JPWO2001048188A1 - Novel guanosine triphosphate-binding protein-coupled receptors and their genes, as well as their production and use - Google Patents

Abstract

(57) [Abstract] Through screening of human tissue cDNA, we have succeeded in isolating nine novel genes that retain the seven transmembrane domains and hydrophobic regions thought to be characteristic of G protein-coupled receptors. These genes and their translational products, the proteins, can be used for screening of ligands, screening of agonists and antagonists useful as pharmaceuticals, and for diagnosing diseases associated with these genes.

Description

Detailed Description of the InventionTechnical Field The present invention relates to novel G protein-coupled receptors and their genes, as well as
This invention relates to the manufacture and use ofBackground technologyG protein-coupled receptors
ors) transmits signals into cells via the activation of trimeric GTP-binding proteins.
G protein-coupled receptors are a group of receptors that bind to the cell membrane.
Due to its structural characteristics of having seven transmembrane domains, it is also called a "seven-transmembrane receptor."
G protein-coupled receptors transmit information about various physiologically active substances to the trimeric GTP-binding protein
White matter activation and its associated changes in intracellular second messengers
It is transmitted from the cell membrane into the cell via . It is controlled by trimeric GTP-binding proteins
The intracellular second messenger that is activated is cA via adenylate cyclase.
MP, Ca via phospholipase C²⁺are well known, but
Many of these include the regulation of channels via GTP-binding proteins and the activation of phosphorylation enzymes.
It has recently become clear that the cellular proteins that are its targets (Annu.
Rev. Neurosci. (97) 20:399). G protein-coupled receptors
The substrates (ligands) for these proteins are extremely diverse, including protein hormones, chemokines, and
kinases, peptides, amines, lipid-derived substances, and even proteases such as thrombin
The number of G protein-coupled receptors whose genes have been identified is currently
Excluding sensory receptors, there are just under 300 G receptors in humans, but only a few G receptors have been identified with ligands.
The number of protein-coupled receptors is only about 140, and there are only about 140 types of protein-coupled receptors with unknown ligands.
There are more than 100 types of "orphan G protein-coupled receptors."
In the actual human genome, there are at least 400 types, and possibly as many as 1,000 types.
It has also been postulated that similar G protein-coupled receptors exist (Trends
Pharmacol. Sci. (97) 18:430). This will be a key factor in the future development of genome editing.
With the rapid progress of molecular biology, the number of orphan G protein-coupled receptors with unknown functions has increased.
This means that the number of drugs that have been developed by pharmaceutical companies around the world will also increase explosively. More than 90% of the drugs developed so far are extracellular.
It targets spatial interactions, particularly those related to G protein-coupled receptors.
The majority of drugs are small molecule drugs that act on G protein-coupled receptors.
Associated diseases include genetic disorders, as well as diseases of the nervous system, circulatory system, digestive system, and immune system.
It is related to many areas, including the immune system, musculoskeletal system, and urogenital system.
Therefore, recently, many pharmaceutical companies have been promoting orphan drugs identified through genome analysis.
They possess G protein-coupled receptors and are competing to discover ligands and elucidate their physiological functions.
Against this background, recently, the physiological role of novel G protein-coupled receptors has been investigated.
Successful examples of gand search have also begun to be reported.
ene-related peptide receptor (J. Biol. Chem. (
96)271:11325), orexin (Cell(98)92:573)
and prolactin-releasing peptide (Nature
e(98)393:272) are also examples of basic research in the field of life science.
This was a case that had a major impact. In particular, orphan G protein-coupled receptors have a high potential to lead to the development of new drugs.
Generally, orphan G protein-coupled receptors are
Since there is no specific ligand for the receptor, its agonists and antagonists
However, in recent years, a rich compound library has been developed.
By combining this with high-throughput screening, orphan G proteins
The creation of drugs targeting coupled receptors has been proposed (Trends Pha
rmacol. Sci. (97) 18:430, Br. J. Pharm. (98
) 125:1387). That is, orphan G identified by genetic engineering
Protein-coupled receptors are used to bind to intracellular second messengers such as cAMP and Ca.^2
+We will discover physiological agonists through functional screening using changes in the
This involves analyzing the functions of the compound in the body.
By increasing the throughput of screening, orphan G protein co-reactivity can be improved.
Specific surrogate agonists and antagonists for steroid receptors
This would theoretically make it possible to discover agonists and ultimately develop drugs to treat specific diseases.Disclosure of the Invention The present invention was made in consideration of the current situation surrounding G protein-coupled receptors.
The object of the present invention is to provide a novel G protein-coupled receptor and its gene, as well as
The purpose of this invention is to provide methods for producing and using these molecules.
The purpose is to provide it as a research target. As a result of intensive research to solve the above problem, the inventors have discovered that human tissue c
G protein-coupled proteins were synthesized by DNA-templated polymerase chain reaction.
It retains the seven transmembrane domains and hydrophobic regions that are characteristic of the α-type receptor.
We succeeded in isolating nine novel genes.
A certain protein may be a target for screening of ligands or for identifying agonists or antagonists that are useful as pharmaceuticals.
Screening for antagonists or diagnosing diseases related to these genes
That is, the present invention relates to novel G protein-coupled receptors and their genes, as well as
More specifically, regarding their production and use, (1) The following (a) encoding a guanosine triphosphate-binding protein-coupled receptor:
) to (d), (a) an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4, 17 to 21,
(b) a code for a base sequence set forth in any one of SEQ ID NOs: 5 to 8 and 22 to 26;
(c) a DNA comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4, 17 to 21;
In the present invention, one or more amino acids are substituted, deleted, added and/or inserted.
(d) DNA encoding a protein consisting of a nucleotide sequence set forth in any one of SEQ ID NOs: 5 to 8 and 22 to 26;
(2) DNA that hybridizes to DNA under stringent conditions; (3) an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4, 17 to 21;
(3) A vector containing the DNA described in (1) or (2); (4) A vector containing the DNA described in (1) or (2) or the vector described in (3).
(5) a protein encoded by the DNA according to (1) or (2); or
(6) Culturing the transformant described in (4) and producing a peptide from the transformant or its culture.
(5) The method according to (5), further comprising recovering the expressed protein or peptide from the supernatant.
