WO1996024846A1 - Human t cell inositol 1,4,5-trisphosphate receptor - Google Patents

Abstract

The present invention relates to the human type 1 inositol 1,4,5-trisphosphate receptor/calcium release channel ('IP3R'). It is based, at least in part, on the cloning and characterization of a cDNA encoding the human IP3R.

Description

Description

HUMAN T CELL INOSITOL 1.4.5.-TRISPHOSPHATE RECEPTOR

1. INTRODUCTION

The present invention relates to the human type l inositol 1,4,5-trisphosphate receptor/calcium release channel ("IPSR") . It is based, at least in part, on the cloning and characterization of a cDNA encoding the human IP3R.

2. BACKGROUND OF THE INVENTION The second messenger inositol 1,4,5-trisphosphate (IP3) triggers intracellular calcium (Ca) release by activating IP3 receptor (IP3R)/calcium release channels on the endoplasmic reticulum of many types of cells.

During T cell activation there is a rapid early rise in cytoplasmic calcium due to intracellular release. It has been proposed that this intracellular calcium release in T cells is triggered by IP3 (Gardner, 1989, Cell, 5_£:15-20). Although this IP3-induced calcium release is presumed to occur via an IP3R in the T cell endoplasmic reticulum, prior to the present invention the complete structure of a T cell IP3R has not been reported. Three forms of IP3R have been identified in non-T cell tissues. The type 1 IP3R has been biochemically characterized in murine brain (cerebellum) , vas deferens and aortic smooth muscle (Ross et al., 1989, Nature, 339:468-470; Ferris et al., 1989, Nature, 342:87-89: Mourey et al., 1990, Biochem. J. , 272:383- 389; Chadwick et al., 1990, Proc. Natl. Acad. Sci., J ZJ2132-2136) . The type I IP3R, purified from bovine aortic smooth muscle, has a MW of approximately 240 kDa, based on polyacrylamide gel electrophoresis (Chadwick et al., 1990, Proc. Natl. Acad. Sci., £2:2132-2136) and a MW of 313 kDa based on cDNA cloning (Furuichi et al., 1989, Nature, 242:32-38). Examination of the single channel properties of the IP3R reconstituted into planar lipid bilayers has shown that it forms an IP3 gated cation channel (Bezprozvanny et al., 1991, Nature, 251:751-754; MayrLeitner et al., 1991, Cell Calcium, 12:505-514). The type 2 IP3R has been cloned from rat cerebellum (Sudhof et al., 1991, EMBO J. , 11:3199-3206) and human endothelial cells (Yamamoto-Hino, et al., 1994, Receptors and Channels, 2 : 9-22) . The type 2 IP3R shares 69% amino acid identity with the type 1 IP3R. A third form (IP3R type 3) shares 64% identity with the amino acid sequence of the type 1 IP3R (Yamamoto-Hino, et al., 1994, Receptors and Channels, 2:9-22; Blondel et al., 1993, J. Biol.

Chem. , 268:11356-11363: Maranto, 1994, J. Biol. Chem. , 269:1222-12301.

A model for the transmembrane topography of the IP3R has been proposed based on hydropathy analyses of its deduced amino acid sequence (Furuichi et al., 1989, Nature, 3_42:32-38; Miyawaki et al., 1990, Neuron, .5:11- 18) . Hydrophobic sequences forming the putative pore are clustered in the carboxy terminal 25% of the linear sequence, similar to the calcium release chan- nels/ryanodine receptors (RyR) of the sarcoplasmic reticulum (Takeshima et al., 1989, Nature, 339:439-445: Zorzato et al., 1990, J. Biol. Chem., 265:2244-2256: Marks et al., 1990, J. Biol. Chem., 26^:13143-13149). Three domains have been proposed for the IP3R: a ligand binding domain near the amino terminus (Mignery et al., 1990, EMBO J. , 9_:3893-3898) , a coupling domain in the middle of the molecule that may link IP3 binding to calcium release (Mori et al., 1991, Nature, 350:398- 402) , and the putative pore region at the carboxy terminus (Mignery et al., 1990, J. Biol. Chem.,

265:12679-12685 . Alternative splicing of the type 1 IP3R transcript (Mignery et al., 1990, J. Biol. Chem., 265:12679-12685: Nakagawa et al., 1991, Proc. Natl. Acad. Sci. U.S.A., £8.:6244-6248) defines neuronal and non-neuronal forms (Danoff et al., 1991, Proc. Natl. Acad. Sci. U.S.A., fi8_:2951-2955) .

Recently it was reported that a polyclonal anti¬ body directed against the complete IP3R protein recog¬ nized a molecule on the plasma membrane of T lympho¬ cytes (Khan et al., 1992, Science, 257:815-818) . Other groups have found IP3R protein on intracellular mem¬ branes corresponding to the endoplasmic reticulum (Fujimoto et al., 1992, J. Cell. Biol., 119:1507-1513: Kume et al., 1993, Cell, 72:555-570; Kijima et al., 1993, J. Biol. Chem., 268:3499-3506: Bourguignon et al.,1993, J. Biol. Chem., 268:7290-7297) .

3. SUMMARY OF THE INVENTION The present invention relates to the human type 1 inositol 1,4,5-trisphosphate receptor/calcium release channel (^MIP3R^M) . It is based, at least in part, on the cloning and characterization of a cDNA encoding the human IP3R, and on studies demonstrating an association between phosphorylation of tyrosine residues of the human IP3R and T cell activation.

In various embodiments, the present invention provides for nucleic acid molecules encoding human IP3R and for human IP3R proteins.

In view of the role of the human IP3R in T cell activation, the present invention also provides for assay systems that may be used to identify agents which either block or enhance the ability of the IP3R to act as a calcium channel. Such assay systems may be used, for example, to identify agents which block the channel and act as suppressants of immune function. 4. DESCRIPTION OF THE FIGURES Figure 1A and B. Deduced amino acid sequence of the human type I inositol 1,4,5-triphosphate receptor.

Comparison of the human and rat (Mignery et al. , 1990, J. Biol. Chem., 265:12679-12685) type 1 IP3R sequences reveals 98% identity. The first line is the amino acid sequence of human IP3R (SEQ ID N0:1), the second line indicates the amino acid residue where there are differences with the rat form. The alterna- tively spliced exon denoted SI (underlined sequence) is preferentially expressed in the thymus and spleen. The alternatively spliced SII exon (underlined sequence; SEQ ID NO:2) is excluded from the present form of the IP3R consistent with the non-neuronal splicing pattern. The amino acid sequence of the human type 1 IP3R was deduced from cDNA cloning as described in "Materials and Methods". Two putative tyrosine phosphorylation sites are denoted by the small asterisks (*) above residues 482-486 and 2617-2621. The large asterisks (*) at serine residues 1589 and 1717 denote putative

PKA phosphorylation sites. Six putative transmembrane sequences are overlined and labeled Ml through M6. The double underlining identifies consensus sequences for nucleotide binding sites at amino acid residues 1689- 1694, 1737-1742, and 1978-1983. Two stretches Of 10 amino acids that are 90% conserved between the two channels are denoted by under- and overlines: residues 2001-2010, SLTEYCQGPC (SEQ ID NO:3), with only one mis¬ match (I for C in the RyR) ; and residues 1931-1940, ILRFLQLLCE (SEQ ID NO:4) , with only one mismatch (F for L in the RyR) • The exclamation marks (!) at amino acid residues 2652-2663 identifies the sequence of the syn¬ thetic peptide used to raise site-specific anti-IP3R antibodies. Figure 2. Northern hybridization analysis of IP3R mRNA in human T lymphocytes

A 1.3 kb human IP3R CDNA was hybridized to 20 μg of total RNA isolated from: Lane l, rat brain; lane 2, rat heart; lane 3 Jurkat; lane 4, PMA stimulated

Jurkat; lane 5, anti-CD3 activated Jurkat. A 10 kb mRNA is detected in each lane. PMA and anti-CD3 had no effect on IP3R mRNA level in Jurkat lymphocytes. Ethidiu bromide staining of the 28s and 18s ribosomal RNAs (after transfer) is shown to indicate that equal amounts of RNA were loaded in each lane.

Figure 3. immunoblot of IP3R in human T lymphocytes and in rat brain.

Rat brain homogenate (50 μg total protein) and crude membrane preparation from Jurkat were size fractionated on a 6% SDS-polyacrylamide gel, blotted to a polyvinylidene difluoride (PVDF) membrane and probed with affinity-purified anti-IP3R antibody. A single band migrating at -300,000 Da is seen in both human T lymphocytes (Jurkat) and rat brain, a non-specific lower molecular weight band is seen in rat brain, but not in human T lymphocytes. Primary antibody was used at a 1:100 dilution. Position of molecular weight markers are indicated: myosin (205 kDa) , β-galac- tosidase (116.5 kDa), bovine serum albumin (prestained, migrates at 80 kDa) and ovalbumin (49.5 kDa).

Figure 4. FλCS analysis of human T lymphocyte (Jurkat) IP3R.

Top panel (4A) , non permeabilized T cells (Jurkat) ; bottom panel (4B) , permeabilized T cells

(Jurkat) . Cells were stained with a polyclonal site- specific anti-IP3R antibody (α-IP3R-l) and a fluorescein isothiocyanate (FITC)-conjugated secondary antibody. IP3R was detected only in permeabilized cells. Normal rabbit serum and secondary antibody alone gave no significant signal.

Figure 5. Xmmunocytochemistry of IP3R in human T lymphocytes (Jurkat) . Permeabilized (100% methanol, 5 min) cells were stained with anti-IP3R antibody (αIP3R-l) and a rho- damine-conjugated secondary antibody. Serial sections through a representative T lymphocyte are shown at loA intervals. IP3R is detected in both the cytoplasm and on the inner surface of the plasma membrane. Staining of the perinuclear membrane is seen in panels c and d. Magnification is 100X.

Figure 6. Tyrosine phosphorylation of the human type l inositol 1,4,5-trisphosphate receptor. Lane 1 is an immunoblot of T cell (Jurkat) lysates using preimmune serum. Lane 2 is an immunoblot of T cell lysate using an anti-IP3R antibody showing the 308 kDa IP3R. Lanes 3 and 4 were immunoblotted with anti- phosphotyrosine antibody. Lane 3 contains the anti- phosphotyrosine antibody immunoprecipitate from activa¬ ted T cells, lane 4 contains similar immunoprecipitate from non activated cells. The band recognized by the anti-IP3R antibody in lane 2 corresponds to the high molecular weight band identified by the anti-phos- photyrosine antibody in lane 3.

Figure 7A-D. Nucleic acid sequence of cDNA encoding human IP3R protein (SEQ ID NO:5).

5. DETAILED DESCRIPTION OF THE INVENTION For purposes of clarity of description, and not by way of limitation, the detailed description of the invention is divided into the following subsections. (1) human IP3R-encoding nucleic acids; (2) human IP3R proteins;

(3) antibodies directed toward human IP3R; and

(4) utilities of the invention.

5.1. HUMAN IP3R-ENC0DING NUCLEIC ACIDS

The present invention provides for a purified and isolated nucleic acid encoding a human IP3R protein. The nucleic acid may be DNA or RNA, and may be prepared using recombinant DNA techniques or by chemical syn- thesis.

