JP2003189876A - Screening method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a screening method or the like of an agonist/an antagonist. <P>SOLUTION: This screening method of a compound changing the bondability of a receptor protein or a salt thereof to a phospholipid compound comprises using a new G-protein conjugate type receptor protein having the same as or the substantially same amino acid sequence as a specific amino acid sequence derived from humans, or a salt thereof, and the phospholipid compound. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a screening method using a human brain-derived G protein-coupled receptor protein (TGR4) or a salt thereof and a phospholipid compound as a ligand, and a novel rat and mouse-derived TGR4.
And DNA encoding the same.

[0002]

BACKGROUND OF THE INVENTION Many physiologically active substances such as hormones and neurotransmitters regulate the functions of the living body through specific receptor proteins present in cell membranes. Many of these receptor proteins are coupled to guanine nucleo
Intracellular signal transduction is carried out through activation of tide-binding protein (hereinafter sometimes abbreviated as G protein), and since it has a common structure with 7 transmembrane regions, it is a G protein-coupled type. Collectively referred to as receptor protein or 7-transmembrane receptor protein (7TMR). G protein-coupled receptor proteins are present on the surface of each functional cell of living cells and organs, and are physiologically used as targets for molecules that regulate the functions of those cells and organs, such as hormones, neurotransmitters, and physiologically active substances. It plays an important role. The receptor transmits a signal into the cell through binding with a physiologically active substance, and this signal causes various reactions such as activation and inhibition of the cell. To elucidate the relationship between substances that regulate complex functions in cells and organs of various living organisms and their specific receptor proteins, particularly G protein-coupled receptor proteins, is the function of cells and organs of various living organisms. And provide a very important means for drug development closely related to these functions.

For example, in various organs of the living body, physiological functions are regulated under the control of many hormones, hormone-like substances, neurotransmitters or physiologically active substances. In particular, physiologically active substances are present in various parts of the body and regulate their physiological functions through corresponding receptor proteins. There are many unknown hormones, neurotransmitters and other physiologically active substances in the body, and many of them have not been reported so far regarding the structures of their receptor proteins. Furthermore, even in known receptor proteins, it is often unknown whether subtypes exist. Clarifying the relationship between substances that regulate complex functions in the living body and their specific receptor proteins is a very important means for drug development. Further, in order to efficiently screen for agonists and antagonists to the receptor protein and to develop a drug, it is necessary to elucidate the function of the gene of the receptor protein expressed in vivo and express them in an appropriate expression system. Was needed. In recent years, as a means for analyzing a gene expressed in vivo, research on randomly analyzing the sequence of cDNA has been actively conducted,
The fragment sequence of the cDNA thus obtained is Expresse
d Sequence Tag (EST) is registered in the database and made public. However, many ESTs have only sequence information, and it is difficult to estimate their function. W
O00 / 22131 and WO00 / 23588
The publication describes the G protein-coupled receptor protein (TGR4) and its DNA used in the present invention, but does not describe the specific ligand for the receptor protein or the function of the receptor protein. WO01 / 77326 describes a G protein-coupled receptor protein (TGR4) and its DNA used in the present invention, wherein lysophosphatidic acid is used as a ligand, and a central disease (for example, Alzheimer's disease) is a related disease. Disease, dementia, eating disorders, etc.), inflammatory diseases (for example, allergy, asthma, rheumatism, etc.),
Cardiovascular disease (for example, hypertension, cardiac hypertrophy, angina, arteriosclerosis, etc.), cancer (for example, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon) Cancer, rectal cancer, etc.), diabetes, immune system diseases (eg, AIDS, atopic dermatitis, allergies, asthma, rheumatoid arthritis, psoriasis, arteriosclerosis, diabetes, Alzheimer's disease, etc.), digestive system diseases (eg, Irritable colitis, ulcerative colitis, diarrhea, ileus, etc.) are exemplified.

[0004]

[Problems to be Solved by the Invention] Conventionally, a substance that inhibits the binding between a G protein-coupled receptor and a physiologically active substance (ie, ligand), or a signal transduction similar to that of a physiologically active substance (ie, ligand) by binding. The substance that causes the above has been utilized as a drug that regulates biological functions as a specific antagonist or agonist of these receptors. Therefore, it is new to find a new G protein-coupled receptor protein that is not only important for physiological expression in vivo, but can also be a target for drug development, and to clone its gene (eg, cDNA). This is a very important means for finding a specific ligand, agonist or antagonist of G protein coupled receptor protein. However, not all G protein-coupled receptors have been found,
At present, there are a large number of unknown G protein-coupled receptors and so-called orphan receptors for which corresponding ligands have not been identified, and there is a strong demand for the search for new G protein-coupled receptors and elucidation of their functions. . The G protein-coupled receptor is useful for searching for a new physiologically active substance (that is, a ligand) using its signal transduction action as an index, and for searching for an agonist or an antagonist to the receptor. On the other hand, even if no physiological ligand is found, it is also possible to prepare an agonist or antagonist for the receptor by analyzing the physiological action of the receptor from an inactivation experiment of the receptor (knockout animal). . Ligands, agonists or antagonists for these receptors can be expected to be utilized as prophylactic / therapeutic agents and diagnostic agents for diseases associated with dysfunction of G protein-coupled receptors. Furthermore, in many cases, a decrease or increase in the function of the G protein-coupled receptor gene in the living body, which is caused by a gene mutation, causes some disease. In this case, in addition to the administration of antagonists and agonists for the receptor, it is applied to gene therapy by introducing the receptor gene into a living body (or a specific organ) or introducing an antisense nucleic acid against the receptor gene. You can also do it. In this case, the nucleotide sequence of the receptor is indispensable information for investigating the presence or absence of a deletion or mutation on the gene, and the gene of the receptor is a prophylactic / therapeutic drug for diseases associated with dysfunction of the receptor. It can also be applied to diagnostic agents. The present invention provides the use of the novel G protein-coupled receptor protein useful as described above. That is, a novel G protein-coupled receptor protein (TGR4) or a partial peptide thereof or a salt thereof,
A polynucleotide containing a polynucleotide (DNA, RNA and derivatives thereof) encoding the G protein-coupled receptor protein or a partial peptide thereof (D
NA, RNA and their derivatives), uses such as antibodies against the G protein-coupled receptor protein or partial peptides thereof or salts thereof, and a method for screening a compound that changes the expression level of the G protein-coupled receptor protein, Method for determining ligand for G protein-coupled receptor, method for screening compound (antagonist, agonist) or salt thereof that alters the binding property between ligand and the G protein-coupled receptor protein, screening kit, screening method or screening Compounds (antagonists, agonists) or salts thereof that change the binding property between the ligand and the G protein-coupled receptor protein obtained by using a kit, and the binding between the ligand and the G protein-coupled receptor protein Compound (antagonists, agonists) of changing the or to provide such pharmaceutical agent comprising the compound or its salt that changes the expression level of said G protein coupled receptor protein.

[0005]

As a result of intensive studies, the present inventors have found that one of the ligands of the human brain-derived G protein-coupled receptor protein (TGR4) described in WO01 / 77326 is It was found to be a phospholipid compound. The present inventors have completed the present invention as a result of further research based on these findings.

That is, the present invention provides [1] (1) a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof or a salt thereof. And (2) a method for screening a compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and a phospholipid compound, characterized by using a phospholipid compound, and [2] a G protein-coupled receptor protein The screening method according to [1] above, which is a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 8 or SEQ ID NO: 10, and [3] the phospholipid compound is (1) 1.
A compound in which a pyrophosphate group is bonded to the terminal of a repeating structure composed of one or more isoprene units, or (2) one phosphate group is bonded to the glycerol skeleton, and one fatty acid or long-chain alcohol is ester-bonded The screening method according to [1] above, which is a compound, wherein [4] the phospholipid compound is geranylgeranyl diphosphate (GGPP), farnesyl diphosphate (FPP) or lysophosphatidic acid (LPA) [5] phospholipid compound Geranylgeranyl diphosphate (GGPP) or farnesyl diphosphate (FPP), the screening method according to [1] above, [6] (1) the same or substantially the same amino acid as the amino acid sequence represented by SEQ ID NO: 1. G protein-coupled receptor protein containing a sequence, its partial peptide or its salt, and 2) A screening kit for a compound or a salt thereof, which contains a phospholipid compound and changes the binding property between the receptor protein or a salt thereof and the phospholipid compound, [7] The screening method according to [1] above Alternatively, a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 with the phospholipid compound, which can be obtained using the screening kit described in [6] above, or A compound or a salt thereof that changes the binding property to the salt, [8] phospholipid compound and SEQ ID NO: 1
A pharmaceutical comprising a compound or a salt thereof that changes the binding property to a G protein-coupled receptor protein or a salt thereof, which contains the same or substantially the same amino acid sequence as that of

[9] The medicine according to the above [8], which is a prophylactic / therapeutic agent for central diseases, inflammatory diseases, cardiovascular diseases, cancer, diabetes, immune system diseases or digestive system diseases,
[10] The drug according to the above [8], which is a prophylactic / therapeutic agent for liver / gallbladder diseases, digestive system diseases, anxiety, pain or obesity, and [11] a test compound together with the amino acid sequence represented by SEQ ID NO: 1. An intracellular Ca ²⁺ concentration-elevating activity or an intracellular cAMP producing activity when contacted with a cell containing a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence, Method for determining ligand for G protein-coupled receptor protein or salt thereof, [12] Ligand obtained by the method according to [11] above, [13]
(I) G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, its partial peptide or its salt, phospholipid compound or phospholipid compound, and said G
When the compound or its salt that changes the binding property to the protein-coupled receptor protein or its salt is contacted, (ii) an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 is obtained. G protein-coupled receptor protein contained therein, partial peptide or salt thereof, phospholipid compound or compound or salt thereof that changes the binding property between phospholipid compound and the G protein-coupled receptor protein or salt thereof, and test compound And an agonist to a G protein-coupled receptor protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, characterized in that Or a method for screening an antagonist, [14]
(I) Change in binding property to a labeled phospholipid compound or a G protein-coupled receptor protein or a salt thereof which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 with a phospholipid compound When the labeled compound or salt thereof is brought into contact with the G protein-coupled receptor protein, a partial peptide thereof or a salt thereof, and (ii) a labeled phospholipid compound or a phospholipid compound and the G protein-coupled receptor protein Or a labeled compound or a salt thereof that changes the binding property to a salt thereof, and a labeled phospholipid compound or phospholipid when a test compound is contacted with the G protein-coupled receptor protein, a partial peptide thereof or a salt thereof Change in the binding property between a compound and the G protein-coupled receptor protein or a salt thereof The amount of the labeled compound or salt thereof bound to the G protein-coupled receptor protein, its partial peptide or its salt is measured and compared, which is the same as or substantially the same as the amino acid sequence represented by SEQ ID NO: 1. For screening agonists or antagonists of G protein-coupled receptor proteins or salts thereof containing the same amino acid sequence, [15] (i) labeled phospholipid compound or phospholipid compound and amino acid represented by SEQ ID NO: 1 A labeled compound or a salt thereof that changes the binding property to a G protein-coupled receptor protein or a salt thereof containing the same or substantially the same amino acid sequence as the sequence is added to cells containing the G protein-coupled receptor protein. When contacted, (ii) labeled phospholipid compound or Is a case where a labeled compound or a salt thereof that changes the binding property between a phospholipid compound and the G protein-coupled receptor protein or a salt thereof, and a test compound are contacted with cells containing the G protein-coupled receptor protein. The binding amount of the labeled compound or its salt, which changes the binding property between the labeled phospholipid compound or the phospholipid compound and the G protein-coupled receptor protein or its salt, to the cells is measured and compared. A method for screening an agonist or an antagonist of a G protein-coupled receptor protein or a salt thereof, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1.
6] (i) Binding property to a labeled phospholipid compound or a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 with a phospholipid compound or a salt thereof When a labeled compound or a salt thereof that changes γ is contacted with a membrane fraction of a cell containing the G protein-coupled receptor protein, and (ii) a labeled phospholipid compound or the G protein-coupled receptor protein or Labeled phospholipid compound or phospholipid in the case where a labeled compound or its salt that changes the binding property to the salt and a test compound are contacted with the membrane fraction of cells containing the G protein-coupled receptor protein A labeled compound or its compound that changes the binding property between the compound and the G protein-coupled receptor protein or a salt thereof. A G protein-coupled receptor containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, characterized in that the amount of salt bound to the membrane fraction of the cells is measured and compared. Method for screening agonist or antagonist for protein or salt thereof, [17] (i)
A labeled phospholipid compound or a labeled phospholipid compound which changes the binding property to a G protein-coupled receptor protein or its salt containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 A G protein expressed on the cell membrane of a transformant obtained by culturing a transformant obtained by transforming a compound or a salt thereof with a recombinant vector containing a DNA containing a DNA encoding the G protein-coupled receptor protein. When contacted with a coupled receptor protein, (i
i) G labeled with a labeled phospholipid compound or a phospholipid compound and a labeled compound or a salt thereof that changes the binding property of the G protein-coupled receptor protein or a salt thereof, and a test compound to the cell membrane of the transductant. When contacted with a protein-coupled receptor protein,
The binding amount of the labeled compound or its salt that changes the binding property between the labeled phospholipid compound or the phospholipid compound and the G protein-coupled receptor protein or its salt is measured and compared. [18] (i) A method for screening an agonist or an antagonist of a G protein-coupled receptor protein or a salt thereof, which comprises the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, ) A phospholipid compound or a compound which changes the binding property to a phospholipid compound or a G protein-coupled receptor protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 A salt containing the G protein-coupled receptor protein And (ii) a phospholipid compound or a compound or a salt thereof that changes the binding property between the phospholipid compound or the phospholipid compound and the G protein-coupled receptor protein or a salt thereof, and a test compound. Identical to the amino acid sequence represented by SEQ ID NO: 1, characterized in that intracellular Ca ²⁺ concentration-elevating activity or intracellular cAMP producing activity is measured and compared when the cells are contacted with a cell containing the receptor protein Alternatively, a method for screening an agonist or antagonist for a G protein-coupled receptor protein or its salt containing substantially the same amino acid sequence, [19] (i) a phospholipid compound or a phospholipid compound and an amino acid represented by SEQ ID NO: 1 G protein containing the same or substantially the same amino acid sequence as the sequence A transformant obtained by transforming a compound or a salt thereof, which changes the binding property to a protein-coupled receptor protein or a salt thereof, with a recombinant vector containing a DNA containing a DNA encoding the G protein-coupled receptor protein. G expressed on the cell membrane of the transformant by culturing
When contacted with a protein-coupled receptor protein,
(Ii) Phospholipid compound or phospholipid compound and the G
Intracellular Ca when a compound or its salt that changes the binding property to a protein-coupled receptor protein or its salt, and a test compound are contacted with the G protein-coupled receptor protein expressed on the cell membrane of the transformant.
G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, characterized by measuring and comparing ²⁺ concentration increasing activity or intracellular cAMP producing activity Or a method for screening an agonist or an antagonist to a salt thereof, wherein the [20] phospholipid compound is (1) 1
A compound in which a pyrophosphate group is bound to the end of a repeating structure composed of one or more isoprene units, or (2) one phosphate group is bound to the glycerol skeleton, and one fatty acid or long-chain alcohol is ester-linked. [21] The screening method according to [1] above, wherein the phospholipid compound is geranylgeranyl diphosphate (GGPP),
Farnesyl diphosphate (FPP) or lysophosphatidic acid (LPA), the screening method according to the above [13] to [20], wherein [22] the phospholipid compound is geranylgeranyl diphosphate (GGPP) or farnesyl diphosphate. The screening method according to the above [13] to [20], which is (FPP), and [23] a G protein-coupled type in which the test compound contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. The screening method according to the above [13] to [20], which is a compound designed to bind to the ligand binding pocket based on the atomic coordinates of the active site of the receptor protein or its salt and the position of the ligand binding pocket.
4] A G protein-coupled receptor containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, which is obtained by using the screening method described in any of [13] to [23] above. Agonists or antagonists for proteins or salts thereof, [2
5] A medicament comprising an agonist or an antagonist to a G protein-coupled receptor protein or a salt thereof which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, [26] SEQ ID NO: A central disease, inflammatory disease, cardiovascular disease, or cancer containing an agonist or antagonist to a G protein-coupled receptor protein or its salt containing the same or substantially the same amino acid sequence as that represented by 1. , A preventive / therapeutic agent for diabetes, immune system diseases or digestive system diseases, [27] a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or the same Liver / gall containing an agonist or antagonist to salt Prophylactic / therapeutic agent for caries disease, digestive system disease, anxiety, pain or obesity, [28] G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. , Central disease, inflammatory disease, cardiovascular disease, cancer, diabetes, immune system disease, liver / gallbladder disease, digestive system disease, anxiety, pain or obesity prevention / comprising a partial peptide or a salt thereof Therapeutic agent, [29] A central disease comprising a DNA containing a DNA encoding a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. , Inflammatory disease, cardiovascular disease, cancer, diabetes, immune system disease, liver / gallbladder disease, digestive system disease, anxiety, pain or obesity preventive / therapeutic agent [30] A central disease, an inflammatory disease, which comprises a polynucleotide containing a polynucleotide encoding a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the number 1 Cardiovascular disease, cancer, diabetes, immune system disease, liver / gallbladder disease, digestive system disease, anxiety, pain or obesity diagnostic agent, the same as or substantially the same as the amino acid sequence represented by [31] SEQ ID NO: 1. A central disease, an inflammatory disease, a cardiovascular disease, characterized by using a polynucleotide containing a polynucleotide encoding a G protein-coupled receptor protein containing the amino acid sequence of
Cancer, diabetes, immune system disease, liver / gallbladder disease, digestive system disease, anxiety, pain or obesity diagnostic method, [32] having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. A central disease, an inflammatory disease, a cardiovascular disease comprising a compound or a salt thereof that changes the expression level of the contained G protein-coupled receptor protein,
Cancer, diabetes, immune system disease, liver / gallbladder disease, digestive system disease, anxiety, pain or obesity preventive / therapeutic agent, [33]
Central diseases, inflammatory diseases, comprising a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof or a salt thereof, Cardiovascular disease, cancer, diabetes, immune system disease, liver / gallbladder disease,
Prophylactic / therapeutic agent for digestive system diseases, anxiety, pain or obesity,
[34] Central diseases and inflammations containing an antibody against a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, its partial peptide or its salt Disease, cardiovascular disease, cancer, diabetes, immune system disease, liver / gallbladder disease, digestive system disease, anxiety, pain or obesity diagnostic agent, [35] identical or substantially to the amino acid sequence represented by SEQ ID NO: 1 Containing identical amino acid sequences
A central disease, inflammatory disease, cardiovascular disease, cancer, diabetes, immunity, characterized by using a method for quantifying the G protein-coupled receptor protein using an antibody against a protein-coupled receptor protein, a partial peptide thereof or a salt thereof. System disease, liver / gallbladder disease, digestive system disease, anxiety, pain or obesity diagnostic method, [36] G protein coupling containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 Central diseases, inflammatory diseases, cardiovascular diseases containing a polynucleotide containing a nucleotide sequence complementary to a polynucleotide containing a polynucleotide encoding a type I receptor protein or a partial peptide thereof, or a polynucleotide containing a part thereof Prevention of cancer, diabetes, immune system diseases, liver and gallbladder diseases, digestive system diseases, anxiety, pain or obesity療剤 for [37] mammals,
Agonist or antagonist to a G protein-coupled receptor protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, the same or substantially the amino acid sequence represented by SEQ ID NO: 1 G protein-coupled receptor protein containing the same amino acid sequence, its partial peptide or salt thereof, G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Alternatively, a polynucleotide containing a polynucleotide encoding the partial peptide, a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof Against Alternatively, a base sequence complementary to a polynucleotide containing a polynucleotide encoding a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof, or Central diseases, inflammatory diseases, cardiovascular diseases, cancer, diabetes, immune system diseases, liver / gallbladder diseases, characterized by administering an effective amount of a polynucleotide containing a part thereof
Gastrointestinal diseases, anxiety, pain or obesity prevention / treatment methods, [38] central diseases, inflammatory diseases, cardiovascular diseases,
The same or substantially the same as the amino acid sequence represented by SEQ ID NO: 1 for producing a prophylactic / therapeutic agent for cancer, diabetes, immune system disease, liver / gallbladder disease, digestive system disease, anxiety, pain or obesity Agonist or antagonist to G protein-coupled receptor protein containing an amino acid sequence or a salt thereof, G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, and a portion thereof A peptide or a salt thereof, a polynucleotide containing a polynucleotide encoding a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof, SEQ ID NO: The same as the amino acid sequence represented by: G protein-coupled receptor protein containing the qualitatively identical amino acid sequence, an antibody or SEQ number for its partial peptide, or a salt thereof:
A nucleotide sequence complementary to a polynucleotide containing a polynucleotide encoding a G protein-coupled receptor protein or a partial peptide thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by 1, or a part thereof. [39] Use of a polynucleotide comprising [39] a G protein-coupled receptor protein comprising the amino acid sequence represented by SEQ ID NO: 8 or SEQ ID NO: 10, a partial peptide thereof or a salt thereof, [40] described in [39] above A polynucleotide containing a polynucleotide encoding the G protein-coupled receptor protein of
[41] A DNA comprising the nucleotide sequence represented by SEQ ID NO: 9 or SEQ ID NO: 11, [42] a recombinant vector containing the polynucleotide according to [40] above, [4]
3] The transformant transformed with the recombinant vector according to [42] above, [44] the transformant according to [43] above is cultured, and the G protein-coupled receptor protein according to [39] above or its transformant A method for producing a G protein-coupled receptor protein or a salt thereof according to the above [39], which produces a salt, [45] A G protein-coupled receptor protein according to the above [39], a partial peptide thereof or a salt thereof [46] A pharmaceutical agent containing the polynucleotide according to [40] above, [47] a diagnostic agent containing the polynucleotide according to [40] above, [48] above [39] ] An antibody against the G protein-coupled receptor protein, its partial peptide or a salt thereof, [49] the G protein-coupled receptor protein CIGNA of the above [39] [48] The antibody described in [48] above, which is a neutralizing antibody that inactivates transmission, [50] a diagnostic agent containing the antibody described in [48] above, [51] containing the antibody described in [48] above. [52] An antisense polynucleotide comprising a nucleotide sequence complementary to the polynucleotide according to [40] or a part thereof, [5]
[3] A diagnostic agent containing the antisense polynucleotide according to [52] above, [54] A pharmaceutical agent containing the antisense polynucleotide according to [52] above, [5]
5] The method for screening an agonist or antagonist to the G protein-coupled receptor protein according to [39] above, which comprises using the G protein-coupled receptor protein according to [39] above, a partial peptide thereof or a salt thereof. ] A kit for screening an agonist or antagonist to the G protein-coupled receptor protein according to [39] above, which comprises the G protein-coupled receptor protein according to [39] above, a partial peptide thereof or a salt thereof. 57] The screening method according to [55] above or [56] above
[58] An agonist or antagonist to the G protein-coupled receptor protein according to [39], which is obtained by using the screening kit according to [39].
[9] A medicament comprising an agonist or an antagonist to the G protein-coupled receptor protein according to [9],
[59] A method for screening a compound or a salt thereof that alters the expression level of the G protein-coupled receptor protein according to [39] above, which comprises using the polynucleotide according to [40] above, [60] above [40] ] The polynucleotide as described above is contained, [3]
[9] A kit for screening a compound that alters the expression level of a G protein-coupled receptor protein or a salt thereof, [61] which is obtained by using the screening method described in [59] above or the screening kit described in [60] above. [39] The compound or a salt thereof that changes the expression level of the G protein-coupled receptor protein, and [62] the compound or salt thereof that changes the expression level of the G protein-coupled receptor protein of [39]. There is provided a medicine comprising:

Furthermore, in the present invention, the [63] receptor protein comprises a) the amino acid sequence represented by SEQ ID NO: 1,
1 or 2 in the amino acid sequence represented by SEQ ID NO: 1
1 or more (preferably about 1 to 30, more preferably about 1 to 10 and even more preferably several (1 to 5))
Amino acid sequence in which the amino acid of SEQ ID NO: 1 has been deleted, b) SEQ ID NO: 1
An amino acid sequence in which 1 or 2 or more (preferably about 1 to 30, more preferably about 1 to 10 and further preferably several (1 to 5)) amino acids are added to the amino acid sequence represented by c) 1 or 2 or more (preferably 1 to 2 in the amino acid sequence represented by SEQ ID NO: 1)
Approximately 30 amino acids, more preferably approximately 1 to 10, more preferably several (1 to 5) amino acids are substituted with other amino acids, or d) an amino acid sequence in which they are combined. The above [1] which is a protein
[64] The screening method described in [6] above, which comprises cells containing the G protein-coupled receptor protein described in [1] above, [65] above described [1] [6] The screening kit according to [6] above, and [66] the G protein-coupled receptor protein according to [1] above, which comprises a membrane fraction of a cell containing the G protein-coupled receptor protein The above-mentioned [containing the G protein-coupled receptor protein expressed in the cell membrane of the transformant by culturing the transformant transformed with the recombinant vector containing the DNA containing the encoding DNA. 6] The screening kit and the like described above are provided.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The G protein-coupled receptor protein of the present invention (hereinafter sometimes abbreviated as a receptor protein) is identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 (FIG. 2). It is a receptor protein containing the amino acid sequence of. The receptor protein of the present invention includes, for example, humans and mammals (eg, guinea pig,
Rat, mouse, rabbit, pig, sheep, cow, monkey, etc.) (for example, spleen cells, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelium) Cell, fibroblast, fiber cell, muscle cell, adipocyte, immune cell (eg, macrophage, T cell, B cell, natural killer cell, mast cell, neutrophil, basophil, eosinophil, monocyte) , Megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary gland cells, hepatocytes or stromal cells, or progenitors of these cells, stem cells or cancer cells, etc.) and hematopoietic cells , Or any tissue in which those cells are present, such as the brain, parts of the brain (eg, olfactory bulb, squamous nucleus, basal sphere, hippocampus, thalamus, hypothalamus, subthalamic nucleus, cerebral cortex, medulla oblongata, cerebellum, Occipital , Frontal lobe, temporal lobe, putamen,
Caudate, brain stain, substantia nigra), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), Blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cells, prostate,
It may be a protein derived from the testis, testis, ovary, placenta, uterus, bone, joint, skeletal muscle, or the like, or may be a synthetic protein.

The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 is, for example, about 50% or more, preferably about 60% or more, more preferably the amino acid sequence represented by SEQ ID NO: 1. About 70% or more, more preferably about 80% or more, and most preferably about 9%.
Examples thereof include amino acid sequences having a homology of 0% or more, and most preferably about 95% or more. SEQ ID NO of the invention:
An example of a protein containing an amino acid sequence substantially the same as the amino acid sequence represented by 1 is SEQ ID NO: 1
A protein and the like having an amino acid sequence substantially the same as the amino acid sequence represented by and having substantially the same activity as the amino acid sequence represented by SEQ ID NO: 1 are preferable. Examples of substantially the same activity include ligand binding activity,
Examples include signal transduction action. "Substantially the same quality" means that their activities are qualitatively the same.
Therefore, activities such as ligand binding activity and signal transduction activity are equivalent (eg, about 0.01 to 100 times, preferably about 0.5 to 20 times, more preferably about 0.5 to 2 times).
However, quantitative factors such as the degree of activity and the molecular weight of the protein may be different. The activity such as ligand binding activity and signal transduction activity can be measured according to a method known per se,
For example, it can be measured according to a screening method described later.

Further, as the receptor protein of the present invention, a) SEQ ID NO: 1, SEQ ID NO: 8 or SEQ ID NO:
1 or 2 or more in the amino acid sequence represented by 10 (preferably about 1 to 30, more preferably 1 to 1)
An amino acid sequence in which about 0, more preferably several (1 to 5) amino acids are deleted, b) 1 or 2 in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 8 or SEQ ID NO: 10. More than one (preferably about 1 to 30,
More preferably about 1 to 10, more preferably several (1 to 5) amino acid sequences added,
c) SEQ ID NO: 1, SEQ ID NO: 8 or SEQ ID NO: 10
1 or 2 or more (preferably about 1 to 30, more preferably about 1 to 10, further preferably several (1 to 5)) amino acids in the amino acid sequence represented by A protein containing a substituted amino acid sequence, or d) an amino acid sequence combining them is also used.

