AU1869802A - Protein binding fragments of gravin - Google Patents

Description

P/00/011 28/5/91 Regulation 3.2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT 0 0.

0 0 oo: Name of Applicant: Actual Inventors Address for service is: ICOS Corporation John D Scott, J Brian Nauert and Theresa M Klauck WRAY ASSOCIATES 239 Adelaide Terrace Perth, WA 6000 Attorney code: WR Invention Title: "Protein Binding Fragments of Gravin" This application is a Divisional Application by virtue of Section 39 of Australian Patent Application 57141/98.

The following statement is a full description of this invention, including the best method of performing it known to me:- -1/2- PROTEIN BINDING FRAGMENTS OF GRAVIN FIELD OF THE INVENTION The present invention relates generally to proteins that maintain the subcellular localization of kinases. More specifically, this invention relates to polypeptide fragments of a protein, gravin, which binds to a regulatory subunit of cAMP-dependent protein kinase or to protein kinase C. The invention also relates to methods of modulating the interaction of gravin and its binding partners.

BACKGROUND OF THE INVENTION Protein kinases are ubiquitous enzymes expressed in all eukaryotic cells and are involved in cellular responses to physiological stimuli.

Protein kinases attach phosphate groups to substrate proteins. Cyclic-AMP 15 (cAMP) dependent protein kinase (PKA) is an enzyme with broad substrate specificity which phosphorylates sutbstrate proteins in response to cAMP.

Protein kinase C (PKC) is an enzyme which phosphorylates substrate proteins in response to intracellular Ca:" and phospholipid.

Many hormones act through common signal transduction 20 pathways that generate the intracellular second messenger cAMP. The predominant action of cAMP is to activate a PKA by binding to the regulatory subunit dimer of the holoenzvym therehy releasing the catalytic subunit.

Free C subunit potentiates honnonal responses by phosphorylating substrate proteins near the site of kinase activation.

Two classes of the R subunit have been identified; RI and RII subunits which respectively fonu the type I and type II PKA holoenzymes. The subcellular distributions of PKA isoforms appear to be distinct. The RI isoforms (RIce and RIP) are reported to be predominantly cytoplasmic and are excluded from the nuclear compartment, whereas up to 75% of the RI isoforms (RIIc or RIIP) are particulate and associate with the plasma membrane, cytoskeletal components, secretory granules, the golgi apparatus, centrosomes and/or possibly nuclei.

Intracellular transduction of signals from the plasma membrane to specific subcellular compartments is a complex and highly regulated series of events which control essential physiological processes. An example of signaling pathway involvment are essential in maintaining cellular homeostasis appears in Hunter, Cell, 80:225-236 (1995) where it was shown that many transforming oncogenes encode signal transduction components such as low 10 molecular weight G proteins, protein kinases, or phosphatases. Phosphatases remove phosphate groups from proteins or other compounds. Kinase and phosphatase activities thus control intracellular signal transduction by phosphorylating and dephosphorylating substrate molecules. Now that many genes encoding these proteins have been identified, research emphasis has begun to focus on how these cnyvmes interface to control cellular events. A critical element in this operation is the subcellular location of each signaling enzyme. For example. Newton. Current Biology, 6:806-809 (1996) showed that the correct intracellular targeting of kinases and phosphatases directs these enzymes to their preferred substrates and reduces indiscriminate background phosphorylation and dephosphorylation.

Kinase and phosphata.sc targeting is achieved through association with targeting proteins or subunits Irevicwcd by Faux and Scott, TIBS, 21:312- 315 (1996b)]. For example, tyrosine kinase (PTK) and tyrosine phosphatase (PTPase) activity are coupled to downstream cytoplasmic enzymes through adaptor proteins that contain SH2 and SH3 domains. SH2 domains recognize certain phosphotyrosyl residues and SH3 domains bind to a PXXP motif found in some kinases and phosphatases. Modular adaptor proteins like Grb2, p 8 5 IRS-1, Crk and Nck comprise a single SH2 domain that recognizes phosphotyrosyl residues of signalling enzymesand two SH3 domains that bind to the PXXP motif on a separate set of target proteins. Similarly, many phospholipases, kinases, phosphatases and heterotrimeric G-proteins are targeted by specific membrane-targeting motifs such as the LIM, C2, pleckstrin homology and lipid anchoring domains [Gill, Structure, 3:1285-1289 (1995); Newton, Current Biology, 5:973-976 (1995)]. Through these interactions, signaling complexes assemble around receptor kinases or scaffold proteins to mediate cellular processes including hormone signaling events and immune cell function [Harrison et al., TIBS, 20:1213-1221 (1995)].

1 0 Until recently. second messenger-stimulated kinases and phosphatases were thought to be localized through association with individual targeting proteis. For example, three classes of phosphatase targeting subunits have been identified which are specific for protein phosphatase I [Chen et al.

FEBS Letters, 356:51-55 (1994))1: protein phosphatase 2A [Csortos et al., J.

Biol. Chem., 271:2578-2588 (10)96]: or protein phosphatase 2B [Shibasaki et al.. Nature, 382:370-373 (1Q96). Likewise, three classes of PKC targeting proteins have been identified in Chapline et al., J. Biol. Chem. 268:6858-6861 (1993): Mochly-Rosen. 1995: and Staudinger et al., J. Cell Biol., 128:263-271 (1995). Compartmentalization of PKA is achieved through interaction of the R subunits with a functionally related family of thirty or so A-Kinase Anchoring Proteins. called AKAPs [reviewed in Scott and McCartney, Molecular Endocrinology. 8:5-13 (1 The present invention contemplates that anchoring proteins confer specificity on serine/threonine kinases and phosphatases by directing these cn/ymes to discrete subcellular sites where they have restricted access to cenain substrates and are optimally positioned to respond to fluctuations in the levels of second messengers.

A variation on this theme was reported in the recent identification of multivalent binding proteins that coordinate the location of serine/threonine kinase and phosphatase signaling complexes. For example, Herskowitz, Cell, 80:187-197 (1995) showed that the pheromone mating response in yeast is initiated through a G-protein linked receptor that activates a yeast MAP kinase cascade. This process proceeds efficiently because each enzyme in the cascade is associated with a scaffold protein called sterile 5 (STE [Choi er al., Cell, 78:499-512, (1994)]. Clustering of successive members in the MAP kinase cascade allows for the tight regulation of the pathway and prevents "cross-talk" between the six functionally distinct MAP kinase modules in yeast [Herskowitz er al., 1995]. Another example of a multivalent binding 10 protein is AKAP79 which targets PKA. PKC and protein phosphatase 2B at the postsynaptic densities of mammalian synapses [Klauck el al., Science, 271:1589-1592 (1996); Coghlan. er al., (1995b). The structure of AKAP79 is modular and resembles STE 5. Deletion analysis, peptide studies and coprecipitation studies of AKAP79 and STE5 have demonstrated that enzymes bind to distinct regions of the anchoring protein [Klauck er al., 1996].

Targeting of the AKAP79 signaling complex to the postsynaptic densities suggests a model for reversible phosphorylation in which the opposing effects of kinase and phosphatase action are co-localized by a common anchoring protein [Coghlan et al.. Advances in Protein Phosphatases, 6:51-61 (1995a)].

AKAPs have been identified in a variety of organisms. At least seven proteins that bind the regulatory subunit of PKA in Aplysia califomica, a marine invertebrate, have been identified [Cheley et al., J. Biol. Chem., 269:2911-2920 (1994)]. One of these proteins is enriched in crude membrane fractions and taxol-stabilized miicrotubules and may thus anchor microtubules to the cell membrane as well as hind PKA. A mammalian AKAP microtubuleassociated protein 2 (MAP2) attaches PKA to the cytoskeleton [DeCamilli et al., J. Cell Biol., 103:189-203 (1986)]. The PKA-binding site on MAP2 is a 31-residue peptide in the amino-terminal region of the MAP2 molecule [Rubino et al., Neuron, 3:631-638 (1989)].

To date, a number of AKAPs have been identified which apparently bind PKA by a common secondary structure motif that includes an amphipathic helix region [Scott and McCartney, 1994]. Binding of PKA to most, if not all, identified AKAPs can be blocked in the presence of a peptide (Ht31) (SEQ ID NO: 8) that mimics the common secondary structure, while a mutant Ht31 peptide containing a single amino acid substitution (SEQ ID NO: 18) that disrupts the secondary structure of the peptide has no effect on PKA/AKAP binding [Carr et al,. J. Biol. Chem., 266:14188-14192 (1991)].

10 Even though PKA/AKAP interaction is effected by a common secondary oeee structure. AKAPs (or homologous AKAPs found in different species) generally have unique primary structure as is evidenced by the growing number of AKAPs that have been identified in a variety of organisms. The unique structure in each anchoring protein confers specificity on a kinase by targeting an AKAP signalling complex to specific intracellular locations.

Chapline and co-workers recently reported the cloning of a PKC binding protein identified as "clone 72" [Chapline et al., J. Biol. Chem., 271:6417-6422 (1996)]. Interestingly. Clone 72 was shown to have substantial homology to a mitogenic regulatory gene identified as "clone 322" [Lin et al., Mol. Cell. Biol., 15:2754-2762 (1995)]. Clone 322 was identified as being the same molecule identified as "SSeCKS" in Lin, et al., J. Biol. Chem.

271:28340-28348 (1996). Clone 322 was shown to be down-regulated in oncogene src, ras. fos and ntyc) transformed cells and thus appears to be a tumor suppressor gene.

Also of interest to the invention is Myasthenia gravis a disease of neuromuscular transmission characterized by weakness and rapid fatigability of the muscles. It is believed that MG is an autoimmune disease in which the patient develops antibodies to the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor controls a cation channel in response to binding of acetylcholine. In addition, development of autoantibodies to other cytoskeletal antigens including alpha actinin, actin, filamin and vinculin is observed in the MG patient. The muscle weakness appears to be caused by a failure of the nicotinic acetylcholine receptor as the autoantibodies apparently participate in destruction of the nicotinic acetylcholine receptors.

A previously unknown MG antigen, gravin, was identified by expression screening of a cDNA library with serum from a patient suffering from MG [Gordon ei al., J. Clin. Invest., 90:992-999 (1992)]. Gordon, er al.

10 disclosed amino acid sequences disclosing 306 C-terminal amino acid residues of gravin and the corresponding polynucleotide. Gravin was shown to be expressed on the cell cortex and was also shown to be expressed in migratory cells such as fibroblasts and neurons, but not in stationary cells such as epithelial cells. In addition. grvin was found to be expressed in adherent cells, but not in non-adherent cells. Thererere. gravin was postulated to play a role in cell migration and!or cellular adhesion [Grove et al., Anar. Rec.,239:231- S242 (1994)].

There continues to exist a need in the art for further insights into .i the nature, function.and distribution of anchoring proteins and the role of anchoring proteins in myasthenia gravis.

SUMMARY OF THE INVENTION This present invention is based on the discovery that gravin is a kinase anchoring protein that hinds to the type II regulatory subunits of PKA and to PKC. The complete amino acid sequence of gravin is provided herein. In binding to protein kinases, gravin localizes kinases to a specific subcellular region(s) and may regulate the function of the kinases and thereby control cellular signalling.

In one aspect, the present invention provides a gravin polypeptide fragment that binds to the type II regulatory subunit of PKA.

Preferably, the polypeptide fragment comprises amino acid residues 1526-1582 (SEQ ID NO: 1) of gravin. More preferably, the polypeptide fragment comprises amino acid residues 1537-1563 (SEQ ID NO: 2) of gravin.

In another aspect, the present invention provides a polypeptide fragment that binds to PKC. Preferably, the polypeptide fragment comprises amino acid residues 265-556 (SEQ ID NO: 3) of gravin.

Yet another aspect of this invention provides polypeptide analogs 10 of such fragments. Analogs are fragments in which additions, substitutions, o* including conservative substitutions, or deletions of amino acid residues have been made in order to increase or decrease the binding affinity of the analog fragment for a protein kinase. These analogs of gravin may be useful for modulating blocking. inhibiting, or stimulating) the interaction between gravin and the kinase.

The polypeptides of the present invention are synthesized in solution or on a solid support in accordance with conventional techniques as described in Stewart and Young. Solid Phase Pepride Synthesis, 2nd ed., Pierce Chemical Company. (1984) or Tam et al., J. Am. Chem. Soc., 105:6442 (1983), both of which are incorporated herein by reference.

The polypeptides of this invention may be modified to facilitate passage into the cell. such as by conjugation to a lipid soluble moiety. For example, the peptides may be conjugated to myristic acid. The peptides inay be myristoylated by standard techniques as described in Eichholtz et al., J.

Biol. Chem. 268:1982-1986 (1993). incorporated herein by. reference.

Alternatively, the peptides may be packaged in liposomes that may fuse with cell membranes and deliver the peptides into the cells. Encapsulation of the peptides in liposomes may also be performed by standard techniques as generally described in U.S. Patent Nos. 4,766,046; 5,169,637; 5,180,713; 5,185,154; 5,204,112; and 5,252,263 and PCT Patent Application No.

92/02244, each of which is incorporated herein by reference.

Another aspect of the invention provides polynucleotides encoding the protein binding fragments of gravin. Polynucleotides of the invention include DNA genomic, complementary, and synthetic) and RNA. Sense and antisense polynucleotides, complementary to coding and noncoding polynucleotides are also contemplated. The polynucleotides of the present invention can be generated and purified by any number of standard, 10 well-known techniques in the art. Also contemplated are polynucleotides which code for the polypeptides of the present invention based upon degeneracy of the genetic code. In addition, polynucleotides which encode gravin degenerate oligomers) useful in polymerase chain reaction (PCR) technologies S are contemplated. Polynucleotides encoding analogs of gravin or structurally related DNAs which hybridize under stringent hybridization conditions to the polynucleotides of the invention arn also contemplated. Those of ordinary skill in the art will understand hybridization conditions described as "stringent." Exemplary stringent hybridization conditions are as follows: hybridization at about 65 0 C in 3X SSC. 20mM NaPO, pH 6.8 and washing at about 65*C in 0.2X SSC. It is understood by those of skill in the art that variation in these conditions occurs ba.sed on the length and GC nucleotide base content of the sequences to he hybridized. Formulas standard in the art are appropriate for determining exact hybridization conditions. See Sambrook ei al., 9.47-9.51 in Molecular Cloningr Cold Spring Harbor Laboratory Press, Cold Spring Harbor. New York (1989).

Polynucleotides of the invention are useful for recombinant production of the kinase binding domain polypeptides. Vectors comprising polynucleotides encoding a kinase binding domain as well as promotor, selectable marker and other well-known vector components origin of replication, multiple cloning sites, etc.) are also contemplated by the invention.

The skilled artisan will understand the various components of vectors, methods for manipulating and the uses of vectors in transforming or transfecting of host cells (prokaryotic and eukaryotic) and expressing the kinase binding domains of the present invention. Host cells, especially unicellular host cells such as procaryotic and eukaryotic cells, are stably or transiently transformed or transfected with DNAs of the invention in a manner allowing expression of the kinase binding fragments of gravin. Host cells of the invention are 10 conspicuously useful in methods for the large scale production of protein binding fragments of gravin wherein the cells are grown in a suitable culture medium and the desired fragments are isolated from the cells or from the m edium in which the cells are grown. Use of mammalian host cells is expected to provide for such post-translational modifications myristoylation, glycosylation, proteolytic processing, lipidation and tyrosine, serine or threonine phosphorylation) as mn:i be needed to confer biological activity on recombinant expression products of the invention.

Another aspect of this invention provides antibody substances polyclonal and monoclonal antibodies, antibody fragments, single chain antibodies, chimeric antibodies. CDR-grafted antibodies, humanized antibodies and the like) specifically immunoreactive with the protein binding domains of gravin. Antibody substances can he prepared by standard techniques using isolated naturally-occurring or recombinant gravin. The antibody substances are useful in modulating blocking. inhibiting, or stimulating) the binding between gravin and the kinase and in detecting gravin in patients suffering from MG. In addition, cell lines. hybridomas), or cell lines transformed with recombinant expression constructs which produce antibody substances of the invention are contemplated.

In another aspect, methods of identifying a modulator compound that inhibits or increases binding between a gravin polypeptide and a gravin binding partner type II regulatory subunit of PKA or PKC) are contemplated. In one method, gravin or a polypeptide fragment thereof such as set out in SEQ ID NOs: 1, 2 or 3 and a binding partner are incubated in the presence and absence of a putative modulator compound under conditions suitable for binding. The amount of binding in the presence and in the absence of the putative test compound is determined and compared. A reduction in the amount of binding observed in the presence of the test compound indicates that 10 the test compound is an inhibitor. An increase in the amount of binding observed in the presence of the test compound indicates that the test compound increases binding between gravin and the binding partner. In one embodiment, either gravin or the binding panner can be immobilized on a solid substrate, and either gravin or the binding partner is detectably labeled. In addition, other assays, such as scintillation proximity assays may also be employed.

.Modulators are useful for example, in inhibiting localization of a gravin binding partner PKA. PKC. or other kinases) to a specific subcellular location. The contemplated modulators include polypeptides, :polypeptide fragments of gravin and other organic and inorganic compounds.