In particular nonlimiting embodiments, the nucleic acid encodes a protein having a sequence consisting essentially of the amino acid sequence set forth in Figures 1A and IB, wherein the SII exon is excluded (the SII exon is neural specific) . In alternative embodiments, the nucleic acid encodes a protein having a sequence consisting essentially of the full amino acid sequence set forth in Figures 1A and IB, including the SII exon. In further nonlimiting embodiments, the nucleic acid comprises a portion having a sequence consisting essentially of the nucleic acid sequence set forth in Figure 7A-7C (SEQ ID N0:5). Three plasmids, TIP3R1A, TIP3RB and TIP3RC, which together contain nucleic acid having the sequence set forth in Figure 7A-7C (SEQ ID NO:5) have been deposited with the ATCC and assigned accession numbers , and . The present invention also provides for portions of the foregoing nucleic acid sequences, said portions comprising at least 10 nucleotides, and more preferably at least 30 nucleotides.

For replication, expression, hybridization or storage, the nucleic acid may be comprised in a vector molecule, such as, but not limited to, a plasmid, phage, cosmid, minichromosome or virus. Such vectors may further comprise elements which function in repli¬ cation and/or expression, including promoter/enhancer elements, ribosome binding sites, polyadenylation sites, secretory sequences, etc.. The present invention further provides for a host cell containing such a vector, where the host cell may be a bacterium, yeast, plant, animal, or insect cell, and for transgenic animals containing a transgene com¬ prising a nucleic acid encoding a human IP3R protein.

5.2. HUMAN IP3R PROTEINS

The present invention provides for human IP3R proteins. In a particular, nonlimiting embodiment, the present invention provides for a purified and isolated human IP3R protein having an amino acid sequence con- sisting essentially of the amino acid sequence set forth in Figures 1A and IB, wherein the SII exon is excluded (the SII exon is neural specific) . In alter¬ native embodiments, the present invention provides for a purified and isolated protein having a sequence con- sisting essentially of the full amino acid sequence set forth in Figures 1A and IB, including the SII exon.

Further, the sequence set forth in Figures 1A and IB may be altered by substitutions, deletions, or addi¬ tions that provide for functionally equivalent mole- cules. For example, the sequence set forth in Figures 1A and IB may be altered by the substitution of one or more amino acid residues within the sequence with a functionally equivalent amino acid, thus producing a silent change. Substitutes for an amino acid within the sequence may be selected from other members of the same polarity class to which the amino acid to be sub¬ stituted belongs. For example, the non-polar (hydro- phobic) amino acids include alanine, leucine, iso- leucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include gly- cine, serine, threonine, cysteine, tyrosine, aspar- agine, and glutamine. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

The present invention further provides for larger proteins comprising such a human IP3R protein, in a fusion protein, as well as for peptides or proteins comprising a biologically active (including immuno- genie) portion of such a human IP3R protein. In a specific, nonlimiting embodiment, the present invention provides for a purified and isolated immunogenic pep- tide or protein having an amino acid sequence com¬ prising the amino acid sequence (Cys)-Glu-Gln-Asn-Glu- Leu-Arg-Asn-Leu-Gln-Glu-Lys-Leu (residues 2652-2663) (SEQ ID NO:6) .

The human IP3R proteins of the invention may be prepared by chemical synthesis or by recombinant DNA associated methods. According to the latter, a human IP3R protein may be expressed using one of the numerous recombinant expression systems known in the art, including expression in cultured mammalian cells (see, for example, Sambrook et al., 1989, in "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory Press, S16.3-S16.73) ; bacteria (see, for example, Sambrook et al., 1989, in "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory Press, S17.3-S17.40) ; insect cells (for example, the baculovirus system) ; yeast; in mould; plants or plant cells; or transgenic animals. A few examples of systems included in the preceding list include those which utilize, as host, Saccharomyces cerevisiae, Kluvero- myces marxianus, Bacillus subtilis , Hansenula poly- morpha , and Pichia pastoris. Protein expressed in such a system may then be purified using standard techniques, including chroma- tography (e.g. affinity chromatography) and preparative polyacrylamide gel electrophoresis.

5.3. ANTIBODIES DIRECTED TOWARD HUMAN IP3R A human IP3R protein, as described in the preceding section, may be used as an immunogen to generate antibodies which specifically bind to the human IP3R protein, or a portion thereof.

To improve the likelihood of producing an immune response, the amino acid sequence of a human IP3R pro- tein may be analyzed in order to identify portions of the molecule which may be associated with increased immunogenicity. For example, the amino acid sequence may be subjected to computer analysis to identify sur¬ face epitopes. Alternatively, the deduced amino acid sequences of IP3R proteins from different species could be compared, and relatively nonhomologous regions iden¬ tified; these non-homologous regions would be more likely to be immunogenic across various species.

For preparation of monoclonal antibodies directed toward a human IP3R protein, any technique which pro¬ vides for the production of antibody molecules by con¬ tinuous cell lines in culture may be used. For example, the hybridoma technique developed by Kohler and Milstein (1975, Nature 256:495-497) . as well as the trioma technique, the human B cell hybridoma technique (Kozbor et al., 1983, Immunology Today J_:72), and the EBV-hybridoma technique to produce monoclonal anti¬ bodies (Cole et al., 1985, in "Monoclonal Antibodies and Cancer Therapy", Alan R. Liss, Inc. pp.77-96), may be used.

The monoclonal antibodies may be human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies may be made by any of numerous techniques known in the art (Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A. fi.ϊ7308-7312; Olsson et al., 1982, Meth. Enzy ol. 92:3- 16) . Chi eric antibody molecules may be prepared con¬ taining a mouse (or other species) antigen-binding domain and human constant regions (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. £1:6851; Takeda et al., 1985, Nature 314:452) .

Various procedures known in the art may be used for the production of antibody. Various host animals may be immunized by injection with human IP3R peptide or protein, and various adjuvants may be used to increase the immunological response, including, but not limited to, Freund's adjuvant (complete or incomplete) , mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitophenol, and BCG.

Antibody molecules may be purified by known tech¬ niques, such as immunoabsorption or immunoaffinity chromatography, HPLC, or a combination thereof. Antibody fragments which contain the idiotype of the molecule can be generated by techniques known in the art, and are within the scope of the present inven¬ tion.

In a specific, nonlimiting embodiment, purified antibody may be prepared which specifically binds to the peptide (Cys)-Glu-Gln-Asn-Glu-Leu-Arg-Asn-Leu-Gln- Glu-Lys-Leu (SEQ ID NO:6).

5.4. UTILITIES OF THE INVENTION The molecules of the invention may be used to produce assay systems for the identification and design of agents which stimulate or suppress the immune sys¬ tem. As demonstrated in Section 6, below, an asso¬ ciation has been demonstrated between phosphorylation of the human IP3R protein and T cell activation. Such phosphorylation, which has been observed in the vicinity of the putative calcium channel region, may be associated with the opening of that channel.

In a first set of embodiments, an assay to identify an agent which inhibits or stimulates T cell activation may be performed as follows. A test agent may be added to a lymphocyte culture comprising T cells previously sensitized to a target antigen, and then the target antigen may be added to the culture, and the level of phophorylation of human IP3R protein may be determined and compared to the amount of phos¬ phorylation achieved in a parallel control culture which does not contain the test agent. If the level of phosphorylation in the test culture is significantly greater than the level of phosphorylation in the con- trol culture, then the test agent may be useful in augmenting the immune response. If, conversely, the level of phosphorylation in the test culture is significantly lower than the level of phosphorylation in the control culture, then the test agent may be useful in suppressing the immune response.

In a second set of embodiments, an assay to identify an agent which inhibits or stimulates T cell activation may be performed as follows. A test agent may be added to a lymphocyte culture comprising T cells previously sensitized to a target antigen, and then the target antigen may be added to the culture, and the level of intracellular calcium release may be deter¬ mined and compared to the amount of intracellular calcium release achieved in a parallel control culture which does not contain the test agent. If the level of intracellular calcium release in the test culture is significantly greater than the level of intracellular calcium release in the control culture, then the test agent may be useful in augmenting the immune response. If, conversely, the level of intracellular calcium release in the test culture is significantly lower than the level of intracellular calcium release in the control culture, then the test agent may be useful in suppressing the immune response.

In further embodiments, the molecules of the invention may be used to design molecules which may block the calcium channel, the configuration of which may be predicted using the amino acid sequence of Figures 1A and IB. Such agents may be used as immuno- suppressants, and may be particularly useful in the treatment of autoimmune diseases or in the facilitation of tissue transplants. Alternatively, such agents may be used to block the channel in vascular smooth muscle, and thereby regulate smooth muscle contraction. Such agents may be useful in the treatment of hypertension. In still further embodiments, the human IP3R protein may be used as a marker for permeabilized T cells. For example, but not by way of limitation, anti¬ body generated toward recombinant human IP3R protein may be allowed to bind to human IP3R protein of human T cells (see Section 6 below) and such binding may be detected using labelling techniques known in the art.

EXAMPLE: CLONING AND CHARACTERIZATION OF

THE HUMAN TYPE I IP3R RECEPTOR FROM T LYMPHOCYTES 6.1. MATERIAL AND METHODS

6.1.1. CELL CULTURE

Jurkat cells were grown in RPMI-1640 medium containing 5% fetal bovine serum, 100 μ/ml penicillin and 100 μg/ml streptomycin. Medium was changed every 48 h.

6.1.2. CDNA LIBRARY SCREENING To isolate the IP3R cDNA, 1X10⁶ recombinants from a human leukemic T cell line (HUT) cDNA library were screened at high stringency (final wash, 0.2X SSC, 55°C) as described in Marks et al., 1989, Proc. Natl. Acad. Sci, U.S.A., ££ 8683-8687. The λgtll HUT cDNA library was screened with the following cDNA probes: (1) a 946 bp mouse aortic smooth muscle IP3R cDNA cor- responding to nucleotides 817 to 1763 of the mouse brain type 1 IP3R sequence (Marks et al., 1990, J. Biol Chem, 265:20719-20722); (2) a 497 bp human T cell type 1 IP3R cDNA corresponding to nucleotides 5270 to 5766 of the mouse brain IP3R sequence; (3) a second -500 bp human T cell type 1 IP3R cDNA corresponding to nucleo¬ tides 4200 to 4700 of the mouse brain IP3R sequence; and (4) a 600 bp type 1 IP3R cDNA amplified from human T cell total RNA by polymerase chain reaction to fill a gap between nucleotides 6500 to 7000. Human T cell type 1 IP3R cDNAs used as probes to screen the cDNA library were amplified from total RNA using reverse transcriptase followed by the polymerase chain reaction as described in Marks et al., 1990, J. Biol Chem, 265:20719-20722. using primers based on the published mouse brain type 1 IP3R sequence (Furuichi et al.,

1989, Nature, 342:32-38) . After tertiary screening, 26 IP3R cDNA clones were isolated and sequenced using dideoxy chain termination methodology (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual., Cold Spring Harbor, N.Y. : Cold Spring Harbor Laboratory Press) . The sequence disclosed herein was obtained entirely from cDNAs isolated from the λgtll cDNA library, with the exception of the 500 nucleotides between bp 6500 and 7000, that were sequenced from three separate amplified cDNAs.

