WO2001070975A1

WO2001070975A1 - Novel melanin concentrating hormone receptor

Info

Publication number: WO2001070975A1
Application number: PCT/JP2001/002343
Authority: WO
Inventors: Takeshi Kurama; Shunichiro Matsumoto; Jun Takasaki; Mitsuyuki Matsumoto; Masazumi Kamohara; Tetsu Saito; Tamaki Oda; Youko Saito
Original assignee: Yamanouchi Pharmaceutical Co., Ltd.; Helix Research Institute
Priority date: 2000-03-24
Filing date: 2001-03-23
Publication date: 2001-09-27
Also published as: AU2001239562A1; JP2005229804A

Abstract

A cDNA encoding a novel melanin concentrating hormone receptor protein is isolated. Provision of this novel melanin concentrating hormone receptor protein enables binding experiments with the use of this protein. By screening a substance modifying the activity of the melanin concentrating hormone receptor based on the binding experiments, drugs targeting this protein can be developed.

Description

Novel melanin concentrating hormone receptor

The present invention relates to a novel melanin concentrating hormone receptor, a gene encoding the receptor, a vector containing the gene, a host cell containing the vector, and a drug screening method using the receptor. The present invention also relates to a substance that modulates the receptor activity, a method for preventing and / or treating obesity and / or eating disorders using the substance, and a pharmaceutical composition. Skill

Melanin-concentrating-hormone (MCH) is a cyclic peptide hormone isolated from the pituitary gland of salmon for the first time (Ka wauchi ₅ H. et al. (1983) Nature 305, 321- 323). In fish, they functionally antagonize the melanocyte stimulating hormone, causing melanophore aggregation and participating in adaptive changes in body color (Bittencourt, J. et al. (1992) J. Comp. Neurol. 319, 218-245). Thereafter, cloning of the MCH precursor cDNA was performed in rats and humans (Nahon, JL et al. (1989) Endocrinology 125, 2056-2065, Presse.et al. (1990) Mo I. Endocrinol. 4 , 632-637), and it has become clear that they perform a wide range of physiological functions, such as regulating the energy homeostasis.

MCH is mainly expressed in the lateral hypothalamus and the uncertain zone in rats and humans (Bittencourt, JC et al. (1992) J. Comp. Neurol. 319, 218-245, Viale ₅ A. et. al. (1997) Brain Res Mol Brain Res 46, 243-255) ₀

Based on the following findings, the lateral hypothalamus has long been known to function as a feeding center. (1) Suppression and disappearance of eating behavior due to destruction of the lateral hypothalamus. (Anand, BK & Brobeck, JR (1951) Yale J Biol Med 24, 123-140)

(2) Elevated feeding behavior and reversible obesity are observed by electrical stimulation of the lateral hypothalamus.

(Steinbaum, E.A. & Miller, N.E. (1965) Am J Physiol 208, 1-5)

(3) Under starvation, neural activity in the lateral hypothalamus is higher than in the ventromedial nucleus, known as the satiety center. (Anand, B.K. & Pillai, R.V. (1967) J Physiol 192, 63-77) Giving a course or inflating the stomach with a balloon placed in the stomach reduces the frequency of nerve firing in the outer hypothalamus. (Anand, BK et al. (1964) Am J Physiol 20 7, 1146-1154; Anand, BK & Pillai, RV (1967) J Physiol 192, 63-7 7) 2-Doxy that reduces glucose utilization Glucose increases the frequency of nerve firing in the outer hypothalamus. (Desiraju, T. et al. (1968) Physio 1 Behav 3, 757-760)

(4) In fasted monkeys, oxygen consumption increases in the lateral hypothalamus, and decreases in the ventromedial nucleus. (Anand, BK et al. (1961) Indian J Med Res 49, 725-732) Increased expression of the MCH gene in the hypothalamus of ob / ob mice and fasted mice with mutations in leptin gene and obese mice (Qu, D. et al. (1996) Nature 380, 243-247). Leptin is a hormone that senses body fat mass and maintains it at an appropriate level.

Indeed, when MCH is administered intraventricularly to rats, a dose-dependent increase in food intake occurs (Qu, D. et al. (1996) Nature 380, 243-247, Ludwig, DS et al. (1998) Am .J. Physiol. 274, E627-E633). MCH-deficient mice showed reduced body weight due to reduced body fat, decreased food intake, and increased oxygen consumption per body weight compared to wild-type mice (Shimada, M. et al.). al. (1998) Nature 396, 670-674). It is also known that MCH has a regulatory relationship with other factors involved in energy homeostasis. The melanocyte stimulating hormone (hereinafter referred to as MSH) is a melanocortin-4 receptor (MC4R) It has been known to exert an antifeeding effect and reduce the amount of energy stored in the body through the body (Friedman, JM (1997) Nature 385, 119-120, Lu, D. et al. (1994) Nature 371, 799-802, Huszar, D. et al. (1997) Cell 88, 131-141).

Similar to the antagonism in fish color change, MCH and MSH also have opposing effects on feeding behavior in mammals. MCH-induced hyperphagia is suppressed by spike-MSH, and conversely, spite-MSH suppresses the antifeedant effect by MCH (Ludwig, DS et al. (1998) Am. J. Physiol. 274 , E627-E633) ₀ However, MCH itself is not affinity MC4R, from the inability inhibit binding of "-MSH and MC4R, has been assumed that exerts its action through its own receptor (Ludwig , DS et al. (199 8) Am. J. Physiol. 274, E627-E633) ₀

In Ay / a mice deficient in the melanocortin system and rats treated with AGRP, an endogenous inhibitor of MC4R, increased expression of the MCH gene occurs in the lateral hypothalamus (Hanada, R. et al. (2000) Biochem Biophys Res Conunun 268, 88-91). In addition, the effect of MCH on feeding enhancement is suppressed by anorexia-suppressing factors such as lebutin, neurotensin, and GLP-1 (Sahu, A. (1998) Endocrinology 139, 4739-4741, Trit osJ.A. et. al. (1998) Diabetes 47, 1687-92).

Based on the above facts, in vivo, MCH is related to various energy homeostasis regulators, but itself exhibits an effect of increasing food intake and suppresses energy consumption, and is responsible for regulating energy balance. Is believed to be

MCH has reported the following effects in addition to the above effects.

-Effect of promoting memory elimination in passive avoidance (McBride, R.B. et al. (1994) Peptides 15, 757-759)

• Effects of sexual receptivity when administered to the medial anterior optic nucleus and ventromedial nucleus of female rats (Gonzalez, MI et al. (1996) Peptides 17, 171-177)

-When administered to the rat medial anterior optic tract, it induces anxiety (Gonzalez, MI et a 1. (1996) Peptides 17, 171-177)

'Suppresses auditory stimulus-induced hippocampal response (Miller, L. (1993) Peptides 14, 431 -440)

• When administered intraventricularly to sheep, it has a diuretic effect and promotes the excretion of sodium and potassium in urine (Parkes, D. G. (1996) J. Neuroendocrinol. 8, 57-63)

Furthermore, SLC-1 has been reported as an MCH receptor in humans (Saito, Y. et al. (1999) Nature 400, 265-269). SLC-1 is a gene originally isolated on the basis of homology with the somatosustin receptor, one of the G protein-coupled receptors (Kolakowski, LF et al. (1996) FEBS Lett. 398, 253-258). The homology with the somatosustin receptor gene was about 40%, and no binding activity with somatosustin was found, but subsequent studies revealed that the ligand was MCH. Disclosure of the invention

It is an object of the present invention to provide a gene encoding a novel human melanin concentrating hormone (MCH) receptor or a protein having a function equivalent to the receptor. Another object of the present invention is to provide a human MCH receptor expressed using the gene or a protein having the same function as the receptor. Another object of the present invention is to provide a method for screening a drug that modifies the activity of the MCH receptor using the MCH receptor of the present invention. In addition, the present invention aims to provide a substance that modulates the receptor activity, a method for preventing and / or treating obesity and / or eating disorders using the substance, and a pharmaceutical composition. I have. As a result of intensive studies, the present inventors have succeeded in isolating a gene encoding a novel human MCH receptor, and have found that the amino acid sequence of human MCH receptor protein and human MCH receptor The nucleotide sequence of DNA encoding was determined. Further, the expression of the MCH receptor, the production of a recombinant protein is enabled, and the vector containing the gene, the vector We have established a method for producing host cells, including Michigan, MCH receptor protein using the host cells, and antibodies against the MCH receptor.

This enables screening of the MCH receptor and a substance that modulates the activity of the MCH receptor, and screening for a disease associated with MCH, such as obesity, an eating disorder prevention and / or therapeutic agent, In the screening, a substance that modifies the activity of the receptor, in particular, a substance having angiogonist activity was obtained, and the present invention was completed.The present inventors further studied the isolation of a homolog of the MCH receptor in monkeys. Succeeded and revealed that it is located in the feeding center.

That is, the present invention relates to the following proteins, genes encoding the same, and a method for screening a substance that enhances the activity of an MCH receptor, particularly a substance that has angiogonist activity, using the protein. The present invention also relates to a substance that modulates the receptor activity, particularly a substance having angiogonist activity, a method for preventing and / or treating obesity and / or eating disorders using the substance, and a pharmaceutical composition. I will consider things.

[1] one or more amino acid residues in one or more amino acid sequences of SEQ ID NO: 2 or one or more in the amino acid sequence of SEQ ID NO: 2; Or a protein having an inserted amino acid sequence and exhibiting melanin-concentrating hormone receptor activity.

[2] the protein of [1], which has the amino acid sequence of SEQ ID NO: 2;

[3] a protein that is encoded by DNA that hybridizes under stringent conditions with the DNA represented by the nucleotide sequence of SEQ ID NO: 1 and that exhibits melanin concentrating hormone receptor activity .

[4] the protein of [3], which has the amino acid sequence of SEQ ID NO: 2;

[5] the protein of [3], which has the amino acid sequence of SEQ ID NO: 8;

[6] a gene encoding the protein of [1] or [3];

[7] the gene of [6], wherein the protein has the amino acid sequence of SEQ ID NO: 2; [8] the gene of [6], wherein the protein has the amino acid sequence of SEQ ID NO: 8; [9] A vector containing the gene of [6].

