CANNABINOID RECEPTOR EXPRESSED IN CELLS OF THE IMMUNE SYSTEM

Field of the Invention

This invention relates to novel nucleotide and amino acid sequences, vectors comprising the novel nucleotide sequence, and a method of screening for therapeutically useful compounds.

Background of the Invention

Delta -Tetrahydrocannabinol (delta -THC), which is the major active ingredient of marijuana, has been used as a psychoactive agent for thousands of years. However

Q marijuana and delta -THC have a wide range of other effects which have attracted attention because of their therapeutic potential. These include analgesia, anti- inflammation, immunosuppression, anti-convulsion, alleviation of intraocular pressure in glaucoma, and attenuation of vomiting, and this has led to interest in the biochemical bases of their action. Progress stemmed initially from the synthesis of potent derivatives of q delta -THC, and more recently from the cloning of a gene encoding a G-protein coupled receptor for cannabinoids

(Matsuda et al. , 1990 Nature 346, 561-564). This receptor gene is expressed in the brain (and is probably therefore responsible for mediating the undesirable psychoactive effects of the drugs), but is not expressed in the periphery except for a low level in testes. Clinical application of cannabinoids has therefore been limited by

their psychoactive effects.

Summary of the Invention

The present inventor has isolated the DNA sequence encoding a novel receptor for cannabinoids that is not expressed in the brain but is expressed in cells of the immune system.

In a first aspect the invention provides a nucleotide sequence encoding a cannabinoid receptor not normally expressed in mammalian brain tissue, or an effective part of such a receptor.

Preferably the receptor of the invention is a human receptor and preferably is expressed in cells of the immune system.

In particular the invention provides a nucleotide sequence encoding a polypeptide having the amino acid sequence shown in Figure 1 or functional equivalents thereof. Desirably the nucleotide sequence comprises substantially nucleotides 127-1206 of the sequence shown in Figure 1 or functional equivalents thereof.

Such functional equivalents include, for example: those sequences which encode the same polypeptide (but which, by virtue of the degeneracy of the genetic code, possess a different nucleotide sequence); sequences which encode substantially the same polypeptide but wherein there may be one or more conserved amino acid substitutions (i.e. the substitution of an amino acid for one with similar properties); and sequences which hybridize under standard conditions to the complement of nucleotides 127-1206.

Preferably such functional equivalents will display at least 70%, more preferably at least 75%, and most preferably at least 80% nucleotide sequence homology with nucleotides 127-1206 of Figure 1.

A particular functional equivalent is the antisense equivalent of the sequence shown in Figure 1. Whilst such antisense sequences are not generally understood to be functional equivalents, the term functional equivalent as used in this application is intended to encompass such sequences.

Preferably the sequence also comprises a suitable 5 ¹ untranslated region, including a promoter, to enable expression in appropriate host cells.

Preferably the sequence also comprises a suitable 3' untranslated region. Thus, for example, when expressed in a eukaryotic host cell the sequence should comprise a polyadenylation signal. Conveniently this can comprise nucleotides 1207-1790 of the sequence shown in Figure 1.

In another aspect the invention provides a vector comprising the nucleotide sequence of the invention. Preferably the vector allows for expression, either in a eukaryotic or prokaryotic host cell, such that the polypeptide encoded thereby may be produced, preferably substantially free of other substances found in the human body.

In a further aspect the invention provides a polypeptide being a cannabinoid receptor not normally expressed in mammalian brain tissue, or an effective part thereof.

Preferably the polypeptide is a human cannabinoid receptor, and preferably is expressed in cells of the immune system.

Desirably the polypeptide has substantially the amino acid sequence shown in Figure 1 , or is a functional equivalent thereof. It is proposed that this receptor be termed CB2, but is referred to herein for present purposes as CX5. It will be appreciated by those skilled in the art that one or more amino acids may be substituted by others having similar properties without significantly altering the function of the polypeptide.

Preferably a functionally equivalent polypeptide will possess at last 50% amino acid homology with the amino acid sequence shown in Figure 1 , more preferably at last 55%, and most preferably at least 60% amino acid homology.

However, it will be apparent to those skilled in the art that those portions of the amino acid sequence which are not highly conserved (from comparison with the sequence of the human brain cannabinoid receptor) may be rather more substantially altered without significantly altering the function of the polypeptide.

Furthermore, comparison of the nucleotide sequence of the present invention with that of the known brain cannabinoid receptor enables the design of oligonucleotide probes to conserved regions of the receptors which could be used to isolate other cannabinoid receptors in humans and possibly in other species.

The novel receptor of the present invention presumably

mediates some of the non-psychoactive effects of cannabinoid drugs. It could thus be used to screen for compounds which bind to the novel receptor but not to the brain receptor. Such compounds would be expected to possess at least some of the known therapeutic activities of cannabinoids without the psychoactive characteristics. Examples of natural and synthetic cannabinoids are disclosed by Snyder (1990, Nature 346, p508).

