WO2001073023A1 - Novel guanosine triphosphate-binding protein-coupled receptor gprv53, gene thereof and production and use of the same - Google Patents

Abstract

A novel gene sustaining hydrophobic domains, which are seemingly seven transmembrane domains characteristic to G-protein-coupled receptors, is successfully isolated by screening human tissue cDNAs. A protein encoded by this cDNA is a histamine receptor having an activity of changing intracellular calcium concentration in response to stimulus with histamine. This gene and the protein which is the translation product thereof are usable in screening a novel ligand and screening an agonist or an antagonist useful as a drug.

Description

 Description Novel guanosine triphosphate binding protein-coupled receptor GPR V53

 And its genes, and their production and use

 The present invention relates to a novel G protein-coupled receptor and its gene, and their production and use. Background art

G protein in-coupled receptors are a general term for a group of cell membrane receptors that transmit signals into cells through activation of trimeric GTP-binding proteins. G protein-coupled receptors are also called “seven-transmembrane receptors” because of their structural characteristics of having seven transmembrane regions in the molecule. G protein-coupled receptors transmit information on various physiologically active substances from the cell membrane into cells through the activation of trimeric GTP-binding proteins and the resulting changes in intracellular second messengers. Intracellular second messengers which are controlled by trimeric GTP-binding proteins, cAMP which via adenylyl two cyclase, although such Ca ^{2 +} which via Fosufuoriba one peptidase C is well known, via trimeric GTP-binding protein Recently, it has become clear that many cellular proteins, such as the control of channels and the activation of kinases, are targets (Annu. Rev. Neurosci. (97) 20: 399). Substrates (ligands) for G protein-coupled receptors are very diverse, including proteinaceous hormones, chemokines, peptides, amines, lipid-derived substances, and proteins such as thrombin. . At present, the number of G protein-coupled receptors for which genes have been identified is less than 300 in humans excluding sensory organ receptors, but the number of G proteins for which ligands have been identified 同 定There are only about 140 types and the ligand is unknown. There are more than 100 types of "fan G protein-coupled receptors". However, it has been assumed that there are at least 400, and in some cases as many as 1000, G-protein coupled receptors in the actual human genome (Trends Pharmacol. Sci. (97) 18: 430). This means that the number of orphan G protein-coupled receptors of unknown function will explode with the rapid progress of genome analysis in the future. Over 90% of drugs created by global pharmaceutical companies so far target interactions in the extracellular space, of which small molecule drugs related to G protein-coupled receptors are mostly Occupy. The basis for this is that diseases associated with G protein-coupled receptors are very rare, including genetic diseases, cerebral nervous system, circulatory system, digestive system, immune system, exercise system, urogenital system, etc. Therefore, many pharmaceutical companies have possessed the orphan G protein-coupled receptor that was revealed by genome analysis, and are now spending less time searching for ligands and elucidating physiological functions. Against this background, there have recently been reports of successful searches for physiological ligands for novel G protein-coupled receptors. For example, calcitonin gene-related peptide receptor (J. Biol. Chem. (96) 271: 11325), orexin (Cell (98) 92: 573) and prolactin-releasing peptide (Nature (98) 393: 272), etc. This case had a great impact as a basic research in the life science field.

In particular, orphan G protein-coupled receptors have received a great deal of attention as targets that are likely to lead to the development of new drugs. In general, since there is no specific ligand for orphan G protein-coupled receptor, it has been difficult to develop its agonist and antagonist. However, in recent years, it has been proposed to create drugs targeting orphan G protein-coupled receptors by combining an extensive compound library and high-throughput screening (Trends Pharmacol. Sci. (97) 18 : 430, Br. J. Pharm. (98) 125: 1387). In other words, the orphan G protein-coupled receptor identified by genetic manipulation is subjected to physiological agonism by functional screening using changes in cAMP and Ca, which are intracellular second messengers. And perform in vivo functional analysis. In this case, by using a compound library to increase the screening throughput, it is possible to discover specific surrogate agonists and orphan gonists for orphan G protein-coupled receptors, and furthermore, to treat specific diseases. Drug development is also theoretically possible.

 One of the G protein-coupled receptors that has attracted attention is the histamine receptor. Histamine is present in mast cells in all peripheral tissues and is one of the major mediators of inflammation-immunity. Histamine also plays an important role in gastric acid secretion of the gastric mucosa. The distribution of Hismin in the brain is in neuronal and non-neuronal cells. Histamine-positive neuronal traits are restricted to the posterior lobe of the hypothalamus, but they are projected to almost the entire brain, including the spinal cord and cerebral cortex. Histamine is thought to be involved in central nervous functions such as arousal response, sexual behavior, and analgesia. It is thought that the physiological activities of these Hismin-mins are expressed through three types of specific receptors, which are called Hl, H2, and H3 (Pharmacol. Rev. (1997) 49: 253). .

The HI receptor (GenBank acc. No. D14436) is expressed in brain, airway muscle, gastrointestinal and digestive organs, micturition-genital tract, circulatory organ, adrenal medulla, other endothelial cells, lymphocytes, etc. In the study of HI receptors, blood vessels and smooth muscle are often targeted. Histamine causes muscle contraction in smooth muscle. It is thought that this reaction is accompanied by an increase in intracellular Ca concentration via inosito 11,4,5_triphosphate by hismin (Eur. J Pharmacol. (1987) 135: 69). In vascular endothelial cells, histamine causes 1) changes in vascular permeability as a result of endothelial cell contraction, 2) proscine cyclin biosynthesis, 3) generation of platelet activating factor (PAT), 4) Von Willebrand factor (VWF) )), And 5) Nitric Oxide (NO) synthesis. The presence of the HI receptor has also been confirmed in T lymphocytes (Gen Pharmacol. (1996) 27: 289). In addition, the physiological functions of HI receptors include catecholamine release in the adrenal medulla (Biochem Pharmacol. (1988) 37: 221), and the inhibitory effect on heart rate in the heart muscle (J Pharmacol. Exp. Ther. (19) 90) 1:71) and the central nervous system (Agents Actions (1990) 30:13). The function of the HI receptor is mediated by a G-protein belonging to the pertussis toxin-insensitive Gq / 11 family, via increasing intracellular inositol phosphate calcium concentration (Br J Pharmacol. (1994) 112: 847). .