[10] A host cell containing the vector of [9].

(11) (10) characterized in that the host cell according to (1) or

[3] The method for producing the protein according to the above.

[12] an antibody that binds to the protein of [1] or [3];

[13] A method for screening for a substance having angiogonist activity of melanin concentrating hormone receptor, comprising the following steps:

(1) contacting the test drug with the protein according to (1) or (3) in the presence of a melanin concentrating hormone,

(2) measuring the binding activity of melanin concentrating hormone to the protein, and

(3) selecting a test drug that reduces the binding activity measured in step (2) as compared with the binding activity of the melanin concentrating hormone to the protein measured in the absence of the test drug

[14] A method for screening for a substance having an angiotensin activity of melanin concentrating hormone receptor, comprising the following steps:

(1) contacting a test agent with a cell expressing the protein of (1) or (3) in the presence of melanin concentrating hormone,

(2) measuring a change in cells caused by binding of the melanin concentrating hormone to the protein, and

(3) selecting a test drug that suppresses the cell change measured in step (2) compared to the cell change measured in the absence of the test drug

[15] The screening method according to [14], wherein the change in the cell is any change selected from the group consisting of a change in GTP binding activity, a change in intracellular Ca ion concentration, and a change in intracellular cAMP concentration. (16) the substance having antagonist activity of melanin concentrating hormone receptor is a substance for preventing and / or treating obesity and / or eating disorders (13) or (14) Screening method. [17] An amino acid gonist of the protein of [1] or [3], which is selected by the method of [13] or [14].

(1 8) obesity, comprising the angyo gonist according to (17) as a main component, and obesity, and

A pharmaceutical composition for preventing and / or treating an eating disorder.

[19] a melanin concentrating hormone receptor gene comprising a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or a DNA complementary to its complementary strand and having a chain length of at least 15 nucleotides Reagent for detection.

[20] DNA containing the base sequence of SEQ ID NO: 1 or DNA complementary to the complementary strand thereof and having a chain length of at least 15 nucleotides is brought into contact with the sample, and the DNA is placed in the sample. A method for detecting a melanin concentrating hormone receptor gene, comprising a step of hybridizing to a DNA of the present invention. The present invention also relates to a method for preventing and / or treating obesity, and Z or an eating disorder, which comprises a step of administering the antagonist according to [17]. Alternatively, the present invention relates to the use of the angonist according to [17] in the manufacture of a pharmaceutical composition for preventing and / or treating obesity and / or eating disorders.

WO 00/49046 published after the priority date describes the base sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2 of the present invention. However, the gene is cloned, the nucleotide sequence is determined, and only the deduced amino acid sequence encoded by the gene is described. A specific method for producing a protein having the amino acid sequence of SEQ ID NO: 2 There is no description of the production of the protein, the specific function and use of the protein, and the description that the protein is an MCH receptor.

As used herein, “melanin concentrating hormone (MCH) receptor” refers to “melanin concentrating hormone (MCH) receptor protein I. Represents

The MCH receptor protein of the present invention includes

(1) an MCH receptor having the amino acid sequence of SEQ ID NO: 2,

(2) an MCH receptor having the amino acid sequence of SEQ ID NO: 8,

(3) one or more amino acid residues are substituted, deleted, and 7 or inserted at any one or more sites in the amino acid sequence described in SEQ ID NO: 2, and A protein having the same activity as the MCH receptor having the amino acid sequence of SEQ ID NO: 2 (hereinafter, referred to as “the same drug” of the protein having the amino acid sequence of SEQ ID NO: 2),

(4) a protein encoded by a DNA that hybridizes under the “stringent conditions” with the DNA represented by the nucleotide sequence of SEQ ID NO: 1 and having the amino acid sequence of SEQ ID NO: 2 Shows the same activity as the body

A protein (ie, functionally equivalent) (hereinafter, referred to as “hybridizing homolog” of a protein having the amino acid sequence of SEQ ID NO: 2), and

(5) an amino acid sequence having 95% or more homology with the amino acid sequence described in SEQ ID NO: 2 and having the same activity as the MCH receptor having the amino acid sequence described in SEQ ID NO: 2 Protein (hereinafter, referred to as "homologous substance" of a protein having the amino acid sequence described in SEQ ID NO: 2)

Is included.

The “equivalent substance” of the protein having the amino acid sequence of SEQ ID NO: 2 is preferably 1 to 10, more preferably 1 to 7, particularly preferably 1 to 10 in the amino acid sequence of SEQ ID NO: 2. And has the same activity as a protein having an amino acid sequence having amino acid substitutions, deletions and / or insertions of up to 5 amino acids and having the amino acid sequence of SEQ ID NO: 2, that is, MCH receptor activity. The proteins shown can be mentioned.

Shows "same activity" as MCH receptor having the amino acid sequence of SEQ ID NO: 2. "MCH receptor activity" means that it can be confirmed, for example, by the method described in Example 3. That is, it can be confirmed that the protein exhibits the same activity as that of the protein using the MCH-dependent increase in intracellular Ca concentration as an index. The “same activity” as the protein having the amino acid sequence of SEQ ID NO: 2 is preferably EC50 = 3 M or less, more preferably EC50 = 300 nM or less under the conditions described in Example 3. Further, the compound has a high binding affinity under the conditions described in Example 2, preferably with a dissociation constant for human MCH of Kd = 50 nM or less, more preferably Kd = 5 nM or less.

The “stringent conditions” under which the DNA encoding the “hybridizing synergistic protein” of the protein having the amino acid sequence of SEQ ID NO: 2 hybridizes with the DNA represented by the nucleotide sequence of SEQ ID NO: 1 The conditions for hybridization are as follows: 5xSSPE, 5xDenhanfs solution, 0.5% SDS, 40% formamide, 200 g / ml salmon sperm DNA, 37 ° C overnight. The conditions were about 5xSSPE, 5xDenhard, s solution, 0.5% SDS, 50% formamide, 200 g / ml salmon sperm DNA, 42 ° C overnight. The conditions for washing are as follows: loose conditions are about 5xSSC, 1% SDS, 42 ° C, and usually about 0.5xSSC, 0.1% SDS, 42 ° C. The condition is about “0.2xSSC, 0.1% SDS, 65 ° C!”.

The homology between the amino acid sequence of the homologous protein j and the amino acid sequence of SEQ ID NO: 2 of the protein having the amino acid sequence of SEQ ID NO: 2 is at least 95% or more. The homology of the amino acid sequence can be identified by the following conditions (parameters) using a BLAST search.

“Same substance” of a protein having the amino acid sequence of SEQ ID NO: 2; “Hybridized same substance” of a protein having the amino acid sequence of SEQ ID NO: 2; "Homologous synonyms" are collectively referred to as "synonymous agents" in the present invention. As a preferable embodiment of the “same drug” in the present invention, for example, a protein having the amino acid sequence described in SEQ ID NO: 8 can be shown. The amino acid sequence set forth in SEQ ID NO: 8 is encoded by the base sequence set forth in SEQ ID NO: 7. The cDNA consisting of the nucleotide sequence of SEQ ID NO: 7 was isolated by the present inventors as a homolog of the MCH receptor in cynomolgus monkeys. The amino acid sequence described in SEQ ID NO: 8 (Rinus cynomolgus monkey) has 97% homology with the amino acid sequence described in SEQ ID NO: 2 (human), and a protein comprising this amino acid sequence has MCH receptor activity. It was confirmed that it had.

The origin of the MCH receptor of the present invention is not limited to humans and monkeys. As long as it corresponds to any of the receptors of the present invention described in the above (1) to (5), for example, a receptor derived from an organism other than humans and monkeys is also a sequence represented by SEQ ID NO: 2 or SEQ ID NO: 8. The receptor of the present invention also includes a receptor artificially modified by genetic engineering based on the above. Further, the receptor of the present invention is preferably a recombinant receptor.

Also, a gene having a nucleotide sequence encoding the protein of the present invention, that is, a MCH receptor represented by the amino acid sequence of SEQ ID NO: 2, or a gene having a nucleotide sequence encoding the “same drug” in the present invention Specifically, as long as it is a gene having a nucleotide sequence encoding a protein selected from the receptors of the present invention described in the above (1) and (5), it is included in the present invention. Preferably, it is a gene having a base sequence encoding the amino acid sequence of SEQ ID NO: 2. More preferably, it is a gene having the 1st to 1023rd of SEQ ID NO: 1. "Genes" of the present invention are DNA and RNA, preferably DNA.

A gene encoding the protein of the present invention, a vector of the present invention, a host cell of the present invention, a protein of the present invention, a method for screening a substance that modifies the activity of the protein of the present invention, an antibody that reacts with the protein of the present invention, and the present invention. The medicaments are described in 1) to 5) below.

1) Method for producing MCH receptor gene of the present invention a) First manufacturing method

MRNA is extracted from cells or tissues capable of producing the MCH receptor protein of the present invention. Next, two types of primers sandwiching the receptor mRNA or a part of the mRNA region are prepared using this mRNA as type III. The MCH receptor cDNA or a part thereof can be obtained by performing a reverse transcriptase-polymerase chain reaction (hereinafter, referred to as RT-PCR). Further, by incorporating the obtained MCH receptor cDNA or a part thereof into an appropriate expression vector, it can be expressed in a host cell to produce the receptor protein.

First, mRNAs including those encoding the protein are extracted from cells or tissues capable of producing the MCH receptor of the present invention, for example, human brain, by a known method. Examples of the extraction method include a guanidine-thiocyanate 'hot' phenol method and a guanidine-thiocyanate-guanidine 'hydrochloric acid method, and preferably a guanidine-thiocyanate cesium chloride method. Cells or tissues having the ability to produce the protein can be obtained by Northern blotting using a gene or a part of a gene having a nucleotide sequence encoding the protein, or Western plotting using an antibody specific to the protein. It can be specified by such as.

