CZ20002315A3 - Novel receptor coupled with G-protein - Google Patents

Abstract

New G-protein coupled receptor

Description

Technical field

The present invention relates to the field of biological receptors and the various uses of such receptors. More specifically, the invention relates to nucleic acids encoding novel G-protein coupled receptors and the receptors themselves.

BACKGROUND OF THE INVENTION

G-protein coupled receptors (GPCRs) are a family of proteins having a common structural organization characterized by an extracellular N-terminal end, seven hydrophobic alpha-helices that appear to form transmembrane domains and an intracellular C-terminal domain. GPCR binds various ligands that trigger intracellular signals by activating carrier G-proteins (Caron et al., Rec. Prog. Horm. Res. 48: 277-290 (1993); Freedman et al., Rec. Prog. Horm. Res. 51: 319-353 (1996)).

To date, more than 300 GPCRs have been cloned and it is believed that there are more than 1000 such receptors. Approximately 50-60% of clinically relevant drugs act through influencing the functions of various GPCRs (Cudermann et al., J. Mol. Med. 73: 51-63 (1995)). Of particular importance are receptors located in the posterior spinal cord ganglia. This region of the central nervous system is densely innervated by primary or afferent sensory neurons involved in the transmission, modulation and perception of pain. Therefore, receptors in this field can be used in assays to identify novel agents for anesthesia and analgesia.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of a novel G-protein coupled receptor that differs from the previously described receptors in its structure and is preferably expressed in the posterior spinal cord ganglia. One dorsal root ganglia receptor (DRR) was isolated and sequenced from rats and six dorsal root ganglia receptors were isolated and sequenced from humans. The rat receptor was designated rDRR-1 and its amino acid sequence is set forth in SEQ ID NO: 1. The human receptors were designated as:

hDRR-1 (SEQ ID NO: 3); hDRR-2 (SEQ ID NO: 5); hDRR-3 (SEQ ID NO: 7); hDRR-4 (SEQ ID NO: 9); hDRR-5 (SEQ ID NO: li); hDRR-6 (SEQ ID NO: 13) .

Unless otherwise indicated, the term DRR as used herein refers to all receptors, both human and rat.

In a first aspect, the invention relates to proteins, excluding natural proteins, comprising the amino acid sequence of a functional rat or human DRR as set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11 or 13. The term comprising the functional sequence includes any a receptor protein in which additions, deletions, or substitutions have been made that do not significantly alter the functional characteristics of the receptor. Thus, the invention encompasses proteins having substantially the same amino acid sequence as listed in the Sequence Listing, as well as proteins with differences that are not essential as they do not alter the basic, qualitative binding characteristics of the DRR receptor. The invention further encompasses substantially pure proteins consisting essentially of the DRR's amino acid sequence, antibodies that specifically bind to DRRs (ie having at least 100-fold higher affinity for DRR than any other non-DRR protein), and antibodies prepared injecting pharmaceutically acceptable formulations of such proteins into an animal capable of producing antibodies. In a preferred embodiment, the monoclonal antibody to human or rat DRR is prepared by injecting a pharmaceutically acceptable preparation of the mouse receptor and then fusing mouse spleen cells with myeloma cells.

The present invention also comprises a substantially pure polynucleotide encoding a protein consisting of an amino acid sequence consisting of a functional sequence of a rat DRR (as set forth in SEQ ID NO: 1) or a human DRR (as set forth in SEQ ID NOs: 3, 5, 7, 9, 11 or 13). This aspect of the invention includes polynucleotides encoding proteins consisting essentially of the amino acid sequences set forth in the Sequence Listing, expression vectors comprising such polynucleotides, and host cells transformed with such vectors. This aspect also includes recombinant rat and human DRR proteins produced by the host cells obtained by this method.

Preferably, the polynucleotide encoding the rat DRR has the nucleotide sequence set forth in SEQ ID NO: 2a and the polynucleotide encoding the human DRR has the nucleotide sequence set forth in SEQ ID NO: 3, 5, 7, 9, or 13. It is also preferred that the vectors and host cells used for these particular polynucleotides contained the expression of DRR.

In another aspect, the present invention relates to a method for testing the ability of a compound to bind to a rat or human DRR The method comprises incubating a source of DRR with a ligand known to bind to a receptor and a test compound. The DRR source should be substantially devoid of other types of G protein-coupled receptors, ie, more than 85% of such contained receptors would be free of other G-protein coupled receptors. ···· «· ····· · · · · · · · ·········································· DRR. Upon completion of incubation, the ability of the test compound to bind to DRR is determined by the extent of ligand binding replacement. The rat DRR should, preferably, correspond to rDRR-1 as set forth in SEQ ID NO: 1. The human receptor should preferably be hDRR-1 (SEQ ID NO: 3); hDRR-2 (SEQ ID NO: 5); hDRR-3 (SEQ ID NO: 7); hDRR-4 (SEQ ID NO: 9); hDRR-5 (SEQ ID NO: 11); or hDRR-6 (SEQ ID NO: 13). Transformed cells expressing recombinant DRR or membranes prepared from such cells may be used in such assays. Although not essential, the assay may also include determining activation of a second messenger pathway, such as an adenylate cyclase pathway. This may help to determine whether a compound that binds to DRR acts as an agonist or antagonist.

An alternative method for determining whether a test compound is an agonist of any of the DRRs described herein is to use a cell signaling assay, for example, an assay measuring either intracellular adenylate cyclase activity or intracellular calcium concentration. The test compound is incubated with cells expressing DRR but substantially free of other G-protein coupled receptors, usually cells transfected with an expression vector encoding DRR. Test compounds that are agonists are identified by causing a statistically significant change in the results obtained in the cell signaling assay compared to control transfectants not exposed to the test compound. For example, cells exposed to a test compound may exhibit a significant increase in adenylate cyclase activity or an increase in intracellular calcium concentration.

The present invention also encompasses a method for determining whether a test compound is a DRR antagonist which consists in the known activation of G-protein coupled receptors that occurs when such receptors are expressed in large amounts. This method requires that the DNA encoding the receptor be incorporated into an expression vector so that it is operably linked to a promoter and this vector. is then used to transfect a suitable host. Preferably, expression systems capable of producing copies of the protein are used to produce sufficient receptors for constitutive receptor activation (i.e., activation in the absence of a natural ligand), for example, DRR DNA can be operably linked to a CMV promoter and expressed in COS or HEK293 cells. After transfection, activated receptor cells are selected according to whether they exhibit increased activity in the cell signaling assay compared to cells that have either not been transfected or that have been transfected with a vector that is unable to express functional DRRs. Typically, such cells will be selected either by showing a statistically significant increase in the intracellular activity of adenyl cyclase or by showing a statistically significant increase in the intracellular calcium concentration. The selected cells are contacted with the test compound and the cell signaling assay is repeated to determine whether the compound causes a decrease in activity relative to control cells not contacted with the test compound. For example, a statistically significant decrease in either adenyl cyclase activity or calcium concentration relative to control cells suggests that the test compound is a DRR antagonist. any of the DRRs described herein may be used in these assays.

Assays for DRR interacting compounds can be performed by incubating a source containing DRR but substantially free of other G-protein coupled receptors (e.g., stably transformed cells) with angiotensin II or III in the presence and absence of the test compound, and then measuring modulation intracellular calcium concentration. A statistically significant increase or decrease in angiotensin-stimulated changes in calcium concentration in response to a test compound indicates an interaction with DRR. Among the receptors that can be used in this are the receptors that can be used in this. The test includes rat DDR-1 and human DRR-1, DRR-2, DRR-3, DRR-4, DRR-5, and DRR-6.

In another aspect, the present invention is directed to a method of testing compounds for their ability to alter the expression of rat or human DRR. This method is performed by culturing cells expressing DRR but substantially free of other G-protein coupled receptors in the presence of a test compound. The cells are then recovered from the culture and the expression of DRR is compared to that of DRR in control cells cultured under essentially the same conditions but in the absence of the test compound. The rat receptor is preferably rDDR-1 and the human receptor is preferably DRR-1, DRR-2, DRR-3, DRR-4, DRR-5 or DRR-6.

A preferred test compound is an oligonucleotide of at least 15 nucleotides in length containing a sequence complementary to the DRR sequence used in the assay.

Description of the figures in the attached drawings

Giant. 1. Nucleotide sequence of rDRR-1: clone 3B-32, encoding rDRR-1, was isolated from the rat genomic library using the Promotor Finder Walking Kit (see Methods, Clontech).

The clone was deposited with the International Microorganism Storage Institute Deutsche Sammlung Von Microorganisms Und Zellkulturen GmbH, address Mascheroder Weg IB, D-3300 Braunschweig, Germany. Storage was performed on 27/11/1997 under accession number DSM 11877.

Giant. 2. Derived amino acid sequence DRR-1: clone 3B-32 encodes a protein of 337 amino acids. The amino acid sequence begins with the first ATG in the nucleotide sequence.

► · »· <<

> ····· «· <I · · · I · · · · ·

Giant. 3. Alignment of the derived amino acid sequence of clone 3B-32 (rDRR-1) with the five most homologous sequences. The frame residues and the stained residues are identical to the residues in the rDRR-1 sequence.

Giant. 4. amino acid sequence of human DRR homologs: the amino acid sequences of all 6 human rDRR-1 homologues (hDRR-1; hDRR-2; hDRR-3; hDRR-4, hDRR-5 and hDRR-6) are aligned. Amino acid residues differing from clone 36 (HUMAN36.PR) are boxed. The degree of identity between these sequences ranges from 77% to nearly 100%.

Definition

The following description uses many terms relating to recombinant DNA technology. For a clear understanding of the present invention and claims, including the scope given by such terms, the following definitions are set forth.

Cloning vector: Plasmid or phage DNA or other DNA sequence that is capable of autonomous replication in a host cell and which is characterized by one or a small number of sites recognized by restriction endonuclease. The foreign DNA fragment can be inserted into the vector at these sites to effect replication and cloning of the fragment. The vector may contain a marker suitable for use in identifying transformed cells.

For example, the marker may confer resistance to tetracycline or ampicillin.

Expression Vector: A vector similar to a cloning vector that can induce expression of DNA that has been cloned into a vector after its transformation into a host. The cloned DNA is usually placed (for example, operably linked to) under the control of certain regulatory sequences, such as promoters and enhancers. Promoter sequences can be constitutive, inducible, or repressible.

Substantially pure: As used herein, the term substantially pure means that the material selected is substantially free of contaminating cellular components. A substantially pure protein or nucleic acid typically makes up at least 85% of the sample, preferably an even higher percentage. Contaminants include proteins, carbohydrates, or lipids. One method for determining purity of a protein or nucleic acid is by electrophoresis of the preparation in a matrix, such as polyacrylamide or agarose. Purity is confirmed by detecting a single strip after staining. Other methods for determining purity are chromatography and analytical centrifugation.

Host: Any prokaryotic or eukaryotic cell that is a recipient of a replicable expression vector or cloning vector is a host for the vector. The term includes prokaryotic or eukaryotic cells that have been engineered to incorporate a selected gene into their chromosome or into their genome. Examples of cells that can serve as hosts are well known in the art, as are cell transformation techniques (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor (1989)).

Promoter: A DNA sequence that is usually located in the 5 'region of the gene, proximal to the start codon. Transcription is initiated in the promoter. If the promoter is inducible, the rate of transcription increases in response to the inducing agent.

Complementary Nucleotide Sequence: The term "complementary nucleotide sequence" as used herein refers to a sequence that results from normal base pairing. For example, the nucleotide sequence of 5-AGAC-3 will have the complementary sequence of 5-GTCT-3.