(7) A method for producing a protein or peptide, (8) A method for screening a ligand that binds to the protein described in (5).
(a) a method comprising the steps of contacting a test sample with the protein or peptide described in (5), (b) selecting a compound that binds to the protein or peptide, (8) a compound having the activity of inhibiting the binding between the protein described in (5) and its ligand.
A method for screening for a compound comprising: (a) ligating to the protein or a partial peptide thereof according to (5) in the presence of a test sample;
and detecting the binding activity of the protein or its partial peptide with the ligand.
(b) comparing the binding activity detected in step (a) with that in the absence of a test sample;
(9) A method comprising screening for compounds that inhibit or promote the activity of the protein described in (5).
A method for screening, comprising: (a) contacting a ligand of the protein with a cell expressing the protein in the presence of a test sample;
(b) detecting changes in the cells due to binding of the ligand to the protein; (c) comparing the changes detected in step (b) with the changes in the cells in the absence of a test sample.
and selecting a compound that suppresses or enhances the change in the selected cells.
(10) The change in the cell is a change in cAMP concentration or a change in calcium concentration.
(11) An antibody that binds to the protein described in (5). (12) An antibody isolated by the screening method described in any one of (7) to (10).
(13) A pharmaceutical composition containing the compound according to (12) as an active ingredient; and (14) A compound for treating a disease selected from the group consisting of cancer, liver cirrhosis, and Alzheimer's disease.
(15) A pharmaceutical composition according to (13) for treating a disease, (16) A pharmaceutical composition according to (14) for treating a disease, (17) A pharmaceutical composition according to (15) for treating a disease, (18) A pharmaceutical composition according to (16) for treating a disease, (19) A pharmaceutical composition according to (19) for treating a disease, (20) A pharmaceutical composition according to (20) for treating a disease, (21) A pharmaceutical composition according to (21)
or a complementary strand thereof,
(16) A nucleotide having a structure similar to that of a ...
A method for diagnosing a disease, comprising: detecting a gene for the disease-related tissue derived from a subject, the gene for the disease-related tissue derived from a subject;
(1) detecting the expression of the DNA described in (1) or the mutation of the DNA described in (1) in a subject
(17) A method comprising:
, a diagnostic agent for a disease selected from the group consisting of cancer, liver cirrhosis, and Alzheimer's disease - Patent application
, and provides. In the present invention, a "G protein-coupled receptor" refers to a receptor that binds to a GTP-binding protein.
In the present invention, a "ligand" refers to a cell membrane receptor that binds to a G protein-coupled receptor and transmits a signal into the cell via activation.
Here, the term "physiological substance" refers to a physiological substance that transmits a signal.
It refers to a compound that binds to a G protein-coupled receptor. In the present invention, an "agonist" refers to a compound that binds to a G protein-coupled receptor and acts intracellularly.
These compounds include physiological substances, artificially synthesized compounds,
This includes naturally occurring compounds. In the present invention, an "antagonist" refers to an agent that binds a ligand to a G protein-coupled receptor.
This refers to compounds that inhibit the binding of or transmission of signals into cells.
These include physiological substances, artificially synthesized compounds, and naturally occurring compounds. The present invention relates to a novel G protein-coupled receptor and DNA encoding the protein.
The present invention provides nine human-derived cDs isolated by the present inventors.
The NA clones were classified as "GPRv8", "GPRv12", "GPRv16", and "G
PRv21”, “GPRv40”, “GPRv47”, “GPRv51”, “G
These clones were named "GPRv71" and "GPRv72" (if necessary, these clones may be used together).
The base sequences of these cDNAs are as follows:
8, 22 to 26 show the amino acid sequence of the protein encoded by the cDNA.
Row numbers: 1 to 4, 17 to 21. As a result of BLAST search, the proteins encoded by GPRv cDNA were all
It showed significant amino acid sequence homology with known G protein-coupled receptors.
"GPRv8" is HUMAN VASOPRESSIN V1B REC
36% homology to EPTOR (P47901, 424 aa) and “GPR
v12” is RAT 5-HYDROXYTRYPTAMINE 6 RECEP
27% homology to TOR (P31388, 436 aa)
6" is MOUSE GALANIN RECEPTOR TYPE 1 (P56
479,348 aa), and "GPRv21" has 28% homology to BOVI.
N NEUROOPEPTIDE Y RECEPTOR TYPE 2 (P79
113,384 aa), and "GPRv40" has 30% homology to OXYT
34% affinity to OCIN RECEPTOR (P97926, 388aa)
Same sex, "GPRv47" is GPRX_ORYLA PROBABLE G P
ROTEIN-COUPLED RECEPTOR (Q91178,428aa
) and "GPRv51" has 43% homology to PROBABLE GPR
OTEIN-COUPLED RECEPTOR RTA (P23749,34
3aa) and "GPRv71" has 37% homology to Chicken P2
Y PURINOCEPTOR 3 (P2Y3) (Q98907, 328aa)
"GPRv72" has 45% homology to ALPHA-1A ADREN
30% against ERGIC RECEPTOR (O02824, 466aa)
The homology between the GPRv cDNA and the protein encoded by the GPRv cDNA isolated by the present inventors (hereinafter referred to as
G protein-coupled receptors (sometimes referred to as "GPRv proteins") are
It retained the seven characteristic transmembrane domains and hydrophobic regions that are thought to be the transmembrane domains.
From these facts, GPRv cDNA is a member of the G protein-coupled receptor family.
It is thought that G protein-coupled receptors encode proteins belonging to the
The action of a ligand activates G proteins, transmitting signals into the cell.
As mentioned above, it has been shown to be effective in treating genetic disorders, as well as the nervous system and circulatory system.
It can affect a wide range of areas, including the digestive system, immune system, musculoskeletal system, and urinary and reproductive systems.
Therefore, the GPRv protein is involved in the regulation of the function of the GPRv protein.
It can be used to screen for agonists and antagonists,
These proteins are important targets for the development of pharmaceuticals for these diseases. The present invention also provides proteins functionally equivalent to the GPRv protein.
"Functionally equivalent" means that the target protein has biological properties equivalent to those of the GPRv protein.