Thus in another aspect the invention provides a method of screening for compounds having a cannabinoid activity but with reduced psychoactive effects, comprising: determining the relative binding affinity of a compound for the human brain cannabinoid receptor and for a receptor encoded by the sequence of the invention; and selecting those compounds which show lower relative binding affinity for the human brain receptor.

Suitable techniques for determining the relative binding affinity of compounds for receptors are well known to those skilled in the art and include that detailed in the examples below.

The invention will be further described by way of illustration, in the following examples and with reference to the accompanying Figures, in which:

Figure 1 shows a nucleotide sequence and the amino acid sequence encoded by it, both comprising sequences in accordance with the present invention, together with part of the nucleotide sequence of a functional equivalent;

Figure 2 shows a comparison between a prior art amino acid sequence and the amino acid sequence of a polypeptide in

accordance, with the invention;

Figure 3a is a graph of amount of compound bound against amount of compound initially added; Figure 3b is a graph of compound remaining bound against the concentration of competitor added;

Figures 4a and 4b show photographs of a Northern blot;

Figures 5a-5c show photographs of in situ hybridisation experiments;

Figure 6 shows the nucleotide sequence of a portion of a functional equivalent of the sequence of the invention and the amino acid sequence encoded thereby; and

Figure 7 shows a comparison of the amino acid sequence of the invention with that of a portion of a functional equivalent.

Example 1

To identify novel G-protein-coupled receptors expressed in myeloid cells, PCR using degenerate primers was performed on cDNA prepared from the human promyelocytic leukemic line HL60. Treating HL60 cells with dimethyl formamide (DMF) induces granulocyte differentiation whilst 12-0- tetradecanoyphorbol 13-acetate (TPA) induces myeloid differentiation.

Briefly, Oligo-dT primed cDNA was synthesised from poly(A)+RNA prepared from HL60 cells induced with 0.5% DMF for 3 days. 5ng of cDNA in 20ul was amplified with Taq polymerase using degenerate primers encoding regions

conserved between many G-protein coupled receptors. These included the primers

GAGGGCCCATYNSNNTNGAYMGNTA (Seq. ID No. 1) and TGAAGCTTSHRTANANSANNGGRTT (Seq. ID No. 2), where each letter has the meaning ascribed in the standard IUPAC-IUB code which correspond to the encoded region shown in bold in Figure 2.

PCR was performed as follows: 40 cycles of 94 ^C C 1 min; 50 ^β C 2min; and 72 ^β C 2min, in 100mM Tris HCI pH8.3, 3mM Mg Cl ₂ , 100mM tetramethylammonium chloride, 0.5% Tween 20, 0,5% NP40, 250uM dNTPs, using 20uM each primer. Gel purified amplification products were digested with Apal and Hindlll and cloned into Bluescript.

The cloned amplification products from DMF-treated cells were sequenced and grouped into 6 classes which showed homology to the G-protein coupled receptor family. Two of these classes corresponded to previously identified receptors; the interleukin 8 receptor-B and the adenosine A3 receptor. Of the remaining four sequences, only one showed particular homology to any published receptors. This clone, CX5, was related to a cannabinoid receptor cloned originally from rat brain. To investigate the functional significance of this homology, the CX5 insert was used to screen 200,000 colonies of a cDNA library from TPA-treated HL60 cells.

Two cDNA clones were obtained, hCX5.1 (Seq. ID No. 5) and hCX5.36 (Seq. ID No. 3), the latter extending the furthest 5*. The complete nucleotide sequence of hCX5.36 and the partial sequence of hCX5.1 is shown in Figure 1. Above the nucleotide sequence of hCX5.36 is indicated the polypeptide (three letter code, Seq. ID No. 4) encoded by

the longest open reading frame. The in-frame stop codon upstream of the most 5' ATG is underlined. The polypeptide encoded by hCX5.36 exhibits 44% identity with the human brain cannabinoid receptor. The transmembrane domains of G-protein coupled receptors are the most highly conserved parts, and the homology rises to 57% if these regions alone are considered (see Figure 2).

Also shown in Figure 1 is the nucleotide sequence of part of hCX5.1 where it diverges from that of hCX5.36 at the poly (A) addition site. The sequence of hCX5.1 is shown from the point equivalent to base 1714 of hCX5.36 onwards. Thus, the 3' untranslated region of hCX5.1 extends beyond that of hCX5.36.

Figure 2 shows the alignment between the proteins encoded by hCX5.36 and the previously reported human cannabinoid receptor (hCB-R). Identities are boxed and the seven putative transmembrane segments are underlined and numbered (Roman numerals). The most 5' ATG in CX5.36 has been assumed to be the initiator codon.