 The H2 receptor (GenBank acc. No. AB023486) is frequently expressed in the basal ganglion, amygdala, cerebral cortex, etc. in the brain, but is also expressed at low concentrations in the cerebellum and hypothalamus ( J Neurochem. (1992) 59: 290). H2 receptors are also expressed in the stomach and heart. The role of the H2 receptor in the stomach plays an important role in gastric acid secretion, which has been clarified by studies using H2 receptor-specific anginists (Br. J Pharmacol. 1985) 86: 571). In the heart, it has been reported that the arrhythmic effect and inotropic effect are exerted on atria and ventricles by H2 receptor-mediated hisminin activity (J Pharmacol. Exp. Ther. (1988) 246: 377). ). In addition, smooth muscle relaxation in the respiratory tract, ovary, and vascular smooth muscle (Br. J CI in Pharmacol (1989) 27: 139) and functions in the immune system are known (Pharmacol. Rev. (1990) 42). : 45). Intracellular signaling through these H2 receptors has been confirmed to accumulate cAMP and increase adenylyl cyclase activity (Br. JCI in. Pharmacol (1987) 91: 213).

The H3 receptor was originally present in histamine-containing neurons and was suggested to exist as a pre-synaptic receptor that controls histamine release (Nature (198 3) 302: 832). Since histamine-containing neurons project to the entire cerebral cortex in the mammalian brain, the H3 receptor was assumed to play an important role in brain function (Trends Pharmacol. Sci. (1998) 19: 177). . In addition, the H3 receptor not only plays a role in histamine release, but also controls the release of various neurotransmitters such as acetylcholine, dopamine, gamma-aminobutyric acid, glutamic acid, noradrenaline, and serotonin at pre-synapse. In addition, it is also involved in peripheral neurotransmission in the digestive system, cardiovascular system, and airway (Pharmacol. Rev. (1997) 49: 253). The gene for the H3 receptor (GenBank acc.No.) 007232) was identified in 1999 (Mol. Pharmacol. (1999) 55: 1101). This receptor has only about 20% homology to the HI and H2 receptors in the entire molecule, but 27% and 33% homology in the transmembrane domain. (Trends Pharmacol. Sci. (2000) 21:11). Experimental properties of the H3 receptor suggest the existence of two receptor subtypes, H3a and H3b (Mol. Pharmacol. (1990) 38: 610). Furthermore, it has been reported that eosinophils have receptors with different properties from the above three types of histamine receptors and act on H3 receptor-specific agonists (Am. J Respir. Crit. Care Med. (1994) 14 9: 1506).

 Based on these facts, histamine receptor among G protein-coupled receptors is considered to be an important target for drug discovery research in combination with the wide range of physiological functions of ligand itself. Disclosure of the invention

 The present invention has been made in view of the current situation surrounding such G protein-coupled receptors, and has as its object a novel G protein-coupled receptor, particularly a histamine receptor, and its gene, and production thereof. It is to provide a method and a use. Furthermore, the purpose is to provide these molecules as targets for drug development research.

 The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, by carrying out the polymerase chain reaction using human tissue cDNA as a 銪 type, the characteristic of G protein-coupled receptor We succeeded in isolating a new gene that retains a hydrophobic region considered to be a transmembrane domain. When a search was made for a ligand for a protein encoded by the isolated gene using the change in intracellular calcium concentration as an index, it was found that histamine was a ligand for the protein. These genes and their translation products can be used for screening for new ligands and for screening of agonists and gonists useful as pharmaceuticals.

That is, the present invention relates to novel G protein-coupled receptors and their genes, and their production and use, and more specifically, (1) the DNA according to any one of the following (a) to (d), which encodes a guanosine triphosphate binding protein-coupled receptor;

 (a) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1.

(b) DNA containing the coding region of the nucleotide sequence of SEQ ID NO: 2.

 (c) a DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 1 in which one or more amino acids have been substituted, deleted, added and / or inserted.

 (d) DNA that hybridizes under stringent conditions to DNA consisting of the nucleotide sequence of SEQ ID NO: 2.

 (2) DNA encoding a partial peptide of a protein consisting of the amino acid sequence of SEQ ID NO: 1,

 (3) a vector containing the DNA of (1) or (2),

 (4) a transformant carrying the DNA of (1) or (2) or the vector of (3),

 (5) a protein or peptide encoded by the DNA of (1) or (2),

 (6) the step of culturing the transformant according to (4) and recovering the expressed protein or peptide from the transformant or a culture supernatant thereof; Production method,

 (7) A method for screening a ligand that binds to the protein according to (5),

 (a) contacting a test sample with the protein or peptide according to (5),

(b) selecting a compound that binds to the protein or peptide;

(8) A method for screening a compound having an activity of inhibiting the binding of the protein according to (5) to a ligand thereof, (a) contacting a ligand with the protein or its partial peptide according to (5) in the presence of a test sample, and detecting the binding activity between the protein or its partial peptide and a ligand;

 (b) selecting a compound that reduces the binding activity detected in step (a) as compared to the binding activity in the absence of the test sample;

 (9) A method for screening a compound that inhibits or promotes the activity of the protein according to (5),

 (a) contacting a cell expressing the protein or a partial peptide thereof with a ligand of the protein in the presence of a test sample;

 (b) detecting a change in a cell due to binding of the ligand to the protein or a partial peptide thereof,

 (c) selecting a compound that suppresses or enhances the change in the cells detected in step (b), as compared to the change in the cells in the absence of the test sample,

 (10) The method according to (8) or (9), wherein the ligand is histamine.

(11) The method according to (8) or (9), wherein the change in the cell is a change in cAMP concentration or a change in calcium ion concentration.

 (12) an antibody that binds to the protein of (5),

 (13) a compound isolated by the screening according to any of (7) to (11),

 (14) A pharmaceutical composition comprising the compound according to (13) as an active ingredient, and

(15) a nucleotide having a chain length of at least 15 nucleotides, which is complementary to DNA consisting of the nucleotide sequence of SEQ ID NO: 2 or a complementary strand thereof,

(16) 8-chloro- 1- (4-methyl-topiperazinyl) -5H-dibenzo [b, e] [l, 4] -dazepine or [(4-chloro-phenyl) methyl] -3- (1H -Imidazole-4-yl) A drug for activating the protein according to (5), which comprises propyl ester rubamidothioic acid as an active ingredient.

 In the present invention, the “G protein-coupled receptor” refers to a cell membrane receptor that transmits a signal into a cell through activation of a GTP-binding protein.

 In the present invention, “ligand” refers to a physiological substance that binds to a G protein-coupled receptor and transmits a signal into a cell. Here, “physiological substance” refers to a compound that binds to a G protein-coupled receptor in a living body.

 In the present invention, “agonist” refers to a compound capable of transmitting a signal into cells by binding to a G protein-coupled receptor, and includes physiological substances, artificially synthesized compounds, and naturally occurring compounds. Including.

 In the present invention, the term “angigonist” refers to a compound that inhibits the binding of a ligand to a G protein-coupled receptor or the transmission of a signal into a cell, and is a physiological substance or an artificially synthesized substance. Compounds, including naturally occurring compounds.