The biological properties of the GPRv protein include:
Transmits signals into cells via activation of trimeric GTP-binding proteins
Trimeric GTP-binding proteins are involved in the type of intracellular signaling pathway they activate.
Therefore, Ca²⁺Gq type that increases cAMP, Gs type that increases cAMP, and
They are classified into three categories of Gi type that suppress AMP (Trends P
Pharmacol. Sci. (99) 20:118). Therefore, the protein of interest
Whether or not a substance has biological properties equivalent to those of the GPRv protein can be determined by, for example,
Changes in intracellular cAMP or calcium concentration due to activation can be detected.
This can be evaluated by one embodiment of a method for preparing a protein functionally equivalent to the GPRv protein.
For example, a method of introducing a mutation into the amino acid sequence of a protein can be mentioned.
Suitable methods include, for example, site-directed mutagenesis (Current Protocols
ls in Molecular Biology edit. Ausubel
et al. (1987) Publish. John Wiley & So
Section 8.1-8.5)) is included.
Mutations of amino acids can occur in nature.
The amino acid sequence of the GPRv protein (SEQ ID NO: 1) whether artificial or naturally occurring.
1 to 4, 17 to 21) in which one or more amino acids are substituted, deleted, or inserted
a protein mutated by insertion and/or addition, and a GPRv protein;
These proteins include functionally equivalent proteins.
There is no limit to the site of mutation as long as the function of the GPRv protein is maintained.
Generally, it is within 10% of the total amino acids, and preferably within 5% of the total amino acids.
It is considered that the amount of amino acids contained in the GPRv protein is more preferably within 1% of the total amino acids. Another embodiment of the method for preparing a protein functionally equivalent to the GPRv protein is
Examples of methods that can be used include hybridization techniques and gene amplification techniques.
That is, those skilled in the art will be familiar with hybridization techniques (Current
t Protocols in Molecular Biology edi
t. Ausubel et al. (1987) Publish. John W.
GPR using the Filey & Sons Section 6.3-6.4
a DNA sequence encoding the v protein (SEQ ID NOs: 5 to 8, 22 to 26); or
Based on a part of it, DNA samples from the same or different species of organisms are extracted to find highly homologous sequences.
and isolating a DNA fragment from the DNA to obtain a protein functionally equivalent to the GPRv protein.
In this way, it is possible to use a DNA fragment encoding a GPRv protein.
A protein encoded by DNA that hybridizes with GPRv
Proteins functionally equivalent to the protein are also included in the proteins of the present invention. Organisms from which such proteins can be isolated include, in addition to humans, rats, for example.
Examples include, but are not limited to, rats, mice, rabbits, chickens, pigs, and cows.
To isolate DNA encoding a protein functionally equivalent to the GPRv protein,
Stringent hybridization conditions are usually 1x SSC,
The conditions are approximately "0.1% SDS, 37°C" and more stringent conditions are "0.5
The conditions are approximately "100x SSC, 0.1% SDS, 42°C" and even stricter conditions are
The conditions are approximately "0.2x SSC, 0.1% SDS, 65°C."
The more stringent the hybridization conditions, the higher the homology with the probe sequence.
However, under the above conditions of SSC, SDS and temperature, it is expected that DNA having the above structure can be isolated.
These combinations are exemplary and those skilled in the art will appreciate that the hybridization sequence
These and other factors (e.g., probe concentration, probe
By appropriately combining the following parameters (length of the probe, hybridization reaction time, etc.),
This makes it possible to achieve the same stringency as above. The DNA isolated using such hybridization techniques is coding for
The corresponding protein usually has high amino acid sequence homology with the GPRv protein.
High homology means at least 40% or more, preferably 60% or more, and more preferably
Preferably, the sequence has a homology of 80% or more (e.g., 90% or more, or 95% or more).
. The identity of amino acid sequences and base sequences is based on the Karlin and Altschu
The BLAST algorithm (Proc. Natl. Acad. Seis. U
SA 90:5873-5877, 1993).
Based on this algorithm, programs called BLASTN and BLASTX are
A system has been developed (Altschul et al. J. Mol. Biol.
215:403-410, 1990).
When analyzing a base sequence, the parameter is, for example, score=100.
, wordlength=12. Also, based on BLAST,
When analyzing an amino acid sequence by X, the parameters are, for example, score
Set e=50 and wordlength=3. BLAST and Gapped BL
When using the AST program, the default parameters of each program are
Specific techniques for these analysis methods are publicly known (http://www
.ncbi.nlm.nih.gov. In addition, gene amplification technology (PCR) (Current protocols
n Molecular Biology edit. Ausubel et
al. (1987) Publish. John Wiley & Sons
The DNA sequence encoding the GPRv protein was isolated using the PCR product (sections 6.1-6.4).
Primers were designed based on a portion of the sequence (SEQ ID NOs: 5 to 8, 22 to 26), and G
A DNA fragment highly homologous to the DNA sequence encoding the PRv protein is isolated, and the DNA fragment is then purified.
It is also possible to obtain a protein functionally equivalent to the GPRv protein based on NA. The present invention also includes a partial peptide of the protein of the present invention.
These include peptides that bind to ligands but do not transduce signals.
Affinity columns based on such peptides are useful for screening ligands.
Furthermore, partial peptides of the protein of the present invention can be suitably used for antibody binding.
The partial peptide of the present invention can be used, for example, in the production of a gene encoding a polypeptide.
The protein of the present invention can be synthesized by a suitable peptide synthesis method, a known peptide synthesis method, or a method for synthesizing the protein of the present invention.
The partial peptide of the present invention can be produced by cleaving the peptide with an enzyme.
Usually, it contains 8 or more amino acid residues, preferably 12 or more amino acid residues (e.g., 15 or more amino acid residues).
The proteins of the present invention can be prepared as recombinant proteins or as natural proteins.
The recombinant protein can be, for example, a recombinant protein containing the protein of the present invention as described below.
The vector into which the DNA to be encoded is inserted is introduced into an appropriate host cell, and the
It can be prepared by purifying the expressed protein.
The protein may be, for example, an affinity antibody bound to the protein of the present invention, as described below.
It can be prepared using a gel column (Current Protocol
ls in Molecular Biology edit. Ausubel
et al. (1987) Publish. John Wiley & So
ns Section 16.1-16.19). Used for affinity purification.