The mRNA may be purified according to a conventional method. For example, the mRNA can be adsorbed and eluted to an oligo (dT) cell source column and purified. Furthermore, mRNA can be further fractionated by sucrose density gradient centrifugation or the like. Alternatively, a commercially available extracted mRNA may be used without extracting the mRNA.

Next, the purified mRNA is subjected to a reverse transcriptase reaction in the presence of a random primer or an oligo dT primer to synthesize a first-strand cDNA. This synthesis can be performed by a conventional method. The obtained first-strand cDNA is subjected to PCR using two types of primers sandwiching a partial region of the target gene to amplify the target MCH receptor DNA. The obtained DNA is fractionated by agarose gel electrophoresis or the like. If desired, cut the above DNA with restricted oxygen or the like and connect The target DNA fragment can also be obtained.

b) Second manufacturing method

The gene of the present invention can be produced by a conventional genetic engineering technique in addition to the above-mentioned production method. First, a single-stranded cDNA is synthesized using reverse transcriptase using the mRNA obtained by the above-mentioned method as a type II, and then a double-stranded cDNA is synthesized from the single-stranded cDNA. The S1 nuclease method (Efstratiadis, A. et al. (1976) Cell, 7, 279-288) ヽ Land method (Land, H. et al. (1981) Nucleic Acids Res., 9) , 2251-2266) s 0. Joon Yoo method (Yoo, 0. J. et al. (1983) Proc. Natl. Acad. Sci. USA, 79, 1049-1053), Okayajna-Berg method (Okayajna, H. and Berg, P. (1982) Mol. Cell. Biol., 2, 16, 170).

Next, the recombinant plasmid obtained by the above-described method is introduced into Escherichia coli, for example, DH5 strain, and transformed, and a recombinant can be selected using tetracycline resistance or ampicillin resistance as an indicator. Transformation of host cells can be performed, for example, by the method of Hanahan (Hanahan, D. (1983) J. Mol. Biol., 166, 557-580) when the host cell is Escherichia coli, ie, CaCl ₂ , MgCl ₂ or RbCl _2. Can be carried out by a method of adding the recombinant DNA to a recombinant cell prepared in the presence of the recombinant DNA. 'As a vector, a phage vector such as a lambda system can be used in addition to a plasmid.

As a method for selecting a strain having the DNA of the target MCH receptor protein from the transformants obtained as described above, for example, the following various methods can be adopted.

(1) Screening method using synthetic oligonucleotide probe

An oligonucleotide corresponding to all or a part of the MCH receptor of the present invention is synthesized (in this case, a plurality of nucleotide sequences obtained by combining a nucleotide sequence derived using codon usage or a possible nucleotide sequence). well, and if the latter, it is also possible to reduce the type by including inosine), this professional one-flop ⁽³² labeled with P or ³³ P), nitrate obtained by modifying fix the DNA of transformants Hybridize with Rocellulose Fill Yuichi, search for the obtained positive strain, and select it.

(2) Screening method using a probe prepared by polymerase chain reaction A sense primer and an antisense primer oligonucleotide corresponding to a part of the MCH receptor of the present invention are synthesized, and these are combined to perform a polymerase chain reaction. (Saiki, RK et al. (1988) Science 239, 487-491) to amplify a DNA fragment encoding all or part of the target MCH receptor. As the type I DNA used here, cDNA or genomic DNA synthesized by reverse transcription from mRNA of cells producing the MCH receptor can be used. The DNA fragment prepared in this way is labeled with ³² P or ³³ P, and the target clone is selected by performing colony priming or plaque hybridization using this as a probe. I do.

(3) Method of screening by producing MCH receptor in other animal cells Transformants are cultured, the gene is amplified, and the gene is transfected into animal cells (in this case, a self-replicating and transcription promoter). It may be a plasmid containing the evening region or a plasmid capable of integrating into the chromosome of an animal cell) ', which produces the protein encoded by the gene on the cell surface. By detecting the protein using an antibody against the MCH receptor protein of the present invention, a strain having cDNA encoding the MCH receptor of interest is selected from the original transformant.

(4) Method for selecting using an antibody against the MCH receptor of the present invention

In advance, the cDNA is integrated into the expression vector, a protein is produced on the surface of the transformant, and a desired MCH receptor-producing strain is prepared using the antibody against the MCH receptor of the present invention and a secondary antibody against the antibody. And select the target strain.

A method for collecting DNA encoding the MCH receptor of the present invention from the obtained transformant of interest can be obtained by a known method (Maniatis, T. et al. (1982): "Molecular Cloning-A Laboratory Manual"). Cold Spring Harbor Laboratory, NY). An example For example, it can be carried out by separating a fraction corresponding to the plasmid DNA from the cells and cutting out the cDNA region from the plasmid DNA.

c) Third manufacturing method

A gene having a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 8 can also be produced by joining a DNA fragment produced by a chemical synthesis method. Each DNA can be synthesized using a DNA synthesizer (for example, Oligo 1000M DNA Synthesizer (Beckman) or 394 DNA / RNA Synthesizer (Applied Biosystems)).

d) Fourth manufacturing method

In order for a substance obtained by a genetic engineering technique using the gene of the present invention to express the function of the MCH receptor of the present invention, the substance necessarily has all of the amino acid sequence shown in SEQ ID NO: 2. No need to be. For example, even if a part of the sequence or another amino acid sequence is added, as long as it shows “the same activity” as the MCH receptor represented by the amino acid sequence shown in SEQ ID NO: 2, However, those proteins are also included in the protein of the present invention.

In general, eukaryotic genes are considered to exhibit polymorphism, as is known in the case of the inferior ferron gene (eg, NishiJ. Et al. (1985) J. Biochem. , 97, 153-159), and one or more amino acids may be replaced by this polymorphism. Therefore, even in a protein in which one or more amino acid residues are substituted, deleted, and / or inserted at one or more sites in the amino acid sequence represented by SEQ ID NO: 2, : Highly likely to have “the same activity” as the MCH receptor represented by the amino acid sequence described in 2. As described above, these proteins are “equivalents” of the protein having the amino acid sequence of SEQ ID NO: 2, and are included in the present invention. All of the genes having the nucleotide sequences encoding these “similar substances” are also included in the present invention. Such various genes of the present invention are, for example, phosphite-based based on the above-mentioned information of the MCH receptor of the present invention. It can also be produced by chemical synthesis of nucleic acids according to a conventional method such as the ester method (Hunkapiller, M. et al. (1984) Nature, 10, 105-111). The codon for the desired amino acid is known per se, and its choice may be arbitrary. For example, it can be determined according to a conventional method in consideration of the codon usage frequency of the host to be used (Crantham, IL et al. (1981) Nu cleic Acids Res., 9 r43-r74) ₀ Further, partial modification of the codons of these base sequences can be performed by a conventional method using a primer comprising a synthetic oligonucleotide encoding the desired modification. The site-specific mutag enesis (Mark, DF et al. (1984) Proc. Natl. Acad. Sci. USA, 81, 5662-5666) can be used.

As another embodiment of the method for preparing the “same substance” in the present invention, there is a method using a hybridization technique or a method utilizing a gene amplification technique. That is, those skilled in the art can use the hybridization technology (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section 6.3-6.4) to synthesize the protein of the present invention. Based on the base sequence of the DNA to be coded (SEQ ID NO: 1) or a part thereof, a DNA highly homologous thereto is isolated from a DNA sample derived from a homologous or heterologous organism. It is usually necessary to obtain a protein functionally equivalent to the protein having the amino acid sequence described in No. 3 in No. 2; that is, having the same activity as the protein having the amino acid sequence described in SEQ ID NO: 2. What you can do. Thus, a protein encoded by DΝ which hybridizes to a protein having the amino acid sequence of SEQ ID NO: 2 and DΝ which hybridizes with the protein having the amino acid sequence of SEQ ID NO: 2, Proteins exhibiting the same activity and DNAs encoding the same are also included in the present invention. The DNA and the protein are preferably a DNA that hybridizes under “stringent conditions” and a protein encoded thereby, and a DNA that hybridizes with the DNA of SEQ ID NO: 1, And the protein encoded thereby is preferred. Non-human organisms can be used to isolate DNA encoding such proteins. Examples include, but are not limited to, monkeys, buses, dogs, and dogs. The stringent hybridization conditions for isolating the DNA encoding the “hybridization equivalent” of the protein having the amino acid sequence of SEQ ID NO: 2 include the following conditions for the hybridization. 5xSSPE, 5xD enhanf s solution, 0.5% SDS, 40% formamide, 200 g / ml salmon sperm DNA, 37 ° C single burn, and more severe conditions are “5xSSPE, 5 Denhard, Solution, 0.5% SDS, 50% formamide, 200 / g / ml salmon sperm DNA, 42 ° C overnight. In addition, as the conditions for washing, loose conditions are `` 5xSSC, 1% SDS ヽ 2 ° Cj, usually `` 0.5xSSC, 0.1 ° SDS, 42 ° Cj, and more severe conditions are `` 0.2xSSC, 0.1% SDS, 65 ° C ”. Thus, as the hybridization conditions become more stringent, isolation of DNA having higher homology to the probe sequence can be expected. However, the combination of the above-mentioned conditions of SS SDS, formamide and temperature is merely an example, and those skilled in the art will understand that the above or other factors (for example, probe concentration, probe concentration, etc.) that determine the stringency of the hybridization. By appropriately combining the length and the hybridization reaction time, the same stringency as described above can be achieved.

A protein encoded by DNA isolated using such a hybridization technique usually has a high homology in the amino acid sequence with the protein having the amino acid sequence of SEQ ID NO: 2. High homology refers to sequence homology of at least 80% or more, preferably 90% or more, more preferably 95% or more, and more preferably 97% or more. Amino acid sequence homology can be determined using the BLAST search algorithm. Specifically, the bl2seq program (Tatiana A. Tatusova, Thomas L. Madden (1999), FEMS Microbiol Lett. 174: 247-250) of the BLAST package (sgi32bit version, Purge Yong 2.0.12, obtained from NCBI) It can be used and calculated according to the default parameters. Pairwise alignment Parame overnight First, use the program name blastp, Gap input Cost value of 0, Gap extension Cost value of 0, SEG as a filter for the query array, and BL0SUM62 as a matrix.