• · · · · · · ·

Expression: Expression is the process by which a polypeptide is produced from DNA. The process requires the gene to be transcribed to mRNA and the mRNA to be translated into a polypeptide.

The present invention is directed to DRR receptor proteins, genetic sequences encoding receptors, methods for testing compounds for binding to DRR receptors, and methods for testing compounds for their ability to alter DRR expression. The receptors and their nucleic acids are defined by their structure (as shown in Figures 1, 2 and 4; and in SEQ ID NOS: 1-14).

It will be appreciated that the present invention encompasses not only sequences identical to those shown in the figures and sequence listing, but also sequences that are substantially the same and sequences that are significantly similar and that retain receptor binding characteristics that are intrinsic DRR. For example, it is well known that techniques such as site-directed mutagenesis can be used to introduce variations into the protein structure. Variations in the DRR protein introduced by this or other similar method are also included in the present invention, with the proviso that the resulting receptor retains the basic qualitative binding characteristics of the unaltered DRR. Therefore, the invention relates to proteins consisting of a functional sequence of SEQ ID NO: 1 (rat) and SEQ ID NO: 3, 5, 7, 9, 11 and 14 (human).

I. DRR encoding nucleic acid sequences

The DNA sequences encoding the DRRs are expressed exclusively, or at least very preferably, in the posterior spinal cord ganglia, and these ganglia may serve as a source for isolating the nucleic acids encoding the receptors. In addition, cells and cell lines that express rat or human DRR may be a source of nucleic acids.

of acids. They may be untransformed cultured cells, or cell lines that have been specifically processed to express recombinant DRRs.

In all cases, the A + mRNA poles are isolated from the posterior spinal cord ganglia, reverse transcribed, and cloned. The cDNA library thus prepared can then be screened using probes derived from the sequences listed in the appended sequence listing as SEQ ID NOs: 2, 4, 6, 8, 10, 12, or 14, depending on which particular DRR is isolated. Probes are typically at least 14 bases in length and should be derived from a portion of the DRR sequence that is poorly conserved (see Figures 3 and 4). Screening can also be performed using genomic libraries with one DRR gene, or part of a gene, as a probe to isolate other DRR genes. For example, complete rDRR-1 can be labeled and used to screen a human genomic library for isolation of hDRR-1, hDRR-2, etc. (see Examples).

Alternatively, genomic DNA libraries can be used to isolate DRR genes by PCR amplification using primers located at one end of the genes (a description of the procedure is given in the Examples). For example, human genomic DNA can be amplified using primers:

5'-GCAAGCTTTCTGAGCATGGATCCAACCGTC, a

5'-CCCTCAGATCTCCAATTTGCTTCCCGACAG.

These primers can be used to amplify all six human DRR genes identified herein as hDRR-1; hDRR-2; hDRR-3; hDRR-4; hDRR-5; and hDRR-6. These genes can then be cloned into a suitable vector, for example pGEM-T (Promega), for DNA sequence analysis.

• ·

II. Antibodies to rats and human DRRs

The present invention also includes antibodies that specifically bind to rat or human DRR and methods for preparing such antibodies. Antibodies that specifically bind to DRR are defined as those having at least 100-fold higher affinity for DRR than any other protein. The method for preparing such antibodies may require either injecting the DRR protein alone into a suitable animal, or more preferably injecting short peptides corresponding to different regions of the DRR. The peptides should be at least 5 amino acids in length and selected from regions believed to be unique to the particular DRR protein to which the antibody is to be directed. Therefore, highly conserved transmembrane regions are generally not used as peptides for the production of antibodies. Methods for preparing and detecting antibodies are well known in the art and are described in standard papers (e.g., Harlow et al., Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1988); Klein, Immunology: The Science of Self-). Nonself Discrimination (1982), Kennett et al., Monoclonal Antibodies and Hybridomas: A New Dimension in Biological Analyzes (1980) and Campbell, Monoclonal Antibody Technology, in Laboratory Techniques in Biochemistry and Molecular Biology (1984).

The term antibody as used herein refers to both an intact molecule and fragments thereof that retain the ability to bind to an antigen (e.g., Fab and F (ab) ₂ fragments). These fragments are usually prepared by proteolytic cleavage of intact antibodies using enzymes such as papain (to prepare Fab fragments) or pepsin (to prepare F (ab) ₂ fragments). The term antibody also refers to both monoclonal and polyclonal antibodies. Polyclonal antibodies are obtained from the serum of animals immunized with antigen. Monoclonal antibodies can be prepared by the hybridoma technique (Kohler et al., Nature 256: 495 (1975); Hammerling et al., In Monoclonal Antibodies and T-cell Hybridomas, Elsevier, MY, pp. 563-681 (1981) )). Generally, this technique requires immunization of an animal, usually a mouse, with either an intact DRR or a DRR-derived fragment. The splenocytes of the immunized animal are extracted and fused with suitable myeloma cells, for example SP2O cells. After fusion, the resulting hybrid cells are selectively maintained in HAT medium and then cloned by limiting dilution (Wands et al.,

Gastroenterology 80: 225-232 (1981)). The cells obtained by such selection are then screened to identify clones that secrete antibodies capable of binding to DRR.

The antibodies, or antibody fragments, of the present invention can be used to detect the presence of a DRR protein using any of a variety of immunoassays. For example, antibodies can be used in radioimmunoassays or immunometric assays, also known as two-digit or sandwich assays (see Chard, T., An Introduction to

Radioimmune Assay and Related Techniques, in Laboratory Techniques in Biochemistry and Molecular Biology, North Holland Publishing Co., N.Y. (1978)). In a typical immunometric assay, the amount of unlabeled antibody is bound to a solid carrier that is insoluble in the test liquid, such as blood, lymph, cell extracts, and the like. After the initial binding of the antigen to the immobilized antibody, an amount of detectably labeled secondary antibody (which may or may not be the same as the first antibody) is added to detect and / or quantify the bound antigen (see, eg, Radioimmune Assay Method, Kirkham et al., Ed. pp. 199-206, E. and S. Livingstone, Edinburgh (1970)). Many variants of these types of assays are known in the art, and these variants can be used to detect DRR.

Antibodies to rat or human DRRs may also be used.

for purification of intact receptors or receptor fragments (generally see Dean et al., Affinity Chromatography, A Practical Approach, IRL Press (1986)). Typically, the antibody is immobilized on the column and the DRR-containing composition passes through the column under conditions promoting binding, for example under low salt concentration conditions. The column is then washed and bound DRR is eluted using a buffer that causes the DRR to dissociate from the antibody, for example a buffer having a different pH or salt concentration. The eluted DRR can be transferred to a selected buffer, for example by dialysis, and then stored or used directly.

III. Radioligand receptor binding assay

One of the major uses of DRR nucleic acids and recombinant proteins is the use in assays designed to identify agents capable of binding to DRR receptors. Such agents may be either agonistic, mimicking the normal effects of receptor binding, or antagonistic, inhibiting the normal effects of receptor binding. Of particular interest is the identification of agents that bind to DRR and modulate adenyl cyclase activity in cells. These agents have potential therapeutic use as analgesics and anesthetics.

In a radioligand binding assay, a source of DRR is incubated with a ligand known to bind to the receptor and a compound that is tested for binding activity. A preferred source of DRR is a cell, preferably a mammalian cell, transformed to recombinantly express the receptor. The selected cell should not express significant amounts of other G-protein coupled receptors that can bind to the ligand and which can misrepresent the results. This can be readily determined by performing cell binding assays on cells obtained from the same tissue or cell line as cells recombinantly expressing DRR, but which were not. · 9 · · 4 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Transformed.

The assay can be performed using intact cells or membranes prepared from cells (see Wang et al., Proc. Natl. Acad. Sci. USA 90: 10230-10234 (1993)). Membranes are incubated with ligands specific for DRR receptor and test compound preparation. Upon completion of binding, the receptor is separated from the solution containing the ligand and test compound, for example by filtration, and the amount of binding is determined. Preferably, the ligand used is detectably labeled with a radioactive isotope such as ^12S . However, if necessary, a fluorescent or chemiluminescent agent may be used instead of the isotope. The most commonly used fluorescent labeling compounds include fluorescein isothiocyne, rhodamine, phycoerythrin, phycocyanine, allophycocyanine, o-phthaldehyde and fluorescamine. Useful chemiluminescent compounds include luminol, isoluminol, a theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester. Any of these reagents can be used to prepare a ligand suitable for this assay.

Non-specific binding can be performed by a coupling reaction in the presence of a large excess of unlabeled ligand. For example, the labeled ligand may be incubated with the receptor and test compound in the presence of a 1000-fold excess of unlabeled ligand. Non-specific binding should be subtracted from total binding, i.e. binding in the absence of unlabeled ligand, to obtain specific binding for each test sample. If necessary, the assay may include additional steps such as washing, mixing, shaking, filtering and the like. Typically, the washing is performed after separating the membrane-bound ligand from the ligand remaining in solution and before quantifying the amount of bound radioligand, for example by means of a radioactive isotope assay. The specific binding obtained in the presence of the test compound is compared to the binding obtained in the presence of the labeled ligand itself to determine the extent of binding of the test compound to the receptor.

When performing binding assays, care should be taken to avoid artifacts that may cause the test compound to appear to interact with the DRR receptor, although in fact binding is inhibited by some other mechanism. For example, test compounds should be in a buffer that does not significantly inhibit ligand binding to DRR by itself and should be tested at several different concentrations. Formulations of the test compound should also be screened for proteolytic activity and it is desirable that the assays contain antiproteases. Finally, it is highly desirable that compounds which have been identified as ligand displacement compounds for binding to the DRR receptor be re-examined at a concentration range sufficient to perform Scatchard analysis of the results. This type of assay is well known in the art and can be used to determine the affinity of test compounds for the receptor (see, for example, Ausubel et al., Current Protocols in Molecular Biology, 11.2.1-11.2.19 (1993); Laboratory Techniques and Biochemistry and Molecular Biology, Work et al., Eds., NY (1978), etc.). Computer programs can be used to facilitate analysis of results (see, e.g., Munson, P., Methods Enzymol., 92: 543-577 (1983); McPherson, GA, Kinetic, EBDA Ligand. Lowry-A Collection of Radioligand Binding Analysis Programs, Elsevier). Biosoft, UK (1985)).

Receptor activation by ligand binding can be monitored using a variety of assays. For example, adenyl cyclase assays may be performed by culturing the cells in the wells of a microtiter plate and then incubating the various wells in the presence or absence of the test compound. cAMP can be extracted in ethanol, lyophilized and resuspended in assay buffer. The determination of the cAMP thus obtained can be carried out using any technique for determining the concentration of cAMP, for example a Biotrack cAMP Enzyme-Immunoassay. System (Amersham) or Cyclic AMP (3 H) Assay System (Amersham). Typically, the adenyl cyclase assays will be performed separately from the binding assays, but it is also possible to perform the binding and adenyl cyclase assays on a single cell preparation. Other cell signaling assays that can be used to monitor receptor activity are described below.

IV. Identification of DRR agonists and antagonists using cell signaling assays

DRRs can also be used to screen drug candidates using cell signaling assays. To identify DRR agonists, the DNA encoding the receptor is incorporated into an expression vector and then transfected into a suitable host. The transformed cells are then contacted with a series of test compounds and the effect of each compound is monitored. Assays that may be used include assays measuring cAMP production (see above), assays measuring activation of the reporter gene, or assays measuring modulation of GTP-gamma-S binding.