The DNA sequence information provided by the present invention also makes possible the development. by homologous recombination or "knockout" strategies see e.g. Capccchi. Science 244:1288-1292 (1989)] of mammals that fail to express functional gravin or that express an analog of gravin. The mammals of the present invention comprise a disruption of the gravin gene of the mammal or the disruption of a homolog of the gravin gene.

The general strategy utilized to produce the mammals of the present invention involves the preparation of a targeting construct comprising DNA sequences homologous to the endogenous gene to be disrupted. The targeting construct is then introduced into embryonic stem cells (ES cells) whereby it integrates into and disrupts the endogenous gene or homolog thereof. After selecting cells which include the desired disruption, the selected ES cells are implanted into an embryo at the blastocyst stage. Exemplary mammals include rodent species.

Numerous additional aspects and advantages of the present invention will be apparent from the following detailed description of illustrative embodiments thereof.

DETAILED DESCRIPTION OF THE INVENTION 10 The present invention is illustrated by the following examples.

Example 1 describes the cloning and characterization of a cDNA encoding gravin. The mapping and identification of a fragment of gravin that binds to the type II regulatory subunit of PKA is disclosed in Example 2. Example 3 describes the expression of full length gravin in COS cells. Example 4 describes the mapping and identification of a PKC binding fragment of gravin.

Example 5 discusses the preparation of monoclonal and polyclonal antibodies.

Experiments describing gravin expression in human erythroleukemia cells (HEL) is provided in Example 6. Example 7 describes experiments identifying tissue distribution of gravin. Example 8 describes the role of gravin in signal transduction and Example 9 describes binding assays utilizing gravin an a binding partner. Example 10 describes gravin's role in cell adhesion. In light of the present disclosure, those of skill in the an will appreciate that the following examples are intended to be illustrative only and that numerous changes, modifications and alterations can be made without departing from the spirit and scope of the invention.

Example 1 To isolate cDNAs encoding potential RII binding proteins, a human fetal brain A-ZAP cDNA library was screened by a modified overlay procedure using radiolabeled RHII as a probe [Lohmann er al., Proc. Natl.

Acad. Sci. 81:6723-6727 (1984)]. Eight RII binding clones were identified, plaque purified and the ends of each insert were sequenced. Two of the clones represented known sequences. One matched MAP2, a previously identified AKAP [Theurkauf et al.. J. Biol. Chem., 257:3284-3290 (1982)].

The 3' end of another clone, designated HF 9, was identical to a previously described partial clone encoding gravin. which was originally isolated by screening a Human Umbilical Vein Endothelial cell (HUVE) cDNA library with serum from a Myasthenia gravis patient [Gordon et al., 1992].

Further sequencing of clone HF 9 showed that the cDNA insert was 3023 base pairs in length and encoded a continuous open reading frame of 651 amino acids. Northern hlt analysis using a 32 P random primed 1676 base pair Eco RI-Spe I fragment of HF l a;s a ponbe indicated that gravin mRNA was selectively expressed in cenain human tissues. Two predominant mRNA S. species of 8.4 kb and 6.7 kb were detected in all tissues but predominated in liver. brain and lung. whereas an additional 5.5 kb message was detected in brain. The larger sizes of all the gri\ in messages, indicated that the HF 9 clone represented a partial cDNA. Thlerctore. the 1676 base pair HF 9 fragment was used to further screen the human fetal brain cDNA library for more complete transcripts. Five additional clones wenre obtained that yielded an additional 600 base pairs of coding region. As an alternative strategy, a human heart cDNA library was screened with the same 1676 base pair HF 9 fragment. Of the five positive clones isolated from the heart cDNA library, the longest clone contained a 4216 base pair insert, which overlapped with the 5' end of HF 9.

This provided a contiguous composite sequence of 6605 base pairs encoding a -13protein of 1780 amino acids. The complete DNA and amino acid sequences of this protein, human gravin, are presented in SEQ ID NO: 4 and 5, respectively.

Example 2 The last 651 amino acids of gravin were demonstrated to contain a binding site for association with the type II regulatory subunit of PKA. It was previously shown that regions of conserved secondary structure which are likely to include amphipathic a-helices are responsible for RIl-binding [Carr et al., SJ. Biol. Chem., 267:13376-13382 (1992)]. Residues 1540-1553 LETKSSKLVQNIIO (SEQ ID NO: 6) of gravin fulfilled these criteria. These residues also show sequence identity with corresponding regions in other AKAPs and a helical-wheel plot suggested that there was a segregation of hydrophobic and hydrophilic side-chains which is compatible with the formation of an amphipathic helix. The RIla binding fragment of gravin also shows some sequence homology to the corresponding RII binding regions in AKAP79 (LIETASSLVKNAIQ) (SEQ ID NO: 7) and in Ht31 (DLIEEAASRIVDAVIEQVKAAGA) (SEQ ID NO: Ht31 is a sequence derived from human thyroid AKAP.

To identify the RII binding site(s) of gravin, a family of recombinant DNAs encoding fragments of gravin were generated by PCR using HF9 as the template. The polynucleotides encoding these fragments were subcloned into the pETI6d pl;asmid which provides nucleotide sequences encoding a histine tag expressed at the amino terminus of the expressed gravin fragment. These constructs were expressed in E. coli and purified using the pETI6d Histag bacterial expression/affinity purification system. Constructs encoding putative RII-binding site residues 1130-1582 (SEQ ID NO: 17) and 1130-1525 (SEQ ID NO: 15) of gravin were generated by utilizing a common primer, CCGCCATGGTGCATATGTCCGAGTCCAGTGAGC, (SEQ ID NO: 9) but utilized distinct 3' primers: GCGCGGATCCGCACTCACTITGACCTCCTG (SEQ ID NO: 10) for residues 1130-1525 (SEQ ID NO: 15) and GCGCGGATCCGCTATCACGTGAGCTTGTGT (SEQ ID NO: 11) for residues 1130-1582 (SEQ ID NO: 14). The 1526-1780 (SEQ ID NO: 16) construct was prepared by using the 5' primer, CCGCCATGGTGCATATGGTAGCAATTGAGGATTTAG (SEQ ID NO: 12) in conjunction with the 3' primer, GGAGGATCCAGAGATTCTGTAGTTCTG (SEQ ID NO: 13) used to subclone the full length clone. Each gravin construct was transfected into E. coli and the expression of recombinant Histag fusion proteins 't 10 was induced by IPTG. Each recombinant protein was purified according to previously published methods [Coghlan et al., Science, 267:108-111 (1995b)].

The gravin fragments were screened for Rfla binding using an overlay procedure essentially as described in Lohman et al., Proc. Nat. Acad.

Sci., 81:6723-6727 (1984). Briefly. the overlay procedure is performed as follows. Protein samples arc separated by SDS polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose by standard electrotransfer techniques. The immobilized protein is partially renatured by S. incubation in a blocking solution containin milk proteins then probed with "Plabelled RII probe. After removal of unbound probe by washing, binding between gravin polypeptide fragments and RII is detected by autoradiography.

To increase sensitivity of the assa (up to ten-fold), bound RH is detected immunologically anti-RII-aniscra and 25 I-protein A, or monoclonal antibodies which specifically rccognize RII.) The 452 residiue fragmeni encompassing residues 1130-1582 (SEQ ID NO: 14) bound ':'P-rnliolabeled RIIa in the overlay, whereas a smaller fragment, residues 1130-1525 (SEQ ID NO: 15), which lacked the RIbinding region was unable to bind RIIa.

Two additional experiments provided evidence that the putative amphipathic helix region was sufficient for RII-binding. The fragment encompassing residues 1526-1780 (SEQ ID NO: 16) of gravin bound RII in the overlay and a synthetic peptide covering residues 1537-1563 (SEQ ID NO: 2) blocked all RII-binding in the overlay. In addition, the anchoring protein inhibitor peptide Ht31 (DLEEAASRIVDAVIEQVKAAGA) (SEQ ID NO: 8) which is a competitive inhibitor of RII/AKAP interactions also blocked RII binding to gravin as assessed by the overlay assay. Control experiments in which overlays were performed in the presence of 0.3 /M inhibitor polypeptide Ht31 (SEQ ID NO: 8) confirmed that the Ht31 inhibited binding between gravin and RIla. In addition a second control peptide, Ht31-pro, UDLIEEAASRPVDAVIEQVKAAGA) (SEQ ID NO: 18) which is unable to block RII/AKAP binding was unable to inhibit binding between gravin and RIIa.

The second control peptide (SEQ ID NO: 18) is the Ht31 peptide in which an isoleucine has been replaced by a proline thereby disrupting the secondary structure. The Ht31 (SEQ ID NO: 8) peptide and Ht31-pro peptide were synthesized. To facilitate laheling and/or tracking of the control peptides. an additional tyrosine (radioiodination) or lysine (biotin/avidin) residue was Ssometimes included at the C-tenninus of the control peptides. This data demonstrates that gravin is an AKAP and its principle RII-binding site is encompassed by residues 1526-1582 (SEQ ID NO: 1).

This finding was tiniher confirmed when the binding affinity of the gravin 1526-1780 (SEQ ID NO: 16) fragment for a recombinant fragment of RIIa was measured by surface plasmon resonance (SPR). SPR is an analytical technique that utilizec evanescent light to examine macromolecular complexes. The binding affinities of one protein to an immobilized binding partner can be measured by SPR. A recombinant fragment encompassing residues 1526-1780 (SEQ ID NO: 16) of gravin was coupled to a carboxymethyldextran IAsys cuvette using standard EDC/NHS coupling -16chemistry [Davies et al., Techniques in Protein Chemistry, 5:285-2992 (1994)].

The cuvette was activated by treating with 0.4 M EDC/0.1 M NHS for 8 min and washed extensively with PBST (PBS 0.05% Tween-20). Coupling of the gravin 1526-1780 fragment (SEQ ID NO: 16) (25 /g/ml) was accomplished in 10 mM formate buffer, pH 3.6 for 10 min at room temperature. Uncoupled protein was washed out with PBST and free amines were blocked with IM ethanolamine, pH 8.5 for 2 min at room temperature. After washing with PBST, a stable baseline was established for 10 min before data collection. All binding experiments were performed with a recombinant fragment of RIIa (RI 1-45) [Scott. et al., Pro. Nat. Acad. Sci. 84:5192-5196 (1987)] which binds AKAPs with a similar affinity as the full-length protein. Previous experiments have indicated that release of RIIa 1-45 from the binding surface can be performed under conditions that are less harmful to the immobilized anchoring protein than studies using full length RII Binding experiments were performed over a range of concentrations from 25 to 150 nM in volumes of 200 Ml. The binding surface was regenerated between binding measurements using ethanol with no decrease in extent measurements over the duration of an experiment. Data collection was done over three second intervals and was .i analyzed using the FastfitTM software which was provided with the IAsys instrument. The binding properties of the immobilized gravin fragment were measured over a range of RIIc 1-45 concentrations from 25 to 150 nM (Figure IA). Uniform first order binding was recorded with a of 160006 9700 M- sec' and with a (of 0.016 0.001 M (Figure IB). These values were used to calculat e a dissociation constant (KD) of 100 nM for the RII/gravin fragment interaction (Figure IB). The nanomolar binding constant for RII/gravin interaction is well within the physiological concentration range of both proteins inside cells and is consistent with the notion that both proteins may associate in situ.

As demonstrated above, residues fragment 1537-1563 (SEQ ID NO: 2) of gravin form a PKA anchoring site. Of note, this sequence is present in the C-terminus of SSeCKS/clone 72 (see Example Interestingly, this shared sequence has ten out of fourteen residues which are conserved in the RII-binding region of another mammalian scaffold protein, called AKAP79, which binds PKA, PKC and protein phosphatase 2B [Coghlan er al., 1995b; Klauck et al.. 1996]. The identification of a conserved RII-binding sequence in gravin, SSeCKS/clone 72 and AKAP79 is the first example of conserved primary structure in known RI binding regions. This finding was unexpected 10 as it was previously proposed that in spite of a lack of sequence identity among the AKAPs there existed a conservation of secondary structure in the RIIbinding motif [Scott er al.. 1994]. Therefore, it is likely that gravin, SSeCKS/clone 72 and AKAP 79 are members of a structurally related subfamily of AKAPs which hind more than one kinase or phosphatase.

Example 3 In order to study the role ot'anchoring of PKA by gravin, full length gravin was expressed in cells that normally do not express the protein.

A plasmid containing, the till length gravin cDNA was prepared as follows.

A 1.7 kb Xbal frament was isolated from HF9 (containing the C-terminal sequece of Gravin) and cloned into pBSIl (Strategene) containing the N-terminal 4216 bp clone (see Example I) which was predigested with Xbal. Clones were screened and sequenced (on the 5' and 3' junctions) for correct orientation. The resulting clone is referred to as pBS/gravin. A EcoRI-Notl fragment of pBS/Gravin was inserted in pcDNA3 predigested with EcoRI and Not 1. Clones were screened for inserts by restriction digests using EcoRI and Notl. Correct clones were confirmed by sequencing the 5' and 3' junctions with primers DCO3 and JHSP6 respectively.

Transfection of recombinant gravin COS cells were grown in 100 mm culture dishes until 20-40% confluent.

Transfection of COS cells was performed as follows. The pcDNA3-gravin vector at a concentration of 10 gg in 150 jl serum free culture media was prepared to which was added 20pl of SuperFect (Qiagen, Chatsworth, CA). Media was removed from the COS cell culture and the cells washed with CMF-PBS. The SuperFect mixture was added to 3 ml of media with 10% FBS and added to the cells for three hours at 37°C. Cells were then washed and fresh media added for an overnight incubation. Next. the cells were washed, trypsin harvested, washed 10 again then lysed as described in Example 5 Transfection procedure for Jurkat ~cells was similar except the SuperFect mixture was added to Jurkat cells and left in the culture overnight Cell lYsates were prepared as described at 24, 48 hours and two weeks after transtfiction Gravin expression was determined by Western blot analysis..

15 Transfected COS cells showed an enhanced gravin signal over baseline expression levels at twenty-four hours post transfection. Jurkat cells, which do not express gravin. showed signiticant expression of the recombinant protein from twenty-four hours post transfection out to two weeks. Recombinant gravin can thus be expressed and maintained in human cell lines.

Exalpl- 4 Further sequence analysis revealed another potential function of gravin. A search of the nucle'tile tkLtahase using the complete gravin sequence showed that the first residues are 69% identical to a murine mitogenic regulatory protein SSeCKS [Lin et al.. 1995)] also identified in the art as "clone 72", which was recently shown io be a protein kinase C binding-protein and also a protein kinase C substrate. [Chapline et al., 1996].

The ability of gravin to bind PKC was therefore examined.

Accordingly, two recombinant gravin polypeptide fragments consisting of amino acids 265-556 (SEQ ID NO: 3) and 1130-1582 (SEQ ID NO: 14) were prepared and overlay analysis similar to the overlay analysis described in Example 2 was performed. The immobilized gravin fragments were incubated with PKC and the bound PKC was detected by using monoclonal antibodies to PKC (Transduction Labs, Lexington, KY) [Klauck, et al., (1996)] The 265- 556 (SEQ ID NO: 3) fragment was prepared by PCR using primers GACGAGATTGTGGAAATCCATGAGG (SEQ ID NO: 19) and GCGCGGATCCAGAGATTCTGTAGTTCTGAC (SEQ ID NO: 20). The 1130-1582 10 (SEQ ID NO: 14) fragment was prepared as described in Example 2. The results showed that PKC bound to the 265-556 fragment (SEQ ID NO: but not to the 1130-1582 fragment (SEQ ID NO: 14). The overlay assay thus showed that the PKC binding fragment of gravin mapped to a region of the sequence between residues 265 to 556 (SEQ ID NO: Neither of the gravin 15 fragments bound PKC in the absence of phosphatidylserine (PS) which is consistent with other reports ihat phospholipid is a co-factor in the PKC/binding Sprotein complex [Chapline et 1996]. It has been suggested that phosphatidylserine (PS) supports a ternary complex of PKC and polybasic regions on the substrate/binding protein [Liao et al., Biochem., 33:1229-1233 (1994)].

Polybasic regions were postulated to participate in formation of a phospholipid bridge between the PKC and its binding proteins [Chapline et al.. 1996; Chapline et al.. 1993.1. In AKAP79, a polybasic region was identified as the PKC binding site [Klauck et al., 1996]. In gravin, there are two polybasic regions in the grvin 265-556 fragment (SEQ ID NO: 3) located between residues 295-316 (FKKFFTQGWAGWRKKTSFRKPK) (SEQ ID NO: 23) and 514-536 (PLKKLFTSTGLKKLSGKKQKGKR) (SEQ ID NO: 24). Both polybasic regions (residues 295-316 and residues 514-536) resemble the PKCbinding site on AKAP79. Synthetic peptides of both polybasic regions of gravin blocked PKC/gravin interactions when assessed by the overlay assay.

These experiments show that protein kinase C binds gravin in vitro at one or more polybasic sites located between residues 265-556 of the protein.