DNA encoding the MCH receptor protein of the present invention using gene amplification technology (PCR) (Current protocols in Molecular Biology edit it. Ausubel et al. (1987) Publish. John Wiley & Sons Section 6.1-6.4). Nucleotide sequence of (SEQ ID NO:

A primer was designed based on a portion of 1), and a DNA fragment containing a nucleotide sequence highly homologous to the nucleotide sequence of the DNA encoding the protein having the amino acid sequence of SEQ ID NO: 2 was isolated. It is also possible to obtain a protein functionally equivalent to the MCH receptor protein having the amino acid sequence of SEQ ID NO: 2 based on DNA. For example, an oligonucleotide having the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 10 is mentioned as a preferred primer. Example 6 is illustrated as an acquisition method using this primer.

As described above, the sequencing of the DNA obtained by a) to d) is carried out, for example, by the chemical modification method of Maxam / Gilbert (Maxam, AM and Gilbert, W. (1980): "Methods in Enzymology" 65, 499). -559) or the dideoxynucleotide chain termination method using M13 (Messing, J. and "Vieira, J (1982)" Gene, 19, 269-276).

The vector of the present invention, the host cell of the present invention, and the MCH receptor of the present invention can be obtained by the following methods.

2) Method for producing vector, host cell and protein of the present invention

The isolated fragment containing the gene encoding the protein of the present invention can be transformed into another eukaryotic host cell by re-incorporation into an appropriate vector DNA. Furthermore, by introducing appropriate promoters and sequences relating to expression into these vectors, the gene can be expressed in each host cell.

Eukaryotic host cells include cells such as vertebrates, insects, and yeast. Examples of vertebrate cells include COS cells, which are monkey cells (Gluzman, Y. (1981) Cell, 2 3, 175-182) and Chinese 'Hams ovary cell (CHO) deficient in dihydrofolate reductase (Urlaub, G. and Chasin ₅ LA (1980) Proc. Atl. Acad. Sci. USA, 77 HEK 293 cells derived from human fetal kidney and 293-EBNA cells (Invitrogen) with the Epstein Barr Virus EBNA-1 gene introduced into the cells are commonly used, but are not limited to these. Not necessarily.

As a vertebrate cell expression vector, one having a promoter located upstream of the gene to be normally expressed, an RNA splice site, a polyadenylation site, a transcription termination sequence, and the like can be used. May further have a replication origin if necessary. Examples of such expression vectors include pSV2dhfr (Subbandi ni, S. et al. (1981) Mol. Cell. Biol., 1, 854-864), which has an initial promoter of SV40, and human el. pEF-BOS (Mizushima, S. and Nagata ₅ S. (1990) Nucleic Acids Res., 18, 5322) having the ongation factor promoter overnight, pCEP4 (Invitrogen) having the cytomegalovirus promoter overnight, etc. It can be exemplified, but not limited to this.

Taking the case where COS cells are used as host cells as an example, the expression vector has an SV40 origin of replication, is capable of autonomous growth in COS cells, and has a transcriptional promoter and a transcription termination signal. And those having an RNA splice site can be used.For example, pME18S (Maruyama, K. and Takebe ₃ Y. (1990) Med. Immunol., 20, 27-32), pEF-BOS (Mizushima , S. and Nagata ₃ S. (1990) Nucleic Acids Res., 18, 5322), pCDM8 (Seed, B. (1987) Nature, 329, 840-842), and the like. The expression vector is a DEAE-dextran method (Luthman, H. and Magnusson, G. (1983) Nucleic Acids Res., 11, 1295-1308), a calcium phosphate-DNA coprecipitation method (GΓaham, FL and van der Ed). , AJ (1973) Virology, 52, 456-457), method using FuGENE6 (Boeringer Mannheim), and electric pulse perforation method (Neumann, E. et al. (1982) EMBO J Li 1, 841-845) Thus, the cells can be incorporated into C0S cells, and thus desired transformed cells can be obtained.

When CHO cells are used as host cells, G A vector capable of expressing the neo gene that functions as a resistance marker, such as pRSVneo (Sambrook, J. et al. (1989): "Molecular Cloning-A Laboratory Manua 1" Cold Spring Harbor Laboratory, ΝΥ) · ^ pSV2 -neo (Southern, PJ and Berg, P. (1982) J. Mol. Appl. Genet., 1, 327-341), etc., and select G4 18 resistant colonies for MCH. A transformed cell stably producing the receptor can be obtained. When 293 cells are used as host cells, a vector capable of expressing a zeocin resistance gene that functions as a zeocin resistance gene together with an expression vector, for example, pcDNA3.1 / Zeo (+) (Invitrogen ) Can be used to transfect and select zeocin-resistant cells to obtain transformed cells that stably produce the MCH receptor. When using 293-EBNA cells as host cells, expression vectors such as pCEP4 (Invitrogen), which has an Epstein Barr Virus replication origin and is capable of self-propagation in 293-EBNA cells, are used. The desired transformed cells can be obtained using the kit.

The desired transformant obtained above can be cultured according to a conventional method, and the culture produces the protein of the present invention in the cell or on the cell surface. The medium used for the culture can be appropriately selected from various conventional ones according to the host cell used. For example, in the case of the above-mentioned COS cells, RPMI-1640 medium or Dulbecco's modified single cell A medium obtained by adding a serum component such as fetal bovine serum (FBS) to a medium such as small essential medium (DMEM) as necessary can be used. For the above-mentioned 293-EBNA cells, use a medium such as Dulbecco's modified minimum essential medium (DMEM) supplemented with serum components such as fetal bovine serum (FBS) plus G418. it can.

As described above, the protein of the present invention produced in the cells of the transformant or on the cell surface is separated and purified by various known separation procedures utilizing the physical properties, chemical properties, and the like of the protein. be able to. Specifically, for example, after solubilizing a membrane fraction containing a receptor protein, treatment with a usual protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ionization Examples thereof include various types of liquid chromatography such as exchanger chromatography, affinity take chromatography, high performance liquid chromatography (HPLC), dialysis methods, and combinations thereof.

The membrane fraction can be obtained according to a conventional method. For example, it can be obtained by culturing cells expressing the MCH receptor of the present invention on the surface thereof, suspending them in a buffer, homogenizing, and centrifuging. In addition, by solubilizing the MCH receptor with a solubilizing agent that is as mild as possible (CHAPS, Triton X-100, dichitonin, etc.), the characteristics of the receptor can be maintained after solubilization.

The fusion of the protein of the present invention with the primary sequence in frame allows expression of the MCH receptor, confirmation of intracellular localization, purification, and the like. Marker sequences include, for example, FLAG epitope ^ Hexa-Histidine tag, Hemagglutinin tag, myc epitope and the like. In addition, by inserting a specific sequence recognized by proteases such as enterokinase, factor-1Xa, and thrombin between the marker sequence and the MCH receptor, the marker-1 sequence is cleaved and removed by these proteases. Things are possible. For example, there is a report that a muscarinic acetylcholine receptor and a Hexa-Hisidine tag are linked by a thrombin recognition sequence (Hayashi, M.K. and Haga, T. (1996) J. Biochem., 120, 1232-1238).

3) Screening method for substances (compounds, peptides, and antibodies) that modify the activity of the protein of the present invention

The present invention includes a method for screening a substance (compound, peptide, and antibody) that modifies the activity of the MCH receptor. Substances that modify the activity of the protein of the present invention include artificially synthesized substances and naturally occurring substances. In addition, it can be an inorganic substance as well as an organic substance. Peptides include peptides composed of more amino acids, such as enzymes, in addition to peptides composed of relatively few amino acids. The screening method comprises the steps of: using the MCH receptor constructed as described above, a system for measuring an index of modification of the receptor protein in accordance with the physiological characteristics of the receptor protein. A means for adding the test drug and measuring the index. As the MCH receptor, a cell in which the receptor has been expressed, a membrane fraction of the cell, a purified preparation of the receptor protein, or the like can also be used. Specific examples of the measurement system include the following screening methods.Test drugs include commercially available compounds, various known compounds and peptides registered in the chemical file, combinatorial chemistry (Terrett technology). ₅ NK, et al. (1995) Tetrahedron, 51, 8135-8137) Compounds and phage display method (Felici, F., et al. (1991) J. Mol. Biol., 222, 301 -310) can be used. In addition, culture supernatants of microorganisms, natural components derived from plants and marine organisms, and animal tissue extracts are also targets for screening. Alternatively, a compound or peptide obtained by chemically or biologically modifying the compound or peptide selected by the screening method of the present invention may be used, but is not limited thereto.

a) Screening method using ligand binding method

• Substances (compounds, peptides, and antibodies) that bind to the MCH receptor of the present invention can be screened by the ligand binding assay. A cell membrane expressing the receptor protein or a purified preparation of the receptor protein is prepared. Optimizing the assay conditions such as buffer, ion and pH, the cell membrane expressing the receptor protein in the optimized buffer or the purified preparation of the receptor protein is used as a labeled ligand, for example. [Phe ¹³ , [ ¹²⁵ I] Tyr ¹⁹ ] ~ MCH is incubated with the test drug for a certain period of time. After the reaction, filter with a glass filter and wash with an appropriate amount of buffer, and measure the radioactivity remaining in the filter with an alcohol filter and the like. A substance (compound, peptide, and antibody) having agonist activity of the receptor protein or a substance (compound, peptide, and antibody) having agonistic activity of the receptor protein, using the obtained inhibition of binding of a radioactive ligand as an index. Can be screened. For example, a substance having an antagonistic activity of the receptor protein selected by the screening method of the present invention is useful for treating or preventing obesity or an eating disorder. The screening method of the present invention is preferably performed under the conditions described in Example 2 or Example 4.

