therein under lesε εtringent conditionε, εuch aε moderately εtringent conditionε, e .g . , waεhing in 0.2xSSC/0.1% SDS at 42°C (Auεubel, et al., 1989, supra ; Sambrook, et al., 1989, supra) , yet which encodes a functionally equivalent gene product. As used herein, the term "functionally equivalent gene product" refers to a gene product that exhibits at least one of the biological functions of the gene product depicted in SEQ ID NOS: 2 and/or 4. Such biological functions can include, but are not limited to, a function (e.g. _f a protein- protein interaction function) as exhibited by at least one of the domains of the SEQ ID NO:2 or 4 gene products.

In another embodiment, DNAs that encode mutant forms of pl90 are alεo included within the εcope of the invention. Such mutant pl90 DNA sequences encompasε deletionε, additionε and/or subεtitutionε of nucleotide residues, or of regionε coding for domainε within the pl90 protein. These mutated pl90 DNAs may encode gene products that are functionally equivalent or which display properties very different from the native forms of pl90. The invention also encompasses l) DNA vectors that contain any of the coding sequences disclosed herein (see SEQ ID NO:l and SEQ ID NO:3), and/or their complements (i.e., antisense) ; 2) DNA expresεion vectorε that contain any of the coding εequences disclosed therein, and/or their complements (i.e., antisense), operatively aεεociated with a regulatory element that directs the expresεion of the coding and/or antisense sequenceε; and 3) genetically engineered host cells

that contain any of the coding sequences disclosed therein, and/or their complements (i.e., antisense), operatively associated with a regulatory element that directs the expresεion of the coding and/or antiεenεe 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 sequenceε discloεed herein. pl90 εequence can be obtained from a variety of sourceε including cDNA librarieε. For example, appropriate cDNA libraries which are good sourceε of pl90 can be obtained from (Clonetech (Palo Alto, CA) , Stratagene (La Jolla, CA) the ATCC Repository (Rockville, MD) . In addition, cDNA libraries may be prepared from mRNA pools collected from mammalian cells which expresε pl90 either conεtitutively or inducibly. By way of example but not by way of limitation, such cellε include rat GH3 cells, as well as CHO, VERO, BHK, HeLa, COS, MDWCK, -293, WI38, etc. The collection of mRNA pools and construction of cDNA libraries from these cells are set forth more fully in the examples described infra .

Any of the cDNA libraries described above may be screened by hybridization or PCR using the pl90 sequenceε described herein as oligonucleotide probeε. Screening can be performed uεing thoεe portions of the pl90 sequence as discussed in the Examples of Section 8, infra.

In addition to cDNA libraries, partial pl90 sequence can be obtained from any genomic library by library screening or from genomic DNA by PCR. Full cDNA sequences can be obtained by PCR of total RNA isolated from any cell or tissue that expresεeε pl90 including, but not limited to, neuronal tissue. Cellular sourceε alεo include, but are not limited to, hematopoietic, fetal, and embryonal tissues.

Alternatively, the cDNA libraries described above can be used to construct expresεion libraries in a cell line such aε CHO, VERO, BHK, HeLa, COS, MDWCK, -293, WI38, etc., or other cellε known in the art to contain little or no autologous

pl90 activity. Theεe expreεεion librarieε can then be εcreened uεing antibodies which are specific to pl90. Expression libraries for antibody screening may also be made in bacteria, such aε E. coli , using phage vectors, such as lambda. These expreεsion libraries may also be screened for pl90 enzyme activity aε εet forth in the exampleε which are described in more detail infra .

Other isoforms of pl90 may exist and may be cloned using the pl90 gene sequence.

5.4 EXPRESSING THE Pl90 GENE PRODUCT In order to expresε a biologically active pl90, the coding εequence for the enzyme, a function equivalent, or a modified sequence, as, e.g.. described in Section 5.3., supra , iε inserted into an appropriate eukaryotic expression vector, i.e., a vector which contains the necessary elements for transcription and translation of the inserted coding sequence in appropriate eukaryotic host cells which posεess the cellular machinery and elementε for the proper processing, i.e., signal cleavage, glycosylation, phosphorylation, sialylation, and protein sorting. Mammalian host cell expresεion εystems are preferred for the expresεion of biologically active enzymes that are properly folded and processed. When administered in humans such expresεion productε may alεo exhibit tissue targeting.

The invention also encompasses peptide fragments of the pl90 gene product. The pl90 gene product or peptide fragments thereof, can be linked to a heterologous peptide or protein as a fusion protein. In addition, chimeric pl90 expressing a heterologouε epitope that iε recognized by a commercially available antibody iε alεo included in the invention. A durable fuεion protein may also be engineered; i.e., a fusion protein which has a cleavage εite located between the pl90 εequence and the heterologouε protein εequence, εo that the pl90 gene product, or fragment thereof, can be cleaved away from the heterologouε moiety. For example, a collagenase cleavage recognition conεenεuε

sequence may be engineered between the pl90 gene product, or fragment thereof, the heterologous peptide or protein. The pl90 domain can be released from this fusion protein by treatment with collagenase.

5

5.4.1 CONSTRUCTION OF EXPRESSION VECTORS AND PREPARATION OF TRANSFECTANTS

Methods which are well-known to those skilled in the art can be used to construct expression vectors containing the ₁₀ pl90 coding sequence and appropriate transcriptional/ translational control signals. These methods include in vitro recombination/genetic recombination. See, for example, the techniques described in Sambook et al., 1987, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, __ N.Y. , Chapter 12. pl90 proteins produced by these methods would be uεeful for in vitro studies on the mechanism of action of pl90 and particularly for further studies on the mechanism of action of any inhibitors that are selective for pl90 that are identified by drug screening with the stably expressing pl90 cell lines, aε infra , or for investigating the mechaniεm of action of existing drugs or of inhibitors that may be identified by other means. The purified pl90 proteins would also be useful for the production of crystalε suitable for X- ray crystallography. Such crystals would be extremely beneficial for the rational design of drugs based on molecular structure. Expression of these chimeric DNA constructs in a baculovirus or yeaεt system and subsequent crystallization of the proteins would yield such data.

A variety of eukaryotic host-expression syεtems may be used to expresε the pl90 coding εequence. Although prokaryotic systemε offer the diεtinct advantage of ease of manipulation and low cost of scale-up, their major drawback in the expresεion of pl90 iε their lack of proper post- translational modifications of expreεsed mammalian proteins. Eukaryotic syεtems, and preferably mammalian expression syεtemε, allow for proper modification to occur. Eukaryotic

cells which possess the cellular machinery for proper procesεing of the primary tranεcript glycoεylation, phosphorylation, and, advantageously secretion of the gene product should be used as host cellε for the expreεsion of pl90. Mammalian cell lines are preferred. Such host cell lines may include but are not limited to CHO, VERO, BHK, HeLa, COS, MDWCK, -293, WI38, etc. Alternatively, eukaryotic host cells which possesε some but not all of the cellular machinery required for optional procesεing of the primary transcript and/or post-translational processing and/or secretion of the gene product may be modified to enhance the host cell's processing capabilities. For example, a recombinant nucleotide sequence encoding a peptide product that performs a procesεing function the host cell had not previously been capable of performing, may be engineered into the host cell line. Such a sequence may either be co-trans- fected into the host cell along with the gene of interest, or included in the recombinant construct encoding the gene of interest. Alternatively, cell lines containing this sequence may be produced which are then tranεfected with the gene of interest.

Appropriate eukaryotic expression vectors should be utilized to direct the expresεion of pl90 in the host cell chosen. For example, at least two baεic approacheε may be followed for the design of vectors based on SV40. The first is to replace the SV40 early region with the gene of interest while the second is to replace the late region (Hammarskjold, et al., 1986, Gene, 43:41-50. Early and late region replacement vectors can also be complemented in vitro by the appropriate SV40 mutant lacking the early or late region. Such complementation will produce recombinants which are packaged into infectious capsidε and which contain the pl90 gene. A permiεsive cell line can then be infected to produce the recombinant protein. SV40-baεed vectorε can alεo be uεed in transient expression εtudieε, where beεt reεults are ob¬ tained when they are introduced into COS (CV-l, origin of SV40) cells, a derivative of CV-l (green monkey kidney cellε)

which contain a εingle copy of an origin defective SV40 genome integrated into the chromoεome. Theεe cellε actively εyntheεize large T antigen (SV40) , thuε initiating replication from any plasmid containing an SV40 origin of replication.

In addition to SV40, almost every molecularly cloned viruε or retrovirus may be used as a cloning or expression vehicle. Viral vectors based on a number of retroviruses (avian and murine) , adenoviruses, vaccinia viruε (Cochran, et al., 1985, Proc. Natl. Acad. Sci. USA, 82:19-23) and polyoma virus may be used for expression. Other cloned viruseε, such as J C (Howley, et al., 1980, J. Virol, 36:878-882), BK and the human papilloma viruses (Heilmsan, et al., 1980, J. Virol, 36:395-407), offer the potential of being used as eukaryotic expresεion vectorε. For example, when using adenovirus expression vectors, the pl90 coding εequence 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-eεεential region of the viral genome (e.g., region El or E3) will reεult in a recombinant viruε that iε viable and capable of expreεsing the human enzyme in infected hosts (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA, 81:3655-3659). Alternatively, the vaccinia virus 7.5K promoter may be used, (e.g., see, Hackett et al., 1982, Proc. Natl. Acad. Sci. USA, 79:7415-7419; Hackett et al., 1994, J. Virol. 49:857-864, Panicali et al., 1982, Proc. Natl. Acad. Sci. USA, 79:4927-4931). Of particular interest are vectors based on bovine papilloma virus (Sarver, et al., 1981, Mol. Cell. Biol., 1:486-496), or Semliki Forest Virus, which provides large quantities of active protein in induced cellε (Olkkohnen et al., 1994, Meth. Cell. Biol., 43 part A:43-53; Lundstrum et al., 1994, Eur. J. Biochem., 224:917- 921) . These vectors have the ability to replicate as extrachromoεomal elementε. Shortly after entry of thiε DNA into mouse cellε, the plaεmid replicateε to about 100 to 200

copieε per cell. Tranεcription of the inεerted cDNA doeε not require integration of the plasmid into the host's chromosome, thereby yielding a high level of expression. These vectorε can be uεed for εtable expression by including a selectable marker in the plasmid, such aε the neo gene. High level expression may alεo be achieved uεing inducible promoters εuch aε the metallothionine IIA promoter, heat shock promoters, etc.

For long-term, high-yield production of recombinant proteins, stable expresεion is preferred. For example, following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days an enriched media, and then are switched to a selective media. Rather than using expresεion vectors which contain viral origins of replication, host cells can be transformed with the pl90 DNA controlled by appropriate expresεion control elementε (e.g., promoter, enhancer, sequences, tranεcription terminators, polyadenylation siteε, etc.), and a selectable marker. 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. A number of selection εyεtemε may be uεed, including but not limited to the herpeε εimplex viruε thymidine kinase (Wigler, et al., 1977, Cell, 11:223-232), hypoxanthine- guanine phosphoribosylatransferaεe (Szybalεka & Szybalεki, 1962, Proc. Natl. Acad. Sci. USA, 48:2026), and adenine phosphoribosyltransferase (Lowy, et al . , 1980, Cell, 22:817- 823) genes can be employed in tk ^" , hgprt" or aprt" cells respectively. Also, antimetabolite resiεtance can be uεed aε the basis of εelection for dhfr, which conferε reεiεtance to methotrexate (Wigler, et al. , 1980, Natl. Acad. Sci. USA 77:3567-3570; O'Hare, et al. , 1981, Proc. Natl. Acad. Sci. USA 78:1527-1531); ygpt, which conferε reεiεtance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA, 78:2072-2076); neo, which conferε reεiεtance to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol.

Biol., 150:1-14); and hygro, which confers resistance to hygromycim (Santerre, et al., 1994, Gene, 30:147-156) genes. 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 & Mulligan, 1988, Proc. Natl. Acad. Sci. USA, 85:8047-8051), and ODC (ornithine decarboxylase) which conferε reεiεtance to the ornithine decarboxylase inhibitor, 2-(difluromethyl)-DL-ornithine, DFMO (McConlogue L. , 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.).

Alternative eukaryotic expression systems which may be used to express the pl90 enzymes are yeast transformed with recombinant yeast expresεion vectors containing the pl90 coding sequence; insect cell system infected with recombinant virus expression vectors (e.g., baculovirus) containing the pl90 coding sequence; or plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic, TMV) or transformed with recombinant plasmid expression vectorε (e.g., Ti plaεmid) containing the pl90 coding εequence.

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. Asεoc. & Wiley Interεcience, Ch. 13; Grant et al., 1987, Expresεion and Secretion Vectorε for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 31987, Acad. Press, N.Y., Vol. 153, pp. 516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., D.C, Ch. 3; Bitter, 1987, Heterologous Gene Expression in Yeast, Methods in

Enzymology, Eds. Berger & Kimmel Acad. Press, N.Y., Vol. 152, pp. 673-694; and The Molecular Biology of the Yeast Saccharomyces, 1982, Eds. Strathem et al., Cold Spring Harbor Press, Volε. I and II. For complementation aεεayε in yeaεt, cDNAε for pl90 may be cloned into yeaεt epiεomal plaεmidε (YEp) which replicate autonomouεly in yeaεt due to the presence of the yeast 2μ circle. The cDNA may be cloned

behind either a constitutive yeast promoter such as ADH or LEU2 or an inducible promoter such as GAL (Cloning in Yeast, Chpt. 3, R. Rothstein In: DNA Cloning Vol. 11, A Practical Approach, Ed. DM Glover, 1986, IRL Press, Wash., D.C). Conεtructε may contain the 5• and 3' non-tranεlated regions of the cognate pl90 mRNA or those corresponding to a yeast gene. YEp plasmids transform at high efficiency and the plasmids are extremely stable. Alternatively, vectors may be used which promote integration of foreign DNA sequenceε into the yeaεt chromosome.

Alternately, active, post-translationally modified pl90 proteins can be obtained using a yeast expresεion εystem εuch aε the Pichia pastoris expression system marketed by Invitrogen (Pichia pastoris is owned and licensed by Research Corporation Technologies, Tucson, AZ; however, all components are available from Invitrogen, San Diego, CA) . In this example, cDNAs encoding human pl90 are independently cloned into the pHIL-D2 Pichia expression vector. After linearization with a restriction endonuclease, these constructs are transfected into spheroblasts of the his 4

Pichia pastoris strain, GS115, and recombinant yeast carrying the cloned pl90 DNA sequenceε are identified by εcreening for yeast clones that grow in the absence of hiεtidine (now supplied by the recombinant vector) , but do not efficiently utilize methanol aε the sole carbon source (due to the presence of pl90 in the place of AOXI gene sequence coding for methanol utilization) . After expansion of such clones in the presence of an alternative carbon source such as glycerol, large quantities of cellε would be transferred to liquid media containing methanol where replication ceaseε. However, cells remain viable for many days during which time human pl90 proteins are specifically expreεεed at high levels under control of the AOXI promoter. The advantageε of this system include very high protein yields and lower expense in the production and maintenance of cultures.

In cases where plant expresεion vectorε are used, the expresεion of the pl90 coding εequence 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 (Briβson et al., 1984, Nature, 310:511-514), or the coat protein promoter of TMV (Takamatεu et al., 1987, EMBO J. , 6:307-311) may be uεed; alternatively, plant promoterε such aε the εmall subunit of RUBISCO (Coruzzi et al., 1994, EMBO J. , 3:1671-1680; Broglie et al., 1984, Science, 224:838-843); or heat shock promoterε, eg., εoybean hεp 17.5-E or hsp 17.3-B (Gurley et al., 1986, Mol. Cell. Biol., 6:559-565) 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, Weisεbach & 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 expreεεion syεtem which could be used to express pl90 is an insect syεtem. In one εuch system, Autographa californica nuclear polyhedrosiε virus (AcNPV) is used as a vector to expreεε foreign geneε. The virus grows in Spodoptera frugiperda cells. The pl90 sequence may be cloned into non-esεential regionε (for example the polyhedrin gene) of the viruε and placed under control of an AcNPV promoter (for example the polyhedrin promoter) . Successful insertion of the coding εequence will result in inactivation of the polyhedrin gene and production of non-occluded re¬ combinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene) . Theεe recombinant viruεeε are then used to infect Spodoptera frugiperda cellε in which the inserted gene is expressed, (e . g. , see Smith et al., 1983, J. Virol., 46:584, Smith, U.S. Pat. No. 4,215,051).

In a specific embodiment of an insect εyεtem, the DNA encoding pl90 can be independently cloned into the pBlueBacIII recombinant tranεfer vector (Invitrogen, San Diego, CA) downstream of the polyhedrin promoter and transfected into Sf9 insect cells (derived from Spodoptera frugiperda ovarian cells, available from Invitrogen, San

Diego, CA) to generate recombinant virus containing pl90. After plaque purification of the recombinant viruε high-titer viral εtocks are prepared that in turn would be used to infect Sf9 or High Five™ (BTI-TN-5B1-4 cellε derived from Trichoplusia ni egg cell homogenateε; available from

Invitrogen, San Diego, CA) insect cellε, to produce large quantitieε of appropriately poεt-tranεlationally modified pl90 proteins. Although it iε poεsible that these cells themselves could be directly useful for drug assays, the pl90 proteins prepared by thiε method can be uεed for in vitro aεεayε of drug potency and εelectivity.

5.4.2 IDENTIFICATION OF TRANSFECTANTS OR TRANSFORMANTS EXPRESSING THE P190 GENE PRODUCT

The hoεt cells which contain the pl90 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 expression of pl90 mRNA transcriptε in the hoεt cell; and (d) detection of the gene product aε meaεured by immunoassay or by itε biological activity.

In the firεt approach, the preεence of the pl90 coding εequence inserted in the expression vector can be detected by DNA-DNA or DNA-RNA hybridization or PCR uεing probeε comprising nucleotide sequences that are homologous the pl90 coding sequence or portions or derivatives thereof.

In the second approach, the recombinant expression vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e .g. , resistance to antibiotics, resiεtance to methotrexate, tranεformation phenotype, occluεion body formation in baculoviruε, etc.). For example, if the pl90 coding εequence iε within a marker gene εequence of the vector, recombinantε containing the pl90 coding εequence can be identified by the

absence of the marker gene function. Alternatively, a marker gene can be placed in tandem with the pl90 sequence under the control of the same or different promoter used to control the expression of the pl90 coding sequence. Expression of the marker in responεe to induction or selection indicates expression of the pl90 coding sequence. In addition, the marker gene may be identified by DNA-DNA or DNA-RNA hybridization or PCR.

In the third approach, transcriptional activity for the pl90 coding region can be aεseεsed by hybridization or PCR assays. For example, RNA can be isolated and analyzed by Northern blot uεing a probe homologouε to the pl90 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 expresεion of the pl90 protein product can be assessed immunologically, for example by Western blots, immunoassays such as radioimmuno- precipitation, enzyme-linked immunoassays and the like. The ultimate test of the succesε of the expresεion syεtem, however, involveε the detection of the biologically active pl90 gene product. Where the host cell secretes the gene product, the cell free media obtained from the cultured transfectant host cell may be assayed for pl90 activity. Where the gene product iε not secreted, cell lyεates may be assayed for such activity. In either case, a number of assays can be used to detect pl90 activity, including but not limited to, those deεcribed in the examples infra or thoεe known in the art.

5.4.3 CELL LINES EXPRESSING P190 The preεent invention alεo relateε to cell lines containing recombinant DNA sequence, preferably a chromosomally integrated recombinant DNA sequence, which comprises the gene encoding pl90 which cell lines further do not expreεε autologouε pl90, apart from that encoded by the recombinant DNA sequence.

A εpecific embodiment of the present invention iε an engineered mammalian cell line which containε a chromoεomally integrated, genetically-engineered ("recombinant") DNA sequence, which DNA sequence expresses mammalian pl90, and wherein said cell line also does not express autologous pl90. The cell line iε preferably of human or primate origin, εuch as the exemplified monkey kidney COS cell line, but cell lineε derived from other species may be employed, including chicken, hamster, murine, ovine and the like; the CHO (Chinese hamster ovary) cell line for example, may be particularly preferred for large scale production.

Any cell or cell line, the genotype of which haε been altered by the presence of a recombinant DNA sequence is encompassed by the invention. The recombinant DNA sequence may also be referred to herein aε "heterologous DNA,"

"exogenous DNA," "genetically engineered" or "foreign DNA," indicating that the DNA waε introduced into the genotype or genome of the cell or cell line by a process of genetic engineering. The invention includes, but iε not limited to, a cell or cell line wherein the native pl90 DNA εequence haε been removed or replaced aε a result of interaction with a recombinant DNA sequence. Such cells are called pl90 knockoutε, herein, if the reεulting cell iε left without a native DNA that encodeε a functional pl90 gene product. Aε uεed herein, the term "recombinant DNA εequence" refers to a DNA sequence that haε been derived or iεolated from any source, that may be εubsequently chemically altered, and later introduced into mammalian cells. An example of a recombinant DNA sequence "derived" from a source, would be a DNA sequence that is identified as a uεeful fragment within a given organism, and which is then chemically synthesized in essentially pure form. An example of εuch DNA εequence "isolated" from a εource would be a DNA εequence that iε excised or removed from εaid εource by chemical means, e . g. , by the uεe of restriction endonucleaεes, so that it can be

further manipulated, e.g., amplified, for use in the invention, by the methodology of genetic engineering.