The present invention provides a novel G protein-coupled receptor and a DNA encoding the protein. The human-derived cDNA clone included in the present invention and isolated by the present inventors was named "GPRv53". The nucleotide sequence of this cDNA is shown in SEQ ID NO: 2, and the amino acid sequence of the protein encoded by the cDNA is shown in SEQ ID NO: 1. As a result of a BLAST search, the protein encoded by GPRv53 cDNA showed significant amino acid sequence homology with a known G protein-coupled receptor. Specifically, "GPRv53" showed 31% homology to MUSCARINIC ACETYLCHOLINE RECEPTOR M3 (P49578, 639aa). Further, the protein encoded by the GPRv53 cDNA isolated by the present inventors retained a hydrophobic region, which is considered to be seven transmembrane domains characteristic of a G protein-coupled receptor. From these facts, GPRv53 cDNA was considered to encode a protein belonging to the G protein-coupled receptor family. Furthermore, when histamine was allowed to act on the GPRv53 protein expressed on the surface of HEK293 cells, a change in the intracellular calcium concentration was observed. From this, GPRv53 cDNA is a G protein-coupled receptor It turned out that it encodes the hissamine receptor in the family. As described above, the GPRv53 protein of the present invention is an important target for drug discovery, because the Hisamine receptor has a wide range of physiological functions, depending on the properties of his ligand His, as described above. Becomes For example, as will be described later, it is possible to screen for the agonist and gonist of the GPRv53 protein, which is a drug candidate, using the change in intracellular calcium ion (Ca ²⁺ ) concentration due to its activation as an index. . In addition, the present inventors have found that clobenpropit acts not only on the histamine H3 receptor but also on the GPRv53 protein. Therefore, in the process of obtaining a drug specific for the histamine H3 receptor, it is effective to detect whether or not a drug candidate compound binds to the GPRv53 protein and to select a compound that does not bind to the protein. That is, the GPRv53 protein is useful in the development of a drug specific to the Hismin H3 receptor.

 The present invention also provides a protein functionally equivalent to the GPRv53 protein. Here, “functionally equivalent” means that the target protein has the same biological properties as the GPRv53 protein. Biological properties of the GPRv53 protein include an activity of transmitting a signal into a cell through activation of a trimeric GTP-binding protein. The activity includes, for example, an activity of changing intracellular calcium concentration or cAMP concentration by activation thereof in response to histamine stimulation. Whether or not the target protein has such an activity is determined by applying histamine to cells expressing the target protein on its surface, and then changing the intracellular calcium concentration or cAMP concentration. It can be evaluated by detection (see Example 4).

One embodiment of a method for preparing a protein functionally equivalent to the GPRv53 protein includes a method of introducing a mutation into an amino acid sequence in a protein. Such methods include, for example, site-directed mutagenesis (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section 8. 1-8.5))) is included. Amino acid mutations in proteins may also occur in nature. In the present invention, one or more amino acids may be substituted, deleted, inserted and / or substituted in the amino acid sequence (SEQ ID NO: 1) of the GPRv53 protein, whether artificial or natural. Includes proteins mutated by addition or the like and functionally equivalent to the GPRv53 protein. The number and location of the amino acid mutations in these proteins are not limited as long as the function of the GPRv53 protein is maintained. The number of mutations will typically be within 10% of all amino acids, preferably within 5% of all amino acids, and more preferably within 1% of all amino acids.

 Another embodiment of a method for preparing a protein functionally equivalent to the GPRv53 protein includes a method using a hybridization technique and a method using a gene amplification technique. That is, if a person skilled in the art uses hybridization technology (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section 6.3-6.4), GPRv53 DNA sequence encoding the protein

(SEQ ID NO: 2) or a portion thereof, a DNA highly homologous thereto is isolated from a DNA sample of the same or different species, and a protein functionally equivalent to the GPRv53 protein is isolated from the DNA. What you get is what you can usually do. Thus, a protein functionally equivalent to a GPRv53 protein, which is a protein encoded by a DNA that hybridizes with a DNA encoding the GPRv53 protein, is also included in the protein of the present invention. Organisms for isolating such proteins include, but are not limited to, rats, mice, egrets, chicks, birds, birds, and the like, in addition to humans.

Stringent hybridization conditions for isolating DNA that encodes a protein functionally equivalent to the GPRv53 protein usually include conditions of `` lxSSC, 0.1¾SDS, 37 ° C ''. Yes, more severe conditions are about 0.5xSSC, 0.1¾SDS, 42 ° C, and more severe conditions are 0.2xSSC, 0.1¾SDS, 6 5 ° C ”. Thus, the isolation of DNA having higher homology to the probe sequence can be expected as the hybridization conditions become more severe. However, the combination of the SSC SDS and the temperature conditions described above is only an example, and those skilled in the art will recognize the above or other factors that determine the stringency of the hybridization (eg, probe concentration, probe length, The same stringency as described above can be achieved by appropriately combining the hybridization reaction times.

 A protein encoded by DNA isolated using such a hybridization technique usually has a high homology in amino acid sequence with the GPRv53 protein. High homology refers to sequence homology of at least 40% or more, preferably 60% or more, more preferably 80% or more (eg, 90% or more and 95% or more).

 Amino acid sequence and nucleotide sequence identity can be determined by Karl in and Altschul by Argoliz BLAST (Proc. Natl. Acad. Sei. USA 90: 5873-5877, 1993). Based on this algorithm, programs called BLASTN and BLASTX have been developed (Altschul et al. J. Mol. Biol. 215: 403-410, 1990). When a nucleotide sequence is analyzed by BLASTN based on BLAST, the parameters are, for example, score: 100 and word length = 12. In the case of analyzing an amino acid sequence by BLA STX based on BLAST, the parameters are, for example, score = 50 and word length = 3. When using BLAST and Gapped BLAST programs, use the default parameters of each program. The specific methods of these analysis methods are known (http: @ www.ncbi.nlm.nih.gov.).

(1987) Publish. John Wiley & Sons Section 6, 1-6.4) Gene sequence amplification technology (PCR) DNA sequence encoding GPRv53 protein. A primer is designed based on a part of SEQ ID NO: 2), and has a high DNA fragment homology with the DNA sequence encoding GPRv53 protein. It is also possible to obtain a protein functionally equivalent to the GPRv53 protein based on the DNA.

 The present invention also includes a partial peptide of the protein of the present invention. This partial peptide includes a peptide which binds to hismin but does not transmit signals (for example, does not cause a change in intracellular calcium ion concentration). Such a peptide can be a competitive inhibitor of the protein of the present invention. The partial peptide of the protein of the present invention can also be used for producing antibodies. The partial peptide of the present invention can be produced, for example, by a genetic engineering technique, a known peptide synthesis method, or by cleaving the protein of the present invention with an appropriate peptidase. The partial peptide of the present invention usually has at least 8 amino acid residues, preferably at least 12 amino acid residues (for example, at least 15 amino acid residues).