The AKAP79 31-52 PKC binding site peptide KASMLCFKRRKKAAKLAKPKAG (SEQ ID NO: 23) blocked PKC binding to gravin. This result demonstrates that both gravin and AKAP79 likely bind to a similar site on PKC. Further similarity to AKAP79 was demonstrated when the gravin 265-556 (SEQ ID NO: 3) fragment was shown to inhibit PKC 10 activity toward peptide substrate VRKRTLRRL (SEQ ID NO: 24) (Sigma Chemical Co.. St. Louis. Mo.) with an ICso of 0.50 0.12 /M (n (Figure y In contrast, the RII binding peptide did not inhibit the kinase. PKC activity was assayed as described [Orr ei al.. J. Biol. Chem., 269:27715-27718, 1994] "in a reaction containing 40 mM HEPES (pH 10 mM MgCl, 0.3 mM CaCI.. I mM DTT. 100 NM1 adenosine triphosphate (ATP) (500 cpm/pmol), phosphatidylserine (20 ygml), and epidermal growth factor receptor peptide (VRKRTLRRL> (SEQ ID NO: 24) as substrate at 30°C for iin. PKC PII (20 ng/pl) was diluted 1:10 in 20 mM Tris (pH 1 mg/ml bovine serum albuimin (BSA) and I mM DTT. Inhibition constants (IC5s) were determined over an inhibition conccntration range of 0.1 to 10 yM gravin 265- 556 fragment (SEQ ID NO: 3).

To date. three classes of PKC-binding proteins have been identified by gel overlay and two-hybrid techniques [Faux et al., Cell, 70:8-12 (1996a)]. PKC substrate!binding proteins [Chapline et al., 1993] and Receptors for Activated C-kinase (RACKs) [Mochly-Rosen et al., Proc. Natl.

Acad. Sci. USA, 88:3997-400(X). (1991)] have been detected by the gel-overlay procedure, while Proteins that Interact with C-kinase (PICKS), have been isolated in two-hybrid screens [Staudinger et al., 1995]. The data provided -21herein shows that a region of approximately 290 amino acids supports PKCbinding and fragments corresponding to that region block kinase activity in vitro.

Example Monoclonal antibodies are prepared by immunizing Balb/c mice subcutaneously with gravin or gravin fragments in complete Freund's adjuvant (CFA). Subsequent immunizations in CFA or incomplete Freund's adjuvant is perforned to increase immune response.

10 The spleen of the immunized animal is removed aseptically and a single-cell suspension is formed by grinding the spleen between the frosted ends of two glass microscope slides submerged in serum free RPMI 1640, supplemented with 2 mM L-glutamine. I mM sodium pyruvate, 100 units/ml penicillin, and 100 ug/ml streptomycin (RPMI) (Gibco, Canada). The cell 15 suspension is filtered through sterile 70-mesh Nitex cell strainer (Becton Dickinson. Parsippany. New Jersey). and washed twice by centrifuging at 200 g for 5 minutes and resuspending the pellet in 20 ml serum free RPMI.

Thymocytes taken from naive Balb/c mice are prepared in the same manner.

2 x 10' spleen cells are combined with 4 x 10' NS-1 cells (kept 20 in log phase in RPMI with 11 fetal bovine serum (FBS) for three days prior to fusion), centrifuged and thie supernatant is aspirated. The cell pellet is dislodged and 2 ml of 37 C PEG 1500 (50% in 75 mM HEPES, pH (Boehringer Mannheim) is added while stirring over the course of one minute, followed by the addition of 14 ml of serum free RPMI over seven minutes.

Additional RPMI can he added and the cells are centrifuged at 200 g for minutes. After discarding the supernatant. the pellet is resuspended in 200 ml RPMI containing 15% FBS. 100 mM sodium hypoxanthine, 0.4 mM aminopterin, 16 mM thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer -22- Mannheim) and 1.5 x 106 thymocytes/ml. The suspension is dispensed into ten 96-well flat bottom tissue culture plates (Corning, United Kingdom) at 200 jl/well. Cells are fed on days 2, 4, and 6 days post-fusion by aspirating 100 il from each well with an 18 G needle (Becton Dickinson), and adding 100 /l/well plating medium containing 10 U/ml IL-6 and lacking thymocytes.

When cell growth reaches 60-80% confluence (day 8-10), culture supernatants are taken from each well and screened for reactivity to gravin by ELISA. ELISAs are performed as follows. Immulon 4 plates (Dynatech, Cambridge, Massachusetts) were coated at 4°C with 50 Al/well with 10 100ng/well of pl 106:GST or GST in 50 mM carbonate buffer, pH 9.6. Plates are washed with PBS with 0.05 Tween 20 (PBST) and blocked 30 minutes at 37 0 C with 0.5% Fish Skin Gelatin. Plates are washed as described above and 50 /l culture supernatant is added. After incubation at 37 0 C for minutes, 50 til of horseradish peroxidase conjugated goat anti-mouse IgG(fc) S 15 (Jackson ImmunoResearch. West Grove. Pennsylvania) [diluted 1:10,000 in PBST] is added. Plates are incubated at 37 0 C for 30 minutes, washed with PBST and 100 ul of substrate. consisting of I mg/ml TMB (Sigma) and 0.15ml/ml 30% H.O. in 100 mM Citrtec. pH 4.5, is added. The color reaction is stopped with the addition of 50 ml of 15 H,SO 4

A

45 0 is read on a plate 20 reader (Dynatech).

Polyclonal antitbodies are prepared by immunizing an animal with an antigen comprising a polypeptidc of the present invention and collecting antisera from the immunized animal. A variety of animal species including rabbit, chicken, mouse. nit. or guincu pig are useful in preparation of polyclonal antibodies. The I130-1780 (SEQ ID NO: 17) gravin fragment was used to prepare polyclonal antibodies in rabbit. Rabbit polyclonal antisera R3698 was produced from the 1130-1780 gravin fragment (SEQ ID NO: 17) by a commercial laboratory (Bethyl Labs, Montgomery, TX). The 1130-1780 -23fragment (SEQ ID NO: 17) was made by preparing and expressing a polynucleotide encoding the 1130-1780 fragment (SEQ ID NO: 9) (generated by PCR using the 5' primer, CCGCCATGGTGCATATGTCCGAGTCCAGTGAGC, (SEQ ID NO: 9) and the 3' primer, GGAGGATCCAGAGAT-TCTGTAGTrCTG

(SEQ

ID NO: 13)) as described in Example 2. In addition, the 265-556 fragment (SEQ ID NO: 3) was used to prepare polyclonal antibodies in rabbit and chicken. Rabbit polyclonal antisera, R4310 and chicken polyclonal antisera were produced from the 265-556 fragment (SEQ ID NO: 3) by Bethyl Labs.

Two additional polyclonal antisera were prepared. Two rabbits (4037J and 10 3548J) were immunized (R&R Rabbitry, Stanwood, WA) with 25-50 pg of recombinant gravin fragment 265-556 for a total of three injections and a final boost. Test sera and pre-immune sera were tested by Western blot analysis.

Recombinant protein (I pg/lane) and lysates from HEL cells (25 pg/lane) grown with or without 40 pg/ml PMA to induce gravin expression were separated by 4- 15 12% SDS-PAGE (Novex. San Diego. CA) and transferred to immobilon by standard techniques Resulting blots were incubated in blocking buffer (TBS with 5% milk proteins) to partially renature immobilized protein, then probed with *o rabbit sera (1:500 dilution in blocking buffer). Unbound antibodies were removed by washing in TBS. followed by incubation with a secondary goat anti-rabbit horse 20 radish peroxidase (HRP) conjugated antibody (1:7500 dilution) in blocking buffer.

Unbound antibody was washed away and the blots were developed by enhanced chemiluminescence (ECL. Dupont-NEN. Boston, MA) and exposed on film.

Sera from both rabbits rcconized the recombinant gravin fragment. A 250 kDa band was detected in the P11A stimulated HEL cell lysate, but not in the Ivsate prepared from unstimulated HEL cells. Serum from rabbit 4037J had the higher sensitivity by Western blot and so this antibody was purified by Protein A affinity chromatography Example 6 Phorbol ester treatment of a human erythroleukemia cell line (HEL) (HEL 92.1.7, ATCC TIB 180) induces morphological, functional and biochemical changes that are characteristic of macrophage-like cells. One hallmark of this process is the robust induction of gravin [Gordon er al., 1992].

Therefore, the PKA and PKC binding protein profile of HEL cells after prolonged exposure to phorbol esters was examined.

.HEL cells were grown in RPMI 1640 containing 12 fetal calf serum and 4 mM glutamine. Gravin expression was induced by culturing with 10 40 nM phorbol myristate acetate (PMA) for 18 hr. Cell lysates were prepared from either adherent cells grown in the presence of PMA, rinsed with PBS and scraped from the interior of 150 cm: flasks or from suspension cultures of HEL cells grown in the absence of PMA. Cell pellets were washed twice with PBS prior to resuspension in 20 mM TrisHCI. pH 7.4, 150 mM NaCI, 10 mM 15 EDTA. 0.25% Triton X-Itl(. 0.05; Tween 20, 0.02% NaN 3 10 mM benzamidine. 21gi.ml pepstatin. 2/ugiml Icupeptin, 4 mM 4 2 -aminoethyl)benzenesulfonyl fluoride hydrochloride (Lysis Buffer) and incubation on ice for min. The extracts were then centrifuged for 10 min at 16,000 x g at 4 C and the cell lysate supernatant was collected. Protein concentrations were 20 measured using the Bio-Rad DC Protein Assay kit.

The extracts from control and treated cells were subjected to western blot analysis with an affinity purified antibody raised against residues 1130 to 1780 of gravin (See Ev.implc PMA treatment caused an induction of a 250 kDa protein that specifically reacted with anti-gravin antibodies.

Subsequent overlay assays delconstrated that PMA treatment induced the expression of a 250 kDa PKC-hinding protein and an RII-binding protein of the same size.

Concomitant with the macrophage-like shift, HEL cells undergoing PMA treatment become adherent and display a considerable cytoplasmic spread [Papayannopoulou et al., Blood, 62:832-845 (1983)]. This sometimes results in the formation of actin stress fibers and causes a general flattening of the cell. In order to establish whether gravin aligned with the actin cytoskeleton, phorbol ester treated HEL cells were stained with rhodamine phalloidin as a marker for actin as described below.

HEL cells were grown on glass coverslips in the presence of nM PMA for 18 hr, rinsed with PBS, fixed in 3.7% formaldehyde and extracted in -20"C absolute acetone. Cells were rehydrated for 1 hr in PBS and 10 0.2% BSA and then incubated with either affinity purified anti-gravin antibody, R3698, at 0.5 Ag/mi or pre-immune IgG at 0.5 A/g/ml. After 1 hr the coverslips were carefully washed in PBS and 0.2% BSA and incubated with either a mixture of FITC conjugated donkey anti-rabbit secondary antibody (1:100 dilution, Jackson ImmlunoRcasearch Laboratories Inc, West Grove, PA) 15 and rhodamine conjugated phalloidin (1 unit/coverslip, Molecular Probes, Inc, Eugene, OR) or secondary antibody alone. In situ RII-overlays were performed essentially as described [Coghlan et al.. J. Biol. Chem., 269:7658-7665 (1994)]. Prior to incubation with primary antibody, cells were incubated with 80 nM recombinant murine RIla for 2 hr and unbound RII removed by washing 20 three times in PBS and 0.27 BSA. The immobilized RIIa was detected immunochemically with affinity purified goat anti-murine RII (1 /g/ml) and Texas red conjugated donkey anti-gioat IgG secondary (1:100 dilution, Jackson ImmunoReasearch Laboratories Inc. West Grove, PA). Control coverslips were treated with the antibody to RII in the absence of exogenous murine RI.

Cells were examined using a Leica confocal laser scanning system equipped with a Leitz Fluoven-FU inverted microscope and an argon/krypton laser.

All of the cells displayed a concentration of actin to the periphery. In contrast. gravin staining was predominantly cytoplasmic and only -26a subset of the cells (approximately 25%) expressed large quantities of the protein. Variable levels of gravin expression were not unexpected as HEL cells represent a heterogeneous population at different stages of differentiation [Papayannopoulou et al., 1983]. Superimposition of images of cells stained for actin and cells stained for gravin showed that both proteins exhibit distinct but partially overlapping subcellular distributions. Control experiments were negative when cells were stained with preimmune serum. More detailed confocal analysis of HEL cells detected gravin staining toward the periphery of the cell and enriched in filopodia at the adherent surface. These findings 10 indicate that gravin functions to enhance HEL cell adhesion to the substratum.

**ooo Co-localization of Gravin and PKA In vitro binding studies described in Examples 2 and 4 indicate that gravin is a kinase scaffold protein. Therefore, co-localization experiments 15 were initiated to detenuine whethicr a gravin signaling complex could be detected in HEL cells. Fixed and penneabilized cells pre-treated with PMA were overlayed with recombinant murine RIIa. RI-binding in situ was detected with antibodies that specifically recognize murine RI and mimicked the staining pattern for gravin. Since control experiments confirmed that the anti-murine RU antibodies did not detect the endogenous human RI, the increased RI staining was due It, direct association with gravin. This conclusion was supported by additional control experiments showing that in situ RII-binding was blocked bh incuhation with the Ht 31 anchoring inhibitor peptide.

Finally. the gr\vin signaling complex was isolated by two complementary biochemical methods: immunoprecipitation and affinity chromatography on cAMP-agarose.

Immunoprecipitation of gravin was performed as follows. HEL cell lysates (200 pl of 15 mg/ml) prepared as described above were incubated with either 15 pg of affinity purified anti-gravin or 15 pg of pre-immune IgG at 4°C for 18 hr. Immune complexes were isolated by the addition of 200 l of 10% Protein A-Sepharose CL-4B (Sigma, St Louis, MO) which had been pre-equilibrated in Lysis Buffer. Following incubation at 4"C for 90 min the beads were washed once in 0.5 M NaCI Lysis Buffer and four times in excess 20 mM TrisHCI, pH 7.4. 150 mM NaCI. The PKA catalytic subunit was released from the immune complex by incubating the Protein-A beads in 200 ,1 1 mM cAMP. 20 mM TrisHCl. pH 7.4, 150 nM NaCI for 15 min. The 10 eluate was TCA precipitated prior to analysis on a 4-15% SDS-PAGE gel, electroblotted onto nitrocellulose and the catalytic subunit was detected, as previously described. For the immunoprecipitation and detection of gravin, elution was accomplished by boiling the washed beads in SDS-PAGE sample buffer. separation of proteins on a 4-15 denaturing PAGE gel 15 transfer to nitrocellulose and analysis by gravin western, PKC overlay and RnI overlay western [as described abo;\ and previously [Klauck et al., 1996].

Gravin was affinity purified by incubating HEL cell lysates (400 ul of 15 mg/ml. prepared as described ah~ve with the addition of 10 mM IBMX to the buffer), with 200 pl packed cAMP-agarose (Sigma, St Louis, MO) which 20 had been equilibrated in Lysis Buffer with 10 mM IBMX. The slurry was gently mixed for 18 hr at 4"C and then washed with 1.5 ml Lysis Buffer with 1 M NaCI followed by four 1.5 ml washes with 20 mM TrisHCl, pH 7.4, 150 mM NaCI. Elution was accomplished by incubating the beads in 0.5 ml mM cAMP, 20 mM TrisHC. pH 7.4. 150 mM NaCI for 30 min at room temperature. The final wash and the chiate were TCA precipitated and the entire sample loaded into a single lane on a 4-15% SDS-PAGE gel. The separated proteins were blotted to nitrocellulose and gravin was identified by western analysis as described above.

-28- Immunoprecipitation with gravin antibodies specifically isolated a 250 kDa protein that could be faintly detected when SDS gels were stained with Coomassie Blue. This 250 kDa protein was present only in immunoprecipitates using the affinity purified gravin antibodies and was not detected in control experiments performed with pre-immune serum. Western blot and overlay assays confirmed that the 250 kDa protein was gravin.

Moreover, co-precipitation of the PKA holoenzyme was demonstrated by detection of the catalytic subunit in fractions eluted from the immunoprecipitate with cAMP but not in experimental fractions treated with pre-immune serum.

10 The R subunit in the immunoprecipitates was undetectable because the 54 kDa protein migrates with the same mobility as the IgG heavy chain. However, the R subunit/gravin complex was purified from PMA induced HEL cell extracts by affinity chromatography on cAMP-agarose. After extensive washing in high salt buffers, gravin was eluted from the affinity resin with 75 mM cAMP.

15 Since free gravin is refractive t the affinity resin, the protein detected in the eluate was associated with the regulatory subunit. Both co-purification techniques strongly suggest that the PKA holoenzyme is associated with gravin in vivo.

20 Example 7 As previously discussed, thus f;r uravin has only been detected in human fibroblasts. neurons and endothelial cells To determine if gravin has a more broad cell distribution-or could he induced im other cell types, antibody 4037J was used against lysates of primary humani cells and a variety of human, monkey, rat and murine cell lines to detect expression hy Western blot analysis.

Preparation of cells Primary peripheral blood mononuclear cells (PBMC) were isolated from heparinzied peripheral blood from adult volunteers. Blood was diluted 1:1 with CMF-PBS and centrifuged over Histopaque (Sigma, St. Louis, MO) at a density of 1.096 g/cm" for thirty minutes at 400 xg. The resulting interface was collected and washed in CMF-PBS. Monocytes were isolated from PBMC (prepared as above) by an one hour incubation in a 100 mm polystyrene culture plate (Coring) in RPMI media supplemented with 10% fetal bovine serum. Non-adherent cells were washed away with CMF-PBS Adherent cells (monocytes) were scraped

S

f rom the plastic with a rubber spatula, then washed in PBS. Polymorphonuclear cells (PMN) were islated from peripheral blood using the red blood 10 cell/ranulocyte fraction of the histopaque procedure described above. The cell pellet was resuspended in an equal volume of CMF-PBS and 3% dextran (Pharmacia. Uppsala. Sweden) in 0 NaCI for thirty minutes to allow for sedimentation of the red blood cells The PMN enriched supernatant was collected and washed three times in CMF-PBS. Between washes residual red 15 blood cells were subjected to hvpotonic lysis in I ml of water for thirty seconds.