Therefore, "recombinant DNA εequence" includeε completely εynthetic DNA, εemi-εynthetic DNA, DNA isolated from biological sources, and DNA derived from introduced RNA. Generally, the recombinant DNA sequence iε not originally resident in the genotype which is the recipient of the DNA sequence, or it is resident in the genotype but is not expressed. The isolated recombinant DNA sequence used for transformation herein may be circular or linear, double- stranded or single-stranded. Generally, the DNA sequence is chimeric linear DNA, or is a plasmid or viral expression vector, that can also contain coding regions flanked by regulatory sequenceε which promote the expression of the recombinant DNA present in the resultant cell line. For example, the recombinant DNA sequence may itself comprise or consist of a promoter that iε active in mammalian cellε, or may utilize a promoter already present in the genotype that iε the transformation target. Such promoterε include, but are not limited to, the CMV promoter, SV 40 late promoter and retroviral LTRε (long terminal repeat elementε) .

The general methods for conεtructing recombinant DNA which can tranεform target cellε are well known to thoεe skilled in the art, and the εame compositions and methods of construction may be utilized to produce the DNA useful herein. For example, J. Sambrook et al.. Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Laboratory Presε (2d ed., 1989), provides εuitable methods of construction. Aside from recombinant DNA sequence that serve as tranεcription unitε for pl90 or other portionε thereof, a portion of the recombinant DNA may be untranεcribed, serving a regulatory or a εtructural function.

The recombinant DNA εequence to be introduced into the cellε further will generally contain either a εelectable marker gene or a reporter gene or both to facilitate identification and εelection of transformed cells.

Alternatively, the εelectable marker may be carried on a εeparate piece of DNA and uεed in a co-tranεformation procedure. Both εelectable markerε and reporter geneε may be flanked with appropriate regulatory sequences to enable expression in mammalian cells. Useful selectable markers are well known in the art and include, for example, anti-biotic and herbicide resiεtance geneε.

Sources of DNA sequences useful in the present invention include Poly-A RNA from mammalian cells, from which the mRNA encoding pl90 can be derived and used for the synthesis of the corresponding cDNA by methods known to the art. Such sourceε include cDNA libraries and mRNA pools made from neuronal, neuroblastoma, embryonic, fetal, and hematopoietic tissues of human, rat or other mammalian origin. Selectable marker genes encoding enzymes which impart reεiεtance to biocidal compoundε are listed in Table 1, below.

Table 3 Selectable Marker Genes

Reεiεtance Conferε Gene or Enzvme Resistance to: Reference

Neomycin phoεpho- G-418, neomycin, Southern et transferase (neo) kanamycin al., 1982, J. Mol.

Appl. Gen., 1:327-341

Hygromycin Hygromycin B Shimizu et al., 1986, phosphotrans- Mol. Cell Biol., ferase (hpt or 6:1074-1087 hyg)

Dihydrofolate Methotrexate Kwok et al., 1986, reductase (dhfr) Proc. Nat'1. Acad. Sci. USA, 4552-4555

Phosphinothricin Phosphinothricin DeBlock et al., 1987, acetyltransferase EMBO J., 6:2513-2518

(bar)

2,2-Dichloropro- 2-2,Dichloropro- Buchanan-Wollaston et pionic acid pionic acid al., 1989, J. Cell. dehalogenase (Dalapon) Biochem. , Supp. 13D, 330

Acetohydroxyacid Sulfonylurea, Anderson et al. (U.S. εynthase imidazolinone and Patent No. triazolopyrimidine 4,761,373) ; G.W. herbicides Haughn et al., 1988 Mol. Gen. Genet., 211:266-271

5-Enolpyruvyl- Glyphosate Comai et al., 1985 shikimatephos- Nature, 317:741-744 phate synthase (aroA)

Haloarylnitrilase Bro oxynil Stalker et al. , published PCT appln. W087/04181

Acetyl-coenzyme A Sethoxydim, Parker et al., 1990 carboxylase haloxyfop Plant Physiol. , 92:1220

Dihydropteroate Sulfonamide Guerineau et al., synthase (sul I) herbicides 1990, Plant Molec. Biol., 15:127-136

32 D photosystem Triazine herbicides Hirschberg et al., II polypeptide 1983, Science, (psbA) 222:1346-1349

Anthrani1 te 5-Methyltryptophan Hibberd et al. (U.S. synthase Patent No. 4,581,847)

Dihydrodipicolin- Aminoethyl cysteine Glasεman et al., ic acid synthase published PCT (dap A) application No. W089/11789

Reporter genes are used for identifying potentially transformed cells and for evaluating the functionality of regulatory sequences. Reporter genes which encode for eaεily assayable marker proteinε are well known in the art. In general, a reporter gene iε a gene which iε not preεent in or expreεsed by the recipient organism or tissue and which encodes a protein whose expression is manifested by some easily detectable property, e . g. , enzymatic activity. Preferred geneε includeε the chloramphenicol acetyl transferase gene (cat) from Tn9 of E. coli , the beta- galactoεidaεe gene of E. coli , the beta-glucuronidaεe gene

(gus) of the uidA locuε of E . coli , and the luciferase gene from firefly Photinus pyralis . Expresεion of the reporter gene is assayed at a suitable time after the DNA haε been introduced into the recipient cells. Other elements εuch aε intronε, enhancers, polyadenylation sequenceε and the like, may alεo be a part of the recombinant DNA εequence. Such elements may or may not be necessary for the function of the DNA, but may provide improved expression of the DNA by affecting transcription, stability of the mRNA, or the like. Such elements may be included in the DNA as desired to obtain the optimal performance of the transforming DNA in the cell.

The recombinant DNA sequence can be readily introduced into the target cells by transfection with an expression vector, such aε a viral expreεεion vector, compriεing cDNA encoding pl90 by the modified calcium phoεphate precipitation procedure of Chen et al. , 1987, Mol. Cell. Biol., 7:2745- 2752. Tranεfection can alεo be accompliεhed by other methods, including lipofection, using commercially available kits, e.g., provided by Life Technologies.

In a preferred embodiment of the invention, the cell lines of the invention are able to expresε a stable pl90 gene product or analog, homologue, or deletion thereof after several passages through cell culture.

5.4.4 PURIFICATION OF THE pl90 GENE

PRODUCT

Once a cell that produces high levelε of biologically active pl90 iε identified, the cell may be clonally expanded and uεed to produce large quantitieε of the enzyme, which may be purified uεing techniques well-known in the art including, but not limited to, immunoaffinity purification, chromatographic methodε including high performance liquid chromatography and the like. Where the enzyme iε εecreted by the cultured cellε, pl90 may be readily recovered from the culture medium.

Where the pl90 coding sequence, or fragment thereof, has been engineered to encode a cleavable fusion protein, the purification of the pl90 gene product, or fragment thereof, may be readily accomplished using affinity purification techniques. For example, an antibody specific for the heterologous peptide or protein can be used to capture the durable fusion protein; for example, on a solid surface, a column etc. The pl90 moiety can be released by treatment with the appropriate enzyme that cleaves the linkage site. cDNA construction using the polymerase chain reaction accompanied by transfection and purification of the expressed protein permits the isolation of sufficient quantities of pl90 for characterization of the enzyme's physical and kinetic properties. Using site-directed mutagenesis or naturally occurring mutant sequences, this system provides a reasonable approach to determine the effects of the altered primary structure on the function of the protein. Fusion constructs of the pl90 protein domain with the marker peptide preceding the amino terminus of pl90 or following the carboxy terminus of pl90 may also be engineered to evaluate which fusion construct will interfere the least, if at all, with the protein's biologic function and the ability to be purified.

Using this aspect of the invention, any cleavage site or enzyme cleavage substrate may be engineered between the pl90 sequence and a second peptide or protein that has a binding partner which could be uεed for purification, e.g, any antigen for which an immunoaffinity column can be prepared.

5.5 ANTIBODIES TO THE P190 GENE PRODUCT

For the production of antibodieε, variouε host animalε may be immunized by injection with the pl90 gene product, or a portion thereof including, but not limited to, portionε of the pl90 gene product in a recombinant protein. Such host animals may include but are not limited to rabbits, mice, and rats, to name but a few. Variouε adjuvants may be used to increase the immunological response, depending on the host

species, including but not limited to Freund's (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 such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

Monoclonal antibodies may be prepared by uεing any technique which provideε for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally deεcribed by Kohler and Milεtein, 1975, Nature, 256:495-497, the human B-cell hybridoma technique (Koεbor et al., 1983, Immunology Today, 4:72, Cote et al. , 1983, Proc. Natl. Acad. Sci., 80:2026-2030) and the EBV-hybrido a technique (Cole et al., 1985, Monoclonal Antibodieε and Cancer Therapy, Alan R. Lisε, Inc., pp. 77-96). In addition, techniqueε 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; Takeda et al., 1985, Nature, 314:452-454) 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 single chain antibodies specific to one of the binding partners.

Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, such fragmentε 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 expresεion librarieε may be conεtructed (Huεe et al. , 1989, Science, 246:1275-1281) to allow rapid and eaεy identification of monoclonal Fab fragmentε with the deεired εpecificity.

5.6 GENE THERAPIES BASED ON THE P190 GENE A variety of gene therapy approacheε may be uεed in accordance with the invention to modulate expreεεion of the pl90 gene in vivo. For example, antiεenεe DNA molecules may be engineered and used to block translation of pl90 mRNA in vivo. Alternatively, ribozyme molecules may be designed to cleave and destroy the pl90 mRNAs in vivo . In another alternative, oligonucleotides deεigned to hybridize to the 5' region of the pl90 gene (including the region upεtream of the coding sequence) and form triple helix structures may be used to block or reduce transcription of the pl90 gene. In yet another alternative, nucleic acid encoding the full length wild-type pl90 message may be introduced in vivo into cells which otherwise would be unable to produce the wild-type pl90 gene product in sufficient quantities or at all.

In a preferred embodiment, the antisenεe, ribozyme and triple helix nucleotides are designed to inhibit the translation or transcription of pl90. To accomplish this, the oligonucleotides used should be designed on the basis of relevant sequences unique to pl90. For example, and not by way of limitation, the oligonucleotides should not fall within those regions where the nucleotide sequence of pl90 is most homologous to that of other known proteins.

Instead, it is preferred that the oligonucleotides fall within the regionε of pl90, which diverge from the εequence of other known proteins.

In the case of antisense molecules, it is preferred that the sequence be chosen from those divergent sequenceε just mentioned above. It iε alεo preferred that the sequence be at least 18 nucleotideε in length in order to achieve sufficiently strong annealing to the target mRNA sequence to prevent translation of the sequence. Izant and Weintraub, 1984, Cell, 36:1007-1015; Rosenberg et al., 1985, Nature, 313:703-706. In the case of the "hammerhead" type of ribozymes, it is also preferred that the target sequences of the ribozymes be chosen from the above-mentioned divergent sequenceε.

Ribozymes are RNA molecules which posseεε highly specific endoribonuclease activity. Hammerhead ribozymes comprise a hybridizing region which is complementary in nucleotide sequence to at least part of the target RNA, and a catalytic 5 region which is adapted to cleave the target RNA. The hybridizing region contains nine (9) or more nucleotides. Therefore, the hammerhead ribozymes of the present invention have a hybridizing region which is complementary to the sequences listed above and is at least nine nucleotideε in

10 length. The conεtruction and production of such ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, 1988, Nature, 334:585-591.

The ribozymes of the preεent invention alεo include RNA endoribonucleaseε (hereinafter "Cech-type ribozymes") such aε

15 the one which occurs naturally in Tetrahymena Thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively deεcribed by Thomaε Cech and collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature, 324:429-

20 433; published International patent application No. WO 88/04300 by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216). The Cech endoribonucleases have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place.

25 The invention encompasεeε those Cech-type ribozymes which target eight baεe-pair active εite εequenceε that are exclusive to pl90.

In the case of oligonucleotides that hybridize to and form triple helix structureε at the 5' terminus of the pl90

30 gene and can be uεed to block tranεcription, it iε preferred that they be complementary to those εequenceε in the 5' terminus of pl90 which are not present in other related proteins. However, it is preferred that the εequenceε not include thoεe regionε of the pl90 promoter which are even

35 εlightly homologous to that of other known proteinε.

The foregoing compounds can be adminiεtered by a variety of ethodε which are known in the art including, but not

limited to the use of liposomeε aε a delivery vehicle. Naked DNA or RNA molecules may also be uεed where they are in a form which is resiεtant to degradation εuch aε by modification of the ends, by the formation of circular molecules, or by the use of alternate bonds including phosphothionate and thiophosphoryl modified bonds. In addition, the delivery of nucleic acid may be by facilitated tranεport where the nucleic acid molecules are conjugated to poly-lysine or transferrin. Nucleic acid may also be transported into cellε by any of the variouε viral carrierε, including but not limited to, retroviruε, vaccinia, AAV, and adenovirus.

Alternatively, a recombinant nucleic acid molecule which encodeε, or iε, εuch antisense, ribozyme, triple helix, or pl90 molecule can be constructed. This nucleic acid molecule may be either RNA or DNA. If the nucleic acid encodes an RNA, it is preferred that the sequence be operatively attached to a regulatory element so that sufficient copies of the desired RNA product are produced. The regulatory element may permit either constitutive or regulated transcription of the sequence. In vivo , that iε, within the cellε or cellε of an organism, a transfer vector such as a bacterial plasmid or viral RNA or DNA, encoding one or more of the RNAs, may be transfected into cells e.g. (Llewellyn et al., 1987, J. Mol. Biol., 195:115-123; Hanahan et al. 1983, J. Mol. Biol.,

166:557-580). Once inside the cell, the transfer vector may replicate, and be transcribed by cellular polymerases to produce the RNA or it may be integrated into the genome of the host cell. Alternatively, a transfer vector containing sequences encoding one or more of the RNAs may be transfected into cells or introduced into cellε by way of micromanipulation techniqueε εuch aε microinjection, such that the transfer vector or a part thereof becomes integrated into the genome of the host cell.

5. 7 DRUG SCREENING ASSAYS

The present invention provides a simple in vitro εyεtem for the screening of drug actionε on pl90, which will be uεeful for the development of drugε that modulate the growth, differentiation or survival of neurons. Assays can be performed on living mammalian cellε, which more closely approximate the effects of a particular serum level of drug in the body, or on microsomal extracts prepared from the cultured cell lines. Studies using microsomal extracts offer the poεεibility of a more rigorouε determination of direct drug/enzyme interactionε.

The pl90-εynthesizing cell lines are useful for evaluating the activity of potential bioactive agents on pl90. The present invention also provides a second mammalian cell line which contains a chromoso ally integrated, recombinant DNA εequence, wherein said DNA sequence expresses mammalian, pl90, and wherein said cell line also preferably does not expresε autologouε pl90 activity. Thiε εecond cell line iε alεo preferably a primate, murine or human cell line. Thuε, the present invention alεo provideε a method to evaluate.

The invention alεo relateε to methodε for the identification of geneε, termed "pathway geneε", which are associated with the pl90 gene product or with the biochemical pathways which extend therefrom. "Pathway gene", as used herein, refers to a gene whose gene product exhibits the ability to interact with the pl90 gene product.

Any method suitable for detecting protein-protein interactions may be employed for identifying pathway gene products by identifying interactions between gene products and the pl90 gene product. Such known gene products may be cellular or extracellular proteins. Those gene products which interact with such known gene products represent pathway gene products and the geneε which encode them repreεent pathway genes.

Among the traditional methods which may be employed are co-immunoprecipitation, crosslinking and co-purification through gradients or chromatographic columns. Utilizing procedures such as these allows for the identification of pathway gene products. Once identified, a pathway gene product may be used, in conjunction with standard techniques, to identify its corresponding pathway gene. For example, at least a portion of the amino acid sequence of the pathway gene product may be ascertained using techniques well known to those of skill in the art, such as via the Edman degradation technique (see, e.g., Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., pp.34-49). The amino acid sequence obtained may be used as a guide for the generation of oligonucleotide mixtures that can be used to screen for pathway gene sequences. Screening made be accomplished, for example by standard hybridization or PCR techniques. Techniqueε for the generation of oligonucleotide mixtures and εcreening are well-known. (See, e.g., Ausubel et al., eds., 1987-1993, Current Protocolε in Molecular Biology, John Wiley & Sons, Inc. New York, and PCR Protocols: A Guide to Methodε and Applicationε, 1990, Inniε, M. et al., edε. Academic Press, Inc. , New York) .

Additionally, methods may be employed which result in the simultaneous identification of pathway genes which encode the protein interacting with the pl90 gene product. These methodε include, for example, probing expression libraries with labeled protein known or suggested to be involved in cardiovascular disease, using this protein in a manner similar to the well known technique of antibody probing of λgtll libraries.

One such method which detects protein interactionε in vivo, the two-hybrid εystem, is described in detail for illustration only and not by way of limitation. One version of this syεtem haε been deεcribed (Chien et al., 1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582) and iε commercially available from Clontech (Palo Alto, CA) .

Briefly, utilizing εuch a εystem, plasmids are constructed that encode two hybrid proteins: one consists of the DNA-binding domain of a transcription activator protein fused to a known protein, and the other consistε of the activator protein's activation domain fused to an unknown protein that is encoded by a cDNA which has been recombined into this plasmid as part of a cDNA library. The plasmids are transformed into a εtrain of the yeaεt Saccharomyces cerevisiae that containε a reporter gene (e.g., lacZ) whoεe regulatory region contains the activator's binding sites. Either hybrid protein alone cannot activate transcription of the reporter gene: the DNA-binding domain hybrid because it does not provide activation function and the activation domain hybrid because it cannot localize to the activator's binding sites. Interaction of the two proteins reconstituteε the functional activator protein and resultε in expression of the reporter gene, which is detected by an assay for the reporter gene product.

The two-hybrid system or related methodology may be used to screen activation domain libraries for proteinε that interact with the pl90 gene product, herein alεo called the known "bait" gene protein. Total genomic or cDNA εequenceε may be fuεed to the DNA encoding an activation domain. Such a library and a plasmid encoding a hybrid of the bait gene protein fuεed to the DNA-binding domain may be cotransformed into a yeast reporter εtrain, and the reεulting tranεformantε may be screened for those that expresε the reporter gene. These colonies may be purified and the library plasmidε responsible for reporter gene expression may be isolated. DNA sequencing may then be used to identify the proteins encoded by the library plasmids.

For example, and not by way of limitation, the bait gene may be cloned into a vector such that it is translationally fused to the DNA encoding the DNA-binding domain of the GAL4 protein.

A cDNA library of the cell line from which proteins that interact with bait gene are to be detected can be made using

methodε routinely practiced in the art. According to the particular syεtem described herein, for example, the cDNA fragments may be inserted into a vector such that they are translationally fused to the activation domain of GAL4. This 5 library may be co-transformed along with the bait gene-GAL4 fusion plasmid into a yeast strain which contains a lacZ gene driven by a promoter which containε the GAL4 activation sequence. A cDNA encoded protein, fused to the GAL4 activation domain, that interacts with bait gene will

10 reconstitute an active GAL4 protein and thereby drive expression of the lacZ gene. Colonies which express lacZ may be detected by their blue color in the presence of X-gal. The cDNA may then be purified from these strains, and used to produce and isolate the bait gene-interacting protein using 5 techniqueε routinely practiced in the art.

Once a pathway gene haε been identified and isolated, it may be further characterized as, for example, discussed herein.

The proteins identified as products of pathway genes may 0 be used to modulate pl90 gene expression, as defined herein, or may themselves be targets for modulation to in turn modulate symptoms associated with pl90 expression.

5.8 COMPOUNDS IDENTIFIED IN THE SCREENS 5 The compounds identified in the εcreen will demonstrate the ability to selectively modulate the expression of pl90. These compounds include but are not limited to nucleic acid encoding pl90 and homologueε, analogues, and deletions thereof, aε well aε antisense, ribozyme, triple helix,

30 antibody, and polypeptide molecules and small inorganic molecules.

5.9 PHARMACEUTICAL FORMULATIONS AND ROUTES OF ADMINISTRATION

__ Any of the identified compounds can be administered to an animal host, including a human patient, by itself, or in pharmaceutical compositionε where it is mixed with εuitable

carriers or excipient(s) at doseε therapeutically effective to treat or ameliorate a variety of disorders, including those characterized by insufficient, aberrant, or excessive pl90 activity or neurite growth, differentiation or survival, including but not limited to: ALS; general ataxia; Parkinson's disease; Alzheimer's diseaee; Huntington's disease; general neuropathy; cerebral palsy; neurologic trauma; and mental retardation. A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms aεsociated with such disorders. Techniques for formulation and administration of the compounds of the instant application may be found in "Remington'ε Pharmaceutical Scienceε," Mack Publishing Co., Easton, PA, latest edition. A number of disorders may be characterized by insufficient, aberrant, or excessive pl90 activity. In addition, several physiological states which may, from time to time be considered undesired, may also be asεociated with pl90 activity. By way of example, but not by way of limitation, such disorders and physiological εtateε which may be treated with the compoundε of the invention include but are not limited to those characterized by insufficient, aberrant, or excessive neurite growth, differentiation or survival, including but not limited to: ALS; general ataxia; Parkinson'ε disease; Alzheimer's diseaεe; Huntington'ε disease; general neuropathy; cerebral palsy; neurologic trauma; and mental retardation.