Example 2

To determine if the receptor encoded by CX5 could bind cannabinoids, the cDNA clone was inserted into an expression vector and transfected into tissue culture cells, from which membrane preparations were obtained and analysed in binding studies together with control membrane preparations from cells expressing an irrelevant receptor.

The method employed was as follows:

The hCX5.36 cDNA was inserted into the vector CDM8 (described by Seed, 1987, Nature 329, 840-842) to make plasmid SC36. Plasmid SCFRM2 is a cDNA for human formyl- peptide receptor (with a peptide-tag) in the same vector. 72 hours after transfection in parallel with the two plasmids, cells were dounce homogenised and the membranes pelleted from the post-nuclear supernatant at 90,000g for 20min, washed and then resuspended in 50mM TrisHCl pH7.4,3mM MgCl ₂ ,1mM EDTA and stored in liquid ₂ « Binding of [ ³ H]-CP55,940 (107 Ci/mmol; New England Nuclear) to membranes (50ug membrane protein per 200ul reaction), was determined as described (Howlett et al. 1988, Mol. Pharmacol. ^1, 297-302), except that silanised 1.5ml polypropylene tubes were used for the binding reactions and 5% ethanol/5% Triton X-100 was used to solubilise the

Q membrane pellets. Displacement by delta -THC (Sigma) was measured in the presence of 0.3nM [ ³ H]-CP55,940. All data were determined in duplicate, error bars denoting the duplicate values. Experiments were repeated at least twice.

The results are shown in Figures 3a and 3b.

Figure 3a shows a graph of amount of radiolabelled CP 55,940 (a synthetic cannabinoid), in femtomoles, bound to CHOP cell membrane preparations against amount of CP 55,940 added. The graph labelled 'specific' shows the specific binding taking place (i.e. binding to hCX5 minus the amount binding to the irrelevant control receptor).

The control cells do not express receptors for cannabinoids, but expression of hCX5.36 causes the appearance of a saturable number of high affinity binding sites for CP55,940.

Q

Furthermore, the archetypal cannabinoid (delta -THC) can compete for these binding sites as revealed by the displacement experiments (data shown in Figure 3b, which is a graph of amount of radiolabelled CP 55,940, in femtomoles, bound to COS cells against concentration of unlabelled delta ⁹ -THC). The Kd for CP55,940 is 1nM (+/- 0.5nM), and the Ki for delta ⁹ -THC, is 1OOnM (+/-50nM). These are comparable to the analagous figures (15nM and 400nM) reported for the previously cloned receptor. Thus it appears that hCX5.36 does indeed encode a high affinity receptor for cannabinoids. In the following text the original receptor is referred to as CB-R and the new receptor as CX5.

Example 3

CX5 was used to probe Northern blots of RNA from HL60 cells and various rat tissues. The method was as follows: HL60 cells were induced with either 20nglml TPA or with 0.7% DMF.

Total RNA was isolated from HL60 cells (at different time- points after induction) by lysis in guanidinium/LiCl, and 7.5 micrograms per sample was separated on a 1.2% agarose/4% formaldehyde gel, transferred to nylon (Hybond- N Amersham [Hybond-N is a trade mark]) and UV-cross linked. Parallel blots were probed at 42 ^β C in 5xSSPE/50% formamide/100ug/ml salmon sperm DNA/5x Denhardts/0.1% SDS with either CX5 or ICAM-1 (disclosed by Simmons et al. 1988, Nature 331, 624-627) labelled with ³² P by random- priming. After washing down to 1 SSC at 42°C, the blot was exposed to Kodak XAR with an intensifying screen either for 8 days [ICAM] or 10 days [hCX5]). The blots

were then stripped according to the manufacturer's instructions and reprobed with human gamma-actin, exposure 6 hours.

The results are shown in Figure 4a. The left hand side shows a blot of RNA from cells induced with TPA (at 0-48 hours post-induction) and the right hand side shows the results with DMF-induced cells (at 0-96 hours post- induction). The blot at the top was probed with CX5, the middle blot probed with the sequence encoding ICAM-1 and the bottom blot was probed with the sequence encoding gamma-actin.

The CX5 probe can be seen to hybridise to two transcripts of about 2.5 and 5.0 kb. The longer transcript probably corresponds to use of a more 3' poly(A) addition site than that used in hCX5.36. The hCX5.36 polyadenylation signal (GAUAAA) is a variant of the AAUAAA consensus that is found in a small fraction of messages and which can be used, albeit inefficiently, in vitro. It is apparent that there is some slight expression of CX5 in uninduced HL60 cells, but transcript levels are markedly increased upon myeloid or granulocyte differentiation. However, the gene does not appear to be expressed in mature neutrophils isolated from blood (data not shown).