6.1.3. RNA ISOLATION AND NORTHERN HYBRIDIZATION Total RNA was isolated from Jurkat T cells using the guanidinium isothiocyanate method as described in Marks et al., 1991, J. Cell. Biol., 114:303-312. RNA samples (20 μg) were size fractionated on a 1% formal- dehyde-agarose gel and transferred to nitrocellulose. Hybridization was at 42°C overnight in 50% formamide, 50 mM Na-phosphate, 5X Denhardt's solution and calf thymus DNA. The final washing was with 0.2X SSC, at 55°C for 10 min. The probe used for Northern hybridi¬ zation was the 5' 1.3 Kb of the human T lymphocyte IP3R cDNA labeled with α-³²P-dCTP to a specific activity of -1 X 10⁹ cpm/μg. Autoradiography was performed with a single intensifying screen at -80°C for 48 h.

6.1.4. IMMUNOBLOTTING

A site specific affinity purified polyclonal anti- IP3R antibody, that had previously been shown to be specific for the IP3R, (Moschella et al., 1993, J. Cell. Biol., 120:1137-1146) was used for both immuno- blotting and i munocytochemistry. This antibody was directed against a synthetic peptide, IP3R2652, based on the deduced amino acid sequence of human T cell type 1 IP3R amino acid residues 2652-2663 [ (Cys)-Glu-Gln- Asn-Glu-Leu-Arg-Asn-Leu-Gln-Glu-Lys-Leu (SEQ ID NO:6), with an amino terminal cysteine added for coupling to keyhole lympet hemocyanin prior to immunization in rabbits as described in Moschella, 1993. Jurkat T cell membranes were prepared using a sucrose gradient essentially as in Peterson et al., 1978, J. Supra- molecular Structure, 9_: 289-298. Fifty μg of membrane protein was size fractionated on 6% SDS/polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes using a Semidry apparatus (Bio- Rad) at 15 V for 1 h followed by 25 V for 1 h, both at 40°C as described in Moschella et al., 1993, J. Cell. Biol., 120:1137-1146. Filters were incubated with blocking buffer (80 mM Na₂HP0₄/ 20mM NaH₂PO₄/100 mM NaCl, 0.05% Tween 20/5% dry milk) for 1 h. Affinity purified rabbit anti-IP3R antibody was added at 1/100 dilution. Filters were washed with PBST and incubated for 1 h with goat anti-rabbit IgG-HRP conjugate at a dilution of 1/7500 in blocking buffer. Signals were detected using the ECL detection reagents (Amersham International Inc.) followed by autoradiography.

6.1.5. IMMUNOCYTOCHEMISTRY

Permeabilized or unpermeabilized Jurkat cells were treated with affinity purified anti-IP3R antibody at 1:50 dilution. Cells were washed three times in phos¬ phate buffered saline (PBS) containing 2% fetal bovine serum (FBS) followed by incubation with donkey anti- rabbit IgG conjugated to biotin. Cells were washed and avidin-rhodamine conjugate was added at a 1/1000 dilu¬ tion, followed by washing and fixation in 2% para- formaldehyde. For permeabilization, cells were treated with 100% methanol at room temperature for 5 minutes, washed and stained as above.

6.1.6. FLUORESCENCE ACTIVATED CELL SORTING ANALYSIS Affinity purified rabbit anti-IP3R antibody was added to 0.5X10⁶ Jurkat cells at a 1/50 dilution in PBS containing 2% FBS for 30 minutes on ice followed by incubation with secondary antibody (donkey anti-rabbit IgG conjugated to FITC) for an additional 30 minutes. Cells were either immediately analyzed using a fluor¬ escence activated cell sorter (FACS) analyzer (Coulter) or fixed in 2% paraformaldehyde and then analyzed within two days. Cells were incubated with either PBS or secondary antibody alone as negative controls. Permeabilization of Jurkat cells was with 100% methanol for 5 min followed by antibody staining as described above. Three thousand cells were analyzed for each sample. 6.1.7. TYROSINE PHOSPHORYLATION Jurkat cells (2.5xl0⁸) were stimulated by incu¬ bating with anti-CD3 (1% culture supernatant, kindly provided by Dr. Asit Panja) at 37°C for 3 minutes. Control cells were treated under identical conditions except that phosphate buffered saline was added instead of the anti-CD3 supernatant. Cells were harvested and lysed in 0.5% Brij 96, 20 mM Tris, pH 7.5, 150 mM NaCl, 10 mM NaF, 1 mM Na orthovanadate, 1 μg/ l leupeptin, 1 mM PMSF, 1 μg/ml pepstatin, 1.8 mg/ml iodoacetamide.

Insoluble material was removed by centrifugation for 10 minutes at 4°C. The anti-phosphotyrosine antibody 4G10 was used for immunoprecipitation and for western blot¬ ting as described (Letourneuer et al., 1992, Science, 255:79: June et al., 1990, J Immunol, 144:1591; Strauss et al., 1992, Cell, 70:585).

6.2. RESULTS

6.2.1. PRIMARY STRUCTURE OF THE HUMAN TYPE 1 INOSITOL 1.4.5-TRIPHOSPHATE RECEPTOR Figure 1 shows the deduced amino acid sequence of the human type 1 IP3R. A consensus sequence for eukaryotic translational start sites (CCGCCATG) (Kozak, M. , 1984, Nucl. Acid Res., 12:857-872) was present before the first ATG. The open reading frame encoded 2712 amino acid residues with a predicted molecular weight of 308 kDa. This value is consistent with the size of the IP3R protein based on immunoblot data (Fig. 3) . There is 98% amino acid identity between the human and rat type 1 IP3R sequences. The neuronal specific SII exon (Nakagawa et al., 1991, Proc. Natl. Acad.

Sci.), £8_:6244-6248 is excluded from the human T cell type I IP3R form, and the alternatively spliced SI exon is retained (Fig. 1) . Three consensus sequences for nucleotide-binding sites (G-X-G-X-X-G-(nX)-K, n=17-21; (Wierenga et al., 1983, Nature, 302:842-844) are conserved at amino acid residues 1689-1694, 1737-1742, and 1978-1983 (Fig. 1) . Two putative PKA phos¬ phorylation sites (R/K-R/K-X-S/T; (Huganir et al., 1984, Proc. Natl. Acad. Sci, £1:6968-6972) are conserved at serine residues 1589 and 1717 (Fig. I) .

The preferred model for the transmembrane topography of the type 1 IP3R identifies six putative transmembrane segments, designated MI through M6 (Fig. 1) , that may form the channel pore clustered near the carboxy terminus (Michikawa et al., 1994 J. Biol Chem, 269:9184-9189) .

The type I IP3R is related to the RyR/Ca release channel from the sarcoplasmic reticulum. These two molecules are members of the intracellular calcium release channel family that is distinct from other known ion channel structures. Several regions of significant homology exist between the IP3R and the RyR. Two stretches of 10 amino acids are 90% conserved between the two channels: residues 2001-2010, SLTEYCQGPC (SEQ ID NO:3), with only one mismatch (I for C in the RyR); and residues 1931-1940, ILRFLQLLCE (SEQ ID NO:4), with only one mismatch (F for L in the RyR). In both channels these sequences are located in the putative cytoplasmic domains and could serve as binding sites for molecules that regulate both channels. Sev¬ eral such agents exist including calcium and caffeine although the modulatory effects of each agent differs markedly between the two channels. For example the channels have differential sensitivities to calcium (Bezprozvanny et al., 1991, Nature, 351:751-754) and caffeine activates the RyR but inhibits the IP3R (Bezprozvanny et al., 1994, Mol Biol Cell, 5:97-103). Two putative tyrosine phosphorylation sites are present at residues 482 (EDLvY) and 2617 (DsTEY) of the human type 1 IP3R. Interestingly, the site at residue 482 is not conserved in human type 2 and 3 receptors (Yamamoto-Hino, et al., 1994, Receptors and Channels, 2: 9-22) , whereas the site at amino acid 2617 is con¬ served in the human type 2 but not the type 3 receptor. The putative tyrosine phosphorylation site at amino acid 482 is near the IP3 binding region identified by Mignery and Sudhof (Mignery et al., 1990, EMBO J. , 2:3893-3898). The putative tyrosine phosphorylation site at amino acid 2617 is near the predicted channel pore region.

6.2.2. IDENTIFICATION OF THE IP3R mRNA AND PROTEIN

IN T LYMPHOCYTES

A single -10 kb mRNA species was identified by Northern hybridization of Jurkat total RNA using a human T lymphocyte cDNA probe (Fig 2) . IP3R mRNA levels determined by Northern hybridization analyses were constant during T cell activation by CD3 (Fig. 2) . Immunoblot analysis was performed using crude homo- genates from rat brain and from human T cell (Jurkat) membranes to determine the specificity of the anti-IP3R antibody. The anti-IP3R antibody recognized a single high molecular weight band (-300 kDa) in these crude homogenates (Fig. 3) . The IP3R protein in brain and T cells had similar mobilities. The sequence of the syn¬ thetic peptide used as the antigen for the anti-IP3R antibody production is 90% identical to the type 2 and 70% identical to the type 3 IP3R. Therefore, we cannot exclude the possibility that the IP3R protein that we detected represents a mixture of types 1, 2 and 3 IP3R.

6.2.3. LOCALIZATION OF THE TYPE 1 INOSITOL 1.4.5-TRISPHOSPHATE RECEPTOR IN HUMAN

LYMPHOCYTES

We used the anti-IP3R antibody to determine the cellular localization of the IP3R in human T lympho¬ cytes (Jurkats) using two approaches. First, we anal- yzed immunostained cells using a FACS. Cells were stained with either preimmune serum or affinity pur¬ ified anti-IP3R antibody, fixed, and then fluorescence intensity was assessed by FACS analysis. Fluorescence signal was observed only on permeabilized cells (Fig. 4) , nonpermeabilized cells gave no signal. These results indicated that the IP3R epitope recognized by our anti-IP3R antibody was cytoplasmic.

Using a second approach, cells were fixed and stained with the same anti-IP3R antibody and analyzed using confocal microscopy (Fig. 5) . Immunofluorescence signals were observed only in permeabilized cells (Fig. 5, panels a through f) but not in nonpermeabilized cells (not shown) indicating that the epitope recog- nized by this antibody was intracellular. Clumps of signal appeared to be associated with the plasma membrane. However, these signals were observed only in permeabilized cells, therefore we concluded that they must be recognizing an epitope inside the cell, rather than on the outside of the plasma membrane In some confocal planes IP3R signal was observed in the peri- nuclear region (panels c and d) .

6.2.4. TYROSINE PHOSPHORYLATION Tyrosine phosphorylation of the IP3R was examined 3 minutes after T cell activation by CD3. Jurkat cell lysates were immunoprecipitated using an anti-phos- photyrosine monoclonal antibody. These immuno- precipitates were size fractionated on SDS-poly¬ acrylamide gels and immunoblotted using the anti- phosphotyrosine antibody. Several proteins were observed to be tyrosine phosphorylated in activated T cells but not in non-activated cells (Fig. 6) . Another group of proteins were tyrosine phosphorylated in both non-activated and in activated T cells but the level of phosphorylation increased following T cell activation (Fig. 6) . One of the high molecular weight phos- photyrosine proteins co-migrated with the IP3R as determined by subsequent immunoblotting of the same filter with an anti-IP3R antibody (Fig. 6) . Phosphory- lated IP3R increased in activated T cells (Fig. 6) .