The compounds of the invention may be designed or administered for tissue specificity. If the compound comprises a nucleic acid molecule, including those compriεing an expreεεion vector, it may be linked to a regulatory sequence which iε specific for the target tisεue, εuch as the brain, skin, joints, bladder, kidney, liver, ovary, etc. by methods which are known in the art including those εet forth in Hart, 1994, Ann. Oncol., 5 Suppl 4: 59-65; Dahler et al., 1994, Gene, 145: 305-310; DiMaio et al., 1994, Surgery, 116:205-213; Weichselbaum et al., Cancer Reε., 54:4266-4269;

Harris et al. , 1994, Cancer, 74 (Suppl. 3) :1021-1025; Rettinger et al., Proc. Nat'l. Acad. Sci. USA, 91:1460-1464; and Xu et al, Exp. Hematol., 22:223-230; Brigham et al., 1994, Prog. Clin. Biol. Res., 388:361-365. The compounds of the invention may be targeted to specific sites of inflammation by direct injection to those siteε, εuch aε joints, in the case of arthritis. Compounds designed for use in the central nervous system should be able to cross the blood brain barrier or be suitable for administration by localized injection. Similarly, compounds specific for the bladder can be directly injected therein. Compounds may alεo be designed for confinement in the gastrointestinal tract for use against disorders such as colorectal carcinoma. In addition, the compounds of the invention which remain within the vascular system may be useful in the treatment of vascular inflammation which might arise as a result of arteriosclerosis, balloon angioplasty, catheterization, myocardial infarction, vascular occlusion, and vascular surgery and which have already been asεociated with pl90 by Pritchard et al., 1994, J. Biol. Chem., 269, 8504-8509. Such compounds which remain within the bloodstream may be prepared by methods well known in the art including those described more fully in Mclntire, 1994, Annals Biomed. Engineering, 22:2-13.

5.9.1 EFFECTIVE DOSAGE Pharmaceutical compositions suitable for use in the present invention include compositionε wherein the active ingredients are contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amounts is well within the capability of those εkilled in the art, especially in light of the detailed disclosure provided herein.

For any compound used in the method of the invention, the therapeutically effective dose can be eεtimated 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 (the dose where 50% of the cells show the desired effects) as determined in cell culture. Such information can be used to more accurately determine useful doseε in humans.

A therapeutically effective dose refers to that amount of the compound that resultε in amelioration of εymptomε or a prolongation of εurvival in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animalε, e.g., for determining the LD50 (the doεe lethal to 50% of the population) and the ED50 (the doεe therapeutically effective in 50% of the population) . The dose ratio between toxic and therapeutic effectε is the therapeutic index and it can be expressed as the ratio between LD50 and ED50. Compoundε which exhibit high therapeutic indiceε are preferred. The data obtained from these cell culture assays and animal studieε can be uεed in formulating a range of dosage for use in human. The doεage 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 desired effects. In caseε of local adminiεtration or εelective uptake, the effective local concentration of the drug may not be related to plasma concentration.

The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

5.9.2 COMPOSITION AND FORMULATION 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 compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks'ε solution. Ringer's solution, or physiological saline buffer. For transmucosal adminiεtration, penetrantε appropriate to the barrier to be permeated are uεed in the formulation. Such penetrantε are generally known in the art.

For oral adminiεtration, the compoundε can be formulated readily by combining the active compoundε with pharmaceutically acceptable carrierε well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capεuleε, liquidε, gelε, εyrups, slurries, suεpenεionε and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral uεe can be obtained solid excipient, optionally grinding a reεulting 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 εuch aε εugarε,

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 carboxymethylcelluloεe, and/or polyvinylpyrrolidone (PVP) . If deεired, diεintegrating agents may be added, such aε the croεs-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 arable, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solventε 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 doseε.

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 capsuleε can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants εuch aε talc or magnesium stearate and, optionally, stabilizerε. In εoft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizerε may be added. All formulations for oral adminiεtration εhould be in doεageε suitable for such administration. For buccal administration,the compositions may take the form of tabletε or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray preεentation from preεεurized packε or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,

dichlorotetrafluoroethane, carbon dioxide or other εuitable gas. In the caee of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsuleε and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder baεe εuch aε lactoεe or εtarch.

The compoundε may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi- dose containers, with an added preεervative. The compoεitionε may take εuch forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agentε such as εuspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suεpenεionε of the active compoundε may be prepared aε appropriate oily injection εuspensions. Suitable lipophilic solventε or vehicleε include fatty oilε 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 suεpension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e . g. , sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositionε such as suppoεitorieε or retention enemaε, e.g., containing conventional εuppoεitory bases such as cocoa butter or other glycerides.

In addition to the formulationε deεcribed previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

A pharmaceutical carrier for the hydrophobic compoundε of the invention iε a coεolvent εyεtem comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied.

Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solventε such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a suεtained-releaεe system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various of sustained- release materialε have been eεtabliεhed and are well known by those skilled in the art. Sustained-releaεe capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed. The pharmaceutical compoεitionε alεo may comprise suitable solid or gel phase carriers or excipientε. Examples of such carriers or excipients include but are not limited to

calcium carbonate, calcium phoεphate, variouε sugars, starches, cellulose derivatives, gelatin, and polymers εuch aε polyethylene glycols.

Many of the compounds of the invention may be provided as saltε 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 solventε that are the correεponding free baεe forms.

5.9.3 ROUTES OF ADMINISTRATION Suitable routes of administration may, for example, include oral, rectal, transmucosal, transdermal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneouε, intramedullary injectionε, aε well aε intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the compound in a local rather than syεtemic manner, for example, via injection of the compound directly into an affected area, 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 an antibody specific for affected cells. The liposomes will be targeted to and taken up selectively by the cells.

5.9.4 PACKAGING

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plaεtic foil, εuch as a blister pack. The pack or dispenεer device may be accompanied by instructions for administration. Compositionε compriεing a compound of the invention formulated in a compatible pharmaceutical carrier may alεo be prepared, placed in an appropriate container, and labelled for treatment of an

indicated condition. Suitable conditionε indicated on the label may include treatment of a disease such as one characterized by insufficient, aberrant, or excesεive neurite growth, differentiation, or εurvival.

6. EXAMPLE: THE INTERACTION BETWEEN

CONTACTIN AND THE CAH DOMAIN OF RPTP3

The subsectionε below describe the biological interaction between contactin and the CAH domain of RPTP3.

The data demonstrate that ligands for RPTP/3 are differentially expresεed in. neuronal and glial cell lineε.

In addition, it is shown that a 140 kDa protein from these cell lineε interacts with the GfcH domain of RPTP/8, and that this 140 kDa protein is contactin. The data also demonstrate that RPTP/8 interacts with both membrane-bound and soluble contactin.

6.1. MATERIALS AND METHODS 6.1.1. CELL CULTURE

SF763T and SF767T human astrocytoma cell lines were grown in athymic nu/nu mice to create a tumor derived cell line. The parental lines (SF763 and SF767) were generously provided by Dr. Michael E. Bernes (The Barrow Neurological Institute, Phoenix, Arizona) . All other cell line used were supplied by the American Type Culture Collection (Rockville, MD) . For culturing of rat sensory neuron, spinal sensory ganglia were dissected from newborn rat pups and dissociated by incubation with trypsin (0.05% for 10 minutes). The ganglia were waεhed εeveral timeε in L15 + 10% fetal calf serum, and triturated with a pasteur pipette. The resulting single cell suspension was not subjected to preplating. The cellε were plated at 15,000 cellε per well in an eight-well chamber slide (Nunc) precoated with 10 mg/ml laminin in PBS. The medium was L15/C0 ₂ with εupplementε aε deεcribed (Hawrot and Patterεon, 1979, Meth. Enzymol., 58:547-584), and nerve

growth factor waε added at 50 ng/ml. The cellε were cultured for two dayε prior to staining.

6.1.2. GENERATION AND PRODUCTION OF FC-FUSIONS

To construct the Fc-fusion molecule, different εubdomainε of RPTP/S extracellular region were amplified using pfu (Stratagene, La Jolla, CA) and cloned into a unique BamHI site upstream from the hinge region of human IgGl-Fc. For the construction of βC and /SCF fusions a DNA fragment waε amplified from position -20, within the Bluescript εequence to poεition 939 and 1245 reεpectively (/3C-Fc aa 1-313, /3CF-Fc aa 1-415) (Levy et al., 1993, J. Biol. Chem., 268:10573-

10581) . In frame fusion was made by creating a BamHI site in the 3' primer maintaining the original amino acidε sequence in the fusion junction. These fragments were further cloned into HindlH-BamHI linearized pC l vector, a modified version of pIGl that contained a cDNA form instead of the genomic fragment of human IgG (Simmons, 1993, in Cellular

Interactions in Development. A Practical Approach, Hartley

(ed.), IRL Press). The same strategy was used to construct human contactin-Fc (Hcon-Fc) fusion molecule. Briefly, total

RNA was prepared from Y79 retinoblastoma cells and converted to single strand cDNA using Superscript II reveres transcriptase (Gibco-BRL) following the suppliers protocol.

This cDNA was use as a template to clone human contactin by three overlapping PCR reactions into EcoRI-BamHI sites of

PC I vector. In order to use these sites, the EcoRI εite at poεition 3173 (Reid et al., 1994, Brain Res. Mol. Brain Res.,

21:1-8), waε eliminated by changing a single base during the

PCR reaction. The final construct contained amino acids 1-

1020 of human contactin fused to the IgG region. To construct /8F-Fc the region between nucleotides 901 to 1242 was amplified with a εet of primerε that introduced SacII and

BamHI εiteε in the endε of the fragment. Thiε fragment waε cloned into pCNγl between the globulin gene and a εequence encoding a εignal peptide derived from TGF/3 gene (Plowman et

al., 1992, J. Biol. Chem., 267:13073-13078). The integrity of all the above constructs was checked by complete nucleotide sequence determination or by restriction enzyme analysiε. Fuεion proteinε were produced tranεiently in C0S7 cellε or by cotransfection with pN1012-Neo into 293 cellε and selecting for individual G418 reεiεtant cloneε as described (Peleε et al., 1991, EMBO J. , 10:2077-2086). Purification of fuεion proteinε was achieved by affinity chromatography on Protein-A Sepharose CL 4B (Pharmacia) . Bound proteins were eluted with 100 mM sodium citrate PH 2.5, IM MgCl ₂ , followed by buffer exchange on a PD-10 desalting column (Pharmacia) . The proteinε were analyzed by gel electrophoreεiε followed by silver staining (ICN, Costa Mesa, CA) . Concentration of the purified proteins waε determined by bradford reagent (BioRad, Richmond, CA) , and by an ELISA assay using peroxidase coupled antibody againεt human IgG (Pierce, Roxford, IL) . The εame antibody waε used to detect the fusion proteins by western blotting followed by chemiluminescence reagent (ECL; Amersham) as described previously (Peles et al., 1992, Cell, 69:205-216) .

6.1.3. EXPRESSION CLONING IN COS CELLS

Total cellular RNA was prepared from GH3 cells using acid guanidinium thiocyanate extraction (Chomczynski and Sacchi, 1987, Anal. Biochem., 162:156-159), and Poly(A) RNA was isolated by two paεsages over an oligo dT cellulose column (Pharmacia) . cDNA waε εyntheεized uεing the Superscript kit (Gibco BRL, Bethesda, MD) by priming with a random primer that contained a Hindlll εite. Following the addition of EcoRI adaptorε the double-εtranded cDNA waε εize selected on agarose gel. cDNAs larger then 2 kb were ligated into a EcoRI and Hindlll-digeεted pcMPl plaεmid vector, a derivative of the pCMV-1 vector (Lammerε et al., 1993, J. Biol. Chem., 168:24456-22462). E. coli DH10B cellε (GIBCO BRL) were transformed by electroporation REF. This procedure generated a cDNA library with 2 X IO ⁶ independent cloneε. Poolε of 3000 bacterial clones were grown for 24 hours and

scraped from plates using, LB containing 15% glycerol. Twenty percent of the cultures were saved aε glycerol stocks at -70 ^C C and plasmid DNA was prepared from the rest using the Wizard plasmid purification kit (Promega) . Plaεmid DNA (10 μg) was transfected into COS7 cell grown on chamber slideε (Nunc) with lipofecta in (GIBCO BRL) . After 72 hourε cellε were incubated for one hour with medium containing 0.5 μg/ml /8CF-Fc. Unbound Fc-fuεion proteinε were removed by three waεheε with cold DMEM/F12 and the cellε were fixed with 4% paraformaldehyd in PBS. Immunostaining waε performed with ABC staining εyεtem (Vector Lab) , uεing biotinylated anti-human IgG antibodieε (Fc specific; Jackεon Labε, West Grove, PA) following by εtreptavidin alkaline phosphatase and NBT/BCIP as substrate according to the protocol provided by the manufacturer. One positive pool

(#54) was subdivided and rescreened until a single clone (F8) was isolated.

DNA sequence determination was carried out uεing the dideoxy-chain termination method (Sanger et al., 1977, Proc. Natl. Acad. Sci., USA 74:5463), with Sequenase 2.0 (United States Biochemical Corporation, Cleveland, OH) . Sequencing was performed on both strands by priming with synthetic oligonucleotides.

6.1.4. CONSTRUCTION OF RPTPS/EGF-

RECEPTOR CHIMERAS

To generate a plasmid for the expression of /SCF/EK chimeras, a portion of the extracellular domain of RPTP/S containing the CAH and the FINIII domains (/SCF, aa 1-418) was fused to the human EGF receptor at position 634, twelve amino acids after the transmembrane domain in its extracellular region. These fragmentε were amplified uεing pfu

(Stratagene, La Jolla, CA) with a εpecific εet of primerε that introduce a BstBI site at the junction between the two genes. The reεulting fragmentε were ligated into Blueεcript

(Stratagene, La Jolla, CA) . Proper fuεion between the two molecules waε verified by nucleotide εequence analysis. This

chimeric gene was then subcloned into a NotI site in the reteroviral vector SRα-SL and viral stockε where prepared by cotranεfecting COS-7 cellε with thiε vector along with a helper viruε plaεmid (Muller et al., 1991, Mol. Cell. Biol., 11:1785-1792). These viruses where used to infect NIH 3T3 (clone 2.2), which lack endogenous EGF-receptor. Following infection, cells where εelected in a medium containing lmg/ml G418 (Gibco-BRL) and reεiεtant colonieε were individually grown and aεsayed for the expression of the chimeric receptor by Western blotting with antibodies against the carboxyl terminus of the EGF-R (Kris et al., 1985, Cell, 40:619-625) as described previously (Peles et al., 1992, Cell, 69:205- 216) .

6.1.5. BINDING OF FC-FUSION PROTEINS

Confluent monolayer of cells were incubated for one hour with conditioned medium containing 0.25-0.5 mg/ml Fc-fusion protein. The unbound proteinε were removed by three waεheε with binding medium (0.1% BSA, 0.2% none fat dry milk in DMEM/F12) and the cells were further incubated with 1 ng/ml [ ^12S I]-Protein A (Amersham) , for 30 minuteε at 4°C Plates were washed three times with cold binding medium and cell bound radioactivity was determined as described previously (Peles et al., 1993, EMBO J. , 12:961-971). Cellular staining using the Fc-fusion proteins was done using the procedure described above for expresεion cloning.

6.1.6. CHEMICAL CROSSLINKING EXPERIMENTS Cells were incubated for four hours with medium containing, the different Fc-fusion proteinε. Following three waεheε with cold PBS/Ca (1 mM CaCl ₂ in PBS) , the cells were incubated for additional 30 minuteε with PBS/Ca containing l mM DTSSP (3,3'-Dithiobiε[εulfoεuccinimidyl— propionate]. Pierce, Rockford, IL) . Free croεε-linker was removed by additional PBS wash followed by quenching with 100 mM glycine in TBS for 10 minutes at 4°C Cell lyεateε were made in SBN lyεiε buffer (Peles et al., 1991, EMBO J. ,

10:2077-2086), and sepharoεe-protein A waε added to the cleared lysates. Following two hours incubation at 4°C, the beads were washed three times with HNTG buffer (Peles et al., 1991, EMBO J. , 10:2077-2086), and the bound proteins were eluted by adding SDS PAGE sample buffer containing 5% /3- mercaptoethanol and further incubation for 10 minutes at 95 ^β C

6.1.7. PROTEIN PURIFICATION AND SEQUENCING Cellular embraneε were prepared from 5X10 ⁸ GH3 cells by homogenization in hypotonic buffer that included 10 mM Hepes pH 7.5, 1 mM EGTA, 1 mM MgCl ₂ , 10 μg/ml aprotinin, 10 μg/ml leupeptin and 2 mM PMSF. Nuclei and unbroken cells were removed by low speed centrifugation (lOOOg x 10 minuteε at 4 ^β C) , and the supernatant was then εubjected to high εpeed centrifugation at 40000g (30 minuteε at 4°C). The membrane pellet waε resuspended in SML solubilization buffer (2% Sodium onolaurate, 2 mM MgC12, 2 mM PMSF in PBS). After one hour incubation on ice the detergent-insoluble materials was removed by centrifugation, and the sample was diluted tenfold with PBS containing 2 mM MgCl ₂ . This sample was loaded on a column of /3CF-FC bound to Sepharose Protein A (200 μg /3CF- Fc/ l beads) at 4°C The column was washed with SML buffer containing 0.15% detergent and the bound proteins were eluted by adding SDS sample buffer and heating to 95°C Proteinε were εeparated on 7.5% gel and electroblotted in CAPS buffer (100 mM CAPS, 10% MeOH) to ProBlott membrane (Applied Biosystems) . The membrane was stained with coomaεεie R-250 and the 140 kDa band waε excised and εubjected to direct microsequencing analysiε. Microεequencing waε performed with an Applied Biosystemε Model 494 sequencer, run using standard reagents and programs from the manufacturer.

To obtain internal peptide εequence the blotted band was moistened with neat acetonitrile, then reduced by the addition of 200 ul of 0.1 M Tris pH 8.5, 10 mM dithiothreitol, 10% acetonitrile. After incubation at 55°C for 30' the εample was cooled to room temperature and 20 ul

of 0.25M 4-vinylpyridine in acetonitrile added. After 30 minuteε at room temperature the blotε were waεhed 5 times with 10% acetonitrile. Digestion waε performed for 16 hourε with 1 ug modified trypεin (Promega) in 50 ul of 0.1M Triε pH 8.0, 10% acetonitrile, 1% octylglucoεide. Digestion waε εtopped by the addition of 2 ul of neat trifluoroacetic acid (TFA) . Peptideε were separated on a 1 mm x 200 mm Reliasil C-18 reverse phase column on a Michrom UMA HPLC run at 50 ul per minute. Solvents uεed were 0.1% TFA in water and 0.085% TFA in 95% acetonitrile/5% water. A linear gradient of 5 to 65% B waε run over 60 minuteε. Absorbance was monitored at 214 nm and peaks were collected manually into a 96 well polyethylene microtitre plate. Purified peptides were sequenced as described above.

6.1.8. TREATMENT WITH PI-PLC Cellε grown to confluency in 90 mm dishes were metabolically labeled with lOOμ Ci/ml [ ^3$ S]-methionine and cysteine mix (NEN, Boston, MA) for four hours at 37 ^β C Labeled cells were washed three timeε with MEM and incubated with 250 mU of phosphatidylinoεitol εpecific phospholipase C (PI-PLC, Boehringer Mannheim or a kind gift from Dr. J. Salzer) for 50 minutes at 37 ^β C The εupernatant waε collected and cleared by centrifugation (lOOOg) , membranes were prepared from the cells and further solubilized in SML buffer as described above. /SCF-Fc bound to Sepharose-protein A beads was added to the supernatant and the membrane fractionε for one hour at 4°C The beadε were waεhed twice with 0.15% sodium monolaurate in PBS and once in PBS before the addition of SDS sample buffer. The precipitated proteins were separated on 7.5% cell and subjected to autoradiography.

For binding experiments, cell were treated with different amounts of PI-PLC (as indicated in the legend to the figures) in MEM containing 0.5% BSA for 30-60 minutes at 37 ^β C Cellε were briefly waεhed and binding of /SCF-Fc waε performed as described above.

6.2 . RESULTS

6.2.1. THE CAH DOMAIN OF RPTP/3 MEDIATES AN INTERACTION WITH NEURONS

To identify cellular ligands for RPTP/S, fusion proteins were constructed between different subdomainε of RPTP/3 and the Fc portion of human IgG. Three chimeric conεtructε were made, one containing both the carbonic anhydraεe and the fibronectin domains (/SCF-Fc) and two others carrying each domain by itself (/8C-FC or /3F-FC) . Initially, /SCF-Fc was used to screen for a membrane bound ligand on the surface of different neuronal and glial cell lineε. Several cell lines that bind this fusion protein were identified. These were the IMR-32 neuroblastoma cellε, the two closely related neuroendocrine derived cell lines GH3 and GH1, and five different glioblastoma cell lines.

The fact that these positive cell lines were derived from glial and neuronal origins raised the poεεibility that RPTP/3 may interact with two different membrane-associated ligands. Alternatively, a single ligand may exist which is expressed by both neurons and glia cellε. To explore these two possibilitieε it waε examined whether a fuεion protein that contained only the CAH domain of RPTP/S (/3C-Fc) will retain the same cell specificity observed with /SCF-FC It was reasoned that in a multidomain receptor like RPTP/8, each domain might function aε an independent unit in terms of its interaction with a specific ligand. Thus, the use of a single domain in binding experiments might allow the identification of a cell type specific ligand. Aε depicted in Fig.2A, thiε fuεion protein, indeed, binds to the same neuronal and neuroendocrine cell lines. In contraεt, none of the glioblaεtomaε were poεitive, εuggesting that there are at least two ligandε for RPTP/3 that are differentially expreεεed on neuronal or glial cells. Thiε reεult alεo implied that the CAH domain mediateε the interaction of RPTP/3 with a specific ligand preεent in neuronε but not in glia cellε.