In the receptor protein in the present specification, the left end is the N-terminal (amino terminal) and the right end is the C-terminal (carboxyl terminal) according to the convention of peptide notation. SEQ ID NO: including receptor protein containing the amino acid sequence represented by 1, the receptor protein of the present invention, C-terminal, usually a carboxyl group (-COOH) or carboxylate (-COO ^-) is a, C-terminal, there may also be an amide (-CONH ₂₎ or ester (-COOR). Here, R in the ester is, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example,
C _3-8 cycloalkyl group such as cyclopentyl and cyclohexyl, for example, C such as phenyl and α-naphthyl
_6-12 aryl groups, for example, phenyl-C _1-2 alkyl groups such as benzyl and phenethyl, or C _7-14 aralkyl groups such as α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl, and oral For example, a pivaloyloxymethyl group commonly used as an ester is used. When the receptor protein of the present invention has a carboxyl group (or carboxylate) other than the C-terminal,
Those in which the carboxyl group is amidated or esterified are also included in the receptor protein of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used. Furthermore, the receptor protein of the present invention is the protein mentioned above, an amino group protecting group of the N-terminal methionine residue (e.g., formyl group, C _1-6 acyl group such as C _{2 -6} alkanoyl group such as acetyl Etc.), those in which the glutamyl group produced by cleavage at the N-terminal side in vivo is oxidized by pyroglutamine, substituents on the side chains of amino acids in the molecule (for example, -O).
H, —SH, amino group, imidazole group, indole group, guanidino group, etc.) is protected by a suitable protecting group (for example, formyl group, C _1-6 acyl group such as C _2-6 alkanoyl group such as acetyl). Also included are complex proteins such as glycoproteins having a sugar chain bound thereto, or so-called glycoproteins. Specific examples of the receptor protein of the present invention include, for example, a G protein-coupled receptor protein (human TGR having the amino acid sequence represented by SEQ ID NO: 1).
4), the amino acid sequence represented by SEQ ID NO: 8 (Fig. 1
0) G protein-coupled receptor protein (rat TGR4), G protein-coupled receptor protein (mouse human TGR4) consisting of the amino acid sequence represented by SEQ ID NO: 10 (FIG. 12) and the like are used. this house,
Rat TGR4 and mouse TGR4 are novel proteins.

The partial peptide of the receptor protein of the present invention (hereinafter sometimes abbreviated as partial peptide) may be any partial peptide of the above-mentioned receptor protein of the present invention. Among the receptor protein molecules of the present invention, those that are exposed outside the cell membrane and that have substantially the same receptor binding activity are used. Specifically, the partial peptide of the receptor protein having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 8 or SEQ ID NO: 10 is an extracellular region (hydrophilic site) in the hydrophobicity plot analysis. Is a peptide containing the portion analyzed to be. In addition, hydrophobicity (Hydrophobi
A peptide containing part of the site c) can be used as well. A peptide containing each domain individually may be used, but a peptide of a part containing a plurality of domains at the same time may be used. The number of amino acids in the partial peptide of the present invention is preferably a peptide having an amino acid sequence of at least 20 amino acids, preferably 50 amino acids or more, and more preferably 100 amino acids or more among the constituent amino acid sequences of the receptor protein of the present invention described above. . A substantially identical amino acid sequence means about 50% or more, preferably about 60% or more, more preferably about 70% or more, further preferably about 80% or more, and particularly preferably about 90% or more with these amino acid sequences. Most preferably, an amino acid sequence having a homology of about 95% or more is shown. Here, the “substantially the same receptor activity” has the same meaning as described above. The “substantially the same quality of receptor activity” can be measured in the same manner as above.

The partial peptide of the present invention has 1 or 2 or more (preferably 1 to 10) amino acids in the above amino acid sequence.
About one, more preferably several (1 to 5) amino acids are deleted, or 1 or 2 or more (preferably about 1 to 20, more preferably 1) in the amino acid sequence.
-10, more preferably several (1-5) amino acids are added, or 1 or 2 or more (preferably 1-10, more preferably several) in the amino acid sequence. , And more preferably about 1 to 5 amino acids) may be substituted with other amino acids. The C-terminal of the partial peptide of the present invention is usually a carboxyl group (-C
OOH) or carboxylate (-COO ^- is a), as the protein of the present invention described above, C-terminal, it may be an amide (-CONH ₂₎ or ester (-COOR). When the partial peptide of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminal, those in which the carboxyl group is amidated or esterified are also included in the partial peptide of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used. Further, in the partial peptide of the present invention, as in the above-mentioned receptor protein of the present invention, a peptide in which the amino group of the N-terminal methionine residue is protected by a protecting group, and the N-terminal side is cleaved in vivo to generate the partial peptide. Also included are those in which Gln is pyroglutamine-oxidized, those in which the substituents on the side chains of amino acids in the molecule are protected by appropriate protecting groups, or complex peptides such as so-called glycopeptides to which sugar chains are bound. Examples of the salt of the receptor protein of the present invention or its partial peptide include physiologically acceptable salts with acids or bases, and physiologically acceptable acid addition salts are particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and salts thereof are used.

The receptor protein of the present invention or a salt thereof can be produced from the above-mentioned human or mammalian cells or tissues by a method known per se for purification of the receptor protein, or can be used as a code for the receptor protein of the present invention described later. It can also be produced by culturing a transformant containing the DNA. In addition, it can also be produced by a protein synthesis method described later or a method similar thereto. When producing from human or mammalian tissues or cells, after homogenizing human or mammalian tissues or cells, extraction is performed with an acid or the like, and the extract is subjected to chromatography such as reverse phase chromatography or ion exchange chromatography. Can be purified and isolated by combining

For the synthesis of the receptor protein of the present invention or its partial peptide or its salt or its amide, a commercially available resin for protein synthesis can be used. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin,
PAM resin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4-
Examples thereof include (2 ', 4'-dimethoxyphenyl-hydroxymethyl) phenoxy resin and 4- (2', 4'-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin according to the sequence of the desired protein according to various condensation methods known per se. At the end of the reaction, the protein is cleaved from the resin and at the same time various protective groups are removed, and then an intramolecular disulfide bond forming reaction is carried out in a highly diluted solution to obtain the target protein or its amide. Regarding the condensation of the protected amino acid described above, various activating reagents that can be used for protein synthesis can be used, but carbodiimides are particularly preferable. As carbodiimides, DC
C, N, N'-diisopropylcarbodiimide, N-ethyl-N '-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, a protected amino acid is added directly to the resin together with a racemization-inhibiting additive (eg, HOBt, HOOBt), or the protected amino acid is previously activated as a symmetrical acid anhydride or HOBt ester or HOOBt ester. After that, it can be added to the resin.

The solvent used for activation of the protected amino acid or condensation with the resin can be appropriately selected from solvents known to be usable for protein condensation reaction. For example,
Acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, sulfoxides such as dimethyl sulfoxide. , Ethers such as pyridine, dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, or an appropriate mixture thereof. The reaction temperature is appropriately selected from the range known to be applicable to protein bond-forming reactions, and is usually appropriately selected from the range of about -20 ° C to 50 ° C. Activated amino acid derivatives are usually 1.5-
Used in 4-fold excess. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation cannot be obtained even after repeating the reaction, unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole.

Examples of the protective group for the amino group of the raw material include Z, Boc, tertiary pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, Trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl oil, Fmoc and the like are used. The carboxyl group is, for example, an alkyl esterified (for example, linear, branched or cyclic alkyl ester such as methyl, ethyl, propyl, butyl, tert-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl and the like. ), Aralkyl esterification (eg benzyl ester, 4-
Nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazide formation, tertiary butoxycarbonyl hydrazide formation, trityl hydrazide formation, etc. . The hydroxyl group of serine can be protected by, for example, esterification or etherification.
Examples of groups suitable for this esterification include lower alkanoyl groups such as acetyl group, aroyl groups such as benzoyl group, groups derived from carbonic acid such as benzyloxycarbonyl group and ethoxycarbonyl group.
Moreover, examples of a group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group. As a protective group for the phenolic hydroxyl group of tyrosine,
For example, Bzl, Cl2-Bzl, ₂ -nitrobenzyl, Br-Z, tertiary butyl and the like are used.
Examples of the protecting group for imidazole of histidine include:
Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bu
m, Boc, Trt, Fmoc, etc. are used.

The activated carboxyl group of the raw material includes, for example, corresponding acid anhydrides, azides, active esters [alcohols (for example, pentachlorophenol, 2,4,5-trichlorophenol, 2,4- Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinide, N
-Hydroxyphthalimide, ester with HOBt)]
Are used. As the activated amino group of the raw material, for example, the corresponding phosphoric acid amide is used. Examples of the method for removing (eliminating) the protecting group include Pd
-Catalytic reduction in a hydrogen stream in the presence of a catalyst such as black or Pd-carbon, or acid with anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, or a mixture thereof. Treatment, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., and reduction with sodium in liquid ammonia are also used. The elimination reaction by the acid treatment is generally carried out at a temperature of about -20 ° C to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol,
The addition of cation trapping agents such as para-cresol, dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol is effective. Further, the 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane group. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

Protection of a functional group which should not be involved in the reaction of the raw material, removal of the protective group, removal of the protective group, activation of the functional group involved in the reaction and the like can be appropriately selected from known groups or known means. . As another method for obtaining an amide form of a protein, for example, first, the carboxy terminal amino acid α
-Amidation of a carboxyl group for protection, extension of the peptide (protein) chain to the amino group side to a desired chain length, and then a protein obtained by removing only the protective group for the α-amino group at the N-terminal of the peptide chain. A protein from which only the protecting group of the C-terminal carboxyl group has been removed is produced, and both proteins are condensed in the mixed solvent as described above. The details of the condensation reaction are the same as above. After purifying the protected protein obtained by condensation, all the protecting groups can be removed by the above method to obtain the desired crude protein. This crude protein can be purified by making full use of various known purification means, and the main fraction can be lyophilized to obtain the amide form of the desired protein. To obtain an ester form of a protein, for example, the α-carboxyl group of a carboxy-terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then an ester form of the desired protein is obtained in the same manner as the amide form of the protein. be able to.

The partial peptide of the protein of the present invention or a salt thereof can be produced by publicly known methods for peptide synthesis, or by cleaving the protein of the present invention with an appropriate peptidase. The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. That is, the target peptide can be produced by condensing a partial peptide or amino acid that can form the protein of the present invention with the remaining portion and removing the protecting group if the product has a protecting group. Examples of known condensation methods and elimination of protective groups include the methods described in a) to e) below. a) M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publisher
s, New York (1966) b) Schroeder and Luebke, The Peptid
e), Academic Press, New York (1965) c) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) d) Haruaki Yajima and Shunpei Sakakibara, Biochemistry Laboratory 1, Protein Chemistry IV, 205, (1977) e) Supervised by Haruaki Yajima, Development of Pharmaceuticals Volume 14 Peptide Synthesis Hirokawa Shoten After the reaction, the partial peptide of the present invention can be purified and isolated by combining usual purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the partial peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method, and conversely, when it is obtained as a salt, it can be converted into a free form by a known method. You can

Any polynucleotide can be used as the polynucleotide encoding the receptor protein of the present invention, so long as it contains the nucleotide sequence (DNA or RNA, preferably DNA) encoding the receptor protein of the present invention. Good. The polynucleotide is DNA such as DNA encoding the receptor protein of the present invention or RNA such as mRNA, and may be double-stranded or single-stranded. When it is double-stranded, it may be double-stranded DNA, double-stranded RNA or a hybrid of DNA: RNA. In the case of a single strand, it may be a sense strand (that is, a coding strand) or an antisense strand (that is, a non-coding strand). Using the polynucleotide encoding the receptor protein of the present invention, for example, the publicly known special edition of Experimental Medicine, “New PC
R and its application ”, 15 (7), 1997, or a method similar thereto, and mRNA of the receptor protein of the present invention.
Can be quantified. The DNA encoding the receptor protein of the present invention includes genomic DNA and genomic DN.
A library, cDNA derived from the above cells / tissues,
Any of the above-mentioned cell / tissue-derived cDNA library and synthetic DNA may be used. The vectors used for the library are bacteriophage, plasmid, cosmid,
It may be any such as phagemid. In addition, reverse Transcriptase Polymer can be directly analyzed by using total RNA or mRNA fraction prepared from the above-mentioned cells / tissues.
It can also be amplified by the raseChain Reaction (hereinafter abbreviated as RT-PCR method). Specifically, the DNA encoding the receptor protein of the present invention includes, for example, D containing the nucleotide sequence represented by SEQ ID NO: 2.
NA or a base sequence represented by SEQ ID NO: 2 which has a base sequence that hybridizes under high stringent conditions and has substantially the same activity as the receptor protein of the present invention (eg, ligand binding activity, signal information). Any DNA may be used as long as it encodes a receptor protein having a transduction action and the like. The DNA capable of hybridizing with the base sequence represented by SEQ ID NO: 2 is, for example, about 70% or more, preferably about 80% or more, more preferably about 90% with the base sequence represented by SEQ ID NO: 2.
As described above, most preferably, a DNA containing a nucleotide sequence having a homology of about 95% or more is used.

Hybridization can be carried out by a method known per se or a method analogous thereto, for example, molecular
Cloning (Molecular Cloning) 2nd (J. Sambrook
et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be carried out under high stringent conditions. The high stringent condition is, for example,
The sodium concentration is about 19-40 mM, preferably about 19
~ 20 mM, temperature about 50-70 ° C, preferably about 6
The conditions of 0 to 65 ° C. are shown. Especially, the sodium concentration is about 1
Most preferred is 9 mM and a temperature of about 65 ° C. More specifically, as the DNA encoding the receptor protein consisting of the amino acid sequence represented by SEQ ID NO: 1, the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2 is the amino acid sequence represented by SEQ ID NO: 8. The DNA encoding the receptor protein consisting of SEQ ID NO: 9 is the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 9, and the DNA encoding the receptor protein consisting of the amino acid sequence represented by SEQ ID NO: 10 is SEQ ID NO: 1
DNA or the like having the nucleotide sequence represented by 1 is used. The polynucleotide containing a part of the base sequence of the DNA encoding the receptor protein of the present invention or a part of the base sequence complementary to the DNA means a DNA encoding the following partial peptide of the present invention. It is used not only to include, but also to include RNA. According to the present invention, an antisense polynucleotide (nucleic acid) capable of inhibiting the replication or expression of a G protein-coupled receptor protein gene encodes a cloned or determined G protein-coupled receptor protein. It can be designed and synthesized based on the nucleotide sequence information of DNA. Such a polynucleotide (nucleic acid) can hybridize with the RNA of the G protein-coupled receptor protein gene, inhibit the synthesis or function of the RNA, or bind to the G protein-coupled receptor protein-related RNA. Expression of the G protein-coupled receptor protein gene can be regulated / controlled through the interaction.
A polynucleotide complementary to a selected sequence of a G protein-coupled receptor protein-related RNA, and a polynucleotide capable of specifically hybridizing with a G protein-coupled receptor protein-related RNA are It is useful for regulating and controlling the expression of the protein-coupled receptor protein gene, and also useful for treating or diagnosing diseases and the like. The term "corresponding" means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. “Corresponding” between a nucleotide (base sequence or nucleic acid) and a peptide (protein) usually refers to an amino acid of a peptide (protein) in a command derived from the nucleotide (nucleic acid) sequence or its complement. .
5'end hairpin loop of G protein-coupled receptor protein gene, 5'end 6-base pair repeat, 5'end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation initiation codon, 3'end non Translation area, 3 '
The terminal palindromic region and the 3'-end hairpin loop can be selected as a preferred target region, but any region within the G protein-coupled receptor protein gene can be selected as a target.

It can be said that the relationship between the target nucleic acid and the polynucleotide complementary to at least a part of the target region, and the relationship between the polynucleotide capable of hybridizing with the target region are "antisense". . Antisense polynucleotides are polydeoxynucleotides containing 2-deoxy-D-ribose, D-
Ribose-containing polydeoxynucleotides, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, or other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acids). Polymer) or other polymer containing a special bond, provided that the polymer contains nucleotides having a configuration that allows base pairing and base attachment as found in DNA and RNA. . They are double-stranded DN
A, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA: RNA hybrid, and unmodified polynucleotide (or unmodified oligonucleotide) as well as addition of known modifications , For example, with a label known in the art, with a cap, methylated, one or more natural nucleotides substituted with an analogue, one with an intramolecular nucleotide modification, For example, those having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.), those having a charged bond or a sulfur-containing bond (eg, phosphorothioate, phosphorodithioate, etc.), eg Proteins (nucleases, nuclease inhibitors, toxins, antibodies, signal peptides Plastid, poly -L- lysine, etc.)
Having side chain groups such as sugars and sugars (eg, monosaccharides), those having intercurrent compounds (eg, acridine, psoralen, etc.), chelate compounds (eg, metals, radioactive metals, boron) , An oxidative metal, etc.), an alkylating agent, or a modified bond (for example, α-anomer type nucleic acid). As used herein, the term "nucleoside", "nucleotide" and "nucleic acid" may include those having not only purine and pyrimidine bases but also other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. The modified nucleotide and the modified nucleotide may also have a modified sugar moiety, for example, one or more hydroxyl groups are substituted with halogen, an aliphatic group, or the like,
Alternatively, it may be converted into a functional group such as ether or amine.

The antisense polynucleotide (nucleic acid) of the present invention is RNA, DNA or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include, but are not limited to, sulfur derivatives and thiophosphate derivatives of nucleic acids, and those resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, it makes the antisense nucleic acid more stable in the cell, increases the cell permeability of the antisense nucleic acid, has a greater affinity for the target sense strand, and if it is toxic, Make sense nucleic acid less toxic. Thus many modifications are known in the art, for example J. Kawakami et al., Pharm Tech Japan, Vo.
l. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; ST Cr
ooke et al. ed., Antisense Research and Applicatio
ns, CRC Press, 1993 etc. The antisense nucleic acid of the present invention has an altered or modified sugar,
It may contain a base or a bond, and may be provided in a special form such as liposome, microsphere, applied by gene therapy, or provided in an added form. Thus, as an additive form, a polycationic substance such as polylysine that acts to neutralize the charge of the phosphate skeleton, a lipid that enhances the interaction with the cell membrane or the uptake of nucleic acid ( Examples thereof include crude aqueous substances such as phospholipid and cholesterol). Preferred lipids to add include cholesterol and its derivatives (eg cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3'-end or 5'-end of the nucleic acid, and can be attached via a base, a sugar, or an intramolecular nucleoside bond. Other groups include
A capping group specifically arranged at the 3'-end or the 5'-end of a nucleic acid, which includes a group for preventing decomposition by nucleases such as exonuclease and RNase. Examples of such a capping group include, but are not limited to, hydroxyl group-protecting groups known in the art including glycols such as polyethylene glycol and tetraethylene glycol. The inhibitory activity of the antisense nucleic acid can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of G protein-coupled receptor protein. it can. The nucleic acid can be applied to cells by various methods known per se.

DNA encoding the partial peptide of the present invention
Any may be used as long as it contains the nucleotide sequence encoding the partial peptide of the present invention. Further, any of genomic DNA, genomic DNA library, cDNA derived from the cells / tissues described above, cDNA library derived from the cells / tissues described above, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, using the mRNA fraction prepared from the cells / tissues described above, the reverse
Transcriptase Polymerase Chain Reaction (hereinafter RT
-Abbreviated as PCR method). Specifically, D encoding the partial peptide of the present invention
Examples of NA include (1) DN having a partial base sequence of DNA having the base sequence represented by SEQ ID NO: 2.
A, or (2) having a base sequence that hybridizes with the base sequence represented by SEQ ID NO: 2 under high stringent conditions, and has substantially the same activity as the receptor protein peptide of the present invention (eg, ligand binding) A DNA having a partial base sequence of a DNA encoding a receptor protein having an activity, a signal transduction action, etc.) is used. The DNA capable of hybridizing with the base sequence represented by SEQ ID NO: 2 is, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably the base sequence represented by SEQ ID NO: 2. Is about 9
A DNA containing a base sequence having a homology of 5% or more is used.

As a means for cloning a DNA which completely encodes the receptor protein of the present invention or a partial peptide thereof (hereinafter sometimes abbreviated as the receptor protein of the present invention), a partial base sequence of the receptor protein of the present invention is used. DNA which is amplified by the PCR method using a synthetic DNA primer which is present, or which is incorporated into an appropriate vector and labeled with a DNA fragment or a synthetic DNA encoding a part or the whole region of the receptor protein of the present invention. It can be selected by hybridization. The method of hybridization is
For example, Molecular Cloning
ing) 2nd (J. Sambrook et al., ColdSpring Harbor L
ab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.

The conversion of the DNA base sequence can be carried out by PCR or a known kit, for example, Mutan ^™ -super Ex.
press Km (Takara Shuzo Co., Ltd.), Mutan ^™ -
ODA-LA PC using K (Takara Shuzo Co., Ltd.)
It can be carried out according to a method known per se such as R method, Gapped duplex method, Kunkel method or a method analogous thereto. The cloned DNA encoding the receptor protein can be used as it is, or if desired after digestion with a restriction enzyme or addition of a linker, depending on the purpose. The DNA has ATG as a translation initiation codon at the 5'-terminal side, and 3 '
The terminal side may have TAA, TGA, or TAG as a translation termination codon. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adaptor. The expression vector of the receptor protein of the present invention includes, for example, (a) a DNA fragment of interest is excised from a DNA encoding the receptor protein of the present invention, and (b) the DNA fragment is placed downstream of a promoter in an appropriate expression vector. It can be manufactured by connecting them.

As the vector, a plasmid derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pBR322) is used.
UC13), a Bacillus subtilis-derived plasmid (eg, pUB11)
0, pTP5, pC194), yeast-derived plasmid (eg, pSH19, pSH15), bacteriophage such as λ phage, retrovirus, vaccinia virus, baculovirus and other animal viruses, and p
A1-11, pXT1, pRc / CMV, pRc / RS
V, pcDNAI / Neo, etc. are used. The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression. For example, when an animal cell is used as a host, SRα promoter, SV40 promoter, L promoter
TR promoter, CMV promoter, HSV-TK
Examples include promoters. Of these, CMV
It is preferable to use a promoter, SRα promoter, or the like. When the host is Escherichia, trp
Promoter, lac promoter, recA promoter, λP _L promoter, lpp promoter, etc., when the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter, etc. When the host is yeast, PHO5 promoter, PGK promoter , GAP promoter, ADH
A promoter and the like are preferable. When the host is an insect cell, polyhedrin promoter, P10 promoter and the like are preferable.

In addition to the above, the expression vector may optionally contain an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori) and the like. Can be used. Examples of selection markers include dihydrofolate reductase (hereinafter, dhfr).
The gene [methotrexate (M
TX) resistance], ampicillin resistance gene (hereinafter referred to as Amp
^r ), and a neomycin resistance gene (hereinafter, sometimes abbreviated as Neo ^r , G418 resistance) and the like. In particular, when the dhfr gene is used as a selection marker using CHO (dhfr ⁻ ) cells, the target gene can also be selected by a thymidine-free medium. If necessary, a signal sequence suitable for the host is added to the N-terminal side of the receptor protein of the present invention. If the host is Escherichia, PhoA
When the host is a genus Bacillus, the signal sequence, OmpA, signal sequence and the like are α-amylase signal sequence and subtilisin signal sequence, and when the host is yeast, MFα, signal sequence and SUC2. When the host is an animal cell such as a signal sequence, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule / signal sequence, etc. can be used, respectively. A transformant can be produced using the vector containing the DNA encoding the receptor protein of the present invention thus constructed.

As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used. Specific examples of Escherichia bacteria include:
Escherichia coli K12 ・ DH
1 [Proc. Natl. Acad. Sci. USA], Volume 60, 160
(1968)], JM103 [Nukuirek Acids.
Research (Nucleic Acids Research), Volume 9, 309
(1981)], JA221 [Journal of Molecular Biolog (Journal of Molecular Biolog
y), 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41.
, 459 (1969)], C600 [Genetics, 39, 440 (1954)] and the like. Examples of the bacterium of the genus Bacillus include Bacillus subtilis MI114 [gene,
24, 255 (1983)], 207-21 [Journal of Biochemis
try), Vol. 95, 87 (1984)] and the like are used.
Examples of the yeast include Saccharomyces cerevisiae AH22 and AH22.
^{R -, NA87-11A, DKD-5D} , 20B-1
2. Schizosaccharomys pombe
ces pombe) NCYC1913, NCYC2036, Pichia pastoris and the like are used.

Examples of insect cells include virus A
In the case of cNPV, larvae of the night-thief moth (Spodop
tera frugiperda cell; Sf cell), Trichoplusia ni
MG1 cells from the midgut of the mouse, H from the eggs of Trichoplusia ni
igh Five ^™ cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used. When the virus is BmNPV, the silkworm-derived cell line (Bombyx
mori N; BmN cells) and the like are used. Examples of the Sf cells include Sf9 cells (ATCC CRL1711), Sf
21 cells (above Vaughn, JL et al., In Vivo
ivo), 13, 213-217, (1977)) is used. Examples of insects include silkworm larvae [Maeda et al., Nature, 315, 592 (198).
5)]. Animal cells include, for example, monkey cells COS-
7, Vero, Chinese hamster cell CHO (hereinafter, abbreviated as CHO cell), dhfr gene-deficient Chinese hamster cell CHO (hereinafter, CHO (dhf
r ⁻ ) cell), mouse L cell, mouse AtT-2
0, mouse myeloma cells, rat GH3, human FL cells and the like are used.

For transforming Escherichia, for example, Proc. Natl. Acad. Sci. USA, Volume 69, Proceedings of the National Academy of Sciences of the USA. , 2110
(1972) and Gene, Volume 17, 107 (19).
82) and the like.
Transformations of Bacillus include, for example, Molecular and General Genetics (Molecula).
r & General Genetics), 168, 111 (197)
It can be performed according to the method described in 9). For transforming yeast, for example, Methods in Enzymology, 194,
182-187 (1991), Procedures of the National Academy of Sciences of the USA (Proc. Natl. Acad.
SA), Vol. 75, 1929 (1978) and the like. For transforming insect cells or insects, for example, bio / technology (Bio /
Technology), 6, 47-55 (1988) and the like. To transform animal cells,
For example, Cell Engineering Separate Volume 8 New Cell Engineering Experimental Protocol.
263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973). In this way, G
A transformant transformed with the expression vector containing the DNA encoding the protein-coupled receptor protein can be obtained. When the host is culturing a transformant that is a bacterium of the genus Escherichia, a bacterium of the genus Bacillus, a liquid medium is suitable as a medium used for the culture, and among them, a carbon source necessary for the growth of the transformant, A nitrogen source, an inorganic substance, etc. are contained. Examples of the carbon source include glucose, dextrin, soluble starch and sucrose, and examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal and potato extract. Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. In addition, yeast extract, vitamins, growth promoting factor and the like may be added. The pH of the medium is preferably about 5-8.

As a medium for culturing Escherichia, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics (Journa
l of Experiments in Molecular Genetics), 431-
433, Cold Spring Harbor Laboratory, New York1
972] is preferable. In order to make the promoter work efficiently if necessary, for example, 3β-indolyl is used.
A drug such as acrylic acid can be added. When the host is a bacterium belonging to the genus Escherichia, the culture is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be added. When the host is a bacterium of the genus Bacillus, the culture is usually carried out at about 30 to 40 ° C. for about 6 to 24 hours, and aeration and stirring can be added if necessary. When culturing a transformant whose host is yeast, examples of the medium include Burkholder's minimum medium [Bostian, KL
Et al., Proc. Natl. Acad. Sci. USA, 77, 4505.
(1980)] and SD medium containing 0.5% casamino acid [Bitter, GA et al., Procedures of the National Academy of Sciences of
The USA (Proc. Natl. Acad. Sci. USA), 8
1, 5330 (1984)]. P of medium
H is preferably adjusted to about 5-8. Culture is usually about 2
It is carried out at 0 ° C to 35 ° C for about 24 to 72 hours, and aeration and stirring are added if necessary.

When culturing a transformant whose host is an insect cell or an insect, the medium is Grace's Insect Mediu
m (Grace, TCC, Nature, 195, 788 (1
962)) to which an additive such as immobilized 10% bovine serum is appropriately added is used. The pH of the medium is about 6.2
It is preferably adjusted to 6.4. Culturing is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added if necessary. When culturing a transformant whose host is an animal cell, the medium is, for example, MEM medium containing about 5 to 20% fetal bovine serum [Science, Volume 122, 50].
1 (1952)], DMEM medium [Virolo
gy), Volume 8, 396 (1959)], RPMI 1640
Medium (The Journal of the American Me
dical Association) Volume 199, 519 (1967)],
199 medium [Proceedi of the Society for the Biological Medicine (Proceedi
ng of the Society for the Biological Medicine), 7
3 volume, 1 (1950)] and the like are used. pH is about 6
It is preferably 8. Culturing is usually performed at about 30 ° C to 40 ° C for about 15 to 60 hours, and aeration and agitation are added if necessary. As described above, the G protein-coupled receptor protein of the present invention can be produced inside the transformant cells, on the cell membrane or outside the cells.