6.3. DISCUSSION The human type 1 IP3R cDNA from T lymphocytes has been cloned, and its cellular localization and phos¬ phorylation at tyrosine have been demonstrated. The human type 1 IP3R is structurally similar to the type 1 receptors from rodents. Based on sequence analysis, the type I IP3R expressed in human T lymphocytes cor¬ responds to the non-neuronal form of the type I IP3R. IP3R heterogeneity is created by alternative splicing, and distinct areas of the brain in rats and mice express different IP3Rs (Nakagawa et al., 1991, Proc Natl Acad Sci, £8:6244-6248). A 15 amino acid sequence near the N-terminus and a 40 amino acid sequence loca¬ ted between two putative cytoplasmic phosphorylation sites determine the brain and nonbrain forms of the type 1 IP3R (expressed predominantly in brain and aortic smooth muscle) (Nakagawa et al., 1991, Proc. Natl. Acad. Sci. U.S..A, £8:6244-6248; Miyawaki et al., 1991, Proc. Natl. Acad. Sci. U.S.A., 88:4911-4915) . The human T cell type 1 IP3R form that we have sequenced includes the alternatively spliced SI exon (amino acid residues 318-332), but not the larger 40 amino acid splice, SII, at residue 1698. Inter¬ estingly, the SI exon is expressed at highest relative levels in tissues that contain T cells and/or hemato- poietic cells, thymus and spleen; whereas the SII exon is expressed almost exclusively in cerebellum (Nakagawa et al., 1991, Proc. Natl. Acad. Sci. U.S.A., ££:6244- 6248) . The type I IP3R in human T lymphocytes may be the intracellular calcium release channel required for T cell activation, and as such, may be constitutively expressed. Indeed, regulation of IP3R mRNA during itogenic activation of T lymphocytes was not observed (Fig. 2) .

In human T lymphocytes the type 1 IP3R is expres¬ sed predominantly in the periphery of the cytoplasm near the plasma membrane, and possibly in the peri- nuclear membrane (Fig. 5) . The site-specific antibody used herein for both FACS analysis and immuno- localization allows us to assign the location of the carboxy terminus of the IP3R to an intracellular site in T lymphocytes. No signal was seen using FACS or immunocytochemistry in non-permeabilized cells, whereas permeabilized cells reproducibly gave a strong signal (Figs 4 and 5) . Therefore, the epitope recognized by our anti-IP3R antibody is apparently cytoplasmic. The intense staining at the inner surface of the plasma membrane suggests that the T lymphocyte IP3R may also be localized to plasmalemma caveolae, as has been reported in endothelial cells (Fujimoto et al., 1992, J. Cell Biol, 119:1507-1513) . and/or to endoplasmic reticulum near the plasmalemma. Khan et al. reported that an IP3R was found in the plasma membrane of human T lymphocytes using a poly- clonal antibody raised against the entire protein (Khan et al., 1992, Science, 257:815-818) . The present disclosure adds information regarding the topography of the IP3R in the membrane because it places the carboxy terminus in the cytoplasm. Bourguignon et al. showed that a monoclonal anti-IP3R antibody stained per¬ meabilized but not non-permeabilized mouse T lymphoma cells (Bourguignon et al., 1993, J. Biol Chem, 268:7290-7297) . However, the location of the epitope for the monoclonal antibody was not identified. The finding that our anti-IP3R antibody does not recognize non-permeabilized cells either by FACS or by immuno- fluorescence staining excludes the possibility that the receptor could be on the plasma membrane facing out- ward. Moreover, if the IP3R were in the plasma mem¬ brane facing outward the IP3 binding site would be extracellular, a localization that is inconsistent with the fact that IP3 is an intracellular second messenger. Thus, two possible transmembrane configurations of the channel are consistent with existing data: 1) the IP3R is on the ER; 2) the IP3R is on the plasma membrane with the bulk of the protein including the IP3 binding site in the cytoplasm. The latter configuration would make the IP3R a calcium influx channel. We believe that this configuration is unlikely because the IP3R is a relatively nonspecific cation channel (Lindsay, et al., 1991, J. Physiol., 429:463-480). On the ER (which under physiological conditions has no gradient for sodium or potassium across its membrane (Somiyo, 1977, J Cell Biol, 24:828-857)), the IP3R functions as a calcium release channel due to the large electrochemical gradient for calcium. If a distinct IP3R does exist on the plasma membrane, as has been proposed (Khan et al., 1992, Science, 2_.52:815-818; FujimotO et al., 1992, J. Cell Biol, 119:1507-1513: Kijima et al., 1993, J Biol Chem, 2££:3499-3506) , it could be another form of IP3R that is more selective for divalent cations than the IP3R on the ER.

Our conclusion regarding the subcellular localiza- tion of the human type 1 IP3R also agrees with func¬ tional data from Mikoshiba and colleagues who demon¬ strated that an antibody which recognizes nearly the same carboxy terminal epitope as our antibody was capable of inhibiting IP3-induced intracellular calcium release in Xenopus oocytes (Miyazaki et al., 1992,

Science, 257:251-255) . Again, these functional results would place the IP3R on the ER. Of interest, however, is the fact that the epitope for the monoclonal anti¬ body used by Mikoshiba and colleagues to block calcium release (Miyazaki et al., 1992, Science, 257:251-255) also overlaps the putative tyrosine phosphorylation site at amino acid residue 2617. Therefore, the possibility exists that the interference with calcium release was due to inhibition of tyrosine phos¬ phorylation that occurs during T cell activation (Fig. 6) .

In T cells, activation of the T cell receptor (TCR)-CD3 complex results in recruitment of tyrosine kinases that are members of the src family including fyn and lck . It has been proposed that the src family of tyrosine kinases activate a phospholipase C isoform (PLC_γl) which in turn stimulates phosphoinositide hydrolysis leading to the generation of IP3 and sub¬ sequent activation of the IP3R. Our data showing that the IP3R is tyrosine phosphorylated during T cell activation are consistent with a role of type 1 T cell IP3R as a substrate for tyrosine kinases during T cell activation. Of interest, the foregoing data is con¬ sistent with that of Khan et al., who showed that the IP3R co-caps with the T cell receptor during T cell activation (Khan et al., 1992, Science, 212:815-818). Co-capping would place the IP3R near the TCR during T cell activation. This association with the TCR may facilitate phosphorylation of the IP3R by tyrosine kinases that are activated during T cell activation. One putative tyrosine phosphorylation site was located near the IP3 binding site. Tyrosine phos¬ phorylation of this site may modulate the affinity of the IP3R for IP3. A decrease in the affinity of the IP3R for the negatively charged IP3 may be induced by the presence of an added negative charge of a phosphate group near the IP3 binding site. Alternatively, tyrosine phosphorylation may increase access to the binding site via a conformational change. A second site is near the putative channel pore region. Phos¬ phorylation at this site may modify channel gating perhaps by inducing a conformational change in the

IP3R, or by increasing the affinity of the channel for calcium due to the added negative charge of a phos¬ phate group located near the channel pore. Thus, IP3 and tyrosine phosphorylation could be co-activators of the IP3R. Alternatively, negative regulation of the IP3R by tyrosine phosphorylation may play a role in shutting off intracellular calcium release via the IP3R after CD3 activation of the T cell receptor. The release of intracellular calcium during T cell activation occurs rapidly during the first few minutes after T cell activation. Activation is mediated by IP3, and inactivation may be due to the subsequent phosphorylation of the IP3R. Both the IP3 generation and the tyrosine phosphorylation may be triggered by activation of the TCR. Thus, T cell activation via the TCR could signal both the activation and the inactiva¬ tion of the IP3R/Ca release channel.

Localization of the IP3R to the perinuclear region (Fig. 5) is of potential significance in T cells. Early events during T cell activation are calcium dependent (Gardner, 1989, Cell, 52:15-20). For exam¬ ple, translocation of NF-AT to the nucleus where it triggers IL2 transcription is dependent on the activity of the Ca-calmodulin dependent protein phosphatase cal- cineurin which is also a target for the immunosup- pressant drugs FK506 and cyclosporin A (Liu et al., 1991, Cell, 6_:807-815) . Phosphoinositide signaling has been localized to the nucleus (Divecha et al., 1991, EMBO J, 10:3207-3214; Martelli et al., 1992, Nature, 358:242-245) . and the IP3R has been reported to be present in the perinuclear region of Xenopus laevis oocytes (Kume et al., 1993, Cell, 21:555-570). Local¬ ization of the IP3R to the perinuclear region in Jurkats suggests that the IP3R could be involved in regulating calcium flux to the nucleus of human T cells. Moreover, cyclic changes in IP3 levels have been linked to cell-cycle changes in calcium transients and inositol polyphosphate levels have recently been shown to vary in a cell-cycle dependent manner (Ciapa et al., 1994, Nature, 368:875-878: Balla et al., 1994, Mol. Bio. Cell., 5:17-27) suggesting a possible role for an IP3R mediated signaling pathway in the regulation of cell cycle progression.

Various publications are cited herein that are hereby incorporated by reference in their entireties.

SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT: Marks, Andrew R.

(ii) TITLE OF THE INVENTION: HUMAN T CELL INOSITOL ,4,5,-TRISPHOSPHATE RECEPTOR

(iii) NUMBER OF SEQUENCES: 8

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Brumbaugh, Graves, Donohue & Raymond

(B) STREET: 30 Rockefeller Plaza

(C) CITY: New York

(D) STATE: NY

(E) COUNTRY: USA

(F) ZIP: 10112-0228

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: DOS

(D) SOFTWARE: FastSEQ Version 1.5

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: 08/386,039

(B) FILING DATE: 09-FEB-1995

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(viii) ATTORNE /AGENT INFORMATION:

(A) NAME: Kole, Lisa B

(B) REGISTRATION NUMBER: 35,225

(C) REFERENCE/DOCKET NUMBER: A30042 - 165/30555

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 212-408-2628

(B) TELEFAX: 212-765-2519

(C) TELEX: (2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2713 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE: (ix) FEATURE:

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

Met Ser Asp Lys Met Ser Ser Phe Leu His lie Gly Asp lie Cys Ser

1 5 10 15

Leu Tyr Ala Glu Gly Ser Thr Asn Gly Phe lie Ser Thr Leu Gly Leu

20 25 30

Val Asp Asp Arg Cys Val Val Gin Pro Glu Thr Gly Asp Leu Asn Asn

35 40 45

Pro Pro Lys Lys Phe Arg Asp Cys Leu Phe Lys Leu Cys Pro Met Asn

50 55 60

Arg Tyr Ser Ala Gin Lys Gin Phe Trp Lys Ala Ala Lys Pro Gly Ala 65 70 75 80

Asn Ser Thr Thr Asp Ala Val Leu Leu Asn Lys Leu His His Ala Ala

85 90 95

Asp Leu Glu Lys Lys Gin Asn Glu Thr Glu Asn Arg Lye Leu Leu Gly

100 105 110

Thr Val lie Gin Tyr Gly Asn Val lie Gin Leu Leu His Leu Lys Ser

115 120 125

Asn Lys Tyr Leu Thr Val Asn Lys Arg Leu Pro Ala Leu Leu Glu Lys

130 135 140

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

Phe Tyr lie Gin Pro Phe Tyr Lys Leu Arg Ser lie Gly Asp Ser Val

165 170 175

Val lie Gly Asp Lys Val Val Leu Asn Pro Val Asn Ala Gly Gin Pro

180 185 190

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

Val Asn Ser Val Asn Cys Asn Thr Ser Trp Lys lie Val Leu Phe Met

210 215 220

Lys Trp Ser Asp Asn Lys Asp Asp lie Leu Lys Gly Gly Asp Val Val 225 230 235 240

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

245 250 255

Arg Lys Lys Gin His Val Phe Leu Arg Thr Thr Gly Arg Gin Ser Ala

260 265 270

Thr Ser Ala Thr Ser Ser Lys Ala Leu Trp Glu Val Glu Val Val Gin

275 280 285

His Asp Pro Cys Arg Gly Gly Ala Gly Tyr Trp Asn Ser Leu Phe Arg

290 295 300

Phe Lys His Leu Ala Thr Gly His Tyr Leu Ala Ala Glu Val Asp Pro 305 310 315 320

Asp Phe Glu Glu Glu Cys Leu Glu Phe Gin Pro Ser Val Asp Pro Asp

325 330 335

Gin Asp Ala Ser Arg Ser Arg Leu Arg Asn Ala Gin Glu Lys Met Val

340 345 350

Tyr Ser Leu Val Ser Val Pro Glu Gly Asn Asp lie Ser Ser lie Phe

355 360 365

Glu Leu Asp Pro Thr Thr Leu Arg Gly Gly Asp Ser Leu Val Pro Arg

370 375 380

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

Ser Thr Asn lie Pro He Asp Lys Glu Glu Glu Lys Pro Val Met Leu

405 410 415

Lys He Gly Thr Ser Pro Val Lys Glu Asp Lys Glu Ala Phe Gly He

420 425 430

Val Pro Val Ser Pro Ala Glu Val Arg Asp Leu Asp Phe Ala Asn Asp

435 440 445

Ala Ser Lys Val Leu Gly Ser He Ala Gly Lys Leu Glu Lys Gly Thr

450 455 460

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

Val Tyr Phe Val Thr Gly Gly Thr Asn Ser Gly Gin Asp Val Leu Glu

485 490 495

Val Val Phe Ser Lys Pro Asn Arg Glu Arg Gin Lys Leu Met Arg Glu

500 505 510

Gin Asn He Leu Lys Gin He Phe Lys Leu Leu Gin Ala Pro Phe Thr

515 520 525

Asp Cys Gly Asp Gly Pro Met Leu Arg Leu Glu Glu Leu Gly Asp Gin 530 535 540 Arg His Ala Pro Phe Arg His He Cys Arg Leu Cys Tyr Arg Val Leu 545 550 555 560

Arg His Ser Gin Gin Asp Tyr Arg Lys Asn Gin Glu Tyr He Ala Lys

565 570 575

Gin Phe Gly Phe Met Gin Lys Gin He Gly Tyr Asp Val Leu Ala Glu

580 585 590

Asp Thr He Thr Ala Leu Leu His Asn Asn Arg Lys Leu Leu Glu Lys

595 600 605

His He Thr Ala Ala Glu He Asp Thr Phe Val Ser Leu Val Arg Lys

610 615 620

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

Met Asn Lye Ser He Pro Val Thr Gin Glu Leu He Cys Lys Ala Val

645 650 655

Leu Asn Pro Thr Asn Ala Asp He Leu He Glu Thr Lys Leu Val Leu

660 665 670

Ser Arg Phe Glu Phe Glu Gly Val Ser Ser Thr Gly Glu Asn Ala Leu

675 680 685

Glu Ala Gly Glu Asp Glu Glu Glu Val Trp Leu Phe Trp Arg Asp Ser

690 695 700

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

Lys Glu Gly Gin Lys Glu Asp Arg Asp Val Leu Ser Tyr Tyr Arg Tyr

725 730 735

Gin Leu Asn Leu Phe Ala Arg Met Cys Leu Asp Arg Gin Tyr Leu Ala

740 745 750

He Asn Glu He Ser Gly Gin Leu Asp Val Asp Leu He Leu Arg Cys

755 760 765

Met Ser Asp Glu Asn Leu Pro Tyr Asp Leu Arg Ala Ser Phe Cys Arg

770 775 780

Leu Met Leu His Met His Val Asp Arg Asp Pro Gin Glu Gin Val Thr 785 790 795 800

Pro Val Lys Tyr Ala Arg Leu Trp Ser Glu He Pro Ser Glu He Ala

805 810 815

He Asp Asp Tyr Asp Ser Ser Gly Ala Ser Lys Asp Glu He Lys Glu

820 825 830

Arg Phe Ala Gin Thr Met Glu Phe Val Glu Glu Tyr Leu Arg Asp Val

835 840 845

Val Cys Gin Arg Phe Pro Phe Ser Asp Lys Glu Lys Asn Lys Leu Thr

850 855 860

Phe Glu Val Val Asn Leu Ala Arg Asn Leu He Tyr Phe Gly Phe Tyr 865 870 875 880

Asn Phe Ser Asp Leu Leu Arg Leu Thr Lys He Leu Leu Ala He Leu 885 890 895

Asp Cys Val His Val Thr Thr He Phe Pro He Ser Lys Met Ala Lys

900 905 910

Gly Glu Glu Asn Lys Gly Ser Asn Val Met Arg Ser He His Gly Val

915 920 925

Gly Glu Leu Met Thr Gin Val Val Leu Arg Gly Gly Gly Phe Leu Pro

930 935 940

Met Thr Pro Met Ala Ala Ala Pro Glu Gly Asn Val Lys Gin Ala Glu 945 950 955 960

Pro Glu Lys Glu Asp He Met Val Met Asp Thr Lys Leu Lys He He

965 970 975

Glu He Leu Gin Phe He Leu Asn Val Arg Leu Asp Tyr Arg He Ser

980 985 990

Cys Leu Leu Cys He Phe Lys Arg Glu Phe Trp Met Lys Ala He Pro

995 1000 1005

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

1010 1015 1020

Asn Val Pro Gly Ala Leu Asp Phe Glu His He Glu Glu Gin Ala Glu 025 1030 1035 1040

Gly He Phe Gly Gly Ser Glu Glu Asn Thr Pro Leu Asp Leu Asp Asp

1045 1050 1055

His Gly Gly Arg Thr Phe Leu Arg Val Leu Leu His Leu Thr Met His

1060 1065 1070

Asp Tyr Pro Pro Leu Val Ser Gly Ala Leu Gin Leu Leu Phe Arg His

1075 1080 1085

Phe Ser Gin Arg Gin Glu Val Leu Gin Ala Phe Lys Gin Val Gin Leu

1090 1095 1100

Leu Val Thr Ser Gin Asp Val Asp Asn Tyr Lys Gin He Lys Gin Asp 1105 1110 1115 1120

Leu Asp Gin Leu Arg Ser He Val Glu Lys Ser Glu Leu Trp Val Tyr

1125 1130 1135

Lys Gly Gin Gly Pro Asp Glu Thr Met Asp Gly Ala Ser Gly Glu Asn

1140 1145 1150

Glu His Lys Lye Thr Glu Glu Gly Asn Asn Lys Pro Gin Lys His Glu

1155 1160 1165

Ser Thr Ser Ser Tyr Asn Tyr Arg Val Val Lys Glu He Leu He Arg

1170 1175 1180

Leu Ser Lys Leu Cys Val Gin Glu Ser Ala Ser Val Arg Lys Ser Arg 1185 1190 1195 1200

Lys Gin Gin Gin Arg Leu Leu Arg Asn Met Gly Ala His Ala Val Val

1205 1210 1215

Leu Glu Leu Leu Gin He Pro Tyr Glu Lys Ala Glu Asp Thr Lys Met 1220 1225 1230 Gln Glu He Met Arg Leu Ala Hie Glu Phe Leu Gin Asn Phe Cys Ala

1235 1240 1245

Gly Asn Gin Gin Asn Gin Ala Leu Leu His Lys His He Asn Leu Phe

1250 1255 1260

Leu Asn Pro Gly He Leu Glu Ala Val Thr Met Gin His He Phe Met 1265 1270 1275 1280

Asn Asn Phe Gin Leu Cys Ser Glu He Asn Glu Arg Val Val Gin His

1285 1290 1295

Phe Val His Cys He Glu Thr His Gly Arg Asn Val Gin Tyr He Lys

1300 1305 1310

Phe Leu Gin Thr He Val Lys Ala Glu Gly Lys Phe He Lys Lys Cys

1315 1320 1325

Gin Asp Met Val Met Ala Glu Leu Val Asn Ser Gly Glu Asp Val Leu

1330 1335 1340

Val Phe Tyr Asn Asp Arg Ala Ser Phe Gin Thr Leu He Gin Met Met 1345 1350 1355 1360

Arg Ser Glu Arg Asp Arg Met Asp Glu Asn Ser Pro Leu Met Tyr His

1365 1370 1375

He His Leu Val Glu Leu Leu Ala Val Cya Thr Glu Gly Lys Asn Val

1380 1385 1390

Tyr Thr Glu He Lys Cys Asn Ser Leu Leu Pro Leu Asp Asp He Val

1395 1400 1405

Arg Val Val Thr His Glu Asp Cys He Pro Glu Val Lys He Ala Tyr

1410 1415 1420

He Asn Phe Leu Asn His Cys Tyr Val Asp Thr Glu Val Glu Met Lys 1425 1430 1435 1440

Glu He Tyr Thr Ser Asn His Met Trp Lys Leu Val Glu Asn Phe Leu

1445 1450 1455

Val Asp He Cys Arg Ala Cys Asn Asn Thr Ser Asp Arg Lys His Ala

1460 1465 1470

Asp Ser He Leu Glu Lys Tyr Val Thr Glu He Val Met Ser He Val

1475 1480 1485

Thr Thr Phe Phe Ser Ser Pro Phe Ser Asp Gin Ser Thr Thr Leu Gin

1490 1495 1500

Thr Arg Gin Pro Val Phe Val Gin Leu Leu Gin Gly Val Phe Arg Val 1505 1510 1515 1520

Tyr His Cys Asn Trp Leu Met Pro Ser Gin Lys Ala Ser Val Glu Ser

1525 1530 1535

Cys He Arg Val Leu Ser Asp Val Ala Lys Ser Arg Ala He Ala He

1540 1545 1550

Pro Val Asp Leu Asp Ser Gin Val Asn Asn Leu Phe Leu Lys Ser His

1555 1560 1565

Ser He Val Gin Lys Thr Ala Met Asn Trp Arg Leu Ser Ala Arg Asn 1570 1575 1580

Ala Ala Arg Arg Aβp Ser Val Leu Ala Ala Ser Arg Aβp Tyr Arg Aβn 1585 1590 1595 1600