Accordingly, if the binding of /3C-FC to neuronal ligand reflectε the interactionε occurring in vivo, one would expect to see similar binding εpecificity on cultureε of primary neurons. The binding of the different fusion proteins to cultured dorεal root ganglion cellε (DRG) , followed by detection of the bound proteinε by immunostaining, was analyzed. /SC-FC and _/ SCF-FC bound to GH3 cells, as well as to the primary neurons. A fuεion protein containing the fibronectin domain alone (/3F-Fc) failed to bind to either GH3 cellε or DRG neuronε. In other experimentε, binding of _/ SF-FC to εeveral glial cell lineε waε detected, but no binding of this domain to neuronal derived cell lineε or neuronε derived from rat DRGε and chick cortex was detected. In addition, it waε examined whether the binding specificity observed with the CAH domain of RPTP _/ S is unique to this receptor by comparing it with the related phosphatase RPTP7 (Barnea et al., 1993, Mol. Cell. Biol., 13:1497-1506). A fusion protein made with the CAH domain of this highly homologous family member did not bind to GH3 cells or to primary neurons. Altogether these resultε suggests that specific ligands for RPTP/S exist on the surface of cellε from neuronal and glial origin. Different subdo ains of the receptor mediate its interaction with those diεtinct ligandε. The CAH mediates an interaction with neurons while the FNIII enables the interaction of RPTP/S with glia cellε. In the work presented here, the identification and molecular characterization of the ligand for the CAH domain is described.

6.2.2. COVALENT CROSSLINKING EXPERIMENTS

REVEAL A 140 KDA PROTEIN THAT INTERACTS WITH THE CAH DOMAIN OF RPTPfl

To characterize ligands for RPTP/3, a reversible cross¬ linker (DSSTP) waε uεed, and proteinε were εought that εpecifically bound to /3C-Fc. Two of the cell lineε that bound /3C-Fc (IMR32 and GH3) , as well as COS7 cells aε a control, were allowed to react with the fuεion proteinε

containing the FNIII or the CAH domains followed by cross¬ linking and precipitation of the complexes. As shown in Fig. 3, a protein of about 140 kilodalton specifically reacted with /3c-Fc in the rat GH3 and human IMR-32 cells. No reactivity was detected in control cellε or in cellε incubated with /SF-Fc. The croεε-linker (DSSTP) uεed, undergoes cleavage in the reducing SDS PAGE conditions and, therefore, permits the identification of the true molecular weight of the putative ligand. This result suggeεted that the same ligand is expressed in the rat GH3 and the human IMR-32 lines.

6.2.3. MOLECULAR CLONING OF A CANDIDATE LIGAND FOR RPTP3 FROM RAT 6H3 CELLS REVEALS THE RAT HOMOLOGUE OF CONTACTIN

An expression cloning strategy was employed in an effort to clone the gene that encodes the 140 kDa candidate ligand. we have employed. Plasmid pools made from a GH3-CDNA library were transfected into COS7 cells and the cells were screened for their ability to bind /3CF-Fc. Positive cellε were detected by immunostaining with biotinylated anti-human IgG antibodieε and sterptavidin alkaline phosphatase. One positive pool was identified that when transfected yielded several stained cells on the elide. Thiε pool waε subdivided and rescreened four timeε until a εingle clone (F8) waε isolated. Transfection of COS7 cellε with thiε plaεmid resulted in positive staining of approximately 25%-50% of the cells, a number that correlates well with the maximum transfection efficiency in our system. DNA εequence analyεeε of clone F8 εhowed that it contained a 3.9 kb insert and a single long open reading frame of 3063 nucleotides. The deduced 1021 amino acid sequence encoded by this clone has been presented elεewhere. Data bank εearch with thiε sequence εhowed that it εhareε 95% and 99% identity at the amino acid level with human and mouεe contactin reεpectively (Berglund and Ranscht, 1994, Genomics, 21:571-582; Gennarini et al., 1989, J. Cell. Biol., 109:755-788; Reid et al. , 1994,

Brain Res. Mol. Brain Res., 21:1-8). It was therefore concluded that the ligand for RPTP/3 cloned from GH3 cellε iε the rat homologue of contactin. Structurally, this protein consiεtε of εix C2 type Ig domainε, four fibronectin type III repeatε and an hydrophobic region that mediateε itε attachment to the membrane by a GPI linkage (Gennarini et al., 1989, J. Cell. Biol., 109:755-788; Reid et al., 1994, Brain Reε. Mol. Brain Reε., 21:1-8). Functionally, it iε a neural cell adhesion molecule that haε been suggested to play a morphogenic role during the development of the nervous system (Rathjen et al., 1987, J. Cell. Biol., 104:343-353; Walsh and Doherty, 1991, Cell. Biol. Int. Rep., 15:1151- 1166) .

In parallel to the expression cloning strategy, and as a complementary approach, a biochemical procedure was employed that utilized the CAH domain as an affinity reagent for protein purification. pl40 was purified from εolubilized membranes prepared from GH3 cellε on a column of /SCF-Fc. After reεolving the eluted protein on SDS/PAGE, the 140 kDa εpecieε waε subject directly to N-terminal sequencing, or was digested with trypsin. Two peptide sequences obtained, one from the N-terminus and the other from an internal peptide after tryptic digeεt. Both sequences matched the translated F8 sequence and confirmed that contactin iε indeed a ligand for the CAH domain of RPTP/S.

6.2.4. BINDING ANALYSIS OF RPTPB AND CONTACTIN

The binding epecificity of different subdomains of RPTP/S towardε contactin waε examined. COS7 cells were transfected with rat contactin (clone F8) and analyzed for their ability to bind fuεion proteinε containing the CAH, FNIII or both domainε. Aε expected, expreεεion of contactin enabled the binding of the CAH domain of RPTP/S to the cells. The FNIII domain alone did not bind to contactin expressing cells. In addition, similar resultε were obtained with a fusion protein that carries most of the extracellular region of the short form of RPTP/3 (aa 1-644; data not εhown).

The reciprocal interaction, namely, whether soluble contactin molecules are able to bind specifically to cells expresεing RPTP/S, was explored next. In these experiments, C0S7 cells were transfected with chimeric receptor constructs that consist of the entire extracellular region of the short form of RPTP/S (/SCFS/EK) , the CAH domain plus the FNIII repeat (/SCF/EK) , or the CAH domain alone (/8C/EK) fused to the transmembrane and intracellular domains of the EGF receptor. A chimeric receptor was used instead of the wild type phosphatase because the wild type phosphataεe waε not able to be expressed in heterologous cells. Human contactin-Fc fusion protein binds to cells transfected with these chimeric receptors but not to control cells. Taken together, these results demonstrate that expression of contactin is both necessary and sufficient for binding to the CAH domain RPTP/S.

6.2.5. SOLUBLE CONTACTIN RELEASED FROM THE

MEMBRANE BY PHOSPHOLIPASE C TREATMENT INTERACTS WITH RPTP/3

Contactin belongs to a family of recognition molecules that TAG-1 and BIG-1, all of which are anchored to the plasma membrane via a glycosyl-phoεphatidylinoεitol (GPI) . Therefore, it waε of intereεt to see how phospholipase C (PI¬ PLC) treatment would effect the interaction between contactin and RPTP/3. When incubated with C0S7 cells expressing contactin (clone F8) , PI-PLC completely abolished the binding of /SCF-Fc to the cells. Similar results were obtained also with GH3 cells.

It haε been demonεtrated that memberε of thiε family and other GPI-linked proteinε may exiεt either in a membrane bound or a εecreted soluble form that iε released from the cell surface (Furley et al. , 1990, Cell, 61:157-170; Theveniau et al., 1992, J. Cell. Biochem., 48:61-72). Hence, it was examined whether the different forms of contactin, including those released after PI-PLC treatment, could interact with RPTP/8. To this aim, GPI-linked proteins were released from metabolically labeled GH3 cells with the enzyme

and purified contactin by bioaffinity precipitation from membrane extracts of the cells or the cell supernatantε. Without PI-PLC treatment, two proteinε pl40 and pl90 from the membrane fraction could εpecifically aεεociate with /8C-Fc. Theεe proteins were not preεent in the supernatant and they were not detected with /8F-Fc. However, after PI-PLC treatment, pl40/contactin could be precipitated from the medium of the cells, indicating that the soluble form produced by phospholipase treatment interacts with RPTP/3. This result may suggest that, in addition to the interaction between the membrane bound forms of these proteins, soluble contactin could potentially interact in vivo with RPTP/3. βC- Fc could precipitate the 190 kilodaltons protein only from membrane fraction and not from the cell εupernatant. PI-PLC treatment did not releaεe this protein from the cells suggesting that it is either an integral membrane protein or a cytoskeletal protein associated with contactin complexes. Since contactin by itself is sufficient to mediate the interaction with RPTPS, the 190 kDa protein may be associated with contactin in the cells and coprecipitated with it during the bioaffinity procedure. One intriguing possibility is that pl90 iε a εignaling unit uεed by contactin on the surface of neurons (see below) .

7. EXAMPLE: THE CAH DOMAIN OF RPTP3

INDUCES CONTACTIN MEDIATED NEURITE OUTGROWTH

The subsections below describe the induction, by the CAH domain of RPTP/S, of contactin mediated neurite outgrowth. It is shown that the CAH domain of RPTP/S is a permissive substrate for neuronal adhesion and neurite growth. In addition, it is alεo εhown that the neurite growth, differentiation and εurvival induced by the carbonic anhydraεe-like domain of RPTP/S iε mediated by neuronal contactin.

7.1. MATERIALS AND METHODS The materials and methods for this example were the same as those set forth in the example described in section 6.1 above, except as supplemented or amended below.

7.1.1. NEURITE OUTGROWTH ASSAYS Neurite outgrowth assays using IMR 32 cells were performed as described previously (Friedlander et al., 1994, J. Cell. Biol., 125:669-680) using 35 mm petri dishes coated with different proteins adsorbed on the substrate. After blocking the dishes with 1% BSA/PBS, the blocking solution was replaced with 3 X 10 ⁴ cells suspended in 140 μl of DMEM/F12/ITS. Following incubation for 3 hrs at 37 ^β C during which time most of the cells adhered to the dish, the medium was removed and replaced with DMEM/FI2/ITS medium containing antibodies (Ig fraction purified by ammonium sulfate precipitation and DE52 chromatography) . Dishes were incubated for 48 hrs and fixed with Hanks/0.3% sucrose 2.5% paraformaldehyde. For PI-PLC treatment, primary tectal neurons (5 X IO ⁴ cells/250 ml) were prepared from E9 chick embryos (Grumet et al., 1984, Proc. Natl. Acad. Sci. USA, 81:267-271) and incubated with 0.25 μl of PIPLC (1.7 u/ml) in DMEM/F12/ITS+ at 37°C for 30 min. The cell suspension was then incubated on dishes coated with different substrateε without changing the medium.

7.2. RESULTS

7.2.1. NEURITE OUTGROWTH INDUCED BY THE CAH DOMAIN OF RPTP3 IS MEDIATED THROUGH CONTACTIN

Contactin haε been εhown to be involved in both poεitive and negative reεponεeε of neuronε to variouε stimuli (Briimmendorf and Rathjen, 1993, J. Neurochem., 61:127-1219). When presented as a ligand to neurons, either as a membrane- bound or a soluble form, contactin induces axonal growth (Brummendorf et al., 1993, Neuron, 10:711-727; Clarke et al.,

1993, Eur. J. Cell. Biol., 61:108-115; Durbec et al., 1992, J. Cell. Biol., 117:877-887; Gennarini et al., 1989, J. Cell. Biol., 109:755-788). Its neural receptor has been identified as the recognition molecule Nr-CAM (Morales et al., 1993, Neuron 11:1113-1122). On the other hand, contactin itself is a receptor preεent on neuronε and mediateε their repuleion by the extracellular matrix protein januεin (Pesheva et al., 1993, Neuron, 10:69-82). The resultε described in the example of Section 6.1 indicate that the CAH domain of RPTP/S can bind to contactin on cells. To analyze effectε of thiε binding on neuronε, chick tectal cellε, known to express contactin, were plated on disheε previouεly coated with SCF- Fc fusion protein or with Ng-CAM or laminin as controls. Cells attached and grow procesβeε on both of theεe εubεtrates. Treatment of the cellε with PI-PLC prior to plating completely abolished cell attachment and neurite extension on RPTP/S. In contrast, PI-PLC did not have a dramatic effect on cellε growing on Ng-CAM or laminin aε substrate. Thuε, it waε concluded that the CAH domain of RPTP/S is a permissive substrate for neuronal adhesion and neurite growth. Moreover, the cell adhesion and axonal elongation induced by RPTP/8 iε mediated through a GPI- anchored receptor.

Next it waε inveεtigated whether contactin could be the neuronal receptor for the CAH domain of RPTP/3. To thiε aim, a human neuroblastoma cell line IMR-32 waε uεed that haε the capacity to differentiate and to elaborate neuriteε in response to different stimuli (Liidecke and Unnsicker, 1990, Cancer, 65:2270-2278) . Theεe cellε have fibroblastic morphology when crown on petri disheε coated with fibronectin, but on laminin εubεtrateε they aεsume a neuronal phenotype and extend processes with growth cones. A similar morphologic differentiation was εeen after plating the cellε on the CAH domain of RPTP/S. In contraεt, the CAH domain of RPTP7 had no effect on cell adhesion and differentiation. These resultε show that IMR-32 cells respond specifically to the carbonic anhydrase domain of RPTP/3. To determine whether

contactin could be acting as a receptor on the IMR-32 cells for RPTP/3, the effects of antibodies against contactin on the growth of cells on different substrateε were teεted. Antibodieε againεt contactin inhibited the growth of procesεeε on /3c-Fc and /3CF-Fc but not on laminin. In the presence of these antibodieε, the IMR-32 cellε also retracted their processes and many cells lifted off the dish yielding fewer cells after 2 days of incubation. No effect was observed with control antibodies. Thus, the neurite growth, differentiation and survival induced by the carbonic anhydrase-like domain of RPTP/8 is mediated by contactin present in the neurons.

8. EXAMPLE: THE CLONING OF p!90 AND THE

INTERACTION BETWEEN IT AND CONTACTIN

The subsectionε below deεcribe the purification and sequencing of pl90 protein and the εubεequent cloning of rat and human pl90 cDNA. The interaction between pl90 and contactin is also demonstrated.

β.l MATERIALS AND METHODS 8.1.1 protein Purification and Sequencing

Solubilized membrane lysate was prepared from 3X10 ⁹ GH3 cellε and loaded on a column of /SCF-Fc bound to Sepharoεe protein A (Pharmacia) aε deεcribed previouεly (Peleε et al., 1995, Cell, 82:251-260). Bound proteinε were separated on 6.5% SDS gel, blotted to ProBlot membrane (Applied Biosyεtem, Inc.) and stained with Coomassie R-250. To obtain internal peptide secpience, the blotted 190 kDa band was moistened with neat acetonitrile and then reduced by the addition of 200 ul of 0.1M Tris pH 8.5, 10 mM dithiothreitol, 10% acetonitrile. Digestion was performed for 16 hourε with 1 μg modified trypεin (Promega) in 50 μl of 0.1M Triε pH 8.0, 10% acetonitrile, 1% octylglucoεide. Digestion was stopped by the addition of 2 μl of neat trifluoroacetic acid (TFA) . Peptides were separated on a 1 mm x 200 mm Reliasil C-18 reverεe phase column on a Michrom UMA HPLC run at 50 ul per

minute. Solvents used were 0.1% TFA in water and 0.085% TFA in 95% acetonitrile/5% water. A linear gradient of 5 to 65 % B was run over 60 minuteε. Absorbance was monitored at 214 nm and peaks were collected manually into a 96 well polyethylene microtiter plate. Purified peptides were sequenced as deεcribed (Peleε et al., 1995, Cell, 82:251- 260) .

8.1.2 Cloninσ of Rat and Human CASPR/P190 CDNA The εequence of one tryptic peptide obtained from the purified protein (QNLPQILEES) was found in a 900 bp EST fragment B102/LF98 from the BRCA1 region on chromosome 17q21 (Friedman et al., 1994, Cancer Reε., 54:6374-6382). Primerε corresponding to this region (5' primer: TCG CAG GCT ATG AGC CTG GCT ACA TCC; 3' primer: GTG GGT AGG GGA GGT TTG CTG CCA GG) were use for RT-PCR to clone this DNA fragment from rat GH3 cells. A 600 bp DNA fragment derived from this region was further used as a probe to screen a ZAPEX-GH3 cDNA library. This cDNA library waε constructed in ZAP-Expresε phage (Stratagene, San Diego, CA) , using oligo dT priming. Plate hybridization and other cloning techniques were performed according to standard procedureε (Sambrook et al., 1989, Molecular cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory)). Clone ZX5 had a 2.5 kb insert that contained in addition to the B102 fragment, a sequence downstream that matched additional peptide sequence. A second cDNA library was made from GH3 mRNA by priming with a specific oligonucleotide GGA GGT CTC CTT TAG according to the sequence that was found in the 5' end of clone ZX5. This cDNA was cloned into ZAP- Express (Stratagene, San Diego, CA) to generate ZB-GH3 library. This library waε uεe to isolate multiple cloneε that overlapped with ZX5 and contained the 5'end of the gene. To clone the human gene a cDNA library wae made from IMR32 neuroblastoma cellε in ZAP-Expresε (ZX-IMR) . Probeε were generated by PCR from the 5' endε of rat clone ZB181 and from IMR32 cDNA according to the B102 εequence aε deεcribed above

for the rat gene. Several clones had a 5 kb insert that contained the full length gene. DNA sequence determination waε carried out uεing the dideoxy-chain termination method with Sequenase 2.0 (United Stateε Biochemical Corporation, Cleveland, OH) . Sequencing was performed on both strands by priming with synthetic oligonucleotides.

8.1.3. Expression Constructs

An EcoRI-Xhol fragment containing the 5' end of rat CASPR/pl90 (from clone ZB161) was ligated with an Xhol-EcoRI fragment containing the 3* end of the gene (from clone ZB181) and cloned into pCMPl (Peles et al., 1995, Cell, 82:251-260) to generate pCM190R. An HA-tagged version of the gene was constructed by replacing an EcoRI-AccI fragment with a PCR- generated fragment containing the HA-tag sequence. This resulted in the addition of the HA sequence to the 3* end of the coding region of rat CASPR/p190 to generate pCM190HA. Construction of contactin expression vectors was previously described (Peles et al., 1995, Cell, 82:251-260). The plasmids pSGT-cSRC and pSGT-fyn, containing human src and fyn genes and the plasmids used for generation of the GST-SH3s fusions were described previously (Erplel et al., 1995, EMBO J., 14:963-975). To generate a GST-fusion protein containing the cytoplasmic tail of rat CASPR/pl90, the corresponding region (aa 1308-1380) was amplified by PCR and cloned into PGEX-4T (Pharmacia) . The sequence of the final construct waε verified by DNA sequencing.

8.1.4. Northern Blot Analysis Multiple tissue northern blots (MTN Blots, Clontech) were Used. A DNA fragment (position 3600-4232 of human CASPR/pl90) waε generated by RT-PCR from IMR32 mRNA. Thiε fragment waε labeled by random priming ("prime it"; Stratagene, San Diego,CA) , purified using PCR-clean column (Qiagen) and used aε a probe. Hybridization was carried out for 16 hours in a buffer containing 5X SSC, 5X Denhart's solution, 50% formamide, 0.2% SDS and 100 ug/ml denatured

salmon sperm DNA at 42oC The blotε were waεhed at 6O0C twice in a buffer containing 0.5X SSC, 0.1% SDS and once with 0.1X SSC, 0.2% SDS. Signalε were detected by autoradiography. The same membraneε were reprobed with a 2 kb human /3-actin cDNA aε a control probe (Clontech, Palo Alto, CA) .

Production of different Ig-fuεion chimeric proteinε and cell binding experiments were done exactly as described previously (Peles et al. , 1995, Cell, 82:251-260). Staining of tiεsue εectionε with antibodieε waε done eesentially aε described (Milev et al., 1994, J. Cell Biol., 127:1703-1715).

8.1.6. Generation of Antibodies

Polyclonal antibodies against CASPR/pl90 were generated according to standard procedures (Hariow, 1990, Antibodies: A Laboratory Manual) . Ab60 was obtained by immunizing rabbits with a GST-fusion protein containing all the cytoplasmic domain of rat CASPR/p190 (GST-190CT) . Affinity purification was achieved first by passing the serum on a column of Sepharose-GST. Then, the unbound material was loaded on a column of GST-190CT Sepharose. Bound antibodies were eluted with 100 mM sodium citrate pH 2.8 and 1.5 M MgCl ₂ . Eluted material waε precipitated with ammonium sulfate, resuspended in DDW and extensively dialysis against PBS. Antibody 87AP was generated against an eight aa long peptide corresponding to the C-terminal sequence of rat CASPR/pl90. Affinity purification on a Sepharose-peptide column was done essentially as described above for Ab60. Antibodies against F3 were previously described (Faivre-Sarrailh et al., 1992, J. Neurosci., 12:257-267). Antibody CST1 that recognize Src, Fyn and Yes waε previously described (Erplel et al., 1995, EMBO J., 14:963-975). Abl8 against Src and Abl6 against Fyn were purchased from Santa Cruz Antibodies (Santa Cruz, CA) . Monoclonal antibody against HA-tag waε purchaεe from Boehringer. Mouεe polyclonal antibody againεt contactin waε

generated by immunization of mice with purified human contactin-Ig fuεion protein according to Yoshihara et al, 1994, Neuron, 13:415-426.

8.1.7. Generation of Anti HCon-lg Sera

Immunoprecipitation and Western blot analysis: COS transfection protocol using Lipofectamine (Gibco-BRL) waε previously described (Peles et al., 1995, Cell, 82:251-260). To detect the association between Contactin and CASPR/pl90 the cells were grown to subconfluency and were metabolically labeled with lOOmCi/ml [ ^3$ S]-methionine and cysteine mix (NEN, Boston, MA) for four hours at 37°C. Membranes were prepared from the cells and further solubilized in SML buffer (2% Sodium onolaurate, 2 mM MgCl ² , 2 mM PMSF in PBS) . /Sc-Fc bound to Sepharose-protein A beads was added to a tenfold diluted supernatant and incubated for two hours at 4°C The beads were washed twice with 0.15% sodium monolaurate in PBS and once in PBS before the addition of SDS sample buffer. The precipitated proteins were separated on 7.5% gel and subjected to autoradiography.