Essentially the same technique was used to investigate expression in various rat tissues (results shown in Figure 4b for, from left to right, nasal tissue, thymus, lung, brain, kidney, spleen and liver). One of the probes used was a rat homologue of CX5 which was cloned from genomic DNA by PCR using primers

GGGCTCGAGGTNRAYTTYCAYGTNTT (Seq. ID No. 6) and GAGGGATCCATNSWRCARAANGCRAA (Seq. ID No. 7, letters

according to IUPAC-IUB convention) that encode sequences in hCX5 which are also found in the cannabinoid receptor but not in other G-protein coupled receptors (amino acid residues VNFHVF (91-96) and FAFCSM (279-284)). Cloning and sequencing of PCR products of 600-650bp produced primarily the rat cannabinoid receptor, but also a second sequence with 87% homology to hCX5, which was termed rCX5. The nucleotide (Seq. ID No. 8) and amino acid (Seq. ID No. 9) sequences of rCX5 are shown in Figure 6. Figure 7 shows a comparison of the human and rat amino acid sequences (identities are indicated by asterisks). Total RNA extracted by guanidinium lysis from various rat tissues (10ug/lane) was blotted and probed as before with rCX5, rat cannabinoid receptor and gamma-actin, exposure times being 5, 4 and 0.5 days respectively.

The results are shown in Figure 4b, which shows the blots of RNA from the various tissues probed with rCX5 (top panel), re-probed with a sequence encoding the rat cannabinoid receptor (rCB-R, middle panel) and subsequently with the sequence encoding gamma actin (bottom panel).

The rat probe (rCX5) detects an mRNA of c2.5kb. in spleen, but not in a variety of other tissues. In particular the rCX5 transcript is not detected in brain even though the 6kb mRNA encoding the rat CB-R can be readily detected in the same sample. The distribution of binding sites for cannabinoids in the brain corresponds well to the expression pattern of the mRNA encoding the brain receptor. However it is possible that CX5 is expressed in a subset of these sites and so its expression level is too low to be detected in total brain mRNA. To investigate this possibility horizontal sections of rat brain were

probed by in situ hybridisation with labelled oligonucleotides corresponding to rat CB-R and to rCX5 as described in Example 4 below.

In situ hybridization experiments were performed on both rat brain and spleen sections. 45bp oligonucleotides were chosen from the anti-sense of the second cytoplasmic loop of CX5 (residues 132-147) or the equivalent region of the rat cannabinoid receptor, as this region is highly divergent between the two genes and between other G- protein coupled receptors, the sequence of the probes being:

GGTGACGAGAGCTTTGTAGGTAGGTGGGTAGCACAGACATAGGTA (Seq. ID No. 10) and

GGTGACGATCCTCTTATAGGCCAGAGGCCTTGTAATGGATATGTA (Seq. ID No. 11). The oligonucleotides were tailed with 35S-dATP and used at 0.5ng/ml to probe 50um sections.

Hybridisations were performed on parallel sections in the absence or presence ("+ cold comp") of an excess of unlabelled oligonucleotide. Sections were exposed to Kodak XAR film for 7 days and then to emulsion for 1 month.

The results are shown in Figure 5a-c.

Figure 5a shows four autoradiographs of in situ hybridisations of labelled oligonucleotides for rat cannabinoid receptor (r-CB-R, top two panels) and rat CX5 (bottom two panels) to horizontal sections of rat brain, in the absence (left hand side) or presence (right hand side, "+ cold comp") of excess unlabelled probe.

Figure 5b shows four autoradiographs of in situ hybridizations of the r-CX5 probe to pairs of transverse sections of the spleen without unlabelled competitor probe (A) or with competitor probe (B).

Figure 5c shows a photomicrograph of an in situ hybridization of the r-CX5 probe to the periphery of the white pulp in the spleen (an arrow headmarks the central arteriole) under bright field illumination with haematoxylin/eosin staining (L) and a parallel section under dark field illumination (D).

Figure 5a shows that as previously reported, the brain receptor has a widespread distribution with high levels of expression in the cortex, hippocampus, striatum and cerebellum. However, when adjacent sections were probed for rCX5, no expression could be detected in these, or any other, regions. The rCX5 oligonucleotide does however hybridise to localised regions of the spleen (Figures 5b- d). The expression appears concentrated in the marginal zone around the outside of the periarteriolar lymphoid sheaths. The expression of hCX5 in HL60 cells differentiated along the myeloid lineage, and absence of substantial expression of rCX5 in thymus, suggests that the expression is probably in macrophages. To confirm this, splenic macrophages/monocytes were purified using FACS and the expression of CX5 examined using PCR. Expression was detected in the macrophage/monocyte population but not in the CD5-positive population used as a control, as described in Example 5 below.