The antibody may be a polyclonal antibody or a monoclonal antibody.
In addition, in vitro translation (e.g., "On the fidelity"
y of mRNA translation in the nuclei
e-treated rabbit reticulocyte lysate
system. Dasso, M. C. , Jackson, R. J. (1989
) NAR 17:3129-3144) or the like.
It is also possible to do so. The present invention also provides DNA encoding the above-mentioned protein of the present invention.
The DNA may be any DNA, regardless of its form, as long as it can encode the protein of the present invention.
There is no limitation to the DNA, and in addition to cDNA, it also includes genomic DNA, chemically synthesized DNA, and the like.
In addition, any base sequence based on the degeneracy of the genetic code may be used as long as it can encode the protein of the present invention.
The DNA of the present invention includes a DNA having a sequence similar to that of the GPRv protein.
DNA sequences encoding proteins (SEQ ID NOs: 5 to 8, 22 to 26) or
Based on the hybridization method using a part of the DNA as a probe and these DNA sequences,
It can be isolated by conventional methods such as PCR using synthesized primers.
The present invention also provides a vector into which the DNA of the present invention is inserted.
There are no particular limitations on the vector, as long as it can stably maintain the inserted DNA.
For example, if E. coli is used as the host, the cloning vector should be
A pBluescript vector (Stratagene) is preferred.
When a vector is used for the purpose of producing the protein of the present invention, the expression
Vectors are useful. Expression vectors can be used in test tubes, E. coli, and cultured cells.
There are no particular limitations on the vector as long as it expresses a protein in an individual organism.
For example, for in vitro expression, use the pBEST vector (Promega), and for E. coli,
pET vector (Invitrogen), and pME18 for cultured cells.
S-FL3 vector (GenBank Accession No. AB009
864), and in the case of an individual organism, the pME18S vector (Mol Cell Bio
1. 8:466-472 (1988)) are preferred.
DNA insertion can be performed by a conventional method, for example, by ligase reaction using a restriction enzyme site.
This can be done by Current Protocols in Molecular Biology (
cular Biology edit. Ausubel et al. (19
87) Publish. John Wiley & Sons. Section 11.4-11.11). The present invention also relates to a transformant carrying the DNA of the present invention or the vector of the present invention.
There are no particular limitations on the host cells into which the vectors of the present invention are introduced.
Various host cells are used depending on the purpose.
Examples of cells include COS cells and CHO cells.
Vectors can be introduced into host cells by, for example, calcium phosphate precipitation, electroporation, etc.
Current protocols in Molecular Bi
logic edit. Ausubel et al. (1987)Publi
sh. John Wiley & Sons. Section 9.1-9.9
), Lipofectamine method (GIBCO-BRL), microinjection
This can be done by known methods such as the DNA encoding the protein of the present invention (SEQ ID NOs: 5 to 8,
27. A DNA having a base sequence according to any one of 22 to 26, or a complementary strand thereof
providing complementary nucleotides having a chain length of at least 15 nucleotides;
Here, the "complementary strand" refers to a base pair of A:T (or U in the case of RNA), G:C.
The term "complementary" refers to one strand of a double-stranded nucleic acid.
, if the sequence is completely complementary over a region of at least 15 consecutive nucleotides
It is not limited to, but may be at least 70%, preferably at least 80%, more preferably 90%.
It is sufficient if the base sequence has a homology of 0%, more preferably 95% or more.
The algorithm for determining the sex may be the one described in this specification.
Such nucleotides can be used as probes for detecting and isolating the DNA of the present invention.
and can be used as a primer for amplifying the DNA of the present invention.
When used as a primer, it is usually 15 bp to 100 bp,
Preferably, the length is 15 bp to 35 bp.
In this case, at least one sequence containing at least a part or all of the sequence of the DNA of the present invention is used.
Nucleotides with a chain length of 15 bp are used. Such nucleotides are preferred.
or those that specifically hybridize to the DNA encoding the protein of the present invention.
"Specifically hybridize" means to hybridize under normal hybridization conditions.
Under these conditions, preferably under stringent conditions,
NA (SEQ ID NOs: 5 to 8, 22 to 26) and other proteins.
This means that they do not hybridize with the DNA that encodes them. These nucleotides can be used to test and diagnose abnormalities in the protein of the present invention.
For example, these nucleotides can be used as probes or primers in
The protein encoding the present invention is detected by hybridization or RT-PCR.
Abnormal expression of DNA can be examined. These nucleotides can be used to detect abnormalities in the expression of DNA, for example, cancer.
, liver cirrhosis, or Alzheimer's disease.
by polymerase chain reaction (PCR) using nucleotides as primers
The DNA encoding the protein of the present invention and its expression control region are amplified and subjected to RFLP analysis.
, SSCP, sequencing, etc. to test and diagnose abnormalities in DNA sequences.
These nucleotides can also contain amino acids that inhibit the expression of the protein of the present invention.
Antisense DNA contains antisense DNA.
For this purpose, the number of nucleotides is at least 15 bp, preferably 100 bp, and more preferably
has a chain length of 500 bp or more, usually 3000 bp or less, preferably 2000 bp or less
Such antisense DNA has a chain length of 100 bp or less.
We are also considering applying this technology to gene therapy for diseases caused by abnormalities (such as functional abnormalities or expression abnormalities).
The antisense DNA is, for example, a DNA encoding the protein of the present invention.
(For example, SEQ ID NOs: 5 to 8, 22 to 26)
Stein, 1988 Physicochemical prop
erties of phosphorothioate oligodeox
ynucleotides. Nucleic Acids Res 16,32
09-21 (1988)). When used in gene therapy, the nucleotides of the present invention can be prepared, for example, by retroviral
vectors, adenovirus vectors, adeno-associated virus vectors, etc.
It is conceivable that the protein of the present invention can be administered to patients by ex vivo or in vivo methods using viral vectors or non-viral vectors such as liposomes. The present invention also provides antibodies that bind to the protein of the present invention.
There are no particular limitations on the form, and it can be a polyclonal antibody, a monoclonal antibody, or an antigen.
Also included are parts of antibodies that have binding properties. All classes of antibodies are included.