Preparation of rat brain membranes: five P7 rat brains were pooled and homogenized in a glass homogenizer in a buffer containing 20 mM Hepes pH 7.4, 0.32 M sucrose, l mM EGTA, 1.5 mM MgS0 ₄ , 10 μg/ml Aprotinin and Leupeptin and 1 mM PMSF. Nuclei and heavy cell debris were removed by low speed centrifugation (3000g x 10 minuteε at 4°C) , and the supernatant waε then subjected to high speed centrifugation at 40,000g for 60 minutes. The membrane pellet was resuspended in SML solubilization buffer. After one hour incubation on ice the detergent-insoluble materials waε removed by centrifugation. The eample was diluted four to tenfold with PBS containing 2 mM MgCl ₂ and subjected to precipitation with antibodieε or Ig-fuεionε.

Biotinylation of cell eurface moleculeε waε carried out for 20 minuteε at 23°C using 50 μg/ml Biotin-LC-NHS (Pierce) . The reaction waε εtopped by adding NH ₄ C1 to final

concentration of 10 mM followed by two waehes with TBS- glycine buffer (50 mM Tris pH 7.4, 150 mM NaCl and 50 mM glycine) on ice prior to solubilization.

Immunoprecipitation and western blotting was performed aε deεcribed previously (Peles et al, 1992, Cell, 69:205- 216) . Blots were reacted with streptavidin-linked peroxidase (Amersham) and detected using chemiluminescence reagent (Pierce) .

8.2. RESULTS

8.2.1. CASPR/P190 Gene and Gene products

The 190 kD protein which associates with the CAH- contactin complex was purified using affinity chromatography with /Sc-Fc, utilizing the techniques described, above, in Section 8.1. Briefly, membrane lysates from GH3 cells were applied to a Sc-Fc column and bound proteins were separated by SDS-PAGE. The protein believed to correspond to pl90 was excised and subjected to trypsin digestion. The amino acid εequenceε of two tryptic peptideε were determined using a gas-phase microsequencer. The amino acid sequences obtained were then utilized to identify corresponding DNA fragments encoding εuch sequences, as described, above, in Section 8.1. The DNA fragments thus obtained were in turn used to isolate cDNA molecules encoding the full length pl90 gene products of both human and rat.

The human CASPR/pl90 nucleic acid sequence is depicted in SEQ ID N0:1, and the human CASPR/p190 amino acid εequence is depicted in SEQ ID NO:2. The rat CASPR/pl90 nucleic acid secpience is depicted in SEQ ID NO:3, and the rat CASPR/pl90 amino acid secpience is depicted in SEQ ID NO:4.

The human and rat CASPR/pi90 transcripts have open reading frames that encode for 1384 and 1381 amino acids, respectively, and share 93% identity at the amino acid level. CASPR/pl90 is a putative type I transmembrane protein with a short proline-rich. cytoplasmic domain. (The transmembrane domain iε marked aε TMD in Figure 1) .

The first pl90 methionine is followed by a stretch of 19-20 amino acid residues rich in hydrophobic residues, which probably acts aε a signal sequence. The extracellular domains of rat and human CASPR/pl90 contain 1281 and 1282 amino acid residues, respectively. The extracellular region of CASPR/pl90 contains 16 potential N-linked glycosylation sites followed by a second hydrophobic stretch that is a typical transmembrane domain.

The CASPR/pl90 extracellular domain is a mosaic of several motifs known to mediate protein-protein interactions. Near the N-terminus of mature CASPR/pl90 (109 amino acid residues) is a domain with 31-33% amino acid identity to the Cl and C2 terminal domains of coagulation factors V and VIII (Jenny et al., 1987, Proc. Natl. Acad. Sci. U.S.A., 84:4846- 50; Wood et al., 1984, Nature, 312:330-37) and 26% identity with the neuronal adhesion molecule neurophilin (previously known as the neuronal A5 antigen) and 20% identity to a region of discoidin I, a lectin from the slime mold Dictyostelium discoideum (Takagi et al., 1991, Neuron, 7:295- 307) . The domain is marked aε DISC in Figure l. The extracellular domain of CASPR/pl90 also containε four repeats, of approximately 140 amino acid residues each, with homology to neurexins, a family of polymorphic neuronal cell surface proteins. These domains are marked as NX1-NX4 in Figure l. There are 6 copieε of the motif in the α- neurexins, one in the 3-neurexins, and one to five in the C- terminal portions of laminin A, agrin, slit, and perlecan (Ushkaryov et al., 1992, Science, 257:50-56). Together, the five motifs in the basement membrane protein laminin A are referred to as the G domain, a region suggested to mediate cell adhesion. The first three neurexin motifs of CASPR/pl90 share 29-32% amino acid identity to regions of rat neurexinlll-α and neurexinll-α, whereas the fourth motif is most similar to agrin (34% identity) . CASPR/pl90 also containε two epidermal growth factor (EGF)-like moduleε (marked aε EGF1-EGF2 in Figure 1) ; both of which are most related to repeats within the drosophila neurogenic proteinε

Notch and εlit (39-46% identity) (Rothberg et al., 1988, Cell, 55:1047-59; Wharton et al., 1985, Cell, 43:567-81). A εingle domain related to the C-terminal region of fibrinogen beta/gamma (marked as FIB in Figure 1) is flanked by an EGF and neurexin motif. Finally, there is a stretch of 47 amino acids, that iε identical between human and rat CASPR/pl90, and containε εeven copieε of Pro-Gly-Tyr-X^ and three additional imperfect repeats of thiε sequence (marked as PGY in Figure 1) . The Pro-Gly-Tyr repeat iε found in a molluscan adhesive protein (SW:A61077, and a putative chicken prior protein (SW:A46280) , whereas the Pro-X-Tyr repeat is present in multiple copies in a soybean cell wall protein (SW:A29324) and the X-Gly-Tyr repeat in heterogeneous nuclear RNP proteins (SW:B41732) . The cytoplasmic domain of human and rat CASPR/p190 contain 78 and 74 amino acids, respectively. These include a 38-42 amino acid proline-rich motif (38% proline) , the majority of which consists of proline residues alternating with alanine, glycine, or threonine residues (marked as PRO in Figure 1) . Alignment of thiε region with the non-redundant protein database revealed several proteinε containing such "PAPA" motifs. Proline-rich domains can serve as binding siteε for SH3-containing protein, yet none of the proteinε that align with thiε domain of CASPR/pl90 are known to interact with an SH3 motif.

8.2.2. CA8PR/P190 EXPreSBJQn Northern blot analysis of mRNA isolated from human tissues reveals that CASPR/pl90 was expreεεed predominantly in the brain as a 6.2 kb transcript. Weak expression of CASPR/pl90 was detected in ovary, as well as in the pancreas, colon, lung, heart, intestine and testiε. Similar results wee obtained for rat tissue hybridized with a rat CASPR/pl90 probe.

A high level of CASPR/pl90 waε detected in different regionε of the adult human nervouε εyεtem, including high expreεεion in the cortex, cerebellum and in the thalamus, while weaker expresεion iε detected in the εpinal cord and in

the corpus callosum. These analyses demonstrated that the CASPR/pl90 gene was expresεed predominantly in the central nervous syεtem.

Polyclonal rabbit antibodies raised against a GST fuεion protein containing the CASPR/pl90 cytoplasmic domain were raised and used to stain permeabilized human IMR-32 neuroblastoma and rat GH3 neuroendocrine cell lines found to express CASPR/pl90. These studies revealed recognition of a 190 kD protein. Similar reεults were obtained εtaining C0S7 cell lysates that had been transfected with an expression vector directing the synthesis of CASPR/pl90. No CASPR/pl90 was detected in ock-transfected or untransfected cells.

Immunohistochemistry studies were then performed which demonstrated that CASPR/pl90 and contactin localized in the rat retina. Specific CASPR/pl90 staining was seen in the ganglion cell fiber layer and in the inner plexiform layers. Similar staining waε observed for contactin, with the highest expression in the nerve fiber layer containing the axons that project from the ganglion cells into the optic nerve. Thus, CASPR/pl90 and contactin colocalize on neuronε in fiber-rich areas of the retina. Further, increased CASPR/pl90 staining was detected in membrane preparations from rat brains from E18 to post-natal day eight, a period of extensive axonal outgrowth and synaptogenesis. A similar temporal expression pattern was detected in this tissue in the same period (Gennarini et al., 1989, J. Cell Biol. 109:755-788).

8.2.3. Lateral Interaction in the Plasma Membrane Between CASPR/pl90 and Contactin

The interaction between contactin, RPTP/3 and CASPR/pl90 waε then investigated using soluble and membrane-associated variants of these proteins. Specifically, the possibility that the interaction between contactin and CASPR/pl90 requires that both proteinε be preεent on the εame cell (ciε interaction) waε εtudied.

To examine this posεibility, COS7 cellε were transfected with expression vectors that expressed either CASPR/pl90 alone or together with contactin. Lysateε of tranεfected cellε were subject to precipitation analysis with the CAH domain of RPTP/3 (/βC-Fc) . The CAH domain of RPTP/3 only precipitated CASPR/pl90 from cells co-expreεεing contactin. Thuε, it appearε that the CAH domain of RPTP/8 can form a ternary complex with contactin and CASPR/pl90 proteins. Similar reεults were obtained using an expression vector expressing tagged CASPR/pl90.

Moreover, soluble contactin molecules did not associate with CASPR/pl90 when RPTP/S and CASPR/pl90 were co-expressed in the same cells.

On the basis of these experiments, it appears that the CAH domain of RPTP/8 does not bind directly to CASPR/pl90 and that contactin and CASPR/pl90 are complexed by means of lateral interactions (ciε) in the membrane, thus explaining the reason why the protein is referred to as CASPR (i.e. , Contactin-associated protein) .

8.2.4. Complex Formation Between CASPR/pl90 and Contactin The role of RPTP/3 in formation of the CASPR/pl90- contactin complex was next examined. IMR-32 cell lyεateε were eubjected to immunoprecipitation with CASPR/pl90 antibodiee followed by immunoblotting with contactin antibodies. These experiments demonstrated that contactin and CASPR/p190 were constitutively associated on the surface of the IMR-32 cellε. In thiε cell line, it appeared that virtually all CASPR/pl90 molecules were asεociated with contactin.

The exietence of an in vivo contactin-CASPR/pl90 complex waε also demonstrated using rat brain tissue. Lyεates of P7 rat brain membraneε were εubjected to precipitation with βc- Fc followed by immunoblotting with antibodies εpecific to either contactin of CASPR/pl90.

Taken together, theεe data demonεtrate that contactin and CASPR/pl90 are conεtitutively complexed in neuronal cell

lines and tissues and that complex formation between these two proteins does not require RPTP/8.

β.2.5. Interaction Between CASPR/pl90 and SH3 Domains of Signaling Molecules .

Experiments described herein demonstrated that the CASPR/pi90 cytoplasmic domain can serve as a binding site for SH3 domains of signalling molecules which will transmit the signal initiated by RPTP/3 binding to the contactin-CASPR/p190 complex.

Specifically, four of seven GST-SH3 domains of signalling molecules were able to bind selectively to the CASPR/pl90 protein, including the SH3 domains of Src, Fyn, p85 and PLC7. Association was not detected with Csk, Grb2 or Gap SH3 domains. CASPR/pl90 did not bind to a mutant Src SH3 domain in which a conserved Trp at position 118 waε replaced with an Ala residue.

Next it was determined that c-src could associate with CASPR/pl90 fusion proteins in transiently infected COS7 cells. Specifically, lyεates of transfected cells were subjected to immunoprecipitation with antibodies against c- Src, followed by immunoblotting with anti-fusion antibodies.

Further, the association between endogenous c-Src and CASPR/pl90 in IMR-32 or GH3 cells was investigated using a similar immunoprecipitation/immunoblotting strategy with Src and CASPR/pl90 antibodies. Resultε of such experimentε detected no association between c-Src and CASPR/pl90.

These experiments raise the possibility that the cytoplasmic domain of CASPR/pl90 can serve as a target for SH3 domains of signalling molecules.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Peles, Elior

(ii) TITLE OF INVENTION: CASPR/pl90, A FUNCTIONAL LIGAND FOR RPTP-BETA AND THE AXONAL CELL RECOGNITION MOLECULE CONTACTIN

(iii) NUMBER OF SEQUENCES: 4

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Pennie & Edmonds LLP

(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 #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US (B) FILING DATE: 27-MAR-1996

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Coruzzi, Laura A.

(B) REGISTRATION NUMBER: 30,742 (C) REFERENCE/DOCKET NUMBER: 7683-111

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (212) 790-9090

(B) TELEFAX: (212) 869-8864/9741

(C) TELEX: 66141 PENNIE

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5294 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 218..4370

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:lι

CAAGAGCGGA GGACCAGGAA CCAGAGAGAG AGAGAGAGAA AAGAGAGAGG AGAGACAGAG 60

CβCTTGGGGG CGAAAGGAGA GAGGGAGGGA AGGGTGGGTA AGGAGGAGAG AGCGGTCTGC 120

TGCAAACCCC AGGAGGAGAG CTTGGAGCCC AAGCCAGAAC TCGAGCCCTA GCCGGAGCCG 180

TTCACAGGGA GGCGGCTGCC GGGACCGTCA GCCCTGC ATG ATG CAT CTC CGG CTC 235 Met Met His Leu Arg Leu

1 5

TTC TGC ATC CTG CTC GCC GCG GTC TCA GGA GCC GAG GGC TGG GGC TAC 283 Phe Cys lie Leu Leu Ala Ala Val Ser Gly Ala Glu Gly Trp Gly Tyr 10 15 20

TAC GGC TGC GAC GAG GAG CTG GTG GGT CCC CTG TAT GCA CGC TCC CTG 331 Tyr Gly Cys Asp Glu Glu Leu Val Gly Pro Leu Tyr Ala Arg Ser Leu 25 30 35

GGC GCC TCC TCC TAC TAC AGT CTC CTT ACT GCG CCG CGA TTC GCC AGG 379 oiy Ala Ser Ser Tyr Tyr Ser Leu Leu Thr Ala Pro Arg Phe Ala Arg

40 45 50

CTG CAC GGC ATA AGC GGG TGG TCA CCA CGG ATT GGG GAT CCG AAT CCC 427 Leu His Gly lie Ser Gly Trp Ser Pro Arg lie Gly Asp Pro Asn Pro 55 60 65 70

TGG CTC CAG ATA GAC TTA ATG AAG AAG CAC CGG ATC CGG GCC GTG GCC 475 Trp Leu Gin lie Asp Leu Met Lys Lye His Arg lie Arg Ala Val Ala

75 80 85

ACA CAG GGC TCC TTT AAT TCT TGG GAC TGG GTC ACA CGT TAC ATG CTA 523 Thr Gin Gly Ser Phe λβn Ser Trp Asp Trp Val Thr Arg Tyr Met Leu 90 95 100

CTC TAC GGC GAC CGA GTG GAC AGC TGG ACA CCG TTC TAC CAG CGA GGG 571 Leu Tyr Gly Asp Arg Val Asp Ser Trp Thr Pro Phe Tyr Gin Arg Gly 105 110 115

CAC AAC TCG ACC TTC TTT GGT AAC GTG AAC GAG TCG GCG GTG GTG CGC 619 His Asn Ser Thr Phe Phe Gly Asn Val Asn Glu Ser Ala Val Val Arg 120 125 130

CAT GAC CTG CAC TTC CAC TTC ACT GCG CGC TAC ATC CGC ATC GTG CCC 667 His Asp Leu His Phe His Phe Thr Ala Arg Tyr lie Arg lie Val Pro 135 140 145 150

CTG GCC TGG AAC CCA CGC GGC AAG ATC GGC CTG AGG CTC GGC CTC TAT 715 Leu Ala Trp Asn Pro Arg Gly Lys lie Gly Leu Arg Leu Gly Leu Tyr 155 160 165

GGC TGC CCA TAC AAG GCC GAC ATA CTC TAT TTC GAC GGC GAC GAT GCC 763 Gly Cys Pro Tyr Lys Ala Asp lie Leu Tyr Phe Asp Gly Asp Asp Ala 170 175 180

ATC TCC TAC CGC TTC CCG CGA GGG GTC AGC CGA AGC CTG TGG GAC GTG 811 lie Ser Tyr Arg Phe Pro Arg Gly Val Ser Arg Ser Leu Trp Asp Val 185 190 195

TTC GCC TTC AGC TTC AAG ACC GAG GAG AAG GAC GGT CTT CTG CTG CAC 859 Phe Ala Phe Ser Phe Lys Thr Glu Glu Lys Asp Gly Leu Leu Leu His 200 205 210

GCC GAG GGC GCC CAG GGC GAC TAC GTG ACG CTC GAG CTG GAG GGG GCA 907 Ala Glu Gly Ala Gin Gly Asp Tyr Val Thr Leu Glu Leu Glu Gly Ala 215 220 225 230

CAC CTG CTG CTG CAC ATG AGC CTG GGC AGC AGC CCT ATC CAG CCA AGA 955 His Leu Leu Leu His Met Ser Leu Gly Ser Ser Pro He Gin Pro Arg 235 240 245

CCA GGT CAC ACC ACC GTG AGC GCA GGC GGA GTC CTC AAT GAC CAG CAC 1003 Pro Gly His Thr Thr Val Ser Ala Gly Gly Val Leu Asn Asp Gin His 250 255 260

TGG CAC TAT GTG CGG GTG GAC CGA TTT GGC CGC GAT GTA AAT TTC ACC 1051 Trp His Tyr Val Arg Val Asp Arg Phe Gly Arg Asp Val Asn Phe Thr 265 270 275

CTG GAC GGC TAT GTG CAG CGC TTT ATT CTC AAT GGA GAC TTC GAG AGO 1099 Leu Asp Gly Tyr Val Gin Arg Phe He Leu Asn Gly Asp Phe Glu Arg 280 285 290

CTG AAC CTG GAC ACT GAG ATG TTC ATC GGA GGT CTG GTG GGC GCC OCG 1147 Leu Asn Leu Asp Thr Glu Met Phe He Gly Gly Leu Val Gly Ala Ala 295 300 305 310

COG AAG AAC CTG GCC TAT CGG CAT AAC TTC CGC GGC TGC ATA GAA AAC 1195 Arg Lys Asn Leu Ala Tyr Arg His Asn Phe Arg Gly Cys He Glu Asn 315 320 325

GTA ATC TTC AAC CGC GTC AAC ATC GCA GAC CTG GCC GTG CGG CGC CAT 1243 Val He Phe Asn Arg Val Asn He Ala Asp Leu Ala Val Arg Arg His 330 335 340

TCC CGG ATC ACC TTC GAG GGT AAG GTG GCT TTT CGT TGC CTG GAC CCG 1291 ser Arg He Thr Phe Glu Gly Lys Val Ala Phe Arg Cys Leu Asp Pro 345 350 355

GTA CCG CAC CCT ATC AAC TTC GGA GGC CCT CAC AAC TTC GTT CAA GTG 1339 Val Pro His Pro He λβn Phe Gly Gly Pro His Asn Phe Val Gin Val 360 365 370

CCC GGT TTC CCA CGC CGT GGC CGC CTG GCA GTC TCA TTT CGC TTC CGC 1387 Pro Gly Phe Pro Arg Arg Gly Arg Leu Ala Val Ser Phe Arg Phe Arg 375 380 385 390

ACC TGG GAC CTC ACC GGG CTT CTC CTT TTC TCC CGT CTG GGG GAC GGG 1435 Thr Trp Asp Leu Thr Gly Leu Leu Leu Phe Ser Arg Leu Gly Asp Gly

395 400 405

CTG GGC CAC GTG GAG CTG ACG CTC AGC GAA GGG CAG GTC AAC GTG TCC 1483 Leu Gly His Val Glu Leu Thr Leu Ser Glu Gly Gin Val Asn Val Ser 410 415 420

ATC GCG CAG AGC GGC CGA AAG AAG CTT CAG TTC GCT GCT GGG TAC CGA 1531 He Ala Gin Ser Gly Arg Lys Lys Leu Gin Phe Ala Ala Gly Tyr Arg 425 430 435

CTG AAT GAC GGC TTT TGG CAC GAG GTG AAT TTT GTG GCA CAG GAA AAC 1579 Leu Asn Asp Gly Phe Trp His Glu Val λβn Phe Val Ala Gin Glu Asn 440 445 450

CAT GCA GTT ATC AGC ATT GAT GAT GTG GAA GGG GCA GAG GTC AGG GTC 1627 His Ala Val He Ser He λβp Asp Val Glu Gly Ala Glu Val Arg Val 455 460 465 470

TCA TAC CCG TTG CTG ATC CGG ACA GGG ACC TCA TAT TTC TTT GGG GGT 1675 ser Tyr Pro Leu Leu He Arg Thr Gly Thr Ser Tyr Phe Phe Gly Gly

475 480 485

TGT CCC AAG CCA GCC AGT CGA TGG GAC TGC CAC TCC AAC CAG ACG GCA 1723 Cys Pro Lys Pro Ala Ser Arg Trp Asp Cys His Ser Asn Gin Thr Ala 490 495 500

TTC CAT GGC TGC ATG GAG CTG CTC AAG GTG GAT GGT CAA CTG GTC AAC 1771 Phe Hie Gly Cys Met Glu Leu Leu Lys Val Asp Gly Gin Leu Val λβn 505 510 515