Remaining cells were returned to isotonic conditions with 50 ml of CMF-PBS.

The following human cell lines were obtained from ATCC (American Type Culture Collection. Rockville. MD) and maintained in RPMI 10% fetal bovine serum: HEL (human ervthroleukemia). A549 (human lung epithelia), HEK 293 20 .(human embryonic kidney). HL60 (human promyelocytic leukemia), KU812 (immature human basophilic leukocvtc). Jurkat (human T cell lymphoma), THP 1.1 (human moncyte). RBL2H3 (rat hasophilic leukemia), COS (monkey fibroblast), RAW309 (murine monocvte). R.A\\V204 7 (murine monocyte), 3T3L1 (murine embryonic fibroblast). L929 (murine tibroblast) and EL4IL-2 (murine thymoma).

The cell cultures were incubated overnight in the presence of 40 ng/ml PMA. 10 ng/ml lipopolysaccharide (LPS), 10-M f-met-leu-phe (fMLP) (all from Sigma, St. Louis, MO) or t1 ng/ml tumor necrosis factor alpha (TNFa, Boehringer Mannheim. Indianapolis. IN) to determine ifgravin expression could be stimulated. Lysates from cell pellets were prepared and protein concentrations determined as previously described in Example 6. Cell lysates were assayed for gravin by western blot as described using Protein-A purified anti-gravin antibody 4037J at 5 pg/ml.

Expression of gravin in various cell lines Western analysis showed that a 250 Kd band was detected in several human cell lines including KU812. HEK293. A549, THPI.1 and murine cell lines 3T3LI and L929. For primary human cells, only adherent monocytes expressed 10 the 250 kDa band. With the exception of PMA stimulation of HEL cells (see Example this band was not induced in any other cells with either PMA, LPS.

tMLP or TNFa. These bands were confirmed to be RII binding proteins by an overlay assay as described in Exaniple 2 The cells that expressed gravin shared a common feature of adherence to plastic or growth in cell clusters.

Co-immunoprecipitation of l'.r;\in and R II HEK293 cells were harvested from culture and a lysate was prepared as described in Exmple 4 HEK2''3 Iksate (100 pOg) was incubated with 10 Pi of either 4037 (anti-uravin polyclonal) or 24 IA (anti-human RJI monoclonal) antibody for two hours on ice eqiual volume of prewashed Protein G- Sepharose beads (Pharnacia I ppsala. Sweden) were added and samples incubated for one hour on a rolo a.11 4 The beads were washed three times in lysis buffer, then once in PBS S)D sample buffer was added to the beads which were then boiled for two minutes a; Ilo) C The samples were spun down and the supernatant recovered The samples were run on to a 4-12% SDS-PAGE gel in duplicate lanes along with antibody (2 pg), and cell lysate (25 ig) as controls.

Proteins were transferred to immobilon by standard techniques. Western analysis -31was performed on the resulting blots by the methods described in 6. One blot was probed with 4037J antibody and detected with a goat anti-rabbit IgG-HRP and the other blot was incubated with 241A antibody and detected with goat anti-mouse IgG-HRP.

In the gravin Western a 250 kDa band was detected in lanes containing the cell lysate, 4037J co-immunoprecipiate. and the 241A co-imunoprecipitate samples. In the RII western a 54 kDa band was detected in the cell lysate. In the lanes containing the 4037J and 241A co-immunoprecipitates, a doublet was detected with one band at 54 kDa and another band running slightly higher. This 10 upper band aligned with the I-G lane indicating the lower band to be RII These results demonstrate that gravin and RII are associated in cells and can be coimmunoprecipitated from cell lysates using either an anti-gravin or an anti-Rll antibody.

Example 8 The nicotinic acetylcholine receptor is a neurotransmitter-gated ion channel comprising five transmembrane polypeptides. The five polypeptides appear to form a transmemnrane aqueous pore through which 20 cations can flow. In response to the binding of acetylcholine, the ion channel "opens" and permits the flow of Na; into the cell (sodium current). The influx of sodium ions causes memhbrane depolarization which signals the muscle to contract. Individual receptors appear to rapidly open and close during the period of time that acetylcholine remains hound to the receptor. Within a few hundred milliseconds of acetylcholine binding, the channel closes and prevents further flow of sodium current and the acetylcholine signal is terminated.

The sensitivity of the nicotinic acetylcholine receptor to acetylcholine is attenuated by the phosphorylation of the transmembrane -32polypeptides (desensitization). Prolonged exposure of the receptor to acetylcholine leads to desensitization of the receptor. PKA appears be involved in desensitization of the nicotinic acetylcholine receptor by phosphorylating serine and tyrosine residues of the five transmembrane polypeptides.

Myasthenia gravis is an autoimmune disease associated with the development of antibodies to the nicotinic acetylcholine receptors The present invention contemplates that gravin functions to localize PKA and PKC to a particular subcellular area of the cell. The role of gravin in coordination of PKA and PKC targeting to cytoskeletal components 10 would be analogous to the role of AKAP79 role in clustering PKA, PKC, and protein phosphatase 2B at the postsynaptic density which is a specialized structure of the dendritic cytoskeleton [Coghlan er al., 1995b; Klauck er al..

1996: Rosenmund er al.. Nature. 368:853-856 (1994)].

Modulators which inhibit or abolish binding between gravin and 15 PKA and/or PKC art useful in modu lating the localization of PKA and/or PKC to particular subcellular regions. These modulators may include polypeptides which specifically hind to gravin or fragments of gravin which bind to PKA and/or PKC. and other non-pcptide compounds isolated or synthetic 00 organic or inorganic molecules) which specifically interact with gravin or fragments of gravin.

An assay to determin te the bmdin of gravin to binding partners was developed. The C-terminal clone. I IFu. containing the RII binding region of gravin, was cloned into a Tlliorcdoxin (Trx) bacterial expression vector as described below. The C-terminal clone. pBSII/HF9 (described in Example 3), containing the RIl binding region ofgravin in was cloned into a Thioredoxin (Trx) bacterial expression vector. Briefly, to construct a thioredoxin expression vector, an XbaI/HindIII thioredoxin fragment was subclonsed into pUC19 containing a lac Z gene and a tacZ promotoer. The resulting plasmid was designated TRX F/S pUC 19. In order to insert the HF9 clone into TRX F/S pUC 19, an NcoI site was created with an oligonucleotide: Met 1153, 5' TACAACCATGGACAGGCTATCCCC.

(The NCO cleavage site is underlined). The 3' oligonucleotide used was T7 (Strategene). Amplification of pBS/HF9 with the two oligonucleotides resulted in a 3 kb fragment which was digested with Ncol and Xhol (the latter provided the polylinker of pBS). Then Ncol/Xhol fragment was ligated in frame with the thioredoxin gene in TRX F/S pUC 19 (predigested with Ncol/XhoI). The fusion 10 protein was expressed in E. Coli. induced with 1 mM IPTG to O.D.

6 0 of 0.7 at 30'C Cells were harvested. lvsed with French Press under standard conditions.], The protein-protein binding assays were performed as follows.

Briefly. anti-Trx mouse monoclonal antibodies were passively captured onto Immulon plates (Dynatech) in PBS -\nti-Trx monoclonal antibody was prepared 15 usingthe methods described in Example 5 Nonspecific sites in the wells were blocked at room temperature lir one hour with a buffer containing 2.5% milk in 50 mM sodiuin citrate and 145 m\l sodium chloride. E coli lysate containing "se* Trx/C-terminal gravin was added to -ells in IBS/0.2% BSA for overnight at 4°C.

Free and non-specific proteins were removed with several washes with PBS.

20 Biotinylated RII (chemically hiotinYlated using standard procedures) was then used as a ligand, and added in PBS 02°o BSA. After three hours of incubation at room temperature. unbound proteins re removed with multiple washes of PBS/0.05% Tween 20 Streptuadin-Eu (Wallac) was diluted 1:1000 in assay buffer (Wallac) and added to detect biotinvlated RII/gravin complexes. After additional washes in PBS/O() 0o Tween to remove non-specifically bound proteins. Enhancement solution (\\allac) diluted 1:1 in water was added and release of europium was measured by increased fluorescence using a DELFIA" Research Fluormeter (Model 1232. Wallac).

This protein-protein binding assay indicated that over 50% of the binding to gravin was pecific. Biotinylated RII bound to gravin in a specific and saturable manner. The Kd for the interaction was found to be approximately 50 nM, similar to that reported in Nauert el al., 1996 using surface plasmon resonance.

Example The amino acid sequence of gravin exhibits some similarity to SSeCKS/clone 322/clone 72 [Chapline er al., 1996]. There is approximately 69% homology in the first 1,000 amino acids of gravin and SSeCKS. Gravin 10 also exhibits some homology in selected regions to myristoylated alanine rich PKC substrate (MARCKS) [Aderem. Cell, 71:713-716 (1992)]. However, the remainder of each of the protein sequences are distinct. Also, gravin is a protein of 1780 amino acids which migrates with a mobility of 250 kDa on SDS gels. whereas SSeCKS/clone 72 is 1687 residues and migrates at 207 kDa [Lin 15 er al., (1995), Chaplin. et al.. (1995) and National Center for Biotechnology Information accession no. 2210332A). In addition, the identification of five prospective nuclear localization signals has led to the idea that SSeCKS is a nuclear protein [Lin el al.. 1995]. whereas immunochemical data clearly shows that gravin is cytoplasmic and likely to he a cytoskeletal component. Clone 322 was described as being a tumor suppressor gene which is down regulated in oncogene transformed cells. Based upon a sequence similarities between gravin and clone 72 (and clone 322). gravin may also function as a tumor suppressor gene.

It is well known in the arn that cancerous cells are non-adhesive cells. The non-adhesive nature ofl malignant cancer cells allows these cells to metastasize. The release or de-adhesion of a cancer cell from matrix proteins or other cells is prerequisite to migration or metastasis to new sites.

Transformed or tumorigenic cells may be converted to a less tumorigenic state by increased expression of cytoplasmic proteins such as alpha actinin or talin that function in cytoskeletal reorganization, adhesion and migration [Gluck et al., Cell Science, 107:1773-1782 (1994)].

A tissue survey has shown that gravin exhibits a restricted cellular distribution and is predominantly expressed in fibroblasts, neurons and cells derived from the neural crest [Grove er al., 1994]. Since each of these cell types participates in adherent, migratory or path-seeking functions, it was postulated that gravin may regulate membrane/cytoskeleton events [Grove er al.. 1994]. This view has been further substantiated by the immunolocalization 10 experiments described in Example 6 which indicates that gravin may concentrate PKA in the ruffles and filopodia of adherent HEL cells. In addition, the data disclosed in Example 6 point toward a role for gravin in cell 'adhesion. Phorbol ester induced adhesion in HEL cells [Papayannopoulou et al.. 1983] is concomitant with the increased gravin expression; whereas loss of 15 an adherent phenotype upon transfonnation of REF 52 fibroblasts with an derivative is coincident with the down-regulation of clone 72 [Chapline et al., 1996]. Since phosphorylation events help to maintain the integrity of the membrane/cytoskeleton it is also tempting to speculate that PKA and PKC anchoring by gravin may play a role in adherent processes.

20 Given that gravin is expressed in adherent cells, but not in nonadherent cells, and given that clone 72 is down-regulated in oncogene transformed cells, gravin is implicated in cancer biology. Similar to its function in localizing kinases near the nicotinic acetylcholine receptor, gravin may also localize one or more kinases near a cell adhesion molecule wherein the response to cellular signals or other stimuli causes the phosphorylation of the cell adhesion molecule. It has been reported that substitution of threonine residues in the cytoplasmic domain of the LFA-1 P subunit abolishes LFA-1 mediated cell adhesion [Hibbs. et al., J.Exp. Med., 174:1227-1238 (1991)].

These threonine residues appear to be phosphorylated during cellular activation [Valmu et al., J. Immunol., 155:1175-1183 (1995)]. These residues are conserved in other integrin P subunits. Thus, phosphorylation may regulate cell adhesion mediated by several distinct integrins.

Modulators which inhibit or abolish binding between gravin and its binding partner are useful in modulating localization of the binding partner by gravin. For example, modulators may interfere with the localization of a kinase near cell adhesion molecules. These modulators may include polypeptides which specifically hind to gravin or fragments of gravin which 10 bind to a gravin binding partner. and other non-peptide compounds (e.g.

isolated or synthetic organic or inorganic molecules) which specifically interact with gravin or fragments of gravin.

Numerous modifications and variations in the practice of this invention are expected to occur to those of skill in the art. Consequently, only 15 such limitations as appear in the appended claims should be placed on the invention.

S* Page(s)bY 69 are claims pages they appear after the sequence listing -37- SEQUENCE LISTING GENERAL INFORMATION: APPLICANTS: ICOS Corporation 22021 2 0 t h Avenue,

S.E.,

Bothell, WA 98201 (ii) TITLE OF INVENTION: Protein Binding Domains of Gravin (iii) NUMBER OF SEQUENCES: 24 (iv) CORRESPONDENCE

ADDRESS:

ADDRESSEE: Marshall, O'Toole, Gerstein, Murray Borun STREET: 6300 Sears Tower/233 South Wacker Drive CITY: Chicago STATE: Illinois COUNTRY: United States of America ZIP: 60606-6402 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION

DATA:

APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: Noland, Greta E.

REGISTRATION NUMBER: 35,302 REFERENCE/DOCKET NUMBER: 27866/33451 (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: 312-474-6300 TELEFAX: 312-474-0448 TELEX: 25-3856 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 57 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Val Ala Ile Glu Asp Leu Glu Pro Glu Asn Gly Ile Leu Glu Leu Glu 1 5 10 -38- Thr Lys Ser Ser Lys Leu Val Gin Asn Ile Ile Gin Thr Ala Val Asp 25 Gin Phe Val Arg Thr Giu Giu Thr Ala Thr Glu Met Leu Thr Ser Giu 40 Leu Gin Thr Gin Ala His Val Ile Lys INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 27 amino acids TYPE: amino acid STPANDEDNESS: single TOPOLOGY: linear (xi SEUNEDSRPIO:.QI O2 Il .e .l e l h y e e y e a l s l l 1 5.1.1 2 INOMTO FORx) EUNC ECRPN SEQ I D NO:2: G Dn T OPOOGr Alla. s inPar a rgTr l 25 3 Ly .h h h l l r l l r r y y h e h INORATO FO4E5I

O

ArgLy Pr Ly) SEQENC CA ATRsTICSalGuAa e luLsLs y LENTH 690mioacd TYuPE: o Gl y acid h l GuApGyLy l l a STA0NES single -39L Ala Ser Glu Lys Leu Thr Ala Ser Glu Gin Ala His Pro Gin Giu Pro Ala Glu Glu LeU Glu Lys 130 Val His 145 Glu Arg Val Pro Glu Pro Glu Asp 2110 Ser Lys 2125 Gin Glu Ser Thr.

Gly'Gly Ser Pro .290 Ser Pro 115 Pro Gin Thr Ala Ala 195 Pro Pro Arg Gly Gly 275 Asp *Ala 100 Ser Ala Glu Glu Glu 180 Lys Thr Pro Met Leu 260 Asp Se r His Glu Pro Glu Glu 165 Glu Glu Gin Glu Lys 245 Ly s Glu Glu Leu Val 150 Gin Leu Le u Gly Gly 230 Val1 Lys Pro Gin Ala 135 Val Lys Val1 Val1 Al a 215 Val1 GIn Leu Arg Val 120 Thr Ala Thr Gly Lys 200 Asp Val1 Gi.