Example 5

PCR analysis of rCX5 expression, rat splenocytes were separated by FACS using an antibody against CD11b (MRC 0X- 42), to label myeloid cells, and an antibody against CD5 (MRC 0X-19) to label T cells (antibodies obtained from Serotec). Cytoplasmic RNA prepared from 3x10 cells by detergent lysis, (20 ug of glycogen added as carrier), was treated with ribonuclease-free DNase (0.5 unit/10 minutes; RQ1, Promega), phenol extracted, ethanol precipitated, resuspended in reverse transcriptase buffer with random primers, divided in two and M-MLV reverse transcriptase was added to one set (GIBCO). After 60 min. at 37 ^β C, PCR amplification was performed with the primer pairs TTTCACGGTGTGGACTCC (Seq. ID No. 12) and TAGGTAGGAGATCAAGCG (Seq. ID No. 13, from rCX5, giving a 214bp product) or, as a positive control, GAAATGCACCATGAAGCT (Seq. ID No. 14) and TTACGATGCATTGTTATC (Seq. ID No. 15) (specific for Elongation factor 1-alpha [EF-1a]) using 94 ^β C, 1 min 54 ^β C, 1 min, 72 ^β C 1 min with the manufacturer's buffer (Promega) .

The results (not shown) indicated that expression could be detected in the macrophage/monocyte population but not in T cells.

The results overall (including those of the in situ hybridization experiments) suggest that expression is concentrated in the macrophages of the marginal zone, a population distinct from that of the rest of the spleen.

In summary, the results indicate that whilst G-protein coupled receptors are generally highly conserved throughout evolution, the sequence of CX5 is considerably divergent from that of CB-R. Of the 162 residues in transmembrane sections of the human CB-R, three are

divergent in rat CB-R, but 68 are different in human CX5. This suggests that the two receptors did not diverge recently and hence the endogenous ligand for these receptors may well be used by wide range of both vertebrate and non-vertebrate species.

Finally, it has been proposed that the peripheral effects of cannabinoids are either indirect effects of central actions, or reflect a direct interaction with non-receptor proteins such as lipoxygenases. The identification of CX5 clearly raises the additional possibility that some of the peripheral effects are mediated by this receptor. Indeed, it has recently been reported that cannabinoids inhibit immune function, and adenylate cyclase activity, in mouse spleen cells and CX5 is a good candidate for the mediator of this effect. The divergence between the CX5 and CB-R indicates that it should be possible to identify receptor- specific cannabinoids.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Medical Resezrch Council

(B) STREET: 20 Park Crescent

(C) CITY: London

(E) COUNTRY: United Kingdom

(F) POSTAL CODE (ZIP): WIN 4AL

(G) TELEPHONE: (071) 6365422 (H) TELEFAX: (071) 3231331

(ii) TITLE OF INVENTION: Improvements in or Relating to Drug Receptors

(Hi) NUMBER OF SEQUENCES: 15

(lv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

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

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

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (ill) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GAGGGCCCAT YNSNNTNGAY MGNTA 25

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (ni) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: TGAAGCπSH RTANANSANN GGRTT 25

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1790 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 127..1206

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

CAGGTCCTGG GAGAGGACAG AAAACAACTG GACTCCTCAG CCCCCGGCAG CTCCCAGTGC 60

CCAGCCACCC ACAACACAAC CCAAAGCCTT CTAGACAAGC TCAGTGGAAT CTGAAGGGCC 120

CACCCC ATG GAG GAA TGC TGG GTG ACA GAG ATA GCC AAT GGC TCC AAG 168 Met Glu Glu Cys Trp Val Thr Glu He Ala Asn Gly Ser Lys 1 5 10

GAT GGC TTG GAT TCC AAC CCT ATG AAG GAT TAC ATG ATC CTG AGT GGT 216 Asp Gly Leu Asp Ser Asn Pro Met Lys Asp Tyr Met He Leu Ser Gly 15 20 25 30

CCC CAG AAG ACA GCT GTT GCT GTG TTG TGC ACT CTT CTG GGC CTG CTA 264 Pro Gin Lys Thr Ala Val Ala Val Leu Cys Thr Leu Leu Gly Leu Leu 35 40 45

AGT GCC CTG GAG AAC GTG GCT GTG CTC TAT CTG ATC CTG TCC TCC CAC 312 Ser Ala Leu Glu Asn Val Ala Val Leu Tyr Leu He Leu Ser Ser His 50 55 60

CAA CTC CGC CGG AAG CCC TCA TAC CTG TTC ATT GGC AGC TTG GCT GGG 360 Gin Leu Arg Arg Lys Pro Ser Tyr Leu Phe He Gly Ser Leu Ala Gly 65 70 75