Separation and purification of the receptor protein of the present invention from the above culture can be carried out, for example, by the following method. When the receptor protein of the present invention is extracted from cultured bacterial cells or cells, after the culture, the bacterial cells or cells are collected by a known method, suspended in an appropriate buffer solution, and then subjected to ultrasonic wave, lysozyme and / or freeze-thawing. After destroying the bacterial cells or cells by, for example, a method of obtaining a crude extract of the receptor protein by centrifugation or filtration is appropriately used. The buffer solution may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ . When the receptor protein is secreted into the culture medium, after the culture is completed, the cells or cells are separated from the supernatant by a method known per se, and the supernatant is collected. Purification of the receptor protein contained in the thus obtained culture supernatant or the extract can be carried out by appropriately combining separation / purification methods known per se. As the known separation and purification methods thereof, a method utilizing solubility such as salting out or solvent precipitation method, a dialysis method, an ultrafiltration method, a gel filtration method, and an SDS-polyacrylamide gel electrophoresis method are mainly used. Difference, charge difference such as ion exchange chromatography, specific relevance such as affinity chromatography, hydrophobicity difference such as reversed-phase high performance liquid chromatography And a method of utilizing a difference in isoelectric points such as an isoelectric focusing method.

When the receptor protein thus obtained is obtained as a free form, it can be converted into a salt by a method known per se or a method analogous thereto, and conversely, when it is obtained as a salt, a method known per se. Alternatively, it can be converted into a free form or another salt by a method similar thereto. The receptor protein produced by the recombinant can be optionally modified or partially removed by reacting it with an appropriate protein-modifying enzyme before or after purification. Examples of the protein-modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like. The activity of the thus-formed receptor protein of the present invention or a salt thereof can be measured by a binding experiment with a labeled ligand, an enzyme immunoassay using a specific antibody, or the like.

The antibody against the receptor protein of the present invention or its partial peptide or its salt may be either a polyclonal antibody or a monoclonal antibody as long as it is an antibody capable of recognizing the receptor protein of the present invention or its partial peptide or its salt. Good. The receptor protein of the present invention or a partial peptide thereof or a salt thereof (hereinafter sometimes abbreviated as the receptor protein of the present invention)
Antibodies against can be produced using the receptor protein of the present invention as an antigen according to a method for producing an antibody or antiserum known per se.

[Preparation of Monoclonal Antibody] (a) Preparation of Monoclonal Antibody-Producing Cell The receptor protein of the present invention is administered to mammals at a site where antibody production is possible by itself or together with a carrier or diluent. . Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration. Administration is usually 2-6
It is performed once a week, about 2 to 10 times in total. Examples of mammals used include monkeys, rabbits, dogs,
Examples include guinea pig, mouse, rat, sheep and goat, and mouse and rat are preferably used. For preparation of monoclonal antibody-producing cells, warm-blooded animals immunized with an antigen, for example, individuals with an antibody titer were selected from mice, and 2 to 5 days after the final immunization, spleens or lymph nodes were collected and By fusing the antibody-producing cells contained therein with myeloma cells, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting the labeled receptor protein described below with the antiserum and then measuring the activity of the labeling agent bound to the antibody. The fusion operation is a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (19).
1975)]. Examples of the fusion accelerator include polyethylene glycol (PEG) and Sendai virus, and PE is preferable.
G is used. Examples of myeloma cells include NS-
1, P3U1, SP2 / 0, etc., but P3U
1 is preferably used. The preferred ratio of the number of antibody-producing cells (spleen cells) to the number of myeloma cells used is 1: 1 to
It is about 20: 1, and PEG (preferably PEG10
(00-PEG6000) is added at a concentration of about 10 to 80%, and the cells can be efficiently fused by incubating at about 20 to 40 ° C, preferably at about 30 to 37 ° C for about 1 to 10 minutes.

Various methods can be used for screening a hybridoma producing a monoclonal antibody. For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which an antigen of a receptor protein is directly or adsorbed with a carrier, Next, an anti-immunoglobulin antibody labeled with a radioactive substance or an enzyme (if the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A is added, and the monoclonal antibody bound to the solid phase is detected. Method, a method in which a hybridoma culture supernatant is added to a solid phase on which anti-immunoglobulin antibody or protein A is adsorbed, a receptor protein labeled with a radioactive substance or an enzyme is added, and a monoclonal antibody bound to the solid phase is detected, etc. Is mentioned. The monoclonal antibody can be selected according to a method known per se or a method analogous thereto, but usually it can be carried out in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. As the medium for selection and breeding, any medium may be used as long as the hybridoma can grow. For example, 1 to 20
%, Preferably 10-20% fetal calf serum
I 1640 medium, GI containing 1-10% fetal bovine serum
T medium (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) can be used. Culture temperature is usually 20-40
℃, preferably about 37 ℃. Cultivation time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture is
Usually, it can be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the above antibody titer measurement in antiserum.

(B) Purification of Monoclonal Antibody The isolation and purification of the monoclonal antibody can be carried out in the same manner as the usual isolation and purification of a polyclonal antibody [eg, salting out method, alcohol precipitation method, isoelectric focusing method, Electrophoresis method, adsorption / desorption method with ion exchanger (eg, DEAE), ultracentrifugation method, gel filtration method, antigen-binding solid phase or active antibody such as protein A or protein G is used to dissociate the antibody. Specific Purification Method of Obtaining Antibody to Obtain Antibody]

[Preparation of Polyclonal Antibody] The polyclonal antibody of the present invention can be manufactured by a method known per se or a method analogous thereto. For example, a mammal is immunized in the same manner as in the above-mentioned method for producing a monoclonal antibody by forming a complex of an immune antigen (antigen of receptor protein) and a carrier protein, and an antibody-containing product against the receptor protein of the present invention Can be collected and the antibody can be separated and purified. Regarding a complex of an immunizing antigen and a carrier protein used for immunizing a mammal, the type of the carrier protein and the mixing ratio of the carrier and the hapten are such that the antibody can be efficiently against the hapten immunized by crosslinking with the carrier. What kind of thing may be cross-linked at any ratio, but, for example, bovine serum albumin, bovine thyroglobulin, keyhole limpet hemocyanin, etc. in a weight ratio of about 0.1 to 20 per hapten. Preferably, a method of coupling at a ratio of about 1 to 5 is used. Also,
For the coupling of the hapten and the carrier, various condensing agents can be used, and glutaraldehyde, carbodiimide, maleimide active ester, active ester reagent containing a thiol group, dithiopyridyl group, etc. are used. The condensation product is administered to a warm-blooded animal at a site where antibodies can be produced, by itself or together with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration. The administration can be performed once every about 2 to 6 weeks, about 3 to 10 times in total. The polyclonal antibody can be collected from blood, ascites, etc., preferably blood of a mammal immunized by the above method. The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-mentioned antibody titer in the serum. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described separation and purification of the monoclonal antibody.

The ligand of the receptor protein of the present invention or a salt thereof is a phospholipid compound, for example, (1) a compound having a pyrophosphate group bonded to the terminal of a repeating structure composed of one or more isoprene units, or (2) A compound in which one phosphate group is bound to the glycerol skeleton, and further one fatty acid or long chain alcohol is ester-bonded is used. Repeated isoprene unit is 1 or more,
The number is preferably 1 to 6, and more preferably 2 to 4. As the fatty acid, for example, a fatty acid having about 6 to 30 carbon atoms is preferable, and specifically, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, oleic acid, linoleic acid, linolenic acid, etc. are used. .
As the long chain alcohol, for example, an alcohol having about 6 to 30 carbon atoms is preferable, and specifically, tetradecanol, pentadecanol, hexadecanol, octadecanol and the like are used. The position of bonding of one phosphate group to the glycerol skeleton is not particularly limited, but it is preferably bonded to the hydroxyl group at the 3-position, for example. The position at which the fatty acid or long-chain alcohol is ester-bonded to the glycerol skeleton is not particularly limited, but for example, it is preferably ester-bonded to the hydroxyl group at the 1-position or 2-position. More specifically, examples of the phospholipid compound include geranylgeranyl diphosphate (GGPP) and farnesyl diphosphate (FP).
P) or lysophosphatidic acid (LPA) and the like are used. Among them, geranylgeranyl diphosphate (GGP
P) or farnesyl diphosphate (FPP) is preferably used. Therefore, the receptor protein of the present invention,
A partial peptide or a salt thereof (hereinafter sometimes abbreviated as a receptor protein of the present invention), a DNA encoding the receptor protein or a partial peptide thereof (hereinafter,
The DNA of the present invention may be abbreviated), and the antibody to the receptor protein or its partial peptide (hereinafter sometimes abbreviated as the antibody of the present invention) has the following uses.

(1) Preventive and / or therapeutic agent for diseases associated with dysfunction of the receptor protein of the present invention a) Receptor protein of the present invention or b) DNA encoding the receptor protein of the receptor protein of the present invention It can be used as a medicine such as a preventive and / or therapeutic agent for diseases associated with dysfunction. For example, when there is a patient in whom the physiological action of the phospholipid compound as a ligand cannot be expected (the deficiency of the receptor protein) due to a decrease in the receptor protein of the present invention in vivo, a) the receptor protein of the present invention To the patient to supplement the amount of the receptor protein, or b) (a) by administering and expressing the DNA encoding the receptor protein of the present invention to the patient, or (b) in the target cell. DNA encoding the receptor protein of the present invention
After inserting and expressing, the cells can be transplanted into the patient, etc., to increase the amount of the receptor protein in the patient's body and sufficiently exert the action of the ligand. That is, the DNA encoding the receptor protein of the present invention is useful as a safe and low-toxicity preventive and / or therapeutic agent for diseases associated with dysfunction of the receptor protein of the present invention. Specifically, the receptor protein of the present invention or the DNA of the present invention is, for example, a central disease (eg, Alzheimer's disease, dementia, eating disorder, etc.),
Inflammatory disease (for example, allergic disease, asthma, rheumatism, etc.), cardiovascular disease (for example, hypertension, cardiac hypertrophy, angina, arteriosclerosis, etc.), cancer (for example, non-small cell lung cancer, ovarian cancer, Prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc., diabetes, immune system diseases (eg autoimmune disease, AIDS, atopic dermatitis, allergic disease, immunodeficiency, asthma) , Rheumatoid arthritis, psoriasis, arteriosclerosis, diabetes, Alzheimer's disease, etc.), liver and gallbladder diseases (eg, cirrhosis, hepatitis, liver failure, cholestasis, stones, etc.), digestive system diseases (eg, ulcer, It is useful for prevention and / or treatment of diseases such as enteritis, dyspepsia, irritable colitis, ulcerative colitis, diarrhea, ileus), anxiety, pain and obesity. When the receptor protein of the present invention is used as the above-mentioned preventive / therapeutic agent, it can be prepared into a pharmaceutical preparation according to a conventional method. On the other hand, when the DNA of the present invention is used as the prophylactic / therapeutic agent, the DNA of the present invention is used alone or after being inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, etc. It can be carried out according to the means. The DNA of the present invention can be administered as it is or together with an auxiliary agent for promoting uptake by a gene gun or a catheter such as a hydrogel catheter. For example, a) the receptor protein of the present invention or b) the DNA of the present invention is orally administered as tablets, capsules, elixirs, microcapsules and the like, optionally coated with sugar, or water or other pharmaceutical agents. It can be used parenterally in the form of an injectable solution such as a sterile solution or a suspension with a physiologically acceptable liquid. For example, a) the receptor protein of the present invention or b) D of the present invention.
Manufactured by admixing NA with known physiologically acceptable carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders and the like in a unit dose form required for generally accepted formulation practice. be able to. The amount of active ingredient in these preparations is such that a suitable amount within the indicated range can be obtained.

Additives that can be mixed with tablets, capsules and the like include, for example, binders such as gelatin, corn starch, tragacanth and acacia, excipients such as crystalline cellulose, corn starch, gelatin and alginic acid. Puffing agents such as and the like, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red oil or cherry, and the like are used. When the unit dosage form is a capsule, a liquid carrier such as fat may be included in addition to materials of the above type. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices such as dissolving or suspending the active substance in a vehicle such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, Polysorbate 80 ^™ , HCO-50) and the like may be used in combination. Examples of the oily liquid include sesame oil and soybean oil,
You may use together with solubilizing agents, such as benzyl benzoate and benzyl alcohol.

The above-mentioned prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers ( For example, human serum albumin, polyethylene glycol, etc.), preservatives (eg,
(Benzyl alcohol, phenol, etc.), antioxidant, etc. The prepared injection solution is usually filled in a suitable ampoule. Since the preparation thus obtained is safe and has low toxicity, for example, humans and mammals (for example, rat, mouse, rabbit, sheep, pig, cow,
(Cats, dogs, monkeys, etc.). The dose of the receptor protein of the present invention is,
Although there are differences depending on the target organs, symptoms, administration method, etc., in the case of oral administration, generally, for example, hypertensive patients (60 k
(as g), about 0.1 to 100 m per day
g, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally,
Although the single dose varies depending on the administration subject, target organ, symptom, administration method, etc., for example, in the form of an injection, usually, for example, in a hypertensive patient (60 kg),
About 0.01 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg.
It is convenient to administer about mg by intravenous injection.
In the case of other animals, the dose converted per 60 kg can be administered. The dose of the DNA of the present invention varies depending on the administration subject, target organ, symptom, administration method and the like, but in the case of oral administration, generally, for example, in hypertensive patients (as 60 kg), the daily dose is about 0.1-
It is 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose is the administration subject, target organ, symptom,
Although it depends on the administration method, for example, in the form of an injection, for example, in a hypertensive patient (as 60 kg), about 0.01 to 30 mg, preferably about 0.1 to 20 mg per day is more preferable. Preferably about 0.1
It is convenient to administer about 10 mg by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

(2) Gene Diagnostic Agent The DNA of the present invention can be used in humans or mammals (eg, rat, mouse, rabbit, sheep, pig, cow, cat, dog, monkey) by using it as a probe. Since it is possible to detect an abnormality (gene abnormality) in the DNA or mRNA encoding the receptor protein of the present invention or a partial peptide thereof, for example, damage, mutation or decreased expression of the DNA or mRNA and increase in the DNA or mRNA. Alternatively, it is useful as a gene diagnostic agent for overexpression. The above-mentioned gene diagnosis using the DNA of the present invention can be carried out, for example, by known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989).
,), Proceedings of the National Academy of Sciences o
f the United States of America), Vol. 86, 276
6 to 2770 (1989)) and the like. For example, when a decrease in the expression of the receptor protein of the present invention is detected by Northern hybridization, for example, it is likely that the disease associated with the dysfunction of the receptor protein of the present invention is high or likely to be affected in the future. Can be diagnosed. Further, when overexpression of the receptor protein of the present invention is detected by Northern hybridization, for example, it is highly possible that the disease is caused by overexpression of the receptor protein of the present invention, or that there is a high possibility of contracting in the future. Can be diagnosed. Examples of diseases associated with dysfunction of the receptor protein of the present invention or diseases caused by overexpression of the receptor protein of the present invention include, for example, central diseases (e.g., Alzheimer's disease, dementia, eating disorders, etc.), inflammatory diseases. Diseases (for example, allergic diseases, asthma, rheumatism, etc.), cardiovascular diseases (for example, hypertension, cardiac hypertrophy, angina,
Arteriosclerosis, etc.), cancer (for example, non-small cell lung cancer, ovarian cancer,
Prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer,
Rectal cancer etc.), diabetes, immune system diseases (eg autoimmune diseases, AIDS, atopic dermatitis, allergic diseases,
Immunodeficiency, asthma, rheumatoid arthritis, psoriasis, arteriosclerosis, diabetes, Alzheimer's disease, etc., liver / cholecysteal disease (eg, cirrhosis, hepatitis, liver failure, cholestasis, stones, etc.), digestive system disease (eg For example, ulcer, enteritis, dyspepsia,
Irritable colitis, ulcerative colitis, diarrhea, ileus, etc.), anxiety, pain, obesity and the like.

(3) Pharmaceutical containing the compound which changes the expression level of the receptor protein of the present invention or its partial peptide The DNA of the present invention is used as a probe to
It can be used for screening a compound that changes the expression level of the receptor protein of the present invention or its partial peptide. That is, the present invention is included in, for example, (i) a) blood of a non-human mammal, b) a specific organ, c) a tissue or cell isolated from an organ, or (ii) a transformant or the like. The present invention provides a method for screening a compound that changes the expression level of the receptor protein of the present invention or its partial peptide by measuring the amount of mRNA of the receptor protein or its partial peptide.

The mRNA amount of the receptor protein of the present invention or its partial peptide is specifically measured as follows. (I) Normal or disease model Non-human mammal (eg, mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, and more specifically, demented rat, obese mouse, arteriosclerotic rabbit, cancer bearing) Mouse)
Blood ( Obtaining a specific organ (eg, brain, liver, kidney, etc.), or tissue or cells isolated from the organ. MRN of the receptor protein of the present invention or its partial peptide contained in the obtained cells
A can be quantified by, for example, extracting mRNA from cells or the like by an ordinary method and using a technique such as TaqMan PCR, or can be analyzed by performing Northern blotting by a method known per se. (Ii) a transformant expressing the receptor protein of the present invention or a partial peptide thereof is prepared according to the above method,
The mRNA of the receptor protein of the present invention or its partial peptide contained in the transformant can be similarly quantified and analyzed.

Screening for a compound that changes the expression level of the receptor protein of the present invention or a partial peptide thereof is carried out by (i) a certain time before a drug or physical stress is applied to a normal or disease model non-human mammal ( 30 minutes to 24 hours, preferably 30 minutes to 1
2 hours before, more preferably 1 hour-6 hours before) or after a certain time (30 minutes-3 days later, preferably 1 hour-2 days later, more preferably 1 hour-24 hours later), or drug Alternatively, the test compound is administered simultaneously with physical stress, and after a lapse of a certain period of time (30 minutes to 3 days later, preferably 1 hour to 2 days later, more preferably 1 hour later).
24 hours later), the amount of mRNA of the receptor protein of the present invention or its partial peptide contained in cells can be quantified and analyzed, and (ii) the test compound when the transformant is cultured according to a conventional method. Mixed in the medium,
After culturing for a certain period of time (after 1 day to 7 days, preferably after 1 day)
After 3 days, more preferably after 2 to 3 days), the mRNA amount of the receptor protein of the present invention or its partial peptide contained in the transformant can be quantified and analyzed.

The compound or its salt obtained by using the screening method of the present invention is a compound having an action of changing the expression level of the receptor protein of the present invention or its partial peptide, and specifically, (a) By increasing the expression level of the receptor protein of the invention or a partial peptide thereof, cell stimulating activity mediated by G protein-coupled receptors (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation,
a compound that enhances the activity of promoting or suppressing a decrease in pH), and (b) a compound that attenuates the cell stimulating activity by reducing the expression level of the receptor protein of the present invention or its partial peptide. . Examples of the compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc. These compounds may be novel compounds or known compounds. As described above, the ligand of the receptor protein of the present invention is a phospholipid compound. Therefore, the compound that changes the expression level of the receptor protein of the present invention or a partial peptide thereof obtained by the above screening method can be used as a prophylactic and / or therapeutic agent for diseases associated with dysfunction of the receptor protein of the present invention. . Specifically, a compound that increases the expression level of the receptor protein of the present invention or a partial peptide thereof and enhances cell stimulating activity is a safe and low-toxic preventive agent for diseases associated with dysfunction of the receptor protein of the present invention.
It is useful as a therapeutic agent. The compound that reduces the expression level of the receptor protein of the present invention or a partial peptide thereof and attenuates the cell stimulating activity is a safe and low-toxic preventive agent against diseases caused by overexpression of the receptor protein of the present invention.
It is useful as a therapeutic agent. Examples of diseases associated with dysfunction of the receptor protein of the present invention or diseases caused by overexpression of the receptor protein of the present invention include, for example, central diseases (e.g., Alzheimer's disease, dementia, eating disorders, etc.), inflammatory diseases (For example, allergic disease, asthma, rheumatism, etc.), cardiovascular disease (for example, hypertension, cardiac hypertrophy, angina, arteriosclerosis, etc.), cancer (for example, non-small cell lung cancer, ovarian cancer, prostate cancer, Gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc., diabetes, immune system disease (eg, autoimmune disease, AIDS, atopic dermatitis, allergic disease, immunodeficiency, asthma, rheumatic) arthritis,
Psoriasis, arteriosclerosis, diabetes, Alzheimer's disease, etc.), liver and gallbladder diseases (eg, cirrhosis, hepatitis, liver failure, cholestasis, stones, etc.), digestive system diseases (eg, ulcer, enteritis, dyspepsia, etc.) Irritable colitis, ulcerative colitis, diarrhea, ileus, etc.), anxiety, pain, obesity and the like.

When the compound or its salt obtained by using the screening method of the present invention is used as a pharmaceutical composition, it can be formulated according to a conventional method. For example, the compound is orally as a sugar-coated tablet, capsule, elixir, microcapsule, or the like, or a sterile solution with water or another pharmaceutically acceptable liquid, Alternatively, it can be used parenterally in the form of injection such as suspension. For example, admixing the compound with a known physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder and the like in a unit dose form generally required for the implementation of the formulation. Can be manufactured by. The amount of active ingredient in these preparations is such that a suitable amount within the indicated range can be obtained. Examples of additives that can be mixed with tablets, capsules, and the like include, for example, binders such as gelatin, corn starch, tragacanth, and gum arabic.
Excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin, alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, peppermint, red oil or cherry. Such a flavoring agent is used. When the unit dosage form is a capsule, a liquid carrier such as fat may be included in addition to materials of the above type. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices such as dissolving or suspending the active substance in a vehicle such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As an aqueous solution for injection,
For example, physiological saline, an isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used, and an appropriate solubilizing agent, such as alcohol (eg, ethanol, is used. ), Polyalcohol (eg, propylene glycol, polyethylene glycol), a nonionic surfactant (eg, Polysorbate 80 ^™ , HCO-50) and the like.
As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.

The above-mentioned prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers ( For example, human serum albumin, polyethylene glycol, etc.), preservatives (eg,
(Benzyl alcohol, phenol, etc.), antioxidant, etc. The prepared injection solution is usually filled in a suitable ampoule. Since the preparation thus obtained is safe and has low toxicity, for example, humans and mammals (for example, rat, mouse, rabbit, sheep, pig, cow,
(Cats, dogs, monkeys, etc.). The dose of the compound or its salt varies depending on the administration subject, target organ, symptoms, administration method, etc., but in the case of oral administration, in general, for example, in hypertensive patients (as 60 kg), About 0.1-100 mg,
Preferably about 1.0 to 50 mg, more preferably about 1.
It is 0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc., but is usually in the form of an injection, for example, in hypertensive patients (60 kg). About 0.01 to 30 mg per day, preferably about 0.1
It is convenient to administer about -20 mg, more preferably about 0.1-10 mg by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

(4) Method for quantifying ligand for G protein-coupled receptor protein of the present invention and diagnostic method The antibody of the present invention specifically recognizes a phospholipid compound which is a ligand for the G protein-coupled receptor protein of the present invention. Therefore, it can be used for quantification of phospholipid compounds in a test solution, particularly for quantification by sandwich immunoassay. That is, the present invention provides (i) the antibody of the present invention, the test solution and the labeled receptor protein of the present invention, in a competitive reaction, and the labeled receptor protein of the present invention bound to the antibody. Method for quantifying the receptor protein of the present invention in a test solution, and (ii) the antibody of the present invention insolubilized on the test solution and a carrier and the labeling of the present invention. And a method for quantifying the receptor protein of the present invention in a test solution, which comprises measuring the activity of a labeling agent on an insolubilized carrier after simultaneous or continuous reaction with the antibody.

In the quantification method of (ii) above, one antibody is an antibody that recognizes the N-terminal portion of the receptor protein of the present invention, and the other antibody is an antibody that reacts with the C-terminal portion of the receptor protein of the present invention. Is desirable. In addition, the receptor protein of the present invention can be quantified using a monoclonal antibody against the receptor protein of the present invention, and can also be detected by tissue staining or the like. For these purposes, the antibody molecule itself may be used, or F (ab ′) ₂ , Fab ′, or Fab of the antibody molecule may be used.
Fractions may be used. The method for quantifying the receptor protein of the present invention using the antibody of the present invention is not particularly limited, and the antibody, the antigen or the antibody corresponding to the amount of the antigen in the liquid to be measured (for example, the amount of the receptor protein of the present invention) is used. −
Any measurement method can be used as long as it is a method of detecting the amount of the antigen complex by chemical or physical means and calculating it from a standard curve prepared using a standard solution containing a known amount of antigen. Good. For example, nephrometry, competitive method, immunometric method and sandwich method are preferably used, but from the viewpoint of sensitivity and specificity, the sandwich method described later is particularly preferably used.

As the labeling agent used in the measuring method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance or the like is used. Examples of radioisotopes include [ ¹²⁵ I], [ ¹³¹ I], [ ³ H],
[ ¹⁴ C] or the like is used. As the above-mentioned enzyme, those having a stable and large specific activity are preferable, and for example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate, etc. are used. Luminol, a luminol derivative, luciferin, lucigenin, etc. are used as a luminescent substance, for example. Furthermore, the biotin-avidin system can be used for binding the antibody or the antigen to the labeling agent. For the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond which is usually used for insolubilizing and immobilizing the receptor protein or enzyme of the present invention may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran and cellulose, synthetic resins such as polystyrene, polyacrylamide and silicone, and glass. In the sandwich method, a test solution is reacted with the insolubilized monoclonal antibody of the present invention (first reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction), and then on the insolubilized carrier. The amount of the receptor protein of the present invention in the test solution can be quantified by measuring the activity of the labeling agent. First reaction and 2
The next reaction may be performed in the reverse order, may be performed at the same time, or may be performed at different times. The labeling agent and the method of insolubilization can be the same as those described above. In the immunoassay by the sandwich method, the antibody used as the solid phase antibody or the labeling antibody is not necessarily 1
It does not have to be a kind, and a mixture of two or more kinds of antibodies may be used for the purpose of improving the measurement sensitivity.

In the method for measuring the receptor protein of the present invention by the sandwich method of the present invention, the monoclonal antibodies of the present invention used in the primary reaction and the secondary reaction are antibodies having different binding sites for the receptor protein of the present invention. Is preferably used. That is, when the antibody used in the secondary reaction recognizes the C-terminal portion of the receptor protein of the present invention, the antibody used in the primary reaction is preferably the antibody used in the primary reaction. An antibody that recognizes an N-terminal other than the C-terminal is used. The monoclonal antibody of the present invention can be used in a measuring system other than the sandwich method, for example, a competitive method, an immunometric method or nephrometry. In competitive law,
After the antigen in the test liquid and the labeled antigen react competitively with the antibody, the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated (B / F Separation), B, F
The amount of any label is measured to quantify the amount of antigen in the test liquid. In this reaction method, a soluble antibody is used as an antibody, and B /
F separation is performed by a liquid phase method using polyethylene glycol, a second antibody against the above antibody, and a solid phase antibody is used as the first antibody, or alternatively, the first antibody is soluble and the solid phase is used as the second antibody. A solid phase immobilization method using a conjugated antibody is used. In the immunometric method, the antigen in the test liquid and the solid-phased antigen are competitively reacted with a fixed amount of the labeled antibody and then the solid phase and the liquid phase are separated, or the antigen in the test liquid is separated. After reacting with an excess amount of the labeled antibody and then adding the immobilized antigen to bind the unreacted labeled antibody to the solid phase, the solid phase and the liquid phase are separated. Next, the labeled amount of either phase is measured to quantify the amount of antigen in the test liquid. In nephrometry, the amount of insoluble precipitate produced as a result of the antigen-antibody reaction in the gel or in the solution is measured. Even when the amount of antigen in the test liquid is small and only a small amount of precipitate can be obtained, laser nephrometry utilizing laser scattering is preferably used. In applying each of these immunological assay methods to the quantification method of the present invention, no special conditions, operations, etc. are required to be set. The assay system for the receptor protein of the present invention may be constructed by adding ordinary technical consideration to those skilled in the art to ordinary conditions and operating methods in each method. For more information on these common technical means,
You can refer to reviews and books. For example, Hiroshi Irie "Radioimmunoassay" (Kodansha, published in 1974), Hiroshi Irie "Radioimmunoassay" (Kodansha, published in 1979), Eiji Ishikawa et al. "Enzyme Immunoassay" (Medical Shoin, Showa Showa) 1993), Eiji Ishikawa et al., "Enzyme Immunoassay" (second edition) (Medical Shoin, published in 1982), Eiji Ishikawa, et al. "Enzyme Immunoassay" (3rd edition) (Medicine Shoin, Showa) Published in 1987), "Methods in ENZYMOLOGY"
Vol. 70 (Immunochemical Techniques (Part A)), ibid
Vol. 73 (Immunochemical Techniques (Part B)), ibid.
Vol. 74 (Immunochemical Techniques (Part C)), ibid.
Vol. 84 (Immunochemical Techniques (Part D: Selecte
d Immunoassays)) ibid.Vol. 92 (Immunochemical Tec
hniques (Part E: Monoclonal Antibodies and General
Immunoassay Methods)), ibid Vol. 121 (Immunochemi
cal Techniques (Part I: Hybridoma Technology and M
onoclonal Antibodies)) (above, published by Academic Press). As described above, the receptor protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention.