For example, under the conditions of the assay described in Example 4, a substance having a binding inhibitory activity having a Ki of 10 μm or less and an IC50 of 16 μm or less, more preferably Ki or less and an IC50 of 1.6 zM or less can be selected. .

b) Screening method using GTPa S binding method

Substances (compounds, peptides, and antibodies) that modify the activity of the MCH receptor of the present invention can be screened by the GTPγS binding method (Lazareno, S. and Birdsall, NJM (1993) Br. J. Pharmacol. 109, 1120-1127). Cell membranes expressing the receptor _{20mM HEPES (pH7.4) 3 lOOniM NaCl} , at lOmM MgCl _2, 50mM .GDP solution., ³⁵ S in mixing with labeled GTP § S 400 pM. After incubation in the presence and absence of the test drug, filter through a glass filter or the like, and measure the radioactivity of bound GTPyS in a liquid scintillation counter or the like. Substances (compounds, peptides, and antibodies) having the agonist activity of the MCH receptor can be screened using an increase in specific GTPyS binding in the presence of the test drug as an index. In addition, screening for substances (compounds, peptides, and antibodies) having an agonistic activity of the receptor protein based on the suppression of increase in GTPaS binding by MCH 'in the presence of the test drug is used as an index. Can be.

c) Screening method using fluctuation of intracellular Ca ⁺⁺ and cAMP concentration

The substances (compounds, peptides, and antibodies) that modify the activity of the MCH receptor of the present invention can be screened by utilizing the fluctuation of intracellular Ca ⁺⁺ or cAMP concentration of cells expressing the MCH receptor. It is possible to The intracellular Ca ⁺⁺ concentration can be measured using fura2, uo3, or the like. The cAMP concentration can be measured using a commercially available cAMP measurement kit (Amersham, etc.).

Alternatively, the concentration of Ca ⁺⁺ and cAMP can be measured indirectly by detecting the transcriptional activity of a gene whose transcription level is regulated depending on the concentration of Ca ⁺⁺ or cAMP. is there. Specifically, Ca ⁺⁺ and cAMP concentrations can be measured by the following method. First, a repo overnight gene linked to a serum responsive element or a cAMP responsive element is introduced into a cell in which the receptor protein has been expressed, and a test drug is added to a culture solution of the cell. Any gene capable of producing a detectable signal can be used as the repo overnight gene. For example, the luciferase gene is desirable as the repo overnight gene. After incubation at 37 ° C for 4 hours, remove the medium, lyse the cells, and measure luciferase activity. Then, substances (compounds, peptides, and antibodies) having the agonist activity of the receptor protein can be screened using the induction of lucif Xase activity upon addition of the test drug as an index. After the test drug is added to the culture of the cells, MCH at a final concentration of 0.4 nM is added, and the luciferase activity is measured in the same manner. Then, substances (compounds, peptides, and antibodies) having angiogonist activity of the receptor protein can be screened using the inhibition of luciferase activity induction by MCH upon addition of the test drug as an index.

In the screening method of the present invention, substances (compounds, peptides, and antibodies) and the like are allowed to act on cells expressing the protein and host cells not expressing the protein (control cells) for a certain period of time to obtain Ca ⁺⁺ and cAMP. Measure the concentration directly or indirectly. Compared with control cells, substances (compounds, peptides, and antibodies) having agonist activity are indicated by an increase in cell-specific Ca ⁺⁺ and / or an increase or decrease in cAMP concentration, which expresses the protein. Can be screened. In addition, a substance (compound, peptide, and antibody) having an antagonistic gonist activity of the MCH receptor based on the inhibitory effect of MCH on Ca ⁺⁺ increase and / or cAMP concentration increase or decrease in the presence of the test drug as an index ) Can be screened. The screening method of the present invention is preferably performed under the conditions described in Example 3 or Example 5.

For example, under the conditions described in Example 3, a substance having an EC50 of 100 / M or less, more preferably a substance having an EC50 of 1 or less is selected as a substance having agonist activity. Can be In addition, by adding the test drug to the Athesay conditions described in Example 3, that is, a substance having an IC50 of 3 or less under the conditions of Example 5, preferably

A substance having 0 of 1 or less, and more preferably a substance having an IC50 of 1 / M or less, can be selected as a substance having angiogenesis activity.

4) Method for producing an antibody that reacts with the MCH receptor of the present invention

The antibody that reacts with the MCH receptor of the present invention, for example, a polyclonal antibody or a monoclonal antibody can be obtained by directly administering the MCH receptor 受容 the fragment of the MCH receptor to various animals. In addition, using the plasmid into which the gene encoding the MCH receptor of the present invention has been introduced, the DNA vaccine method (Raz, E. et al. (1994) Proc. Natl. Acad.

Sci. USA, 91, 9519-9523; Donnelly, J. J. et al. (1996) J. Infect. Dis., 173,

314-320).

The polyclonal antibody is prepared by emulsifying the protein or a fragment thereof in a suitable adjuvant such as Freund's complete adjuvant and immunizing the peritoneal cavity, subcutaneously or intravenously, and sensitizing animals such as serum of rabbits, rats, goats, chickens or the like. Manufactured from eggs. The polyclonal antibody can be separated and purified from the serum or eggs thus produced by a conventional protein isolation and purification method. Examples of such methods include centrifugation, dialysis, salting out with ammonium sulfate, and chromatographic methods using DEAE-cell mouth, nose, needle mouth, xiapatite, and protein A agarose.

A preferred example for preparing an antibody is the method described in Example 8.

Monoclonal antibodies can be easily produced by those skilled in the art by the cell fusion method of Kerayl and Milstein (Kohler, G. and Milstein, C. (1975) Nature, 256, 495-497).

Mice are immunized by inoculating the intraperitoneal, subcutaneous, or vein several times with an emulsion prepared by emulsifying the protein of the present invention or a fragment thereof in an appropriate adjuvant such as Freund's complete adjuvant every few weeks. After the final immunization, remove the spleen cells and A hybridoma is produced by fusing with an erotic cell.

Myeloma cells that have markers such as hypoxanthine, guanine, phosphoribosyltransferase deficiency, and thymidine kinase deficiency, such as Mau's Smieloma cells, Use strain P3X63Ag8.Ul. In addition, polyethylene glycol is used as a fusion agent. Furthermore, 10 to 30% fetal bovine serum is added as appropriate to commonly used mediums such as Eagle's minimum essential medium, Dullco's modified minimum essential medium, and RPMI-1640 as the medium for hybridoma production. Used. Fusion strains are selected by the HAT selection method. Hybridoma screening is performed using the culture supernatant by a well-known method such as ELISA, immunohistochemical staining, or the above-mentioned screening method, and a clone of the hybridoma secreting the desired antibody is selected. Repeated subcloning by limiting dilution guarantees the monoclonality of the hybridoma. The thus obtained hybridomas can be purified in an amount that can be purified by culturing them in the medium for 2-4 days or in the abdominal cavity of BALB / c mice pre-treated with prismin for 10-20 days. Produced.

The monoclonal antibody thus produced can be separated and purified from the culture supernatant or ascites by a conventional protein isolation and purification method. Examples of such methods include centrifugation, dialysis, salting out with ammonium sulfate, and chromatographic methods using DEAE-cellulose, hydroxyapatite, protein Aagaguchi, and the like. Alternatively, an antibody fragment containing a monoclonal antibody or a part thereof can be produced by incorporating all or a part of a gene encoding the antibody into an expression vector and introducing the gene into E. coli, an enzyme, or an animal cell.

The antibody separated and purified as described above is digested with a protease such as pepsin, papain, etc. by a conventional method, and then separated and purified by a conventional protein isolation and purification method. An antibody fragment containing, for example, F (ab ') 2, Fa Fab', and Fv can be obtained. Furthermore, an antibody that reacts with the MCH receptor of the present invention can be obtained by the method of Clackson et al., Zebedee et al. (Clackson, T. et al. (1991) Nature, 352, 624-628; (1992) Proc. Natl. Acad. Sci. USA, 89, 3175-3179) to obtain a single chain Fv or Fab. It is also possible to obtain a human antibody by immunizing a transgenic mouse (Lonberg. Et al. (1994) Nature, 368, 856-859) in which the mouse antibody gene is replaced with a human antibody gene.

5) Pharmaceutical of the present invention

The present invention includes a medicament comprising, as an active ingredient, an MCH receptor or a substance (compound, peptide, or antibody) selected by the above-mentioned screening method, which significantly modifies the activity of the protein.

A preparation containing the MCH receptor activity-modifying substance (compound, peptide, and antibody) of the present invention as an active ingredient, depending on the type of the active ingredient, a pharmacologically acceptable carrier usually used for the preparation of the active ingredient. It can be prepared as a pharmaceutical composition by using a excipient and other additives. The pharmaceutical composition of the present invention is preferably a pharmaceutical composition for preventing and / or treating obesity and eating disorders, which contains a substance having an agonist activity of the receptor as an active ingredient. Alternatively, the present invention is a method for preventing and / or treating obesity and eating disorders, which comprises a step of administering the substance having the antagonist activity.

For oral administration, such as tablets, pills, capsules, granules, fine granules, powders, oral solutions, injections such as intravenous and intramuscular injections, suppositories, transdermal preparations, and transmucosal Parenteral administration by administration agents and the like can be mentioned. Parenteral administration, such as intravenous injection, is particularly desirable for peptides digested in the stomach.

The solid compositions for oral administration according to the present invention may contain one or more active substances of at least one inert diluent, such as lactose, mannitol, glucose, microcrystalline cellulose, hydroxypropylcellulose, starch, polyvinylpyrrolid. Don, mixed with magnesium metasilicate and aluminate. The composition may be prepared in a conventional manner using additives other than inert diluents, such as lubricants, disintegrants, stabilizers, It may contain a solubilizer or the like. Tablets and pills may be coated with a sugar coating or a film of a gastric or enteric substance, if necessary.

Liquid compositions for oral use include emulsions, solutions, suspensions, syrups, and elixirs, and include commonly used inert diluents, such as purified water, ethanol. The compositions may contain additives other than inert diluents, such as wetting agents, suspending agents, sweetening agents, flavoring agents, preservatives.