Cell signaling assays can also be used to identify DRR antagonists. G-protein coupled receptors can be activated even in the absence of their ligand by being expressed at very high concentrations in a heterologous system. For example, the receptor may be overexpressed using baculovirus infection of insect Sf9 cells, or the DRR gene may be operably linked to a CMV promoter and may be expressed in COS or HEK293 cells. In this activated constitutive state, a receptor antagonist can be identified in the absence of a ligand to measure the ability of the test compound to inhibit constitutive cellular signaling activity. Appropriate assays for this are, again, cAMP assays, activation ♦ * · «· 9 9 tf '« «* • ·· ·· · · ·" and • * · · · »· ₉ *»> • »» ··· «· * *» »

Reporter gene or GTP-gamma-S binding measurement assays.

One preferred cell signaling assay is based on the observation that cells stably transfected with DRR show changes in intracellular calcium concentration in response to incubation in the presence of angiotensin II or III (see Example 5). Thus, the procedure that can be used to identify DRR agonists or antagonists is similar to the radioreceptor assay described above except that angiotensin II or III is used instead of labeled ligand and calcium concentration is measured instead of bound radioactivity. The concentration of calcium in the presence of the test compound and angiotensin II or III is compared to the concentration in the presence of only angiotensin II or III to determine whether the test compound interacts with the DRR receptor. A statistically significant increase in intracellular calcium in response to the test compound indicates that the test compound acts as an agonist, while a statistically significant decrease in intracellular calcium in response to the test compound indicates that the test compound acts as an antagonist.

V. Assays for the ability to modulate DRR expression

One way to increase or decrease the biological effects of DRR is by altering the expression of receptors in cells. Therefore, assays identifying compounds that inhibit or enhance receptor expression are significant. These assays are performed by culturing cells expressing DRR in the presence of a test compound and then comparing the expression of the receptors in these cells with the expression of the receptors in cells cultured under substantially identical conditions but in the absence of the test compound. As in the binding assays described above, it is desirable for the cells to be substantially free of competing G-protein coupled receptors. One way to quantify receptor expression is by fusing the DRR sequence to the coding sequence

4 «« · 4 · 4 4 ···· 4 4 4 4 4 4 · 4 · * »44 44444 ·· 4 ····· * 4 · · 4 4 4 * 444

A 4 * peptide or protein that can be easily quantified.

For example, a DRR sequence can be ligated to a sequence encoding haemagglutinin as described in Example 5, and this sequence can be used for stable transfection of cells. After incubation with the test compound, the haemagglutinin / receptor complex may be processed by immunoprecipitation or Western blotting with an anti-haemagglutinin antibody. Alternatively, Scatchard analysis of labeled ligand binding assays can be performed to determine the number of receptors. Binding assays can be performed as described above and will preferably utilize cells that have been engineered to recombinantly express DRR.

A preferred group of test compounds for use in DRR expression assays consists of oligonucleotides complementary to different segments of the DRR nucleic acid sequence. These oligonucleotides should be at least 15 bases long and be derived from the non-conserved regions of the receptor nucleic acid sequence. The sequences may be derived from the sequences shown as SEQ ID NOs: 2, 4, 6, 8, 10, 12 or 14.

Oligonucleotides that have been found to reduce receptor expression can be derivatized or conjugated to further enhance their potency. For example, nucleoside phosphorothioates may be substituted for their natural counterparts (see Cohen, J., Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press (1989)). Oligonucleotides may be administered to a patient in vivo to inhibit DRR expression. When such administration is performed, it is preferred that the oligonucleotide be administered in a form that enhances uptake by the cells. For example, the oligonucleotide may be administered in liposomes or may be conjugated to a peptide that is digested by cells (see, for example, U.S. Patent Nos. 4897355 and 4394448; see also non-US patent applications WO 8903849 and EP 0263740). Other methods for increasing the efficiency of administration of oligonucleotides are well known in the art and are also useful in the present invention.

Having previously described the present invention, the present invention will be further elucidated in the following examples, which are provided to illustrate the invention in no way limiting its scope.

DETAILED DESCRIPTION OF THE INVENTION

Example 1: Cloning of Rat DRR-1

Isolation of the cDNA fragment

Degenerate oligonucleotides were synthesized to highly conserved regions of G-protein coupled receptors (transmembrane bridging domains 2 and 7) with the following nucleotide sequences:

5 'GG CCG TCG TCA TCG TC (A / T) (A / C) (T / C) C TI (G / T) CI (T / C) TIG C (A / C / G / T) G 3 (TM2: encoding) SEQ ID NO: 15; and

5 '(A / G) (C / A / T) (A / G) (A / G) TA IATIATIGG (A / G) TT 3' (TM7: antisense) SEQ ID NO: 16 .

A + mRNA poles were isolated from cultured fetal rat spinal ganglion ganglia (Sprague-Dawley). mRNA was reverse transcribed using First strand cDNA Synthesis kit (Pharmacia Biotech), was amplified by polymerase chain reaction (PCR) using Ampli-Taq DNA (Perkin-Elmer Cetus) polymerase under the following conditions: 3 minutes at 94 ° C, 40 cycles by 1 minute at 94 ° C, 45 ° C and 72 ° C. A cDNA PCR fragment corresponding to approximately 650 bp was isolated and this fragment was subcloned into the pGEM-T-vector (Promega Corporation). The nucleotide sequence of the recombinant clone was determined using the T7-dideoxy Chain termination sequencing kit (Pharmacia Biotech) and was found to be unique in a Genbank / EMBL database search.

The complete rat DRR-1 sequence was obtained from rat genomic DNA using a 650 bp fragment and the Promotor Finder DNA Walking Kit (Clontech, Cat. No. K1806-1). The kit contains five libraries of uncloned, adapter-coupled genomic DNA fragments.

The procedure requires two sequential PCR reactions. Both reactions were performed using Advantage tth Polymerase Mix, which was also obtained from Clontech, according to the manufacturer's recommended procedure. The first PCR reaction was performed using an external adapter primer (API) contained in the kit and an external gene specific primer (GSP1) derived from the DRR-1 PCR fragment sequence. Primary PCR mixtures were diluted and used as templates for the secondary (nested) PCR reaction with nested adapter primer (AP2) and (nested) gene specific primer (GSP2). The following oligonucleotides were used to obtain the sequence of the rat DRR-1 gene before the original PCR fragment sequence:

GSP1: oligo YF3B59-B, 5‘-CGCAGATGAGGTAGTACAGCATCAC of SEQ ID NO: 17

GSP1: oligo MML-R.1,5‘- CTGTGAGAGAGATGGTAACATACAG SEQ ID NO: 18

The 1.9 kb AP2-MMLR1 fragment was obtained from the first library and the approximately 1.0 kb fragment was obtained from the third library. The following primers were used to obtain the sequence after the known sequence:

GSP1: YF3B59-F2 oligo, 5‘-GCATCCTTGACTGGTTCTTCTCAG SEQ ID NO: 19 GSP2: MML-F1 oligo, 5 GGGTGAGACTCATCATCATTTGTGG. SEQ ID NO: 20

An approximately 1 kb MMLF1-AP2 fragment was obtained from the first library, and an approximately 600 bp fragment was obtained from the third library. A composite sequence of 1154 nucleotides containing the complete putative open reading frame of DRR-1 is shown in Figure 1. The open reading frame encodes a protein of 337 amino acids (Figure 2) with an estimated molecular weight of 38.7 kDa. The protein sequence contains all the characteristic properties of G-protein coupled receptors: seven hydrophobic helices probably representing transmebrane domains, a potential glycosylation site at the N-terminal extracellular domain (position 30), and a conserved NPXXY sequence at position 285-289.

Example 2: Cloning of genes for human DRR receptors

Two approaches were used to identify and clone new human DNA sequences homologous and / or related to the rat DRR-1 gene. In the first approach, the human genomic library was screened in a lambda vector, Fix II (STratagene cat.

946203). Approximately 10 6 human genomic clones were plated and transferred to nitrocellulose membranes for hybridization with the complete, ³² P-labeled rat DRR-1 sequence as a probe. Hybridization was performed at 42 ° C overnight. The filters were washed at ambient temperature at low stringency (1X SSC / 0.1% SDS) to allow detection of related but not necessarily identical sequences.

The inserted human DNA present in the positive phages was amplified by PCR using an Expand PCR kit from Boehringer Mannheim under conditions allowing accurate amplification of very large DNA fragments. These long DNA fragments were digested with various restriction enzymes and were subcloned into a plasmid vector. Portions of these clones that hybridized to the rat DRR-1 gene probe were sequenced using an ABI cycle sequencing kit.

A second approach for identifying new human DRR-1 related sequences is to use a polymerase chain reaction (PCR) performed on total human DNA. The primers were synthesized according to the human genomic clones described above and had the following sequences:

HML.H, 5'-GCAAGCTTTCTGAGCATGGATCCAACCGTC, SEQ ID 21 and HML.Bg, 5'-CCCTCAGATCTCCAATTTGCTTCCCGACAG. SEQ ID NO: 22.

After amplification, approximately 1 kb fragments were obtained containing the entire coding sequence of human genes. These obtained fragments were subcloned into pGEM-T (Promega) DNA sequence analysis vector.

Six human clones were isolated using the above strategies:

clone 7, SEQ ID NO: 3a 4; clone 18, SEQ ID NO: 5 and 6; clone 23, SEQ ID NO: 7 and 8; clone 24, SEQ ID NO: 9 a 10; clone 36, SEQ ID NO: 11 and 12; clone 40, SEQ ID NO: 13 and 14.

None of these clones contained introns and their alignment is shown in Figure 3.

At the amino acid level, the rat DRR-1 clone is 47% to 49% identical to human clones.

At the nucleic acid level, the rat DRR-1 clone is from 56% to 58%. · Identical to human clones. · · 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 identical to human clones. The level of sequence identity between human clones (7, 18, 23, 24, 36, 40) is very high, from 77% to 98% at the amino acid level. All human sequences were used as templates to search for homologous sequences in published databases (Genbank, Swissprot, EST). No identical sequences were detected. The most related sequences were proteins of the oncogene mass family. The overall amino acid sequence homology between rat DRR-1 and any of the isolated human genes ranged from 47% to 50%. However, certain regions showed much higher sequence homology, especially regions encoding putative transmembrane domains III and VII (TM3 and TM7) and intracellular loops 2 and 3, where the homologies between the rat sequence and the human homologue were approximately 80%.

Example 3: In situ hybridization experiments

Tissue Preparation: Male adult Sprague-Dawley rats (approximately 300 g, Charles River, S. Constant, Quebec) were sacrificed by decapitation. Brains and spinal cord with posterior root ganglia were removed, were rapidly frozen in isopentane at -40 ° C for 20 seconds and stored at -80 ° C. Frozen human brains, spinal cord and posterior spinal ganglion were obtained from the Brain and Tissue Bank for Devolopmental Disorders, University of Maryland, Baltimore, in accordance with strict ethical regulations. The frozen tissue was sectioned at 14 m on a Microm HM 500 M cryostat (Germany) and was thawed on ProbeOn Plus slides (Fisher Scientifics, Montreal, Quebec). Sections were stored at -80 ° C until in situ hybridization.

Ribon probe synthesis: Plasmid pGemT-3b32 GPCR was linearized using either SacII or NotI restriction enzyme. The coding and antisense DRR ribosondes were transcribed in vitro using either T7 or SP6 RNA polymerase (Pharmacia Biotech), in the appropriate.

order, in the presence of (35S) UTP (approximately 800 Ci / mmol; Amersham, Oakville, Ontario). Plasmid pGemT-Clone 36 GPCR was linearized using the restriction enzymes SacII and PstI. The coding and antisense Clon36 ribosondes were transcribed in vitro using either SP6 or T7 RNA polymerase (Pharmacia Biotech), respectively, in the presence of (35S) UTP. After transcription, the DNA template was digested with DNAse I (Pharmacia). Ribosondes were purified by phenol / chloroform / isoamyl / alcohol extraction and were precipitated in 70% ethanol containing ammonium acetate and tRNA. The quality of the labeled ribosondes was confirmed by polyacrylamide-urea gel electrophoresis.