Furthermore, when a decrease in the concentration of the receptor protein of the present invention is detected by quantifying the concentration of the receptor protein of the present invention using the antibody of the present invention,
For example, it can be diagnosed as a disease associated with dysfunction of the receptor protein of the present invention, or likely to be affected in the future. Further, when an increase in the concentration of the receptor protein of the present invention is detected, it can be diagnosed that, for example, it is a disease caused by overexpression of the receptor protein of the present invention, or is likely to be affected in the future. .
Examples of diseases associated with dysfunction of the receptor protein of the present invention or diseases caused by overexpression of the receptor protein of the present invention include, for example, central diseases (e.g., Alzheimer's disease, dementia, eating disorders, etc.), inflammatory diseases (For example, allergic disease, asthma, rheumatism, etc.), cardiovascular disease
(For example, hypertension, cardiac hypertrophy, angina, arteriosclerosis, etc.),
Cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer,
Bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc., diabetes, immune system diseases (eg, autoimmune disease, AIDS, atopic dermatitis, allergic disease, immunodeficiency, asthma,
Rheumatoid arthritis, psoriasis, arteriosclerosis, diabetes, Alzheimer's disease, etc., liver / cholecystic diseases (eg, cirrhosis, hepatitis, liver failure, cholestasis, stones, etc.), digestive system diseases (eg, ulcer, enteritis) , Dyspepsia, irritable colitis,
Ulcerative colitis, diarrhea, ileus, etc.), anxiety, pain, obesity, etc.

(5) Method for Determining Ligands Other Than Phospholipid Compound for Receptor Protein of the Present Invention By binding phospholipid compound to the receptor protein of the present invention, intracellular Ca ²⁺ release, intracellular cAMP
Since production is observed, the receptor protein of the present invention searches for ligands other than phospholipid compounds for the receptor protein of the present invention using this intracellular signal as an index,
Or it is useful as a reagent for determination. That is, the present invention relates to intracellular Ca ²⁺ release activity (intracellular Ca ²⁺ concentration increasing activity) or cell mediated by the receptor protein of the present invention when a test compound is brought into contact with cells containing the receptor protein of the present invention. Provided is a method for determining a ligand for a receptor protein of the present invention, which comprises measuring the internal cAMP producing activity. Examples of test compounds include known ligands (eg, angiotensin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxytocin, PACAP (eg, PACAP27, PACAP3.
8), secretin, glucagon, calcitonin, adrenomedullin, somatostatin, GHRH, CRF, A
CTH, GRP, PTH, VIP (vasoactive intestinal and related polypeptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene relayed peptide), leukotriene, pancreatatin, prostaglandin, thromboxane. , Adenosine, adrenaline, chemokine superfamily (eg, IL-8, GROα, GROβ, GROγ, NA
P-2, ENA-78, GCP-2, PF4, IP-1
0, Mig, PBSF / SDF-1 and other CXC chemokine subfamilies; MCAF / MCP-1, MCP-
2, MCP-3, MCP-4, eotaxin, RAN
TES, MIP-1α, MIP-1β, HCC-1, M
IP-3α / LARC, MIP-3β / ELC, I-3
09, TARC, MIPF-1, MIPF-2 / eot
axin-2, MDC, DC-CK1 / PARC, SL
CC chemokine subfamily such as C; lympho
C chemokine subfamily such as tactin; fr
CX3C chemokines subfamily such as actalkine), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA), sphingosine 1-phosphate, etc.),
For example, a tissue extract of human or mammals (for example, mouse, rat, pig, cow, sheep, monkey, etc.), cell culture supernatant, etc. are used. For example, the tissue extract,
A cell culture supernatant or the like can be added to the receptor protein of the present invention and fractionated while measuring cell stimulating activity and the like to finally obtain a single ligand.

Specifically, the method for determining the ligand of the present invention comprises constructing an expression system for recombinant GRP40, and using a receptor binding assay system using the expression system to determine cells mediated by the receptor protein of the present invention. Within Ca ²⁺
It is a method for determining a compound or a salt thereof having a free activity (intracellular Ca ²⁺ concentration increasing activity) or an intracellular cAMP producing activity. More specifically, the present invention provides the following determination method. (1) Intracellular Ca ²⁺ releasing activity (intracellular Ca ²⁺ concentration increasing activity) or intracellular cAM when a test compound is brought into contact with cells containing the receptor protein of the present invention
A method for determining a ligand for the receptor protein of the present invention, which comprises measuring the P-producing activity, and (2) culturing a transformant containing a test compound containing a DNA encoding the receptor protein of the present invention to produce a cell membrane. The present invention, which is characterized by measuring intracellular Ca ²⁺ release activity (intracellular Ca ²⁺ concentration increasing activity) or intracellular cAMP producing activity mediated by the receptor protein of the present invention when contacted with the receptor protein expressed above. A method for determining a ligand for the receptor protein of Escherichia coli is provided. In particular, it is preferable to carry out the above test after confirming that the test compound binds to the receptor protein of the present invention.

When cells containing the receptor protein of the present invention are used in the ligand determination method of the present invention, the cells may be immobilized with glutaraldehyde, formalin or the like. The immobilization method can be performed according to a known method. The membrane fraction of cells containing the receptor protein of the present invention refers to a fraction containing a large amount of cell membrane obtained by a known method after crushing the cells. As a method of crushing cells, a method of crushing cells with a Potter-Elvehjem type homogenizer, crushing with a Waring blender or polytron (manufactured by Kinematica), crushing with ultrasonic waves,
Examples include crushing by ejecting cells from a thin nozzle while pressurizing with a French press or the like. For the fractionation of the cell membrane, a fractionation method by centrifugal force such as a fractionation centrifugation method or a density gradient centrifugation method is mainly used. For example,
The cell lysate is centrifuged at a low speed (500 rpm to 3000 rpm) for a short time (usually about 1 minute to 10 minutes), and the supernatant is further processed at a higher speed (15000 rpm to 30000 rpm) for 3 times.
Centrifuge for 0 minutes to 2 hours, and use the resulting precipitate as a membrane fraction. The membrane fraction is rich in the expressed receptor protein of the present invention and membrane components such as cell-derived phospholipids and membrane proteins.

The amount of the receptor protein of the present invention in the cells containing the receptor protein of the present invention or the cell membrane fraction thereof is preferably 10 ³ to 10 ⁸ molecules per cell, and is 10 ⁵ to 10 ⁷ molecules. Preferably. In addition,
The higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, which enables not only the construction of a highly sensitive screening system but also the measurement of a large amount of samples in the same lot. In order to carry out the method for determining the ligand of the present invention, intracellular Ca ²⁺ release activity (intracellular Ca ²⁺ concentration increasing activity) mediated by the receptor protein of the present invention
Alternatively, the intracellular cAMP production activity can be measured using a known method or a commercially available measurement kit. Specifically, first, cells containing the receptor protein of the present invention are cultured in a multiwell plate or the like. To determine the ligand, exchange with fresh medium or an appropriate buffer that does not show toxicity to cells in advance, add a test compound etc. and incubate for a certain period of time, then extract the cells or collect the supernatant and generate The amount of the obtained product is quantified according to each method. When the production of substances (eg, Ca ²⁺ , cAMP, etc.) used as an indicator of cell stimulating activity is difficult to assay by the degrading enzyme contained in the cells, an inhibitor may be added to the degrading enzyme to perform the assay. .. Regarding activities such as inhibition of cAMP production,
It can be detected as an inhibitory effect on the production of cells whose basic production has been increased by forskolin or the like. The ligand determination kit of the present invention contains cells containing the receptor protein of the present invention or a cell membrane fraction thereof. Since the ligand for the receptor protein of the present invention thus determined binds to the receptor protein of the present invention and regulates its physiological function, a prophylactic / therapeutic agent for diseases associated with the function of the receptor protein of the present invention Can be used as

(6) A method for screening a compound (agonist, antagonist, etc.) that changes the binding property between the G protein-coupled receptor protein of the present invention and a ligand,
And a medicament containing a compound that alters the binding property between the G protein-coupled receptor protein of the present invention and a ligand, the receptor protein of the present invention is used, or an expression system of a recombinant receptor protein is constructed and the expression system is used. By using the receptor binding assay system used, a compound that changes the binding property between the phospholipid compound or the like as a ligand and the receptor protein of the present invention (for example, a peptide,
Proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc.) or salts thereof can be efficiently screened. Such compounds include (a) cell-stimulating activity (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, via G protein-coupled receptor,
Intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, phosphorylation of intracellular proteins, activation of c-fos, activity promoting or suppressing pH decrease, etc. A compound having a Ca ²⁺ releasing activity (intracellular Ca ²⁺ concentration increasing activity) and an intracellular cAMP producing activity (so-called an agonist for the receptor protein of the present invention), (b) a compound having no cell stimulating activity (so-called, present) An antagonist to the receptor protein of the present invention), (c) a compound that enhances the binding force between the ligand and the G protein-coupled receptor protein of the present invention, or (d) a binding force between the ligand and the G protein-coupled receptor protein of the present invention The compound of the above (a) is used to determine the ligand. It is preferable to screening by law). That is, the present invention provides (i) a case where the receptor protein of the present invention or a partial peptide thereof or a salt thereof is contacted with a ligand, and (ii) a receptor protein of the present invention or a partial peptide thereof or a salt thereof, a ligand and Provided is a method for screening a compound or a salt thereof, which changes the binding property between a ligand and the receptor protein of the present invention or a partial peptide thereof or a salt thereof, which is characterized by comparing with the case of contacting with a test compound. In the screening method of the present invention, in the cases (i) and (ii), for example, the binding amount of a ligand to the receptor protein, the cell stimulating activity, etc. are measured and compared.

More specifically, the present invention relates to a) the case where a labeled ligand is brought into contact with the receptor protein of the present invention, and the case where the labeled ligand and a test compound are brought into contact with the receptor protein of the present invention. Measuring the amount of binding of the labeled ligand to the receptor protein and comparing them, a method for screening a compound or a salt thereof which changes the binding property between the ligand and the receptor protein of the present invention, b) the labeled ligand When the cells containing the receptor protein of the present invention or the membrane fraction of the cells are contacted, the labeled ligand and the test compound are contacted with the cells containing the receptor protein of the present invention or the membrane fraction of the cells. In this case, the amount of labeled ligand bound to the cells or the membrane fraction is measured and compared. Method for screening a compound or its salt that changes the binding property between the target ligand and the receptor protein of the present invention, c) The labeled ligand is cultivated on the cell membrane by culturing a transformant containing the DNA of the present invention. In the case of contacting with the expressed receptor protein and the case of contacting the labeled ligand and the test compound with the receptor protein of the present invention expressed on the cell membrane by culturing the transformant containing the DNA of the present invention, A method for screening a compound or a salt thereof which changes the binding property between the ligand and the receptor protein of the present invention, characterized in that the amount of labeled ligand bound to the receptor protein is measured and compared.

D) When a compound which activates the receptor protein of the present invention (for example, a ligand for the receptor protein of the present invention) is brought into contact with cells containing the receptor protein of the present invention, and when the receptor protein of the present invention is When a compound that activates and a test compound are contacted with cells containing the receptor protein of the present invention, the cell-stimulating activity mediated by the receptor is measured and compared with the ligand and the receptor protein of the present invention. Method for screening a compound or salt thereof that changes the binding property of e.g., and e) a transformant containing the compound of the present invention with a compound that activates the receptor protein of the present invention (eg, a ligand for the receptor protein of the present invention) To the receptor protein of the present invention expressed on the cell membrane by culturing When touched, and when a compound that activates the receptor protein of the present invention and a test compound are contacted with the receptor protein of the present invention expressed on the cell membrane by culturing a transformant containing the DNA of the present invention In the present invention, there is provided a method for screening a compound or a salt thereof which changes the binding property between a ligand and the receptor protein of the present invention, which comprises measuring and comparing the cell stimulating activity mediated by the receptor. As the ligand, the above-mentioned phospholipid compound such as geranylgeranyl diphosphate (GGPP), farnesyl diphosphate (FPP) or lysophosphatidic acid (LPA) is used. Furthermore, as the ligand, a compound that changes the binding property between the phospholipid compound and the receptor protein of the present invention or a salt thereof can also be used. The compound or a salt thereof that changes the binding property between the phospholipid compound and the receptor protein of the present invention can be obtained, for example, by using the phospholipid compound as a ligand and carrying out the screening method of the present invention described later. .

Before the receptor protein of the present invention was obtained, when screening for G protein-coupled receptor agonists or antagonists, first, cells, tissues or cell membrane fractions thereof containing G protein-coupled receptor proteins such as rat were used. After obtaining a candidate compound (primary screening), the candidate compound is actually human G
A test (secondary screening) for confirming whether or not the binding between the protein-coupled receptor protein and the ligand is inhibited was necessary. If the cell, tissue or cell membrane fraction is used as it is, other receptor proteins are also mixed, so that it is difficult to actually screen an agonist or antagonist for the target receptor protein. However, for example, by using the human-derived receptor protein of the present invention, the need for primary screening is eliminated, and compounds that inhibit the binding between the ligand and the G protein-coupled receptor protein can be efficiently screened. Furthermore, whether the screened compound is an agonist or an antagonist can be easily evaluated. A detailed description of the screening method of the present invention is given below. First, the receptor protein of the present invention used in the screening method of the present invention may be any one as long as it contains the above-described receptor protein of the present invention. Cell membrane fractions of animal organs are preferred. However, since human-derived organs are extremely difficult to obtain, as those used for screening,
A human-derived receptor protein that is expressed in large quantities using a recombinant is suitable.

The above-mentioned method is used to produce the receptor protein of the present invention, but it is preferable to express the DNA of the present invention in mammalian cells or insect cells. Although complementary DNA is used for the DNA fragment encoding the target protein portion, it is not necessarily limited thereto. For example, a gene fragment or synthetic DNA may be used. D encoding the receptor protein of the present invention
In order to introduce NA fragments into host animal cells and express them efficiently, nuclear polyhedrosis virus (nuclear) belonging to the baculovirus using the DNA fragment as an insect host
Polyhedrosis virus (NPV) polyhedrin promoter, SV40-derived promoter, retrovirus promoter, metallothionein promoter, human heat shock promoter, cytomegalovirus promoter, SRα promoter and the like are preferably incorporated downstream. The amount and quality of the expressed receptor can be examined by a method known per se. For example, the literature [Na
mbi, P. The Journal of Biological
Chemistry (J. Biol. Chem.), Volume 267, 19555-19559.
Page, 1992]. Therefore, in the screening method of the present invention, the receptor protein containing the present invention may be a receptor protein purified according to a method known per se, or cells containing the receptor protein may be used. Alternatively, a membrane fraction of cells containing the receptor protein may be used.

When cells containing the receptor protein of the present invention are used in the screening method of the present invention,
The cells may be fixed with glutaraldehyde, formalin and the like. The immobilization method can be performed according to a method known per se. The cell containing the receptor protein of the present invention refers to a host cell expressing the receptor protein. Examples of the host cell include Escherichia coli, Bacillus subtilis,
Yeast, insect cells, animal cells and the like are preferable. The cell membrane fraction refers to a fraction containing a large amount of cell membrane obtained by a method known per se after crushing cells. As a method of crushing cells, a method of crushing cells with a Potter-Elvehjem type homogenizer, crushing with a Waring blender or polytron (Kinematica), crushing with ultrasonic waves, ejection of cells from a thin nozzle while applying pressure with a French press, etc. For example, crushing by allowing it to occur. For the fractionation of the cell membrane, a fractionation method by centrifugal force such as a fractionation centrifugation method or a density gradient centrifugation method is mainly used. For example, the cell lysate may be fed at a low speed (500 rpm to 3000 rp).
m) for a short time (usually about 1 minute to 10 minutes), and the supernatant is further centrifuged at a higher speed (15000 rpm to 30000 rpm) for usually 30 minutes to 2 hours, and the resulting precipitate is used as a membrane fraction. The membrane fraction is rich in expressed receptor proteins and membrane components such as cell-derived phospholipids and membrane proteins. The amount of the receptor protein in cells or membrane fractions containing the receptor protein is preferably 10 ³ to 10 ⁸ molecules per cell, and more preferably 10 ⁵ to 10 ⁷ molecules. It should be noted that the higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, making it possible not only to construct a highly sensitive screening system, but also to measure a large number of samples in the same lot. .

Screening for compounds that alter the binding of the ligand to the receptor protein of the invention described above.
To carry out a) to c), for example, a suitable receptor protein fraction and a labeled ligand are required. As the receptor protein fraction, a natural receptor protein fraction or a recombinant receptor protein fraction having an activity equivalent to that is desirable. Here, the equivalent activity refers to equivalent ligand binding activity, signal transduction action, and the like. As the labeled ligand, a labeled ligand, a labeled ligand analog compound or the like is used. For example, [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [
^For example, a ligand labeled with ³⁵ S] or the like is used.
Specifically, in order to screen a compound that changes the binding property between the ligand and the receptor protein of the present invention, first, cells or a membrane fraction of cells containing the receptor protein of the present invention are treated with a buffer suitable for screening. A receptor protein preparation is prepared by suspending the protein in the solution. The buffer should have a pH of 4 to 10 (preferably a pH of 6).
Any buffer may be used as long as it does not inhibit the binding between the ligand and the receptor protein, such as the phosphate buffer and Tris-hydrochloric acid buffer described in 8). Further, for the purpose of reducing non-specific binding, CHAPS, Tween-80
Surfactants such as ^TM (Kao-Atlas), digitonin and deoxycholate may be added to the buffer. Furthermore, PMSF, leupeptin, E-6 for the purpose of suppressing the degradation of the receptor and ligand by protease.
4 (manufactured by Peptide Institute), a protease inhibitor such as pepstatin can also be added. 0.01 ml-1
A constant amount (5000 cpm) was added to 0 ml of the receptor solution.
˜500,000 cpm) labeled ligand is added,
At the same time, 10 ⁻⁴ M to ^{10 −10} M test compound is allowed to coexist. A reaction tube containing a large excess of unlabeled ligand is also prepared in order to know the amount of non-specific binding (NSB). The reaction is carried out at about 0 to 50 ° C, preferably about 4 to 37 ° C and about 20
Minutes to 24 hours, preferably about 30 minutes to 3 hours.
After the reaction, the mixture is filtered through a glass fiber filter paper or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter paper is measured with a liquid scintillation counter or a γ-counter. Count when there is no competing substance
_{(B 0)} when the amount of non-specific binding count obtained by subtracting the _(NSB) (B 0 -NSB) was 100%, the specific binding amount (B-NSB) of, for example, a test compound comprising 50% or less Can be selected as a candidate substance capable of competitive inhibition.

Compounds that alter the binding between the ligand and the receptor protein of the present invention are screened as described above.
For carrying out the methods of d) to e), for example, cell stimulating activity mediated by a receptor protein (eg, arachidonic acid release, acetylcholine release, intracellular Ca release, intracellular c release)
AMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-
In particular, intracellular Ca-releasing activity (intracellular C
a2 ⁺ concentration increasing activity) and intracellular cAMP producing activity) can be measured using a known method or a commercially available measurement kit. Specifically, first, cells containing the receptor protein of the present invention are cultured in a multiwell plate or the like. In screening, the medium was exchanged with a fresh medium or an appropriate buffer that did not show toxicity to cells in advance, test compounds were added and incubated for a certain period of time, and then cells were extracted or the supernatant was collected,
The product produced is quantified according to the respective method. When the production of a substance that serves as an index of cell stimulating activity (for example, arachidonic acid) is difficult to assay with a degrading enzyme contained in cells, an inhibitor for the degrading enzyme may be added to the assay. Further, the activity of suppressing cAMP production and the like can be detected as a production suppressing action on cells in which the basic production amount of cells has been increased by forskolin or the like. In order to measure the cell stimulating activity and perform screening, cells expressing an appropriate receptor protein are required. As a cell expressing the receptor protein of the present invention, a cell line having the natural receptor protein of the present invention, a cell line expressing the above recombinant receptor protein, and the like are preferable. As the test compound, for example, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. are used, and these compounds may be novel compounds. It may be a known compound. Further, as the test compound, a compound designed to bind to the ligand binding pocket based on the atomic coordinates of the active site of the receptor protein of the present invention and the position of the ligand binding pocket is preferably used. The atomic coordinates of the active site of the receptor protein of the present invention and the position of the ligand binding pocket can be measured by using a known method or a method similar thereto.

A screening kit for a compound or a salt thereof that alters the binding property between a ligand and the receptor protein of the present invention comprises a receptor protein of the present invention, cells containing the receptor protein of the present invention, or the receptor protein of the present invention. For example, those containing the membrane fraction of the containing cells. Examples of the screening kit of the present invention include the following. 1. Screening Reagent a) In a measurement buffer and a washing buffer Hanks' Balanced Salt Solution (manufactured by Gibco), add 0.
To which 05% bovine serum albumin (manufactured by Sigma) was added. Sterilize by filtration with a 0.45μm filter at 4 ℃
It may be stored at or prepared at the time of use. b) G protein-coupled receptor preparation CHO cells expressing the receptor protein of the present invention are
Transfer to 12-well plate at 5 × 10 ⁵ pieces / hole, 37 ℃,
What was cultured for 2 days in 5% CO ₂ and 95% air. c) Labeled ligand commercially available [ ³ H], [ ¹²⁵ I], [ ¹⁴ C],
An aqueous solution of a ligand labeled with [ ³⁵ S] or the like is stored at 4 ° C. or −20 ° C., and diluted to 1 μM with a measurement buffer at the time of use. d) ligand standard solution 0.1% bovine serum albumin (manufactured by Sigma)
Dissolve to 1 mM in PBS containing and store at -20 ° C.

2. Assay method a) CHO cells expressing the receptor protein of the present invention cultivated on a 12-well tissue culture plate were added with 1 ml of a measurement buffer.
After washing twice with, 490 μl of measuring buffer is added to each well. b) 5 μl of 10 ⁻³ to 10 ⁻¹⁰ M test compound solution
After the addition, 5 μl of labeled ligand is added and the reaction is allowed to proceed at room temperature for 1 hour. In order to know the amount of non-specific binding, 5 μl of 10 ⁻³ M ligand was added in place of the test compound. c) Remove the reaction solution and wash 3 times with 1 ml of wash buffer. The labeled ligand bound to the cells was treated with 0.2 N NaO.
It is dissolved in H-1% SDS and mixed with 4 ml of liquid scintillator A (manufactured by Wako Pure Chemical Industries). d) The radioactivity was measured using a liquid scintillation counter (manufactured by Beckman) and Percent Maximum Bind
ing (PMB) is calculated by the following formula. _{PMB = [(B-NSB)} / (B 0 -NSB)] × 10
0 PMB: Percent Maximum Binding B: Value when a sample is added NSB: Non-specific Binding B ₀ : Maximum binding amount

The compound or its salt obtained by using the screening method or screening kit of the present invention is a compound having an action of changing the binding property between the ligand and the receptor protein of the present invention, and specifically,
(A) a compound having a cell stimulating activity via a G protein-coupled receptor (so-called an agonist for the receptor protein of the present invention), (b) a compound having no such cell stimulating activity (a so-called antagonist for the receptor protein of the present invention) And (c) a compound that enhances the binding force between the ligand and the G protein-coupled receptor protein of the present invention, or (d) a compound that decreases the binding force between the ligand and the G protein-coupled receptor protein of the present invention. . Examples of the compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc. These compounds may be novel compounds or known compounds. Further, the compound may be a compound designed based on the atomic coordinates of the active site and the position of the ligand binding pocket of the receptor protein of the present invention described above. Since the agonist for the receptor protein of the present invention has the same physiological activity as that of the phospholipid compound that is a ligand for the receptor protein of the present invention, it is safe and has low toxicity depending on the physiological activity of the phospholipid compound. It is useful as a new medicine. Since the antagonist for the receptor protein of the present invention can suppress the physiological activity of the phospholipid compound that is a ligand for the receptor protein of the present invention, a safe and low-toxic pharmaceutical agent for suppressing the physiological activity of the phospholipid compound Is useful as The compound that enhances the binding force between the ligand and the G protein-coupled receptor protein of the present invention can enhance the physiological activity of the ligand for the receptor protein of the present invention, and therefore, depending on the physiological activity of the phospholipid compound. It is useful as a safe and low-toxic drug. Since the compound that reduces the binding force between the ligand and the G protein-coupled receptor protein of the present invention can reduce the physiological activity of the ligand for the receptor protein of the present invention, it suppresses the physiological activity of the phospholipid compound. It is useful as a safe and low-toxic pharmaceutical agent. Specifically, the compound or its salt obtained by using the screening method or the screening kit of the present invention,
For example, central diseases (for example, Alzheimer's disease, dementia, eating disorders, etc.), inflammatory diseases (for example, allergic diseases, asthma, rheumatism, etc.), cardiovascular diseases (for example, hypertension, cardiac hypertrophy, angina). , Arteriosclerosis, etc.), cancer (for example, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer,
Breast cancer, cervical cancer, colon cancer, rectal cancer, etc., diabetes, immune system diseases (eg, autoimmune disease, AIDS, atopic dermatitis, allergic disease, immunodeficiency, asthma, rheumatoid arthritis, psoriasis, arteries Sclerosis, diabetes, Alzheimer's disease, etc., liver and gallbladder diseases (eg cirrhosis, hepatitis,
Liver failure, cholestasis, stones, etc., digestive system diseases (eg, ulcer, enteritis, dyspepsia, irritable colitis, ulcerative colitis, diarrhea, ileus, etc.), anxiety, pain, obesity, etc. It is useful as a prophylactic / therapeutic agent for

When the compound or its salt obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned pharmaceutical composition, it can be formulated according to a conventional method. For example, the compound is orally as a sugar-coated tablet, capsule, elixir, microcapsule, or the like, or a sterile solution with water or another pharmaceutically acceptable liquid, Alternatively, it can be used parenterally in the form of injection such as suspension. For example, admixing the compound with a known physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder and the like in a unit dose form generally required for the implementation of the formulation. Can be manufactured by. The amount of active ingredient in these preparations is such that a suitable amount within the indicated range can be obtained. Additives that can be mixed with tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, acacia, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Puffing agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red oil or cherry and the like are used. When the unit dosage form is a capsule, a liquid carrier such as fat may be included in addition to materials of the above type. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices such as dissolving or suspending the active substance in a vehicle such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. , For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80)
You may use together with ^TM , HCO-50), etc. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.

The above-mentioned prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers ( For example, human serum albumin, polyethylene glycol, etc.), preservatives (eg,
(Benzyl alcohol, phenol, etc.), antioxidant, etc. The prepared injection solution is usually filled in a suitable ampoule. Since the preparation thus obtained is safe and has low toxicity, for example, humans and mammals (for example, rat, mouse, rabbit, sheep, pig, cow,
(Cats, dogs, monkeys, etc.). The dose of the compound or its salt varies depending on the administration subject, target organ, symptoms, administration method, etc., but in the case of oral administration, in general, for example, in hypertensive patients (as 60 kg), About 0.1-100 mg,
Preferably about 1.0 to 50 mg, more preferably about 1.
It is 0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc., but is usually in the form of an injection, for example, in hypertensive patients (60 kg). About 0.01 to 30 mg per day, preferably about 0.1
It is convenient to administer about -20 mg, more preferably about 0.1-10 mg by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

(7) Pharmaceutical containing a compound which changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane The antibody of the present invention specifically recognizes the receptor protein of the present invention or its partial peptide or its salt. Therefore, it can be used for screening a compound that alters the amount of the receptor protein of the present invention or its partial peptide in the cell membrane. That is, the present invention is
For example, (i) a) blood of a non-human mammal, b) a specific organ, c) a tissue or cell isolated from an organ is destroyed, and then a cell membrane fraction is isolated, A method for screening a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane by quantifying the receptor protein of the present invention or its partial peptide, (ii) expressing the receptor protein of the present invention or its partial peptide After disrupting the transformant, etc., the cell membrane fraction is isolated, and the amount of the receptor protein of the present invention or its partial peptide in the cell membrane is determined by quantifying the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction. (Iii) a non-human mammal a) blood, b) a specific organ, c) Confirm the protein on the cell membrane by quantifying the degree of staining of the receptor protein on the cell surface by using immunostaining method after sectioning the tissue or cells isolated from the organ. Provides a method for screening a compound that alters the amount of the receptor protein of the present invention or a partial peptide thereof in the cell membrane. (Iv) By quantifying the degree of staining of the receptor protein on the cell surface by using an immunostaining method after sectioning a transformant or the like expressing the receptor protein of the present invention or a partial peptide thereof, The present invention also provides a method for screening a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane by confirming the protein on the cell membrane.