Parenteral injections include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Aqueous solutions and suspensions include, for example, distilled water for injection and physiological saline as diluents. Diluents for non-aqueous solutions and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, polysorbate 80 and the like. The composition may further contain a wetting agent, an emulsifying agent, a dispersing agent, a stabilizer, a solubilizing or solubilizing agent, a preservative and the like. The composition may be sterilized, for example, by filtration through a nocturnal retention filter, combination with a bactericide, or irradiation. In addition, a sterile solid composition can be produced and dissolved in sterile water or another sterile injectable medium before use.

The dose is appropriately determined in consideration of the activity intensity, symptoms, age, sex, and the like of the active ingredient selected by the screening method. For example, in the case of oral administration, the dose is generally about 0.1 to 100 mg, preferably 0.;! To 50 ing per day for an adult (assuming a body weight of 60 kg). In the case of parenteral administration, the dosage is 0.01 to 50 mg / day, preferably 0.01 to 10 m / day in the form of injection.

The invention also encompasses the use of DNA encoding the MCH receptor as a diagnostic. Detection of variants of the MCH receptor gene associated with dysfunction can be used to diagnose diseases or susceptibility caused by under-, over- or altered expression of MCH receptors. That is, the present invention relates to a DNA that specifically hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 and has a chain length of at least 15 nucleotides. In the present invention, a DN consisting of the nucleotide sequence of SEQ ID NO: 1 A includes its complementary strand. Therefore, the DNA of the present invention includes a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 or a DNA having a chain length of at least 15 nucleotides, which is complementary to the complementary strand thereof. The term “specifically hybridize” with the DNA of the present invention means that the DNA hybridizes with the DNA of the present invention under ordinary hybridization conditions, preferably under strict conditions, and with other DNA. Does not hybridize. Such a DNA can be used as a probe for detecting and isolating the DNA of the present invention and as a primer for amplifying the DNA of the present invention. When used as a primer, it usually has a chain length of 15 nucleotides to 100 nucleotides, preferably 15 nucleotides to 40 nucleotides. Examples of primers for PCR for amplifying DNA consisting of the nucleotide sequence of SEQ ID NO: 1 include, for example, SEQ ID NO: 3 (forward primer) and SEQ ID NO: 0.4 (river primer), SEQ ID NO: 5 (forward primer) and SEQ ID NO: 6 (reverse primer), SEQ ID NO: 11 (forward primer) and SEQ ID NO: 12 (reverse primer) can be used. When used as a probe, a DNA having at least a part or all of the DNA of the present invention or its complementary sequence) and a chain length of at least 15 'nucleotides is used. Preferably, a DNA having the 1st to 1023rd of the base sequence described in SEQ ID NO: 1 is used.

Diagnostic DNA can be obtained from a subject's cells, such as blood, urine, saliva, tissue biopsy or autopsy material. Deletion and insertion mutations can be detected by a change in the size of the amplified product when compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled MCH receptor nucleotides. Perfectly matched sequences can be distinguished from mismatched strands by RNase digestion or by differences in melting temperatures. In addition, a difference in the nucleotide sequence of DNA can be detected based on a change in the electrophoretic mobility of a DNA fragment in gel electrophoresis due to the presence or absence of a denaturing agent. Alternatively, you can detect nucleotide sequence differences by direct DNA sequencing. (Myers, RM et al. (1985) Science. 230, 1242-1246). Sequence changes at specific positions can also be confirmed by nuclease protection assays (eg, RNase and S1 protection) or by chemical cleavage (Cotton et al. (1 985) Proc. Natl. Acad. Sci. USA, 85, 4397-4401).

In addition, an array of oligonucleotide probes containing the nucleotide sequence of the MCH receptor or a fragment thereof can be constructed. This array technique is known and has been used to analyze gene expression, genetic linkage and genetic variability (Chee, M. et al. (1996) Science, 274, 610-613). In addition, methods for measuring abnormally decreased or increased levels of MCH receptors from samples obtained from subjects can be used to determine whether a disease or susceptibility to disease resulting from under-, over- or altered expression of MCH receptors. Used for diagnosis. Decrease or increase in expression can be determined by any of the polynucleotide quantification methods known to those skilled in the art, for example, PCH,! ^: ー:? ^^! ^ It can be measured at the RNA level by case protection, northern plot, and other hybridization methods.

In addition, a decrease or increase in the level of a protein such as MCH receptor in a sample obtained from a subject can be measured by an assay method known to those skilled in the art. Examples of such methods include radioimmunoassay, competitive binding assay, western plotting, and ELISA assay.

In addition, “a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or a DNA complementary to a complementary strand thereof and having a chain length of at least 15 nucleotides” includes expression of the protein of the present invention. Antisense DNA for suppression is included. The antisense DNA has a chain length of at least 15 nucleotides or more, preferably 100 nucleotides, more preferably 500 nucleotides or more, and usually has a length of not more than 3000 nucleotides, preferably not more than 2000 nucleotides in order to cause an antisense effect. Have a length. Such antisense DNA is also considered to be applicable to gene therapy for diseases caused by abnormality (abnormal function or expression) of the protein of the present invention. You. The antisense DNA is, for example, based on the sequence information of the DNA encoding the protein of the present invention (for example, the DNA described in SEQ ID NO: 1) based on the phosphorothioate method (Stein, 1988 Physicochemical properties of phosphorothioate). It can be prepared using oligodeox ynucleotides. Nucleic Acids Res 16, 3209-21 (1988)). By knocking out the MCH receptor gene using the antisense DNA according to the present invention, the elucidation of a disease involving the MCH receptor can be promoted.

When the DNA or antisense DNA of the present invention is used for gene therapy, for example, a virus vector such as a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, or a non-viral vector such as a ribosome is used. It is administered to patients by the e iw method or the i / 3 method.

All prior art documents cited in the present specification are incorporated herein by reference. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a photograph showing the result of analyzing the distribution of GPRv17 protein in the monkey lateral hypothalamus by immunohistological staining.

1 Monkey hypothalamus including lateral hypothalamus

2 Enlarged photo of lateral hypothalamus

3 Neurons expressing GPRvl 7 protein in the lateral hypothalamus

4 GPRvl 7 protein-expressing neurons in the lateral hypothalamus

Abbreviation

Fx Brainbow

LH lateral hypothalamus Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to the examples. Unless otherwise specified, it can be carried out according to a known method (Maniatis, T. et a 1. (1982). "Molecular Cloning-A Laboratory Manual" Cold Spring Harbor Laboratory, NY).

Example 1 Isolation of Gene Encoding New G Protein-Coupled Receptor GPRv17 The full-length cDNA encoding the novel G protein-coupled receptor GPRv17 was obtained from PCI. The amplification of the gene encoding GPRvl7, Marathon Ready cDNA from human fetal brain (Clontech) to錡型c DNA, the forward primer one as ^5, - ATGAATCCATTTCATGCATCTTGTTGGA-3 5 (SEQ ID NO: 3), and a reverse primer one 5,-CTAAAAGTGTGATTTCAGAGTGTTTCCG-3 (SEQ ID NO: 4). PCR was performed using Pyrobest DNA polymerase (Takara Shuzo) at 94 ° C (2.5 minutes). 5 cycles of C (5 seconds) / 72 ° C (4 minutes), 5 cycles of 94 ° C (5 seconds) / 70 ° C (4 minutes), 94 ° C (5 seconds) / 68 ° The cycle of C (4 minutes) was repeated 25 times. As a result, a DNA fragment of about 1 kbp was amplified. This fragment was cloned using pCR2.1 plasmid (Invitrogen). The nucleotide sequence of the obtained clone was analyzed using the ABI377 DNA Sequencer (Applied Biosystems II :) according to the dideoxy Yuichi Mine-Yuichi method. The elucidated sequence is shown in SEQ ID NO: 1.

The sequence has an open reading frame of 1023 bases (SEQ ID NO: 1 from 1st to 1023rd). The amino acid sequence (340 amino acids) predicted from the open reading frame is shown in SEQ ID NO: 2. Since the predicted amino acid sequence has seven transmembrane domains that are likely to be characteristic of G protein-coupled receptors, this gene should encode G protein-coupled receptors. There was found.

Further, a BLAST search for SWISS-PR0T using the amino acid sequence of “GPRvl7” was performed. As a result, GPRvl7 showed the highest phase at 38% of HUMAN PROBABLE G PROTEIN-COUPLED RECEPTOR SLC-1 (Q99705, 402aa) among known G protein-coupled receptors. Showed the same sex. This proved that “GPRvl7” is a novel G protein-coupled receptor. SLC-1 has been clarified to be a protein using MCH as a ligand similarly to the protein of the present invention (SaitoJ. Et al. (1999) Nature 400, 265-269). However, the homology in both amino acid sequences was only 38, indicating that the protein of the present invention was novel.

(Example 2) The expression of GPRvl7 protein by 293 cells and the following experiment with [Phe ⁱ³ , [I] Tyr] -MCH confirmed the MCH receptor activity of the protein encoded by GPRvl7. First, in order to express the GPRvl7 protein encoded by the cDNA, the cDNA was obtained by HT-PCR using human brain-derived poly (A) + RNA (Clontech) as a type III, and expressed in an expression vector. Incorporated.

In fact, the amplification of the full-length c DNA encoding GPRvl7 protein, the forward primer one as ^5, -GGTCTAGAATGAATCCATTTCATGCATCTTGTT-3 5 (SEQ ID NO: 5), as a reverse Bly Ma one ^5, -GGTCTAGACTAAAAGTGTGATTTCAGAGTGTTT-3 5 (SEQ ID No .: 6) was used (an Xbal site was added to each 5 'end). RT--PCR was performed using Ex Taq DNA polymerase (Takara Shuzo) in the presence of 5% DMS0, and repeated 34 cycles of 94 ° C (30 seconds) Z55 ° C (30 seconds) / 72 ° C (2 minutes) 34 times . As a result, a DNA fragment of about 1.0 kbp was amplified. After digesting this fragment with Xbal, it was cloned using pEF-BOS plasmid (Mizushima, S. and Nagata, S. (1990) Nucleic Acids Res., 18, 5322). The nucleotide sequence of the obtained clone was analyzed using the ABI377 DNA Sequencer (Applied Biosystems) according to the dideoxy-mine method. The obtained plasmid was designated as pEF-BOS-GPRv17.