CTG ACT CTG GTG GAG GGC CGG CGG CTT GGA TTC TAT GCT GAG GTC CTC 1819 Leu Thr Leu Val Glu Gly Arg Arg Leu Gly Phe Tyr Ala Glu Val Leu 520 525 530

TTT GAT ACA TGT GGC ATC ACT GAT AGG TGC AGC CCT AAC ATG TGT GAG 1867 Phe Asp Thr Cys Gly He Thr Asp Arg Cys Ser Pro Asn Met Cys Glu 535 540 545 550

CAT GAT GGA CGC TGC TλC CAG TCT TGG GAT GAC TTC ATT TGC TAC TGC 1915 His Asp Gly Arg Cys Tyr Gin Ser Trp λβp λβp Phe He Cys Tyr Cys 555 560 565

GAA CTG ACG GGC TAC AAG GGA GAG ACC TGC CAC ACA CCT TTG TAT AAG 1963 Glu Leu Thr Gly Tyr Lys Gly Glu Thr Cys His Thr Pro Leu Tyr Lys 570 575 580

GAA TCC TGT GAG GCT TAT CGG CTC AGT GGG λλλ ACT TCT GGA AλC TTC 2011 Glu Ser Cys Glu λla Tyr λrg Leu Ser Gly Lys Thr Ser Gly λβn Phe 585 590 595

ACC ATT GAT CCT GAT GGC AGT GGC CCC CTG AAG CCA TTT GTA GTG TAC 2059 Thr He λβp Pro λβp Gly Ser Gly Pro Leu Lye Pro Phe Val Val Tyr 600 605 610

TGT GλT ATC CGA GAG AAC CGA GCG TGG ACA GTT GTG CGG CAT GλC AGG 2107 Cys λβp He λrg Glu λβn λrg λla Trp Thr Val Val λrg Hie λβp λrg 615 620 625 630

CTG TGG λCλ λCT CGA GTG ACA GGT TCC AGC ATG GAG CGG CCA TTC CTG 2155 Leu Trp Thr Thr λrg Val Thr Gly Ser Ser Met βlu λrg Pro Phe Leu 635 640 645

GGG GCT λTC CAG TAC TGG AAT GCA TCC TGG GAG GAA GTC AGT GCC CTT 2203 Gly λla He Gin Tyr Trp λβn λla Ser Trp Glu Glu Val Ser λla Leu 650 655 660

GCC AλT GCT TCC CAG CAT TGT GAA CAG TGG ATC GAG TTC TCC TGC TAC 2251 λla λβn λla Ser Gin Hie Cys Glu Gin Trp He Glu Phe Ser Cys Tyr 665 670 675

AAT TCC CGG CTG CTC AλC λCT GCA GGA GGC TAC CCC TAC AGC TTT TGG 2299 λβn Ser λrg Leu Leu λβn Thr λla Gly Gly Tyr Pro Tyr Ser Phe Trp 680 685 690

ATT GGC CGA AAT GAG GAG CAG CAC TTC TAC TGG GGA GGC TCC CAG CCT 2347 He Gly λrg λβn Glu Glu Gin Hie Phe Tyr Trp Gly Gly Ser Gin Pro 695 700 705 710

GGG ATC CAG CGC TGT GCC TGT GGT CTG GAC CGG AGC TGT GTG GλC CCT 2395 Gly He Gin λrg Cys λla Cyβ Gly Leu λβp λrg Ser Cys Val λβp Pro 715 720 725

GCC TTG TλC TGC λλC TGT GλC GCT GλC CλG CCC CλG TGG λGλ λCT GλC 2443 λla Leu Tyr Cyβ λsn Cyβ λβp λla λβp Gin Pro Gin Trp λrg Thr λβp 730 735 740

AAG GGA CTG CTG ACC TTT GTG GAC CAT CTG CCT GTC ACT CAG GTA GTG 2491 Lye Gly Leu Leu Thr Phe Val Aβp Hie Leu Pro Val Thr Gin Val Val 745 750 755

ATA GGG GAT ACG AAC CGC TCC ACT TCT GAG GCC CAG TTC TTC CTG AGG 2539 He Gly Aβp Thr Aβn λrg Ser Thr Ser Glu λla Gin Phe Phe Leu λrg 760 765 770

CCT CTG CGC TGC TλT GGC GλT CGA AλT TCC TGG λλC λCC λTT TCC TTC 2587

Pro Leu λrg Cyβ Tyr Gly λβp λrg λβn Ser Trp λβn Thr He Ser Phe 775 780 785 790

CAC ACC GGG GCT GCA CTA CGC TTC CCC CCA ATC CGT GCC AλC CAC AGC 2635 Hie Thr Gly Ala Ala Leu λrg Phe Pro Pro He λrg λla λβn Hie Ser 795 800 805

CTG GλT GTC TCC TTC TλC TTC AGG λCC TCT GCT CCC TOG GGG GTC TTC 2683 Leu λβp Val Ser Phe Tyr Phe λrg Thr Ser λla Pro Ser Gly Val Phe 810 815 820

CTA GAG AAT ATG GGG GGC CCT TAC TGC CAG TGG CGC CGA CCT TAT GTG 2731 Leu Glu λβn Met Gly Gly Pro Tyr Cyβ Gin Trp λrg λrg Pro Tyr Val 825 830 835

CGG GTG GAA CTC AAC ACλ TCC CGG GλT GTG GTC TTC GCC TTT GλT GTG 2779 λrg Val Glu Leu λβn Thr Ser λrg λsp Val Val Phe λla Phe λsp Val 840 845 850

GGG λλT GGG GλT GλG AAC CTC ACλ GTA CAC TCA GλC GλC TTT GAG TTC 2827 Gly λβn Gly λβp Glu λβn Leu Thr Val Hie Ser λβp λsp Phe Glu Phe 855 860 865 870

λλT GAT GAC GλG TGG CAC CTG GTC CGG GCT GAA ATC AλC GTG λλG CλG 2875 λsn λβp λβp Glu Trp Hie Leu Val λrg λla Glu He λβn Val Lye Gin 875 880 885

GCC CGG CTC CGA GTG GAT CAC CGG CCC TGG GTT CTG CGG CCT ATG CCA 2923 λla λrg Leu λrg Val λβp His λrg Pro Trp Val Leu λrg Pro Met Pro 890 895 900

CTG CλG λCC TλC λTC TGG λTG GλG TλT GλC CAG CCC CTC TAT GTG GGA 2971 Leu Gin Thr Tyr He Trp Met Glu Tyr Aap Gin Pro Leu Tyr Val Gly 905 910 915

TCT GCA GAG CTT λλG λGλ CGC CCC TTT GTG GGT TGC TTG AGG GCC ATG 3019 Ser λla Glu Leu Lye λrg λrg Pro Phe Val Gly Cyβ Leu λrg λla Met 920 925 930

CGT CTG AAC GGA GTG ACT CTG AAC CTG GAG GGC CGT GCC λλT GCC TCT 3067 λrg Leu λβn Gly Val Thr Leu λβn Leu Glu Gly λrg λla λβn λla Ser 935 940 945 950

GλG GGT λCC TCλ CCC AAC TGC ACλ GGC CAC TGT GCC CAC CCT CGG CTC 3115 Glu Gly Thr Ser Pro λβn Cyβ Thr Gly Hie Cyβ λla Hie Pro λrg Leu 955 960 965

CCC TGT TTC CλT Gβλ GGC CGC TGC GTG GλG CGC TλT λβC TλC TλC ACG 3163 Pro Cyβ Phe Hie Gly Gly Arg Cyβ Val Glu λrg Tyr Ser Tyr Tyr Thr 970 975 980

TGT GλC TGT GλC CTC λCG GCT TTT GAT GGG CCA TAC TGC AAC CAC GAT 3211 cyβ λβp Cyβ Aβp Leu Thr λla Phe λβp Gly Pro Tyr Cyβ λβn Hie λβp 985 990 995

λTT GGT GOT TTC TTT GλG CCG GGC λCC TGG λTG CGC TλT AAC CTA CλG 3259 He Gly Gly Phe Phe Glu Pro Gly Thr Trp Met λrg Tyr λβn Leu Gin 1000 1005 1010

TCλ GCG CTG CGC TCT GCA GCC AGG GAG TTC TCC CλC λTG CTG λGC CGG 3307 Ser λla Leu λrg Ser λla λla λrg Glu Phe Ser Hie Met Leu Ser λrg 1015 1020 1025 1030

CCA GTG CCA GGC TAT GAG CCT GGC TAC ATC CCG GGC TAT GAT ACT CCG 3355 Pro Val Pro Gly Tyr Glu Pro Gly Tyr He Pro Gly Tyr λβp Thr Pro

1035 1040 1045

GGC TAT GTG CCT GGC TAC CAT GGC CCC GGG TAC CGC CTG CCC GAC TAC 3403 Gly Tyr Val Pro Gly Tyr Hie Gly Pro Gly Tyr Arg Leu Pro Aβp Tyr 1050 1055 1060

CCC CGG CCT GGT CGG CCT GTG CCC GGT TAC CGT GGG CCT GTC TAC AAC 3451 Pro Arg Pro Gly Arg Pro Val Pro Gly Tyr Arg Gly Pro Val Tyr Aβn 1065 1070 1075

GTT ACG GGA GAG GAG GTC TCC TTC AGC TTC AGC ACC AGC TCC GCC CCT 3499 Val Thr Gly Glu Glu Val Ser Phe Ser Phe Ser Thr Ser Ser λla Pro 1080 1085 1090

GCT GTC CTG CTC TλC GTC λGT TCC TTT GTT CGT GλC TAC ATG GCT GTG 3547 Ala Val Leu Leu Tyr Val Ser Ser Phe Val Arg λsp Tyr Met λla Val 1095 1100 1105 1110

CTC λTC λλG GλT GλT GGG λCC CTT CλG CTG CGλ TλT CλG CTG GGC λCC 3595 Leu He Lys λsp λsp Gly Thr Leu Gin Leu λrg Tyr Gin Leu Gly Thr

1115 1120 1125

λGT CCC TλC GTG TλC CλG CTA ACC ACT CGA CCA GTG ACC GAT GGC CAG 3643 Ser Pro Tyr Val Tyr Gin Leu Thr Thr Arg Pro Val Thr Asp Gly Gin 1130 1135 1140

CCC CλT λGC λTC λλT λTC λCC CGT GTT TλC CGG AAC CTC TTC ATC CAG 3691 Pro His Ser He Aβn He Thr λrg Val Tyr λrg λβn Leu Phe He Gin 1145 1150 1155

GTG GλC TλC TTC CCA CTG ACA GλG CAG λλG TTC TCG CTG TTG GTG GAC 3739 val λβp Tyr Phe Pro Leu Thr Glu Gin Lye Phe Ser Leu Leu Val λβp 1160 1165 1170

λGC CλG TTG GλC TCλ CCC λλG GCC TTG TλT TTA GGG CGT GTG ATG GλG 3767 Ser Gin Leu λβp Ser Pro Lye λla Leu Tyr Leu Gly λrg Val Met βlu 1175 1180 1185 1190

ACA GGA GTC ATT βλC CCG GAG ATC CAG CGC TAC AλC λCC CCA GGT TTC 3835 Thr Gly Val He Asp Pro Glu He Gin λrg Tyr λβn Thr Pro Gly Phe 1195 1200 1205

TCλ GGC TGC CTG TCT GGT GTT CGλ TTC AAC AλC GTG GCT CCC CTC λλG 3883 ser Gly Cyβ Leu Ser Gly Val λrg Phe λβn λβn Val λla Pro Leu Lye 1210 1215 1220

λCC CλC TTC CGλ λCC CCT CGλ CCC λTG ACT GCT GAG CTA GCT GλG GCC 3931 Thr Hie Phe λrg Thr Pro λrg Pro Met Thr λla Glu Leu λla Glu λla 1225 1230 1235

CTT CGλ GTT CλG GGλ Gλλ CTG TCC GAA TCT AλT TGC GGλ GCT λTG CCλ 3979 Leu λrg Val Gin Gly Glu Leu Ser Glu Ser λβn Cyβ Gly Ala Met Pro 1240 1245 1250

CGT CTT GTT TCΛ GλG GTG CCλ CCT GλG CTT GλT CCC TGG TλT CTG CCC 4027 λrg Leu Val Ser Glu Val Pro Pro Glu Leu λβp Pro Trp Tyr Leu Pro 1255 1260 1265 1270

CCλ GλC TTC CCC TλC TλC CλT GλT Gλλ ββλ TGG GTT GCC λTλ CTT TTA 4075 Pro λβp Phe Pro Tyr Tyr Hie λβp βlu Gly Trp Val λla He Leu Leu

1275 1280 1285

GβC TTT TTG GTG GCC TTT CTG CTG CTG GGG CTG GTG GGλ λTG TTG GTG 4123 Gly Phe Leu Val λla Phe Leu Leu Leu Gly Leu Val Gly Met Leu Val 1290 1295 1300

CTC TTC TλT CTG CAA λλT CλT CGC TλT λλG GGC TCC TλC CλT λCC λλT 4171 Leu Phe Tyr Leu Gin λβn Hie λrg Tyr Lye Gly Ser Tyr Hie Thr λβn 1305 1310 1315

GλG CCC λλG GCT GCC CλC GλG TλC CAT CCT GGC AGC λλλ CCT CCC CTA 4219 Glu Pro Lye λla λla Hie Glu Tyr Hie Pro Gly Ser Lye Pro Pro Leu 1320 1325 1330

CCC ACT TCA GGC CCT GCC CAG GTC CCC ACC CCT ACλ GCA GCT CCC AλC 4267 Pro Thr Ser Gly Pro λla βln Val Pro Thr Pro Thr λla λla Pro λβn 1335 1340 1345 1350

CAA GCT CCA GCC TCA GCC CCA GCC CCA GCC CCλ ACT CCA GCC CCλ GCC 4315 Gin λla Pro λla Ser λla Pro λla Pro Ala Pro Thr Pro λla Pro λla 1355 1360 1365

CCT GGC CCC CGG GλT CλG AAC CTA CCC CλG λTC CTG GλG GλG TCC AGG 4363 o Gly Pro Arg Aβp Gin λβn Leu Pro Gin He Leu Glu Glu Ser λrg 1370 1375 1380

TCT Gλλ T GAGTCAGAAG GGCTTCTGGG λCCλλTTCCλ GCTCCTGλCA TTCCCCCAGT 4420 Ser Glu

CCTGCCTCTC CCCCλTCCTA TCAGGGλCλT TTGGCTCCTC TTλGCTGGCT CTGCTCλTCC 4480

AGAGGATATT CCCCCATCCC CCCCCCλTCλ λGTTTGGTGG GCAGAGCTλC λGλTGGGλCC 540

CAAGGGAGTG GCCGAGCCTC λCTGCCTλAA CCλλTGCCCT TCTCATCCCT GTTTCCCCAG 4600

GCTCCTGGCT GTTTλTCTGC CCCλλλGGλG λλGCCTCATG GGGTTGλCλT λGGTCCTTTC 4660

TGCCλTCTCT GTTCCAGCTG CTGTCAGGGA TTλλCλλCλG AGTGTλGGGG λGλTTλλCTG 4720

CCTCCCTTCC AλTλGλCλCT λTCλGCλGGG λCλGλTGTGT GGGλGTGCAG GGCTGCλGAG 4780

GGTλTGGGGG GλGGλGGCTG CTλAACCCTA TCCCCCAGCC TCCCCCCTGC CCTGλλGλTC 4840

TTCCλTTTGC TTCCλCTCλG CTGGλGGCTC λλGλGGGCTT GλTGGCTGTC CCCTGCCCCC 4900

CTCCTTTTGT TTTGTλCACA GλGλCCλAGA GGCCTCAGTT TAGCACCTTA GTACCTCCGC 4960

TGCTTCλCTT GCTTTλGCCλ AλGCCλTλλA AλACCTGCλA CGTAGAGAAA ATλλTGCλGλ 5020

TACCCTGλCT AGCCAGCCCT CTACTCCTCC λλCCTTTTCC λλGλTλTGCλ λTβGCCTTTG 5080

TGCCTGCCCλ λλGGCTTCGC CCCCTCCAGT GCATGAGGAA CCCTCTTTCC TCCGCTCAGλ 5140

GATGCTGCTT CλTTTλCCCλ GGAGGTCATA TTCTTTATλT λTATTTTTTG TTGCλλλGTG 5200

TCTCTCTλGλ GλλλCTCTλT λTλTTλTTCG λATTTTTAλλ TTATTTGTTT ATATATAAAA 5260

GλλλλGCTCλ ATTGGCAAAA AAAAAAAAAA AAAA 5294

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1384 amino acidβ

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

(ii) MOLECULE TYPE: protein

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

Met Met Hie Leu Arg Leu Phe Cys He Leu Leu Ala Ala Val Ser Gly i 5 10 15

Ala Glu Gly Trp Gly Tyr Tyr Gly Cys λsp Glu Glu Leu Val Gly Pro 20 25 30

Leu Tyr λla λrg Ser Leu Gly λla Ser Ser Tyr Tyr Ser Leu Leu Thr 35 40 45

λla Pro λrg Phe λla λrg Leu His Gly He Ser Gly Trp Ser Pro λrg

50 55 60

He Gly λβp Pro λβn Pro Trp Leu Gin He λβp Leu Met Lys Lys Hie 65 70 75 80

λrg He λrg λla Val λla Thr Gin Gly Ser Phe λβn Ser Trp λβp Trp

85 90 95

Val Thr λrg Tyr Met Leu Leu Tyr Gly λβp λrg Val λβp Ser Trp Thr 100 105 110

Pro Phe Tyr Gin λrg Gly Hie λβn Ser Thr Phe Phe Gly λβn Val λβn 115 120 125

Glu Ser λla Val Val λrg His Asp Leu Hie Phe His Phe Thr λla λrg 130 135 140

Tyr He λrg He Val Pro Leu λla Trp Asn Pro Arg Gly Lye He Gly 145 150 155 160

Leu λrg Leu Gly Leu Tyr Gly Cys Pro Tyr Lye λla λβp He Leu Tyr 165 170 175

he λβp Gly λβp λsp λla He Ser Tyr λrg Phe Pro λrg Gly Val Ser 180 185 190

λrg Ser Leu Trp λsp Val Phe λla Phe Ser Phe Lys Thr Glu Glu Lye 195 200 205

Aβp Gly Leu Leu Leu Hie Ala Glu Gly λla Gin Gly λβp Tyr Val Thr 210 215 220

Leu Glu Leu Glu Gly λla Hie Leu Leu Leu Hie Met Ser Leu Gly Ser 225 230 235 240

ser Pro He Gin Pro λrg Pro Gly His Thr Thr Val Ser λla Gly Gly

245 250 255

Val Leu λβn λβp Gin Hie Trp His Tyr Val λrg Val λsp λrg Phe Gly 260 265 270

Arg Asp Val Aβn Phe Thr Leu Asp Gly Tyr Val Gin λrg Phe He Leu 275 280 285

λβn Gly λβp Phe Glu λrg Leu λβn Leu λβp Thr Glu Met Phe He Gly 290 295 300

Gly Leu Val Gly λla λla λrg Lye λβn Leu λla Tyr λrg Hie λβn Phe 305 310 315 320

λrg Gly Cyβ He Glu Asn Val He Phe Aβn λrg Val λβn He λla λβp 325 330 335

Leu λla Val λrg λrg Hie Ser λrg He Thr Phe Glu Gly Lye Val λla 340 345 350

Phe λrg Cyβ Leu λβp Pro Val Pro Hie Pro He λβn Phe Gly Gly Pro 355 360 365

Hie λβn Phe Val Gin Val Pro Gly Phe Pro λrg λrg Gly λrg Leu λla 370 375 380

Val Ser Phe λrg Phe λrg Thr Trp λβp Leu Thr Gly Leu Leu Leu Phe 385 390 395 400

Ser λrg Leu Gly λβp Gly Leu Gly Hie Val Glu Leu Thr Leu Ser Glu 405 410 415

Gly Gin Val λβn Val Ser He λla Gin Ser Gly λrg Lye Lye Leu Gin 420 425 430

Phe λla λla Gly Tyr λrg Leu λβn λβp Gly Phe Trp Hie Glu Val λen 435 440 445

Phe Val λla βln βlu λβn Hie λla Val He Ser He λβp λβp Val βlu 450 455 460

βly λla βlu Val λrg Val Ser Tyr Pro Leu Leu He λrg Thr βly Thr 465 470 475 480

Ser Tyr Phe Phe Gly Gly Cyβ Pro Lye Pro λla Ser λrg Trp λβp Cyβ 485 490 495

Hie Ser λβn Gin Thr λla Phe Hie Gly Cys Met Glu Leu Leu Lye Val 500 505 510

λβp Gly Gin Leu Val λβn Leu Thr Leu Val βlu Gly λrg λrg Leu βly 515 520 525

Phe Tyr λla βlu Val Leu Phe λβp Thr Cys Gly He Thr λsp λrg Cyβ 530 535 540

Ser Pro λβn Met Cyβ Glu His λβp βly λrg Cyβ Tyr Gin Ser Trp λβp 545 550 555 560

λβp Phe He Cyβ Tyr Cyβ Glu Leu Thr Gly Tyr Lye βly βlu Thr Cyβ 565 570 575

Hie Thr Pro Leu Tyr Lye βlu Ser Cyβ βlu λla Tyr λrg Leu Ser βly 580 585 590

Lye Thr Ser Gly λβn Phe Thr He λβp Pro λβp Gly Ser Gly Pro Leu 595 600 605

Lye Pro Phe Val Val Tyr Cyβ λβp He λrg Glu λβn λrg λla Trp Thr 610 615 620

Val Val λrg Hie λβp λrg Leu Trp Thr Thr λrg Val Thr Gly Ser Ser 625 630 635 640

Met Glu λrg Pro Phe Leu βly λla He βln Tyr Trp λβn λla Ser Trp 645 650 655

βlu Glu Val Ser λla Leu λla Aβn λla Ser Gin Hie Cyβ Glu Gin Trp 660 665 670

He βlu Phe Ser Cyβ Tyr λβn Ser λrg Leu Leu λβn Thr λla Cly βly 675 680 685

Tyr Pro Tyr Ser Phe Trp He Gly λrg λβn Glu Glu Gin Hie Phe Tyr 690 695 700

Trp Gly Gly Ser βln Pro βly He βln λrg Cyβ λla Cyβ βly Leu λβp 705 710 715 720

λrg Ser Cyβ Val λβp Pro λla Leu Tyr Cyβ λβn Cyβ λβp λla λβp βln 725 730 735

Pro Gin Trp λrg Thr λβp Lye Gly Leu Leu Thr Phe Val λβp His Leu 740 745 750

Pro Val Thr Gin Val Val He Gly λβp Thr λβn λrg Ser Thr Ser Glu 755 760 765

λla βln Phe Phe Leu λrg Pro Leu λrg Cyβ Tyr Gly λβp λrg λβn Ser 770 775 780

Trp λβn Thr He Ser Phe His Thr Gly λla λla Leu λrg Phe Pro Pro 785 790 795 800

He λrg λla λβn Hie Ser Leu λβp Val Ser Phe Tyr Phe λrg Thr Ser 805 810 815

λla Pro Ser Gly Val Phe Leu Glu Asn Met βly βly Pro Tyr Cyβ βln 820 825 830

Trp λrg λrg Pro Tyr Val λrg Val βlu Leu λβn Thr Ser λrg λβp Val 835 840 845

Val Phe Ala Phe λβp Val βly λβn Gly λβp Glu λβn Leu Thr Val Hie 850 855 860

Ser λβp λβp Phe Glu Phe Aβn Aβp Aβp Glu Trp Hie Leu Val λrg λla 865 870 875 880

Glu He λβn Val Lye Gin λla λrg Leu λrg Val Asp Hie Arg Pro Trp 885 890 895

Val Leu Arg Pro Met Pro Leu Gin Thr Tyr He Trp Met Glu Tyr λβp 900 905 910

Gin Pro Leu Tyr Val Gly Ser λla Glu Leu Lys λrg λrg Pro Phe Val 915 920 925

βly Cys Leu λrg λla Met λrg Leu λβn Cly Val Thr Leu λβn Leu βlu 930 935 940

βly λrg λla λβn λla Ser βlu βly Thr Ser Pro λβn Cyβ Thr Cly Hie 945 950 955 960

Cyβ λla Hie Pro λrg Leu Pro Cyβ Phe Hie Gly Gly λrg Cyβ Val Glu 965 970 975

λrg Tyr Ser Tyr Tyr Thr Cyβ λβp Cyβ λβp Leu Thr Ala Phe Aβp Gly 980 985 990

Pro Tyr Cyβ λβn Hie λβp He Gly Gly Phe Phe Glu Pro Gly Thr Trp 995 1000 1005

Met λrg Tyr λβn Leu βln Ser λla Leu λrg Ser Ala Ala Arg Glu Phe 1010 1015 1020

Ser Hie Met Leu Ser Arg Pro Val Pro Gly Tyr Glu Pro Gly Tyr He 1025 1030 1035 1040