The receptor protein of the present invention or its partial peptide contained in the cell membrane fraction is quantified specifically as follows. (I) Normal or disease model Non-human mammal (eg, mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, and more specifically, demented rat, obese mouse, arteriosclerotic rabbit, cancer bearing) Mouse)
Blood ( Obtaining a specific organ (eg, brain, liver, kidney, etc.), or tissue or cells isolated from the organ. The obtained organ, tissue, cell or the like is suspended in, for example, an appropriate buffer solution (for example, Tris-HCl buffer solution, phosphate buffer solution, Hepes buffer solution, etc.),
The organs, tissues or cells are destroyed, and a cell membrane fraction is obtained by using a surfactant (eg, Triton X100 ^™ , Tween 20 ^™, etc.) and a method such as centrifugation, filtration or column fractionation. The cell membrane fraction refers to a fraction containing a large amount of cell membrane obtained by a method known per se after crushing cells. The cells are disrupted by crushing the cells with a Potter-Elvehjem homogenizer, Waring blender or polytron (Kinema).
(manufactured by tica), disruption by ultrasonic waves, disruption by ejecting cells from a thin nozzle while applying pressure with a French press, and the like. For the fractionation of the cell membrane, a fractionation method by centrifugal force such as a fractionation centrifugation method or a density gradient centrifugation method is mainly used. For example, the cell lysate is centrifuged at low speed (500 rpm to 3000 rpm) for a short time (usually about 1 minute to 10 minutes), and the supernatant is further centrifuged at high speed (15000 rpm to 30000 rpm) for usually 30 minutes to 2 hours to obtain The precipitate is used as the membrane fraction. The membrane fraction is rich in expressed receptor proteins and membrane components such as cell-derived phospholipids and membrane proteins. The receptor protein of the present invention or its partial peptide contained in the cell membrane fraction can be quantified by, for example, a sandwich immunoassay using the antibody of the present invention, Western blot analysis, or the like. The sandwich immunoassay can be performed in the same manner as the above method, and the Western blot can be performed by a method known per se. (Ii) a transformant expressing the receptor protein of the present invention or a partial peptide thereof is prepared according to the above method,
The receptor protein of the present invention or its partial peptide contained in the cell membrane fraction can be quantified.

Screening for a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane is carried out by (i) a certain period of time before a drug or physical stress is applied to a normal or disease model non-human mammal. (30 minutes to 24 hours ago, preferably 3 minutes
0 minutes to 12 hours before, more preferably 1 hour to 6 hours before) or after a fixed time (30 minutes to 3 days later, preferably 1 hour to 2 days later, more preferably 1 hour to 24 hours later). ), Or a compound to be tested is administered at the same time as a drug or physical stress, and after a lapse of a predetermined time (30 minutes-
3 days, preferably 1 to 2 days later, more preferably 1 to 24 hours later), by quantifying the amount of the receptor protein of the present invention or its partial peptide in the cell membrane, (ii) When the transformant is cultured according to a conventional method, the test compound is mixed in the medium, and after culturing for a certain time (1 day to 7 days later, preferably 1 day to 3 days later, more preferably 2 days to 3 days later), This can be performed by quantifying the amount of the receptor protein of the present invention or its partial peptide in the cell membrane. Specifically, the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction is confirmed as follows. (Iii) Normal or disease model non-human mammal (eg, mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, and more specifically, demented rat, obese mouse, arteriosclerotic rabbit, cancer-bearing) Mouse)
Blood ( Obtaining a specific organ (eg, brain, liver, kidney, etc.), or tissue or cells isolated from the organ. The obtained organs, tissues, cells or the like are cut into tissue sections according to a conventional method and immunostained with the antibody of the present invention. By quantifying the degree of staining of the receptor protein on the cell surface, by confirming the protein on the cell membrane, the amount of the receptor protein of the present invention or its partial peptide on the cell membrane can be quantitatively or qualitatively determined. You can check. (Iv) It can also be confirmed by the same procedure using a transformant expressing the receptor protein of the present invention or a partial peptide thereof.

The compound or its salt obtained by using the screening method of the present invention is a compound having an action of changing the amount of the receptor protein of the present invention or its partial peptide in the cell membrane, and specifically, (a) By increasing the amount of the receptor protein of the present invention or a partial peptide thereof in the cell membrane, a cell stimulating activity mediated by a G protein-coupled receptor (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, Intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation,
a compound that enhances the activity of activating c-fos, promoting or suppressing pH lowering, etc., and (b) reducing the amount of the receptor protein of the present invention or its partial peptide in the cell membrane, It is a compound that reduces stimulatory activity. Examples of the compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc. These compounds may be novel compounds or known compounds. A compound which enhances the cell stimulating activity by increasing the amount of the receptor protein of the present invention or its partial peptide in the cell membrane is a safe and low toxic prophylactic / therapeutic agent for diseases associated with dysfunction of the receptor protein of the present invention. Is useful as By reducing the amount of the receptor protein of the present invention or its partial peptide in the cell membrane,
The compound that attenuates the cell stimulating activity is useful as a safe and low toxic prophylactic / therapeutic agent for diseases caused by overexpression of the receptor protein of the present invention. Specifically, the compound having the action of changing the amount of the receptor protein or its partial peptide of the present invention or a salt thereof is, for example,
Central diseases (for example, Alzheimer's disease, dementia, eating disorders, etc.), inflammatory diseases (for example, allergic diseases, asthma, rheumatism, etc.), cardiovascular diseases (for example, hypertension,
Cardiac hypertrophy, angina, arteriosclerosis, etc.), cancer (for example, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.), diabetes, Immune system diseases (eg, autoimmune diseases, AIDS, atopic dermatitis, allergic diseases, immunodeficiency, asthma, rheumatoid arthritis, psoriasis, arteriosclerosis, diabetes, Alzheimer's disease, etc.), liver / gallbladder diseases (eg, Cirrhosis, hepatitis, liver failure, cholestasis, stones, etc., digestive system diseases (eg,
Ulcer, enteritis, dyspepsia, irritable colitis, ulcerative colitis,
It can be used as a prophylactic / therapeutic agent for diseases such as diarrhea, ileus), anxiety, pain and obesity. When the compound or its salt obtained by using the screening method of the present invention is used as a pharmaceutical composition, it can be formulated according to a conventional method. For example, the compound is orally as a sugar-coated tablet, capsule, elixir, microcapsule, or the like, or a sterile solution with water or another pharmaceutically acceptable liquid, Alternatively, it can be used parenterally in the form of injection such as suspension. For example, admixing the compound with a known physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder and the like in a unit dose form generally required for the implementation of the formulation. Can be manufactured by. The amount of active ingredient in these preparations is such that a suitable amount within the indicated range can be obtained.

Additives that can be mixed with tablets, capsules and the like include, for example, binders such as gelatin, corn starch, tragacanth and acacia, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid. Puffing agents such as and the like, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red oil or cherry, and the like are used. When the unit dosage form is a capsule, a liquid carrier such as fat may be included in addition to materials of the above type. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices such as dissolving or suspending the active substance in a vehicle such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, Polysorbate 80 ^™ , HCO-50) and the like may be used in combination. Examples of the oily liquid include sesame oil and soybean oil,
You may use together with solubilizing agents, such as benzyl benzoate and benzyl alcohol. In addition, the above-mentioned preventive / therapeutic agents are
For example, buffer (eg, phosphate buffer, sodium acetate buffer), soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizer (eg, human serum albumin, polyethylene glycol, etc.), preservation You may mix | blend with agents (for example, benzyl alcohol, phenol, etc.), antioxidants, etc. The prepared injection solution is usually filled in a suitable ampoule. Since the preparation thus obtained is safe and has low toxicity, it should be administered to, for example, humans or mammals (eg, rat, mouse, rabbit, sheep, pig, cow, cat, dog, monkey). You can The dose of the compound or its salt varies depending on the administration subject, target organ, symptoms, administration method, etc., but in the case of oral administration, generally, for example, in hypertensive patients (as 60 kg), About 0.1-
It is 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose is the administration subject, target organ, symptom,
Although it depends on the administration method, for example, in the form of an injection, for example, in a hypertensive patient (as 60 kg), about 0.01 to 30 mg, preferably about 0.1 to 20 mg per day is more preferable. Preferably about 0.1
It is convenient to administer about 10 mg by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

(8) A medicine comprising an antibody against the receptor protein of the present invention, a partial peptide thereof or a salt thereof. The neutralizing activity of an antibody against the receptor protein of the present invention or a partial peptide thereof or a salt thereof means the receptor. It means an activity that inactivates a signal transduction function involving a protein. Therefore, when the antibody has neutralizing activity, signal transduction involving the receptor protein, for example, cell stimulating activity mediated by the receptor protein (eg, arachidonic acid release, acetylcholine release, intracellular C
a ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, activity promoting or suppressing pH decrease, etc. ) Can be inactivated. Therefore, the receptor protein of the present invention, a neutralizing antibody against the partial peptide or a salt thereof is a disease caused by overexpression of the receptor protein (for example, a central disease (for example, Alzheimer's disease,
Dementia, eating disorders, etc., inflammatory diseases (for example, allergic diseases, asthma, rheumatism, etc.), cardiovascular diseases (for example,
Hypertension, cardiac hypertrophy, angina, arteriosclerosis, etc.), cancer (for example, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer,
Breast cancer, cervical cancer, colon cancer, rectal cancer, etc., diabetes, immune system diseases (eg, autoimmune disease, AIDS, atopic dermatitis, allergic disease, immunodeficiency, asthma, rheumatoid arthritis, psoriasis, arteries Sclerosis, diabetes, Alzheimer's disease, etc., liver and gallbladder diseases (eg cirrhosis, hepatitis,
Liver failure, cholestasis, stones, etc., digestive system diseases (eg, ulcer, enteritis, dyspepsia, irritable colitis, ulcerative colitis, diarrhea, ileus, etc.), anxiety, pain, obesity, etc. ) Can be used as a preventive / therapeutic agent.

(9) Pharmaceutical comprising the antisense polynucleotide of the present invention The antisense polynucleotide of the present invention is a disease caused by overexpression of the receptor protein of the present invention (for example, central diseases (for example, Alzheimer's disease, dementia, eating disorders, etc., inflammatory diseases (for example, allergic diseases,
Asthma, rheumatism, etc., cardiovascular disease (for example, hypertension, cardiac hypertrophy, angina, arteriosclerosis, etc.), cancer (for example, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer,
Cervical cancer, colon cancer, rectal cancer, etc., diabetes, immune system diseases (eg autoimmune disease, AIDS, atopic dermatitis, allergic disease, immunodeficiency, asthma, rheumatoid arthritis, psoriasis, arteriosclerosis) , Diabetes, Alzheimer's disease, etc.), liver / cholecystic diseases (eg, cirrhosis, hepatitis, liver failure, cholestasis, stones, etc.), digestive system diseases (eg,
Ulcer, enteritis, dyspepsia, irritable colitis, ulcerative colitis,
Diarrhea, ileus, etc.), anxiety, pain, obesity, etc.). For example, when the antisense polynucleotide is used, the antisense polynucleotide may be used alone or after inserting it into an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector, according to a conventional method. it can.
The antisense polynucleotide can be administered as it is or in the form of a preparation with a physiologically acceptable carrier such as an auxiliary agent for promoting ingestion, and can be administered by a gene gun or a catheter such as a hydrogel catheter. Further, the antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for investigating the presence of the DNA of the present invention or the expression state thereof in tissues or cells.

(10) Preparation of DNA-Introduced Animal of the Present Invention The present invention is directed to the foreign DNA of the present invention (hereinafter abbreviated as the foreign DNA of the present invention) or its mutant DNA (exogenous mutant DNA of the present invention). Sometimes abbreviated). That is, the present invention provides [1]
A non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof, [2] the animal of [1], wherein the non-human mammal is a rodent, and [3] the rodent is a mouse or rat. A recombinant vector containing the animal described in [2] and [4] the exogenous DNA of the present invention or a mutant DNA thereof and capable of being expressed in a mammal is provided. The non-human mammal having the exogenous DNA of the present invention or the mutant DNA thereof (hereinafter, abbreviated as the DNA-transferred animal of the present invention) is used for embryo cells including unfertilized eggs, fertilized eggs, sperms and their progenitor cells. Preferably, at the stage of embryogenesis in development of non-human mammals (more preferably at the stage of single cells or fertilized egg cells and generally before the 8-cell stage), the calcium phosphate method, electric pulse method, lipofection method, agglutination method, It can be produced by transferring the target DNA by the microinjection method, particle gun method, DEAE-dextran method, or the like. In addition, by the DNA transfer method, the target foreign DNA of the present invention can be transferred to somatic cells, living organs, tissue cells and the like and used for cell culture, tissue culture and the like. The DNA-transferred animal of the present invention can also be produced by fusing the above-mentioned embryo cells with a cell fusion method known per se. Examples of the non-human mammal include cow, pig, sheep, goat, rabbit, dog, cat, guinea pig, hamster, mouse, rat and the like. Among them, rodents that have relatively short ontogeny and biological cycle from the viewpoint of creating a pathological animal model system and are easy to reproduce, especially mice (for example, as a pure system, C57BL / 6 strain, DB
As a hybrid system such as A2 strain, B6C3F ₁ strain, BD
F ₁ system, B6D2F ₁ system, BALB / c system, IC
R strain) or rat (eg, Wistar, S
D etc.) and the like are preferable. Examples of the “mammal” in the recombinant vector that can be expressed in mammals include humans in addition to the above-mentioned non-human mammals.

The exogenous DNA of the present invention does not mean the DNA of the present invention originally possessed by non-human mammals, but the DNA of the present invention once isolated and extracted from mammals.
The mutant DNA of the present invention is a DNA having a mutation (eg, mutation) in the base sequence of the original DNA of the present invention,
Specifically, DNA in which a base is added, deleted, or replaced with another base is used, and abnormal DNA is also included. The abnormal DNA means a DNA that expresses an abnormal receptor protein of the present invention, and for example, a DNA that expresses a receptor protein that suppresses the function of the normal receptor protein of the present invention can be used. The exogenous DNA of the present invention may be derived from mammals of the same or different species as the target animal. When transferring the DNA of the present invention to a target animal, the DN
It is generally advantageous to use A as a DNA construct linked downstream of a promoter that can be expressed in animal cells. For example, when transferring the human DNA of the present invention, expression of DNA derived from various mammals (for example, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.) having the DNA of the present invention having high homology with the human DNA The human DNA of the present invention is provided downstream of various promoters
DNA constructs that bind DNA (eg, vectors, etc.)
DNA of the present invention by microinjecting a fertilized egg of a target mammal, for example, a mouse fertilized egg.
It is possible to produce a DNA-transferred mammal that highly expresses.

The expression vector of the receptor protein of the present invention includes Escherichia coli-derived plasmid, Bacillus subtilis-derived plasmid, yeast-derived plasmid, bacteriophage such as λ phage, retrovirus such as Moloney leukemia virus, vaccinia virus or baculovirus. Animal viruses such as are used. Of these, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, and the like are preferably used. Examples of the above-mentioned promoter for controlling DNA expression include promoters of DNA derived from viruses (eg, simian virus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, poliovirus, etc.), various mammals (human , Rabbit, dog,
(Cat, guinea pig, hamster, rat, mouse, etc.)
Derived promoters such as albumin, insulin II, uroplakin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acidic protein, glutathione S-transferase, platelet derived growth factor β, keratins K1, K10 and K14, collagen I Type and type II, cyclic AMP-dependent protein kinase βI subunit, dystrophin, tartrate-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor tyrosine kinase (generally abbreviated as Tie2), sodium potassium adenosine 3 phosphatase ( Na, K-ATP
a), neurofilament light chain, metallothionein I and IIA, metalloproteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ra.
s, renin, dopamine β-hydroxylase, thyroid peroxidase (TPO), peptide chain elongation factor 1α (EF
-1α), β actin, α and β myosin heavy chains, myosin light chains 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, Promoters such as troponin C, smooth muscle α-actin, preproenkephalin A and vasopressin are used. Among them, cytomegalovirus promoter, human peptide chain elongation factor 1α (EF-1α) promoter, human and chicken β-actin promoter, which can be highly expressed systemically, are preferable.
The vector preferably has a sequence that terminates the transcription of the messenger RNA of interest in a DNA-transferred mammal (generally called a terminator). SV40 terminator of simian virus and the like can be used.

In addition, for the purpose of further highly expressing the desired foreign DNA, the splicing signal of each DNA, the enhancer region, a part of the intron of eukaryotic DNA and the like are provided 5 ′ upstream of the promoter region, between the promoter region and the translation region. Alternatively, it may be linked to the 3'downstream of the translation region depending on the purpose. A normal translation region of the receptor protein of the present invention includes liver-, kidney-, thyroid-cell, fibroblast-derived DNA derived from human or various mammals (eg, rabbit, dog, cat, guinea pig, hamster, rat, mouse) As all or part of genomic DNA from various commercially available genomic DNA libraries,
Or RNA derived from liver, kidney, thyroid cells, fibroblasts
The complementary DNA prepared by a more known method can be obtained as a raw material. In addition, extraneous abnormal DNA
Can produce a translation region in which the translation region of the normal receptor protein obtained from the above cells or tissues is mutated by the point mutagenesis method. The translation region can be prepared as a DNA construct that can be expressed in a transgenic animal by a conventional DNA engineering method in which it is linked to the downstream of the promoter and, if desired, the upstream of the transcription termination site. The transfer of the foreign DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germinal cells and somatic cells of the target mammal. In the embryonic cells of the created animal after DNA transfer, the exogenous DN of the present invention is used.
The presence of A means that all progeny of the producing animal retain the exogenous DNA of the invention in all of its germ cells and somatic cells. The offspring of this type of animal that inherits the exogenous DNA of the present invention has the exogenous DNA of the present invention in all of its germ cells and somatic cells. The non-human mammal to which the exogenous normal DNA of the present invention has been transferred can be subcultured in a normal breeding environment as the DNA-bearing animal after confirming that the exogenous DNA is stably retained by mating. . Exogenous D of the present invention at the fertilized egg cell stage
NA transfer is ensured to be present in excess in all germinal and somatic cells of the subject mammal. Excessive presence of the exogenous DNA of the present invention in the germinal cells of the producing animal after DNA transfer means that all the progeny of the producing animal have the exogenous DNA of the present invention in all of their germinal cells and somatic cells. Means The offspring of this type of animal that inherited the exogenous DNA of the present invention have the exogenous DNA of the present invention in excess in all of their germ cells and somatic cells. Introduction D
By obtaining a homozygous animal having NA on both homologous chromosomes and mating the male and female animals, all the progeny can be passaged so as to have the DNA in excess.

The non-human mammal having the normal DNA of the present invention has a high expression of the normal DNA of the present invention, and by promoting the function of the endogenous normal DNA, the receptor protein of the present invention is finally expressed. It may develop hyperfunction and can be used as a model animal for its pathological condition. For example, using the normal DNA-transferred animal of the present invention, elucidation of the pathophysiological mechanism of hyperfunction of the receptor protein of the present invention and diseases related to the receptor protein of the present invention, and examination of treatment methods for these diseases It is possible. In addition, since the mammal to which the exogenous normal DNA of the present invention has been transferred has an increased symptom of the released receptor protein of the present invention, it can be used for screening test of therapeutic agents for diseases related to the receptor protein of the present invention. It is possible. On the other hand, the non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured as a DNA-carrying animal in a normal breeding environment after confirming that the exogenous DNA is stably retained by mating. Furthermore, the foreign DNA of interest can be incorporated into the above-mentioned plasmid and used as a protozoa. The DNA construct with the promoter can be prepared by a usual DNA engineering technique. Abnormal DN of the present invention at the fertilized egg cell stage
The A transfer is ensured to be present in all germinal and somatic cells of the subject mammal. The presence of the abnormal DNA of the present invention in the germinal cells of the producing animal after DNA transfer means that all the progeny of the producing animal have the abnormal DNA of the present invention in all of its germinal cells and somatic cells.
The offspring of this type of animal that inherited the exogenous DNA of the present invention has the abnormal D of the present invention in all of its germ cells and somatic cells.
Has NA. By obtaining a homozygous animal having the introduced DNA in both homologous chromosomes and mating the male and female animals, all the progeny can be passaged so that they have the DNA.

The non-human mammal having the abnormal DNA of the present invention has a high expression of the abnormal DNA of the present invention, and finally inhibits the function of the endogenous normal DNA to finally obtain the receptor protein of the present invention. It may be functionally inactive refractory, and can be used as a model animal for its pathological condition. For example, by using the abnormal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of the functionally inactive refractory disease of the receptor protein of the present invention and to examine the treatment method for this disease. In addition, as a specific applicability, the animal with high expression of abnormal DNA of the present invention is characterized by the inhibition of function of normal receptor protein by the abnormal receptor protein of the present invention in the case of functionally inactive refractory disease of the receptor protein of the present invention (dominant negative). It becomes a model to clarify the action. Further, since the mammal to which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of the released receptor protein of the present invention, it can be used in a therapeutic drug screening test for the receptor protein of the present invention or a functionally inactive refractory disease thereof. Is also available. In addition, the above-mentioned two types of DNA of the present invention
Other uses of metastatic animals include, for example, use as a cell source for tissue culture, DNA or R in tissue of a DNA transgenic animal of the invention.
Analysis of the relationship with the receptor protein specifically expressed or activated by the receptor protein of the present invention by directly analyzing NA or by analyzing the tissue of the receptor protein expressed by DNA, tissue having DNA Cells are cultured by standard tissue culture techniques and used to study the function of cells from tissues that are generally difficult to culture, screening for agents that enhance cell function by using the cells described above, and It is conceivable to isolate and purify the mutant receptor protein of the present invention and to prepare its antibody. Furthermore, the DNA-transferred animal of the present invention can be used to investigate clinical symptoms of diseases related to the receptor protein of the present invention, including functionally inactive refractory disease of the receptor protein of the present invention, and also the present invention. More detailed pathological findings in each organ of a disease model related to the receptor protein of Escherichia coli can be obtained, which can contribute to the development of new therapeutic methods and further to the research and treatment of secondary diseases caused by the diseases. Further, it is possible to take out each organ from the DNA-transferred animal of the present invention, cut it into small pieces, and then use a protease such as trypsin to obtain released DNA-transferred cells, culture the cells, or systematize the cultured cells. . Furthermore, the receptor protein-producing cells of the present invention can be identified, their relation to apoptosis, differentiation or proliferation, or their signal transduction mechanism can be investigated, and their abnormalities can be investigated. It will be an effective research material for elucidation. Furthermore, in order to develop a therapeutic agent for a disease related to the receptor protein of the present invention, including a functionally inactive refractory disease of the receptor protein of the present invention, using the DNA-transferred animal of the present invention,
It becomes possible to provide an effective and rapid screening method for the therapeutic drug for the disease by using the above-mentioned test method and quantitative method. It is also possible to study and develop a DNA therapeutic method for diseases associated with the receptor protein of the present invention, using the DNA-transferred animal of the present invention or the foreign DNA expression vector of the present invention.

(11) Knockout Animal The present invention provides a non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated and a DNA expression-deficient non-human mammal of the present invention. That is, the present invention provides [1] a non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated,
[2] The embryonic stem cell according to [1], wherein the DNA is inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from Escherichia coli), [3] Neomycin resistant [1] [4] The embryonic stem cell according to item [1], wherein the non-human mammal is a rodent, [5] The embryonic stem cell according to item [4], wherein the rodent is a mouse, [4] 6] The DNA expression-deficient non-human mammal in which the DNA of the present invention is inactivated, [7] The DNA is inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from Escherichia coli), The non-human mammal according to [6], wherein the reporter gene can be expressed under the control of a promoter for the DNA of the present invention,
[8] The non-human mammal according to [6], wherein the non-human mammal is a rodent.

[9] A test compound is administered to the non-human mammal according to the item [8], wherein the rodent is a mouse, and the animal according to the item [10] [7], and the expression of the reporter gene is detected. D of the present invention characterized by
Provided is a screening method for a compound or its salt that promotes or inhibits the promoter activity against NA. The non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated means that the expression ability of the DNA is suppressed by artificially mutating the DNA of the present invention possessed by the non-human mammal. By substantially abolishing the activity of the receptor protein of the present invention encoded by the DNA,
The term refers to a non-human mammalian embryonic stem cell (hereinafter abbreviated as ES cell) in which DNA has substantially no ability to express the receptor protein of the present invention (hereinafter sometimes referred to as knockout DNA of the present invention). As the non-human mammal, the same ones as described above are used. The method of artificially mutating the DNA of the present invention can be carried out, for example, by deleting a part or all of the DNA sequence or inserting or replacing another DNA by a genetic engineering technique. Due to these mutations, the knockout DNA of the present invention may be prepared, for example, by shifting the reading frame of the codon or disrupting the function of the promoter or exon.

Specific examples of the non-human mammalian embryonic stem cells in which the DNA of the present invention is inactivated (hereinafter abbreviated as the DNA inactivated ES cells of the present invention or the knockout ES cells of the present invention) include, for example: The DNA of the present invention contained in the target non-human mammal is isolated, and its exon portion has a neomycin resistance gene, a drug resistance gene represented by a hygromycin resistance gene, or lacZ (β-galactosidase gene), cat (chloram). A DNA sequence (eg, polyA) that disrupts the function of an exon by inserting a reporter gene typified by a phenicol acetyl transferase gene) or terminates gene transcription at an intron portion between exons.
Insert additional signals, etc., and complete messenger R
By disabling NA synthesis, a DNA chain having a DNA sequence constructed so as to destroy the gene as a result (hereinafter abbreviated as targeting vector) is introduced into the chromosome of the animal by, for example, homologous recombination method. , D on the Southern hybridization analysis or targeting vector of the obtained ES cells using the DNA sequence of the present invention or a DNA sequence in the vicinity thereof as a probe
The knockout E of the present invention was analyzed by the PCR method using the NA sequence and the DNA sequence of the neighboring region other than the DNA of the present invention used for the preparation of the targeting vector as a primer.
It can be obtained by selecting S cells. Also,
As the original ES cells for inactivating the DNA of the present invention by the homologous recombination method, for example, those already established as described above may be used, or newly established according to the method of known Evans and Kaufma. You can also use it. For example, in the case of mouse ES cells, currently, 129 ES cells are generally used.
Although cells have been used, the immunological background is not clear. For the purpose of obtaining an ES cell whose immunological genetic background is clear in a pure system as an alternative, for example, C
C57BL / 6 mice and C57BL / 6 egg number of lack of such even better ones were established using _{(F 1} of C57BL / 6 and DBA / 2) BDF ₁ mice improved by crossing with DBA / 2 Can be used. The BDF ₁ mouse has a large number of eggs and has the advantage that the eggs are strong, and since it has the C57BL / 6 mouse as a background, the ES cells obtained by using the BDF ₁ mouse are , C57BL / 6 mice can be advantageously used in that their genetic background can be changed to that of C57BL / 6 mice. In addition, when ES cells are established, blastocysts on the 3.5th day after fertilization are generally used, but other than this, 8-cell stage embryos are ovulated and cultured up to the blastocysts for efficient and large numbers. The early embryos of can be obtained. Although either male or female ES cells may be used, male ES cells are usually more convenient for producing a germ line chimera. In addition, it is desirable to distinguish males and females as soon as possible in order to reduce troublesome culturing.