GCT GAC πc CTG GCC AGT GTG GTC TTT GCA TGC AGC TTT GTG AAT TTC 408 Ala Asp Phe Leu Ala Ser Val Val Phe Ala Cys Ser Phe Val Asn Phe 80 85 90

CAT Gπ πC CAT GGT GTG GAT TCC AAG GCT GTC πC CTG CTG AAG Aπ 456 His Val Phe His Gly Val Asp Ser Lys Ala Val Phe Leu Leu Lys He 95 100 105 110

GGC AGC GTG ACT ATG ACC πC ACA GCC TCT GTG GGT AGC CTC CTG CTG 504 Gly Ser Val Thr Met Thr Phe Thr Ala Ser Val Gly Ser Leu*pl636&Ueu 115 120 125

ACC GCC Aπ GAC CGA TAC CTC TGC CTG CGC TAT CCA CCT TCC TAC AAA 552 Thr Ala He Asp Arg Tyr Leu Cys Leu Arg Tyr Pro Pro Ser Tyr Lys 130 135 140

GCT CTG CTC ACC CGT GGA AGG GCA CTG GTG ACC CTG GGC ATC ATG TGG 600 Ala Leu Leu Thr Arg Gly Arg Ala Leu Val Thr Leu Gly He Met Trp 145 150 155

GTC CTC TCA GCA CTA GTC TCC TAC CTG CCC CTC ATG GGA TGG ACT TGC 648 Val Leu Ser Ala Leu Val Ser Tyr Leu Pro Leu Met Gly Trp Thr Cys 160 165 170

TGT CCC AGG CCC TGC TCT GAG Cπ πC CCA CTG ATC CCC AAT GAC TAC 696 Cys Pro Arg Pro Cys Ser Glu Leu Phe Pro Leu He Pro Asn Asp Tyr 175 180 185 190

CTG CTG AGC TGG CTC CTG πC ATC GCC πC CTC Tπ TCC GGA ATC ATC 744 Leu Leu Ser Trp Leu Leu Phe He Ala Phe Leu Phe Ser Gly He He 195 200 205

TAC ACC TAT GGG CAT Gπ CTC TGG AAG GCC CAT CAG CAT GTG GCC AGC 792 Tyr Thr Tyr Gly His Val Leu Trp Lys Ala His Gin His Val Ala Ser 210 215 220 πG TCT GGC CAC CAG GAC AGG CAG GTG CCA GGA ATG GCC CGA ATG AGG 840 Leu Ser Gly His Gin Asp Arg Gin Val Pro Gly Met Ala Arg Met Arg 225 230 235

CTG GAT GTG AGG πG GCC AAG ACC CTA GGG CTA GTG πG GCT GTG CTC 888 Leu Asp Val Arg Leu Ala Lys Thr Leu Gly Leu Val Leu Ala Val Leu

240 245 250

CTC ATC TGT TGG πC CCA GTG CTG GCC CTC ATG GCC CAC AGC CTG GCC 936 Leu He Cys Trp Phe Pro Val Leu Ala Leu Met Ala His Ser Leu Ala 255 260 265 270

ACT ACG CTC AGT GAC CAG GTC AAG AAG GCC TTT GCT πC TGC TCC ATG 984 Thr Thr Leu Ser Asp Gin Val Lys Lys Ala Phe Ala Phe Cys Ser Met 275 280 285

CTG TGC CTC ATC AAC TCC ATG GTC AAC CCT GTC ATC TAT GCT CTA CGG 1032 Leu Cys Leu He Asn Ser Met Val Asn Pro Val He Tyr Ala Leu Arg 290 295 300

AGT GGA GAG ATC CGC TCC TCT GCC CAT CAC TGC CTG GCT CAC TGG AAG 1080 Ser Gly Glu He Arg Ser Ser Ala His His Cys Leu Ala His Trp Lys 305 310 315

AAG TGT GTG AGG GGC CTT GGG TCA GAG GCA AAA GAA GAA GCC CCG AGA 1128 Lys Cys Val Arg Gly Leu Gly Ser Glu Ala Lys Glu Glu Ala Pro Arg 320 325 330

TCC TCA GTC ACC GAG ACA GAG GCT GAT GGG AAA ATC ACT CCG TGG CCA 1176 Ser Ser Val Thr Glu Thr Glu Ala Asp Gly Lys He Thr Pro Trp Pro 335 340 345 350

GAT TCC AGA GAT CTA GAC CTC TCT GAT TGC TGATGAGGCC TCπCCCAAT 1226 Asp Ser Arg Asp Leu Asp Leu Ser Asp Cys 355 360 πAAACAACT CAAGTCAGAA ATCAGπCAC TCCCTGGAAG AGAGAGAGGG GTCTTGGCAC 1286