Pro Gly Tyr λβp Thr Pro Gly Tyr Val Pro βly Tyr Hie βly Pro βly 1045 1050 1055

Tyr λrg Leu Pro λβp Tyr Pro λrg Pro βly λrg Pro Val Pro βly Tyr 1060 1065 1070

λrg βly Pro Val Tyr λβn Val Thr βly βlu βlu Val Ser Phe Ser Phe 1075 1080 1085

Ser Thr Ser Ser λla Pro λla Val Leu Leu Tyr Val Ser Ser Phe Val 1090 1095 1100

λrg λβp Tyr Met λla Val Leu He Lye λβp λβp βly Thr Leu βln Leu 1105 1110 1115 1120

λrg Tyr βln Leu βly Thr Ser Pro Tyr Val Tyr βln Leu Thr Thr λrg 1125 1130 1135

Pro Val Thr λβp βly βln Pro Hie Ser He λβn He Thr λrg Val Tyr 1140 1145 1150

λrg Aβn Leu Phe He βln Val Aβp Tyr Phe Pro Leu Thr βlu βln Lye 1155 1160 1165

Phe Ser Leu Leu Val λβp Ser βln Leu Asp Ser Pro Lye λla Leu Tyr 1170 1175 1180

Leu βly λrg Val Met βlu Thr Gly Val He λβp Pro βlu He βln λrg 1185 1190 1195 1200

Tyr λβn Thr Pro βly Phe Ser Gly Cyβ Leu Ser Gly Val λrg Phe λβn 1205 1210 1215

λsn Val λla Pro Leu Lys Thr His Phe λrg Thr Pro λrg Pro Met Thr 1220 1225 1230

λla βlu Leu λla Glu λla Leu λrg Val Gin Gly Glu Leu Ser Glu Ser 1235 1240 1245

λβn Cyβ βly λla Met Pro λrg Leu Val Ser βlu Val Pro Pro βlu Leu 1250 1255 1260

λβp Pro Trp Tyr Leu Pro Pro λβp Phe Pro Tyr Tyr His Aβp βlu βly 1265 1270 1275 1280

Trp Val Ala He Leu Leu βly Phe Leu Val λla Phe Leu Leu Leu βly 1285 1290 1295

Leu Val Gly Met Leu Val Leu Phe Tyr Leu βln λβn His λrg Tyr Lye 1300 1305 1310

βly Ser Tyr Hie Thr λβn βlu Pro Lye λla λla Hie βlu Tyr Hie Pro 1315 1320 1325

βly Ser Lye Pro Pro Leu Pro Thr Ser βly Pro λla βln Val Pro Thr 1330 1335 1340

Pro Thr λla λla Pro λβn βln λla Pro λla Ser λla Pro λla Pro λla 1345 1350 1355 1360

Pro Thr Pro λla Pro λla Pro βly Pro λrg λβp βln λβn Leu Pro βln 1365 1370 1375

He Leu βlu βlu Ser λrg Ser βlu 1380

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5350 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ϋ) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Rattus norvegicuβ

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 154..4297

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

GATTTTCλCT GGGGβTAGGA GAAAβGGAλG GβTGGGTGAG GACGGλλλAA GCλβCλTCβG 60

TCλβCCβCβλ λCCCCAGGAβ AλλλGCTββG GGCCTGAGCC λGλACCGGAG CCCTAGCGGC 120

ACAAGGCAGA CACCCAGGGT TGGTCλGCTC CGC λTG λTG λGT CTC CGG CTT TTC 174

Met Met Ser Leu λrg Leu Phe 1 5

λGC ATT CTG CTC GCC CCT βTβ GTC TCT Gβλ GCC CλG GβC TGG ββC TλC 222 Ser He Leu Leu λla λla Val Val Ser Gly λla Gin βly Trp βly Tyr 10 15 20

TλT GGC TGC λλT GAG GAG CTG GTG GGG CCT CTG TAT GCA CGG TCT CTG 270 Tyr Gly Cyβ λβn βlu Glu Leu Val βly Pro Leu Tyr λla λrg Ser Leu 25 30 35

GGT GCT TCC TCC TλC TλT GGλ CTC TTT λCC λCλ GCC CCC TTT βCC CGG 318 Gly λla Ser Ser Tyr Tyr Gly Leu Phe Thr Thr λla λrg Phe λla λrg 40 45 50 55

CTA CAC GβC ATC AGT GGA TGG TCG CCC CGG ATT GGG GλC CCG λλT CCC 366 Leu Hie Gly He Ser Gly Trp Ser Pro λrg He Gly λβp Pro λβn Pro 60 65 70

TGG CTC CλG λTλ βλC TTλ λTβ λλβ AAG CAT CGA ATC Cββ GCT GTG GCC 414 Trp Leu βln He Aβp Leu Met Lye Lye Hie Arg He Arg λla Val λla 75 80 85

λCλ Cλβ ββλ βCC TTT λλT TCT Tββ βλT TGG GTC λCλ CGT TλC λTG CTG 462 Thr Gin Gly λla Phe λβn Ser Trp λβp Trp Val Thr λrg Tyr Met Leu go 95 100

CTC TλC GGG GλC CGT GTG GλC λβC TGG ACA CCA TTC TAC CAA CAA GGG 510 Leu Tyr Gly Aβp λrg Val λβp Ser Trp Thr Pro Phe Tyr Gin Gin Gly 105 110 115

CAC AλC GCG λCC TTC TTC GβT λλT βTC λλC βλC TCG GCG GTβ βTλ CGC 558 His λβn λla Thr Phe Phe Gly λβn Val λβn λβp Ser λla Val Val λrg

120 125 130 135

CλT GλC CTG CλC TλC CλT TTT λCG CCT CGC TλC λTC CGC λTC GTG CCλ 606 His λβp Leu His Tyr His Phe Thr λla λrg Tyr He λrg He Val Pro

140 145 150

CTG GCC TGG AAC CCC CGC ββC AAG ATT GβC TTG AGG CTG GβC ATC TAC 654 Leu Ala Trp λsn Pro λrg Cly Lys He Gly Leu λrg Leu Gly He Tyr 155 160 165

GGT TGT CCC TλC λCG TCC λλC λTC CTG TλT TTT GλC GβC βλT βλT βCC 702 βly Cyβ Pro Tyr Thr Ser λβn He Leu Tyr Phe λβp Gly λβp λβp λla 170 175 180

ATT TCλ TλC CGC TTC CλG CGλ Gββ βCC λCT CAA AGT CTT Tββ GλC βTβ 750 He Ser Tyr λrg Phe βln λrg βly λla Ser βln Ser Leu Trp λβp Val 185 190 195

TTC CCT TTT AGT TTC AAG ACλ GAG GAG λλG GλC Gββ CTG CTG TTG CλC 798 Phe λla Phe Ser Phe Lye Thr Glu Glu Lye λβp Gly Leu Leu Leu Hie 200 205 210 215

λCC GAA GGC TCC CAG GGG GAT TAT GTG ACG CTT GAA CTG CAA GGA GCA 846 Thr Glu Gly Ser Gin Gly Aβp Tyr Val Thr Leu Glu Leu Gin βly λla 220 225 230

CλC CTG CTG CTG CλC λTG λGC CTG GGC λGC λGC CCC λTC CλG CCG λGλ 894 Hie Leu Leu Leu Hie Met Ser Leu Gly Ser Ser Pro He Gin Pro λrg 235 240 245

CCT GGT CλC λCC ACG βTβ λβC CCT GGT ββC βTλ CTT λλT CλC CTA ACC 942 Pro βly Hie Thr Thr Val Ser λla βly βly Val Leu λβn λβp Leu Ser 250 255 260

TGG CλT TλT GTG CGG GTG GλC CGλ TAC GGC CGA GAA GCA AλT CTC λCC 990 Trp Hie Tyr Val λrg Val λβp λrg Tyr Gly λrg Glu λla λβn Leu Thr 265 270 275

CTG GλT GGT TλC GTλ CAT CGC TTT GTG CTC AλC GGC GAC TTT GAA AβG 1038 Leu λβp Gly Tyr Val Hie λrg Phe Val Leu Asn Gly λβp Phe βlu λrg 280 285 290 295

CTG λλT CTC Gλλ λλT βλβ λTλ TTC λTC ββλ GGT CTA βTβ ββC GCA GCG 1086 Leu λsn Leu βlu λsn βlu He Phe He βly βly Leu Val βly Ala Ala 300 305 310

CGT AAG AλC CTG GCC TλC CGC CλT AAC TTC CGT GGC TGT ATA GAA AλC 1134 λrg Lye λsn Leu λla Tyr λrg His λβn Phe λrg Gly Cyβ He βlu λβn 315 320 325

βTβ λTC TλC AAC CGG ATC AλC λTλ βCT GAA ATG GCA GTβ CAG CGC CλT 1182 Val He Tyr λβn λrg He λen He λla Glu Met λla Val Gin λrg Hie 330 335 340

TCG CGG λTC λCC TTC GλG GβT λλT βTβ GCT TTC CGG TGC TTG βλT CCC 1230 Ser λrg He Thr Phe Clu βly λβn Val Ala Phe λrg Cyβ Leu λβp Pro 345 350 355

GTT CCλ CAC CCC ATC λλC TTC GGλ GGC CCT CAC AAC TTC GTC CAA GTG 1278 Val Pro Hie Pro He λβn Phe Gly βly Pro Hie λβn Phe Val βln Val 360 365 370 375

CCT ββC TTT CCλ CCT Cβλ GGC CGC CTT GCT GTC TCC TTC CGC TTC CGC 1326 Pro Gly Phe Pro λrg λrg Gly λrg Leu λla Val Ser Phe λrg Phe λrg 380 385 390

ACC TGG βλC CTC λCλ GGG CTG CTC CTT TTC TCC CGC TTG GGG GλT Gββ 1374 Thr Trp Aβp Leu Thr βly Leu Leu Leu Phe Ser Arg Leu βly λβp βly 395 400 405

CTC GGT CλT GTβ βλβ CTG λTG CTT λGT GAA GGG CAA GTC λλT GTλ TCC 1422 Leu βly Hie Val βlu Leu Met Leu Ser βlu βly Gin Val λβn Val Ser 410 415 420

ATC GCG Cλβ λCT ββC CGC λλβ λλβ CTT CλC TTT GCT GCG GGG TλC CGC 1470 He λla Gin Thr Gly λrg Lye Lye Leu βln Phe λla λla βly Tyr λrg 425 430 435

CTC λλT CλT GGC TTC TGG CλT GλG GTG AAC TTT GTG GCA CAG GAA AAC 1518 Leu Aβn Aβp Gly Phe Trp Hie Glu Val Aβn Phe Val Ala Gin Glu λβn 440 445 450 455

CAT GCG GTC ATC λGT λTT GλT GAT GTG GAG GGG GCA GAG GTC AGG GTA 1566 Hie Ala Val He Ser He Aβp Aβp Val βlu Gly Ala Glu Val λrg Val

460 465 470

TCλ TλC CCλ CTβ CTC λTC CβC λCλ βββ λCT TCλ TλC TTC TTT ββT ββT 1614 Ser Tyr Pro Leu Leu He λrg Thr βly Thr Ser Tyr Phe Phe βly βly 475 480 485

TGT CCC λλλ CCλ GCC λGT CGA TGG ββC TCC CAC TCC AλC CλG λCλ GCA 1662 cyβ Pro Lye Pro λla Ser λrg Trp Gly Cye Hie Ser λβn Gin Thr λla 490 495 500

TTC CλT GGC TGC λTG GλG CTG CTC λλG βTβ βλT ββT CAA CTG GTC AAC 1710 Phe Hie Gly Cyβ Met βlu Leu Leu Lye Val Aβp βly βln Leu Val λβn 505 510 515

CTC λCT CTC CTλ βλβ TTT CGG λλG CTT GGT TλC TTT GCT GλG GTC CTC 1758 Leu Thr Leu Val Glu Phe λrg Lye Leu Gly Tyr Phe λla βlu Val Leu 520 525 530 535

TTT βλC λCA TCT ββC λTC ACA CAC λβλ TGC λGC CCT λλT λTC TGT GλG 1806 phe λβp Thr Cyβ Gly He Thr λβp λrg Cys Ser Pro λβn Met Cyβ Glu

540 545 550

CλT GλT GGG CGC TGC TλC CλG TCT TGG GλT GAC TTC λTC TGC TAC TGC 1854

Hie λβp Gly λrg Cyβ Tyr Gin Ser Trp λβp λβp Phe He Cye Tyr Cyβ 555 560 565

Gλλ CTC λCC GCC TλC λλβ ββλ GTT λCC TGC CλT Gλλ CCλ TTG TλT λλG 1902 βlu Leu Thr βly Tyr Lye βly Val Thr Cyβ Hie Clu Pro Leu Tyr Lye 570 575 580

βλβ TCC TGT Gλλ GCC TλT CGC CTC λGC GGG λλλ TλT TCT GGλ λλT TλC 1950 Glu Ser Cyβ Glu λla Tyr λrg Leu Ser Gly Lye Tyr Ser Gly λβn Tyr 585 590 595

λCC λTT GλT CCT βλT GβC λβT ββλ CCC CTβ λλλ CCλ TTT CTλ GTG TλT 1998 Thr He λβp Pro λβp βly Ser βly Pro Leu Lye Pro Phe Val Val Tyr 600 605 610 615

TGT GλT λTC CGλ Gλβ AAC CCA GCG TGG ACA GTT GTG CGA CλT GλC AGG 2046 Cyβ Aβp He λrg Glu λβn λrg λla Trp Thr Val Val λrg Hie λβp λrg 620 625 630

CTλ TGG λCC ACT CGA GTG ACA GβT TCC AGC ATG GAC Cββ CCC TTT CTG 2094 Leu Trp Thr Thr Arg Val Thr Gly Ser Ser Met λβp λrg Pro Phe Leu 635 640 645

Gββ βCC λTC CAA TAC Tββ λλT GCC TCC TGG'GλG Gλλ GTC λGT GCT CTG 2142 βly λla He Gin Tyr Trp λβn λla Ser Trp βlu βlu Val Ser λla Leu 650 655 660

βCC λλT GCT TCC CλG CλC TGT βλβ CλC Tββ λTC CλG TTC TCC TGC TλC 2190 λla λβn λla Ser Gin Hie Cyβ Glu Gin Trp He Glu Phe Ser Cyβ Tyr 665 670 675

λλT TCC CGG CTβ CTC AAC ACT GCA GGA GGC TAC CCC TAC AGC TTT TGG 2238 λβn Ser λrg Leu Leu λβn Thr λla Gly βly Tyr Pro Tyr Ser Phe Trp 680 685 690 695

λTT GGC CGλ λλT Gλλ Gλλ CλG CAT TTC TλC TGG Gβλ GβC TCC Cλβ CCT 2286 He βly λrg λβn Glu Glu Gin Hie Phe Tyr Trp Gly Gly Ser βln Pro

700 705 710

βββ λTC CAA CCC TGT GCC TGT Gββ CTG GAC CAG AGC TGT ATA GAC CCT 2334 Gly He Gin λrg Cyβ λla Cyβ βly Leu λβp βln Ser Cyβ He λβp Pro 715 720 725

GCA CTβ CλC TCC AAC TCC GAT GCT GλC CλG CCA CλG TGG λGλ ACA GAC 2382 λla Leu Hie Cyβ λβn Cyβ λβp λla λβp Gin Pro Gin Trp λrg Thr λβp 730 735 740

λλG Gββ CTC CTG λCC TTT GTG GλC CλT CTG CCT OTC ACT CAG GTA GTG 2430 Lye Gly Leu Leu Thr Phe Val Asp Hie Leu Pro Val Thr βln Val Val 745 750 755

ATA ββT βλC λCA λλC CβC TCC λGC TCT Gλλ GCT CλG TTC TTC CTG λββ 2478 He βly λβp Thr λβn λrg Ser Ser Ser βlu λla Gin Phe Phe Leu λrg 760 765 770 775

CCT CTG CGC TGT TλT GβT βλC CCC λλT TCC Tββ λλC λCT λTC TCC TTC 2526 Pro Leu λrg Cyβ Tyr βly λβp λrg λβn Ser Trp λβn Thr He Ser Phe 780 785 790

CGC λCT βGA GCT GCA CTG CGT TTC CCT CCA ATC CGT GCC AAC CAC AGC 2574 Arg Thr Gly Ala λla Leu λrg Phe Pro Pro He Arg Ala Aβn His Ser 795 800 805

CTT CΛT βTC TCC TTC TAC TTC AGG ACC TCG GCT CCC TCλ GGλ GTC TTC 2622 Leu λβp Val Ser Phe Tyr Phe λrg Thr Ser λla Pro Ser Gly Val Phe 810 815 820

CTλ GλG λλC λTG GGG GGT CCT TTC TGC CλG TGG CGC CGλ CCT TλC GTβ 2670 Leu Glu λβn Met Gly Gly Pro Phe Cyβ Gin Trp λrg λrg Pro Tyr Val 825 830 835

λGλ GTG Gλβ CTC λλC λCA TCC CGG GAT GTβ βTC TTT βCC TTT βλT λTT 2718 λrg Val βlu Leu λβn Thr Ser λrg λβp Val Val Phe λla Phe Asp He 840 845 850 855

GGC AAT GGG GAT GAG AAC CTG ACλ GTG CAC TCλ βλT βλC TTC GλG TTC 2766 Gly λβn Gly λβp Glu λβn Leu Thr Val Hie Ser λβp λβp Phe βlu Phe 860 865 870

λλT βλT βλC βλβ TGG CλT TTG GTC CGG GCT Gλλ λTC AAC GTG λλG CλC 2814 λβn λβp λβp Glu Trp Hie Leu Val λrg λla Glu He λβn Val Lye βln 875 880 885

βCC CGG CTG CGλ GTG GλC CλT CGG CCC Tββ βTβ CTλ λββ CCC λTβ CCC 2862 λla λrg Leu λrg Val λβp Hie λrg Pro Trp Val Leu λrg Pro Met Pro 890 895 900

CTC CλG ACG TAC λTC TGG CTG GλG TλT βλC CAA CCC CTC TλT GTG Gβλ 2910 Leu βln Thr Tyr He Trp Leu βlu Tyr λβp βln Pro Leu Tyr Val βly 905 910 915

TCT CCA βλβ CTT λλG AGG CGC CCA TTT GTG GGG TGC TTG λββ GCC λTβ 2958 ser λla βlu Leu Lye λrg λrg Pro Phe Val βly Cyβ Leu λrg λla Met 920 925 930 935