As a method for determining the sex of ES cells, for example, a method of amplifying and detecting a gene in the sex determining region on the Y chromosome by the PCR method can be mentioned. When this method is used, the number of ES cells required for karyotype analysis has conventionally been about 10 ⁶ cells, whereas the number of ES cells per colony (about 50 cells) is sufficient. It is possible to perform the primary selection of ES cells in the early stage by discriminating males and females, and the early selection of male cells can significantly reduce the labor required in the initial stage of culture.
The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method. The number of chromosomes of ES cells obtained is 100% of the normal number
However, if it is difficult because of physical manipulations during establishment, the gene of ES cell is knocked out, and then cloned again into normal cell (for example, cell having 2n = 40 chromosomes in mouse). Is desirable. The embryonic stem cell line thus obtained usually has a very good proliferative property, but since it is easy to lose the ability to develop ontogeny, it is necessary to carefully subculture. For example, on suitable feeder cells such as STO fibroblasts, LIF (1 to
Incubate in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5% oxygen, 5% carbon dioxide, 90% air) in the presence of 10,000 U / ml at about 37 ° C. However, at the time of subculture, for example, trypsin / EDTA
Solution (usually 0.001-0.5% trypsin / 0.1-5)
mM EDTA, preferably about 0.1% trypsin / 1m
(M EDTA) treatment to obtain single cells and seeding on newly prepared feeder cells. Such subculture is usually performed every 1 to 3 days, but it is desirable to observe the cells at this time and to discard the cultured cells when morphologically abnormal cells are found. ES cells are differentiated into various types of cells such as parietal muscle, visceral muscle, and myocardium by performing monolayer culture until reaching a high density or suspension culture until forming a cell clump under appropriate conditions. Is possible [MJ Evans and MH
Kaufman, Nature Volume 292, 154, 1981.
Year; GR Martin Proceedings of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) 78, 7634, 1
981; TC Doetschman et al., Journal of Embiology and Experimental Morphology, Vol. 87, p. 27, 1985], deficient DNA expression of the present invention obtained by differentiating ES cells of the present invention. The cells are useful in vitro for cell biological studies of the receptor proteins of the invention or of the receptor proteins of the invention.

The non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA level of the animal using a known method and indirectly comparing the expression level. is there. As the non-human mammal,
The same as the above is used. The non-human mammal deficient in DNA expression of the present invention can be obtained by, for example, introducing the targeting vector prepared as described above into mouse embryonic stem cells or mouse egg cells, and inactivating the DNA of the present invention in the targeting vector. The DNA of the present invention can be knocked out by homologous recombination in which the sequence replaces the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by homologous recombination. A cell in which the DNA of the present invention has been knocked out is a DNA on or near the DNA of the present invention.
A DNA sequence on a Southern hybridization analysis or targeting vector using the sequence as a probe,
It can be determined by an analysis by the PCR method using the DNA sequence of the neighboring region other than the mouse-derived DNA of the present invention used for the targeting vector as a primer. When a non-human mammal embryonic stem cell is used, a cell line in which the DNA of the present invention is inactivated by gene homologous recombination is cloned, and the cell is cloned at an appropriate time, for example, at the 8-cell stage. It is injected into an animal embryo or blastocyst, and the produced chimeric embryo is transplanted into the uterus of the pseudopregnant non-human mammal. The produced animal is a chimeric animal composed of both cells having the normal DNA locus of the present invention and cells having the artificially mutated DNA locus of the present invention. When a part of the germ cells of the chimeric animal has the mutated DNA locus of the present invention, all tissues are artificially mutated from the population obtained by mating such a chimeric individual with a normal individual. It can be obtained by selecting an individual composed of the cells having the added DNA locus of the present invention, for example, by judging the coat color. The individual thus obtained is usually an individual deficient in heteroexpression of the receptor protein of the present invention. Individuals heterozygous for deficient expression of the receptor protein of the present invention are crossed to obtain the receptor protein of the present invention from their offspring. An individual deficient in homo expression of can be obtained.

When egg cells are used, for example, a transgenic non-human mammal having the targeting vector introduced into its chromosome can be obtained by injecting a DNA solution into the nucleus of the egg cell by the microinjection method. It is obtained by selecting those having a mutation in the DNA locus of the present invention by gene homologous recombination as compared with a transgenic non-human mammal. In this way, an individual whose DNA of the present invention has been knocked out can be passaged in a normal breeding environment after confirming that the DNA of the animal individual obtained by mating is also knocked out. Furthermore, the acquisition and maintenance of the germ line may be carried out according to the conventional method. That is, by mating male and female animals carrying the inactivated DNA,
A homozygous animal having the inactivated DNA on both homologous chromosomes can be obtained. The homozygous animals obtained are
It can be efficiently obtained by breeding a mother animal in a state where there are one normal individual and a plurality of homozygotes. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred. DNA of the present invention
The non-human mammal embryonic stem cells inactivated by the method are very useful for producing the non-human mammal deficient in DNA expression of the present invention. Further, the non-human mammal deficient in expression of the DNA of the present invention lacks various biological activities that can be induced by the receptor protein of the present invention, so that diseases caused by inactivation of the biological activity of the receptor protein of the present invention Since it can be used as a model of the disease, it is useful for investigating the causes of these diseases and investigating treatment methods.

(11a) Method for Screening Compound Having Curative / Preventive Effect Against Disease Caused by Defect or Damage of DNA of the Present Invention The non-human mammal deficient in DNA expression of the present invention is D
It can be used for screening a compound having a therapeutic / preventive effect on diseases caused by NA deficiency or damage. That is, the present invention results from a defect or damage of the DNA of the present invention, which comprises administering a test compound to a non-human mammal deficient in DNA expression of the present invention and observing and measuring the change in the animal. Provided is a method for screening a compound or a salt thereof having a therapeutic / prophylactic effect against diseases. Examples of the non-human mammal deficient in DNA expression of the present invention used in the screening method include the same ones as described above. Examples of test compounds include peptides, proteins, non-peptidic compounds, synthetic compounds,
Examples include fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, and the like, and these compounds may be novel compounds or known compounds. Specifically, the non-human mammal deficient in DNA expression of the present invention is treated with a test compound and compared with an untreated control animal, and tested using changes in each organ, tissue, disease symptoms, etc. of the animal as an index. The therapeutic / prophylactic effect of a compound can be tested. As a method of treating a test animal with a test compound, for example,
Oral administration, intravenous injection, etc. are used to
It can be appropriately selected depending on the properties of the test compound. Further, the dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.

In the screening method, when the test compound is administered to the test animal, the blood glucose level of the test animal decreases by about 10% or more, preferably by about 30% or more, more preferably by about 50% or more, the test is conducted. The compound can be selected as a compound having a therapeutic / prophylactic effect against the above diseases. The compound obtained by using the screening method is a compound selected from the test compounds described above, and is a disease caused by a deficiency or damage of the receptor protein of the present invention (for example, a central disease (for example,
Alzheimer's disease, dementia, eating disorders, etc.), inflammatory diseases
(For example, allergic diseases, asthma, rheumatism, etc.),
Cardiovascular disease (for example, hypertension, cardiac hypertrophy, angina, arteriosclerosis, etc.), cancer (for example, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon) Cancer, rectal cancer, etc., diabetes, immune system diseases (eg autoimmune diseases,
AIDS, atopic dermatitis, allergic diseases, immunodeficiency, asthma, rheumatoid arthritis, psoriasis, arteriosclerosis,
Diabetes mellitus, Alzheimer's disease, etc., liver / cholecystic disease (eg, cirrhosis, hepatitis, liver failure, cholestasis, stones, etc.), digestive system diseases (eg, ulcer, enteritis, dyspepsia, etc.)
Since it has a therapeutic / preventive effect on irritable colitis, ulcerative colitis, diarrhea, ileus, etc.), anxiety, pain, obesity), it is used as a drug such as a safe and low toxic therapeutic / prophylactic agent for the disease. can do. Furthermore, compounds derived from the compounds obtained by the above screening can be used as well. The compound obtained by the screening method may form a salt, and the salt of the compound may be a physiologically acceptable acid (eg, inorganic acid, organic acid, etc.) or base (eg, alkali metal, etc.). ) And the like are used, and physiologically acceptable acid addition salts are particularly preferable. Examples of such salts include salts with inorganic acids (for example, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (for example, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.) and the like salts are used. The drug containing the compound or its salt obtained by the screening method can be produced in the same manner as the drug containing the compound which changes the binding property between the receptor protein of the present invention and the ligand. Since the preparation thus obtained is safe and has low toxicity, for example, humans or mammals (for example, rat, mouse, guinea pig, rabbit,
Sheep, pigs, cows, horses, cats, dogs, monkeys, etc.). The dose of the compound or its salt varies depending on the target disease, the administration subject, the administration route, etc., but, for example, when the compound is orally administered,
Generally, in a hypertensive patient (as a weight of 60 kg), the compound is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to about 100 mg per day.
Administer 20 mg. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, etc., but for example, the compound is usually administered in the form of an injection to a hypertensive patient (60 kg body weight). In that case, the amount of the compound is about 0.01 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 mg.
It is convenient to administer about 0 mg intravenously. In the case of other animals, the dose converted per 60 kg can be administered.

(11b) Method for Screening Compound for Promoting or Inhibiting Promoter Activity for DNA of the Present Invention The present invention provides a non-human mammal deficient in DNA expression of the present invention,
Provided is a method for screening a compound or its salt that promotes or inhibits the activity of a promoter for the DNA of the present invention, which comprises administering a test compound and detecting the expression of a reporter gene. In the above screening method, the non-human mammal deficient in DNA expression of the present invention, among the non-human mammal deficient in DNA expression of the present invention, the DNA of the present invention is inactivated by introducing a reporter gene, A reporter gene that can be expressed under the control of a promoter for the DNA of the present invention is used. Examples of the test compound include the same ones as described above. As the reporter gene, the same ones as described above are used, and the β-galactosidase gene (lacZ), the soluble alkaline phosphatase gene, the luciferase gene and the like are preferable. DN of the present invention in which the DNA of the present invention is replaced with a reporter gene
In a non-human mammal deficient in A expression, the reporter gene exists under the control of the promoter for the DNA of the present invention, and thus the activity of the promoter can be detected by tracing the expression of the substance encoded by the reporter gene. For example, when a part of the DNA region encoding the receptor protein of the present invention is replaced with the β-galactosidase gene (lacZ) derived from E. coli,
In tissues where the receptor protein of the present invention is expressed, β-galactosidase is expressed instead of the receptor protein of the present invention. Thus, for example, 5-bromo-4-chloro-3
-Indolyl-β-galactopyranoside (X-gal)
By staining with such a reagent as a substrate for β-galactosidase, the expression state of the receptor protein of the present invention in an animal body can be easily observed. Specifically, the receptor protein-deficient mouse of the present invention or a tissue section thereof is fixed with glutaraldehyde or the like, washed with phosphate buffered saline (PBS), and then treated with X-g.
staining solution containing al at room temperature or around 37 ° C, about 30
After reacting for 1 minute to 1 hour, the tissue sample was treated with 1 mM ED.
The β-galactosidase reaction may be stopped by washing with a TA / PBS solution, and the coloration may be observed. In addition, mRNA encoding lacZ may be detected according to a conventional method. The compound or its salt obtained by using the above screening method is a compound selected from the above-mentioned test compounds, and is a compound that promotes or inhibits the promoter activity for the DNA of the present invention. The compound obtained by the screening method may form a salt,
The salt of the compound includes a physiologically acceptable acid (eg,
A salt with an inorganic acid or the like) or a base (eg, an organic acid or the like) is used, and a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, inorganic acids (for example,
Salts with hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc., or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid) Acid, methanesulfonic acid, benzenesulfonic acid, etc.) and the like are used.

The compound or its salt that promotes the promoter activity for the DNA of the present invention can promote the expression of the receptor protein of the present invention and the function of the receptor protein. Therefore, for example, the receptor protein of the present invention can be used. It is useful as a medicine such as a prophylactic / therapeutic agent for diseases associated with dysfunction of the. The compound or its salt that inhibits the promoter activity for the DNA of the present invention can inhibit the expression of the receptor protein of the present invention and inhibit the function of the receptor protein. It is useful as a medicine such as a prophylactic / therapeutic agent for diseases related to urine. Specifically, compounds or salts thereof that promote or inhibit the promoter activity for DNA of the present invention include, for example, central diseases (for example, Alzheimer's disease, dementia, eating disorders, etc.),
Inflammatory diseases (for example, allergic disease, asthma, rheumatism, etc.), cardiovascular diseases (for example, hypertension, cardiac hypertrophy, angina, arteriosclerosis, etc.), cancer (for example, non-small cell lung cancer, ovarian cancer, Prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc., diabetes, immune system diseases (eg autoimmune disease, AIDS, atopic dermatitis, allergic disease, immunodeficiency, asthma) , Rheumatoid arthritis, psoriasis, arteriosclerosis, diabetes, Alzheimer's disease, etc.), liver and gallbladder diseases (eg, cirrhosis, hepatitis, liver failure, cholestasis, stones, etc.), digestive system diseases (eg, ulcer, Enteritis, dyspepsia, irritable colitis, ulcerative colitis, diarrhea, ileus, etc.), anxiety, pain, obesity and other diseases. Furthermore, compounds derived from the compounds obtained by the above screening can be used as well. The drug containing the compound or its salt obtained by the screening method can be prepared in the same manner as the drug containing the compound which changes the binding property between the receptor protein of the present invention or its salt and the ligand. . Since the preparation thus obtained is safe and has low toxicity, it is, for example, a human or mammal (for example, rat, mouse, guinea pig, rabbit, sheep,
Pig, cow, horse, cat, dog, monkey, etc.). Although the dose of the compound or its salt varies depending on the target disease, administration subject, administration route, etc., for example, when a compound that promotes the promoter activity for the DNA of the present invention is orally administered, it is generally used in patients with hypertension. In a body weight of 60 kg, the compound is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day.
Administer. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, etc.
For example, when a compound that promotes the promoter activity for the DNA of the present invention is usually administered in the form of an injection to a hypertensive patient (with a body weight of 60 kg), the compound is administered in an amount of about 0.01 to 30 mg, preferably about 30 mg per day. About 0.1-2
It is convenient to administer about 0 mg, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

As described above, the non-human mammal deficient in expression of the DNA of the present invention is extremely useful for screening a compound or its salt that promotes or inhibits the activity of the promoter for the DNA of the present invention, and the DNA of the present invention. It can greatly contribute to the investigation of the cause of various diseases caused by the expression failure or the development of preventive / therapeutic drugs. Further, DN containing the promoter region of the receptor protein of the present invention
By using A, genes encoding various proteins are ligated to the downstream thereof and injected into animal egg cells to produce a so-called transgenic animal (transgenic animal), which specifically synthesizes the receptor protein. Then, it becomes possible to examine the action in the living body. Furthermore, if a suitable reporter gene is bound to the above promoter portion and a cell line capable of expressing the same is established, a low molecular weight molecule that specifically promotes or suppresses the in vivo production ability of the receptor protein of the present invention It can be used as a compound search system.

In this specification and the drawings, when an abbreviation is used for a base or amino acid, IUPAC-IUB is used.
It is based on the abbreviations by the Commission on Biochemical Nomenclature or the abbreviations commonly used in this field, and examples are given below. When amino acids may have optical isomers, the L form is shown unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine tri Phosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate

[0099]   Gly: Glycine   Ala: Alanine   Val: Valine   Leu: Leucine   Ile: Isoleucine   Ser: Serine   Thr: Threonine   Cys: Cysteine   Met: Methionine   Glu: Glutamic acid   Asp: Aspartic acid   Lys: Lysine   Arg: Arginine   His: Histidine   Phe: Phenylalanine   Tyr: Tyrosine   Trp: Tryptophan   Pro: Proline   Asn: Asparagine   Gln: Glutamine   pGlu: pyroglutamic acid   *: Corresponds to the stop codon   Me: Methyl group   Et: ethyl group   Bu: butyl group   Ph: Phenyl group   TC: thiazolidine-4 (R) -carboxamide group

The substituents, protecting groups and reagents commonly used in the present specification are represented by the following symbols. Tos: p-toluenesulfonyl CHO: formyl Bzl: benzyl Cl ₂ Bzl: 2,6-dichlorobenzyl Bom: benzyloxymethyl Z: benzyloxycarbonyl Cl-Z: 2-chlorobenzyloxycarbonyl Br-Z: 2-bromobenzyl Oxycarbonyl Boc: t-butoxycarbonyl DNP: dinitrophenol Trt: trityl Bum: t-butoxymethyl Fmoc: N-9-fluorenylmethoxycarbonyl HOBt: 1-hydroxybenztriazole HOOBt: 3,4-dihydro-3-hydroxy -4-Oxo-1,2,3-benzotriazine HONB: 1-hydroxy-5-norbornene-2,3-dicarboximide DCC: N, N'-dicyclohexylcarbodiimide Fam: 6-carboxy Fluorescein Tamra: 6- carboxy - tetramethyl - rhodamine

The sequence numbers in the sequence listing of the present specification show the following sequences. SEQ ID NO: 1 This shows the amino acid sequence of human-derived novel G protein-coupled receptor protein TGR4 of the present invention. SEQ ID NO: 2 This shows the base sequence of cDNA encoding the human-derived novel G protein-coupled receptor protein TGR4 of the present invention. SEQ ID NO: 3 Shows the base sequence of primer 1 used in the PCR reaction in Reference Example 1 below. SEQ ID NO: 4 Shows the base sequence of primer 2 used in the PCR reaction in Reference Example 1 below. SEQ ID NO: 5 Shows the base sequence of the probe used in the PCR reaction in Reference Example 2 below. SEQ ID NO: 6 Shows the base sequence of the primer used in the PCR reaction in Reference Example 2 below. SEQ ID NO: 7 This shows the base sequence of the primer used in the PCR reaction of Reference Example 2 below. SEQ ID NO: 8 This shows the amino acid sequence of rat novel G protein-coupled receptor protein TGR4 of the present invention. SEQ ID NO: 9 This shows the base sequence of cDNA encoding the rat-derived novel G protein-coupled receptor protein TGR4 of the present invention. SEQ ID NO: 10 This shows the amino acid sequence of mouse-derived novel G protein-coupled receptor protein TGR4 of the present invention. SEQ ID NO: 11 This shows the base sequence of cDNA encoding the novel mouse G-derived protein-coupled receptor protein TGR4 of the present invention. SEQ ID NO: 12 Shows the base sequence of the primer used in the PCR reaction in Example 4 below. SEQ ID NO: 13 This shows the base sequence of the primer used in the PCR reaction of Example 4 below. SEQ ID NO: 14 Shows the base sequence of the primer used in the PCR reaction in Example 5 below. SEQ ID NO: 15 This shows the base sequence of the primer used in the PCR reaction of Example 5 below. SEQ ID NO: 16 This shows the base sequence of the primer used in the PCR reaction of Example 8 below. SEQ ID NO: 17 This shows the base sequence of the primer used in the PCR reaction in Example 8 below. SEQ ID NO: 18 This shows the base sequence of the probe used in the PCR reaction in Example 8 below.

The transformant Escherichia coli DH5α / pCR obtained in Example 1 below was obtained.
-Blunt-TGR4 is 2000 4
From the 3rd of March, 1-1-1, Higashi 1-1-1, Tsukuba, Ibaraki Prefecture, Central Japan 6th National Institute of Advanced Industrial Science and Technology, Patent Biological Depository Center (Former Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (NIBH), Deposit Number FERM) Since March 23, 2000, BP-7115 has been deposited under the deposit number IFO 16409 at the Institute for Ionic Fermentation (IFO), 2-17-85, 13-3 Honcho, Yodogawa-ku, Osaka-shi, Osaka Prefecture. . The transformant Escherichia coli JM109 / p obtained in Example 4 below.
Since August 8, 2002, TArTGR4 has been deposited under the deposit number FERM BP-8147 at the Patent Biological Depositary Center, Central Research Institute, National Institute of Advanced Industrial Science and Technology, 1-1-1 East, Tsukuba City, Ibaraki Prefecture. . The transformant Escherichia coli JM109 / pTAmTGR4 obtained in Example 5 below.
From August 8, 2002, the deposit number FERM B at the Patent Biological Depositary Center, National Institute of Advanced Industrial Science and Technology, Central No. 6 1-1-1 East, Tsukuba City, Ibaraki Prefecture
Deposited as P-8146.

[0103]

The present invention will be described in more detail with reference to Reference Examples and Examples below, but these do not limit the scope of the present invention. In addition, the gene using E. coli is
The method described in Molecular cloning was followed.

Reference Example 1 Cloning of cDNA encoding G protein-coupled receptor protein TGR4 and determination of nucleotide sequence Using human brain cDNA (GIBCO BRL) as a template, two primers, primer 1 (5'-GGG TCG ACA) TGT TAG CC
A ACA GCT CCT CAA CCA AC-3 '; SEQ ID NO: 3) and primer 2 (5'-GGA CTA GTT CAG AGG GCG GAA TCC
PCR reaction was performed using TGG GGA CAC-3 '; SEQ ID NO: 4). The composition of the reaction solution in the reaction is the above cDNA
Using 1/50 of the amount as a template, Pfu Turbo DNA Po
1/50 amount of lymerase (STRATAGENE), primer 1
And 0.8 μM each of primer 2 and 40 dNTPs
0 μM, and the buffer attached to the enzyme was added to give a total of 50
μl. PCR reaction at 94 ° C for 2 minutes, then 94 ° C
The cycle of 15 seconds, 56 ° C., 15 seconds, 72 ° C., 100 seconds was repeated 40 times, and finally the extension reaction was carried out at 72 ° C. for 5 minutes. The reaction product after the PCR reaction is the Zero Blunt TOPO
Subcloning into the plasmid vector pCR-Blunt Vector (Invitrogen) was performed according to the prescription of PCR Cloning Kit (Invitrogen). This was introduced into E. coli DH5α,
The clone containing the cDNA of interest was cloned into L containing kanamycin.
After selection in B agar medium, the sequences of individual clones were analyzed, and as a result, a cDNA sequence of 1116 base pairs encoding a novel G protein-coupled receptor protein (SEQ ID NO:
2) was obtained. A novel G protein-coupled receptor protein having an amino acid sequence (SEQ ID NO: 1) derived from this cDNA was named TGR4, and a transformant containing the DNA represented by SEQ ID NO: 2 was transformed into Escherichia coli.
coli) DH5α / pCR-Blunt-TGR4. A hydrophobicity plot of TGR4 is shown in FIG.

Reference Example 2 Analysis of Tissue Distribution of TGR4 Expression Tissue distribution of G protein-coupled receptor protein TGR4 was analyzed by real-time quantitative P analysis.
The CR method (TaqMan method) was used. TaqMa
The n method uses TaqMan for the specific PCR strand amplified by PCR.
It is based on the principle of detecting and quantifying by SDS7700 in real time by the fluorescence intensity of a fluorescent probe called a probe. Taq that specifically recognizes TGR4
Man probe and primers are Primer E
xpress (PE Applied Biosystem
Ems software) was used for the design and synthesis.
The reaction composition in the TaqMan PCR reaction was Hum.
an MTC Panel I & II (CLONTECH
2x
TaqMan Universal PCR Master
r Mix (PEApplied Biosystems)
12.5 μl, 200 nM TaqMan probe (SEQ ID NO: 5), TaqMan primer (SEQ ID NO: 5)
6 and SEQ ID NO: 7) 100 nM was added to each to give a total volume of 25 μl. PCR reaction at 50 ℃ ・ 2
Minutes, 95 ° C / 10 minutes later, 95 ° C ・ 15 seconds, 62 ° C ・ 1
The cycle of 40 minutes was performed 40 times, and simultaneously with the completion of the reaction, quantitative automatic analysis of PCR was performed. The standardization is TaqMan
GAPDH Control Reagents (PE
The same system was performed using Applied Biosystems. The results are shown in Fig. 3.

Example 1 Measurement of reporter gene expression level by phospholipid compounds in CHO cells transiently expressing TGR4 Expression plasmid vector pAKKO-111 for animal cells
H (Biochem. Biophys. Acta, H
inuma, S .; et al. Volume 1219, 251-
259, the same plasmid vector as pAKKO-1.11H described in 1994) by known methods.
Expression vector plasmids pAK-TGR4, pAK-Gi prepared by inserting R4 cDNA or inhibitory G protein α subunit Gi cDNA, and original pAKKO-111H into which no specific gene was inserted were prepared by the following method. CHO cells were transfected. First, using these vectors, E. coli JM10
After transforming 9 and isolating and culturing the resulting colonies, Q
The plasmid was prepared using the IAGEN Plasmid Maxi Kit (Qiagen). Also, c
PCRE in which the luciferase gene is linked as a reporter downstream of the AMP response element (CRE)
-Luc (Clontech) plasmid was similarly prepared. CHO (dhfr ⁻ ) cells were treated with pAKKO.
96 CHO-mock cells transformed with -111H.
-Well black plate (Costar) 40,000
Cells / well and 100 μl of culture solution were seeded and cultured overnight. The medium for culturing on a plate is DMEM (D
ulbecco's modified Eagle's
Medium, GibcoBRL) 10% fetal bovine serum (FBS, GibcoBRL) was added alone. In order to detect the increase in the expression level of the reporter gene more clearly, pAK-Gi was co-transfected with pAK-TGR4. Each plasmid is pAK
-TGR4, pAK-Gi and reporter plasmid pCRE-Luc at a ratio of 7: 3: 1 with 240 μl of O.
It was added to pti-MEM-I (GibcoBRL). This is also 240 μl of Opti-MEM-I.
10 μl of Lipofectamine 2000 (Gibco
BRL) was added in an equal amount, and a liposome-plasmid complex was formed according to the method described in the manual attached to Lipofectamine 2000. 25 μl / well of each of these was added to the culture solution of CHO-mock cells and cultured at 37 ° C. for 4 hours, and then the culture solution was exchanged for an assay buffer (DMEM supplemented with 0.1% bovine serum albumin) to introduce the plasmid. I went. These cells were further cultured overnight at 37 ° C. under 5% CO ₂ . CHO-mock transfected above
Geranylgeranyl diphosphate (GGPP, Sigma) diluted in assay medium, farnesyl 2 was added to the cells.
Phosphoric acid (FPP, Sigma), lysophosphatidic acid (LPA, Sigma) 1, 0.1, 0.01 μ
M was added thereto, and the mixture was cultured at 37 ° C. under 5% CO ₂ for 4 hours. The culture supernatant is discarded, 50 μl of Picagene LT2.0 (Toyo Ink Mfg. Co., Ltd.), which is a substrate for luciferase activity measurement, is added, and a plate reader (ARVO sx multi-label counter, Walla) is added.
(Company c) was used to measure the luminescence amount of luciferase. As a result, in cells into which the TGR4 gene was introduced, 1,
An increase in luciferase activity was observed with 0.1 μM GGPP and FPP, 1 μM LPA (FIG. 4), T
It was shown that CHO cells expressing GR4 were activated by the above phospholipid compounds.

Example 2 TGR4 with phospholipid compounds
Measurement of intracellular calcium ion mobilization activity of expressing CHO cells Stable expression of TGR4 CHO by a method known per se using the plasmid for TGR4 expression prepared in Example 1
Cells, CHO-TGR4, were created. CHO-TGR4
Alternatively, CHO-moc into which no receptor gene is introduced
BlaK cells at a concentration of 30,000 cells / well
Seeding in a 96-well plate (Costar) of ck plate, Clear bottom, 37 ° C, 5% C
An overnight culture of O ₂ was used for the assay. Hank's balanced sa as a washing buffer
lt solution (HBSS, GibcoBRL
20 mM HEPES pH 7.4, 2.5 mM
The one to which probenecid (Sigma) was added was prepared. 0.1% for wash buffer for sample buffer
% Bovine serum albumin (BSA, Sigma) was used. 1% FBS in wash buffer, 4μ
M Fluo 3-AM (Wako Pure Chemical Industries, Ltd.), 0.04% Pl
uronic acid (Molecular Pro
bes) was used as a dye loading solution.
Discard the cell culture medium and add the dye loading solution to 37 ° C.
After culturing for 1 hour, the cells were washed with a washing buffer using a plate washer. Fluorometr
ic Imaging Plate Reader (F
The cells were set in LIPR (Molecular Devices) and the change in intracellular calcium ion concentration was measured for about 3 minutes after the addition of the sample. The sample is FPP,
LPA was added to 2.5 μM. As a result, as shown in FIGS. 5 and 6, it was found that when FPP and LPA were added, the intracellular calcium ion mobilization activity was specifically increased in CHO-TGR4.