He He Glu Arg Leu Gin Aβp He Val Ser Ala Leu Glu Aβp Arg Leu

1605 1610 1615

Arg Pro Leu Val Gin Ala Glu Leu Ser Val Leu Val Asp Val Leu His

1620 1625 1630

Arg Pro Glu Leu Leu Phe Pro Glu Asn Thr Asp Ala Arg Arg Lye Cys

1635 1640 1645

Glu Ser Gly Gly Phe He Cys Lye Leu He Lye His Thr Lys Gin Leu

1650 1655 1660

Leu Glu Glu Asn Glu Glu Lys Leu Cys He Lye Val Leu Gin Thr Leu 1665 1670 1675 1680

Arg Glu Met Met Thr Lys Asp Arg Gly Tyr Gly Glu Lys Gly Glu Ala

1685 1690 1695

Leu Arg Gin Val Leu Val Asn Arg Tyr Tyr Gly Asn Val Arg Pro Ser

1700 1705 1710

Gly Arg Arg Glu Ser Leu Thr Ser Phe Gly Aβn Gly Pro Leu Ser Ala

1715 1720 1725

Gly Gly Pro Gly Lys Pro Gly Gly Gly Gly Gly Gly Ser Gly Ser Ser

1730 1735 1740

Ser Met Ser Arg Gly Glu Met Ser Leu Ala Glu Val Gin Cys His Leu 1745 1750 1755 1760

Aβp Lye Glu Gly Ala Ser Aβn Leu Val He Asp Leu He Met Asn Val

1765 1770 1775

Ser Ser Asp Arg Val Phe His Glu Ser He Leu Leu Ala He Ala Leu

1780 1785 1790

Leu Glu Gly Gly Asn Thr Thr He Gin His Ser Phe Phe Cys Arg Leu

1795 1800 1805

Thr Glu Asp Lye Lys Ser Glu Lys Phe Phe Lys Val Phe Tyr Asp Arg

1810 1815 1820

Met Lye Val Ala Gin Gin Glu He Lys Ala Thr Val Thr Val Asn Thr 1825 1830 1835 1840

Ser Asp Leu Gly Asn Lys Lys Lye Asp Asp Glu Val Asp Arg Asp Ala

1845 1850 1855

Pro Ser Arg Lys Lys Ala Lys Glu Pro Thr Thr Gin He Thr Glu Glu

1860 1865 1870

Val Arg Asp Gin Leu Leu Glu Ala Ser Ala Ala Thr Arg Lys Ala Phe

1875 1880 1885

Thr Thr Phe Arg Arg Glu Ala Asp Pro Asp Asp His Tyr Gin Pro Gly

1890 1895 1900

Glu Gly Thr Gin Ala Thr Ala Asp Lys Ala Lys Asp Asp Leu Glu Met 1905 1910 1915 1920 Ser Ala Val He Thr He Met Gin Pro He Leu Arg Phe Leu Gin Leu

1925 1930 1935

Leu Cyβ Glu Aβn Hie Aβn Arg Aβp Leu Gin Aβn Phe Leu Arg Cyβ Gin

1940 1945 1950

Aβn Aβn Lys Thr Asn Tyr Aβn Leu Val Cyβ Glu Thr Leu Gin Phe Leu

1955 1960 1965

Asp Cys He Cys Gly Ser Thr Thr Gly Gly Leu Gly Leu Leu Gly Leu

1970 1975 1980

Tyr He Aβn Glu Lys Asn Val Ala Leu He Asn Gin Thr Leu Glu Ser 1985 1990 1995 2000

Leu Thr Glu Tyr Cys Gin Gly Pro Cys Hie Glu Aβn Gin Asn Cys He

2005 2010 2015

Ala Thr His Glu Ser Asn Gly He Asp He He Thr Ala Leu He Leu

2020 2025 2030

Asn Asp He Asn Pro Leu Gly Lys Lys Arg Met Asp Leu Val Leu Glu

2035 2040 2045

Leu Lys Asn Aβn Ala Ser Lye Leu Leu Leu Ala He Met Glu Ser Arg

2050 2055 2060

Hie Asp Ser Glu Asn Ala Glu Arg He Leu Tyr Asn Met Arg Pro Lys 2065 2070 2075 2080

Glu Leu Val Glu Val He Lys Lys Ala Tyr Met Gin Gly Glu Val Glu

2085 2090 2095

Phe Glu Asp Gly Glu Asn Gly Glu Aβp Gly Ala Ala Ser Pro Arg Asn

2100 2105 2110

Val Gly His Asn He Tyr He Leu Ala His Gin Leu Ala Arg His Asn

2115 2120 2125

Lys Glu Leu Gin Ser Met Leu Lys Pro Gly Gly Gin Val Asp Gly Asp

2130 2135 2140

Glu Ala Leu Glu Phe Tyr Ala Lys His Thr Ala Gin He Glu He Val 2145 2150 2155 2160

Arg Leu Asp Arg Thr Met Glu Gin He Val Phe Pro Val Pro Ser He

2165 2170 2175

Cys Glu Phe Leu Thr Lys Glu Ser Lys Leu Arg He Tyr Tyr Thr Thr

2180 2185 2190

Glu Arg Asp Glu Gin Gly Ser Lys He Asn Asp Phe Phe Leu Arg Ser

2195 2200 2205

Glu Asp Leu Phe Asn Glu Met Asn Trp Gin Lys Lys Leu Arg Ala Gin

2210 2215 2220

Pro Val Leu Tyr Trp Cyβ Ala Arg Aβn Met Ser Phe Trp Ser Ser He 2225 2230 2235 2240

Ser Phe Asn Leu Ala Val Leu Met Asn Leu Leu Val Ala Phe Leu Tyr

2245 2250 2255

Pro Leu Lys Gly Val Arg Gly Gly Thr Leu Glu Pro His Trp Ser Gly 2260 2265 2270

Leu Leu Trp Thr Gly Met Leu He Ser Leu Gly He Val He Gly Leu

2275 2280 2285

Pro Asn Pro Hie Gly He Arg Ala Leu He Gly Ser Thr He Leu Arg

2290 2295 2300

Leu He Phe Ser Val Gly Ser Gin Pro Ala Leu Phe Leu Leu Gly Ala 2305 2310 2315 2320

Phe Aβn Val Cyβ λβn Lys He He Phe Leu Met Ser Phe Val Gly Asn

2325 2330 2335

Cys Gly Thr Phe Thr Arg Gly Tyr Arg Ala Met Val Leu Val Leu Asp

2340 2345 2350

Val Glu Phe Leu Tyr His Leu Leu Tyr Leu Val He Cys Ala Met Gly

2355 2360 2365

Leu Phe Val His Val Phe Phe Tyr Ser Leu Leu Leu Leu Asp Leu Val

2370 2375 2380

Tyr Arg Glu Glu Ser Leu Leu Asn Val He Lys Ser Val Thr Arg Asn 2385 2390 2395 2400

Gly Arg Ser He He Leu Thr Ala Val Leu Ala Leu He Leu Val Tyr

2405 2410 2415

Leu Phe Ser He Val Gly Tyr Leu Phe Phe Lys Aβp Aβp Phe He Leu

2420 2425 2430

Glu Val Aβp Arg Leu Pro Aβn Glu Thr Ala Val Pro Glu Thr Gly Glu

2435 2440 2445

Ser Leu Ala Ser Glu Phe Leu Phe Ser Aβp Val Cyβ Arg Val Glu Ser

2450 2455 2460

Gly Glu Asn Cys Ser Ser Pro Ala Pro Arg Glu Glu Leu Val Pro Ala 2465 2470 2475 2480

Glu Glu Thr Glu Gin Asp Lye Glu His Thr Cys Glu Thr Leu Leu Met

2485 2490 2495

Cys He Val Thr Val Leu Ser His Gly Leu Arg Ser Gly Gly Gly Val

2500 2505 2510

Gly Aβp Val Leu Arg Lye Pro Ser Lye Glu Glu Pro Leu Phe Ala Ala

2515 2520 2525

Arg Val He Tyr Asp Leu Leu Phe Phe Phe Met Val He He He Val

2530 2535 2540

Leu Asn Leu He Phe Gly Val He He Asp Thr Phe Ala Asp Leu Arg 2545 2550 2555 2560

Ser Glu Lys Gin Lys Lys Glu Glu He Leu Lys Thr Thr Cys Phe He

2565 2570 2575

Cys Gly Leu Glu Arg Asp Lys Phe Asp Asn Lys Thr Val Thr Phe Glu

2580 2585 2590

Glu His He Lys Glu Glu His Asn Met Trp His Tyr Leu Cys Phe He 2595 2600 2605 Val Leu Val Lye Val Lye Aβp Ser Thr Glu Tyr Thr Gly Pro Glu Ser

2610 2615 2620

Tyr Val Ala Glu Met He Lye Glu Arg Aβn Leu Aβp Trp Phe Pro Arg 2625 2630 2635 2640

Met Arg Ala Met Ser Leu Val Ser Ser Aβp Ser Glu Gly Glu Gin Asn

2645 2650 2655

Glu Leu Arg Asn Leu Gin Glu Lys Leu Glu Ser Thr Met Lye Leu Val

2660 2665 2670

Thr Aβn Leu Ser Gly Gin Leu Ser Glu Leu Lys Asp Gin Met Thr Glu

2675 2680 2685

Gin Arg Lys Gin Lys Gin Arg Met Gly Leu Leu Gly His Pro Pro His

2690 2695 2700

Met Asn Val Aβn Pro Gin Gin Pro Ala 2705 2710

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTISENSE: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(ix) FEATURE: neural exon

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

Gin He Ser He Asp Glu Leu Glu Asn Ala Glu Leu Pro Gin Pro Pro

1 5 10 15

Glu Ala Glu Asn Ser Thr Glu Glu Leu Glu Pro Ser Pro Pro Leu Arg

20 25 30

Gin Leu Glu Asp His Lys Arg 35

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (ix) FEATURE:

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

Ser Leu Thr Glu Tyr Cyβ Gin Gly Pro Cyβ 1 5 10

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (ix) FEATURE:

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

He Leu Arg Phe Leu Gin Leu Leu Cys Glu 1 5 10

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8791 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE: (ix) FEATURE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