Furthermore, the antibodies of the present invention also include special antibodies such as humanized antibodies. In the case of polyclonal antibodies, the antibodies of the present invention can be prepared by ligating the proteins of the present invention in accordance with conventional methods.
By synthesizing an oligopeptide corresponding to the amino acid sequence of
It is possible to obtain (Current protocols in Molecular Biology, Vol.
cular Biology edit. Ausubel et al. (19
87) Publish. John Wiley & Sons. Section 11.12-11.13). In the case of monoclonal antibodies, the colon is irradiated according to conventional methods.
Mice were immunized with the purified protein expressed in bacteria, and their spleen cells and myeloma cells were then cultured.
and preparing hybridoma cells by cell fusion of the two, and obtaining
(Current protocols in Molecules
r Biology edit. Ausubel et al. (1987) P.
publish. John Wiley & Sons. Section 11.
4-11.11). Antibodies that bind to the proteins of the present invention can be used, for example, to purify the proteins of the present invention.
The present invention may also be used to test and diagnose abnormalities in the expression or structural abnormalities of the protein.
Specifically, proteins are extracted from tissues, blood, or cells, and then purified by Western blotting.
Detection of the protein of the present invention by methods such as immunoblotting, immunoprecipitation, and ELISA.
Through this, it is possible to test and diagnose whether or not there is an abnormality in expression or structure.
The body could be used, for example, to test for cancer, cirrhosis, or Alzheimer's disease.
Furthermore, antibodies that bind to the proteins of the present invention can be used to treat diseases associated with the proteins of the present invention.
The antibody of the present invention can be used for the purpose of treatment of the protein of the present invention.
They can act as agonists or antagonists. The antibodies can be used to treat patients.
In such cases, human antibodies or humanized antibodies are preferred because of their low immunogenicity.
The body is a mouse with a human immune system (e.g., "Functiona
l transp lant of megabase human immu
noglobulin loci recapitulates human
Antibody response in mice, Mendez, M. J
．． et al. (1997) Nat. Genet. 15:146-156”
Humanized antibodies can be prepared by immunizing a monoclonal antibody against a humanized antibody.
It can be prepared by genetic recombination using the hypervariable region of a clonal antibody (
Methods in Enzymology 203, 99-121 (199
1)) The present invention also provides a ligand that binds to the protein of the present invention, using the protein of the present invention.
The screening method includes: (a) screening the strain of the present invention;
(b) contacting a test sample with the protein or a partial peptide thereof;
The test sample is not particularly limited, and examples include the selection of compounds that bind to the protein or its partial peptides.
The ligand activity of known compounds or peptides (e.g., chemical peptides) is unknown.
(registered in J. Mol. Biol. 2004) or phage display method (J. Mol. Biol. 2004).
iol. (1991) 222, 301-310)
Dam peptides can be used.
Natural ingredients derived from marine organisms are also subject to screening.
Examples include biological tissue extracts, cell extracts, and expression products of gene libraries.
The protein of the present invention used for screening may be, for example, in a form expressed on the cell surface.
, in the form of a cell membrane fraction of the cell, or in the form bound to an affinity column.
Specific screening methods include, for example, affinity screening of the protein of the present invention.
The test sample is contacted with the Tee column to purify compounds that bind to the protein of the present invention.
Many known methods, such as the Western blotting method, can be used.
When using these methods, the test sample is appropriately labeled, and this label
Binding to the protein of the present invention can be detected by utilizing these methods.
In addition, cell membranes expressing the protein of the present invention are prepared and immobilized on a chip.
The dissociation of the trimeric GTP-binding protein upon ligand binding was observed by surface plasmon
Detected by changes in surface plasmon resonance
(Nature Biotechnology (99) 17: 1105)
It is also possible to use a protein of the present invention. The binding activity between a test sample and the protein of the present invention can be measured by measuring the binding activity of the test sample expressed on the cell surface.
The changes in cells caused by binding to the protein of the present invention were examined as indicators.
Such changes include, for example, the increase in intracellular Ca²⁺Rebbe
These include, but are not limited to, changes in the ATP level and changes in cAMP levels.
Specifically, the agonist activity against G protein-coupled receptors was measured by the GTPγS binding method.
As an example of this method, a cell membrane expressing a G protein-coupled receptor is
20mM HEPES (pH 7.4), 100mM NaCl, 10mM Mg
Cl₂, in a 50 μM GDP solution,³⁵S-labeled GTPγS 400p
M, and incubated in the presence and absence of a test sample, followed by filtration (fil
The radioactivity of the bound GTPγS was compared.
G protein-coupled receptors can also activate trimeric GTP-binding proteins, which can activate cells.
They share a system for transmitting signals into the cytoplasm. Trimeric GTP-binding protein
Depending on the type of intracellular signaling system that is activated,²⁺Gq type, which increases
They are classified into three types: Gs type, which increases AMP, and Gi type, which suppresses cAMP.
This fact can be applied to the Gq protein α subunit and other G protein α subunits.
and a chimera or promiscuous Gα protein, Gα15, G
Using α16 to detect positive signals during ligand screening via intracellular transduction of Gq
Pathway, Ca²⁺This can be attributed to an increase in Ca²⁺Re
The bell is a TRE (TPA responsive element) or MR
E (multiple responsive element) upstream
reporter gene systems, dye indicators such as Fura-2 and Fluo-3, and fluorescent dyes
It can be detected by using changes in the photoprotein aequorin as an indicator.
The white α subunit was chimerized with other G protein α subunits, and a positive signal was observed.
The intracellular pathway of Gs results in an increase in cAMP, which is CRE (cAMP-r
In a reporter gene system having an upstream responsive element
It is also possible to use changes in the blood glucose level as an index (Trends Pharmacol. Sc
i. (99) 20:118). In this screening system, the host cells used to express the protein of the present invention are
There are no particular limitations, and various host cells can be used depending on the purpose. For example, COS
Examples of the protein of the present invention include cells, CHO cells, and HEK293 cells.
As a vector for expressing the protein in vertebrate cells,
The promoter located upstream of the gene being read, the splice site of RNA, and the polynucleotides
It is preferable to use a DNA having an adenylation site, a transcription termination sequence, a replication origin, etc.