Example 3 TGR4 with phospholipid compounds
Measurement of cAMP production amount in expressing CHO cells 2500 CHO-TGR4 or CHO-mock cells into which no receptor gene was introduced prepared in Example 2.
A 96-well plate (Becton Dickinson) was seeded at a concentration of 0 cells / well, and 37 ° C., 5% CO ₂
The one that was cultured overnight in the above was used for the assay. Sample buffer is 0.1% BSA and 0.2 mM in HBSS
The one to which IBMX (Sigma) was added was used.
Wash cells twice with sample buffer and incubate at 37 ° C for 30
After preincubation for min, the cells were washed twice more, sample was added and incubated for 30 min at 37 ° C. Farnesyl diphosphate and lysophosphatidic acid were added to the sample at 2 μM. Intracellular cAMP after incubation with sample
The production amount is based on the cAMP Screen System (AB
It was measured using I). As a result, as shown in FIGS. 7 and 8, when FPP and LPA were added, CHO-TGR
It was found that the intracellular cAMP production increased in a 4-specific manner.

Example 4 PCR from rat brain cDNA
Acquisition of rat type TGR4 receptor gene by the method Using the rat brain cDNA as a template, the following two synthetic D
Amplification by PCR was performed using NA. rF: 5'-GTCGACATGCCTCAGACTAGTTTCTCTCCCCAC-3 '(SEQ ID NO: 12) rR: 5'-GCTAGCCTTTCAGAGGGCTGAATCTTGGGGGCC-3' (SEQ ID NO: 13) PCR reaction solution was cDNA solution 1 microl, 0.5
micro rF (10 microM), 0.5 mi
crol rR (10 microM), 2.5 micro
ol attached 10x reaction solution, 2.5 micro dNT
P (10 mM), 0.5 micron KlenTaq
(Clontech) and 17.5 micron Otsuka distilled water were added to make a total of 25 micron. Thaw the reaction solution
PCR reaction was performed using the armalCycler9600. The condition of PCR is 98 after denaturation at 95 ° C for 2 minutes.
The cycle of ℃ · 10 seconds, 60 ℃ · 20 seconds, 72 ℃ · 60 seconds was repeated 40 times. After confirming the amplification of the PCR product of about 1000 bp by electrophoresis using a part of the PCR product, the PCR product was subjected to Quiagen PCR purif.
The product was purified using the ication Kit and was directly sequenced to obtain the sequence shown in FIG. DN in Figure 9
The amino acid sequence predicted from the A sequence is shown in FIG. Next, the PCR product recovered from the gel was subcloned into Escherichia coli JM109 using the TA cloning kit (Invitrogen) to obtain E. coli JM1.
09 / pTArTGR4 was obtained. Plasmid pTArTGR4 was extracted from Escherichia coli obtained by subcloning using a plasmid extractor (Kurabo), the nucleotide sequence of the inserted fragment was determined, and it was confirmed that the sequence was rat TGR4 receptor cDNA. The rat type and human type showed 79% homology.

Example 5 PCR from mouse brain cDNA
Acquisition of Mouse TGR4 Receptor Gene by Method Using the mouse brain cDNA as a template, the following two synthetic D
Amplification by PCR was performed using NA. mF: 5'-GTCGACATGCCTCAGACTAATTTCTCTTCCCAC-3 '(SEQ ID NO: 14) mR: 5'-GCTAGCTCAGAGAGCAGAATCCTGGGAGCAGTT-3' (SEQ ID NO: 15) PCR reaction solution was cDNA solution 1 microl, 0.5
microl mF (10 microM), 0.5 mi
crol mR (10 microM), 2.5 micro
ol attached 10x reaction solution, 2.5 micro dNT
P (10 mM), 0.5 micron KlenTaq
(Clontech) and 17.5 micron Otsuka distilled water were added to make a total of 25 micron. Thaw the reaction solution
PCR reaction was performed using the armalCycler9600. The condition of PCR is 98 after denaturation at 95 ° C for 2 minutes.
The cycle of ℃ · 10 seconds, 60 ℃ · 20 seconds, 72 ℃ · 60 seconds was repeated 40 times. After confirming the amplification of the PCR product of about 1000 bp by electrophoresis using a part of the PCR product, the PCR product was subjected to Quiagen PCR purif.
The product was purified using the Icationation Kit and subjected to direct sequencing to obtain the sequence shown in FIG. The amino acid sequence predicted from the DNA sequence in FIG. 11 was as shown in FIG. Next, the PCR product recovered from the gel is treated with T
Subcloning into E. coli JM109 using A cloning kit (Invitrogen)
M109 / pTAmTGR4 was obtained. Plasmid pTAmTGR4 from E. coli obtained by subcloning
Was extracted using a plasmid extractor (Kurabo) to determine the nucleotide sequence of the inserted fragment, and the sequence was determined to be mouse TGR.
It was confirmed to be 4 receptor cDNA. Further, the mouse type and the human type showed 81% homology.

Example 6 TGR expressed in CHO cells
Intracellular transfer of 4-GFP fusion protein by addition of farnesyl diphosphate Green Fluorescent Protein (GF isolated from Owan jellyfish with translational frame at C-terminal of TGR4
P) An expression plasmid was constructed to express the fusion protein ligated with the cDNA. At that time, GFP cDN
For A, a fragment cut out from the GFP expression vector pQBI25 (Takara Shuzo) was used. TGR4 has its stop codon modified by a PCR method into a recognition sequence of a restriction enzyme NheI, and a GFP fragment is ligated to the expression vector pAK.
It was inserted into KO-111H. TGR thus obtained
The CHO-mock cells were transfected with the plasmid of the fusion protein of 4 and GFP (hereinafter, TGR4-GFP) expression vector by the following method. CHO-mock cells are grown in growth medium [DMEM (Dulbecco's Mod
if the Eagle Eagle Medium) (GIBCO
10% fetal bovine serum (GIBCO BRL)
Added), 0.6 × 10 ⁵ cells
Lab- with 4 chambers at a concentration of s / chamber
TekII Cover Glass Chamber (Nalgen Nu
nc) and cultured overnight at 37 ° C. under 5% CO ₂ condition and then transfected. Lipofectamine ^™ 2000 Re for transfection
The agent (GIBCO BRL) was used. First, Lipof
ectamine ^™ 2000 Reagent 2 μl
And OPTI-MEM-I (GIBCO BRL) (50 μl) were mixed and allowed to stand at room temperature for 20 minutes to form a complex of DNA and lipofectamine, and then 100 μl was added to the chamber in which the CHO cells were cultured. And then 3
It was cultured overnight at 7 ° C. under 5% CO ₂ . The medium is a medium for confocal microscope observation [Hanks' Balanced Sa]
lt Solution (GIBCO BRL) to 0.
1% bovine albumin (Essentily Fat)
ty Acid Free, GIBCO BRL) was replaced with a suspension] and a fluorescent image of GFP was observed with a confocal microscope (Leica). At that time, the excitation of GFP is 4
It was performed at 88 nm. As a result, the TGR4-GFP fusion protein was observed on the cell membrane. It was found that 30 minutes after the farnesyl diphosphate was added to the medium at a ^{concentration} of 10 ⁻⁶ M, the fluorescence of GFP migrated to the cytoplasm rather than to the cell membrane. This means that TGR4 is a G protein-coupled receptor expressed on the cell membrane, and
It was shown that TGR4 translocated to the cytoplasm, that is, internalized in response to farnesyl diphosphate.

Example 7 Analysis of Human TGR4 mRNA Expression Distribution ABI PRISM 770 was used to quantify the expression level of mRNA.
0 Sequence Detector (Applied Biosystems) was used. The primers and probes used for quantifying the expression level were ABI PRISM 7700 Se based on the nucleotide sequence of human TGR4 (SEQ ID NO :).
Software Pr dedicated to the sequence detector
It was designed using imager Express (Applied Biosystems). The template cDNA is
1 μg of polyA + RNA (Clontech) derived from various human tissues was synthesized at 42 ° C. using random primers. SuperScriptII reverse transcriptase (GIBCO BRL) was used for the reverse transcription reaction, and the reaction was performed according to the attached manual. After the reaction was completed, ethanol precipitation was performed and dissolved in 100 μl. The fractionated human blood cell-derived cDNA is a Multiple Tissue c
DNA (MYTC ^™ ) panels Human Blo
od Fractions (Clontech) was used. ABI PRISM 7700 SequenceD
The reaction liquid of the detector is TaqManUnivers
According to the manual of al PCR Master Mix (Applied Biosystems), 12.5 μl of master mix, 0.9 μM of primer, 0.25 μM of probe, and 1 μl of cDNA solution of each sample
And mixed with distilled water to prepare 25 μl of distilled water. AB
I PRISM 7700 SequenceDetec
The reaction at tor was carried out by repeating a cycle of 50 ° C. for 2 minutes, 95 ° C. for 10 minutes, 95 ° C. for 15 seconds and 60 ° C. for 1 minute 40 times. TGR4 mRNA in various human tissues
The expression distribution of is shown in FIG. High expression was found in tissues involved in immunity such as spleen and lymph nodes. The expression level of TGR4 mRNA in human blood cells is shown in FIG.

Example 8 Analysis of Rat TGR4 mRNA Expression Distribution Various organs were isolated from Wistar rat and total.
RNA was prepared using Isogen (Nippon Gene Co., Ltd.), and mRNA p was prepared from the obtained total RNA.
Poly (A) + RNA was prepared by the purification kit (Pharmacia). All were prepared according to their respective manuals. 1 μg of the obtained poly (A) + RNA was added to DnaseI.
(Amplification Grade, GIB
CO BRL), 160 ng of RNA PC
CDNA was synthesized at 42 ° C. according to the manual using R Kit (Takara). Synthesized cDNA
Was a solution of 4 ng / μl in terms of poly (A) + RNA and used as a template for the subsequent RT-PCR. RT-
PCR is Sequence Detection Sy
system Prism 7700 (PE Biosystem
5'-CACCTGCAAGTACGAGAACGT-3 '(SEQ ID NO: 1) as a primer for amplification and detection using
6), 5'-TGCCCTTCCACAGTTCATC-3 '(SEQ ID NO: 17) and 5'as TaqMan probe.
-(Fam) -TGAGCCTGTGCTTCGAGAGCTTCA- (Tamr
a) -3 '(SEQ ID NO: 18) was used. RT-PC
R reaction solution is TaqMan Universal PCR
Master Mix (PE Biosystems
12.5 μl each with 100 μM primer solution, 0.05 μl, 5 μM TaqMan probe
0.5 μl of e and 0.5 μl of the cDNA solution prepared above were added to make the total reaction solution volume 25 μl with distilled water. PCR reaction at 50 ° C for 2 minutes, 95 ° C for 10 minutes,
A cycle of 95 ° C. for 15 seconds and 60 ° C. for 1 minute was repeated 40 times. The mRNA expression level of GPR7 ligand in various rat tissues obtained was poly (A) + RNA 1n.
It was calculated as the number of copies per gram. Rat TGR4
As shown in FIG. 15, mRNA expression was highly expressed in the spleen, lymph nodes, and intestinal tissues involved in immunity.

Example 9 Activation of MAP Kinase Specific to TGR4-expressing CHO Cells Human TGR4-expressing CHO cells and mock cells were mixed in 3 cells.
6-well pla at 10 ⁵ cells / well
The seeds were inoculated into te and cultured overnight. 0.1 for serum-free
% -BSA-added nucleic acid-free α-MEM medium was exchanged, the cells were further cultured overnight, and pre-incubation was further performed for 2 hours in the same medium immediately before the assay. 0.
After 1 μM farnesyl diphosphate was added to the medium to start the treatment, the medium was removed by suction at regular intervals, and SDS-
The reaction was stopped by adding a sample buffer for PAGE. SDS-PAGE was performed according to a standard method to transfer the protein to a nitrocellulose filter, and then Pho
spoPlus p44 / 42 MAPK (Thr
202 / Tyr204) Antibody Kit
Phosphorylated p44 / 42 MAP ki according to the method described in the manual (Cell Signaling).
nase was detected. As a result, farnesyl 2
Phosphorylation of MAP kinase was specifically detected in human TGR-expressing CHO cells by phosphoric acid treatment, and it was revealed that the enzyme was activated.

Example 10 Concentration Dependence of TGR4-Specific Intracellular Calcium Ion Mobilizing Activity by Various Phospholipid Compound-Related Substances [0115] As in the method described in Example 7, TGR4-expressing CHO cells and mock CHO cells were used to analyze phosphorus. The concentration dependence of intracellular calcium ion mobilization activity by lipid compound-related substances was examined. As phospholipid compound-related substances, geranylgeranyl diphosphate, farnesyl diphosphate, geranyl diphosphate, lysophosphatidic acid, sphingosine 1 phosphate, nordeoxycholic acid, deoxycholic acid, geranylgeraniol, and farnesol were used. As a result, as shown in FIG. 16, by adding phospholipids such as geranylgeranyl diphosphate, farnesyl diphosphate, and lysophosphatidic acid to CHO-TGR4, concentration-dependent intracellular calcium ion mobilization activity was observed. It was On the other hand, as shown in FIG. 17, no activity was observed in mock CHO cells that did not express TGR4. From this, the above phospholipid compound is
It was revealed to act as a 4-specific agonist. Table 1 shows EC ₅₀ values of intracellular calcium ion mobilization activity against TGR4 of related compounds including compounds other than those shown in the figure.

[Table 1] *: Calculated with the reaction when adding farnesyl diphosphate 2 × 10 ⁻⁶ M as 100%. **: Calculated with the reaction when adding 115 × 10 ⁻⁶ M of farnesyl diphosphate as 100%.

Example 11 Concentration Dependence of TGR4-Specific cAMP Production-Increasing Activity by Various Phospholipid Compound-Related Substances Using various phospholipids using TGR4-expressing CHO cells and mock CHO cells in the same manner as in Example 8. The concentration dependence of the cAMP production increasing activity by the compound-related substances was examined. As a result, as shown in FIG. 18, by adding phospholipids such as geranylgeranyl diphosphate, farnesyl diphosphate, and lysophosphatidic acid to CHO-TGR4, concentration-dependent intracellular calcium ion mobilization activity was observed. It was See Example 9 for compound abbreviations. On the other hand, as shown in FIG. 19, no activity was observed in mock CHO cells that did not express TGR4. From this, it became clear that the above phospholipid compound acts as an agonist specific to TGR4. In addition, ECs of related compounds including those other than the compounds shown in the figure, showing the activity of increasing cAMP production for TGR4.
_{The 50} value was as shown in Table 1 of Example 10.

[0117]

By using the G protein-coupled receptor protein of the present invention, a partial peptide thereof or a salt thereof and a phospholipid compound as a ligand, the phospholipid compound and the G protein-coupled receptor protein of the present invention, a portion thereof It is possible to efficiently screen for a compound that changes the binding property with a peptide or a salt thereof.

[0118]

[Sequence list] [SEQUENCE LISTING] <110> Takeda Chemical Industries, Ltd. <120> Novel Screening Method <130> B02272 <150> JP 2001-258479 <151> 2001-08-28 <160> 18 <210> 1 <211> 372 <212> PRT <213> Human <400> 1 Met Leu Ala Asn Ser Ser Ser Thr Asn Ser Ser Var Leu Pro Cys Pro                   5 10 15 Asp Tyr Arg Pro Thr His Arg Leu His Leu Val Val Tyr Ser Leu Val              20 25 30 Leu Ala Ala Gly Leu Pro Leu Asn Ala Leu Ala Leu Trp Val Phe Leu          35 40 45 Arg Ala Leu Arg Val His Ser Val Val Ser Val Tyr Met Cys Asn Leu      50 55 60 Ala Ala Ser Asp Leu Leu Phe Thr Leu Ser Leu Pro Val Arg Leu Ser  65 70 75 80 Tyr Tyr Ala Leu His His Trp Pro Phe Pro Asp Leu Leu Cys Gln Thr                  85 90 95 Thr Gly Ala Ile Phe Gln Met Asn Met Tyr Gly Ser Cys Ile Phe Leu             100 105 110 Met Leu Ile Asn Val Asp Arg Tyr Ala Ala Ile Val His Pro Leu Arg         115 120 125 Leu Arg His Leu Arg Arg Pro Arg Val Ala Arg Leu Leu Cys Leu Gly     130 135 140 Val Trp Ala Leu Ile Leu Val Phe Ala Val Pro Ala Ala Arg Val His 145 150 155 160 Arg Pro Ser Arg Cys Arg Tyr Arg Asp Leu Glu Val Arg Leu Cys Phe                 165 170 175 Glu Ser Phe Ser Asp Glu Leu Trp Lys Gly Arg Leu Leu Pro Leu Val             180 185 190 Leu Leu Ala Glu Ala Leu Gly Phe Leu Leu Pro Leu Ala Ala Val Val         195 200 205 Tyr Ser Ser Gly Arg Val Phe Trp Thr Leu Ala Arg Pro Asp Ala Thr     210 215 220 Gln Ser Gln Arg Arg Arg Lys Thr Val Arg Leu Leu Leu Ala Asn Leu 225 230 235 240 Val Ile Phe Leu Leu Cys Phe Val Pro Tyr Asn Ser Thr Leu Ala Val                 245 250 255 Tyr Gly Leu Leu Arg Ser Lys Leu Val Ala Ala Ser Val Pro Ala Arg             260 265 270 Asp Arg Val Arg Gly Val Leu Met Val Met Val Leu Leu Ala Gly Ala         275 280 285 Asn Cys Val Leu Asp Pro Leu Val Tyr Tyr Phe Ser Ala Glu Gly Phe     290 295 300 Arg Asn Thr Leu Arg Gly Leu Gly Thr Pro His Arg Ala Arg Thr Ser 305 310 315 320 Ala Thr Asn Gly Thr Arg Ala Ala Leu Ala Gln Ser Glu Arg Ser Ala                 325 330 335 Val Thr Thr Asp Ala Thr Arg Pro Asp Ala Ala Ser Gln Gly Leu Leu             340 345 350 Arg Pro Ser Asp Ser His Ser Leu Ser Ser Phe Thr Gln Cys Pro Gln         355 360 365 Asp Ser Ala Leu     370 <210> 2 <211> 1116 <212> DNA <213> Human <400> 2 atgttagcca acagctcctc aaccaacagt tctgttctcc cgtgtcctga ctaccgacct 60 acccaccgcc tgcacttggt ggtctacagc ttggtgctgg ctgccgggct ccccctcaac 120 gcgctagccc tctgggtctt cctgcgcgcg ctgcgcgtgc actcggtggt gagcgtgtac 180 atgtgtaacc tggcggccag cgacctgctc ttcaccctct cgctgcccgt tcgtctctcc 240 tactacgcac tgcaccactg gcccttcccc gacctcctgt gccagacgac gggcgccatc 300 ttccagatga acatgtacgg cagctgcatc ttcctgatgc tcatcaacgt ggaccgctac 360 gccgccatcg tgcacccgct gcgactgcgc cacctgcggc ggccccgcgt ggcgcggctg 420 ctctgcctgg gcgtgtgggc gctcatcctg gtgtttgccg tgcccgccgc ccgcgtgcac 480 aggccctcgc gttgccgcta ccgggacctc gaggtgcgcc tatgcttcga gagcttcagc 540 gacgagctgt ggaaaggcag gctgctgccc ctcgtgctgc tggccgaggc gctgggcttc 600 ctgctgcccc tggcggcggt ggtctactcg tcgggccgag tcttctggac gctggcgcgc 660 cccgacgcca cgcagagcca gcggcggcgg aagaccgtgc gcctcctgct ggctaacctc 720 gtcatcttcc tgctgtgctt cgtgccctac aacagcacgc tggcggtcta cgggctgctg 780 cggagcaagc tggtggcggc cagcgtgcct gcccgcgatc gcgtgcgcgg ggtgctgatg 840 gtgatggtgc tgctggccgg cgccaactgc gtgctggacc cgctggtgta ctactttagc 900 gccgagggct tccgcaacac cctgcgcggc ctgggcactc cgcaccgggc caggacctcg 960 gccaccaacg ggacgcgggc ggcgctcgcg caatccgaaa ggtccgccgt caccaccgac 1020 gccaccaggc cggatgccgc cagtcagggg ctgctccgac cctccgactc ccactctctg 1080 tcttccttca cacagtgtcc ccaggattcc gccctc 1116 <210> 3 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 gggtcgacat gttagccaac agctcctcaa ccaac 35 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 ggactagttc agagggcgga atcctgggga cac 33 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 5 ttcgtctctc ctactacgca ctgcacca 28 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 ggtggtgagc gtgtacatgt gt 22 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 catgttcatc tggaagatgg cg 22 <210> 8 <211> 384 <212> PRT <213> Rat <400> 8 Met Pro Gln Thr Ser Phe Ser Pro His Leu Asp Ala Met Phe Ala Asn                  5 10 15 Ser Ser Ala Asn Thr Ser Ser Thr Asp Ser Ser Val Pro Gln Cys Arg              20 25 30 Asp Tyr Arg Ser Thr His Arg Leu His Met Val Val Tyr Ser Leu Val          35 40 45 Leu Ala Thr Gly Leu Pro Leu Asn Ala Leu Ala Leu Trp Val Phe Leu      50 55 60 Arg Val Leu Arg Val His Ser Val Val Ser Val Tyr Met Cys Asn Leu  65 70 75 80 Ala Ala Ser Asp Leu Leu Phe Thr Leu Ser Leu Pro Leu Arg Leu Ser                  85 90 95 Tyr Tyr Ala Arg His His Trp Pro Phe Pro Asp Phe Leu Cys Gln Thr             100 105 110 Ser Gly Ala Ile Phe Gln Met Asn Met Tyr Gly Ser Cys Ile Phe Leu         115 120 125 Met Leu Ile Asn Val Asp Arg Tyr Ala Ala Ile Val His Pro Leu Arg     130 135 140 Leu Arg His Leu Arg Arg Pro Leu Val Ala Arg Arg Leu Cys Leu Gly 145 150 155 160 Val Trp Ala Leu Ile Leu Leu Phe Ala Val Pro Ala Ala Arg Val His                 165 170 175 Ser Pro Thr Thr Cys Lys Tyr Glu Asn Val Thr Leu Ser Leu Cys Phe             180 185 190 Glu Ser Phe Ser Asp Glu Leu Trp Lys Gly Arg Leu Leu Pro Leu Leu         195 200 205 Leu Leu Ala Glu Thr Leu Gly Phe Leu Leu Pro Leu Ala Ala Val Val     210 215 220 Tyr Ser Ser Gly Arg Val Phe Trp Thr Leu Ala Arg Pro Asp Ala Thr 225 230 235 240 Gln Ser His Arg Arg Gln Lys Thr Val Arg Leu Leu Leu Ala Asn Leu                 245 250 255 Ile Ile Phe Leu Leu Cys Phe Val Pro Tyr Asn Ser Thr Leu Ala Val             260 265 270 Tyr Gly Leu Leu Arg Ala Asn Leu Val Lys Asn Ser Leu Gln Ala Arg         275 280 285 Asp Gln Val Arg Gly Val Leu Met Ile Met Val Leu Leu Ala Gly Ala     290 295 300 Asn Cys Val Leu Asp Pro Leu Val Tyr Tyr Phe Ser Ala Glu Gly Phe 305 310 315 320 Arg Asn Thr Leu Arg Asn Leu Gly Ala Pro Phe His Thr Arg Pro Leu                 325 330 335 Pro Thr Asn Gly Thr Arg Gly Glu Leu Thr Glu Pro Pro Ser Glu Ser             340 345 350 Thr Gln Asn Thr Gly Gln Asp Ala Thr Ser Gln Arg Leu Leu Gln Pro         355 360 365 Ala Asn Leu Gly Thr Arg Thr Asp Asn Gly Pro Gln Asp Ser Ala Leu     370 375 380 <210> 9 <211> 1152 <212> DNA <213> Rat <400> 9 atgcctcaga ctagtttctc tccccacctg gacgccatgt ttgccaattc ttcagccaac 60 acctcttcta ccgacagctc tgtgccccag tgccgtgact atcggagtac acatcgtttg 120 catatggtgg tctacagctt ggtgttggca accggtcttc ctctcaacgc tctggctctc 180 tgggtcttcc tgcgtgtact gcgcgtacac tcggtggtga gtgtgtacat gtgcaacctg 240 gcagccagcg acttgctctt caccctgtca cttcccctgc gcctctccta ctatgcacgg 300 caccactggc ccttcccaga cttcctgtgc cagacgtcgg gcgccatctt ccagatgaac 360 atgtatggca gctgtatctt tctgatgctc atcaacgtgg accgctatgc ggccatcgtg 420 catccgctga gactgcgcca tctgcggcgg cccctcgtgg cgcggcggct ctgcctgggc 480 gtgtgggctc tcatcctgct gttcgccgtg cccgccgccc gcgtgcacag cccgaccacc 540 tgcaagtacg agaacgtcac cctgagcctg tgcttcgaga gcttcagcga tgaactgtgg 600 aagggcaggc tgctgccact cctgctgctg gccgagaccc tgggctttct gctgcccctg 660 gcggctgtcg tctattcgtc cggcagggtc ttctggacac tggcgaggcc cgacgccact 720 cagagccacc ggcgacagaa gaccgtgcgc ctcctactgg ccaacctcat catctttctg 780 ctgtgcttcg tgccctacaa ctccacgctg gcagtttatg ggttgctgcg ggccaacctg 840 gtaaagaaca gccttcaggc ccgcgatcag gtgcgcggag tgctgatgat aatggtgctg 900 ctggccggtg ccaactgcgt gctggatccg ctggtttact acttcagcgc cgagggtttt 960 cgtaacactc ttcgaaacct gggcgctccg tttcatacca ggcctttgcc taccaatggg 1020 actagagggg agctcaccga accaccctca gaaagcaccc aaaacactgg gcaggatgcc 1080 accagccaga ggctacttca acctgccaat ctgggtacac gcacggacaa cggcccccaa 1140 gattcagccc tc 1152 <210> 10 <211> 384 <212> PRT <213> Mouse <400> 10 Met Pro Gln Thr Asn Phe Ser Ser His Leu Asp Met Met Phe Ala Asn                  5 10 15 Ser Ser Ala Asn Thr Thr Ser Thr Asn Ser Ser Val Leu Gln Cys Pro              20 25 30 Asp Tyr Arg Asp Thr His Arg Leu His Met Val Val Tyr Ser Leu Val          35 40 45 Leu Ala Thr Gly Leu Pro Leu Asn Ala Leu Ala Leu Trp Val Phe Leu      50 55 60 Arg Val Leu Arg Val His Ser Val Val Ser Val Tyr Met Cys Asn Leu  65 70 75 80 Ala Ala Ser Asp Leu Leu Phe Thr Leu Ser Leu Pro Leu Arg Leu Ser                  85 90 95 Tyr Tyr Ala Gln His His Trp Pro Phe Pro Gly Phe Leu Cys Gln Thr             100 105 110 Ser Gly Ala Ile Phe Gln Met Asn Met Tyr Gly Ser Cys Leu Phe Leu         115 120 125 Met Leu Ile Asn Val Asp Arg Tyr Ala Ala Ile Val His Pro Leu Arg     130 135 140 Leu Arg His Leu Arg Arg Pro Arg Val Ala Arg Arg Leu Cys Leu Gly 145 150 155 160 Val Trp Ala Leu Ile Leu Leu Phe Ala Val Pro Ala Ala Arg Val His                 165 170 175 Ser Pro Ser His Cys Thr Tyr Lys Asn Ile Thr Val Arg Leu Cys Phe             180 185 190 Glu Ser Phe Ser Asp Glu Leu Trp Lys Gly Arg Leu Leu Pro Leu Leu         195 200 205 Leu Leu Ala Glu Ile Leu Gly Phe Leu Leu Pro Leu Ala Ala Val Val     210 215 220 Tyr Ser Ser Gly Arg Val Phe Trp Thr Leu Ala Arg Pro Asp Ala Thr 225 230 235 240 Gln Ser Gln Arg Arg Arg Lys Thr Val Arg Leu Leu Leu Ala Asn Leu                 245 250 255 Ile Ile Phe Leu Leu Cys Phe Val Pro Tyr Asn Ser Thr Leu Ala Val             260 265 270 Tyr Gly Leu Leu Arg Ala Asn Leu Val Lys Asn Ser Ile Gln Asp Arg         275 280 285 Asp Gln Val Arg Gly Val Leu Met Ile Met Val Leu Leu Ala Gly Ala     290 295 300 Asn Cys Val Leu Asp Pro Leu Val Tyr Tyr Phe Ser Ala Glu Gly Phe 305 310 315 320 Arg Asn Thr Leu Arg Asn Leu Gly Ala Pro Leu Asn Thr Arg Pro Leu                 325 330 335 Ala Thr Asn Gly Ala Ala Gly Val Leu Thr Glu Leu Pro Ser Glu Ser             340 345 350 Thr Gln Asn Thr Gly Gln Asp Ala Thr Ser Gln Val Leu Leu Gln Pro         355 360 365 Ala Thr Leu Gly Thr Pro Pro Asp Asn Cys Ser Gln Asp Ser Ala Leu     370 375 380 <210> 11 <211> 1152 <212> DNA <213> Mouse <400> 11 atgcctcaga ctaatttctc ttcccacctg gacatgatgt ttgccaattc ttcagccaac 60 acgacttcta ccaacagctc tgtgctccag tgccctgact atcgagatac acatcgtttg 120 catatggtgg tctacagcct ggtattggcg actggtctcc ctctcaacgc tctggctctc 180 tgggtcttcc tgcgtgtact gcgcgtacac tcagtggtga gcgtgtacat gtgcaacctg 240 gcagccagcg acttgctctt caccctgtca cttcccctgc gcctctccta ctatgcacag 300 caccactggc cttttccagg cttcctgtgc cagacgtcgg gcgccatctt ccagatgaac 360 atgtacggca gctgtctctt tctgatgctc atcaacgtgg accgctatgc ggccatcgtg 420 cacccgctga gactgcgcca cctacggcgg ccccgtgtgg cacggcggct ctgcctgggc 480 gtgtgggctc tcatcctgct gttcgctgtg cccgccgccc gcgtgcacag cccgtcccac 540 tgcacgtaca agaacatcac tgtgcgcctg tgcttcgaga gcttcagcga tgaactgtgg 600 aagggcaggc tgctgccgct cctgctgctg gccgagatac taggctttct gctgcccctg 660 gcggctgtcg tctattcgtc tggcagagtc ttctggacac tggcgaggcc cgacgccact 720 cagagccaac ggcgacggaa gaccgtgcgc ctcctgctgg ccaatctcat catcttcctg 780 ctgtgcttcg tgccctataa ctccacgctg gctgtatatg ggttgctacg ggccaacttg 840 gtgaagaaca gcattcagga ccgcgatcag gtgcgcgggg tgctgatgat aatggtgctg 900 ctggccggcg ccaactgcgt gctggatcca ctggtttact acttcagtgc cgagggtttc 960 cgtaacaccc ttcgcaacct gggcgccccg ctgaatacca ggcctttggc taccaatggg 1020 gctgcaggcg tgctcaccga actaccctca gaaagcaccc aaaacactgg gcaggatgcc 1080 acaagtcagg ttctactcca gcctgccact ctgggtacac ccccggacaa ctgctcccag 1140 gattcggctc tc 1152 <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 12 gtcgacatgc ctcagactag tttctctccc cac 33 <210> 13 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 13 gctagccttt cagagggctg aatcttgggg gcc 33 <210> 14 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 14 gtcgacatgc ctcagactaa tttctcttcc cac 33 <210> 15 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 15 gctagctcag agagcagaat cctgggagca gtt 33 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 16 cacctgcaag tacgagaacg t 21 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 17 tgcccttcca cagttcatc 19 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 18 tgagcctgtg cttcgagagc ttca 24

[Brief description of drawings]

FIG. 1 is a hydrophobicity plot of TGR4.