TCTCπcπA CTTAAACCAG TCCCAGACAC CTAGACACGG ACCCCTππ GCTGATGAGT 1346

GπGGGACTG ACTCCTGGAA GACAGCCTGG CCTTGCCCAC CTGCACACAG TCTGπGGAT 1406

AGGTAGGGCC ACGAGGAGTA GCCAGGTAGG CGAGACACAA AAAGGCCTGG GACAGGGTCA 1466

GTACAAGTCA GGACAGGCπ CATGCCTGCA TCCTCCAGAG ACCACCAGGA GCCAAAGCGA 1526

GCCTCCAGGC CCAGCAATGA GGGACTTGGG AGAAATCTGA GAAGAATGGG πGπCTCπ 1586

GGGAAGTCAG GGTATCAGAT GGGATGGACA TCCAGGTCTT CTCTCTGCCT AAπGTCAAG 1646

GCCTCCπGG CTCTGGAGCT ATGAAAGGCC CCACTπCAA GTCACCCTTG CCACTGAGGA 1706

CCGAGGACTA TGCTATGATG AGGAπAAGG TGπGAOTG CCTCTTTCAG AGATAAATGA 1766

CAAGCCπCA AAAAAAAAAA AAAA 1790

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 360.amino acids

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

(ii) MOLECULE TYPE: protein

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

Met Glu Glu Cys Trp Val Thr Glu He Ala Asn Gly Ser Lys Asp Gly 1 5 10 15

Leu Asp Ser Asn Pro Met Lys Asp Tyr Met He Leu Ser Gly Pro Gin 20 25 30

Lys Thr Ala Val Ala Val Leu Cys Thr Leu Leu Gly Leu Leu Ser Ala 35 40 45

Leu Glu Asn Val Ala Val Leu Tr Leu He Leu Ser Ser His Gin Leu 50 55 60

Arg Arg Lys Pro Ser Tyr Leu Phe He Gly Ser Leu Ala Gly Ala Asp 65 70 75 80

Phe Leu Ala Ser Val Val Phe Ala Cys Ser Phe Val Asn Phe His Val 85 90 95

Phe His Gly Val Asp Ser Lys Ala Val Phe Leu Leu Lys He Gly Ser 100 105 110

Val Thr Met Thr Phe Thr Ala Ser Val Gly Ser Leu Leu Leu Thr Ala 115 120 125

He Asp Arg Tyr Leu Cys Leu Arg Tyr Pro Pro Ser Tyr Lys Ala Leu 130 135 140

Leu Thr Arg Gly Arg Ala Leu Val Thr Leu Gly He Met Trp Val Leu 145 150 155 160

Ser Ala Leu Val Ser Tyr Leu Pro Leu Met Gly Trp Thr Cys Cys Pro 165 170 175

Arg Pro Cys Ser Glu Leu Phe Pro Leu He Pro Asn Asp Tyr Leu Leu 180 185 190

Ser Trp Leu Leu Phe He Ala Phe Leu Phe Ser Gly He He Tyr Thr 195 200 205

Tyr Gly His Val Leu Trp Lys Ala His Gin His Val Ala Ser Leu Ser 210 215 220

Gly His Gin Asp Arg Gin Val Pro Gly Met Ala Arg Met Arg Leu Asp 225 230 235 240

Val Arg Leu Ala Lys Thr Leu Gly Leu Val Leu Ala Val Leu Leu He 245 250 255

Cys Trp Phe Pro Val Leu.Ala Leu Met Ala His Ser Leu Ala Thr Thr 260 265 270

Leu Ser Asp Gin Val Lys Lys Ala Phe Ala Phe Cys Ser Met Leu Cys 275 280 285

Leu He Asn Ser Met Val Asn Pro Val He Tyr Ala Leu Arg Ser Gly 290 295 300

Glu He Arg Ser Ser Ala His His Cys Leu Ala His Trp Lys Lys Cys 305 310 315 320

Val Arg Gly Leu Gly Ser Glu Ala Lys Glu Glu Ala Pro Arg Ser Ser 325 330 335

Val Thr Glu Thr Glu Ala Asp Gly Lys He Thr Pro Trp Pro Asp Ser 340 345 350

Arg Asp Leu Asp Leu Ser Asp Cys 355 360

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 84 base pairs 311X (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: TATGCTATGA TGAGGAπAA GGTGπGACT TGCCTCTTTC AGAGATAAAT GACAAGCCπ 60 CAGπTGGGG CATCCTGπG TfTG 84

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

GGGCTCGAGG TNRAYπYCA YGTNπ 26

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GAGGGATCCA TNS RCARAA NGCRAA 26

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 540 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(ix) FEATURE: 261X (A) NAME/KEY: CDS