CGGGAGAGAA AGCGCACGCC GAGAGGAGGT GTGGGTGTTC CGCTTCCATC CTAACGGAAC 60

GAGCTCCCTC TTCGCGGACA TGGGATTACC CAGCGGCTGC TAACCCCTCT CCTCGCCCTG 120

CTCCCCCAAA CCGGCGTGGC TCCCCGGGCA CCAAGGAGCT GACTACAGAG GAGCAGGATT 180

TGCACCCCTC GCTGGGCTTG CTTTGGCAAC AGAGTGCCTG ACCCAGGTCA GGATTTTCAA 2 0

GAAAGACATG TCTGACAAAA TGTCTAGCTT CCTACATATT GGAGACATTT GTTCTCTGTA 300

CGCGGAGGGA TCGACAAATG GATTTATTAG CACCTTGGGC CTGGTTGATG ATCGTTGTGT 360

TGTACAGCCA GAAACCGGGG ACCTTAACAA TCCACCTAAG AAATTCAGAG ACTGCCTCTT 420

TAAGCTATGT CCCATGAACC GCTACTCTGC CCAAAAGCAG TTCTGGAAAG CCGCTAAGCC 480

TGGGGCCAAC AGCACCACAG ACGCAGTGCT ACTCAACAAA CTGCACCACG CTGCAGACTT 540

GGAAAAGAAG CAGAATGAGA CAGAAAACAG GAAATTGCTG GGGACCGTAA TCCAGTATGG 600

CAATGTGATC CAGCTCCTGC ATTTGAAAAG TAATAAATAC CTAACAGTGA ATAAGAGGCT 660

TCCTGCTCTG TTGGAGAAGA ATGCCATGAG AGTCACATTG GACGAGGCTG GAAATGAAGG 720

GTCCTGGTTT TATATTCAGC CATTCTACAA GCTGCGATCC ATTGGAGACA GCGTGGTCAT 780

AGGTGACAAG GTGGTTCTGA ACCCCGTCAA TGCTGGTCAG CCCCTACATG CTAGCAGCCA 840

TCAACTGGTA GATAACCCAG GCTGCAATGA GGTCAATTCC GTCAACTGCA ATACAAGCTG 900

GAAAATAGTC CTTTTCATGA AATGGAGTGA TAACAAAGAC GACATATTAA AGGGGGGTGA 960

CGTGGTGAGG CTGTTTCATG CTGAGCAGGA GAAGTTTCTC ACCTGTGACG AACACAGGAA 1020

GAAGCAGCAC GTCTTCCTGA GAACCACGGG CCGGCAGTCG GCCACATCTG CCACCAGTTC 1080

AAAAGCCCTG TGGGAGGTGG AGGTGGTCCA GCATGACCCA TGTCGGGGCG GAGCAGGGTA 1140

TTGGAACAGC CTTTTCCGTT TCAAGCATCT GGCCACGGGG CATTACTTGG CAGCAGAGGT 1200

AGACCCTGAC TTTGAGGAAG AATGCCTGGA GTTTCAGCCC TCAGTGGACC CTGATCAGGA 1260

CGCCTCTCGA AGTAGGTTGC GGAATGCCCA AGAAAAGATG GTATACTCCC TGGTCTCTGT 1320

GCCTGAAGGC AATGACATCT CCTCCATTTT CGAGCTAGAT CCCACCACTC TGCGTGGAGG 1380

TGACAGCCTT GTCCCAAGGA ACTCTTATGT TCGGCTCAGA CACCTATGTA CTAATACCTG 1440

GGTTCACAGC ACAAATATTC CTATTGACAA GGAAGAAGAA AAGCCCGTGA TGCTGAAAAT 1500

TGGCACCTCT CCTGTGAAGG AGGATAAGGA AGCATTTGGC ATAGTTCCGG TTTCTCCTGC 1560

TGAAGTTCGG GACCTGGACT TTGCCAATGA TGCCAGCAAG GTGCTGGGCT CCATTGCTGG 1620

GAAGCTAGAG AAGGGCACCA TCACCCAGAA TGAAAGGAGG TCTGTAACCA AGCTGCTAGA 1680

AGATTTGGTT TACTTCGTCA CTGGTGGAAC TAATTCTGGT CAAGATGTTC TCGAAGTTGT 17 0

CTTCTCCAAG CCCAACAGAG AACGGCAGAA ACTGATGAGA GAACAGAATA TTCTCAAGCA 1800

GATCTTCAAG TTGTTACAAG CCCCATTCAC AGACTGCGGT GATGGCCCAA TGCTTCGGCT 1860

GGAAGAGCTC GGGGACCAGC GGCACGCTCC TTTCAGACAC ATCTGCCGGC TCTGCTACAG 1920

GGTGCTGAGA CACTCGCAGC AAGACTACAG GAAGAACCAG GAGTATATAG CCAAGCAGTT 1980

TGGCTTCATG CAGAAGCAGA TTGGCTATGA TGTGTTGGCT GAAGACACTA TCACTGCCCT 2040 GCTCCACAAT AATCGGAAAC TCCTGGAAAA ACACATTACC GCGGCAGAGA TTGACACATT 2100

TGTCAGCCTG GTGCGAAAGA ACAGGGAGCC CAGATTCTTA GATTACCTCT CCGACCTCTG 2160

TGTCTCCATG AACAAATCAA TTCCAGTGAC CCAGGAACTG ATATGTAAAG CTGTGCTGAA 2220

CCCCACCAAC GCTGACATCC TGATTGAGAC CAAATTGGTT CTTTCTCGTT TTGAATTTGA 2280

AGGTGTCTCT TCCACTGGAG AGAATGCTCT GGAGGCAGGA GAAGACGAGG AAGAGGTGTG 2340

GCTGTTTTGG AGGGACAGCA ACAAAGAGAT TCGCAGCAAG AGTGTGAGGG AATTGGCTCA 2400

GGATGCTAAA GAAGGGCAGA AGGAGGACCG AGACGTTCTC AGCTACTACA GATATCAGCT 2460

GAACCTCTTT GCGAGGATGT GTCTGGACCG CCAATACCTG GCCATCAACG AAATCTCAGG 2520

CCAGCTGGAT GTCGATCTCA TTCTCCGCTG CATGTCTGAC GAGAACCTGC CCTATGACCT 2580

CAGGGCGTCC TTCTGCCGCC TCATGCTTCA CATGCATGTG GACCGAGATC CCCAGGAACA 2640

AGTCACCCCC GTGAAATATG CCCGCCTCTG GTCGGAGATT CCCTCGGAGA TCGCCATTGA 2700

CGACTATGAT AGTAGTGGAG CTTCCAAAGA TGAAATTAAG GAGAGATTTG CTCAGACCAT 2760

GGAGTTTGTG GAGGAGTATT TAAGAGATGT GGTTTGTCAG AGGTTCCCTT TCTCTGATAA 2820

AGAGAAGAAT AAGCTTACGT TTGAGGTTGT AAATTTAGCT AGGAATCTCA TATACTTTGG 2880

TTTCTACAAC TTCTCTGACC TTCTACGATT AACTAAGATC CTTCTGGCCA TATTGGACTG 2940

TGTACATGTG ACAACAATCT TCCCCATTAG CAAGATGGCG AAAGGAGAAG AGAATAAAGG 3000

CAGTAACGTG ATGAGATCTA TTCATGGCGT GGGAGAGCTG ATGACCCAGG TGGTGCTCCG 3060

GGGAGGAGGC TTTTTGCCCA TGACTCCCAT GGCTGCTGCC CCTGAAGGCA ATGTGAAGCA 3120

GGCAGAGCCT GAGAAGGAGG ACATCATGGT CATGGACACC AAGCTGAAGA TCATTGAGAT 3180

ACTCCAGTTT ATTTTGAATG TGAGGTTGGA TTATAGGATC TCCTGCCTCC TGTGTATATT 3240

TAAGCGAGAG TTTTGGATGA AAGCAATTCC CAGGACTTCA GAAACATCCT CCGGAAACAG 3300

CAGCCAAGAA GGGCCAAGTA ATGTACCAGG TGCTCTTGAC TTTGAACACA TTGAAGAACA 3360

AGCAGAAGGC ATCTTTGGAG GAAGTGAGGA GAACACCCCA CTGGACTTGG ATGACCACGG 3420

CGGCAGAACC TTTCTCCGTG TCCTGCTCCA CTTGACGATG CATGACTACC CACCCCTGGT 3480

GTCAGGGGCC CTGCAGCTCC TCTTCCGGCA CTTCAGCCAG AGGCAGGAGG TGCTCCAGGC 3540

CTTCAAACAG GTTCAACTGC TGGTTACCAG CCAAGATGTG GACAACTACA AACAGATCAA 3600

ACAAGACTTG GATCAACTGA GGTCCATCGT GGAAAAGTCA GAGCTTTGGG TGTACAAAGG 3660

GCAGGGCCCC GATGAGACTA TGGATGGTGC ATCTGGAGAA AATGAACATA AGAAAACGGA 3720

GGAGGGAAAT AACAAGCCAC AAAAGCATGA AAGCACCAGC AGCTACAACT ACAGAGTGGT 3780

CAAAGAGATT TTGATTCGGC TTAGCAAACT CTGTGTTCAA GAGAGTGCCT CAGTGAGAAA 3840

GAGCAGGAAG CAGCAACAGC GTCTGCTCCG GAACATGGGC GCGCACGCCG TGGTGCTGGA 3900

GCTGCTGCAG ATTCCCTATG AGAAGGCCGA AGATACCAAG ATGCAAGAGA TAATGAGGTT 3960

GGCTCATGAA TTTTTGCAGA ATTTCTGCGC AGGCAACCAG CAGAATCAAG CTTTGCTACA 4020

TAAACACATA AACCTGTTTC TCAACCCAGG GATCCTGGAG GCAGTAACCA TGCAGCACAT 4080

CTTCATGAAC AATTTCCAGC TTTGCAGTGA GATCAACGAG AGAGTTGTTC AGCACTTCGT 4140

TCACTGCATA GAGACTCACG GTCGGAATGT CCAGTATATA AAGTTCTTAC AGACAATTGT 4200

CAAGGCAGAA GGGAAATTTA TTAAAAAATG CCAAGACATG GTTATGGCCG AGCTGGTCAA 4260

TTCGGGAGAG GATGTCCTCG TGTTCTACAA CGACAGAGCC TCTTTCCAGA CTCTGATCCA 4320

GATGATGCGG TCAGAACGGG ATCGGATGGA TGAGAACAGC CCTCTCATGT ACCACATCCA 4380

CTTGGTCGAG CTCCTGGCTG TGTGCACGGA GGGTAAGAAT GTCTACACAG AGATCAAGTG 4440

CAACTCCCTG CTCCCGCTGG ATGACATCGT TCGCGTTGTG ACCCACGAGG ACTGCATCCC 4500

TGAGGTTAAA ATTGCATACA TTAACTTCCT GAATCACTGC TATGTGGATA CAGAGGTGGA 4560

AATGAAGGAG ATTTATACCA GCAATCACAT GTGGAAATTG GTTGAGAATT TCCTTGTAGA 4620 CATCTGCAGG GCCTGTAACA ACACTAGTGA CAGGAAACAT GCAGACTCGA TTTTGGAGAA 4680