FIG. 2 shows the amino acid sequence of SEQ ID NO: 1 in one-letter code.

FIG. 3 shows the expression distribution of TGR4 in each tissue.

FIG. 4 CHO-mo introduced with TGR4 expression vector
8 shows an increase in luciferase activity by phospholipid compounds in ck cells. The horizontal axis represents the concentration of phospholipid compound. GGPP is geranylgeranyl diphosphate, FPP is farnesyl diphosphate, and LPA is lysophosphatidic acid. Base indicates the case where no phospholipid compound is added. The vertical axis represents luciferase activity.

FIG. 5: FPP (farnesyl diphosphate) or LPA
By adding (lysophosphatidic acid), CHO-mo
It shows an increase in intracellular calcium ion mobilization activity in ck cells. The horizontal axis represents time (seconds). The vertical axis represents changes in calcium concentration. ◇ indicates no addition, □ indicates FPP addition, and △ indicates LPA addition.

FIG. 6 FPP (farnesyl diphosphate) or LPA
Fig. 7 shows an increase in intracellular calcium ion mobilization activity in TGR4-expressing CHO cells by the addition of (lysophosphatidic acid). The horizontal axis represents time (seconds). The vertical axis represents changes in calcium concentration. ◇ indicates no addition, □ indicates FPP addition, and △ indicates LPA addition.

FIG. 7: FPP (farnesyl diphosphate) or LPA
By adding (lysophosphatidic acid), CHO-mo
It shows an increase in intracellular cAMP production in ck cells.
n = 3. Base shows the case without addition, FPP shows the case with FPP addition, and LPA shows the case with LPA addition. The vertical axis represents the amount of cAMP produced.

FIG. 8: FPP (farnesyl diphosphate) or LPA
Fig. 8 shows an increase in intracellular cAMP production in TGR4-expressing CHO cells due to the addition of (lysophosphatidic acid). n = 3. Base is the case without addition, FPP is FP
The case of P addition shows the case of LPA addition. The vertical axis represents the amount of cAMP produced.

FIG. 9 shows the nucleotide sequence of cDNA encoding rat TGR4.

FIG. 10 shows the amino acid sequence of rat TGR4.

FIG. 11 shows the nucleotide sequence of cDNA encoding mouse TGR4.

FIG. 12 shows the amino acid sequence of mouse TGR4.

FIG. 13 shows the expression distribution of human TGR4 mRNA. The vertical axis represents human TG per 1 ng poly (A) + RNA.
The copy number of R4 mRNA is shown, and the horizontal axis shows the analyzed tissue.

FIG. 14 shows the expression distribution of human TGR4 mRNA. The vertical axis represents the copy number of human TGR4 mRNA per 1 μl, and the horizontal axis represents the analyzed cells and tissues.

FIG. 15 shows expression distribution of rat TGR4 mRNA.
The vertical axis represents the copy number of rat TGR4 mRNA per 1 ng poly (A) + RNA, and the horizontal axis represents the analyzed tissue.

FIG. 16 shows the results of examining the effect of various phospholipid compounds on intracellular calcium ion mobilization activity in TGR4-expressing CHO cells. Conc. (NM) on the horizontal axis indicates the concentration of various phospholipid compounds. ▲ is geranylgeranyl diphosphate, ■ is farnesyl diphosphate, ◆ is geranyl diphosphate, ● is lysophosphatidic acid, ○ is sphingosine 1 phosphate, △ is nordeoxycholic acid, □ Indicates deoxycholic acid, ⋄ indicates geranylgeraniol, and × indicates farnesol. Change on the vertical axis
of fluorescence (%) shows calcium ion mobilization activity. The calculation method is Farnesyl 2
Calculated as a percentage value relative to the reaction when 2 × 10 ⁻⁶ M phosphoric acid was added to CHO-TGR4. Average of n = 3.

FIG. 17 shows the results of examining the effect of various phospholipid compounds on intracellular calcium ion mobilization activity in mock CHO cells. Conc. (NM) on the horizontal axis indicates the concentration of various phospholipid compounds. ▲ is geranyl geranyl 2
Phosphoric acid, ■ is farnesyl diphosphate, ◆ is geranyl diphosphate, ● is lysophosphatidic acid, ○ is sphingosine 1 phosphate, and △ is nordeoxycholic acid.
□ indicates deoxycholic acid, ◇ indicates geranylgeraniol, and × indicates farnesol. Change on the vertical axis
of fluorescence (%) shows calcium ion mobilization activity. The calculation method was calculated as a percentage value with respect to the reaction when farnesyl diphosphate 2 × 10 ⁻⁶ M was added to CHO-TGR4. Average of n = 3.

FIG. 18 shows the results of examining the effect of various phospholipid compounds on the cAMP production increasing activity in TGR4-expressing CHO cells. Conc. (NM) on the horizontal axis indicates the concentration of various phospholipid compounds. ▲ is geranylgeranyl diphosphate, ■ is farnesyl diphosphate, ◆ is geranyl diphosphate, ● is lysophosphatidic acid, ○ is sphingosine 1 phosphate, △ is nordeoxycholic acid, □ Indicates deoxycholic acid, ⋄ indicates geranylgeraniol, and × indicates farnesol. The vertical axis represents cAMP production increasing activity. Farnesyl diphosphate 1 is a cAMP production increasing activity
Calculated as a percentage value relative to the reaction when 15 × 10 ⁻⁶ M was added to CHO-TGR4. Average of n = 3.

FIG. 19 shows the results of examining the influence of various phospholipid compounds on the activity of increasing cAMP production in mock CHO cells. Conc. (NM) on the horizontal axis indicates the concentration of various phospholipid compounds. ▲ is geranylgeranyl diphosphate,
■ is farnesyl diphosphate, ◆ is geranyl diphosphate, ● is lysophosphatidic acid, ○ is sphingosine 1 phosphate, △ is nordeoxycholic acid, □ is deoxycholic acid, and ◇ is Geranyl geraniol, x indicates farnesol. The vertical axis represents cAMP production increasing activity. Farnesyl diphosphate 11
Calculated as a percentage of the reaction when 5 × 10 ⁻⁶ M was added to CHO-TGR4. Average of n = 3.

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 48/00 A61P 1/16 4C084 A61P 1/00 3/04 4C085 1/16 3/10 4C086 3/04 9/00 4H045 3/10 25/00 9/00 25/04 25/00 25/22 25/04 29/00 25/22 35/00 29/00 37/00 35/00 C07K 14/705 37/00 16/28 C07K 14/705 C12N 1/15 16/28 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 C12Q 1/02 5/10 1/68 A C12P 21/02 G01N 33 / 15 Z C12Q 1/02 33/50 Z 1/68 C12N 15/00 ZNAA G01N 33/15 5/00 A 33/50 A61K 37/02 (72) Inventor Yugo Habata 3-17 Namiki, Tsukuba, Ibaraki 1 Roy Yarkopo Yokota 606 (72) Inventor Yuji Kawamata 4-22 Matsushiro, Tsukuba-shi, Ibaraki No. 203 Building No. 2 Building No. 2, Matsushiro 4-chome housing complex No. 203 (72) Inventor Masaki Hosoya 83-71, Itaya, Tsuchiura-shi, Ibaraki ( 72) Inventor Shoji Fukuzumi Namiki, Tsukuba City, Ibaraki Prefecture 17th Street 6 Roy Yar City Namiki 302 (72) Inventor Hidetoshi Komatsu 1-7th Street Kasuga, Tsukuba, Ibaraki Prefecture 9 7th Takeda Kasuga Heights 803 F term (reference) 2G045 AA34 AA35 BB20 BB50 BB51 CB01 DA13 DA36 FB02 FB12 GC15 4B024A AA11 BA43 BA63 CA01 CA04 CA09 CA11 DA02 DA05 DA11 EA02 EA03 EA04 FA02 GA01 GA11 HA01 HA03 HA11 HA12 HA15 4B063 QA01 QA08 QA18 QQ05 QQ13 QQ42 QQ52 QR08 QR33 QR42 QR64 QR24 QR48 QR01 QR20 QR74 QR80 QS05 QS05 QS05 QS05 QS25 QS05 QS05 QS05 QS05 QS05 QS05 QS25 QS05 QS25 QS05 QS05 QS25 QS05 QS05 QS05 QS25 QS05 QS05 QS25 QS05 QS05 QS25 QS05 QS25 QS25 QS25 QS05 QS05 QS05 QS05 QSQ QSQA QAQ QSQA QAQQA QSQA QA QAQA DA13 4B065 AA01X AA57X AA90X AA91Y AA93Y AB01 AB02 BA01 BA08 CA24 CA25 CA44 CA46 4C084 AA02 AA07 AA13 AA17 BA01 BA08 BA22 CA18 NA14 ZA02 ZA05 MA01 A01 A14 A35 A25 A13 A35 A25A25A25A25A25A25A25A25A25A25A25A25A25A25A25A25 ZA02 ZA08 ZA36 ZA66 ZA70 ZA75 ZB01 ZB05 ZB11 ZB26 ZC35 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA50 DA76 EA20 EA50 FA72 FA74

Claims (62)

[Claims] (1) A G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof or a salt thereof, and (2) a phospholipid compound. A method for screening a compound or a salt thereof, which changes the binding property between the receptor protein or a salt thereof and a phospholipid compound, which comprises using 2. The screening method according to claim 1, wherein the G protein-coupled receptor protein is a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 8 or SEQ ID NO: 10. 3. A phospholipid compound (1) a compound having a pyrophosphate group bonded to the terminal of a repeating structure composed of one or more isoprene units, or (2) a phosphate group bonded to a glycerol skeleton, The screening method according to claim 1, which is a compound in which one fatty acid or long-chain alcohol is ester-bonded. 4. The screening method according to claim 1, wherein the phospholipid compound is geranylgeranyl diphosphate (GGPP), farnesyl diphosphate (FPP) or lysophosphatidic acid (LPA). 5. The phospholipid compound is geranylgeranyl diphosphate (GGPP) or farnesyl diphosphate (FPP).
The screening method according to claim 1, wherein (6) A G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof or a salt thereof, and (2) a phospholipid compound. A kit for screening a compound or a salt thereof which changes the binding property between the receptor protein or a salt thereof and a phospholipid compound, the kit comprising: 7. An amino acid which is obtainable by using the screening method according to claim 1 or the screening kit according to claim 6 and which is the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 1 with the phospholipid compound. A compound or a salt thereof that changes the binding property to a G protein-coupled receptor protein containing a sequence or a salt thereof. 8. A compound which changes the binding property between a phospholipid compound and a G protein-coupled receptor protein or its salt, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a salt thereof. A medicine containing a salt. 9. Central diseases, inflammatory diseases, cardiovascular diseases,
The medicament according to claim 8, which is a prophylactic / therapeutic agent for cancer, diabetes, immune system diseases or digestive system diseases. 10. The medicine according to claim 8, which is a preventive / therapeutic agent for liver / gallbladder diseases, digestive system diseases, anxiety, pain or obesity. 11. An intracellular Ca when a test compound is contacted with a cell containing a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. ²⁺
A method for determining a ligand for the G protein-coupled receptor protein or a salt thereof, which comprises measuring a concentration increasing activity or an intracellular cAMP producing activity. 12. A ligand obtained by the method according to claim 11. (I) A G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, its partial peptide or its salt, and a phospholipid compound or phosphorus. When a lipid compound is contacted with a compound or a salt thereof that changes the binding property of the G protein-coupled receptor protein or a salt thereof, (ii) the same or substantially the same amino acid sequence as represented by SEQ ID NO: 1 A G protein-coupled receptor protein containing the same amino acid sequence, a partial peptide thereof or a salt thereof, a compound that changes the binding property between a phospholipid compound or a phospholipid compound and the G protein-coupled receptor protein or a salt thereof, or a compound thereof SEQ ID NO: characterized by making a comparison with the case of contacting with a salt and a test compound G protein-coupled receptor protein or screening method of agonist or antagonist to a salt thereof comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by 1. 14. A G protein-coupled receptor protein or a salt thereof which comprises (i) a labeled phospholipid compound or a phospholipid compound containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. When a labeled compound or a salt thereof that changes the binding property of G is contacted with the G protein-coupled receptor protein, a partial peptide thereof or a salt thereof, and (ii) a labeled phospholipid compound or a phospholipid compound and the G The labeled compound or its salt, which changes the binding property to the protein-coupled receptor protein or its salt, and the test compound are
A labeled compound that changes the binding property between a labeled phospholipid compound or a phospholipid compound and the G protein-coupled receptor protein or a salt thereof when brought into contact with a protein-coupled receptor protein, a partial peptide thereof or a salt thereof SEQ ID NO: 1, characterized in that the binding amount of the salt to the G protein-coupled receptor protein, its partial peptide or its salt is measured and compared.
A method for screening an agonist or an antagonist to a G protein-coupled receptor protein or a salt thereof, which contains the same or substantially the same amino acid sequence as that of 15. (i) A labeled phospholipid compound or a G protein-coupled receptor protein or a salt thereof which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 with the phospholipid compound. When a labeled compound or a salt thereof that changes the binding property of the G protein is contacted with cells containing the G protein-coupled receptor protein, and (ii) a labeled phospholipid compound or a phospholipid compound and the G protein-coupled type Labeled phospholipid compound or phospholipid in the case where a labeled compound or a salt thereof that changes the binding property to a receptor protein or a salt thereof and a test compound are brought into contact with cells containing the G protein-coupled receptor protein Labeling to change the binding property between a compound and the G protein-coupled receptor protein or a salt thereof G-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, characterized in that the amount of a substance or a salt thereof bound to the cell is measured and compared. Alternatively, a method for screening an agonist or an antagonist to a salt thereof. 16. A G protein-coupled receptor protein or a salt thereof which comprises (i) a labeled phospholipid compound or a phospholipid compound containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. When a labeled compound or a salt thereof that changes the binding property of the protein is brought into contact with the membrane fraction of cells containing the G protein-coupled receptor protein, and (ii) the labeled phospholipid compound or the G protein-coupled type Labeled phospholipid compound in the case of contacting a labeled compound or a salt thereof, which changes the binding property with a receptor protein or a salt thereof, and a test compound with a membrane fraction of a cell containing the G protein-coupled receptor protein Alternatively, a labeled compound that changes the binding property between a phospholipid compound and the G protein-coupled receptor protein or a salt thereof. Alternatively, the amount of the salt bound to the membrane fraction of the cells is measured and compared, and a G protein-coupled protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 is characterized. For screening agonists or antagonists for type I receptor proteins or salts thereof. 17. A G protein-coupled receptor protein or a salt thereof which comprises (i) a labeled phospholipid compound or a phospholipid compound containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. The labeled compound or salt thereof that changes the binding property of the above is transformed with a recombinant vector containing a DNA containing a DNA encoding the G protein-coupled receptor protein, thereby culturing the transformant. When contacted with a G protein-coupled receptor protein expressed on the cell membrane of the body, and (ii) a label which changes the binding property between the labeled phospholipid compound or the phospholipid compound and the G protein-coupled receptor protein or a salt thereof. Protein or a salt thereof and a test compound expressed on the cell membrane of the transfectant G protein-coupled A labeled compound or a salt thereof which changes the binding property between a labeled phospholipid compound or a phospholipid compound and the G protein-coupled receptor protein or a salt thereof when brought into contact with a receptor protein Agonist or antagonist to G protein-coupled receptor protein or its salt containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, characterized by measuring and comparing the amount of binding to Screening method. 18. (i) Binding to a phospholipid compound or a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 with a phospholipid compound or a salt thereof. A case where a compound or a salt thereof that changes sex is brought into contact with cells containing the G protein-coupled receptor protein;
(Ii) Phospholipid compound or phospholipid compound and the G
Intracellular Ca ²⁺ concentration-elevating activity when a compound or a salt thereof that changes the binding property to a protein-coupled receptor protein or a salt thereof, and a test compound are contacted with a cell containing the G protein-coupled receptor protein or G containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, characterized by measuring and comparing intracellular cAMP producing activity
A method for screening an agonist or antagonist for a protein-coupled receptor protein or a salt thereof. (I) Binding to a phospholipid compound or a G protein-coupled receptor protein or a salt thereof containing a phospholipid compound or an amino acid sequence identical to or substantially the same as the amino acid sequence represented by SEQ ID NO: 1. A compound or a salt thereof that changes sex is cultivated in a cell membrane of the transformant by culturing the transformant transformed with a recombinant vector containing the DNA containing the DNA encoding the G protein-coupled receptor protein. When contacted with the expressed G protein-coupled receptor protein,
(Ii) Phospholipid compound or phospholipid compound and the G
Intracellular Ca when a compound or its salt that changes the binding property to a protein-coupled receptor protein or its salt, and a test compound are contacted with the G protein-coupled receptor protein expressed on the cell membrane of the transformant.
G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, characterized by measuring and comparing ²⁺ concentration increasing activity or intracellular cAMP producing activity Alternatively, a method for screening an agonist or an antagonist to a salt thereof. 20. A compound in which a phospholipid compound is (1) a compound having a pyrophosphate group bonded to the terminal of a repeating structure composed of one or more isoprene units, or (2) 1 in a glycerol skeleton.
The screening method according to claim 1, which is a compound in which one phosphoric acid group is bound, and further one fatty acid or long-chain alcohol is ester bound. 21. The phospholipid compound is geranylgeranyl diphosphate (GGPP), farnesyl diphosphate (FPP) or lysophosphatidic acid (LPA).
The screening method according to any one of 3 to 20. 22. The phospholipid compound is geranylgeranyl diphosphate (GGPP) or farnesyl diphosphate (FP).
The screening method according to claim 13, which is P). 23. An atomic coordinate and a ligand binding pocket of the active site of a G protein-coupled receptor protein or a salt thereof, in which the test compound contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. The screening method according to claims 13 to 20, which is a compound designed to bind to a ligand binding pocket based on position. 24. A G protein-coupled receptor containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 obtained by using the screening method according to any one of claims 13 to 23. An agonist or antagonist for a protein or a salt thereof. 25. A medicine comprising an agonist or an antagonist to a G protein-coupled receptor protein or its salt, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. 26. A central disease or inflammation comprising an agonist or an antagonist to a G protein-coupled receptor protein or its salt containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. A preventive / therapeutic agent for sexually transmitted diseases, cardiovascular diseases, cancer, diabetes, immune system diseases or digestive system diseases. 27. A liver comprising an agonist or an antagonist to a G protein-coupled receptor protein or a salt thereof which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1.
A prophylactic / therapeutic agent for gallbladder diseases, digestive system diseases, anxiety, pain or obesity. 28. A central disease or inflammation comprising a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof or a salt thereof. Prophylactic / therapeutic agent for sexual diseases, cardiovascular diseases, cancer, diabetes, immune system diseases, liver / gallbladder diseases, digestive system diseases, anxiety, pain or obesity. 29. A central disease comprising a DNA containing a DNA encoding a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Prevention of inflammatory diseases, cardiovascular diseases, cancer, diabetes, immune system diseases, liver / gallbladder diseases, digestive system diseases, anxiety, pain or obesity
Therapeutic agent. 30. Centrality comprising a polynucleotide containing a polynucleotide encoding a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Disease, inflammatory disease, cardiovascular disease, cancer, diabetes, immune system disease, liver / gallbladder disease, digestive system disease, anxiety, pain or obesity diagnostic agent. 31. Use of a polynucleotide containing a polynucleotide encoding a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof. A method for diagnosing central disease, inflammatory disease, cardiovascular disease, cancer, diabetes, immune system disease, liver / gallbladder disease, digestive system disease, anxiety, pain or obesity characterized by: 32. A central compound comprising a compound or a salt thereof which changes the expression level of a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Disease / inflammatory disease, cardiovascular disease, cancer, diabetes, immune system disease, liver / gallbladder disease, digestive system disease, anxiety, pain or obesity preventive / therapeutic agent. 33. A central disease comprising an antibody against a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, its partial peptide or its salt. , A prophylactic / therapeutic agent for inflammatory diseases, cardiovascular diseases, cancer, diabetes, immune system diseases, liver / gallbladder diseases, digestive system diseases, anxiety, pain or obesity. 34. A central disease comprising an antibody against a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, its partial peptide or its salt. , Inflammatory disease, cardiovascular disease, cancer, diabetes, immune system disease, liver / gallbladder disease, digestive system disease, anxiety, pain or obesity diagnostic agent. 35. A G protein-coupled receptor protein comprising an antibody against a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof or a salt thereof. A method for diagnosing central disease, inflammatory disease, cardiovascular disease, cancer, diabetes, immune system disease, liver / gallbladder disease, digestive system disease, anxiety, pain or obesity, which is characterized by using a protein quantification method. 36. Complementary to a polynucleotide containing a polynucleotide encoding a G protein-coupled receptor protein or a partial peptide thereof which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Diseases containing polynucleotides containing various base sequences or parts thereof, inflammatory diseases, cardiovascular diseases, cancer, diabetes, immune system diseases, liver / gallbladder diseases, digestive system diseases, anxiety , A preventive or therapeutic agent for pain or obesity. 37. An agonist or antagonist to a G protein-coupled receptor protein or its salt, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 for mammals, SEQ ID NO: A G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by 1, or a partial peptide thereof or a salt thereof, the same as or substantially the same as the amino acid sequence represented by SEQ ID NO: 1. A polynucleotide containing a polynucleotide encoding a G protein-coupled receptor protein containing an amino acid sequence or a partial peptide thereof,
An antibody against a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 1, a partial peptide thereof or a salt thereof, or the same as the amino acid sequence shown in SEQ ID NO: 1 An effective amount of a polynucleotide containing a base sequence complementary to a polynucleotide containing a polynucleotide encoding a G protein-coupled receptor protein containing substantially the same amino acid sequence or a partial peptide thereof, or a part thereof A method for preventing / treating central nervous system diseases, inflammatory diseases, cardiovascular diseases, cancer, diabetes, immune system diseases, liver / gallbladder diseases, digestive system diseases, anxiety, pain or obesity, which is characterized in that: 38. Central diseases, inflammatory diseases, cardiovascular diseases,
The same or substantially the same as the amino acid sequence represented by SEQ ID NO: 1 for producing a prophylactic / therapeutic agent for cancer, diabetes, immune system disease, liver / gallbladder disease, digestive system disease, anxiety, pain or obesity Agonist or antagonist to G protein-coupled receptor protein containing an amino acid sequence or a salt thereof, G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, and a portion thereof A peptide or a salt thereof, a polynucleotide containing a polynucleotide encoding a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof, SEQ ID NO: The same as the amino acid sequence represented by: G protein-coupled receptor protein containing the qualitatively identical amino acid sequence, an antibody or SEQ number for its partial peptide, or a salt thereof:
A nucleotide sequence complementary to a polynucleotide containing a polynucleotide encoding a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by 1 or a partial peptide thereof, or a part thereof. Use of the polynucleotide comprising. 39. A G protein-coupled receptor protein consisting of the amino acid sequence represented by SEQ ID NO: 8 or SEQ ID NO: 10, a partial peptide thereof or a salt thereof. 40. A polynucleotide containing a polynucleotide encoding the G protein-coupled receptor protein according to claim 39. 41. A DNA consisting of the nucleotide sequence represented by SEQ ID NO: 9 or SEQ ID NO: 11. 42. A recombinant vector containing the polynucleotide according to claim 40. 43. A transformant transformed with the recombinant vector according to claim 42. 44. A transformant according to claim 43 is cultured,
The method for producing a G protein-coupled receptor protein or a salt thereof according to claim 39, which comprises producing the G protein-coupled receptor protein or a salt thereof according to claim 39. 45. A medicine comprising the G protein-coupled receptor protein according to claim 39, a partial peptide thereof or a salt thereof. (46) A pharmaceutical comprising the polynucleotide according to (40). 47. A diagnostic agent comprising the polynucleotide according to claim 40. 48. An antibody against the G protein-coupled receptor protein according to claim 39, a partial peptide thereof or a salt thereof. 49. The antibody according to claim 48, which is a neutralizing antibody that inactivates signal transduction of the G protein-coupled receptor protein according to claim 39. 50. A diagnostic agent comprising the antibody according to claim 48. (51) A pharmaceutical comprising the antibody according to (48). 52. An antisense polynucleotide comprising a base sequence complementary to the polynucleotide according to claim 40 or a part thereof. 53. A diagnostic agent comprising the antisense polynucleotide according to claim 52. 54. A medicine comprising the antisense polynucleotide according to claim 52. 55. The method for screening an agonist or antagonist for the G protein-coupled receptor protein according to claim 39, which comprises using the G protein-coupled receptor protein according to claim 39, a partial peptide thereof, or a salt thereof. 56. A kit for screening an agonist or antagonist for the G protein-coupled receptor protein according to claim 39, which contains the G protein-coupled receptor protein according to claim 39, a partial peptide thereof or a salt thereof. .. 57. An agonist or antagonist to the G protein-coupled receptor protein according to claim 39, which is obtained by using the screening method according to claim 55 or the screening kit according to claim 56. 58. A pharmaceutical comprising the agonist or antagonist for the G protein-coupled receptor protein according to claim 39. 59. A method for screening a compound or a salt thereof which alters the expression level of a G protein-coupled receptor protein according to claim 39, which comprises using the polynucleotide according to claim 40. 60. A screening kit for a compound or a salt thereof which changes the expression level of the G protein-coupled receptor protein according to claim 39, which comprises the polynucleotide according to claim 40. 61. A compound or a salt thereof which alters the expression level of the G protein-coupled receptor protein according to claim 39, which is obtained by using the screening method according to claim 59 or the screening kit according to claim 60. 62. A medicine comprising a compound or a salt thereof which changes the expression level of the G protein-coupled receptor protein according to claim 39.