Se r Leu 105 Ser Glu Glu Glu Met 185 Leu Leu Ser Se r Gly 265 90 Ser Gly Val Val Val 170 Asp Lys Ser Glu Pro ,50 Lys Ala Ser Phe His 155 Glu Al a Glu Pro Val 235 Le u Lys Glu Gin Asp 140 Val Giu Glu Thr Asp 220 Glu Lys Gin Tyr Gly 125 Glu Ser Thr Pro Cys 205 Glu Met Lys Lys G16 110 Pro Lys Thr Ala Gin 190 Val Lys Leu Leu Gly 270 Lys Ser Ile Val Gly 175 Glu Ser Val Ser Phe 255 Lys Val Glu Glu Glu 160 Ser Al a Gly Leu Ser 240 Thr Arg Glu Glu Ser Gly Glu His Thr Gin Val Pro Ala Asp 280 285 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 6605 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 192. .5531 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CCTrCTTTTA AGGAGTGC CGCGAGCGCG TCTCCTTCAT TCGCAGGCTG GGCGCGTrCG CAGTCGGCTG GCGGCGAAGG AAGGCGCTCT CGGGACCTCA CGGGCGCGCG

TCTTTT-GGCT

CTT GCCCCTG TCCCTGCGGC ITGGGGAAAG CGTAACCCGG CGGCTAGGCG

CGGGAGAAGT

GCGGAGGAGC C ATG GGC GCC GGG AGC TCC ACC GAG CAG CGC AGC CCG GAG Met Gly Ala Gly Ser Ser Thr Glu Gin Arg Ser Pro Glu 120 180 230 CAG CCG CCC GAG GGG AGC TCC ACG CCG GCT Gin Pro Pro Giu Gly Ser Ser Thr Pro Ala GAG CCC Glu Pro GAG CCC AGC GGC Giu Pro Ser Gly

GGC

Gly 30 GGC CCC TCG GCC Gly Pro Ser Ala GCG GCG CCA GAC Ala Ala Pro Asp

ACC

Thr 40 ACC GCG GAC CCC Thr Ala Asp Pro

GCC

Al a 326 374 ATC GCT GCC TCG Ile Ala Al~a Ser CCC GCC ACC AAG Pro Ala Thr Lys

CTC

Leu CTA CAG AAG AAT Leu Gin Lys Asn GGT CAG Gly Gin

S

CTG TCC ACC Leu Ser Thr GAG GGT GAC Giu Gly Asp AAT GGC GTA GCT Asn Gly Val Ala

GAG

Giu -70 CAA GAT GAG CTC Gin Asp Giu Leu AGC CTC GAG Ser Leu Gin CAA GGA GCC Gin Gly Ala CTA AAT GGC CAC Leu Asn Gly Gin GGA CCC CTG AAC Gly Ala Leu Asn

GGT

Gly 422 470 518 566 CTA AAC Leu Asn AGC CAG GAG GAA Ser Gin Ciu Giu .GAA G77 ATT GTC Giu Val lie Val

ACG

Thr 105 GAG GTT GGA GAG Giu Val Gly Gin

AGA

Arg 110 GAC TCT GAA CAT Asp Ser Giu Asp

GTG

Val1 115 AGC GAA AGA GAC Ser Glu Ary Asp

TCC

Ser 120 GAT AAA GAG ATG Asp Lys Giu Met

GCT

Ala 12S ACT AAG TGA GCG Thr Lys Ser Ala

GTT

Val 130 GTT CAC GAC ATC Val His Asp Ilie

ACA

Th r 135 CAT GAT GGG GAG Asp Asp Gly Gin GAG GAG Giu Giu 140 AAC CGA AAT Asn Arg Asn

ATC

Ile 145 GAA GAG ATr CCT Ciu Gin Ile Pi-o T77 Sev '50 TCA GAA AGC Sei- Ciu Ser AAT TTA GAA GAG Asn Leu Giu Giu 155 GGA TTT AAG AAG Gly Phe Lys Lys 170 CTA ACA CAA CCC Leu Thr Gin Pro 160 ACT GAG TCC Thr Giu Ser

GAG

Gin 165 GCT AAT CAT ATT Ala Asn Asp Ile GTG TTT AAG TITT GIT GC Val Phe Lys Phe Val Gly 175 TTT AAA TTC ACT GTG AAA AAG GAT AAG AGA Phe Lys Phe Thr Val Lys Lys Asp Lys Thr

GAG

Glu 190 AAG CCT GAC ACT GTC CAG CTA CTC ACT GTG AAG AAA GAT GAA GGG Lys Pro Asp Thr Gin Leu Leu Thr Val 200 Lys Lys Asp Glu Gly 205 GAG GGA GCA GCA GGG GCT GGC GAC Glu Gly Ala Ala Gly 210 Ala Gly Asp CAC CAG His Gin 215 GAC CCC AGC CTr Asp Pro Ser Leu GGG GCT Gly Ala 220 GGA GAA GCA Gly Glu Ala CCC GAA GAG Pro Glu Glu 240

GCA

Ala 225 TCC AAA GAA AGC Ser Lys Glu Ser

GAA

Glu 230 CCC AAA CAA TCT Pro Lys Gin Ser ACA GAG AAA Thr Glu Lys 235 ATT TCT CCC Ile Ser Pro ACC CTG AAG CGT Thr Leu Lys Arg CAA AGC CAC GCA Gin Ser His Ala

GAA

Glu 250 CCA GCC Pro Ala 255 GAA TCT GGC CAA Glu Ser Gly Gin

GCA

Ala 260 GTG GAG GAA TGC Val Glu Glu Cys

AAA.

Lys 265 GAG GAA GGA GAA Glu Glu Gly Glu 998

GAG

Glu 270 AAA CAA GAA AAA Lys Gin Giu Lys

GAA

Glu 27c CCT AGC AAG TCT Pro Ser Lys Ser

GCA

Ala 280 GAA TCT CCG ACT Glu Ser Pro Thr

AGT

Ser 285 CCC GTG ACC AGT Pro Val Thr Ser

GAA

Glu 290 ACA GGA TCA ACC Thr Gly Ser Thr AAA AAA TTC TTC Lys Lys Phe Phe ACT CAA Thr Gin 300 1046 1094 1142 1190 GGT TGG GCC Gly Trp Ala GAT GAA GTG Asp Giu Val 320

GGC

Gly 305 TGG CGC AAA AAG Trp Arg Lys Lys

ACC

Thr 310 AGT rTC AGG AAG Ser Phe Arg Lys CCG AAG GAG Pro Lys Glu 315 CCA GAA AAA Pro Glu Lys GAA GCT TCA GAG Glu Ala Ser Glu

AAG

Lys 325 AAA AAG GAA CAA Lys Lys Glu Gin

GAG

Glu 330 GTA GAC Val Asp 335 ACA GAA GAA GAC Thr Glu Glu Asp

GGA

Gly 340 AAG GCA GAG GTT Lys Ala Giu Val

GCC

Ala 345 TCC GAG AAA CTG Ser Glu Lys Leu 1238

ACC

Thr 350 GCC TCC GAG CAA GCC CAC CCA CAG GAG Ala Ser Glu Gin Ala His Pro Gin Glu 355

CCG

Pro 360 GCA GAA AGT GCC Ala Giu Ser Ala

CAC

His 365 1286 GAG CCC CGG TTA Glu Pro Arg Leu

TCA

Ser 370 GCT GAA TAT GAG Ala Giu Tyr Glu GTT GAG CTG CCC Val Giu Leu Pro TCA GAG Ser Glu 380 1334 1382 GAG CAA GTC Glu Gin Val

AGT

Ser 385 GGCTCG CAG GGA CCT TCT GAA GAG Gly Ser Gin Gly Pro Ser Glu Glu 390 AAA CCT GCT CCG Lys Pro Ala Pro 395 TTG GCG ACA GAA GTG TTT GAT GAG Leu Ala Thr Giu Val Phe Asp Glu 400 405 AAA ATA GAA GTC CAC CAA GAA GAG Lys Ile Glu Val His Gin Glu Glu 410 1430 -42- GTT GTG Val Val 415 GCC GAA GTC CAC Ala Glu Val His GTC AGC Val Ser 420 ACC GTG GAG Thr Val Glu GAG AGA Glu Arg 425 ACC GAA GAG Thr Glu Glu 1478

CAG

Gin 430 AAA ACG GAG GTG Lys Thr Glu Val

GAA

Glu 435 GAA ACA GCA GGG TCT GTG CCA GCT GAA Glu Thr Ala Gly Ser Val Pro Ala Glu 440

GAA

Glu 445 1526 TTG GTT GGA ATG Leu Val Gly Met

GAT

Asp 450 GCA GAA CCT CAG Ala Glu Pro Gin

GAA

Glu 455 GCC GAA CCT GCC Ala Glu-Pro Ala AAG GAG Lys Glu 460 1574 CTG GTG AAG Leu Val Lys GGA GCT GAC Gly Ala Asp 480

CTC

Leu 465 AAA GAA ACG TGT Lys Glu Thr Cys

GTT

Val 470 TCC GGA GAG GAC Ser Gly Glu Asp CCT ACA CAG Pro Thr Gin 475 CCC CCC GAA Pro Pro Glu 0* CTC AGT CCT GAT Leu Ser Pro Asp

GAG

Glu 485 AAG GTG CTG TCC Lys Val Leu Ser

AAA

Lys 490 GGC .GTT Gly Val 495 GTG AGT GAG GTG Val Ser Glu Val

GAA

Glu 500 ATG CTG TCA TCA Met Leu Ser Ser

GAG

Gin 505 GAG AGA ATG AAG Glu Arg Met Lys

GTG

Va1 510 GAG GGA AGT CCA Gin Gly Ser Pro AAG AAG TTT Lys Lys Leu Phe

ACC

Thr 520 AGC ACT GGC TTA Ser Thr Gly Leu

AAA

Lys 525 1622 1670 1718 1766 1814 1862 1910 AAG CTT TCT GGA Lys Leu Ser Gly

AAG

Lys 530 AAA CAG GGC Lys Gin Lys G>.

AAA

535 AGA GGA GGA GGA Arg Gly Gly Gly GAC GAG Asp Glu 540 GAA TCA GGG Glu Ser Gly GAG GAG CAA Glu Giu Gin 560

GAG

Glu 545 CAC ACT GAG GT7 His. Thr Gin Val

CCA

Pro 550 GCC GAT TCT CCG Ala Asp Ser Pro GAC AGC GAG Asp Ser Gin 555 GAG CCC GAG Glu Pro Glu AAG GGC GAG AGC Lys Gly Giu Ser GCC TCA TCC CCT Ala Ser Ser Pro

GAG

Glu 570 GAG ATC Glu Ile 575 ACG TGT CTG GAA Thr Cys Leu Glu

AAG

Lys 580 CCC 7,A GCC GAG CGl Leu Ala Glu

GTG

Val 585 GAG GAG GAT GGG Gin Gin Asp Gly

GAA

Glu 590 GCT GAA GAA GGA Ala Giu Giu Gly ACT TCV C A GGA Thr Se: CGly

GAG

Glu 600 AAA AAA AGA GAA Lys Lys Arg Glu

GGT

Gly 605 1958 2006 2054 GTC ACT CCC TGG Val Thr Pro Trp

GCA

Ala 610 TCA TTC AAA AAC Ser Phe Lys Lys

ATG

Met 615 GTG ACG CCC AAG Val Thr Pro Lys AAG CGT Lys Arg 620 GTT AGA CGG Val Arg Arg

CCT

Pro 625 TCG GAA AGT GAT Ser Giu Ser Asp AAA GAA Lys Glu 630 GAT GAG CTG Asp Glu Leu GAC AAG GTC Asp Lys Val 635 2102 -43- AAG AGC GCT Lys Ser Ala 640 ACC TTG TCT TCC Thr Leu Ser Ser ACC GAG Thr Glu 645 AGC ACA 0CC TCT GAA ATG CAA Ser Thr Ala Ser Glu Met Gin 650 2150 GAA GAA ATG AAA GGG AGC GTG GAA GAG CCA AAG Glu Glu 655 Met Lys Gly Ser Val 660 Glu Giu Pro Lys

CCG

Pro 665 GAA GAA CCA AAG Glu Giu Pro Lys

CGC

Arg 670 AAG GTG GAT ACC Lys Val Asp Thr

TCA

Ser 675 GTA TCT TGG GAA Val Ser Trp Glu

GCT

Ala 680 TTA ATT TGT GTG Leu Ile Cys Val

GGA

Gly 685 2198 2246 2294 TCA TCC AAG AAA Ser Ser Lys Lys

AGA

Arg 690 GCA AGG AGA AGG Ala Arg Arg Arg

TCC

Ser 695 TCT TCT GAT GAG Ser Ser Asp Glu GAA GGG Glu Gly 700 0* *00* 0* 0e 0 0000 0 GGA CCA AAA Gly Pro Lys AAA GAC AAA Lys Asp Lys 720

GCA

Ala 705 ATG GGA GGA GAC Met Gly Gly Asp

CAC

His 710 CAG AAA GCT GAT Gin Lys Ala Asp GAG GCC GGA Glu Ala Gly 715 TCC CAA GAA Ser Gin Glu 2342 2390 GAG ACG GGG ACA Glu Thr Gly Thr

GAC

Asp 725 GGG ATC CTT GCT Gly Ile Leu Ala

GGT

Gly 730 CAT OAT His Asp 735 CCA GGG CAG GGA Pro Gly Gin Gly

AGT

Ser 740 TCC TCC CCG GAG Ser Ser Pro Glu

CAA

Gin 745 GCT GGA AGC CCT Ala Gly Ser Pro @0 .0 GAA GGG GAG GGC Glu Gly Glu Gly TCC ACC TGG GAG Ser Thr Trp Glu

TCA

Ser 760 TTT AAA AGG TTA Phe Lys Arg Leu

GTC

Val 765 2438 2486 2534 ACG CCA AGA AAA Thr Pro Arg Lys

AAA

Lys 770 TCA AAG TCC AAG Ser Lys Ser Lys

CTG

Leu 775 GAA GAG AAA AGC Olu Glu Lys Ser GAA GAC Olu Asp 780 TCC ATA OCT Ser Ile Ala GGT AAA GAA Gly Lys Glu 800 TCT GGT GTA GAA Ser Gly Val Glu

CAT

His 790 TCC ACT CCA GAC Ser Thr Pro Asp ACT GAA CCC Thr Glu Pro 795 CCT GGA CGA Pro Gly Arg 2582 2630 GAA TCC TGG GTC Glu Ser Trp Val

TCA

Sex 805 ATC AAG AAG TTT Ile Lys Lys Phe

ATT

Ile 810 AGO AAO Arg Lys 815 AAA AGO CCA GAT Lys.Arg Pro Asp

GGG

Gly 820 AAA CA, GAA CAA Lys Gin Olu Gin 0CC CCT GTT GAA GAC Ala Pro Val Giu Asp 825 2678

OCA

Ala 830 GGG CCA ACA GGG Oly Pro Thr Gly 0CC Ala 835 AAC GAA GAT GAC TCT OAT GTC CCG 0CC Asn Glu Asp Asp Ser Asp Val Pro Ala 840

GTG

Val 845 2726 OTC CCT CTO TCT Val Pro Leu Ser GAO TAT OAT OCT OTA Glu Tyr Asp Ala Val 850 OAA AGO GAG AAA ATO Glu Arg Glu Lys Met 855 GAG OCA Olu Ala 860 2774 -44- CAG CAA GCC CAA AAA GGC GC.A GAG G-AG Gin.Gin Ala GAG GTG TCC Glu Val Ser 880 CCC GAG CAG AAG Pro Giu Gin Lys Lys Gly Ala Giu Gin 870 GCA GCC ACT Ala Ala Thr 875 ATG GCA GCA Met Ala Ala 28222 2870 AAG GAG CTC AGC Lys Giu Leu Ser

GAG

Giu 885 AGT GAG GTr CAT Ser Gin Val His

ATG

Met 890 GCT GTC Ala Val 895 GCT GAG GGG ACG Ala Asp Gly Thr

AGG,

Arg 900 GCA GCT ACC ATT Ala Ala Thr Ile

ATT

Ile 905 GAA GAA AGG TCT Giu Glu Arg Ser

CCT

Pro 910 TCT TGG ATA TCT Ser Trp Ile Ser

GCT

Al a 915 TCA GTG AC-A GAA Ser Val Thr Giu

CCT

Pro 920 CTT GAA CAA GTA Leu Giu Gin Val

GAA

Glu 925 GCT GAA GCC GCA Ala Glu Ala Ala

CTG

Leu 930 TTA ACT GAG GAG Leu Thr Glu G lu

GTA

Val1 935 TTG GAA AGA GAA Leu Giu Arg Giu GTA ATT Val Ile 940 GCA GAA GAA Ala Giu Glu GAG CCC CCC Glu Ala Arg 960

GAA

Giu 94.5 CCC CCC ACG GT-r Pro Pro Thr Val

ACT

Thr 950 GAA CCT CTG CCA Glu Pro Leu Pro GAG AAC AGA Glu Asn Arg 955 ACC CCC GAA Thr Pro Glu 2918 2966 3014 3062 3110 3158 3206 GGC GAG ACG GTC Gly Asp Thr Val

GTT

Val1 965 ACT GAG GCG GAA Ser Glu Ala Giu GCT GTG Ala Val 975 ACA GCT GCA GAA Thr Ala Ala Glu