In situ hybridization: Sections were post-fixed in 4% paraformaldehyde (BDH, Poole, England) in 0.1 M phosphate buffer (pH 7.4) for 10 minutes at ambient temperature and were washed three times with 2X standard citrate buffer (SSC). 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0). The sections were then equilibrated in 0.1 M triethanolamine, treated with 0.25% acetic anhydride in triethanolamine, rinsed in 2X SSC and dehydrated in ethanol series (50-100%). Hybridization was performed in a buffer containing 75% formamide (Sigma, St. Louis, MO), 600 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, 1X Denhart's solution (Sigma), 50 g / ml denatured salmon sperm DNA (Sigma), 50 µg / ml yeast tRNA (Sigma), 10% dextran sulfate (Sigma), 20 mM dithiothreitol and ( ³⁵ S) UTP-labeled cRNA probes (10 x 10 ⁶ cpm / ml) at 55 ° C for 18 hours in a humidified chamber. After hybridization, slides were washed in 2X SSC at ambient temperature, treated with 20 g / ml RNAse IA (Pharmacia) in RNAse buffer (10 ml Tris, 500 mM NaCl, 1 mM EDTA, pH 7.5) for 45 minutes and was washed to a final stringency of 0.1xSSC at 65 ° C. Sections were then dehydrated and exposed to Kodak Biomax MR film for 21 days and / or immersed in Kodak NTB2 emulsion diluted 1: 1 with distilled water and exposed for 4-6 weeks at 4 ° C prior to development and treatment. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • -acetate (Sigma).

Results: Of all regions examined in the nervous system of rats, DRR-1 mRNA was expressed exclusively in the posterior root ganglia. Emulsive autoradiography with high resolution showed the accumulation of silver grains exclusively in small and medium size neurons. This unique and highly restricted character of the DRR-1 distribution was confirmed in the rat embryo. A rat fetal sagittal section of E17 showed that DRR-1 mRNA is restricted to the posterior spinal cord ganglia. No specific hybridization signal was detected in any other rat embryo structures, confirming the highly selective pattern of DRR-1 expression.

Expression of the human clone 36 receptor was present in the fetal ganglia of the posterior spinal cord, but not in the spinal cord. The specific hybridization signal for clone 36 was not detected in any human adult CNS tissue examined so far. These tissues include spinal cord, cerebral cortex, hippocampus, thalamus, substantia nigra and periaquaductal gray matter (data not shown). The presence of clone 36 mRNA in the posterior root ganglia of spinal cord adults has yet to be investigated. Standard controls in which additional sections of the spinal cord ganglion were hybridized with DRR-1 antisense or clone 36 coding probes labeled with ³⁵ S showed no specific hybridization signal.

Example 4: Northem transmissions

Commercial rat and human Northern blot membranes containing 2 g poles of A RNA from various tissues (Clontech) were used to determine expression and distribution of rat DRR-1 mRNA and its human homologs. Radiolabeled probes were prepared as follows: 25 ng of the 650 bp 3b-32 PCR fragment obtained from rat DRR-1 (see Example 1) or human knee 36 was randomized. Labeled using a Ready-to-Go DNA labeling kit (Pharmacia Biotech) and ( ³² P) CTP (3000 Ci / mmol / Amersham). The membrane was prehybridized for 1 hour at 68 ° C using Expresshyb (Clontech) and then hybridized (2x10 6 cpm / ml probe) for 1 hour using the same conditions. The membranes were washed at ambient temperature in 2X SSC, 0.05% SDS for 30 minutes and then 3 washes were performed in 0.2X SSC, 1% SDS at 50 ° C for 60 minutes and the membranes were exposed at -80 ° C on Kodak Biomax film for 6 days.

Rat DRR-1 Expression and Distribution: All rat rats (heart, brain, spleen, lung, skeletal muscle, kidney and testes) were negative for DRR-1 expression after 2 weeks exposure, whereas southern analysis of the rat genome showed 1.1 kb band when probed with the same cDNA fragment.

Expression and distribution of human clone 36: Northern membranes containing RNA from various human tissues were probed with radiolabeled DNA fragment from clone 36. All human tissues studied (human fetal brain, lung, liver and kidney and adult human cerebellum, cerebral cortex, spinal cord, occipital lobe, frontal lobe, temporal lobe, putamen, spinal cord, amygdala, caudatus nucleus, callosum corpus, hippocampus, whole brain, subtalamic nucleus and thalamus) were negative for expression of this receptor after 2 weeks exposure.

Example 5: Calcium signaling in response to angiotensin I-III

The coding sequence of human clone 24 was transferred to the pcDNA3 vector and was modified to add the haemagglutinin terminal to the C-terminus of the receptor sequence. This clone, designated pcDNA3-HML-HA24, was transfected into HEK293 cells using a modified CaCl ₂ method (Maniatis, Molecular Cloning:

Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)). Cells were cultured in culture medium at 37 ° C, 5% CO ₂ and were diluted 10-fold every 3 days.

Cells were plated on 90 mm tissue culture discs (5 x 10 5 cells per flask) in Dulbecco's modified essential medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum (FBS), 100 U / ml penicillin, 100 g / ml streptomycin and 0.25 g / ml fungizone. One day after seeding, cells were transiently transfected with 30 g of plasmid DNA per disc. Cells were harvested 48 hours after transfection and analyzed. Gene expression was first confirmed by immunoprecipitation and western blotting with an anti-haemagglutinin antibody. A protein of approximately 43 kD was detected in stable as well as transiently transfected HEK293 cells, but not in control cells.

Stably transfected HEK293 cells were obtained after approximately 21 days of selection in culture medium containing 800 g / ml G418. Measurement of calcium signaling was performed using one of these stably transfected cell lines,

293 / pcDNA3-HML-HA24. Cells were cultured on 24 mm round slides to 50-70% confluency. After washing the cells with 1.8 NBS buffer (135 mM Naci, 5 mM KCl, 1.2 mM MgCl ₂ , 1.8 mM CaCl ₂ , 5 mM glucose and 10 mM HEPES, pH 7.3), the cells were incubated for 1 hour. hours at ambient temperature in the presence of 0.5 ml of 3.5 M FURA-2AM (Molecular Probe, F-1221) diluted in 1.8 NBS. The cells were then washed three times with 1.8 NBS and incubated for an additional 30 minutes at ambient temperature. Calcium removal was measured using a PTI (Photon Technology International) D104 photometer equipped with a PTI Delta RAM High Speed Illuminator with multiple wavelengths, a PTI SC500 Shutter Controller, a PTI LPS220 ARC light source, and a PTI FELIX software, v. 1.2. Groups of 2 to 8 cells were selected and isolated using a photometer membrane. Cells were exposed. • 4 4 44 44 •• 4 · 4 4 · 4 4 · 4

44 »4 44 4 ····

4944944 4 44444 94 4 • 4 4 94 4 444

No. 4,944,444, 4 light 340 and 380 nm and light of 510 nm wavelengths emitted by the cells were sensed. Angiotensin I, II and II were gradually added - in different order in different experiments - and then bradykinin was added as a positive control. A significant response was obtained with angiotensin II and III stimulation. The addition of angiotensin I produced no response.

All publications cited herein are hereby incorporated by reference. From the complete description of the invention, it will be apparent to those skilled in the art that the invention may be practiced using various conditions, parameters, and the like without departing from the scope of the invention or any embodiment thereof.

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·· · · · · · · · ·

List of Sequences (1) General Information (i) Applicant: Astra Pharma Inc, Canada (ii) Title of the invention: New G-protein coupled receptor (iii) Number of sequences: 22 (iv) Mailing address:

(A) Address: Astra AB, Patent Department (B) Street: S-151 85 Sodertalje (C) City: Sodertalje (D) Country:

(E) Country: Sweden (F) ZIP: no (v) Computer read form:

(A) Media Type: Floppy Disk (B) Computer: IBM PC Compatible (C) Operating System: PC-DOS / MS-DOS (D) Software: PatentIn Release # 1.0, Version # 1.30 (vi) Data ^ Current Applications:

(A) Application Number:

(B) Filing Date:

(C) Classification:

(ix) Telecommunication Information:

(A) Telephone: 46-8 553 26000 (B) Telephone: 46-8 553 28820 (2) Information for SEQ ID NO: 1:

(i) Sequence characteristics:

(A) Length: 337 amino acids (B) Type: amino acid (C) Chain: not relevant (D) Topology: not relevant (ii) Molecule type: protein (iii) Hypothetical: no (iv) Antisense: no (xi) Description SEQ ID NO: 1:

• 9 • 99 • 99 • 99 • • 99 · · · · · · · · · · · · · · · · · · · ·

MeC Val Cys Val Phu Val Thu Thr Thr Thr Thr Thr Gly Arg Phe Val Ser Met Asp

10 15

Pro Thr Ile Ser Ser. Leu Ser Thr Glu Ser Thr Thr Leu Asn Lys Thr

25 30

Gly His Pro Ser Cys Arg Pro Ile Leu Thr Leu Ser Phe Leu Val Pro

40 45

Ile Ile Thr Leu Gly Leu Ala Gly Asn Thr Ile Val Leu Trp Leu

55 60

Leu Gly Phe Arg Met Arg Arg Lys Ala Ile Val Val Tyr Val Leu Asn

71 15 80

Leu Ser Leu Ala Asp Ser Phe Phe Leu Cys His Phe Ile Asp Ser

90 95

Leu Met Arg Asle Phe Tyr Gly Ile Tyr Ala His Lys Leu Ser

100 105 110

Lys Glu Leu Gly Asn Val Ala Phe Ile For Tyr Ile Ser Gly Leu

115 120 125

Ser Ile Leu Ser Ilu Ser Thr Glu Arg Cys Leu Ser Val Leu Trp

130

135

140 • ·

Pro Ile Trp His Cys His Arg

145 150

Cys Val Leu Ile Trp Val Leu Ser

165

Phe Phe Ser Gly Phe Leu Gly Glu

180

Val Thr Ala Phe

195 200

Phe Gly Ser Ser Leu Ala Leu Leu

210 215

Arg Lys For Leu Ser

225 230

Val Tyr Leu Ile Cys

245

Tyr Trp Phe Gly

260

Val Thr Val Val Leu Ser Cys Val

For Arg Asn Met Ser Ala Ile Ile

155 160

Phe Leu Met Gly lele Leu Asp Trp

170 175

Thr His His His Leu Trp Lys Asn

185 190

Leu Ile Phe Leu

205

VaL Ile Leu Cys Gly Ser Arg

220

Val Thr Ile Ser Leu Thr Val Met

235 240

Leu Gly Leu

250 255

Tyr Pro Phe Cys. His Ile Tyr Gin

265 270

Asn Ser Ser Ala Asn For Ile Ile

275

280

2B5 »4 4 4

Tyr Phe Leu Val Gly Ser

290 295 300

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

305 310 315 320

Tyr Thr Asp Ser Val Val Gin Lys Pro Thr Glu Arg Arg

325 330 335 (2) Information for SEQ ID NO: 2:

(i) Sequence characteristics:

(A) Length: 1011 base pairs (B) Type: nucleic acid (C) String: double (D) Topology: linear (ii) Molecule type: DNA (genomic) (iii) Hypothetical: no (iv) Antisense: no ( (xi) Sequence description: SEQ ID NO: 2:

ATGGTTTGTG TTCTCAGGGA CACTACTGGA AGATTTGTGA GCATGGATCC AACCATCTCA 60 TCCCTCAGTA CAGAATCTAC AACACTGAAT AAAACTGGTC ATCCCAGTTG CAGGCCAATC 120 CTCACCCTGT CCTTCCTGGT CCCCATCATC ACCCTGCTTG GATTGGCAGG AAACACCATT 180 GTACTCTGGC TCTTGGGATT CCGCATGCGC AGGAAAGCCA TCTCAGTCTA CGTCCTCAAC 240 CTGTCTCTGG CAGACTCCTT CTTCCTCTGC TGCCATTTTA TTGACTCTCT GATGCGGATC 300 ATGAACTTCT ATGGCATCTA TGCCCATAAA TTAAGCAAAG AAATCTTAGG CAATGTAGCA 360 TTCATTCCCT ATATCTCAGG CCTGAGCATC CTCAGTGCTA TCAGCACGGA GCGCTGCCTG 420

• · ··· · · · ··· · ··· · ···

33 • · · • · · · TCTGTATTGT GGCCAATCTG GTACCACTGC CACCGCCCAA GAAACATGTC AGCTATTATA 480 TGTGTTCTAA TCTGGGTTCT GTCCTTTCTC ATGGGCATCC TTGACTGGTT TTTCTCAGGA 540 TTCCTGGGTG AGACTCACCA TCATTTGTGG AAAAATGTTG ACTTTATTGT AACTGCATTT 600 CTGATTTTTT TATTTATGCT TCTCTTTGGG TCCAGTCTGG CGCTACTGGT GAGGATCCTC 660 TGTGGTTCCA GACGGAAAGC ACTGTCCAGG CTGTACGTTA CAATCTCTCT CACAGTGATG 720 GTCTACCTCA TCTGCGGCCT GCCTCTCGGG CTTTACTTGT TCCTGCTATA TTGGTTTGGG 780 ATCCATTTAC attatccctt TTGTCACATT TACCAAGTTA CTGTGCTCCT GTCCTGTGTG 840 AACAGCTCTG CCAACCCCAT CATTTACTTC CTTGTAGGGT CCTTTAGGCA CCGTAAAAAG 900 CATCGGTCCC TCAAAATGGT TCTTAAAAGG GCTCTGGAGG AGACTCCTGA GGAGGATGAA 960 TATACAGACA GCCATGTTCA GAAACCCACT GAGATCTCAG AAAGGAGATG T 1011

(2) Information for SEQ ID NO: 3:

(i) Sequence characteristics:

(A) Length: 322 amino acids (B) Type: amino acid (C) String: not relevant (D) Topology: not relevant β · · · • · · · · · · · · · · · · · · · · · · · · · · · · · · · .

• · · · · · «· ···· .. ♦ _β • · · ♦ · • · · · · · (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) antisense: no (xi) Sequence Description: SEQ ID NO: 3:

Met Asp For Thr Ile For Val Leu Gly Thr Lys Leu Thr For Ile Asn

5 10 15

Gly Arg Glu Glu Thr Pro Cys Tyr Asn Gin Thr Leu Phe Thr Gly

25 30

Leu Thr Cys Ile Ile Ser

40 45

Leu Trp Leu Gly Cys Arg Arg Arg Arg Asn Ala Val Ser Ile Tyr

55 60

Ile Leu Asn Leu Val Ala Ala Asn Phe Leu Phe Leu Ser Gly His Ile

70

Ile Phe Ser Pro Leu Pro Leu Ile Asn Ile Arg His Pro Ile Ser Lys

90 95

Ile Leu Ser Thr Phe Thr Phe Ile Gly Leu Ser Met

100 ALIGN!

105

110

Leu Ser Ala Ile

115 120

Trp Tyr His Cys Arg For Arg Tyr Leu

130 135

Leu Leu Trp Ala Leu Ser

145 150

Cys Asp Phe Leu Phe Ser Gly Ala Asn Ser

165 170

Asp Phe Ile Thr. Ile Ala Trp Leu Val Phe

180 185

Gly Ser Ser Leu Val Leu Val Arg Ile

195 200

Met Leu Thr Arg Leu Thr Val Thr Ile

210 215

Phe Leu Cys Gly Leu For Phe Gly Ile

225 230

Arg Ile His Leu Php

245 250

Ser Ile Phe Leu Ser Ala Leu Asn Ser Ser * · Ser Ser Ser · Ser Ser v v v v v v v v v v v v v v v ···························

Ser Ile Leu Trp For Ile

125

Ser Cys Val Cys Val

140

Ile Leu Glu

155 160

Val Trp, Cys Glu Thr Ser

175

Leu Cys Val Val Leu Cys

190

Leu Cys - Gly Ser Arg Lys

205

Leu Val Le Th Val Val Le Val

220

Gin Trp Ala Leu Ph. Ser

235 240

Cys His Val His Leu Val ²⁵⁵

Ala Asn For Ile Ile Tyr

260

265

270 • · · · · · · · · · · · · · · · · · · · · · · ·

Phe Phe Val Gly Ser Phe Arg Gin As G Arg As G Arg As G Leu Lys 275 280 ²⁸⁵

Leu Val Le Gin Arg Ala Leu Gin Glu Glu 290 295 ⁸⁰⁰

Gly Gly Trp Leu Glu Glu Thr Leu Glu Leu Ser Gly Ser Lys Leu 305 31 ° 315 ³²⁰

Glu Gin (2) Information for SEQ ID NO: 4:

(i) Sequence characteristics:

(A) Length: 969 bp (B) Type: nucleic acid (C) String: double (D) Topology: linear (ii) Molecule type: DNA (genomic) (iii) Hypothetical: no (iv) Antisense: no ( (xi) Sequence description: SEQ ID NO: 4:

ATGGATCCAA CCATCCCAGT CTTGGGTACA AAACTGACAC CAATCAACGG ACGTGAGGAG 50

ACTCGTTGCT ACAACCAAAC CCTGAGCTTC ACGGGGCTGA CGTGCATCAT TTCCCTTGTC 120

GCGCTGACAG GAAACGCGGT TGTGCTCTGG CTCCTGGGCT GCCGCATGCG CAGGAACGCT 180

GTCTCCATCT ACATCCTCAA CCTGGTCGCG GCCAACTTCC TCTTCCTTAG CGGCCACATT 240

ATATTTTCGC CGTTACCCCT CATCAATATC CGCCATCCCA TCTCCAAAAT CCTCAGTCCT 300 GTGATGACCT TTCCCTACTT TATAGGCCTA AGCATGCTGA GCGCCATCAG CACCGAGCGC 360 TGCCTGTCCA TCCTGTGGCC CATCTGGTAC CACTGCCGCC GCCCCAGATA CCTGTCATCG 420 GTCATGTGTG TCCTGCTCTG GGCCCTGTCC CTGCTGCGGA GTATCCTGGA GTGGATGTTC 480 TGTGACTTCC TGTTTAGTGG TGCTAATTCT GTTTGGTGTG AAACGTCAGA TTTCATTACA 540 ATCGCGTGGC TGGTTTTTTT ATGTGTGGTT CTCTGTGGGT CCAGCCTGGT CCTGCTGGTC 600 AGGATTCTCT GTGGATCCCG GAAGATGCCG CTGACCAGGC TGTACGTGAC CATCCTCCTC 660 ACAGTGCTGG TCTTCCTCCT CTGTGGCCTG CCCTTTGGCA TTCAGTGGGC CCTGTTTTCC 720 AGGATCCACC TGGATTGGAA AGTCTTATTT TGTCATGTGC ATCTAGTTTC cattttcctg 780 TCCGCTCTTA ACAGCAGTGC CAACCCCATC ATTTACTTCT TCGTGGGCTC CTTTAGGCAG 840 CGTCAAAATA GGCAAAACCT GAAGCTGGTT CTCCAAAGGG CTCTGCAGGA CACGCCTGAG 900 GTGGATGAAG GTGGAGGGTG GCTTCCTCAG GAAACCCTGG AGCTGTCGGG AAGCAAATTG 960

GAGCAGTGA

969 »-» - - - - -

· Β β β i i i i i i i i i i i i i

* · ^R '·· ·· (2) Information for SEQ ID NO: 5:

(i) Sequence characteristics:

(A) Length: 322 amino acids (B) Type: amino acid (C) Chain: not relevant (D) Topology: not relevant (ii) Molecule type: protein (iii) Hypothetical: no (iv) Antisense: no

(xi) Description sequence: SEQ ID NO: 5: Met Asp Pro Thr Val For Val Leu Gly Thr Glu Leu Thr For Ile Asn 1 5 10 15 Dec Gly Arg Glu Glu Thr For Cys Tyr Lys Gin Thr Leu Ser Phe Thr Gly 20 May 25 30 Leu Thr Cys Ile Val Ser Ala Leu Thr Gly Asn 35 40 45 Leu Trp Leu Gly Cys Arg Met Arg Arg Asn Ala Val Ser Ile Tyr 50 55 60 Ile Leu Asn Leu Val Ala Ala Asp Phe Leu Phe Leu - Ser Gly His Ile 65 70 75 80 Ile Cys Ser For Leu Arg Leu Ile Asn Ile Ser His Pro Ile by Ser Lys 55 90 95 Badly Leu Ser For Val Met Thr Phe For Tyr Phe Ile Gly Leu

100 ALIGN!

105

110 «

# 9 - φ « _β

Leu Asn. Ala Ile Ser Thr Glu Cys Leu Ser Ile Leu Trp Pro Ile

115 120 125

Trp Tyr His Cys Arg Arg

130

Leu Leu Trp Ala Ser Ser Leu Leu Arg Ser Ile Leu Glu Trp Met Phe

145 150 155 160

Cys Asp Phe Leu Phe Ser Cly Glu Thr Ser

165 170 175

Asp Phe Ile Thr Ile Ala Trp Phu Leu Phu Leu Arg Val Leu Cys

180 185 190

Gly Ser Ser Lys Ile Leu Cys Gly Ser Lys

195 200 205

Met Leu Thr Arg Leu Thr Val Thr Ile Leu Thr Val Leu Thr

210 215 220

Phe Leu Cys Gly Leu Pro Phe Gly Ile Gin Trp Ala Leu Phe Ser ²²⁵ 230 235 240

Arg Ile His Leu Asp Trp

245

250

255 · • 0 * 0 255 0 0 0 0 0 0 0 0 · · · · · · ♦ «... ....

and

Ser Ile Phe Leu Ser Ala Leu As Ser Ser Ala Asn For Ile Ile Tvr!

and

250 265 270

Phe Phe Mec Gly Ser Phe Gin Leu Gin Asn Arg Lys Thr Leu Lys

275 280 285

Leu Gin Glu Asp Leu Gin Glu Thr For Glu Glu Glu

290 295 300

Gly Trp Trp Leu Glu Glu Thr Leu Glu Leu Ser Gly Ser Lys Leu

305 310 315 320

Glu Ile (2) Information for SEQ ID NO: 6:

(i) Sequence characteristics:

(A) Length: 969 bp (B) Type: nucleic acid (C) String: double (D) Topology: linear (ii) Molecule type: DNA (genomic) (iii) Hypothetical: no (iv) Antisense: no ( (xi) Sequence description: SEQ ID NO: 6:

ATGGATCCAA CCGTCCCAGT CTTGGGTACA GAACTGACAC CAATCAACGG ACGTGAGGAG 60

ACTCCTTGCT ACAAGCAGAC CCTGAGCTTC ACGGGGCTGA CGTGCATCGT TTCCCTTGTC 120

GCGCTGACAG GAAACGCGGT TGTGCTCTGG CTCCTGGGCT GCCGCATGCG CAGGAACGCT 180

GTCTCCATCT acatcctcaa CCTGGTCGCG GCCGACTTCC TCTTCCTTAG CGGCCACATT 240

ATATGTTCGC CGTTACGCCT CATCAATATC AGCCATCCCA TCTCCAAAAT CCTCAGTCCT 300

GTGATGACCT TTCCCTACTT TATAGGCCTA AGCATGCTGA ACGCCATCAG CACCGAGCGC 360

TGCCTGTCCA TCCTGTGGCC CATCTGGTAC CACTGCCGCC GCCCCAGATA CCTGTCATCG 420 GTCATGTGTG TCCTGCTCTG GGCCCCGTCC CTGCTGCGGA GTATCCTGGA GTGGATGTTC 480 TGTGACTTCC TGTTTAGTGG TGCTGATTCT gttcggtgtg AAACGTCAGA TTTCATTACA 540 ATCGCGTGGC TGGTTTTTTT ACGTGTGGTT CTCTGTGGGT CCAGCCTGGT CCTGCTGGTC 500 aggattctct GTGGATCCCG GAAGATGCCG CTGACCAGGC TGTACGTGAC CATCCTCCTC 660 ACAGTGCTGG rp <»tr *« τ 'r · ** (· * r * * τ » CTGTGGCCTG CCCTTTGGCA TTCAGTGGGC 720 aggatccacc TGGATTGGAA AGTGTTATTT TGTCATGTGC ATCTAGTTTC CATTTTCCTC · 730 TCCGCTCTTA ACAGCAGTGC CAACGCCATC ATTTACTTCT TCATGGGCTC CTTTAGGCAG 840 CTTCAAAACA GGAAGACCCT CAAGCTGGTT CTCCAGAGGG ATCTGCAGGA CAC3CCTGAG 900 GTGGATGAAG GTGGATGGTG gcttcctcag GAAACCCTGG AGCTGTCGGG AAGCAAATTG 960

GAGA7C7GA 969 (2) Information for SEQ ID NO: 7:

(i) Sequence characteristics:

(A) Length: 322 amino acids (B) Type: amino acid (C) String: not relevant (D) Topology: not relevant • · «· · ······· · ··· (Ii) Type of molecule: protein (iii) Hypothetical: no (iv) Antisense: no (xi) Sequence description: SEQ ID NO: 7:

Mac Asp Thr Val Ser Thr Leu Asp Thr Glu Leu Thr Pro Press Asn

10 15

Glu Thr Glu Glu Thr Leu Cys Tyr Lys

25 30

Leu Thr Cys Xle Val Ser

40 45

Leu Trp Leu Gly Cys Arg Arg Arg Arg Asn Ala Phe Ser Ile Tyr

S0 55 60

Ile Leu Asn Leu

70

Ile Tyr Ser Leu Leu Ser Phe Ile Ser Ile For His Thr Ile Ser Lys

90 95

Ile Leu Tyr For Val Met Met Phe Ser Tyr Phe Ala Gly Leu Asn Phe

100 105 110

Leu Ser Val Val Thr Asp Arg Cys Leu Ser Val Leu Trp Pro Ile

115 120 125

Trp Tyr Arg Cys His Arg

130

135

140 · · · · · · · · · · · · · · · · · ·

Leu Leu Trp Ala Leu

145 150

Cys Gly Phe Leu

165

Asp Phe Ile Thr

ISO

Gly Ser Ser Leu Val Leu Leu Ile

195 200

Arg Ile Leu Glu Trp Mec Leu

155 160

Asp Ser Ala Trp, Cys Gin Thr Ser

170 _ 175

Ile Phe Leu Cys

185 190

Arg Ile Leu

205

Ile Leu Le Thr Arg Leu Le Thr Ile Leu Le Thr Val Leu Val

210 215 220

Phe Leu Cys Gly Leu Phe Leu Phe Leu Phe Leu

(+420) 225 230 235 240

Trp Ile His Val

245 250 255

Ser Ile Phe Leu Ser Ala Leu As Ser Ser Ala Asn For Ile Ile Tyr

250 265 270

Phe Phe Val Gly Ser. Leu Arg Gin Arg Gin Asn Arg Gin Asn Leu Lys

275 280 285

Leu Val Le Gin Arg Ala Leu Gin Glu Glu ²⁹⁰ 295 300

Gly Gly Trp Leu For Gin Glu Thr Leu Glu Leu Ser Gly Ser Arg Leu

305

310

320

Glu Gin (2) Information for SEQ ID NO: 8:

(i) Sequence characteristics:

(A) Length: 969 base pairs (B) Type: nucleic acid (C) Chain: double (D) Topology: linear (ii) Molecule type: DNA (genomic) (iii) Hypothetical: no (iv) Antisense: no ( (xi) Sequence description: SEQ ID NO: 8:

ATGGATCCAA CCGTCTCAAC CTTGGACACA GAACTGACAC CAATCAACGG AACTGAGGAG

ACTCTTTGCT ACAAGCAGAC CTTGAGCCTC ACGGTGCTGA CGTGCATCGT TTCCCTTGTC

GGGCTGACAG GAAACGCAGT TGTACTCTGG CTCCTGGGCT GCCGCATGCG CAGGAACGCG 130

TTCTCCATCT ACATCCTCAA CTTGGCCGCA GCAGACTTCG TCTTCCTCAG CGGCCGCCTT 240

ATATATTCCC TGTTAAGCTT CATCAGTATC CCCCATACCA TCTCTAAAAT CCTCTATCCT 300

GTGATGATGT TTTCCTACTT TGCAGGCCTG AACTTTCTGA GTGCCGTGAG CACCGATCGG 3o0

TGCCTGTCCG TCGTGTGGCC CATCTGGTAC CGCTGCCACC GCCCCACACA CCTGTCAGCG 420

GTGGTGTGTG TCCTGCTCTG GGCCCTGTCC CTGCTGCGGA GCATCCTGGA ATGGATGTTA 480 · · · · · ··· ···· · · · · ···· · ······ · ······ · ······ · · »

TGTGGCTTCC TGTTCAGTGG TGCTGATTCT GCTTGGTGTC AAACATCAGA TTTCATCACA 540

GTCGCGTGGC TGATTTTTTT ATGTGTGGTT CTCTGTGGGT CCAGCCTGGT CCTGCTGATC 600

AGGATTCTCT GTGGATCCCG GAAGATACCG CTGACCAGGC TGTACGTGAC CATCCTGCTC 660

ACAGTACTGG TCTTCCTCCT CTGTGGCCTG CCCTTTGGCA TTCAGTTTTT CCTATTTTTA '20

TGGATCCACG TGGACAGGGA AGTCTTATTT TGTCATGTGC ATCTAGTTTC CATTTTCCTG 780

TCCGCTCTTA ACAGCAGTGC CAACCCCATC ATTTACTTCT TCGTGGGCTC CCTTAGGCAG 840

CGTCAAAATA GGCAGAACCT GAAGCTGGTT CTCCAGAGGG CTCTGCAGGA CACGCCTGAG 900

GTGGATGAAG GTGGAGGGTG GCTTCCTCAG GAAACCCTGG AGCTGTCGGG AAGCAGATTG 960

GAGCAGTGA 969 (2) Information for SEQ ID NO: 9:

(i) Sequence characteristics:

(A) Length: 322 amino acids (B) Type: amino acid (C) Chain: not relevant (D) Topology: not relevant (ii) Molecule type: protein (iii) Hypothetical: no (iv) Antisense: no (xi) Description SEQ ID NO: 9:

• · · · ««

Met Thr Glu Le Ser Thr Glu Leu Thr Pro Ile Asn

10 15

Glu Thr Glu Glu Thr Leu Cys Tyr Lys

Leu Thr Cys Ile Val Ser

40 45

Leu Trp Leu Gly Cys Arg Arg Arg Arg Asn Ala Phe Ser Ile Tyr

55 60

Ile Leu Asn Leu

70

Ile Tyr Ser Leu Leu Ser Phe Ile Ser Ile For His Thr Ile Ser Lys

90 95

Ile Leu Tyr For Val Met Met Phe Ser Tyr Phe Ala Gly Leu Ser Phe

100 105 110

Leu Ser Ala Val Ser Thr Glu Arg Cys Leu Ser Val Leu Trp Pro Ile

115 120 125

Trp Tyr Arg Cys His Arg

130

Leu Leu Trp Ala Leu Ser Leu Leu Arg Ile Leu Glu Trp Met Leu

145 150 155 160

Cys Gin Phr Leu Phe Ser Gly Ala Asp Ser Ala Trp Cys Gin Thr Ser

165

170

175

Asp Phe lie Thr Wall Ala Trp Leu Ile Phe 180 185 Gly Ser Ser Leu Wall Leu Leu Ile Arg Ile 195 200 lie For Leu Thr Arg Leu Tyr Wall Thr Ile 210 215 Phe Leu Leu Cys C-ly Leu For Phe Gly Ile 225 230 Trp Ile His Wall Asp Arg Glu Wall Leu Phe 245 250 Ser Ile Phe Leu Ser Ala Leu Asn Ser Ser 260 265 Phe Phe Wall Gly Ser Phe Arg Gin Arg Gin 275 230 Leu Wall Leu Gin Arg Ala Leu Gin Asp Ala 290 295 Gly Gly Gin Leu For Gin Glu Thr Leu Glu

305 310 <4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 ·· · · 4 4 4 · ·· · ·

Val Val Leu Cys i

190

Cys Gly Ser Arg Lys

205

Leu Val Leu Val

220

Phe Phu Leu Phe Phu Leu

240

His Val His Leu Val

255

Asn Pro Ile Ile Tyr

270

Arg Gin Asn Leu Lys

285

Glu Val Asp

300

Ser Gly Ser Arg Leu

320

Glu Gin 4 «4 4 · <4 β (2) Information for SEQ ID NO: 10:

(i) Sequence characteristics:

(A) Length: 969 bp (B) Type: nucleic acid (C) String: double (D) Topology: linear (ii) Molecule type: DNA (genomic) (iii) Hypothetical: no (iv) Antisense: no ( (xi) Sequence description: SEQ ID NO: 10:

ATGGATCCAA CGGTCTCAAC CTTGGACACA GAATTGACAC CAATCAACGG AACTGAGGAG 60 ACTCTTTGCT ACAAGCAGAC CTTGAGCCTC ACGGTGCTGA CGTGCATCGT TTCCCTTGTC 120 GGGCTGACAG GAAACGCGGT TGTGCTCTGG CTCCTGGGCT GCCGCATGCG CAGGAACGCC 180 TTCTCCATCT ACATCCTCAA CTTGGCCGCA GCAGACTTCC TCTTCCTCAG CGGCCGCCTT 240 ATATATTCCC TGTTAAGCTT CATCAGTATC CCCCATACCA TCTCTAAAAT CCTCTATCCT 300 GTGATGATGT TTTCCTACTT TGCAGGCCTG AGCTTTCTGA GTGCCGTGAG CACCGAGCGC 360 TGCCTGTCCG TCCTGTGGCC CATCTGGTAC CGCTGCCACC GCCCCACACA CCTGTCAGCG 420 GTGGTGTGTG TCCTGCTCTG GGCCCTGTCC CTGCTGCGGA GCATCCTGGA GTGGATGTTA 480 TGTGGCTTCC TGTTCAGTGG TGCTGATTCT GCTTGGTGTC AAACATCAGA TTTCATCACA 540 GTCGCGTGGC TGATTTTTTT ATGTGTGGTT CTCTGTGGGT CCAGCCTGGT CCTGCTGATC 600 AGGATTCŤCT GTGGATCCCG GAAGATACCG CTGACCAGGC TGTACGTGAC CATCCTGCTC 660