For example, pSV2dhfr (
Mol. Cell. Biol. (1981) 1,854-864), pEF-
BOS (Nucleic Acids Res. (1990) 18, 5322)
, pCDM8 (Nature (1987) 329, 840-842), pCEP
4 (Invitrogen) and others are used to express G protein-coupled receptors.
The DNA of the present invention can be inserted into the vector by conventional methods using restriction enzymes.
This can be done by a ligase reaction using a primer site (Current product
otocols in Molecular Biology edit. Au
Subel et al. (1987) Publish. John Wiley
& Sons. Section 11.4-11.11).
The vector can be introduced by, for example, calcium phosphate precipitation, electroporation (Cu
rrent protocols in Molecular Biology
edit. Ausubel et al. (1987) Publish. Jo
Wiley & Sons. Section 9.1-9.9), Lipov
Ectamine method (GIBCO-BRL), FuGENE 6 reagent (Boehringer
This can be done by known methods such as injection (Mannheim) and microinjection.
By the above-mentioned method for screening for a ligand that binds to the protein of the present invention,
Once the band is isolated, the compound that inhibits the interaction between the protein and the ligand of the present invention can be used.
Therefore, the present invention also provides the protein of the present invention and its
A method for screening a compound having an activity of inhibiting binding to a ligand is provided.
This screening method comprises: (a) detecting the protein or protein of the present invention in the presence of a test sample;
contacting the partial peptide with a ligand, and
(b) detecting the binding activity to the ligand; and (c) comparing the binding activity in the absence of a test sample with that in the absence of a test sample.
and selecting a compound that reduces the binding activity detected in step (a).
, etc. The test sample is not particularly limited, and examples thereof include those obtained by combinatorial chemistry.
-technology (Tetrahedron (1995) 51, 8135-8137)
or a group of compounds obtained by the phage display method (J. Mol. Biology
ol. (1991) 222, 301-310) and other methods.
In addition, the culture supernatant of microorganisms, plants, marine
Natural components derived from living organisms are also subject to screening.
tissue extracts, cell extracts, gene library expression products, and synthetic small molecules
These include, but are not limited to, compounds, synthetic peptides, and natural compounds.
The protein of the present invention used for screening may be expressed on the cell surface, for example.
, in the form of a cell membrane fraction of the cells, or bound to an affinity column.
Specific screening methods include, for example, using a radioisotope as the ligand.
and the like, and contacting the protein of the present invention in the presence of a test sample,
The binding activity between the protein of the present invention and a ligand is compared to that detected in the absence of a substrate.
A compound that reduces the activity is detected based on the label attached to the ligand.
Furthermore, screening of ligands that bind to the protein of the present invention can be carried out.
As with the case of HIV-1, it is possible to screen using intracellular changes as an indicator.
That is, cells expressing the protein of the present invention are contacted with a ligand in the presence of a test sample.
and the changes in the cells are reduced compared to those detected in the absence of the test sample.
By selecting a compound that inhibits the binding between the protein of the present invention and a ligand,
It is possible to screen for compounds that express the protein of the present invention.
The cells were subjected to the same screening as in the case of the above-mentioned screening of a ligand that binds to the protein of the present invention.
Compounds isolated by this screening can be prepared in the same manner as described above.
These compounds are candidates for agonists or antagonists of the protein of the present invention. The present invention also provides a screening method for compounds that inhibit or promote the activity of the protein of the present invention.
The screening method includes (a) detecting the presence of a test sample.
contacting a cell expressing the protein of the present invention with a ligand for the protein in the presence of the protein;
(b) detecting changes in cells due to binding of the ligand to the protein of the present invention;
(c) comparing the change in the cells in the absence of the test sample with the change in the cells in step (b);
selecting a compound that suppresses or enhances the detected changes in the cells;
The test sample includes a compound that inhibits the binding between the protein of the present invention and its ligand.
Similar to the screening method for materials, combinatorial chemistry techniques
The resulting compound group and random vectors created using phage display methods, etc.
peptides, microbial culture supernatants, natural components derived from plants and marine organisms, and biomolecules
tissue extracts, cell extracts, expression products of gene libraries, synthetic low molecular weight compounds,
Synthetic peptides, natural compounds, etc. can be used.
Compounds isolated by screening for compounds that inhibit the binding of proteins to ligands
The cells expressing the protein of the present invention can also be used as a test sample.
The preparation was carried out in the same manner as in the case of screening for the ligand that binds to the protein of the present invention.
The changes in the cells after contact with the test sample can be measured by the above-mentioned screening method.
As with the rinsing method, intracellular Ca²⁺Changes in the levels of ATP and cAMP were used as indicators.
In addition, when detecting intracellular signal transduction,
Use a measurement system such as a reporter assay system that uses enzymes as a reporter gene.
It is also possible to detect the presence of a ligand in the absence of a test sample.
Changes in cells when contacted with a test sample compared to changes in
If the activity is suppressed, the test sample used is a compound that inhibits the activity of the protein of the present invention.
Conversely, if the test sample enhances the change in the cell,
The compound is determined to be a compound that promotes the activity of the protein of the present invention.
The term "promoting or inhibiting the activity of the protein of the present invention" used herein means
The result, whether direct or indirect, is a reduction in white matter.
This means that the activity of the protein of the present invention is promoted or inhibited by the
The compounds isolated by screening include those that act on the protein or ligand of the present invention.
By inhibiting or promoting these bindings, the activity of the protein of the present invention is inhibited.
In addition to compounds that inhibit or promote these bindings, there are also compounds that do not inhibit or promote these bindings themselves but
The present invention also includes compounds that inhibit or promote the activity of the protein of the present invention.
Suitable compounds include, for example, compounds that do not inhibit the binding of the protein of the present invention to a ligand, but do not inhibit the binding of cells.
This includes compounds that inhibit or promote intracellular signal transduction pathways. When compounds isolated by the screening method of the present invention are used as pharmaceuticals,
In this case, the isolated compound itself can be directly administered to a patient, or the compound can be administered by a known pharmaceutical method.
It is also possible to administer the compound as a pharmaceutical composition formulated by a pharmaceutical method.