ACT

Th r 980 GCA GGG CCA FFG Ala Cly Pro Leu

GGT

Gly 985 TCC GAA GAA GGA Ser Glu Glu Gly GTG TCA GCA GTC Val Ser Ala Val 1005

ACC

Thr 990 GAA GCA TCT GCT Glu Ala Ser Ala

GCT

Ala 995 GAA GAG ACC ACA Glu Glu Thi, Thr GAA ATG Glu Met 1000 TCC GAG TTA Ser Gin Leu GAG GAG GTG Gin Glu Val ACC GAG TCC Thr Asp Ser 1010 CCA GAG ACC' Pro Asp Thr ACA GAG Thr Glu 1015 GAG CCC ACT Glu Ala Thr CCG GTG Pro Val 1020 3254 GAA GGT GGC GTA CC7 GA'- Glu Gly Cly Val Pro 1025 ATA GAA GAG CAA u1tf Glu Giu Gin GAG AGG CGG Giu Arg Arg 1035 3302 ACT CAA GAG Thr Gin Glu 1040 GTC CTC GAG GCA GTG GCA; GAA A AA GTG AAA GAG GAA TCC Val Leu Gin Ala Val Glu Lys.Vai Lys Glu Giu Ser 3350 104 5 1050 GAG CTG CCT GGC ACC GGT Gin Leu Pro Gly Thr Gly 1055 GGG CCA GAA CAT GTG Gly Pro Glu Asp Val 1060 CTT GAG CCT GTG GAG Leu Gin Pro Val Gin 1065 3398 AGA GGA GAG GGA GAA AGA CGA GAA GAG GAG GCT GAA CC TCG GGT CTG Arg Ala Glu Ala Giu Arg Pro Glu Glu Gin Ala Glu Ala Ser Giy Leu 3446 1070 1075 1080 1085 AAG AAA GAG ACG GAT GTA GTG TTG AAA GTA GAT Lys Lys Glu Thr Asp Val Val Leu Lys Val Asp 1090 1.095 ACT GAG CCT TTT ACA Thr Giu Pro Phe Thr 1105 AGC TTT GAA AAA GCT Ser Phe Glu Lys Ala 1120 CAA GGG AAG GTG GTG GGG Gin Gly Lys Val Val Gly 11210 CCT CAA GTC ACA GAG AGC Pro Gin Val Thr Giu Ser 11225 GCT CAG GAG GCA AAA Ala Gin Glu Ala Lys 1100 CAG ACC ACC CCA GAA Gin Thr Thr Pro Giu 1115 ATA GAG TCC AGT GAG Ile Giu Ser Ser Glu 1130 3494 3542 3590 9* a Crr GTA ACC ACT TGT CAA GCC GAA ACC TIA GCT GGG GTA AAA TCA CAG Leu Val Thr Thr Cys Gin Ala Glu Thr Leu Ala Gly Val Lys Ser Gin 1135 1140 1.145 GAG ATG GTG ATG GAA CAG GCT ATC CCC CCT GAC TCG GTG GAA ACC CCT Giu Met Val Met Giu Gin Ala Ile Pro Pro Asp Ser Val Giu Thr Pro 1150 .1155 1160 1165 ACA GAC AGT GAG ACT GAT GGA AGC ACC CCC GTA GCC GAC 'rrr GAC GCA Thr Asp Ser Glu Thr Asp Gly Ser Thr Pro Val Ala Asp Phe Asp Ala 1170 1175 1180 CCA GGC ACA ACC CAG AAA GAC GAG ATT GTG GAA ATC CAT GAG GAG AAT Pro Gly Thr Thr Gin Lys Asp Giu Ile Val Giu Ile His Giu Glu Asn 1185 i190 1195 3638 3686 3734 3782 3830 3878 GAG GTG CAT CTG Giu Val His Leu 1200 GTA CCA GTC AGG GGC Vai Pro Val Arg G1-., 1205 ACA GAA GCA GAG GCA GTT CCT Thr Glu Ala Giu Aia Val Pro 1210

OCA

Al a CAG AAA Gin Lys 1215 GAG AGG CCT Glu Arg Pro CCA GCA Pro Ala 1220 CCTI TCC AGT Pro Ser Ser 'ITT GTG 'ITC CAG GAA Phe Val-he Gin Giu 1225 GAA ACT AAA Giu Thr Lys 1230 GAA CAA TCA AAG Giu Gin Ser Lys 1235 ATG GAA GAC Met Glu Asp ACT CTA GAG CAT Thr Leu Giu His 1240 ACA GAT Thr Asp 1245 GAG GGG Giu Gly 1260 3926 AAA GAG GTG TCA Lys Glu Val Ser GTG GAA ACT GTA TCC Val Giu Thr Val Sei 1250 ATT CTG Ile Leu 12155 TC.A AAG ACT Ser Lys Thr 3974 ACT CAA GAG Thr Gin Giu GCT GAC CAG TAT Ala Asp Gin TPyr 1265 GCT GA7 GAG AAA Ala Asp Giu Lys 1270 ACC AAA GAC GTA CCA Thy Lys Asp Vai Pro 1275 4022 4070 TTT TTC GAA GGA CTT GAG GGG TCT ATA GAC ACA GGC ATA ACA GTC AGT Phe Phe Glu Gly Leu Giu Gly Ser Ile Asp Thr Gly Ile Thr Val Ser 1280 1285 1290 CGG GAA AAG GtC ACT GAA Arg Glu Lys Val Thy Giu 1295 GTT GCC CT? AAA GGT Val Aia Leu Lys Gly 1300 GAA GGG ACA GAA GAA Giu Gly Thr Glu Glu 1305 4118 GCT GAA TGT Ala Giu Cys 1310 TCT C-CT CCA Ser Pro Pro GAG AAA AC-A Glu Lys Thr AAA AAG GAT GAT GCT CTT GAA CTG C-AG AGT C-AC GC-T AAG Lys Lys Asp Asp Ala Leu Glu Leu Gin Ser His Ala Lys 1315 1320 1325 4166 TCC C-CC GTG GAG Ser Pro Val Glu 1330 AGA GAG ATG GTA GTT CAA GTC GAA AGG Arg Giu Met Val Val Gin Val Glu Arg 1335 1340 4214 GAA GC-A GAG Glu Ala Glu 1345 CCA ACC C-AT GTG AAT Pro Thr His Val Asn 1350 G.AA GAG AAG C-rr GAG Glu Glu Lys Leu Glu 1355 4262 C-AC GAA AC-A GCT His Glu Thr Ala 1360 GTT ACC GTA Val Thr Val TC-T GAA Ser Glu 1365 GAG GTC AGT .AAG C-AG CTC C-TC Giu Val Ser Lys Gin Leu Leu 1370 4310 to .9 0 9* C-AG AC-A GTG AAT Gin Thr Val Asn- 1375 TTG GAA GGA AGC Leu Giu Gly Ser 1390 GTG CCC ATC ATA Val Pro Ile Ile 1380 GAT GGG GC-A Asp Gly Ala AAG GAA Lys Glu 1385 GTC AGC AGT Val Ser Ser GC-A GTA TGC Ala Vai C-ys 1405 4358 4406 C-CT C-CT C-C-C Pro Pro Pro 1395 TGC C-TA GGT Cys Leu Gly C-AA GAG GAG Gin Glu Glu 1400 ACC AAA ATT C-AA GTT C-AG AGC Thr Lys Ile Gin Val Gin *Ser 1410 TC-T GAG GC-A TC-A TTC ACT C-TA ACA GCG Ser Glu Ala. Ser Phe Thr Leu Thr Ala 1415 1420 W. 9 90~ 000 9 to9 GC-T GC-A GAG Ala Ala Giu

GAG

Giu 142! AC-A GGT GAA AC-G Thr Gly Giu Thr 1440 AAA TCC TC-T GAA Lys Ser Ser Giu 145S GAA AAG GTC TTA Giu Lys Val Leu TTG GAG C-CT GCA Leu Glu Pro Ala 1445 AAA AAT GAA GAC Lys Asn Glu Asp 1460 GGA GAA G1. Glu 14 3 AC-T GCC AAC ATT TTA GAA Thr Ala Asn Ile Leu Glu 1435 4454 4502 4550 4598 GGT GC-A CAT

G

3 1 y His flT CC GC-T Phe Ala Ala TTA GTT C-TG GAA GAG Leu Val Leu Glu Glu 1450 C-AT CCA GGG GAA GAT His Pro Gly Glu Asp 1465 GC-T GTG C-CC Ala Val Pro 1470 ACA GGG C-C-C GAC Thr Gly Pro Asp 1475 TGT C-AG GCA C-ys Gin Ala AAA TC-G ACA Lys Ser Thr 1480 C-C-A GTG ATA Pro Val Ile 1485 GAA GGA GAG Giu Gly Glu 1500 4646 GTA TC-T GC-T ACT Val Ser Ala Thr ACC AAG AAA Thr Lys Lys 1490 GC 7-.A AGT TCC GAC CTG Cly L.2,u Ser Ser Asp Leu 1495 4694 AAA ACC ACA TCA CTG AAG TGG AAG TCA GAT GAA GTC GAT GAG CAG GTT Lys Thr Thr Ser Leu Lys Trp Lys Ser Asp Giu Val Asp Glu Gin Val 4742 1505 1510 1515 GC-T TGC C-AG GAG GTC AAA GTG AGT Ala C-ys Gin Glu Val Lys Val Ser 1520 1525 GTA GC-A ATT GAG GAT TTA GAG C-CT Val Ala Ile Giu Asp Leu Giu Pro 1530 4790 -47- GAA AAT GGG Glu Asn Gly 1535 ArT TTG GAA CTT GAG Ile Leu Giu Leu Glu 1540 ACC AAA AGC AGT AAA CIT GTC CAA Thr Lys Ser Ser Lys Leu Val Gin 1545 4838 AAC ATC Asn Ile 1550 ATC CAG ACA GCC G'T GAC Ile Gin Thr Ala Vai Asp 1555 CAG TTT GTA CGT Gin Phe Val Arg 1560 ACA GAA GAA ACA Thr Giu Giu Thr 1565 4886 GCC ACC GAA ATG Ala Thr Gi'u Met TTG ACG TCT Leu Thr Ser 1570 GAG TTA GAG ACA Glu Leu Gin Thr 1575 CAA GCT CAC Gin Ala.His GTG ATA Val Ile 1580 4934 AAA GCT GAC Lys Ala Asp AGC GAG Ser Gin 1585 GAC GCT GGA Asp Ala Gly GAG GAA Gin Glu 1590 ACG GAG AAA GAA GGA GAG Thr Giu Lys Giu Giy Glu 1595 4982 0* GAA CCT GAG GCC Glu Pro Gin Ala 1600 GAG GAG TCA GAG Glu Glu Ser Glu 1615 TCT GCA CAG Ser Ala Gin GAT GAA Asp Glu 1605 ACA CGA ATT Thr Pro Ile ACT TGA GCC AAA Thr Ser Aia Lys 1610 5030 5078 TCA ACC GCA GTG Ser Thr Ala Val 1620 GGA CAA GCA Gly Gin Ala CAT TCT GAT ATT TCC His Ser Asp Ile Ser 1625 AAA GAC ATG Lys Asp Met 1630 AGT GAA GCC TCA GAA Ser Giu Ala Ser Glu 1635 AAG ACC ATG ACT GTT GAG Lys Thr Met Thr Val Glu 1640 GTA GAA Val Glu 1645 S. S

S

*5 GGT TCC ACT GTA AAT GAT GAG GAG CTG GAA GAG GTC GTC CTC CCA TCT Gly Ser Thr Val Asn Asp Gin Gin Leu Giu Giu Val Val Leu Pro Ser 1650 1655 1660 GAG GAA GAG GGA GGT Glu Glu Giu Gly Gly 1665 GGA GCT GGA ACA AAG Gly Ala Gly Thr Lys 1670 TCT GTG CCA GAA GAT GAT Ser Val Pro Giu Asp Asp 1675 5126 5174 5222 5270 5318 GGT CAT GCC TTG Gly His Ala Leu 1680 TTA GCA GAA Leu Ala Glu AGA ATA Arg Ile 1685 GAG AAG TCA Glu Lys Ser CTA GTT GAA CCG Leu Val Giu Pro 1690 AAA GAA GAT Lys Giu Asp 1695 GAA AAA GGT Glu Lys Gly GAT GAT GTT GAT GAC Asp Asp Val Asp Asp 1700 CCT GAA AAC GAG AAC Pro Glu Asn Gin Asn 1705

TCA

Ser 1710 GCC CTG GCT GAT ACT GAT GCC TCA GGA Ala Leu Ala Asp Thr Asp Ala Ser Gly GGC TTA ACC AAA Gly Leu Thr Lys 1720 GAG TCC Glu Ser 1725 1715 CCA GAT AGA AAT Pro Asp Thr Asn GTA GAA TTG GAG Val Giu Leu Gin 1745 GGA CCA AAA CAA AAA GAG AAG GAG GAT GCC GAG GAA Gly Pro Lys Gin Lys Giu Lys Giu Asp Ala Gin Glu 1730 1735 1740 GAA GGA AAA GTG GAC AGT GAA TGA GAT AAA GCG ATC Glu Gly Lys Val His Ser Giu Ser Asp Lys Ala Ile 1750 1755 5366 5414 5462 -48- ACA CCC CAA GCA CAG GAG GAG TrA CAG AAA CAA GAG AGA GAA TCT GCA Thr Pro Gin Ala Gin Glu Giu Leu Gin Lys Gin Glu Arg Giu Ser Ala.

1760 1765 1770 AAG TCA GAA CTr ACA GAA TCT TAAAACATCA TGCAGII'AAA

CTCATTGTCT

Lys Ser Glu Leu Thr Glu Ser 1775 1780 G'r-GGAAGA CCAGAATGTG AAGACAAGTA GTAGAAGAAA ATGAATGCTG

CTGCTGAGAC

*se Geof 0..

Oe 5*

S

06S

TGAAGACCAG

TCTAGAGAGC

TCTTTCCAAG

CTAAATTCTT

ATCATTCTTT

TTTTTCTTAA

ATGATGGGGC

ACAAAAAACA

ACTAATGATT

GAGTGTGCCA

ATTTTGCTTT

TTACATCCTT

GCAAAGTAGT

CTACTCTTAT

ATGGTAT7T

CAATGTTGCA

TATTTCAGAA

CCCTGACAAT

ACCAACCTAC

AACCTGGAAC

ACATATI-rAT

TGTTTAAGGA

ATGTGCCATA

GAGCCTCCTA

GAGGTCCATA

AAACTAAAAA

CTAACCCAGT

TTTCCTAC1'

GAATATGTT

ATGCTGGACT

GATAGATACT

ACAC-ATrCAT

CTITGAGAAT

CCTGAGGC TT

ATTTITCCCT

TGGAGTTGGC

ATGTATG'TT7

AATGTGCAGG

GTGCAGGC7T

GATGTAAC;AT

TTAGTGGTA

GCATTTG;,;

GGAGGTTAGA

GTTATGGTTCG

TATATG'rrAT

GCATTCACAC

GGATTGTG1rT

TGGAGAGCAG

CATCAGGAGC

TGATAACCAT

AATACCTAGT

TAAGTAGTCC

ATACTACATG

GGGGAGCTTT

TCC-TGATCAA

C'TCTGAAATT

,-CATACAGAA

Zc3AAGT TAT =7GGACCGA

GAAGAAAAGA

ATG GCATGAA

TGTGCCATAT

GC-ACATCAAC

TAGAGCCrr

ATAAATTCTG

TCTGCTTCTG

TCCTGTATCT

AAGCCTCAGT

GGTACAArrC GGTCAC'rrTC

TGTTCTATTG

ATTCTGGTAG

TAAGTGTGCT

ATTGTTGTAA

ATAAGTCAGG

TTGTGCCATT

TGATCTCATr

TAACATTTCC

ATTTAAGGTC

AAACTGGAGT

ATTGTATArr

CACACAGTAT

TATATAACCC

TTTAAAATTC

CTATTACACG

TCATTGGGAA

CAAATTAACT

TAATCCTGAG

GTTTTTGATT

TTCTTTACAA

CCTTTAAGAA

5510 5561 5621 5681 5741 5801 5861 5921 5981 6041 6101 6161 622."1 6281 6341 6401 6461 6521 6581 TTGGATAAG7 TGTGArn-GA CGACTGATTT AAATAAAATA TrTGCTTCAC TAAAAAAAA

AAAA

INFORMATION FOR SEQ ID NO:c SEQUENCE

CHAP.ACTERIST:CS:

LENGTH: 1780 aminc acids TYPE: amfino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein 6605 -49- DESCRIPTION: SEQ ID (xi) SEQUENCE Met Gly Ala Gly Ser Ser Thr Glu Gin Arg Ser Prc 00 .0 0 09 e g.

0S @0 0

OC

06 S

OS

0e 0 0* 9 0O

S

00@0

S.

00 0 0000 1 Glu Ser Ser Ile 65 Leu Gin Glu Ala Ile 145 Pro Phe Asp Ala Ala 225 Thr Ser Gl Alz Asp Asn Asn Glu Asp Va1 130 Glu Thr Val Thr Gly 210 Ser Leu fly Sex Glu Prc Gly Gly Glu Va1 115 Va1 Gin Glu Gly Val 195 Ala Lys Lys Gin Ser Ala Ala Val Gin Glu 100 Ser His Ile Ser Phe 180 Gln Gly Glu Arg Ala 260 Thi Ala Thr Ala Lys 85 Glu Glu Asp Pro Gln 165 Lys Leu Asp Ser Glu 245 Jal Pro Pro Lys Glu 70 Gly Val Arg Ile Ser 150 Ala Phe Leu His Glu 230 Gin Glu Ala Asp Leu 55 Gin Ala Ile Asp Thr 135 Ser Asn Thr Thr fin 215 Pro Ser 3lu Glu Thr 40 Leu Asp Leu Val Ser 120 Asp Glu Asp Va 1 Val 200 Asp Lys His Cys I Prc 25 Thr Gin Glu Asn Thr 105 Asp Asp Ser Ile Lys 185 Lys Pro GIn Ala Lys.