GTATGTCACC GAAATCGTCA TGAGTATTGT TACTACTTTC TTCAGCTCTC CCTTCTCAGA 4740

CCAGAGTACG ACTTTGCAGA CTCGCCAGCC TGTCTTTGTG CAACTGCTGC AAGGCGTGTT 4800

CAGGGTTTAC CACTGCAACT GGTTAATGCC AAGCCAAAAA GCCTCCGTGG AGAGCTGTAT 4860

TCGGGTGCTG TCTGATGTAG CCAAGAGCCG GGCCATTGCC ATTCCCGTGG ACCTGGACAG 4920

CCAAGTCAAC AACCTCTTTC TCAAGTCCCA CAGCATTGTG CAGAAAACAG CCATGAACTG 4980

GCGGCTCTCA GCCOGCAATG CCGCACGCAG GGACTCTGTT CTGGCAGCTT CCAGAGACTA 5040

CCGGAATATC ATTGAGAGAT TGCAGGACAT CGTCTCCGCG CTGGAGGACC GTCTCAGGCC 5100

CCTGGTGCAG GCAGAGTTAT CTGTGCTCGT GGATGTTCTC CACAGACCCG AGCTGCTTTT 5160

CCCAGAGAAC ACAGACGCCA GAAGGAAATG TGAAAGTGGC GGTTTCATTT GCAAGTTAAT 5220

AAAGCATACA AAACAGCTGC TAGAAGAAAA TGAAGAGAAG CTCTGCATTA AGGTCCTACA 5280

GACCCTGAGG GAAATGATGA CCAAAGATAG AGGCTATGGA GAAAAGGGTG AGGCGCTCAG 5340

GCAAGTTCTG GTCAACCGTT ACTATGGAAA CGTCAGACCT TCGGGACGAA GAGAGAGCCT 5400

TACCAGCTTT GGCAATGGCC CACTGTCAGC AGGAGGACCC GGCAAGCCCG GGGGAGGAGG 5460

GGGAGGTTCC GGATCCAGCT CTATGAGCAG GGGTGAGATG AGTCTGGCCG AGGTTCAGTG 5520

TCACCTTGAC AAGGAGGGGG CTTCCAATCT AGTTATCGAC CTCATCATGA ACGTATCCAG 5580

TGACCGAGTG TTCCATGAAA GCATTCTCCT GGCCATTGCC CTTCTGGAAG GAGGCAACAC 5640

CACCATCCAG CACTCCTTTT TCTGTCGCTT GACAGAAGAT AAGAAGTCAG AGAAATTCTT 5700

TAAGGTGTTT TATGACCGGA TGAAGGTGGC CCAGCAAGAA ATCAAAGCAA CAGTGACAGT 5760

GAACACCAGT GACTTGGGAA ATAAAAAGAA AGACGATGAG GTAGACAGGG ATGCCCCATC 5820

ACGGAAAAAA GCTAAAGAGC CCACAACACA GATAACAGAA GAGGTCCGGG ATCAGCTCCT 5880

GGAGGCCTCC GCTGCCACCA GGAAAGCCTT CACCACTTTC AGGAGGGAGG CTGATCCCGA 5940

CGACCACTAC CAGCCTGGAG AGGGCACCCA GGCCACTGCC GACAAGGCCA AGGACGACCT 6000

GGAGATGAGC GCGGTCATCA CCATCATGCA GCCCATCCTC CGCTTCCTTC AGCTCCTGTG 6060

TGAAAACCAC AACCGAGACC TGCAGAACTT CCTCCGTTGC CAAAATAACA AGACCAACTA 6120

CAATTTGGTA TGTGAGACCC TGCAGTTTCT GGACTGTATT TGTGGAAGCA CAACTGGAGG 6180

CCTTGGTCTT CTGGGCTTGT ATATAAATGA AAAGAACGTA GCGCTTATCA ACCAAACCCT 6240

GGAAAGTCTG ACCGAATACT GTCAAGGACC TTGCCATGAG AACCAGAACT GCATAGCCAC 6300

CCATGAATCC AATGGCATTG ACATCATCAC AGCCCTGATC CTCAATGATA TCAATCCTTT 6360

GGGAAAGAAG AGGATGGACC TTGTGTTAGA ACTGAAGAAC AATGCCTCGA AGTTGCTCCT 6420

GGCCATCATG GAAAGCAGGC ACGACAGTGA AAACGCAGAG AGGATACTTT ATAACATGAG 6480

GCCCAAGGAA CTGGTGGAAG TGATCAAGAA AGCCTACATG CAAGGTGAAG TGGAATTTGA 6540

GGATGGAGAA AACGGTGAGG ATGGGGCGGC GTCCCCCAGG AACGTGGGGC ACAACATCTA 6600

CATATTAGCC CATCAGTTGG CTCGGCATAA CAAAGAACTT CAGAGCATGC TGAAACCTGG 6660

TGGCCAAGTG GACGGAGATG AAGCCCTGGA GTTTTATGCC AAGCACACGG CGCAGATAGA 6720

GATTGTCAGA TTAGACCGAA CAATGGAACA GATAGTCTTT CCCGTGCCCA GCATATGTGA 6780

ATTCCTAACC AAGGAGTCAA AACTACGAAT TTACTATACT ACAGAGAGAG ACGAACAAGG 6640

CAGCAAAATC AATGATTTCT TTCTGCGGTC TGAAGACCTC TTCAATGAAA TGAATTGGCA 6900

GAAGAAACTG AGAGCCCAGC CCGTGTTGTA CTGGTGTGCC CGCAACATGT CTTTCTGGAG 6960

CAGCATTTCG TTTAACCTGG CCGTCCTGAT GAACCTGCTG GTGGCGTTTC TCTACCCGCT 7020

TAAGGGAGTC CGAGGAGGAA CCCTGGAGCC CCACTGGTCG GGACTCCTGT GGACAGGCAT 7080

GCTCATCTCT CTGGGCATCG TCATTGGCCT CCCCAATCCC CATGGCATCC GGGCCTTAAT 7140

TGGCTCCACT ATTCTACGAC TGATATTTTC AGTCGGGTCA CAACCCGCGT TGTTTCTTCT 7200 GGGCGCTTTC AATGTATGCA AGAAAATCAT CTTTCTAATG AGCTTTGTGG GCAACTGTGG 7260

GACATTCACA AGAGGCTACC GAGCCATGGT TCTGGTTCTG GATGTCGAGT TCCTCTATCA 7320

TTTGTTGTAT CTGGTGATCT GTGCCATGGG GCTCTTTGTC CATGTATTCT TCTACAGTCT 7380

GCTGCTTTTA GATTTAGTGT ACAGAGAAGA GTCTTTGCTT AATGTCATTA AAAGTGTCAC 7440

TCGCAATGGA CGGTCCATCA TCCTGACAGC AGTTCTGGCT CTGATCCTCG TTTACCTGTT 7500

CTCAATAGTG GGCTATCTTT TCTTCAAGGA TGACTTTATC TTGGAAGTAG ATAGGCTGCC 7560

CAATGAAACA GCTGTTCCAG AAACCGGCGA GAGTTTGGCA AGCGAGTTCC TGTTCTCCGA 7620

TGTGTGTAGG GTGGAGAGTG GGGAGAACTG CTCCTCTCCT GCACCCAGAG AAGAGCTGGT 7680

CCCTGCAGAA GAGACGGAAC AGGATAAAGA GCACACATGT GAGACGCTGC TGATGTGCAT 7740

TGTCACCGTG CTGAGTCACG GGCTGCGGAG CGGGGGTGGA GTAGGAGATG TACTCAGGAA 7800

GCCGTCCAAA GAGGAACCCC TGTTTGCTGC TAGAGTTATT TATGACCTCT TGTTCTTCTT 7860

CATGGTCATC ATCATTGTTC TTAACCTGAT TTTTGGGGTT ATCATTGACA CTTTTGCTGA 7920

CCTGAGGAGT GAGAAGCAGA AGAAGGAAGA GATCTTGAAG ACCACGTGCT TTATCTGTGG 7980

CTTGGAAAGA GACAAGTTTG ACAACAAGAC TGTCACCTTT GAAGAGCACA TCAAGGAAGA 8040

ACACAACATG TGGCACTATC TGTGCTTCAT CGTCCTGGTG AAAGTAAAGG ACTCCACCGA 8100

ATATACTGGG CCTGAGAGTT ACGTGGCAGA AATGATCAAG GAAAGAAACC TTGACTGGTT 8160

CCCCAGGATG AGAGCCATGT CATTGGTCAG CAGTGATTCT GAAGGAGAAC AGAATGAGCT 8220

GAGAAACCTG CAGGAGAAGC TGGAGTCCAC CATGAAACTT GTCACGAACC TTTCTGGCCA 8280

GTTGTCGGAA TTAAAGGATC AGATGACAGA ACAAAGGAAG CAGAAACAAA GAATGGGTCT 8340

TCTTGGACAT CCTCCTCACA TGAATGTCAA CCCACAACAA CCAGCATAAG CAAATGAAAG 8400

AAAGGAATTG TATTTACCTT TTATAATTAT TATTAGTGTG GGTATGGCTA ATGAGTTCTG 8460

ATTCACCCAC GAAGGTTACA TTTATGCTGA ATACATTTGT AAATACTCAG TTTTATACTG 8520

TATGTATATG ATTGCTACTC TAAAGGTTTG GATATATGTA TTGTAATTAG AATTGTTGGC 8580

ATGATGACAT TTCATTTGTG CCAAAAATAT TAAAAATGCC TTTTTTGGAA GGACTAACAG 8640

AAAGCACCTG ATTTGCACTT GAACCAGTCC GGAATTCCTG CAGCCCGGGG GGATCCACTA 8700

GTTCTAGAGC GGCCGCCACC GCGGTGGAGC TCCAGCTTTG GTTCCCTTTA GTGAGGGTTA 8760

ATTGCGCGCT TGGCGTAATC ATGGTCATAG T 8791

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Cyβ Glu Gin Aβn Glu Leu Arg Aβn Leu Gin Glu Lys Leu 1 5 10

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (ix) FEATURE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

1 5

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (ix) FEATURE:

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

1 5

Claims

WHAT IS CLAIMED IS:

1. A purified and isolated nucleic acid encoding a human type 1 inositol 1,4,5-trisphosphate receptor/calcium release channel protein having an amino acid sequence consisting essentially of the sequence set forth in Figures 1A and IB (SEQ ID NO:l).

2. The purified and isolated nucleic acid encoding a human type 1 inositol 1,4,5-trisphosphate receptor/calcium release channel protein of claim 1, wherein exon SII is excluded.

3. The nucleic acid of claim 2 which is DNA.

4. The nucleic acid of claim 2 which is RNA.

5. A vector comprising the nucleic acid of claim 2.

6. A host cell containing the vector of claim 5.

7. A purified and isolated nucleic acid comprising a portion having a nucleic acid sequence consisting essentially of the nucleic acid sequence set forth in Figure 7A-7C (SEQ ID NO:5).

8. A vector comprising the nucleic acid of claim 7.

9. A host cell containing the vector of claim 8.

10. A purified and isolated human type 1 inositol 1,4,5-trisphosphate receptor/calcium release channel protein having an amino acid sequence consisting essentially of the amino acid sequence set forth in Figures 1A and IB (SEQ ID NO:l).

11. The human type 1 inositol 1,4,5-tris- phosphate receptor/calcium release channel protein of claim 10, wherein the SII exon is excluded.

12. A fusion protein comprising the human type 1 inositol 1,4,5-trisphosphate receptor/calcium release channel protein of claim 11.

13. An immunogenic peptide fragment of the human type 1 inositol 1,4,5-trisphosphate recep- tor/calcium release channel protein of claim 11, having an amino acid sequence comprising the sequence (Cys)-Glu- Gln-Asn-Glu-Leu-Arg-Asn-Leu-Gln-Glu-Lys-Leu (SEQ ID N0:6).

14. A purified antibody which specifically binds to a human type 1 inositol 1,4,5-trisphosphate receptor/calcium release channel protein.

15. The antibody of claim 14 which specifically binds to the sequence (Cys)-Glu-Gln-Asn-Glu-Leu-Arg-Asn- Leu-Gln-Glu-Lys-Leu (SEQ ID NO:6).