293 cells were seeded with lxlO ⁶ cells on a 10 cm culture dish (Asahi Techno Glass Co.) coated with Type I Collagen, cultured for 24 hours, and then 9 g of pEF-B0S-GPRvl7 and lg pcDNA3.1 / Zeo (+ ) (Invitrogen) using FuGENE6 (Boeringer Mannheim) The gene was introduced. 30 hours after transfection, the cells were newly seeded on a 10-cm culture dish, selected with 40 // g / ml zeocin (Invitrogen), and the cells that survived and formed colonies were collected, and the GPRvl7 protein of the present invention was recovered. 293 cells with stable expression were obtained.

The cells were collected, washed, suspended in 0.32M sucrose, and homogenized with a Dounce homogenizer. The mixture was centrifuged at 300 xg for 10 minutes to denucleate, and the supernatant was centrifuged again at 12, OOOxg for 15 minutes to obtain a precipitate fraction. This was suspended in 50 mM Tris.HCl (pH 7.4) containing 0.0075% Tron-X100, stirred slowly at 4 ° C for 30 minutes, and then centrifuged at 12,000 xg for 15 minutes to obtain a suspension. The precipitate was washed once with 5 mM Tris.HCl (pH 7.4) and twice with 50 mM Tris.HCl (pH 7.4), 10 mM MgCl ₂ , 2 mM EGTA, aprotinin, and pepstatin A, and this was used as a membrane fraction.

The membrane fraction 20〃G ^{^{[Phe 13, [125 I]}} Tyr 19] -MCH (NEN Life Science Products, Inc) was added to a final concentration 0.07~5.7xl0- ⁹ M, 50mM Tris.HCl (pH7 .4), lOmM gCl ₂₅ 2mM E6TA, lmg / ml bacitracin, lmg / ml ovalbumin, 10KIU / ml aprotinin, l / g / ml pepstatin A It was collected at Vesu Yuichi at Grass Fill Yuichi. The radioactivity recovered in the glass filter was measured at the gamma level and the total amount bound to the membrane fraction was determined.

Furthermore, the amount of non-specific binding to the membrane fraction was determined by adding MCH (Bachem) at a final concentration of 1〃Μ or 2〃Μ to the above test. Specific binding was detected based on the total and non-specific binding of [Phe ¹³ , [ ¹²⁵ I] Tyr ¹⁹ ] -MCH to the membrane fraction of 293 cells into which pEF-B0S-GPRvl7 was stably introduced. In each case, specific binding was observed at concentrations of 0.07 to 5.7xl ([Phe ¹³ , [ ¹²⁵ I] Tyr ¹⁹ ] -MCH in T ⁹ M).

As a result of Scatchard analysis of this binding when MCH (Bachem) at a final concentration of 2〃M was added, [Phe ¹³ , [Pe ¹³ , [ The dissociation constant for ^lz5 I] Tyr ¹⁹ ] -MCH binding was Kd = 1.8 nM, and the maximum binding was Bmax = 890 fmol / mg protein. As described above, it was confirmed that the GPRv17 protein of the present invention has a strong affinity for MCH.

(Example 3) GPRvl7 protein stable expression Changes in intracellular Ca ⁺⁺ concentration by MCH in 293 cells

GPRvl7 protein stable expression 293 cells are seeded on 96-well Black / clear bottom plate, collagen I coated (manufactured by BECTON DICKINSON) at 2xl0 ⁴ cells / well, cultured for 24 hours, and the medium is discarded. , AM (manufactured by Molecular Probe), DMEM containing 0.004 acid pluronic acid and 10% FBS were added at 100〃 per 1 ゥ, and incubated at 37 ° C for 1 hour. After the incubation, the cells were washed four times with Hanks BSS (manufactured by GIBCO) containing 20 mM HEPES, and Hanks BSS containing 100〃1 of 20 mM HEPES per well was added.

Changes in intracellular Ca ⁺⁺ concentration were measured over time using FLIPR (Moleucular Device). That, MCH and (Bachem, Inc.) was added Caro so the final concentration 2Xl0- ⁶ M in lxlO- ¹² M after the measurement after 10 seconds, after the addition, the every second 5 0 seconds, the additional 4 minutes 6 seconds The fluorescence intensity was measured every time. GPRvl7 protein stably expressing 293 cells, 2 XLO one ^6-change in the capacitance dependent intracellular Ca ⁺⁺ concentration MCH concentration of 1 xl (r ^1Q M was observed. On the other hand, the empty base Kuta one introduced gene No change in intracellular Ca ⁺⁺ concentration due to MCH was observed in the cells, and the maximum fluorescence intensity of the intracellular Ca ⁺⁺ concentration change data was plotted on the vertical axis, and the MCH concentration was plotted on the horizontal axis. Analysis of the dose dependence by the regression method revealed that EC50 was 130 nM.

As described above, it was confirmed that cells transformed with this GPRvl7 protein induced a change in intracellular Ca ⁺⁺ concentration in response to MCH in a dose-dependent manner, confirming that GPRvl7 protein is an MCH receptor. Was. By measuring changes in intracellular Ca ⁺⁺ concentration, screening of agonists and gonists was possible. (Example 4) GPRvl7 using protein stably expressing 293 cells (¾¾Vl7 and protein ^{^{^{[Phe 13, [1 25 I}}} ] Tyr 19] Screening of a substance that inhibits the binding of -MCH

Using the membrane fraction of 293 cells stably expressing GPRvl7 protein prepared in Example 2, [Phe ⁱ³ ,

To ^[lz5 I] Tyr ^19] indicator activity to inhibit the binding of -MCH tested study drug. As the test drug, a commercially available compound was used. Basically, the method of Example 2 was used with some changes. The test drug was added to the solution 1001 described in Example 2 containing 15 μg of the membrane fraction, and lxlO- ⁹ M [Phe ¹³ , [ ^i25I ] Tyr ¹⁹ ] -MCH was added to perform the binding reaction. went. The recovered radioactivity was measured by adding a glass scintillator to a micro scintillator and using a top count (Packard). At the same time, the radioactivity was measured as the total binding amount and the non-specific binding amount in the above-mentioned test in which the test drug was not added and in which the unlabeled ligand was added, respectively. Under these conditions, compounds A (FAB-MS (M + H) ⁺ 307), B (FAB-MS (M + H) + 383), C (FAB -MS (M + H) + 365) was obtained. Compound A, B, IC50 of C are 0.22, 3.0, 3.3 / a M, both the GPRvl7 protein ^{^{[Phe 13, [1 I]}} Tyr 19] strong binding inhibition that inhibits in a dose-dependent manner the binding of -MCH Showed activity.

(Example 5) Screening of a substance that inhibits an increase in intracellular Ca ⁺⁺ concentration by MCH using 293 cells stably expressing GPRvl7 protein

The test drug was added to the conditions of Example 3, and after 5 minutes, 75 nM MCH was added, and the change in intracellular Ca ⁺⁺ concentration was measured under the same conditions as in Example 3. For example, the IC50s of the compounds A, B, and C selected in Example 4 are 1.7 and 13, respectively, and the increase of the intracellular Ca ⁺⁺ concentration due to MCH in 293 cells stably expressing GPRvl7 protein was dose-dependent. It was found to be a strong antagonist of the GPRvl7 protein, which suppresses it.

(Example 6) Cloning of monkey GPRvl7 gene

Full length cDNA encoding monkey GPRvl7 was obtained from PCI. Monkey GPRvl7 To amplify the gene encoding, the cDNA derived from the monkey brain frontal lobe is converted into type II cDNA, the forward primer is 5, -ATGAATCCATTTCACTCATCTTGTTGGA-3 '(SEQ ID NO: 9), and the reverse primer is 5, -CTAAAAGTGTGATTTCAGAGTGTTTCCC -3 ⁵ (SEQ ID NO: 1 0) was used. PCR was performed under the conditions of Example 1.

The elucidated sequence is shown in SEQ ID NO: 7. The sequence has an open reading frame of 1023 bases (1st to 1023rd of SEQ ID NO: 7). The amino acid sequence (340 amino acids) predicted from the open reading frame is shown in SEQ ID NO: 8. This amino acid sequence had a high homology of 97% with the amino acid sequence of human GPRvl7 protein, and it was clarified that it encodes the monkey GPRvl7 protein.

Monkey GPRvl7 protein was transiently expressed in 293 cells, and the change of intracellular Ca ⁺⁺ concentration by MCH was examined. As in Example 2, cDNA encoding monkey GPRv17 was inserted into pEF-BOS plasmid to obtain pEF-BOS-monkey GPRv17. Incubate 293 cells per 96 μl / clear bottom plate, collagen I coated with lxlO ⁴ cells per 1 μl and culture overnight, then add 20 ng per 1 μl of pEF-BOS-GPRvl7 or pEF-BOS-monkeyGPRvl7. The gene was introduced using FuGENE6 (Boeringer Mannheim). The medium was discarded 24 hours after gene introduction, and the change in intracellular Ca ⁺⁺ concentration was measured under the conditions of Example 3. Similar to human GPRvl7-transient 293 cells, MCH capacity-dependent changes in intracellular Ca ⁺⁺ concentration were observed in 293 cells of monkey GPRvl7 transiently expressing. The EC50 at that time was 171 nM. Thus, it was revealed that the monkey GPRvl7 gene is also a functional MCH receptor.

(Example 7) Gene expression distribution of human GPRvl7 in tissues

The distribution of human GPRv17 gene expression was analyzed by Northern blot hybridization. As a probe for the human GPRvl7 gene, a cDNA fragment (first to 1023th of SEQ ID NO: 1) was used. PolyA A (2 / g) from human organs Hybridization of the prepared membrane (Clontech) and the probe was performed at 42 ° C (18 hours) in a solution containing 5X SSPE, 5x denhaldt, s, 0.5% SDS, 50% formamide, and 200 µg salmon sperm DNA. ). Membrane may be used once in a solution containing 2X SSPE, 0.1% SDS, once in a solution containing lx SSPE, 0.1% SDS, and finally twice in a solution containing 0.5x SSPE, 0.1% SDS. Also washed at 65 ° C for 15 minutes.