265 10 Glu Ala Lys Leu Gly 90 Glu Lys Gly Asn Gly 170 Lys Lys Se r Ser Glu 250 Glu Pro Asp Asn Ser 75 Gin Val Glu Gin Leu 155 Phe Asp Asp Leu Thr 235 Ile Glu Ser Pro Gly Leu Gly Gly Met Glu 140 Glu Lys Lys Glu Gly 220 flu Ser ily Glu .Gln Gly Gly Ala Ile Gin Leu Gin Glu Ala Leu Gin Arg 110 Ala Thr 125 Glu Asn Glu Leu Lys Val Thr Glu 190 Gly Glu 205 Ala Gly Lys Pro Pro Pro Glu Glu 270 Pro Gly Ala Ser Gly Asn Asp Lys Arg Thr Phe 175 Lys Gly Glu Glu Ala 255 Lys Pro Pro Ala Thr Asp Ser Ser Ser Asn Gin 160 Lys Pro Ala Ala Glu 240 Glu Gin Glu Lys Giu Pro Ser Lys Ser Ala Glu Ser Pro Thr Ser Pro Val Thr Ser Glu Thr Gly Ser Thr Phe Lys Lys Phe Phe Thr Gin Gi: Gi' Git Gli Glu Le Ser 385 Glu Glu Glu Met Leu 465 Leu Se r Ser Gly Glu 545 Lys 290 1. Trp i Ala Glu Gin Ser 370 Gly Val Val Val Asp 450 Lys Ser Glu Pro Lys 530 His I Gly C Arc Ser Asp Ala 355 Ala Ser Phe His Glu 435 Ala Glu Pro Val Leu 515 Lys rhr flu Lye Gli Gl 34C His Glu Gln Asp Val 420 Glu Glu Thr Asp Glu 500 Lys Gin Gin Ser s Lys I Lys 325 P Lys Pro Tyr Gly Glu 405 Ser Thr Pro Cys Glu 485 Met Lys Lys Val I Ser 2 565 Thr 310 Lys Ala Gin Glu Pro 390 Lys Thr Ala Gln Val 470 Lys Leu Leu 31 y ?ro 550 1 a 295 Ser Lys Glu Glu Lys 375 Ser Ile Va1 Gly Glu 455 Ser Va1 Ser Phe Ls 535 Ala J Ser Phe Glu Val Pro 360 Va1 Glu Glu Glu Ser 440 Ala Gly Leu Se- Thi %sp er Arg Gin Ala 345 Ala Glu Glu Val Glu 425 .a.

Glu Glu Se r Gln Ser I Pro C Lys Glu 330 Ser Glu Leu Lys His 410 Arg Pro Prc Asp Lys 490 Glu Pro flu 570 Pro 315 Pro Glu Ser Pro Pro 395 Gin Thr Ala Ala Pro 475 Pro Arg Gly Gly Asp 555 Glu 300 Lys Glu Lys Ala Ser 380 Ala Glu Glu Glu Lys 460 Thr Pro Met Leu Asp 540 Ser Pro Glu Lys Leu His 365 Glu Pro Glu Glu Glu 445 Glu Gin Glu Lys Lys 525 Glu Gin C Glu C Asi Val Thi Glu Glu Leu Vai Gin 430 Leu Leu Gly Gly Val 510 Lys .lu flu flu r Trp Glu Asp 335 Ala Pro Gin Ala Val 415 Lys Val Val Ala Val 1 495 Gin C Leu I Ser C Glu C lie T 575 Ala Val 320 Thr Ser Arg Val Thr 400 Al a Thr Gly Lys Asp 480 Jal 1 y jer fly ;ln 'hr Cys Leu Giu Lys Gly Leu Ala Giu Vai Gin Gin Asp Gly Glu Ala Glu 580 585 qqn Glu Gly Ala Thr Ser Asp Gly Giu Lys Lys 595 a a a.

a a a .0 a a.o *a.

0 0 Trp Pro 625 Thr Lys Asp Lys Al a 705 Giu Gly Glu Lys Gly 785 Glu Arg Thr Se r Gin 865 Ala 610 Ser Leu Gly Thr Arg 690 Met Thr Gin Gly Lays 770 Se r Se r Pro Giy Giu 850 Lys Ser Glu Se r Ser Ser 675 Ala Gly Gly Giy Val1 755 S er Giy Trp Asp Ala 835 T'yr Gly Phe Ser Ser Val1 660 Val1 Arg Gly Thr Ser 740 Ser Lys Val1 Val1 Gly 820 Asn Asp Ala Lys Asp Thr 645 Giu Se r Arg Asp Asp 725 Ser Th r Ser Giu Ser 805 Lys Giu Al a Glu Lys ILys 630 *Glu Giu Trp Arg His 710 *G ly Ser Trp Lys His 790 Ile Gin Asp Val1 Gin 870 Met 615 Giu Ser Pro Giu Se r 695 Gin Ile Pro Giu Le u 775 Se r Lys Glu Asp Giu 855 Pro Lys Le u Giu Se r 760 Giu Th r Lys G 1 n Set- 840 Ar 9 Glu Al a Ala Gi1n '74 5 P he Giu Pro Phe Al1a 825 Asp Giu Gin Asp Gly 730 Ala Lys Ly s Asp Ilie 810 Pro Val1 Lys 600 Val Thr Prc Asp Giu Leu Thr Aia Ser 650 Lys Pro Giu 665 Aia Leu Ile 680 Ser Ser Asp *Arg ILys Asp 635 Glu Glu Cys Glu Giu 715 Ser Gly Arg Ser Thr 795 Pro Val Pro Met Ala.

875 Glu Lys 620 Lys Met Pro Vai Giu 700 Ala Gln Ser Leu Giu 780 Giu Gly Giu Al a Glu 860 Al a Gly 605 Arg Val Gln Lys Gly 685 Gly Gly Giu Pro Val 765 Asp Pro Arg Asp Val 845 Al a Thr Val Val Lys Glu Arg 670 Ser Gly Lys His Thr 750 Thr Ser Gly Arg Ala 830 Val Gin Glu Thr Arg Ser Glu 655 Lys Ser Pro Asp Asp 735 Giu Pro Ile Lys Lys 815 Gly Pro Gin Val *Pro Arg Ala 640 X1e t Val Lys Lys Lys 720 Pro Gly Arg Al a Giu 800 Lys Pro Leu Ala.

Ser 880 Lys Giu Leu Ser Glu Ser Gin Val His Met Met Ala Ala Ala Val Ala 885 890 gqc; -52- Glu Glu Asp Gly Thr Arg Ala Ala Thr Ile Ile 900 0*

C

C.

C.

C

C.

Ile Ser Ala Ser Val Th 915 Ala Leu Leu Thr Glu Gi 930 Glu Pro Pro Thr Val Th 945 95' Gly Asp Thr Val Val Se: 965 Ala Ala Glu Thr Ala Gl 980 Ser Ala Ala Glu Glu Th~ 995 Thr Asp Ser Pro Asp Th2 1010 Glu Gly Gly Val Pro Asp 1025 103 Val Leu Gin Ala Val Ala 1045 Gly Thr Gly Gly Pro Glu 1060 Ala Giu Arg Pro Glu Giu 1075 Thr Asp Val Val Leu Lys 1090 Phe Thr Gin Gly Lys Val Lys Ala Pro Gin Val Thr 1125 Thr Cys Gin Ala Glu Thr 1140 Met Glu Gin Ala 1ie Pro 1155 Glu Thr Asp Gly Ser Thr 1170 905 r Glu Pro Leu Glu Gin 920 Val Leu Glu Arg Glu 935 Glu Pro Leu Pro Glu 0 955 r Giu Ala Glu Leu Thr 970 ~Pro Leu Gly Ser Glu 985 Thr Giu Met Vai Ser 1.000 Thr Glu Glu Ala Thr 1015 Ile Giu Giu Gin Glu .0 1035 Giu Lys Val Lys Giu 1050 Asp Val Leu Gin Pro 1065 Gin Ala Glu Ala Ser 1080 Val Asp Ala Gin Glu 1095 Val Giy Gin Thr Thr 0 1115 Giu Ser Ilie Giu Ser 1130 Leu Ai ;.iv Val Lys 'i4s Pro Asp Ser Val Giu 1160 Pro Val Ala Asp Phe 1175 *Arg Val Val 940 Asn Ser Pro 910 Glu Ala 925 Ile Ala Arg Glu Ser Glu Giu Ala 960 Pro Glu Ala Val Thr 975 Glu Gly Thr Glu Ala 990 Ala Vai Ser Gin Leu 1005 Pro Val Gin Glu Val 1020 Arg Arg Thr Gin Glu 1040 Glu Ser Gin Leu Pro loss Val Gin Arg Ala Glu 1070 Gly Leu Lys Lys Giu 1085 Ala Lys Thr Glu Pro 1100 Pro Giu Ser Phe Glu 1120 Ser Glu Leu Val Thr 1135 Ser Gin Glu Met Val 1150 rhr Pro Thr Asp Ser 1165 %sp, Ala Pro Gly Thr 1180 Trp, Al a Glu Thr Gin Lys Asp Glu Ile Val Glu Ile His Giu Glu Asn Glu Val 1.185 1190 1195 His 1200 -53- Ala Glu Ala 1210 Leu Val Pro Val Arg Giy Thr Giu 1205 Val Pro Ala Gin Lys 1215 Glu Arg' Pro Pro Ala Pro Ser Ser Phe Val Phe Gin Glu Giu Thr Lys 1220 1225 1230 Giu Gln Ser Lys Met Giu Asp Thr Leu Glu His Thr Asp Lys Glu Val 1235 1240 1245 Ser Val Giu Thr Val Ser Ile Leu Ser Lys Thr Glu Gly Thr Gin Glu 1250 1255 1260 Ala Asp Gin Tyr Ala Asp Glu Lys Thr Lys Asp Val Pro Phe Phe Glu 1265 1270 1275 1280 Gly Leu Giu Gly Ser Ile Asp Thr Gly Ile Thr Val Ser Arg Giu Lys 1285 1290 1295 Val Thr Giu Val Ala Leu Lys Giy Glu Gly Thr Giu Giu Ala Glu Cys 1300 1305 1310 Lys'Lys Asp Asp Ala Leu Giu Leu Gin Ser His Ala Lys Ser Pro Pro 1315 1320 1325 Ser Pro Val Giu Arg Giu Met Vai Vai Gin Val Giu Arg Glu Lys Thr 1330 1335 1340 Giu Ala Giu Pro Thr His Val Asn Glu Giu Lys Leu Glu His Giu Thr 1345 1350 1355 1360 Ala Val Thr Val Ser Giu Giu Vai Ser Lys Gin Leu Leu Gin Thr Val 1365 1370 1375 Asn Val Pro Ile Ile Asp Gly Ala Lys Giu Val Ser Ser Leu Giu Gly 1380 1385 1390 Ser Pro Pro Pro Cys Leu Gly Gi'n Giu Glu Ala Val Cys Thr Lys.Ile 1395 1400 1405 Gin Val Gin Ser Ser Giu Ala Ser* Phe Thr Leu Thr Ala Ala Ala Giu 1410 1415 1420 Giu Glu Lys Val Leu Gly Giu Thr Ala Asn Ile Leu Glu Thr Gly Glu 1425 1430 1435 1440 Thr Leu Giu Pro Ala Gly Ala His Leu Val Leu Glu Giu Lys Ser Ser 1445 -1450 1455 Glu Lys Asn Glu Asp Phe Ala Ala His Pro Gly Glu Asp Ala Val Pro 1460 1465 1470 Thr Gly Pro Asp Cys Gin Ala Lys Ser Thr Pro Val Ile Val Ser Ala 1475 1480 1485 Thr Thr Lys Lys Gly Leu Ser Ser Asp Leu Glu Gly Glu Lys Thr Thr 1490 1495 1500 -54- Ser Leu Lys Trp Lys Ser Asp Glu Val Asp Glu Gin Val Ala Cys Gin 1505 1510 1515 1520 Giu Vai Lys Val Ser Val Ala Ile Giu Asp Leu Giu Pro Giu Asn Gly 1525 1530 1535 Ile Leu Giu Leu Glu Thr Lys Ser Ser Lys Leu Val Gin Asn Ile Ile 1540 1545 1550 Gin Thr Ala Val Asp Gin Phe Val Arg Thr Glu Giu Thr Ala Thr Giu 1555 1560 1565 Met Leu Thr Ser Giu Leu Gin Thr Gin Ala His Vai Ile Lys Ala Asp 1570 1575 1580 Ser Gin Asp Ala Gly Gin Glu Thr Glu Lys Giu Gly Giu Giu Pro Gin .1585 1590 1595 1600 Ala Ser Ala Gin Asp Giu Thr Pro Ile Thr Ser Ala Lys Glu Glu Ser 1605 1610 1615 Giu Ser Thr Ala Val Gly Gin Ala His Ser Asp Ile Ser Lys Asp Met 1620 1625 1630 Ser Giu Ala Ser Giu Lys Thr Met Thr Val Giu Val Glu Gly Ser Thr .*.1635 164C 1645 *Val Asn Asp Gin Gin Leu Giu Giu 1.al Val Leu Pro Ser Glu Glu Glu 1650 1655 1660 *..Gly Gly Gly Ala Gly Thr Lys S e i Va 1.Pro Giu Asp Asp Gly His Ala 1665 1670 1675 1680 Leu Leu Ala Giu Arg Ile Giu Lys Ser Leu Val Giu Pro Lys Glu Asp 1685 1690 1695 .Giu Lys Gly Asp Asp Val Asp Asp Pro Giu Asn Gin Asn Ser Ala Leu 1700 1'705 1710 Ala Asp Thr Asp Ala Set- Gly- Glyj Leu Thu, Lys Giu Ser Pro Asp Thr 1715 172,: 1725 Asn Gly Pro Lys Gin Lys Giu Lys Gu Asp Ala Gin Glu Val Glu Leu 1730 1735 1740 Gin Glu Gly Lys Val His Set, Gil: Asp Lys Ala Ile Thr Pro Gin 1745 1750 1755 1760 Ala Gin Giu Giu Leu Gin Lys Gin Giu Arg Glu Ser Ala Lys Ser Glu 1765 1770 1775 Leu Thr Giu Ser 1780 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Leu Glu Thr Lys Ser Ser Lys Leu Val Gin Asn Ile Ile Gin 1 5 INFORMATION FOR SEQ ID NO:7: i) SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Leu Ile Glu Thr Ala Ser Ser Leu Val Lys Asn Ala Ile Gin 1 5 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 23 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SE- ID NO:8: Asp Leu Ile Glu Glu Ala Ala Ser Arg Ile Val Asp Ala Val Ile 1 5 10 Glu Gin Val Lys Ala Ala Gly Ala INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid -56- STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CCGCCATGGT GCATATGTCC GAGTCC.AGTG AGC 33 INFORMATION FOR SEQ ID NO:l0: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi SEUNEDSCITO:SE

DN:

GC C GA C .C C CA T .G C T C G3 (2 INOMTONFR.Q DN:1 (i EQECECAACEISIS (xi) SEQUENCE DESCRIPTION: SEQ: ID NO:lO: GCGCGGATCC GCACTCACTT GACTCTG INFORMATION FOR SEQ ID NO:ll: i) SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single& TOPOLOGY: linear (xi SEUNEDSRPIN.E DN:2 SEQUMATINCEO DECIPI O: SQ 3 O SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GGAGGATCCA GAGATTCTGT AGTTCTG 2 INFORMATION FOR SEQ ID NO:14: 00 SEQUENCE

CHARACTERISTICS:

0 0(A) LENGTH: 453 amino- acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear o.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: *000 Glu Ser Ser Glu Leu Val Thr Thr Cys Gln Ala Glu Thr Leu Ala Gly 1 5 10 Val Lys Ser Gln Glu Met Val Met Glu Gin Ala Ile Pro Pro Asp Ser.

25 Val Glu Thr Pro Thiy Asp Sei- Glu Thiy Asp Gly Ser Thr Pro Val Ala 40 Asp Phe Asp Ala Pro Gly Th*. 7j.. Gin Lys Asp Glu Ile Val Glu Ile so 5 c) His Glu Giu Asn Giu Val His Leu Val Pro Val Arg Gly Thr Giu Ala 70 75 Giu Ala Val Pro Ala Gin Lys Giu Arg Pro Pro Ala Pro Ser Ser Phe 90 Val Phe Gin Giu Giu Thr Lys Glu Gln Ser Lys Met Glu Asp Thr Leu 100 105 110 Giu His Thr Asp Lys Glu Val Ser Val Glu Thr Val Ser Ile Leu Ser 115 120 125 -58- Lys Thr Glu Gly Thr Gin Giu Ala Asp Gin Tyr Ala Asp Giu Lys Thr 130 135 14A Lys 145 Ile Gly Ser Gin Glu 225 Lys Glu Glu Thr Asn 305 Val Pro Thr Leu Asp 385 Asp As Thl Th Hi Val 210 Lys Gin Vai Ala Leu 290 Ile Leu Gly Pro lu 370 3lu eu p Val r Val G1L Ala 195 Glu Leu Leu Ser Val 275 Thr Leu Glu Glu Val.