The protein of the present invention can be prepared as a recombinant protein or as a natural protein. The recombinant protein can be prepared, for example, by introducing a vector into which a DNA encoding the protein of the present invention is inserted into an appropriate host cell as described below, and purifying the protein expressed in the transformant. . On the other hand, a natural protein can be prepared using, for example, an affinity column to which an antibody against the protein of the present invention described below is bound (Current Protocols in Molecular Biology edit. Ausubel et al. (1987)). Publish. John Wiley & Sons Section 16. 16.19). The antibody used for affinity purification may be a polyclonal antibody or a monoclonal antibody. In addition, in vitro translation (for example, see “0n the fidelity of mRNA translation in the nuclease-treated rabbit reticulocyte lysate system. Dasso, MC, Jackson, RJ (1989) NAR 17: 3129-3144”) It is also possible to prepare the protein of the present invention. The present invention also provides a DNA encoding the protein of the present invention. The form of the DNA of the present invention is not particularly limited as long as it can encode the protein of the present invention, and includes genomic DNA, chemically synthesized DNA, etc. in addition to cMA. In addition, the present invention DNAs having an arbitrary base sequence based on the degeneracy of the genetic code are included as long as they can encode the above protein. The DNA of the present invention encodes a GPRv53 protein as described above! ) NA sequence (SEQ ID NO: 2) or a part thereof can be isolated by a conventional method such as a hybridization method using a probe or a PCR method using a primer synthesized based on these DNA sequences. .

 The present invention also provides a vector into which the DNA of the present invention has been inserted. The vector of the present invention is not particularly limited as long as it can stably maintain the inserted DNA. For example, if Escherichia coli is used as a host, a vector for cloning is a pBluescript vector (manufactured by Stratagene). Is preferred. When a vector is used for producing the protein of the present invention, an expression vector is particularly useful. As an expression vector, a vector that expresses a protein in a test tube, in a bacterial cell (for example, Escherichia coli), in a cultured cell, or in an individual organism can be used. For example, pBEST for expression in a test tube Vector (Promega), E. coli for pET vector (In vitrogen), cultured cells for pME18S-FL3 vector (GenBank Accession No. AB009864), for living organisms, pME18S vector (Mol Cell l) Biol. 8: 466-472 (1988)). Insertion of the DNA of the present invention into a vector can be carried out in a conventional manner, for example, by a ligase reaction using a restriction enzyme site (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley

& Sons. Section 11.4-11.11).

 The present invention also provides a transformant carrying the DNA of the present invention or the vector of the present invention. The host cell into which the vector of the present invention is introduced is not particularly limited, and various host cells may be used depending on the purpose. Examples of eukaryotic cells for highly expressing a protein include COS cells and CH0 cells. Vectors can be introduced into host cells by, for example, calcium phosphate precipitation, pulsed electroporation, etc.

(Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), Lipofecumin method (GIBC0-BR L (manufactured by L Company) and a known method such as a microinjection method.

 Further, the present invention provides a nucleotide having a chain length of at least 15 nucleotides, which is complementary to DNA encoding the protein of the present invention (DNA comprising the base sequence of SEQ ID NO: 2 or a complementary strand thereof). I do. Here, the “complementary strand” refers to one strand of a single-stranded nucleic acid composed of A: T (but U for RNA) and G: C base pairs with respect to the other strand. Further, the term "complementary" is not limited to a case where the sequence is completely complementary in at least 15 contiguous nucleotide regions, but is at least 70%, preferably at least 80%, more preferably 90%, and still more preferably It suffices to have 95% or more homology on the base sequence. The algorithm for determining homology may use the algorithm described in this specification. Such nucleotides can be used as a probe for detecting and isolating the MA of the present invention, and as a primer for amplifying the DNA of the present invention. When used as a primer, it usually has a chain length of 15 bp to 100 bp, preferably 15 bp to 35 bp. When used as a probe, a nucleotide having a chain length of at least 15 bp containing at least a part or the entire sequence of MA of the present invention is used. Such nucleotides preferably hybridize specifically to DNA encoding the protein of the present invention. "Specifically hybridize" refers to a DNA that hybridizes with a DNA encoding the protein of the present invention (SEQ ID NO: 2) under ordinary hybridization conditions, preferably under stringent conditions, and DNA encoding a protein means not hybridized.

These nucleotides can be used for testing and diagnosing abnormalities of the protein of the present invention. For example, abnormal expression of the DNA encoding the protein of the present invention can be examined by Northern hybridization or RT-PCR using these nucleotides as probes or primers. DNA encoding the protein of the present invention is obtained by polymerase chain reaction (PCR) using these nucleotides as primers. And its expression control region can be amplified and DNA sequence abnormalities can be examined and diagnosed by methods such as RFLP analysis, SSCP, and sequencing.

 In addition, these nucleotides include antisense DNA for suppressing the expression of the protein of the present invention. The antisense DNA has a chain length of at least 15 bp or more, preferably 100 bp, more preferably 500 bp or more, and usually has a chain length of 3000 bp or less, preferably 2000 bp or less in order to cause an antisense effect. Such antisense DNA may also be applied to gene therapy for diseases caused by abnormalities (abnormal function or abnormal expression) of the protein of the present invention. The antisense DNA is prepared, for example, by the phosphorothioate method (Stein, 1988 Physicochemical properties of phosphorothioate oligodeoxynucieotides. Nucleic Acids Res.) Based on the sequence information of the DNA encoding the protein of the present invention (eg, SEQ ID NO: 2). 16, 3209-21 (1988)).

 When used for gene therapy, the nucleotides of the present invention can be used, for example, by using exvl V, a viral vector such as a retrovirus vector, an adenovirus vector, or an adeno-associated virus vector, or a non-viral vector such as a ribosome. It may be possible to administer to patients by the 0 method or the in vivo method.

 The present invention also provides an antibody that binds to the protein of the present invention. The form of the antibody of the present invention is not particularly limited, and includes a polyclonal antibody, a monoclonal antibody, and a part thereof having antigen-binding properties. Also, all classes of antibodies are included. Furthermore, the antibodies of the present invention also include special antibodies such as humanized antibodies.

In the case of a polyclonal antibody, the antibody of the present invention can be obtained by synthesizing an oligonucleotide corresponding to the amino acid sequence of the protein of the present invention according to a conventional method and immunizing a rabbit (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11. 12-11. 13). In the case of a monoclonal antibody, a mouse is immunized with a protein expressed and purified in Escherichia coli according to a conventional method, and a hybridoma cell obtained by fusing the spleen cell and myeloma cell of the mouse. A cell can be prepared and obtained from the hybridoma cell (Current protocols in Molecular Biology edit. Ausube 1 et al. (1987) Publish. John Wiley & Sons. Section 11.4-11.11).