Analysis of human organs (brain, heart, placenta, lung, liver, skeletal muscle, kidney, knee, spleen, thymus, prostate, testis, ovary, small intestine, large intestine, large intestine, peripheral blood leukocytes); 4.0 kb in brain MRNA was strongly detected. Expression in other peripheral tissues was below the detection sensitivity.

Furthermore, the expression distribution in the brain was analyzed in detail by PCR. In the PCR, a cDNA derived from each part of the human brain (amygdala, caudate nucleus, hippocampus, substantia nigra, cerebellum, frontal lobe, hypothalamus, pituitary gland, and whole brain) is shown as SEQ ID NO. Oligonucleotide 5, -TGC AATCCCAGTGTACCAAAACAGAGAG-3 'was used as a forward primer and the oligonucleotide shown by SEQ ID NO: 12 was used as a 5'-CAGTGAGGCCACAGTGTGGAGGGCAAGG-3' reverse primer. Ex Taq (Takara Shuzo) was used for PCR, and the cycle of 94 ° C (30 seconds) Ζ60 ° σ (30 seconds) (1 minute) was repeated 40 times.

As a result, it was found that expression was observed in the hypothalamus, which controls the eating function, as one of the functions. Expression was also observed in the cerebral cortex (frontal lobe), hippocampus, amygdala, caudate nucleus, and substantia nigra, but not in the cerebellum or pituitary.

(Example 8) Preparation of anti-human GPRvl7 antiserum

The peptide CEKEINNMGNTLKSHF corresponding to the C-terminus of GPRvl7 was synthesized using a multi-item simultaneous solid phase method automated peptide synthesizer PSSM-8 (Shimadzu Corporation). The synthesized peptide was purified using SepPakC18 (Waters). Mosinine (KLH) was bound to 2 mg of purified peptide and 2 mg of keyhole phosphate by dry weight using an Inject Maleimide Activated mcKLH kit (Pierce) to obtain a KLH conjugate. note Emulsion was prepared by mixing 0.2 mg of KLH conjugate (0.25 ml) and 0.25 ml of TiterMax Gold (Funakoshi) in a launch tube by sonication. The above-mentioned emulsion was administered subcutaneously to the back of two Japanese White Egrets several times to immunize. This immunization operation was performed eight times every two weeks. After eight immunizations, blood was collected from the ear vein to prepare antiserum.

Using COS cells transiently expressing human and monkey GPRvl7 proteins, antibody reactivity was confirmed by the indirect fluorescent antibody method. After inoculating COS cells with lxlO ⁶ cells in a 10 cm culture dish (Asahi Techno Glass) and culturing for 24 hours, ZOug pEF-B0S-GPRvl7 or 20 μg pEF-BOS-monkey GPRvl7 Was used for gene transfer. Again seeded type I collagen after gene transfer 18 hours co one you encountered a 8 Uweru culture slides (Becton- Dickinson Co.) with 2Xl0 ⁴ cells / Ueru were incubated further 22 hours.

Fix with lOOmM phosphate buffer (pH 7.4) containing 4% paraformaldehyde for 30 minutes at room temperature, wash with phosphate buffer containing 0.1M glycine, and then wash with phosphate buffer containing 0.2% TritonX-100 at room temperature. Incubated for a minute. After blocking with a phosphate buffer containing 2% goat serum for 30 minutes at room temperature, it was reacted with the anti-human GPRv17 antiserum diluted 100-1000-fold with the same solution for 1 hour at room temperature. After washing with a phosphate buffer, the mixture was reacted with a FITC-labeled goat anti-pseudoimyoglobulin antibody (Biosource) for 1 hour at room temperature. After washing with phosphate buffer and distilled water, the cells were sealed and observed with a confocal microscope.

As a result, the antiserum diluted 100- to 1000-fold gave a signal in COS cells transiently transformed with human GfPIlvl7 and monkey GPRvl7. On the other hand, similarly, no signal was given in COS cells transiently transformed with human SLC-1. Accordingly, antisera that specifically detect human and monkey GPRv17 proteins were obtained.

(Example 9) Distribution analysis of GPRvl7 protein in monkey hypothalamus by anti-human GPRvl7 antiserum Using the anti-human GPRv17 antiserum prepared in Example 8, the distribution of GPRv17 protein in the monkey hypothalamus was analyzed.

Male cynomolgus monkeys (6.3 years old, weighing 9.65 kg, purchased from Hamley Co., Ltd.) were killed by exsanguinating the carotid artery under deep anesthesia with pentobarbi sodium sodium. Immediately thereafter, the brain was removed, sliced into 5 thighs, and infiltrated and fixed at 4 ° C for 2 days using lOOmM phosphate buffer (PH7.4) containing 4 paraformaldehyde. After soaking in lOOmM phosphate buffer (PH7.4) containing 16% sucrose for 2 days, the brain tissue including the hypothalamus was cut out, rapidly frozen with dry ice, and thickened with a microtome. Frozen sections of 20 / m2 were prepared.

A series of monkey hypothalamic sections prepared in 500〃M every treated for 30 minutes at room temperature 0.3% Triton X- 100, lOOmM phosphate buffer (pH7.4) containing _2% ¾0 2, flop locking in 10% skim milk Then, it was reacted with the anti-human GPRvl7 antiserum described in Example 8 diluted 3000-fold with the same solution containing 1% skim milk at 4 ° C for 3 days. The antigen-antibody reaction was detected by ABC method (Vector Laboratories kit). Identification of monkey hypothalamus nuclei was performed by Nissl staining of serial sections and referring to The Rhesus Monkey Brain in Stereotaxic Goordinates (Paxinos ₃ G. et al (2000) Academic Press, NY).

The analysis results are shown in FIG. As a result, in the hypothalamus, the signal was almost localized to some neurons in the lateral hypothalamus, and staining of the nerve cell body surface and dendrites was observed. Glial cells were not stained. As a result of the test using the preimmune serum under the same conditions, no staining of the lateral hypothalamus nerve cells was observed. Therefore, it was found that GPRV17 exists mainly in some neurons in the lateral hypothalamus in the monkey hypothalamus. The lateral hypothalamus has long been known as a feeding center, and GPRV17 protein was found to mediate the feeding control function of MCH in the lateral hypothalamus. Industry = t ^ Possibility of use The present invention provides a novel MCH receptor. The receptor of the present invention is useful for the prevention of diseases associated with MCH, obesity and eating disorders such as keratia, anorexia nervosa, bulimia and the search and evaluation of drugs that modify the activity of the receptor as Z or therapeutic agents. It is useful and can provide a therapeutic agent for a disease associated with the receptor. Further, the DNA encoding the MCH receptor of the present invention is useful not only for producing the MCH receptor, but also for diagnosing a disease caused by mutation or abnormal fluctuation of the expression of the MCH receptor. The polyclonal or monoclonal antibody of the receptor is useful as a drug acting on the receptor, a diagnostic agent or a means for separating and purifying a protein.

Claims

Claims In the amino acid sequence of SEQ ID NO: 2, or at one or more sites in the amino acid sequence of SEQ ID NO: 2, one or more amino acid residues are substituted, deleted, and Z or inserted. A protein having the specified amino acid sequence and exhibiting melanin-concentrating hormone receptor activity.

2. The protein according to claim 1, which has the amino acid sequence of SEQ ID NO: 2.

A protein encoded by DNA that hybridizes with DNA represented by the nucleotide sequence of SEQ ID NO: 1 under stringent conditions, and that exhibits melanin concentrating hormone receptor activity.

4. The protein according to claim 3, which has the amino acid sequence of SEQ ID NO: 2.

4. The protein according to claim 3, which has the amino acid sequence of SEQ ID NO: 8.

A gene encoding the protein according to claim 1 or 3.

7. The gene according to claim 6, wherein the protein has the amino acid sequence of SEQ ID NO: 2. 7. The gene according to claim 6, wherein the protein has the amino acid sequence of SEQ ID NO: 8. A vector comprising the gene according to claim 6. '

A host cell comprising the vector according to claim 9.

The method for producing a protein according to claim 1 or claim 3, wherein the host cell according to claim 10 is cultured.

An antibody that binds to the protein according to claim 1 or 3.

A method for screening a substance having an agonistic activity of a melanin concentrating hormone receptor, comprising the following steps:

(1) a step of contacting the test drug with the protein according to claim 1 or 3 in the presence of a melanin concentrating hormone,

(2) measuring the binding activity of melanin concentrating hormone to the protein, and (3) selecting a test drug that reduces the binding activity measured in step (2) as compared with the binding activity of melanin concentrating hormone to the protein measured in the absence of the test drug

14. A method for screening for a substance having an agonist activity of melanin concentrating hormone receptor, comprising the following steps:

(1) contacting a cell expressing the protein according to claim 1 or 3 with a test drug in the presence of a melanin concentrating hormone,

(2) measuring the change in cells due to the binding of melanin concentrating hormone to the protein, and

15. The screening method according to claim 14, wherein the cell change is any change selected from the group consisting of a change in GTP binding activity, a change in intracellular Ca ion concentration, and a change in intracellular cAMP concentration.

16. The screening method according to claim 13 or claim 14, wherein the substance having angiogonist activity of melanin concentrating hormone receptor is a substance for preventing and / or treating obesity, Z or eating disorders.

17. An angonist of the protein of claim 1 or 3 selected by the method of claim 13 or claim 14.

18. A pharmaceutical composition for the prevention and / or treatment of obesity and / or eating disorders, comprising the angonist of claim 17 as a main component.

19. A reagent for detecting a melanin concentrating hormone receptor gene, comprising a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or a DNA complementary to a complementary strand thereof and having a chain length of at least 15 nucleotides.

20. Contacting a sample with a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or a DNA complementary to its complementary strand and having a length of at least 15 nucleotides A method for detecting a melanin concentrating hormone receptor gene, comprising the step of hybridizing said DNA to DNA in a sample.