Physically acceptable carriers (excipients, binders, disintegrants, flavoring agents, odorants, emulsifiers, diluents,
Pharmaceutical compositions obtained by mixing with other ingredients (e.g., diluents, solubilizers, etc.) or tablets, pills, powders,
Granules, capsules, lozenges, syrups, liquids, emulsions, suspensions, injections (
As preparations such as liquids, suspensions, suppositories, inhalants, transdermal absorbents, eye drops, and eye ointments
It is formulated in a form suitable for oral or parenteral administration.
For example, it can be administered by methods known to those skilled in the art, such as intraarterial injection, intravenous injection, or subcutaneous injection.
The dosage varies depending on the patient's weight and age, the method of administration, etc., but it is within the skill of the art to determine the dosage.
In addition, if the compound binds to DNA, it is possible to select an appropriate dose.
If the gene can be encoded by the gene, the DNA is incorporated into a vector for gene therapy,
Gene therapy may also be considered.
The compounds have applications in the treatment of, for example, cancer, cirrhosis of the liver, or Alzheimer's disease.
It is expected that the present invention also provides a method for detecting the expression of a gene encoding the GPRv protein of the present invention.
and a method for diagnosing cancer, liver cirrhosis, or Alzheimer's disease, the method comprising:
In this example, the gene encoding the GPRv protein of the present invention is used to treat cancer, liver cirrhosis, and other liver diseases.
in affected tissues associated with Alzheimer's disease compared with normal tissue
The expression levels were found to be significantly different in these tissues of the subjects.
By detecting the expression of the gene encoding the GPRv protein of the present invention in
This makes it possible to diagnose these diseases.
This includes both transcription and translation. The diagnostic method of the present invention can be carried out, for example, as follows: RNA is extracted from a portion of tissue collected by biopsy or a blood sample using standard methods.
The extracted fragments were subjected to quantitative PCR, Northern hybridization, and other methods as described in the Examples.
GPRv mRNA was determined by dot blot hybridization or other methods.
It is also possible to extract proteins from the tissue and use them for diagnosis.
GPRv protein using methods such as western blotting, immunoprecipitation, and ELISA.
As a non-invasive method, compounds that bind to GPRv protein
The labeled antibody is administered to the patient, and PET (positron emission tomography) is performed.
It is also possible to diagnose by detecting the gene expression in the tissues of the subject.
The expression of the gene in tissues derived from the same patient is compared with that in normal tissues.
If the subject exhibits a significant increase or decrease in the expression level of the compared genes, the subject is diagnosed as suffering from the disease.
It is determined that a person has or is at risk of having the disease. For example, GPRv8 is expressed in the colon, but this expression is significantly reduced in colon cancer.
Therefore, high levels of GPRv8 expression are observed in the colonic tissue of the subject.
If present, the subject is suspected of colon cancer.
Expression was not detected in the uterus, but moderate expression was observed in the cancerous tissue.
If GPRv8 expression is found in the pancreas or uterus, the subject is diagnosed with pancreatic cancer.
There is suspicion of cancer or uterine cancer. GPRv12 expression was not detected in normal ovaries or testes, but it was expressed in cancerous tissue.
Furthermore, expression in the hippocampus was decreased in patients with Alzheimer's disease.
If expression of GPRv12 is found in the ovaries or testes of the subject,
of subjects are suspected of having ovarian or testicular cancer.
If a lower level of GPRv12 expression than normal is found, the subject
Alzheimer's disease is suspected. GPRv16 is expressed in the colon, but expression became undetectable after canceration.
In the brain, expression increased with cancer. In the liver, expression became undetectable with cirrhosis.
In Alzheimer's disease brains, expression was increased in the hippocampus.
If a subject has a lower level of GPRv16 expression than normal,
Colon cancer is suspected. Also, higher than normal levels of GPRv16 are found in the brain.
If expression is observed, the subject is suspected of having brain cancer.
If a lower level of GPRv16 expression than the normal value is found, the subject is diagnosed with liver cirrhosis.
In addition, the expression of GPRv16 in the hippocampus is higher than normal.
If this is found, the subject is suspected of having Alzheimer's disease. GPRv21 expression in the colon and testis becomes undetectable due to canceration.
Therefore, the expression of GPRv21 in the colon or testis of the subject is lower than normal.
If this phenomenon is observed, the subject is suspected of having colon cancer or testicular cancer. GPRv40 expression increases in the brain and testis due to canceration, and expression decreases due to liver cirrhosis.
Therefore, the expression of GPRv40 in the brain and testis was higher than normal.
If any of these is found, the subject is suspected of having brain or testicular cancer.
If a subject is found to have a lower level of GPRv40 expression than normal, the subject
He is suspected of having cirrhosis of the liver. GPRv47 expression increases in the brain and kidneys and decreases in the testes as the patient becomes cancerous.
The expression in the liver was undetectable in patients with liver cirrhosis.
If a higher level of GPRv47 expression than normal is found, the subject will
Cancer or kidney cancer is suspected. Also, lower than normal levels of GPR in the liver
If expression of v47 is detected, the subject is suspected of having liver cirrhosis. GPRv51 expression decreases in the colon and testis due to canceration. Liver in cirrhosis
Expression was also reduced in the hippocampus compared to normal subjects.
Therefore, the expression of GPRv51 in the colon and testis at lower than normal levels was
If any of these symptoms are present, the subject is suspected of having colon or testicular cancer.
If a lower level of GPRv51 expression than normal is observed in the liver,
The examiner suspected liver cirrhosis. Also, higher than normal levels of GPRv were found in the hippocampus.
If expression of GPRv71 is detected, the subject is suspected of having Alzheimer's disease. Expression of GPRv71 decreases in the colon and kidney due to cancer, and in the liver in cirrhosis.
In Alzheimer's disease, expression in the frontal lobe is reduced.
Therefore, the expression of GPRv71 in the colon or kidney was lower than normal.
If any of the above is found, the subject is suspected of having colon or kidney cancer.
If a lower level of GPRv71 expression than normal is observed in this subject,
Patients are suspected of having liver cirrhosis. Also, patients with lower than normal levels of GPRv in the frontal lobe are
If expression of GPRv72 is found, the subject is suspected of having Alzheimer's disease. GPRv72 is strongly expressed in the colon, but its expression becomes undetectable due to cancer.
Expression was increased in the hippocampus of Alzheimer's disease patients.