 Antibodies that bind to the protein of the present invention may be used, for example, for the examination and diagnosis of abnormal expression or structural abnormality of the protein of the present invention, in addition to purification of the protein of the present invention. Specifically, for example, proteins are extracted from tissues, blood, or cells, and abnormalities in expression and structure are detected through detection of the proteins of the present invention by methods such as Western blotting, immunoprecipitation, and ELISA. Inspection / presence can be diagnosed.

 It is also conceivable to use an antibody that binds to the protein of the present invention for the purpose of treating a disease associated with the protein of the present invention. The antibody of the present invention can act as an agonist of the protein of the present invention. When the antibody is used for the purpose of treating a patient, a human antibody or a humanized antibody is preferred because of its low immunogenicity. Human antibodies include mice in which the immune system has been replaced with that of a human (e.g., "Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice, endez, MJ et al. (1997) Nat. Genet. 15 : 146-156 "). In addition, humanized antibodies can be prepared by genetic recombination using the hypervariable region of a monoclonal antibody (Methods in Enzymology 203, 99-121 (1991)).

 The present invention also provides a method for screening for a new ligand that binds to the protein of the present invention, using the protein of the present invention. This screening method

 (a) contacting a test sample with the protein of the present invention or a partial peptide thereof,

(b) selecting a compound that binds to the protein or a partial peptide thereof. The test sample is not particularly limited. For example, known compounds and peptides whose ligand activities of various G protein-coupled receptors are unknown (eg, those registered in chemical files) or phage display Biol. (1991) 222, 301-310). Can be. In addition, culture supernatants of microorganisms and natural components derived from plants and marine organisms are also targets for screening. Other examples include, but are not limited to, biological tissue extracts including the brain, cell extracts, and expression products of gene libraries.

 The protein of the present invention used for screening may be, for example, a form expressed on a cell surface, a form as a cell membrane fraction of the cell, or a form bound to an affinity column.

 Specific screening techniques include, for example, a method of contacting a test sample with an affinity column for the protein of the present invention to purify a compound that binds to the protein of the present invention, and a number of known methods such as a West Western plotting method. Methods are available. When these methods are used, the test sample is appropriately labeled, and the binding to the protein of the present invention can be detected using the label. In addition to these methods, a cell membrane expressing the protein of the present invention is prepared and immobilized on a chip, and the separation of the trimeric GTP-binding protein upon ligand binding is determined by surface plasmon resonance.

It is also possible to use (Nature Biotechnology (99) 17: 1105), which is a method of detecting by surface plasmon resonance.

Further, the binding activity between the test sample and the protein of the present invention can be detected by using, as an index, a change in cells caused by binding of the test sample to the protein of the present invention expressed on the cell surface. Such changes include, but are not limited to, changes in intracellular Ca ²⁺ levels and changes in cAMP levels. Specifically, agonist activity for G protein-coupled receptors can be measured by the GTP-S binding method.

As one example of this method, the cell membrane expressing the protein of the present invention was labeled with ³⁵ S in a solution of 20 mM HEPES (pH 7.4), 100 mM NaCl, 10 mM MgCl ₂ , 50 / zM GDP. Mix with GTP yS 400 pM and incubate in the presence and absence of the test sample. After the filtration, filtration can be performed to compare the radioactivity of the bound GTPas.

Further, the G protein of the present invention shares a system for transmitting a signal into a cell through activation of a trimeric GTP-binding protein. Trimeric GTP-binding proteins are classified into three types, depending on the type of intracellular signaling system that activates, Gq type that increases Ca ²⁺ , Gs type that increases cAMP, and Gi type that suppresses cAMP. You. Applying this fact, Gq protein subunits are chimerized with other G protein subunits, and a positive signal during ligand screening results in an increase in Ca2 ⁺ , a Gq intracellular transduction pathway. Is possible. The elevated Ca ²⁺ level can be detected using the repo overnight gene system having TRE (TP A response element) upstream, a staining indicator such as Fluor-3, and a change in the fluorescent protein aequorin as indicators. Similarly, Gs protein subunits are chimerized with other G protein subunits, and a positive signal results in an increase in cAMP, a Gs intracellular transduction pathway, and a CRE (cAMP-responsive element) is upstream. It is also possible to use the change in the reporter gene system in

(Trends Pharmacol. Sci. (99) 20: 118).

 There are no particular restrictions on the host cells that express the protein of the present invention in this screening system, and various host cells may be used depending on the purpose.Examples include COS cells, CH0 cells, HEK293 cells, and the like. it can. Examples of the vector for expressing the protein of the present invention in vertebrate cells include a promoter located upstream of a gene encoding the protein of the present invention, an RNA splice site, a polyadenylation site, a transcription termination sequence, and an origin of replication. Those having the above can be preferably used. For example, pSV2dhfr (Mol. Cell. Biol. (1981) 1, which has the SV40 early promoter,

854-864), pEF-BOS (Nucleic Acids Res. (1990) 18, 5322), pCDM8 (Nature (1987) 329, 840-842), pCEP4 (Invitrogen), and others are G protein-coupled receptors. Is a vector that is useful for expressing Insertion of the MA of the present invention into a vector can be carried out by a ligase reaction using a restriction enzyme site in a conventional manner (Curr Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.4-11. U) Vector introduction into host cells can be performed, for example, by calcium phosphate precipitation, electropulse perforation (Current (1987) Publish. John Wiley & Sons. Section 9.1-9.9), ribofectamine method (GIBCO-BRL), FuGENE6 reagent (Behringer Mannheim), microinjection method. It can be performed by a known method such as

 The present invention also provides a method for screening a compound having an activity of inhibiting the binding between the protein of the present invention and its ligand. This screening method

 (a) a step of contacting a ligand with the protein of the present invention or its partial peptide in the presence of a test sample and detecting the binding activity between the protein or its partial peptide and the ligand; (b) no test sample present Selecting a compound that reduces the binding activity detected in step (a) as compared to the binding activity below.

 The test sample is not particularly limited. For example, a compound group obtained by combinatorial chemistry—technology (Tetrahedron (1995) 51, 8135-8137), or a phage display method (J. Mol. Biol. (1991) 222, 301-310) can be used. In addition, culture supernatants of microorganisms and natural components derived from plants and marine organisms are also targets for screening. Other examples include, but are not limited to, brain and other biological tissue extracts, cell extracts, expression products of gene libraries, synthetic low molecular weight compounds, synthetic peptides, and natural compounds.

 The protein of the present invention used for screening may be, for example, a form expressed on a cell surface, a form as a cell membrane fraction of the cell, or a form bound to an affinity column. Histamine can be used as the ligand.