355 Gly Gin Glu Pro Ser Glu 180 Lys Arg Glu Leu Ser 260 Cys Ala Glu Glu Asp J 340 Ile I Glu I Val Pro G 4 Phe ArS 165 Ala Ser Glu His Gin 245 Leu Thr Ala Thr Lys 325 kla Ial .ys la ;lu

OS

PhE 151 Git Gli Prc Lys Glu 230 Thr Glu Lys Ala Gly 310 Ser Va1 Ser Thi Cys 390 Asn Glu a Lys i Cys Pro Thr 215 Thr Val G iv Ile Glu 295 Glu Ser Pro Ala Thi 375 Gin Gly Gi3 Val Lys Ser 200 Glu Ala Asn Ser G Ir.

28 c) Glu Thr Glu Th r Thr 3 C 3lu Ile r Leu Thr Lys 185 Pro Ala Val Val Pro 265 Val Glu Leu Lys Gly 345 Thr Leu Vai Leu Git Git 17( Asy Val Glu Thr Pro 250 Pro Gin Lys Glu Asn 330 Pro Lys Lys Lys ,lu L10 I Gly 155 a Val Asp Glu Pro Val 235 Ile Pro Ser Val Pro 315 .Glu Asp I Lys Trp Val 395 Leu C Se~ Al Ala Arc Thr 220 Ser Ile Cys Ser Leu 300 Ala Asp Dys Gly Lys 380 3er ;lu r Ile i Leu 1 Leu Glu 205 His Glu Asp Leu Glu 285 Gly Gly Phe Gin 2 Leu 1 365 Ser Val Thr I As Ly Git 19( Met Val Glu Gly Gly 270 Ala Glu Ala 4. a kla 350 er sp l a ,ys p Thr s Gly 175 a Leu Val Asn Val Ala 255 Gin Ser Thr His Ala 335 Lys Ser Glu Ile C 4 Ser S 415 Gly 160 Glu Gin Val Glu Ser 240 Lys Glu Phe Ala Leu 320 His Ser 4sp Ia 1 lu kOO er Lys Leu Val Gin 420 Asn Ile Ile Gin Thr 425 Ala Val Asp Gin Phe Val Arg 430 -59- Thr Glu Giu Thr Ala Thr Giu Met Leu Thr Ser Giu Leu Gin Thr Gin 435 440 445 Ala His Val Ile Lys 450 INFORMATION FOP, SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 396 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID Glu.Ser Ser Glu Leu Val Thr Thr Cys Gin Ala Giu Thr Leu Ala Gly 1 5 10 Vai Lys Ser Gin Giu Met Val Met Glu Gin Ala Ile Pro Pro Asp Ser 25 Val Giu Thr Pro Thr Asp.Ser Giu Thr Asp Gly Ser Thr Pro Val Ala 40 Asp Phe Asp Ala Pro Gly Thi- Thr Gin Lys Asp Glu Ile Val Giu Ile 55 His Giu Glu Asn Glu Val His Leu Vai Pro Val Arg Gly Thr Glu Ala 70 75 *Giu Ala Val Pro Ala Gin Lys Giu Arg Pro Pro Ala Pro Ser Ser Phe as 8 90 Val Phe Gin Giu Giu Thr Lys Giu Gin Ser Lys Met.Glu Asp Thr Leu 100 105 110 Glu His Thr Asp Lys Giu Val Ser Val Glu Thr Val Ser Ile Leu Ser 115 120 125 Lys Thr Glu Gly Thr Gin Giu Ala Asp Gin Tyr Ala Asp Glu Lys Thr 130 135 140 Lys Asp Val. Pro Phe Phe Giu Gly Leu Giu Giy Ser Ile Asp Thr Gly 145 -150 155 160 Ile Thr Val Ser Arg Giu Lys Val Thr Glu Val Ala Leu Lys Gly Glu 165 170 175 Gly Thr Glu Glu Ala Giu Cys Lys Lys Asp Asp Ala.Leu Glu Leu Gin 180 185 .190.

Ser Pro Ser His Ala Lys Ser Pro Pro 195 200 Val Glu Arg Glu Met Val Val 205 0* Gin Glu 225 Lys Glu Glu Thr Asn 305 Val Pro Thr Leu Asp 385 Val 210 Lys Gin Va1 Ala Leu 290 Ile Leu Gly Pro Glu 370 Glu Glu Leu Leu Ser Val 275 Thr Leu Glu Glu Val 355 Gly Gin Arg Glu Leu Ser 260 Cys Ala Glu Glu Asp 340 Ile Slu Glu His Gin 245 Leu Thr Ala Thr Lys 325 Ala Val Lys Lys Glu 230 Thr Glu Lys Ala Gly 310 Ser Va1 Ser Thr Thr 215 Thr Val Gly Ile Glu 295 Glu Ser Pro Ala Th r 375 Gin Glu Ala Asn Ser Gin 280 Glu Thr Glu Thr Th r 360 Ser Glu Ala Val Val Pro 265 Val Glu Leu Lys *Gly 345 Th x, Leu Val Glu Thr Pro 250 Pro Gin Lys Glu Asn 330 Pro Lys Lys Lys Pro Val 235 Ile Pro Ser Va1 Pro 315 Glu Asp Lys Trp Val 395 Thr 220 Ser Ile Cys Ser Leu 300 Ala Asp Cys Gly Lys 380 Ser His Glu Asp Leu Glu 285 Gly Gly Phe Gin Leu 365 Ser Val Asn Glu Glu Val Ser 240 Gly Ala Lys 255 Gly Gin Glu 270 Ala Ser Phe Glu Thr Ala Ala His Leu 320 Ala Ala His 335 Ala Lys Ser 350 Ser Ser Asp Asp Glu Val *fee 0000 6.

4000 a 09 e Val Ala Cys INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTIC! LENGTH: 255 aminc dcids TYPE: amino acid CC) STRANfEDNESS: singi- TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Val Ala Ile Giu Asp Leu Giu Pro Giu Asn Gly Ile Leu Giu Leu Glu 1 5 10 Thr Lys Ser Ser Lys Leu Val Gin Asn Ile Ile Gin Thr Ala Val Asp 25 Gln Phe Val Arg Thr Giu Giu Thr Ala Thr Glu Met Leu Thr Ser Glu 40 -61- Leu Gin Thr. Gin Ala His Val Ile Lys Ala Asp Ser Gin Asp Aia Giy 55' Gin Giu Thr Giu Lys Giu Gly Gii.

00 S. S 0* 00 @0 0 S 0 *000 *0 U S 00 0 00 0.00

S

Giu Giy Lys Leu Thr 145 Ile Vai Ser Lys His 225 Gin I Thr Gin Thr Glu 130 Lys Giu Asp Giy 3Au 210 3er .ys Pro Ala Me t 115 Giu Ser Lys Asp Gly 195 Lys Glu Gin I JE His 100 Thr Val Vai Ser Pro 180 Leu Giu Se r Glu Thr Ser Ser Asp Val Giu Val Leu Pro Giu 150 Leu Vai 165 Giu Asn Thr Lys Asp Aia Asp Lys 230 Arg GiuI Ala Ile Val Pro 135 Asp Giu Gin Glu Gln 215 kl a er Lys Ser Giu i2 0 Ser Asp Pro Asn Se r 200 Giu Ile Gl.

Glu Lys 105 Gly Giu Gly Lys Ser 185 Pro Vali rhr Prc Giu 90 Asp Ser Glu His Glu 170 Al a Asp Giu Pro Gin Aia Ser Ser Glu Ser Met Ser Giu Thr Val Asn Giu Gly Gly i4 0 Ala Leu Leu 155 Asp Giu Lys Leu Ala Asp Thr Asn Gly 205 Leu Gin Giu 220 Gin Ala Gin 235 Ala Thr Ala 110 Asp Gly Al a Gly Thr 190 Pro Giy Glu *Gin Asp Ala Val Ser Giu Gin Gin Ala Gly Giu Arg 160 Asp Asp 175 Asp Ala Lys Gin Lys Val Glu Leu 240 0S 0 0* 00 0050 0 0000 @0 0 00 0000 0 0005 C 00 0 0 0000 @0 0 @0 O 00 Ala Lys Ser Giu Leu Thr Giu Ser .250 ;9 INFORMATION FOR SEQ ID NO:17: SEQUENCE

CHARACTERISTICS:

LENGTH: 651 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i7: Giu Set Ser Gi~u Leu Val Thr Thr Cys Gin Ala Glu Thr Leu Ala Gly 1 5 10 Val Lys Ser Gin Giu Met Val Met Giu Gin Ala Ile Pro Pro Asp Ser 25 -62- Val Giu Thr Pro Thr Asp Ser Glu Thr Asp Gly Ser Thr Pro Val Ala

C

Asp His Glu Val Glu Lys Lys 145 Ile Gly Ser Gin Glu 225 Lys Glu Glu Thr I Asn 305 Phe Glu Ala Phe His Thr 130 Asp Thr Thr His Val 210 Lys ln Val ,eu 290 le I Asp Glu Val Gin Thr 115 Glu Vai Vai Glu Ala 195 Glu Leu Leu Ser Jal 275 [hr eu Ala Asn Pro Glu 100 Asp Gly Pro Ser Glu 180 Lys Arg Glu Leu Ser 260 Cys Ala J Glu 'J Pro Glu Ala Glu Lys Thr Phe Arg 165 Ala Ser Glu His Gln 245 Leu rhr la rhr Gly Val 70 Gin Thr Glu Gin Phe 150 Glu Glu Pro Lys Glu 230 Thr Glu Lys Ala Gly 310 Thm 55 His Lys Lys Val Glu 235 Glu Lys Cys Pr c Thr 215 Thr Va1 G ly Ile 3lu 295 ulu 40 Thr Leu Glu Glu Ser 220 Ala Gly Val Lv s Sei 200 Glu Ala Asn Sei Gin Glu Thr Gir Val Arg Gin 205 Val Asp Leu Thr Lys 185 Pro Ala Val Val Pro 265 Val Glu Leu 1 Lye Pro Pro 90 Ser Glu Gin Glu Glu 170 Asp Va1 Glu Thr Pro 250 Pro Gin Lys Glu Asp Val 75 I Pro Lys Thr Tyr Gly 155 Val Asp Glu Pro Val 235 Ile Pro Ser Val Pro 2 315 Glu Arg Ala Met Val Ala 140 Ser Ala Ala Arg Thr 220 Ser Ile -ys Ser Leu 300 kla Ile Gly Pro Glu Ser 125 Asp Ile Leu Leu Glu 205 His Glu Asp Leu Glu 285 Gly Gly Val Thr Ser Asp 110 Ile Glu Asp Lys Glu 290 Met Val Glu Gly Gly 270 Ala Glu 1 Ala I GlL Glu Ser Thr Leu Lys Thr Gly 175 Leu Val Asn Va1 4.1a 255 31n 3er 'hr 'is 1 Ile Ala Phe Leu Ser Thr Gly 160 Glu Gln Va1 Glu Ser 240 Lys Glu Phe Ala Leu 320 Val Leu Glu Glu Lys Ser Ser Glu Lys Asn Glu Asp Phe Ala Ala His 325 330 '19 -63- Pro Gly Glu Asp Ala Val Pro Thr Gly Pro Asp Cys Gin Ala Lys Ser Thr Leu Asp 385 Asp Lys Thr Al a Lys 465 Thr Ser Val1 Val1 Pro 545 Le u Glu Thr Asp Pro Giu 370 Giu Leu Leu Glu His 450 Glu Ser Asp Glu Leu 530 Glu Val1 Asn Lys Al a 610 Val 355 Gly Gin Giu Val1 Giu 435 Val1 Gly Al a Ile Val1 515 Pro Asp Glu Gin Giu 595 Gin 340 Ile Giu Val Pro Gin 420 Thr Ile Giu Lys Ser 500 Glu Ser Asp Pro Asn 580 Ser Glu Val Lys Ala Giu 405 Asn Al a Lys Giu Glu 485 Lys Gly Glu Gly Lys 565 Ser Pro Val1 Ser Thr Cys 390 Asn Ile Thr Al a Pro 470 Glu Asp Se r Glu His 550 Glu Ala Asp Glu Ala Thr 375 Gin Gly Ile Glu Asp 455 Gin Se r Met Tb r- Glu 535 Ala Asp Le u Tb r Leu 615 Thr 360 Ser Glu Ile Gin Met 440 Se r Al a Glu SeLi Val1 520 Gl1y Le u G 1-, Ala Asn 600 Gin 345 Thr Leu Val Le u Thr 425 Leu Gin Ser Ser Glu 505 Asn Gly Le u Ly s Asp 585 G ly Glu Lys Lys Lys Glu 410 Al a Thr Asp Al a Thr 490 Al a Asp Gly Al a Gly 570 Thr Pro Gly Lys Trp Val 395 Leu Val Ser Al a Gin .475 Al a Ser Gin Al a Glu 555 Asp Asp Lys Lys Giu 635 Gly Lys 380 Se r Glu Asp Glu Gly 460 Asp Val Glu Gin Gly 540 Arg Asp Ala Gin Val 620 Leu 365 Ser Val Thr Gin Leu 445 Gin Glu Gly Lys Leu 525 Thr Ile Val1 Ser Lys 605 His 350 Ser Asp Al a Lys Phe 430 Gin Giu Thr Gin Thr 510 Giu Lys Glu Asp Gly 590 Glu Ser Ser Glu Ile Ser 415 Val Thr Thr Pro Ala 495 Met Glu Ser Lys Asp 575 Gly Lys Glu Asp Val Glu 400 Ser Arg Gin Giu Ile 480 His Thr Val1 Val Ser 560 Pro Leu Giu Ser Giu 640 Asp Lys Ala Ile Thr Pro Gin Ala Gin Glu Leu Gin Lys Gin -64- Arg Glu Ser Ala Lys Ser Glu Leu Thr Glu Ser 645 650 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 23 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Asp Leu Ile Glu Glu Ala Ala Ser Arg Pro Val Asp Ala Val Ile 1 5 10 Glu Gln Val Lys Ala Ala Gly Ala INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs *i TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GACGAGATTG TGGAAATCCA TGAGG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID GCGCGGATCC AGAGATTCTG TAGTTCTGAC INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 22 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Phe Lys Lys Phe Phe Thr Gin Gly Trp Ala Gly Trp Arg Lys Lys Thr 1 5 10 Ser Phe Arg Lys Pro Lys INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 23 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: e* o* Pro Leu Lys Lys Leu Phe Thr Ser Thr Gly Leu Lys Lys Leu Ser Gly 1 5 10 Lys Lys Gin Lys Gly Lys Arg INFORMATION FOR .SEQ ID NO:23: SEQUENCE CHARACTERISTICS: S. LENGTH: 22 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Lys Ala Ser Met Leu Cys Phe Lys Arg Arg Lys Lys Ala Ala Lys Leu 1 5 10 Ala Lys Pro Lys Ala Gly INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Val. Arg Lys Arg Thr Leu Arg Arg Leu 1

Claims (16)

1. A polypeptide fragment of gravin capable of binding to a type II regulatory subunit of cAMP-dependent protein kinase, said fragment comprising the amino acid sequence set out in SEQ ID NO: 1.

2. The polypeptide fragment of claim 1, said fragment comprising the amino acid sequence set out irn SEQ ID NO: 2.

3. A polypeptide fragment of gravin capable of binding to protein kinase C (PKC). said fragment comprising the amino acid sequence set out in SEQ ID .NO: 3.

4. A polynucleotide encoding a polypeptide fragment of claims 1, 2, or 3.

5. A vector comprising the polynucleotide according to claim 4.

6. The vector according to claim 5 wherein said polynucleotide is operatively linked to an expression control DNA sequence.

7. A host cell stably transformed or transfected with a polynucleotide according to claim 4.

8. A method of prolucing ;a polypcptide fragment of gravin comprising the steps of growing a host cell according to claim 7 in a suitable nutrient medium and isolating the expressed polypcptide from the cell or the nutrient medium.

9. An antibody substance specifically immunoreactive with the polypeptide fragment of claims 1. 2. or 3. The antibody substance of claim 9 wherein said antibody substance is a polyclonal antibody. I1. The antibody substance of claim 9 wherein said antibody substance is a monoclonal antibody.

12. A cell line producing a monoclonal antibody specifically immunoreactive with a polypeptide fragment of claims 1, 2 or 3.

13. A method of identifying a compound that is a modulator of binding between a gravin polypeptide and a binding partner, said method comprising the steps of: a) determining the level of binding between the gravin polypeptide and the binding partner in the absence and presence of the compound; b) comparing the levels of binding observed in step and c) identifying the compound as a modulator of the binding between the gravin polypeptide and the binding panrner on the basis of differences in binding observed in step

14. The method of claim 13 wherein said binding partner or gravin S polypeptide is labeled with a detectable label. The method of claim 14 wherein said detectable label is a radiolabel.

16. The method of claim 13 wherein said gravin polypeptide is a fragment comprising the amino acid sequence set out in SEQ ID NO: 1

17. The method of claim 13 wherein said gravin polypeptide is a fragment comprising the amino acid sequence set out in SEQ ID NO: 2.

18. The method of claim 13 wherein said gravin polypeptide is a fragment comprising the amino acid sequence set out in SEQ ID NO: 3. -69-

19. The method of claim 13 wherein said binding partner is a type II regulatory subunit of cAMP-dependent protein kinase. The method of claim 13 wherein said binding partner is protein kinase C. DATED this TWENTY-SIXTH day of FEBRUARY 2002 ICOS Corporation Applicant Wray Associates Perth, Western Australia Patent Attorneys for the Applicant o* *go* *ooo* ooo o•* *g*