CGT TTG λλT Gβλ CTG ACT CTG AAC TTG βλβ GGT CGT GCC λλT GCC TCC 3006 λrg Leu λβn Gly Val Thr Leu λβn Leu Glu Gly λrg λla λβn λla Ser 940 945 950

Gλβ GGC λCC TTC CCC λλC TGC λCG GGC CλC TGC λCC CλC CCC CGG TTC 3054 Glu Gly Thr Phe Pro λβn Cyβ Thr Gly His Cyβ Thr His Pro λrg Phe 955 960 965

CCC TGT TTC CAC GGA GGA CGC TGT GTG GAG CGA TAC AGC TAC TAC ACG 3102 pro Cyβ Phe Hie Gly Gly λrg Cyβ Val βlu λrg Tyr Ser Tyr Tyr Thr 970 975 980

TβT βλC TCT βλC CTC λCλ CCT TTT CλT ββλ CCλ TλT TCT λλT CλC GλT 3150 Cyβ λβp Cyβ λβp Leu Thr λla Phe λβp Gly Pro Tyr Cyβ λβn Hie λβp 985 990 995

ATT GGT GβA TTC TTT βλβ λCT GGC λCA TGG ATG CGC TAT AλC CTC CλG 3198 He Gly Gly Phe Phe Glu Thr βly Thr Trp Met λrg Tyr λen Leu βln 1000 1005 1010 1015

TCλ GCλ CTG CGT TCT GCG GCC CλG GλG TTC TCT CλC λTG CTG λGC CGG 3246 Ser λla Leu λrg Ser λla λla Gin Glu Phe Ser Hie Met Leu Ser λrg 1020 1025 1030

CCG GTλ CCG GGC TλT βλβ CCT ββC TλT λTC CCλ ββC TλC βλC ACT CCT 3294 Pro Val Pro Gly Tyr Glu Pro Gly Tyr He Pro Gly Tyr λβp Thr Pro 1035 1040 1045

GGT TλC GTβ CCT GGG TλC CλT GGT CCT GGG TλC CGC CTλ CCC GλC TλC 3342 βly Tyr Val Pro βly Tyr Hie Gly Pro Gly Tyr λrg Leu Pro λβp Tyr 1050 1055 1060

CCλ λGG CCT GGC Cββ CCλ GTG CCC Gβλ TλC CGG GβG CCC GTG TλC λλT 3390 Pro λrg Pro Gly λrg Pro Val Pro Gly Tyr λrg βly Pro Val Tyr λβn 1065 1070 1075

βTT λCT ββλ βλβ βλβ GTC TCC TTT λGC TTC λCC λCC λGC TCT GCT CCT 3438 Val Thr Gly βlu Glu Val Ser Phe Ser Phe Ser Thr Ser Ser λla Pro 1080 1085 1090 1095

GCλ GTC CTG CTC TλC GTC λGC TCC TTT βTβ COT GλC TλC λTG GCC GTG 3486 Ala Val Leu Leu Tyr Val Ser Ser Phe Val λrg λβp Tyr Met λla Val

1100 1105 1110

CTC λTC λλG GAA βλT GGG λCC CTλ CλG CTT CβC TλT CλG CTG GβC λCC 3534 Leu He Lye Clu λβp βly Thr Leu βln Leu λrg Tyr βln Leu βly Thr 1115 1120 1125

AGT CCC TAT GTG TλC CλG CTλ λCC λCC CGG CCλ GTG λCC GλT GβC Cλλ 3582 Ser Pro Tyr Val Tyr Gin Leu Thr Thr λrg Pro Val Thr λβp Gly Gin 1130 1135 1140

CCC CλT λβT βTC λλC λTC λCC Cββ GTC TλC CGλ λλC CTC TTT λTC CλG 3630 Pro Hie Ser Val λβn He Thr λrg Val Tyr λrg λβn Leu Phe He Cln 1145 1150 1155

βTβ βλC TλC TTC CCG CTG λCλ Gλλ CλG λλG TTC TCT CTC CTG GTG GλC 3678 Val λβp Tyr Phe Pro Leu Thr Glu Gin Lye Phe Ser Leu Leu Val λβp 1160 1165 1170 1175

λGC CλG CTG GλC TCC CCC λλG GCC TTG TλT CTλ GGG CGT GTG λTG βλβ 3726 Ser βln Leu λβp Ser Pro Lye λla Leu Tyr Leu βly λrg Val Met βlu 1180 1185 1190

λCA GGA GTC ATT GAC CCλ Gλβ λTT Cλβ Cββ TλC AAC ACC CCλ ββT TTC 3774 Thr Cly Val He λβp Pro βlu He βln λrg Tyr λβn Thr Pro βly Phe 1195 1200 1205

TCλ ββC TGC CTG TCT GGT GTC CGG TTC AAC AAT GTβ GCT CCT CTC λλG 3822 Ser Gly Cyβ Leu Ser Gly Val λrg Phe λβn λβn Val λla Pro Leu Lye 1210 1215 1220

λCC CλT TTC CGλ λCC CCT CGC CCC λTG ACT GCT Gλβ CTC GCG GAG GCC 3870 Thr Hie Phe λrg Thr Pro λrg Pro Met Thr λla Glu Leu λla βlu λla 1225 1230 1235

λTβ Cββ GTT Cλλ GGλ Gλλ CTG TCG GλG TCT λλC TGT GGC GCT λTG CCλ 3918 Met λrg Val Gin Gly Glu Leu Ser βlu Ser Asn Cys Cly Ala Met Pro 1240 1245 1250 1255

CβC CTT GTC TCC GAG βTβ CCA CCA GλG CTT GλT CCC TGG TλC CTG CCC 3966 Arg Leu Val Ser Glu Val Pro Pro Glu Leu Aβp Pro Trp Tyr Leu Pro

1260 1265 1270

CCλ βλT TTC CCλ TλC TλC CλT GλC GλC GGλ TGG λTT GCC λTλ CTC TTλ 4014 Pro λβp Phe Pro Tyr Tyr Hie λβp λβp Gly Trp He λla He Leu Leu 1275 1280 1285

GGT TTT TTG GTβ βCC TTC CTG CTG CTG GβG CTT GTG Gβλ λTβ CTG GTG 4062 Gly Phe Leu Val λla Phe Leu Leu Leu Gly Leu Val Gly Met Leu Val 1290 1295 1300

CTG TTC TλT CTG Cλλ λλT CλT CGλ TλC λλG ββC TCC TλT CAC λCC AAC 4110 Leu Phe Tyr Leu Gin Aβn Hie Arg Tyr Lye Gly Ser Tyr Hie Thr λβn 1305 1310 1315

GAG CCC λλG GCC λCC CAT GλT TCC CAC CCT GGC GGC AAA GCT CCC CTA 4158 Glu Pro Lye Ala Thr Hie Aβp Ser His Pro Gly Gly Lys Ala Pro Leu 1320 1325 1330 1335

CCT CCC TCA GβC CCT CCC CAG GCC CCT GCC CCC ACT CCA GCT CCC ACC 4206 pro Pro Ser Gly Pro λla Gin λla Pro λla Pro Thr Pro λla Pro Thr

1340 1345 1350

CλG GTT CCG λCC CCλ GCC CCλ GCC CCλ GCC TCT GGC CCλ GGC CCC λGG 4254 βln Val Pro Thr Pro λla Pro λla Pro λla Ser βly Pro βly Pro λrg 1355 1360 1365

βλC CλG AAC CTC CCC CAG ATC TTG GλG βλβ TCC λββ TCT GAA T 4297 λβp Gin λβn Leu Pro βln He Leu βlu βlu Ser λrg Ser βlu 1370 1375 1380

GλGTCACAAG GβCTTCλGGβ λCCAAGGCCA λCTCCTCTλλ GTCCCTTCλG CTCCTGCCTC 4357

TCCTCTCCCC TGTCAGGGAC λTTTGGCTCT TCTTλβCλβG CTCTCTTCAC CλGβλββλTC 4417

CCCTCTTGCC AλGTTTGGTG TGCAGλGCTA CAGATGGGAC CAAAGGGAGT GGCCGAGTCT 4477

CλCTGCCTλλ ACCAATGCCC TβCCCCCλCC CCCCλCCCCλ GCTCCTGGCT GTTTGCCTGC 4537

CCTACGGGAG AAAGCTCATG GAGCTGAGGC βGGCCTTTCC TβCCλTCTCT GTCCCAGCTG 4597

CTGGCλλβGλ TTλλCAλCCλ AβββCλββGG AGGTGAACTG CCTCCCTTCC TGTGGβTATT 4657

ATCAGCAGββ λCAGATGTGG GGGATCGAGG GGCTGCλCAG GβCAβGCAGG GAGGGAGGGA 4717

GβλβGCTGCT λλλλCλCλCC CTββλGCCTC CCCCCTGCCC TGCTGλCCββ CTGTCTTCCA 4777

TCTGCTTCCT CTCAGCTGGG GTTGAGGGAA GAλCTTCλTC CCCλCCCCCC λCCTCλCCCλ 4837

ACCCTTTTTG TTCTTACλβλ GACCAAGAGG CCTCλGCTTλ GCλCTTTλGT λCCTCCλCTG 4897

CTTCACATGC TTTAGCCAAA GCCATAAAAA GCCTβCλλGT AGλλβλλATλ ATGCAGACCC 4957

TGCCCλGCCA GTCCTCTGCT CCTCCλCCCC TTTCCAλλAT λCGCλATAGC CTGGGGTGCC 5017

TGTβTβCAββ CCTGGCCCCT GCGTGCλTGA GGAGCCCCTC CCGCTCλGλG λTGCTGCβλG 5077

TTCGTCCλGG λGGTCλTλTT CTTTλTλTλT λTTTTTTGTT GCλλAGCCTC TCTCTAβλGA 5137

ACTATλTλTT λCTCTλλTTT TTTλλTTλTC TGTTTλTλTλ TλAAAGAλCT CλGTGGGCCG 5197

TCCTGTGCTG TGCCCAGTTT GTAGTGAGCT CCTTCTGTTG GATGTCTCAT GAGTCCTGCC 5257

AGCCACTCAC CCGCCTGCCG GGCCTCCATT CTAGAGCAGG CAGAGCCCGC TGTGCCCTCA 5317

CCTGAGCλGG TTCAATAAAA GCAGAGTGGC AGA 5350

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1381 amino acidβ

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

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Met Ser Leu λrg Leu Phe Ser He Leu Leu λla λla Val Val Ser 1 5 10 15

Gly λla Gin Gly Trp βly Tyr Tyr Gly Cyβ λβn Glu Glu Leu Val Gly 20 25 30

Pro Leu Tyr λla λrg Ser Leu Gly λla Ser Ser Tyr Tyr Gly Leu Phe 35 40 45

Thr Thr λla λrg Phe λla λrg Leu His Gly He Ser Gly Trp Ser Pro 50 55 60

λrg He Gly λβp Pro λβn Pro Trp Leu Gin He λβp Leu Met Lye Lye 65 70 75 80

Hie λrg He Arg Ala Val λla Thr Gin Gly Ala Phe Aβn Ser Trp Aβp

85 90 95

Trp Val Thr Arg Tyr Met Leu Leu Tyr Gly Aβp λrg Val Asp Ser Trp 100 105 110

Thr Pro Phe Tyr Gin Gin Gly His Asn Ala Thr Phe Phe Gly Aβn Val 115 120 125

λβn λβp Ser λla Val Val λrg Hie λβp Leu Hie Tyr Hie Phe Thr λla

130 135 140

λrg Tyr He λrg He Val Pro Leu λla Trp λβn Pro λrg Gly Lye He 145 150 155 160

Gly Leu λrg Leu Gly He Tyr βly Cyβ Pro Tyr Thr Ser λβn He Leu 165 170 175

Tyr Phe λβp βly λβp λβp λla He Ser Tyr λrg Phe βln λrg βly λla 180 185 190

Ser βln Ser Leu Trp λβp Val Phe λla Phe Ser Phe Lye Thr Glu Glu 195 200 205

Lyβ λβp Gly Leu Leu Leu Hie Thr βlu βly Ser βln βly λβp Tyr Val 210 215 220

Thr Leu βlu Leu βln βly λla Hie Leu Leu Leu Hie Met Ser Leu βly 225 230 235 240

Ser Ser Pro He βln Pro λrg Pro βly Hie Thr Thr Val Ser λla βly 245 250 255

βly Val Leu λβn λβp Leu Ser Trp Hie Tyr Val λrg Val λβp λrg Tyr 260 265 270

βly λrg βlu λla λβn Leu Thr Leu λβp βly Tyr Val Hie λrg Phe Val 275 280 285

Leu λβn βly λβp Phe βlu λrg Leu λβn Leu βlu λβn Glu He Phe He 290 295 300

Gly βly Leu Val βly λla λla λrg Lyβ λβn Leu λla Tyr λrg Hie Aβn 305 310 315 320

Phe Arg βly Cyβ He Glu Aβn Val He Tyr Aβn λrg He λβn He λla 325 330 335

Glu Met λla Val Gin λrg Hie Ser λrg He Thr Phe Glu Gly λβn Val 340 345 350

λla Phe λrg Cyβ Leu λβp Pro Val Pro Hie Pro He λβn Phe Gly Gly 355 360 365

Pro Hie λβn Phe Val βln Val Pro Gly Phe Pro λrg λrg Gly λrg Leu 370 375 380

λla Val Ser Phe λrg Phe λrg Thr Trp λβp Leu Thr Gly Leu Leu Leu 385 390 395 400

Phe Ser λrg Leu Gly λβp Gly Leu Gly Hie Val Glu Leu Met Leu Ser 405 410 415

Glu Gly Gin Val λβn Val Ser He λla Gin Thr βly λrg Lyβ Lyβ Leu 420 425 430

Gin Phe λla λla Gly Tyr λrg Leu λβn λβp Gly Phe Trp Hie Glu Val 435 440 445

λβn Phe Val λla Gin Glu λβn Hie λla Val He Ser He Asp Aβp Val 450 455 460

Glu Gly λla Glu Val λrg Val Ser Tyr Pro Leu Leu He λrg Thr Gly 465 470 475 480

Thr Ser Tyr Phe Phe Gly Gly Cye Pro Lyβ Pro λla Ser λrg Trp βly 485 490 495

Cyβ Hie Ser λβn Cln Thr λla Phe Hie βly Cyβ Met Clu Leu Leu Lyβ 500 505 510

Val λβp βly Gin Leu Val λβn Leu Thr Leu Val Glu Phe λrg Lyβ Leu 515 520 525

βly Tyr Phe λla βlu Val Leu Phe λβp Thr Cyβ βly He Thr λβp λrg 530 535 540

Cyβ Ser Pro λβn Met Cyβ Clu Hie λβp Gly λrg Cyβ Tyr Gin Ser Trp 545 550 555 560

λβp λβp Phe He Cyβ Tyr Cyβ Glu Leu Thr Gly Tyr Lyβ Gly Val Thr 565 570 575

Cyβ Hie Glu Pro Leu Tyr Lyβ Glu Ser Cyβ Glu λla Tyr Arg Leu Ser 580 585 590

Gly Lyβ Tyr Ser βly Aβn Tyr Thr He Aβp Pro λβp βly Ser Gly Pro 595 600 605

Leu Lyβ Pro Phe Val Val Tyr Cyβ Aβp He λrg Glu λβn λrg λla Trp 610 615 620

Thr Val Val λrg Hie λβp λrg Leu Trp Thr Thr λrg Val Thr Gly Ser 625 630 635 640

Ser Met λβp λrg Pro Phe Leu βly λla He βln Tyr Trp λβn λla Ser 645 650 655

Trp Glu Glu Val Ser λla Leu λla λβn λla Ser βln Hie Cyβ Clu βln 660 665 670

Trp He Glu Phe Ser Cyβ Tyr λβn Ser λrg Leu Leu λβn Thr λla βly 675 680 685

βly Tyr Pro Tyr Ser Phe Trp He Gly λrg Aβn βlu βlu βln Hie Phe 690 695 700

Tyr Trp βly βly Ser Gin Pro Gly He Gin λrg Cyβ λla Cyβ Gly Leu 705 710 715 720

λβp Gin Ser Cyβ He λβp Pro λla Leu Hie Cyβ λβn Cyβ λβp λla λβp 725 730 735

Gin Pro βln Trp λrg Thr λβp Lye βly Leu Leu Thr Phe Val λβp His 740 745 750

Leu Pro Val Thr βln Val Val He βly λβp Thr λβn λrg Ser Ser Ser 755 760 765

βlu λla βln Phe Phe Leu λrg Pro Leu λrg Cyβ Tyr Gly λβp λrg λβn 770 775 780

Ser Trp λβn Thr He Ser Phe λrg Thr Gly λla λla Leu λrg Phe Pro 785 790 795 800

Pro He λrg λla λβn Hie Ser Leu λβp Val Ser Phe Tyr Phe λrg Thr 805 810 815

Ser λla Pro Ser Gly Val Phe Leu Glu λβn Met Gly Gly Pro Phe Cyβ 820 825 830

Gin Trp λrg λrg Pro Tyr Val λrg Val Glu Leu λβn Thr Ser λrg λβp 835 840 845

Val Val Phe λla Phe λβp He βly λβn βly λβp βlu λβn Leu Thr Val 850 855 860

Hie Ser λep λβp Phe βlu Phe λβn λβp λβp βlu Trp His Leu Val λrg 865 870 875 880

λla βlu He λsn Val Lyβ βln λla λrg Leu λrg Val λβp Hie λrg Pro 885 890 895

Trp Val Leu λrg Pro Met Pro Leu βln Thr Tyr He Trp Leu βlu Tyr 900 905 910

λβp βln Pro Leu Tyr Val βly Ser λla βlu Leu Lyβ λrg λrg Pro Phe 915 920 925

Val βly Cyβ Leu λrg λla Met λrg Leu λβn Cly Val Thr Leu λβn Leu 930 935 940

βlu βly λrg λla λβn λla Ser Glu βly Thr Phe Pro λβn Cyβ Thr Gly 945 950 955 960

Hie Cyβ Thr Hie Pro λrg Phe Pro Cyβ Phe Hie Gly Gly λrg Cyβ Val 965 970 975

Glu λrg Tyr Ser Tyr Tyr Thr Cyβ λβp Cyβ λβp Leu Thr λla Phe λβp 980 985 990

Gly Pro Tyr Cyβ λβn Hie λβp He Gly Gly Phe Phe Glu Thr Gly Thr 995 1000 1005

Trp Met λrg Tyr λβn Leu βln Ser λla Leu λrg Ser λla λla βln βlu 1010 1015 1020

Phe Ser Hie Met Leu Ser λrg Pro Val Pro βly Tyr βlu Pro βly Tyr 1025 1030 1035 1040

He Pro βly Tyr λβp Thr Pro βly Tyr Val Pro Gly Tyr Hie Gly Pro 1045 1050 1055

Gly Tyr λrg Leu Pro λβp Tyr Pro λrg Pro Gly λrg Pro Val Pro Gly 1060 1065 1070

Tyr λrg Gly Pro Val Tyr λβn Val Thr Gly Glu Glu Val Ser Phe Ser 1075 1080 1085

Phe Ser Thr Ser Ser λla Pro λla Val Leu Leu Tyr Val Ser Ser Phe 1090 1095 1100

Val λrg λβp Tyr Met λla Val Leu He Lyβ Glu λβp Gly Thr Leu Gin 1105 1110 1115 1120

Leu λrg Tyr Gin Leu Gly Thr Ser Pro Tyr Val Tyr Gin Leu Thr Thr 1125 1130 1135

λrg Pro Val Thr λβp Gly βln Pro Hie Ser Val λan He Thr λrg Val 1140 1145 1150

Tyr λrg λβn Leu Phe He βln Val λβp Tyr Phe Pro Leu Thr βlu Gin 1155 1160 1165

Lyβ Phe Ser Leu Leu Val λβp Ser Gin Leu λβp Ser Pro Lyβ λla Leu 1170 1175 1180

Tyr Leu Gly λrg Val Met Glu Thr Gly Val He λβp Pro βlu He Gin 1185 1190 1195 1200

λrg Tyr λβn Thr Pro Gly Phe Ser βly Cyβ Leu Ser Gly Val λrg Phe 1205 1210 1215

λβn λβn Val λla Pro Leu Lye Thr Hie Phe λrg Thr Pro λrg Pro Met 1220 1225 1230

Thr λla Glu Leu λla βlu λla Met λrg Val βln βly βlu Leu Ser Glu 1235 1240 1245

Ser λβn Cyβ Gly λla Met Pro λrg Leu Val Ser Glu Val Pro Pro Glu 1250 1255 1260

Leu λβp Pro Trp Tyr Leu Pro Pro λβp Phe Pro Tyr Tyr Hie λβp λβp 1265 1270 1275 1280

βly Trp He λla He Leu Leu βly Phe Leu Val λla Phe Leu Leu Leu 1285 1290 1295

βly Leu Val βly Met Leu Val Leu Phe Tyr Leu Gin Asn Hie Arg Tyr 1300 1305 1310

Lyβ βly Ser Tyr Hie Thr λβn βlu Pro Lyβ λla Thr His λβp Ser Hie 1315 1320 1325

Pro βly βly Lyβ λla Pro Leu Pro Pro Ser βly Pro λla Gin λla Pro 1330 1335 1340

λla Pro Thr Pro λla Pro Thr Gin Val Pro Thr Pro λla Pro Ala Pro 1345 1350 1355 1360

Ala Ser Gly Pro Gly Pro Arg Aβp Gin Aβn Leu Pro Gin He Leu βlu

1365 1370 1375

Glu Ser λrg Ser Glu 1380

The present invention iε not to be limited in scope by the exemplified embodiments which are intended aε illustration of single aspects of the invention, and any clones, DNA or amino acid sequences which are functional equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

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

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