As a specific screening method, for example, a ligand is labeled with a radioisotope, and the ligand is contacted with the protein of the present invention in the presence of a test sample. A method of detecting a compound that reduces the binding activity between the protein of the present invention and a ligand, based on a label attached to the ligand, as compared with the case where the compound is detected in the absence of a reagent, can be used. As in the case of the screening of the ligand binding to the protein of the present invention, screening can be performed using intracellular changes as an index: that is, the cells expressing the protein of the present invention can be screened. By contacting the ligand in the presence of the test sample and selecting a compound that reduces the change in the cells as compared with the case where detection is performed in the absence of the test sample, the binding between the protein of the present invention and the ligand can be determined. It is possible to screen for compounds that inhibit. Cells expressing the protein of the present invention can be prepared in the same manner as in the above-described screening for a ligand that binds to the protein of the present invention. The compound isolated by this screening is a candidate for the agonist of the protein of the present invention.

 The present invention also provides a method for screening a compound that inhibits or promotes the activity of the protein of the present invention. This screening method comprises the steps of (a) contacting a cell expressing the protein of the present invention with a ligand of the protein in the presence of a test sample, and (b) a change in cells caused by binding of the ligand to the protein of the present invention. And (c) selecting a compound that suppresses or enhances the change in the cells detected in step (b) as compared to the change in the cells in the absence of the test sample.

As a test sample, a group of compounds obtained by combinatorial chemistry technology, a phage display method, etc. are applied in the same manner as in the above-described screening method of a compound that inhibits the binding of a protein to a ligand of the present invention. Created random peptides, culture supernatants of microorganisms, natural components derived from plants and marine organisms, biological tissue extracts, cell extracts, expression products of gene libraries, synthetic low-molecular compounds, synthetic peptides And natural compounds. In addition, a compound isolated by screening a compound that inhibits the binding between the protein of the present invention and a ligand can be used as a test sample. Use histamine as the ligand. Can be. Cells expressing the protein of the present invention can be prepared in the same manner as in the above-described screening for a ligand that binds to the protein of the present invention. Changes in the cells after contact with the test sample can be detected using changes in intracellular Ca ²⁺ levels and cAP levels as indices, as in the screening method described above. When detecting intracellular signal transduction, it is also possible to detect using a measurement system such as a repo overnight assay system using luciferase or the like as a reporter gene.

 As a result of this detection, if the change in cells when the test sample is contacted is suppressed compared to the change in cells when the ligand is contacted in the absence of the test sample, The sample is determined to be a compound that inhibits the activity of the protein of the present invention. Conversely, if the test sample enhances the change in the cells, the compound can be determined to be a compound that promotes the activity of the protein of the present invention. Here, “promoting or inhibiting the activity of the protein of the present invention” means that the action of the protein of the present invention may be either direct or indirect. Indicates that the activity of the protein is promoted or inhibited. Accordingly, compounds isolated by this screening include compounds that act on the protein or ligand of the present invention to inhibit or promote their binding and thereby inhibit or promote the activity of the protein of the present invention. However, compounds that do not inhibit or promote these bindings per se but result in inhibiting or promoting the activity of the protein of the present invention are also included. Such compounds include, for example, compounds that do not inhibit the binding of the protein of the present invention to the ligand, but inhibit or promote intracellular signaling pathways.

The present invention also relates to clozapine (8-clomouth-l- (4-methyl-1-piperazinyl) -5H-diben [b, e] [l, 4] -dazepine) or clobenpropit ([(4-clo The present invention provides an agent for activating the protein of the present invention, which comprises, as an active ingredient, phenyl) methyl] _3- (13--imidazole-4-yl) propyl ester carbamide thionate. The present inventors have found that clozapine, which has been reported to have affinity for the histamine H3 receptor, also has agonist activity for GPRv53. Furthermore, Hiss Evening We found that clobenpropit, a selective synthetic antagonist of the min H3 receptor, showed agonist activity against GPRv53. Therefore, these agents can be used as agents for activating GPRv53. Here, “drug” includes both reagents used for test and research purposes and drugs used for the purpose of preventing or treating diseases.

 When a compound isolated by the screening method of the present invention or clozapine or clobenpropit is used as a drug, the isolated compound itself is directly administered to a patient, and a drug formulated by a known pharmaceutical method is used. Administration can also be carried out as a composition. For example, it is conceivable to formulate and administer a suitable combination with a pharmacologically acceptable carrier or vehicle, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspension and the like. Administration to a patient can be generally performed by a method known to those skilled in the art such as, for example, intraarterial injection, intravenous injection, and subcutaneous injection. The dose varies depending on the weight and age of the patient, the administration method, and the like, but those skilled in the art can appropriately select an appropriate dose.

 If the compound can be encoded by DNA, the DNA may be incorporated into a vector for gene therapy to perform gene therapy. As the gene therapy vector, a viral vector such as a retrovirus vector or an adenovirus vector and a non-viral vector such as a ribosome can be used. The vector can be administered to a living body by an ex vivo method or an in vivo method. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a view showing the results of performing a BLAST search on the entire sequence of SWISS-PR0T using “GPRv53” amino acid sequence as ΓQuery j. MUSCARINIC ACETYLCHOLINE RECEPTOR M3 (P49578) showed the highest homology at 31%.

FIG. 2 is a photograph showing the result of analyzing the expression distribution of the GPRv53 gene. A DNA fragment of about 0.6 kbp was amplified in the thymus, small intestine, peripheral leukocytes, spleen, and large intestine. FIG. 3 is a diagram showing the results of measuring changes in intracellular Ca ²⁺ concentration over time using FLIPR (Molecular Device). Fluorescence intensity was measured when HISTAMINES (-)-Hy-METHYL-, DIHYDR0CHL0iUDE (RaMeHA) (SIGMA), which is a specific histamine and H3 receptor agonist, was added. The maximum value of the fluorescence intensity of the results obtained in the Y-axis, is plotted ligand concentration in the X-axis, GPRv53 at a concentration of 10- ⁶ -10- ⁹ M against His evening Min, for R shed MeHA 10- ⁵ - concentration in dose-dependent changes in intracellular Ca ^{z +} concentration of 10 ⁸ M was observed.

FIG. 4 is a diagram showing the results of changes in intracellular Ca ²⁺ concentration measured over time using FLIPR. The fluorescence intensity was measured when clozapine, a neuroleptic drug, and clobenpropit (SIGMA), a specific antagonist of the H3 receptor, were added. When the obtained fluorescence intensity was plotted on the Y-axis and the reaction time was plotted on the X-axis, specific changes in the intracellular Ca ²⁺ concentration of GPRv53-expressing cells were observed by treatment with both drugs. BEST MODE FOR CARRYING OUT THE INVENTION

 The present invention will be described more specifically with reference to examples, but the present invention is not limited to the following examples. Unless otherwise specified, experiments in this example can be performed according to a known method (Maniatis J. et al. (1982): "Molecular Cloning-A Laboratory Manual" Cold Spring Harbor Laboratory, NY).