• • •

ACAGTACTGG * P Γ * £ f * rp CTGTGGCCTG CCCTTTGGCA TTCAGTTTTT 720 TGGATCCACG TGGACAGGGA. AGTCTTATTT TGTCATGTTC ATCTAGTTTC TATTTTCCTG 7S0 TCCGCTCTTA ACAGCAGT GC CAACCCCATC ATTTACTTCT TCGTGGGCTC CTTTAGGCAG. 340 CGTCAAAATA GGCAGAACCT GAAGCTGGTT CTCCAGAGGG CTCTGCAGGA CGCGTCTGAG 900 GTGGATGAAG GTGGAGGGCA GCTTCCTGAG GAAATCCTGG AGCTGTCGGG AAGCAGATTG 9O0

GAGCAGTGA 959 (2) Information for SEQ ID NO: 11:

(i) Sequence characteristics:

(A) Length: 322 amino acids (B) Type: amino acid (C) Chain: not relevant (D) Topology: not relevant (ii) Molecule type: protein (iii) Hypothetical: no (iv) Antisense: no (xi) Description SEQ ID NO: 11:

Mec Asp Pro Thr Lys Leu Thr Pro Ile Asn

I 5 10 .15

Gly Arg Glu Glu Thr For Cys Tyr Lys Gin Thr Leu Ser Phe Thr Val · · · · ··· · ··············· · · ····· · · ·····················

Leu Thr Cys Asle Ala Val val

40 45

Leu Trp Leu Gly Cys Arg Arg Arg Arg Asn Ala Val Ser Ile Tyr

55 60

Ile Leu Asn Leu Ala Ala. Asp Phe Leu Phe Leu Ser Phe Gin Ile

70 5 80

Ile Arg For Leu Arg Leu Ile Asn Ile Ser His Leu Ile Arg Lys

90 95

Ile Leu Val Ser Thr Phe Pro Tyr Phe Thr Gly Leu Ser Met

100 105 110

Leu Ser Ala Ile Ser Thr Glu Cys Leu Ser Val Leu Trp Pro Ile

115 120 125

Trp Tyr Arg. Cys Arg Arg

130

Leu Leu Leu Leu Leu Leu Leu Le Phe Ser Met Leu Glu Trp Are Phe

1 ^ 5 ISO 155 ISO

Cys Asp Phe Leu Phe Ser Gly Ala Ser Ser Trp Cys Glu Thr Ser

165 170 175

Asp Phe Ile For Val Ala Trp Leu Ile Phe Le Cys Val Val Leu Cys

130

185

190 • *

Val Ser Ser Leu Val Ser Leu Val Ser Ile Leu Cys Gly Ser Arg Lys

195 200 205

Met Leu Thr Arg Leu Thr Val Thr Ile Leu Thr Val Leu Thr

210 215 220

Phy Leu Cys Gly Leu Phe Gly Leu Gly Ala Leu Ile Tyr

(+420) 225 230 235 240

Arg Met Leu Asn Leu Glu Val Leu Tyr Cys His Val Tyr Leu Val

245 250 255

Cys Met Ser Leu Ser Ser Leu Asn Ser Ser Ala Asn For Ile Ile Tyr

260 265 270

Phe Phe Val Gly Phu Phe Arg Gin Arg Gin Asn Arg Gin Asn Leu Lys

275 280 285

Leu Val Le Gin Arg Ala Leu Gin Asp Lys For Glu Val Asp Lys Gly

290 295 300

Glu Gly Gin Leu Glu Glu Ser Leu Glu Leu Ser Gly Arg Arg Leu

305 310 315 320

Gly Pro (2) Information for SEQ ID NO: 12:

(i) Sequence characteristics:

(A) Length: 969 pairs of basses (B) Type: nucleic acid (C) String: double • · «· · · · · · ···························· (D) Topology: linear (ii) Molecule type: DNA (genomic) (iii) Hypothetical: no (iv) Antisense: no (xi) Description SEQ ID NO: 12:

ATGGATCCAA CCGTCCCAGT CTTGGGTACA AAACTGACAC CAATCAACGG ACGTGAGGAG 60 ACTCCTTGCT ACAAGCAGAC CCTGAGCTTC ACGGTGCTGA CGTGCATCAT TTCCCTTGTC 120 GGACTGACAG GAAACGCGGT TGTGCTCTGG CTCCTGGGCT GCCGCATGCG CAGGAACGCT 180 GTCTCCATCT ACATCCTCAA CCTGGCCGCA GCAGACTTCC TCTTCCTCAG CTTCCAAATT 240 ATACGTTCGC CATTACGCCT CATCAATATC AGCCATCTCA T C CG CAAAAT £ i ~ rr * Λ ^ η-ifjirrMRirR 300 GTGATGACCT TACAGGCCTG AGTATGCTGA GCGCCATCAG CACCGAGCGC 360 TGCCTGTCTG TTCTGTGOCC CATCTGGTAC CGCTGCCGCC GCCCCACACA cctgtcagcg 420 GTCGTGTGTG TCCTGC7CTG GGGCCTGTCC CTGCTGTTTA GTATGCTGGA GTGGAGGTTC 430 TGTGACTTCC TGTTTAGTGG TGCTGATTCT AGTTGGTGTG AAACGTCAGA TTTCATCCCA 540 GTCGCGTGGC ATGTGTGGTT CTCTGTGTTT CCAGCCTGGT CCTGCTGGTC 600 AGGATCCTCT GTGGATCCCG GAAGATGCCG CTGACCAGGC TGTATGTGAC CATCCTGCTC 660 ACAGTGCTGG TCTTCCTCCT CTGCGGCCTG CCCTTCGGCA TTCTGGGGGC CCTAATTTAC 720

• ·

AGGATGCACC TGAATTTGGA AGTCTTATAT TGTCATGTTT ATCTGGTTTG CATGTCCCTG 780 TCCTCTCTAA ACAGTAGTGC CAACCCCATC ATTTACTTCT TCGTGGGCTC CTT7AGGCAG 840 CGTCAAAATA GGCAGAACCT GAAGCTGGTT CTCCAGAGGG CCTGCAGGA CAAGCCTGAG 900 GTGGATAAAG GTGAAGGGCA GCTTCCTGAG GAAAGCCTGG AGCTGTCGGG AAGGAGATTG 950

GGGCCATGA (2) Information for SEQ ID NO: 13:

(i) Sequence characteristics:

(A) Length: 322 amino acids (B) Type: amino acid (C) Chain: not relevant (D) Topology: not relevant (ii) Molecule type: protein (iii) Hypothetical: no (iv) Antisense: no (xi) Description SEQ ID NO: 13:

Mec Asp Pro Thr Lys Leu Thr Pro Ile Asn

5 10 15

Gly Arg Glu Glu Glu Thr Pro Cys Tyr Gn Thr Leu Ser Phe Thr Val

25 30

Leu Thr Cys Asle Ala Val Val

40 45

Leu Trp Leu Gly Tyr Arg Mec Arg Arg Asn Ala Val Ser Ile Tyr · ·

> ♦ ·· ·· ···································

Ile Leu Asn Leu Ala Ala. Asp Phe Leu Phe Leu Ser Phe Gin Ile

70

Ile Arg Ser Leu Arg Leu Ile Asn Ile Ser His Leu Ile Arg Lys

90 95

Ile Leu Val Ser Thr Phe Pro Tyr Phe Thr Gly Leu Ser Met

100 105 110

Leu Ser Ala Ile Ser Thr Glu Cys Leu Ser Val Leu Trp Pro Ile

115 120 125

Trp Tyr Arg. Cys Arg Arg

130

Leu Leu Trp Gly Leu Leu Leu Phe Ser Met Leu Glu Trp Arg Phe

145 150 155 160

Cys Asp Phe Leu Phe Ser Gly Ala Ser Ser Trp Cys Glu Thr Ser

165 170 175

Asp Phe Ile For Val Val Trp Leu Ile Phe Le Cys Val Val Leu Cys

180 185 190

Val Ser Ser Leu Val Ser Leu Val Ser Ile Leu Cys Gly Ser Arg Lys

195 200 205

Met Leu Thr Arg Leu Thr Val Thr Ile Leu Thr Val Leu Thr

210

215

220>

Phy Leu Cys Gly Leu Phe Gly Leu Gly Ala Leu Ile Tyr

215 230 235 240

Arg Met Leu Asn Leu Glu Val Leu Tyr Cys His Val Tyr Leu Val

245 250 _ 255

Cys Met Ser Leu Ser Ser Leu Asn Ser Ser Ala Asn For Ile Ile Tyr

250 265 270

Phe Phe Val Gly Phu Phe Arg Gin Arg Gin Asn Arg Gin Asn Leu Lys

275 2B0 235

Leu Val Le Gin Arg Ala Leu Gin Asp Lys For Glu Val Asp Lys Gly

290 295 300

Glu Gly Gin Leu For Glu Glu Ser Leu Glu Leu Ser Gly Ser Lys Leu

305 310 315 320

Gly Pro (2) Information for SEQ ID NO: 14:

(i) Sequence characteristics:

(A) Length: 969 bp (B) Type: nucleic acid (C) String: double (D) Topology: linear (ii) Molecule type: DNA (genomic) (iii) Hypothetical: no • 99 99 99 9 9 9 9 9 9 9 99999 9 9 9 9999 9 99 99 99 (Iv) Antisense: no (xi) Sequence description: SEQ ID NO: 14:

ATGGATCCAA CCGTCCCAGT CTTCGGTACA AAACTGACAC CAATCAACGG ACGTGAGGAG 60 ACTCCTTGCT ACAATCAGAC CCTGAGCTTC ACGGTGCTGA CGTGCATCAT TTCGCTTGTC 120 GGACTGACAG GAAACGCGGT TGTGCTCTGG CTCCTGGGCT ACCGCATGCG CAGGAACGCT 180 GTCTCCATCT ACATCCTCAA CCTGGCCGCA GCAGACTTCC TCTTCCTCAG CTTCCAAATT 240 ATACGTTCGC CATTACGCCT CATCAATATC AGCCATCTCA TCCGCAAAAT CCTCGTTTCT 300 GTGATGACCT TTCCCTACTT TACAGGCCTG AGTATGCTGA GCGCCATCAG CACCGAGCGC 360 TGCCTGTCTG TTCTGTGGCC CATCTGGTAC CGCTGCCGCC GCCCCACACA CCTGTCAGCG 420 GTCGTGTGTG TCCTGCTCTG GGGCCTGTCC CTGCTGTTTA GTATGCTGGA GTGGAGGTTC 480 TGTGACTTCC TGTTTAGTGG TGCTGATTCT AGTTGGTGTG AAACGTCAGA TTTCATCCCA 540 GTCGTGTGGC TGATTTTTTT ATGTGTGGTT CTCTGTGTTT CCAGCCTGGT CCTGCTGGTC 600 AGGATCCTCT GTGGATCCCG GAAGATGCCG CTGACCAGGC TGTACGTGAC CATCCTGCTC 66 0 ACAGTGCTGG CTGCGGCCTG CCGTTCGGCA TTGTGGGGGC CCTAATTTAC 723 AGGATGCACC TGAATTTGGA AGTCTTATAT TGTCATGTTT ATCTGGTTTG CATGTCCCTG 730