(B) LOCATION: 1..540

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

CAC GGT GTG GAC TCC AGG AAT ATC πC CTG πG AAG ATC GGC AGC GTG 48 His Gly Val Asp Ser Arg Asn He Phe Leu Leu Lys He Gly Ser Val 1 5 10 15

ACC ATG ACC πc ACG GCC TCT GTG GGC AGC CTG CTG CTG ACT GCT Gπ 96 Thr Met Thr Phe Thr Ala Ser Val Gly Ser Leu Leu Leu Thr Ala Val 20 25 30

GAC CGA TAC CTA TGT CTG TGC TAC CCA CCT ACC TAC AAA GCT CTC GTC 144 Asp Arg Tyr Leu Cys Leu Cys Tyr Pro Pro Thr Tyr Lys Ala Leu Val 35 40 45

ACC CGT GGG AGG GCA CTG GTG GCC CTT GGT GTC ATG TGG GTC CTC TCG 192

Thr Arg Gly Arg Ala Leu Val Ala Leu Gly Val Met Trp Val Leu Ser 50 55 60

GCG πG ATC TCC TAC CTA CCG CTC ATG GGG TGG ACT TGT TGT CCT AGT 240 Ala Leu He Ser Tyr Leu Pro Leu Met Gly Trp Thr Cys Cys Pro Ser 65 70 75 80

CCC TGT TCT GAA Cπ πC CCC CTG ATC CCC AAC GAC TAC CTC CTG GGC 288 Pro Cys Ser Glu Leu Phe Pro Leu He Pro Asn Asp Tyr Leu Leu Gly 85 90 95

TGG cπ cπ πc Aπ GCC ATC CTC TΓT TCT GGC ATC ATC TAT ACC TAT 336

Trp Leu Leu Phe He Ala He Leu Phe Ser Gly He He Tyr Thr Tyr 100 105 110

GGG TAT GTC CTC TGG AAA GCA CAC CAA CAT GTA GCC AGC πG ACT GAG 384 Gly Tyr Val Leu Trp Lys Ala His Gin His Val Ala Ser Leu Thr Glu 115 120 125

CAC CAG GAC AGG CAG GTG CCT GGG ATA GCT CGG ATG CGG CTA GAC GTG 432 His Gin Asp Arg Gin. Val Pro Gly He Ala Arg Met Arg Leu Asp Val 130 135 140

AGG πG GCC AAG ACT CTG GGC CTG GTC ATG GCT Gπ CTG CTC ATA TGC 480 Arg Leu Ala Lys Thr Leu Gly Leu Val Met Ala Val Leu Leu He Cys 145 150 155 160

TGG πc CCT GCA CTG GCT CTC ATG GGC CAT AGC CTG GTC ACC ACA CTG 528 Trp Phe Pro Ala Leu Ala Leu Met Gly His Ser Leu Val Thr Thr Leu 165 170 175

AGT GAC AAG GTC 540

Ser Asp Lys Val 180

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 180 amino acids

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

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

His Gly Val Asp Ser Arg Asn He Phe Leu Leu Lys He Gly Ser Val 1 5 10 15

Thr Met Thr Phe Thr Ala Ser Val Gly Ser Leu Leu Leu Thr Ala Val 20 25 30

Asp Arg Tyr Leu Cys Leu Cys Tyr Pro Pro Thr Tyr Lys Ala Leu Val 35 40 45

Thr Arg Gly Arg Ala Leu Val Ala Leu Gly Val Met Trp Val Leu Ser

50 55 60

Ala Leu He Ser Tyr Leu Pro Leu Met Gly Trp Thr Cys Cys Pro Ser 65 70 75 80

Pro Cys Ser Glu Leu Phe Pro Leu He Pro Asn Asp Tyr Leu Leu Gly 85 90 95

Trp Leu Leu Phe He Ala He Leu Phe Ser Gly He He Tyr Thr Tyr 100 105 110

Gly Tyr Val Leu Trp Lys Ala His Gin His Val Ala Ser Leu Thr Glu 115 120 125

His Gin Asp Arg Gin Val Pro Gly He Ala Arg Met Arg Leu Asp Val 130 135 140

Arg Leu Ala Lys Thr Leu Gly Leu Val Met Ala Val Leu Leu He Cys 145 150 155 160

Trp Phe Pro Ala Leu Ala Leu Met Gly His Ser Leu Val Thr Thr Leu 165 170 175

Ser Asp Lys Val 180

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GGTGACGAGA GCTπGTAGG TAGGTGGGTA GCACAGACAT AGGTA 45

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GGTGACGATC CTCTTATAGG CCAGAGGCCT TGTAATGGAT ATGTA 45

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: TTTCACGGTG TGGACTCC 18

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: TAGGTAGGAG ATCAAGCG 18

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GAAATGCACC ATGAAGCT 18

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI -SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: πACGATGCA πGπATC 18