[Example 1] Isolation of gene encoding novel G protein-coupled receptor GPRv53 For amplification of novel G protein-coupled receptor GPRv53, Marathon Ready cDNA (Clontech) derived from human fetus was forwarded to 鎵 -type cDNA. 5, -GAATTGTC TGGCTGGATTAATTTGCTAATTTG-3 '(SEQ ID NO: 3) was used as a primer, and 5'-TTAAGAAGATACTGACCGACTGTGTTGT-3' (SEQ ID NO: 4) was used as a reverse primer. PCR was performed using TaKaRa La Taq (Takara Shuzo) at 94 ° C (30 minutes) / 55 after 94 ° C (2.5 minutes). C (30 seconds) / 72. The cycle of C (2 minutes) was repeated 40 times. As a result, a DNA fragment of about 1.2 kbp was amplified. This fragment is cloned using PCR2.1 plasmid (Invitrogen). -I did it. The nucleotide sequence of the obtained clone was analyzed using the ABI377 DNA Sequencer (Applied Biosystems) by the dideoxy-one-mine-one-one method. The clarified sequence is shown in SEQ ID NO: 2.

 This sequence has an open reading frame of 1173 bases (SEQ ID NO: 2). The amino acid sequence (390 amino acids) predicted from the open reading frame is shown in SEQ ID NO: 1. Since the predicted amino acid sequence has seven transmembrane domains that are likely to be characteristic of G protein-coupled receptors, the gene may encode a G protein-coupled receptor. found.

 [Example 2] BLAST search for SWISS-PR 0T in the amino acid sequence of GPRv53, a novel G protein-coupled receptor

 BLAST (Basic local alignment search tool) for SWISS-PR0T in the amino acid sequence of “GPRv53” [SF Altschul et al., J. Mol. Biol., 15: 403-410 (199 0)] search The results are shown in FIG. “GPRv53” is not the same among known G protein-coupled receptors, and showed the highest homology at 31% with MUSCARINIC ACETYLCHOLINE RECEPTOR M3 (P49578, 639a a). This proved that "GPRv53" was a novel G protein-coupled receptor.

 [Example 3] GPRv53 gene expression distribution in tissues

The expression distribution of the GPRv53 gene was analyzed by PCR using Multiple Tissue cDNA Panels (Clontech). 5,-GMTTGTCTGGCTGGA TTAATTTGCTAATTTG-3 '(SEQ ID NO: 3) as the forward primer and 5,-MGMTG ATGTGATGGCAAGGATGTACC-3' (SEQ ID NO: 5) as the reverse primer, and PCR using TaKaRa La Taq (Takara Shuzo) After 94 ° C. (2.5 minutes), a cycle of 94 ° C. (30 seconds) / 55 ° C. (30 seconds) / 72 ° C. (30 seconds) was repeated 40 times. As a result, a DNA fragment of about 0.6 kbp was amplified with cDNA derived from thymus, small intestine, peripheral leukocytes, spleen, and large intestine, and cDNA derived from other organs (lung, prostate, brain, heart, placenta, ovary, testis, Kidney, skeletal muscle, kidney, W

No amplification was observed in the liver, fetal heart, fetal kidney, fetal lung, fetal thymus, fetal skeletal muscle, fetal brain, fetal spleen, or fetal liver (Fig. 2).

[Example 4] Changes in intracellular Ca ²⁺ concentration due to histamine in 293 cells expressing GPRv53 protein

 The following experiment confirmed the histamine receptor activity of the protein encoded by GPRv53. First, in order to express the GPRv53 protein encoded by this cDNA, the cDNA was obtained by PCR and incorporated into an expression vector.

 Actually, for amplification of the full-length cDNA encoding the GPRv53 protein, 5'-CTAGTCTAGAATGCCAGATACTAATAGCACAATCAATTTATC-3 '(SEQ ID NO: 6) as a formal primer and 5, -CTAGTCTAGATTAAGAAGATACTGACCGACTGTG TTGTG-3' (for a reverse primer) SEQ ID NO: 7) was used (an Xbal site was added to the 5 'end of each). For PCR, use Pyrobest DNA polymerase (Takara Shuzo), and for type I DNA, use GPRv53 incorporated into the PCR2.1 plasmid shown in Example 1. After 94 ° C (2 minutes), Sec) / 55. The cycle of C (30 seconds) / 72 ° C (1.5 minutes) was repeated 25 times. As a result, a DNA fragment of about 1.2 kbp was amplified. After digesting this fragment with Xbal, it was inserted into pEF-BOS plasmid (Nucleic Acids Res. (1990) 18: 5322). The nucleotide sequence of the obtained clone was analyzed using the ABI 377 DNA Sequencer by the dideoxy-one-mine-one-one method, and this plasmid was named pEF-BOS-GPRv53.

After plating HEK293 cells on lx10 ⁴ cells on a 96-well Black / clear bottom plate, col laten I coated (BECT0N DICKINSON) and culturing for 24 hours, pEF-BOS-GPRv53 and 0. mouse G-hyper 15 (Proc Natl Acad Sci USA (1991) 88: 10049), one of the trimeric GTP-binding protein subunits inserted into l / g pEF-BOS ¾ Using FuGENE6 (Boeringer Mannheim) The gene was introduced. After culturing for another 24 hours, the medium is discarded, and DMEM containing 4 / M Fluo-3, AM (Molecular Probe), 0.004X pluronic acid and 10% FBS is added at 100〃 / 〃. 1 was added and incubated at 37 ° C for 1 hour. After the incubation, the cells were washed four times with Hanks BSS (GIBC0) containing 20 mM HEPES, and Hanks BSS containing 100〃1 per milliliter of 20 mM HEPES was added.

Changes in intracellular Ca ²⁺ concentration were measured over time using FLIPR (Molecular Device). That is, HISTAMINE, R (-)-H-METHYL-, DIHYDR0CHL0RIDE (Rc MeHA), which is a specific agonist of histamine and hissamine H3 receptor 10 seconds after the start of measurement

(SIGMA Corp.) was added to a final concentration of 10- ^4-10-12 M, after the addition, the 50 seconds every 1 second, the additional 4 minutes and fluorescence intensity was measured every 6 seconds. The maximum value in the Y-axis fluorescence intensity of the results obtained, when plotting the ligand concentration in the X-axis, GPRv53 is 10- ^fi against Hisutami down - at a concentration of 10- ⁹ M, for R shed MeHA 10 - ⁵ -. 1 (concentration capacity Yi change in presence intracellular Ca ²⁺ concentration of gamma ⁸ Micromax was observed (Fig. 3) as described above GPRv53 is confirmed that a human scan evening Min receptor did it.