· · · · · · · · · · · · · · · · · · · · · · · ·

TCCTCTCTAA ACAGTAGTGC CAACCCCATC ATTTACTTCT TCGTGGGCTC CTTTAGGCAG 840

CGTCAAAATA GGCAGAACCT GAAGCTGGTT CTCCAAAGGG CTCTGCAGGA CAAGCCTGAG 900

GTGGATAAAG GTGAAGGGCA GCTTCCTGAG GAAAGCCTGG AGCTGTCGGG AAGCAAATTG 960

GGGCCATGA 969 (2) Information for SEQ ID NO: 15:

(i) Sequence characteristics:

(A) Length: 35 pairs (B) Type: nucleic acid (C) String: single (D) Topology: linear (ii) Molecule type: other nucleic acid (A) Description: / description = synthetic PCR primer (iii) Hypothetical: no (iv) Antisense: no (xi) Sequence description: SEQ ID NO: 15:

GGCCGTCGAC TTCA7CGTCW IGCNG MYCTICCIYT 35 (2) Information for SEQ ID NO: 16:

(i) Sequence characteristics:

(A) Length: 15 pairs (B) Type: nucleic acid (C) String: single (D) Topology: linear (ii) Molecule type: other nucleic acid (A) Description: / description = synthetic PCR primer (iii) Hypothetical: no (iv) Antisense: no (xi) Sequence description: SEQ ID NO: 16:

RHWRCARTAI ATIAT 15 (2) Information for SEQ ID NO: 17:

(i) Sequence characteristics:

(A) Length: 25 pairs (B) Type: nucleic acid (C) String: single (D) Topology: linear (ii) Molecule type: other nucleic acid (A) Description: / description = synthetic PCR primer (iii) Hypothetical: no (iv) Antisense: no (xi) Sequence description: SEQ ID NO: 17:

CGCAGATGAG GTAGTACAGC ATCAC 2:

(2) Information for SEQ ID NO: 18:

(i) Sequence characteristics:

(A) Length: 25 pairs (B) Type: nucleic acid (C) String: single (D) Topology: linear (ii) Molecule type: other nucleic acid (A) Description: / description = synthetic PCR primer (iii) Hypothetical: no (iv) Antisense: no (xi) Sequence description: SEQ ID NO: 18:

CTGTGAGAGA GATGGTAACA TACAG ²² (2) Information for SEQ ID NO: 19:

(i) Sequence characteristics:

(A) Length: 24 pairs «4 · 4»

4 4 4 4 4 (B) Type: nucleic acid (C) String: single (D) Topology: linear (ii) Molecule Type: other nucleic acid (A) Description: / description = synthetic PCR primer (iii) Hypothetical: no (iv) Antisense: no (xi) Sequence description: SEQ ID NO: 19:

GCATCCTTGA CTGGTTCTTC TCAG 24 (2) Information for SEQ ID NO: 20:

(i) Sequence characteristics:

(A) Length: 25 pairs (B) Type: nucleic acid (C) String: single (D) Topology: linear (ii) Molecule type: other nucleic acid (A) Description: / description = synthetic PCR primer (iii) Hypothetical: no (iv) Antisense: no (xi) Sequence description: SEQ ID NO: 20:

GGGTGAGACT CATCATCATT TGTGG (2) Information for SEQ ID NO: 21:

(i) Sequence characteristics:

(A) Length: 30 pairs (B) Type: nucleic acid (C) String: single (D) Topology: linear (ii) Molecule type: other nucleic acid (A) Description: / description = synthetic PCR primer (iii) Hypothetical: no (iv) Antisense: no (xi) Sequence description: SEQ ID NO: 21:

GCAAGCTTTC TGAGCATGGA TCCAACCGTC (2) Information for SEQ ID NO: 22:

(i) Sequence characteristics:

(A) Length: 30 pairs (B) Type: nucleic acid (C) String: single (D) Topology: linear (ii) Molecule type: other nucleic acid (A) Description: / description = synthetic PCR primer (iii) Hypothetical: no (iv) Antisense: no (xi) Sequence description: SEQ ID NO: 22:

CCCTCAGATC TCCAATTTGC TTCCCGACAG

Claims (47)

A protein, other than a natural protein, retaining the qualitative ligand binding characteristics of rat DRR-1, comprising an amino acid sequence consisting of SEQ ID NO: 1. The substantially pure protein of claim 1, wherein the amino acid sequence consists essentially of the amino acid sequence set forth in SEQ ID NO: 1. A substantially pure polynucleotide encoding a protein retaining the qualitative ligand binding characteristics of rat DRR-1 having the amino acid sequence set forth in SEQ ID NO: 1. The polynucleotide of claim 3, which encodes a protein consisting essentially of the amino acid sequence of SEQ ID NO: 1. The polynucleotide of claim 4, having the nucleotide sequence set forth in SEQ ID NO: 2. A protein, other than a natural protein, retaining the qualitative ligand binding characteristics of human DRR-1, comprising an amino acid sequence consisting of SEQ ID NO: 3. The substantially pure protein of claim 6, wherein the amino acid sequence consists essentially of the amino acid sequence set forth in SEQ ID NO: 3. A substantially pure polynucleotide encoding a protein retaining the qualitative ligand binding characteristics of human DRR-1, having the amino acid sequence shown in SEQ ID NO: 3. • (spare page) • · · · ti · * · · · The polynucleotide of claim 8, which encodes a protein consisting essentially of the amino acid sequence of SEQ ID NO: 3. The polynucleotide of claim 9, having the nucleotide sequence set forth in SEQ ID NO: 4. A protein, other than a natural protein, retaining the qualitative ligand binding characteristics of human DRR-2, comprising an amino acid sequence consisting of SEQ ID NO: 5. The substantially pure protein of claim 11, wherein the amino acid sequence consists essentially of the amino acid sequence set forth in SEQ ID NO: 5. A substantially pure polynucleotide encoding a protein retaining the qualitative ligand binding characteristics of human DRR-2, having the amino acid sequence shown in SEQ ID NO: 5. The polynucleotide of claim 13, which encodes a protein consisting essentially of the amino acid sequence of SEQ ID NO: 5. The polynucleotide of claim 14, having the nucleotide sequence set forth in SEQ ID NO: 6. A protein, other than a natural protein, retaining the qualitative ligand binding characteristics of human DRR-3, comprising an amino acid sequence consisting of SEQ ID NO: 7. The substantially pure protein of claim 16, wherein the amino acid sequence consists essentially of the amino acid sequence set forth in SEQ ID NO: 7. A substantially pure polynucleotide encoding a protein retaining (spare side) the qualitative ligand binding characteristics of human DRR-3 having the amino acid sequence shown in SEQ ID NO: 7. The polynucleotide of claim 18, which encodes a protein consisting essentially of the amino acid sequence of SEQ ID NO: 7. 20. The polynucleotide of claim 19 having the nucleotide sequence set forth in SEQ ID NO: 8. A protein, other than a natural protein, retaining the qualitative ligand binding characteristics of human DRR-4, comprising an amino acid sequence consisting of SEQ ID NO: 9. The substantially pure protein of claim 21, wherein the amino acid sequence consists essentially of the amino acid sequence set forth in SEQ ID NO: 9. A substantially pure polynucleotide encoding a protein retaining the qualitative ligand binding characteristics of human DRR-4 having the amino acid sequence shown in SEQ ID NO: 9. The polynucleotide of claim 23, which encodes a protein consisting essentially of the amino acid sequence of SEQ ID NO: 9. The polynucleotide of claim 24, having the nucleotide sequence set forth in SEQ ID NO: 10. A protein, other than a natural protein, retaining the qualitative ligand binding characteristics of human DRR-5, comprising an amino acid sequence consisting of SEQ ID NO: 11. The substantially pure protein of claim 26, wherein the amino acid sequence consists essentially of the amino acid (surrogate side) sequence shown in SEQ ID NO: 11. A substantially pure polynucleotide encoding a protein retaining the qualitative ligand binding characteristics of human DRR-5, having the amino acid sequence shown in SEQ ID NO: 11. The polynucleotide of claim 28, which encodes a protein consisting essentially of the amino acid sequence of SEQ ID NO: 11. 30. The polynucleotide of claim 29 having the nucleotide sequence set forth in SEQ ID NO: 12. A protein, other than a natural protein, retaining the qualitative ligand binding characteristics of human DRR-6, comprising an amino acid sequence consisting of SEQ ID NO: 13. The substantially pure protein of claim 31, wherein the amino acid sequence consists essentially of the amino acid sequence set forth in SEQ ID NO: 13. A substantially pure polynucleotide encoding a protein retaining the qualitative ligand binding characteristics of human DRR-6, having the amino acid sequence shown in SEQ ID NO: 13. 34. The polynucleotide of claim 33, which encodes a protein consisting essentially of the amino acid sequence of SEQ ID NO: 13. 35. The polynucleotide of claim 34 having the nucleotide sequence set forth in SEQ ID NO: 14. 36. An antibody that specifically binds to the DRZR-1 protein of SEQ ID NO: 1, prepared by a method comprising the step of injecting a pharmaceutically acceptable preparation comprising (spare side). · »♦» 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 A protein according to any one of claims 1 or 2, to an animal capable of producing said antibody. An antibody that specifically binds to the DRR-1 protein of SEQ ID NO: 3, prepared by a method comprising the step of injecting a pharmaceutically acceptable composition comprising the protein of any one of claims 6 or 7 to an animal capable of producing said antibody. An antibody that specifically binds to the DRR-2 protein of SEQ ID NO: 5, prepared by a method comprising the step of injecting a pharmaceutically acceptable composition comprising the protein of any one of claims 11 or 12 to an animal capable of producing said antibody. An antibody that specifically binds to the DRR-3 protein of SEQ ID NO: 7, prepared by a method comprising the step of injecting a pharmaceutically acceptable composition comprising the protein of any one of claims 16 or 17 to an animal capable of producing said antibody. An antibody that specifically binds to the DRR-4 protein of SEQ ID NO: 9, prepared by a method comprising the step of injecting a pharmaceutically acceptable composition comprising the protein of any one of claims 21 or 22 to an animal capable of producing said antibody. An antibody that specifically binds to the DRR-5 protein of SEQ ID NO: 11, prepared by a method comprising the step of injecting a pharmaceutically acceptable composition comprising the protein of any one of claims 26 or 27 to an animal capable of producing said antibody. • (spare page) or later. . í * ..! · · · · · · · · · · · · · · · · · · · · · · · · · · · · · An antibody that specifically binds to the DRR-6 protein of SEQ ID NO: 13, prepared by a method comprising the step of injecting a pharmaceutically acceptable composition comprising the protein of any one of claims 31 or 32 to an animal capable of producing said antibody. An antibody that specifically binds to any of the proteins of claims 1, 2, 6, 7, 11, 12, 16, 17, 21, 22, 26, 27, 31 or 32. An expression vector for the expression of rat DRR-1, comprising the polynucleotide of any one of claims 3 or 4. 45. An expression vector for the expression of any of: (i) human DRR-1 comprising the polynucleotide of claims 8 or 9; (ii) human DRR-2 comprising the polynucleotide of claims 13 or 14; (iii) human DRR-3 comprising the polynucleotide of claims 18 or 19; (iv) human DRR-4 comprising the polynucleotide of claims 23 or 24; (v) human DRR-5 comprising the polynucleotide of claims 28 or 29; (vi) human DRR-6 comprising the polynucleotide of claims 33 or 34. 46. A host cell transformed with the vector of claim 44 or 45. 47. Recombinant rat DRR-1, human DRR-1, human DRR-2, human DRR-3, human DRR-4, human DRR-5, human DRR-6, produced by the host cell of claim 46.