[Example 5] GPRv53 protein expression Changes in intracellular Ca ²⁺ concentration of 293 cells by clozapine and clobenpropit

Changes in intracellular Ca ²⁺ concentration were measured temporarily using FLIPR in the same manner as in Example 4. Ten seconds after the start of measurement, the neuroleptic clozapine [8-Chloro-l l- (4-methyl-l-pipe razinyl) -5H-dibenzo [b, e] [l, 4] -diazepine (CAS Registry No. 5786-2 00)] and clobenpropit ([(4-Chlorophenyl) methyl] -3- (lH-imidazol-4-yl) propyl ester carbamimidothioic acid (CAS), a specific antagonist of the H3 receptor Registry No. 145231-45-4) (SIGMA)) was added to a final concentration of 33.3〃M. After the addition, the fluorescence intensity was increased every 1 second for 50 seconds and every 6 seconds for 4 minutes. It was measured. The resulting fluorescence value was plotted on the Y-axis and the reaction time was plotted on the X-axis (Figure 4). As a result, GPRv53-expressing cells were found to undergo a specific change in intracellular Ca ²⁺ concentration when treated with both drugs. From this, it was confirmed that clozapine and clobenpropit had agonist activity against GPRv53. The results obtained with clozapine are in good agreement with previous papers, “clozapine has affinity for the histamine H3 receptor” (Rodrigues, A 丄, Jansen, FP, Leurs, R., Timmerman , H., and Prell, GD Br. J. Pharmacol. (1995) 114,

1523-1524), (Kathmann, M., Schlicker, E., and Gothert, M. Psychopharmacology (Berl) (1994) 116, 464-468). Clinically, treatment with clozapine has the risk of developing severe granulocytopenia as a side effect (Κπιρρ, Ρ., And Barnes, P. Br. J. Psychiatry Suppl. (1992) 17, 38 -40), (Alphs, LD, and Anand, RJ Clin. Psychiatry (1999) 60 Suppl. 12, 39-42). Given that GPRv53 expression was relatively localized to peripheral leukocytes, clozapine's agonist activity against GPRv53 may have some association with drug-induced granulocytopenia.

 More than 15 years ago, the presence of the histamine H3 receptor was suggested as an "autoreceptor" in the central presynapse that inhibits the synthesis of histamine.

(Arrang, J.M., Garbarg, M., and Schwartz, J. C. Nature. (1983) 302, 832-83 7). Subsequently, pharmacological research using selective synthetic agonists and enzymatic agonists proceeded prior to gene identification of the H3 receptor. Clobenprop was reported in 1992 as a synthetic derivative of the histamine H3 receptor selective synthetic agonist imetit (van Der Goot, H., Schepers, MJP, Sterk, GJ, and Timmerman, H. Eur.J. Med. Chem. (1992) 27, 511-517). This drug is 10 times more effective in in iro than thioperamide, an early synthetic gonist, but has low brain permeability.

From the results of this example, clobenpropit, a histamine receptor-selective synthetic agonist, functioned as an agonist in GPRv53. Therefore, in creating a histamine-selective drug, it is useful to examine the effect of a candidate compound of the drug on GPRv53. For example, a histamine H3-selective drug candidate compound group is allowed to act on GPRv53-expressing cells or a cell membrane fraction expressing GPRv53, and the activation of GPRv53-expressing cells is detected, and then GPRv53 is selected from the candidate compound group. Activity of expressing cells By removing the compound that induces the oxidization (this compound is also considered to bind to histamine H3), negative selection of a compound selective for histamine H3 can be performed. Industrial applicability

 The present invention provides a novel histamine receptor, a gene encoding the protein, a vector containing the gene, a host cell containing the vector, and a method for producing the protein. Furthermore, a method for screening a compound that modifies the activity of the protein was provided. The protein of the present invention, its gene, or a compound that modifies the activity of the protein of the present invention is expected to be used for the development of new preventive or therapeutic agents for diseases involving the histamine receptor of the present invention.

Claims

The scope of the claims 1. The DNA according to any one of the following (a) to (d), which encodes a guanosine triphosphate binding protein-coupled receptor.  (a) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1. (b) DNA containing the coding region of the nucleotide sequence of SEQ ID NO: 2.  (c) a DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1 in which one or more amino acids have been substituted, deleted, added and / or inserted.  (d) a DNA consisting of the nucleotide sequence of SEQ ID NO: 2 that hybridizes to MA under stringent conditions.  2. DNA encoding a partial peptide of the protein consisting of the amino acid sequence of SEQ ID NO: 1.  3. A vector containing the DNA according to claim 1 or 2.  4. A transformant carrying the DNA according to claim 1 or 2 or the vector according to claim 3.  5. A protein or peptide encoded by the DNA according to claim 1 or 2 6. The method according to claim 5, comprising culturing the transformant according to claim 4, and recovering the expressed protein or peptide from the transformant or a culture supernatant thereof. Production method.  7. A method for screening a ligand that binds to a protein according to claim 5,  (a) contacting a test sample with the protein or peptide according to claim 5, (b) selecting a compound that binds to the protein or peptide. 8. A method for screening a compound having an activity of inhibiting binding between a protein and a ligand thereof according to claim 5, (a) a step of contacting a ligand with the protein or a partial peptide thereof according to claim 5 in the presence of a test sample, and detecting a binding activity between the protein or a partial peptide thereof and a ligand;  (b) selecting a compound that reduces the binding activity detected in step (a) as compared to the binding activity in the absence of the test sample.  9. A method for screening a compound that inhibits or promotes the activity of the protein according to claim 5, comprising:  (a) contacting a cell expressing the protein or a partial peptide thereof with a ligand of the protein in the presence of a test sample;  (b) detecting a change in a cell due to binding of the ligand to the protein or a partial peptide thereof,  (c) selecting a compound that suppresses or enhances the change in the cell detected in step (b) as compared to the change in the cell in the absence of the test sample.  10. The method according to claim 8 or 9, wherein the ligand is Hismin.  1 1. The method according to claim 8 or 9, wherein the change in the cell is a change in cAP concentration or a change in calcium ion concentration.  12. An antibody that binds to the protein of claim 5.  13. A compound isolated by the screening according to any one of claims 7 to 11.  14. A pharmaceutical composition comprising the compound according to claim 13 as an active ingredient.  15. A nucleotide having a chain length of at least 15 nucleotides, which is complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 2 or a complementary strand thereof. 16.8-Curo- 11- (4-methyl-topiperazinyl) -5H-dibenzo [b, e] [l, 4] _dazepine or [(4-clo-mouth phenyl) methyl] -3- ( 1H-imidazole-4-yl) prop 6. The agent for activating a protein according to claim 5, comprising an ester of carbamide thioic acid as an active ingredient.