If a lower level of GPRv72 expression is found, the subject is suspected of having colon cancer.
Furthermore, higher than normal levels of GPRv72 expression were observed in the hippocampus.
If this is the case, the subject is suspected of having Alzheimer's disease. Furthermore, the above disease may be caused by a mutation in the gene encoding the GPRv protein of the present invention.
Therefore, the gene encoding the GPRv protein of the present invention
It is thought that it may be possible to diagnose the above diseases by detecting mutations in the offspring.
Such genetic diagnosis can be carried out, for example, as follows: The diagnostic nucleic acid can be prepared by directly analyzing genomic DNA or cDNA or by PCR or
Other amplification methods may also be used.
Deletions and insertions can be detected by size changes of the product.
DNA encoding GPRv is hybridized and the difference in melting temperature is used to identify the target site.
The differences in DNA sequence can be traced back to the presence or absence of altered material.
The change in electrophoretic mobility of DNA fragments in a gel can be detected by
This can be detected by direct DNA sequencing. This diagnosis shows that the gene encoding the GPRv protein in the subject is normal.
If the mutation is found in the gene encoding the GPRv protein, the subject is determined to be suspected of having the disease. That is, if the mutation is found in the gene encoding the GPRv protein by the method described herein,
By detecting increases or decreases in mRNA and protein expression, we can identify cancer, cirrhosis, and
The present invention provides a method for diagnosing Alzheimer's disease or a susceptibility to such disease.
It is served.Best Mode for Carrying Out the InventionNext, the present invention will be explained in more detail using examples.
Unless otherwise specified, the method is not limited to the above.
iatis, T. at al. (1982): “Molecular Clone
ing-A Laboratory Manual"Cold Spring
This can be performed according to the method described in the literature (Harbor Laboratory, NY). [Example 1] Isolation of genes encoding novel G protein-coupled receptors The novel G protein-coupled receptors (GPRv8, GPRv12, GPRv1) of the present invention are
6, GPRv21, GPRv40, GPRv47, GPRv51, GPRv71
The full-length cDNA encoding the novel G protein-coupled receptor GPRv8 was obtained by PCR. Marathon DNA from human fetuses was used to amplify the novel G protein-coupled receptor GPRv8.
On Ready cDNA (Clontech) was used as the template cDNA, and forward
The primer was 5'-ATGCCAGCCAACTTCACAGAGGG
CAGCT-3' (SEQ ID NO: 9), 5'-CTAG as a reverse primer
ATGAATTCTGGCTTGGACAGAATC-3' (SEQ ID NO: 10)
PCR was performed using Pyrobest DNA polymerase (Takara Shuzo).
) was used, and the mixture was heated at 94°C (2.5 minutes), followed by a cycle of 94°C (30 seconds)/60°C (30 seconds)/7
The cycle of 2°C (1 min) was repeated 25 times. As a result, a DNA fragment of about 1.1 kbp was obtained.
The A fragment was amplified. This fragment was inserted into pCR2.1 plasmid (Invitrogen).
The nucleotide sequence of the obtained clone was determined by using a DNA fragment encoding the nucleotide sequence of the clone.
The ABI377 DNA Sequencer (Ap
The sequence was analyzed using a DNA sequence analysis software (Plied Biosystems).
is shown in SEQ ID NO: 5. This sequence contains an open reading frame of 1,116 bases (the first sequence of SEQ ID NO: 5).
The open reading frame contains the predicted sequence.
The predicted amino acid sequence (371 amino acids) is shown in SEQ ID NO: 1.
The sequence contains a sparse sequence that appears to contain seven transmembrane domains characteristic of G protein-coupled receptors.
Since this gene contains an aqueous domain, it is believed to encode a G protein-coupled receptor.
It was found that amplification of the novel G protein-coupled receptor GPRv12 involves Mara derived from human fetal brain.
The template cDNA was Thon Ready cDNA (Clontech).
Forward primer: 5'-ATGGGCCCCGGCGAGGCGCTG
CTGGCGG-3' (SEQ ID NO: 11), 5'-T as a reverse primer
CAGTGTGTCTGCTGCAGGCAGGAATCA-3' (SEQ ID NO:
PCR was performed using Pyrobest DNA polymerase (
Takara Shuzo) in the presence of 5% formamide, and then heated at 94°C (2.5 minutes).
5 cycles of 94°C (5 seconds)/72°C (4 minutes), 5 cycles of 94°C (5 seconds)/70°C (4 minutes),
5 cycles, 94°C (5 seconds) / 68°C (4 minutes) cycle repeated 25 times
As a result, a DNA fragment of approximately 1.1 kbp was amplified.
Cloning was performed using the .1 plasmid (Invitrogen).
The nucleotide sequence of the obtained clone was determined by the dideoxy terminator method.
7 DNA Sequencer (Applied Biosystems)
The sequence identified is shown in SEQ ID NO: 6. This sequence contains an open reading frame of 1092 bases (the first base of SEQ ID NO: 6).
The open reading frame contains the predicted sequence.
The predicted amino acid sequence (363 amino acids) is shown in SEQ ID NO: 2.
The sequence contains a sparse sequence that appears to contain seven transmembrane domains characteristic of G protein-coupled receptors.
Since this gene contains an aqueous domain, it is believed to encode a G protein-coupled receptor.
It was found that amplification of the novel G protein-coupled receptor GPRv16 involves the Marathon gene derived from human brain.
On Ready cDNA (Clontech) was used as the template cDNA, and forward
The primer was 5'-ATGCTGGCAGCTGCCTTTGCAGA
CTCTAAC-3' (SEQ ID NO: 13), 5'-C as a reverse primer
TATTTAACACCTTCCCCCTGTCTCTTGATC-3' (SEQ ID NO:
PCR was performed using Pyrobest DNA polymerase (No. 14).
After 94°C (2 minutes), the mixture was heated at 94°C (30 seconds)/60°C (30 seconds).
The cycle of 72°C (1 minute)/72°C (1 second) was repeated 30 times.
The DNA fragment of p was amplified.
Cloning was performed using a DNA sequencer (Vitrogen).
ABI377 DNA Sequence was cloned by the dideoxy terminator method.
The results were analyzed using a r (Applied Biosystems).
The sequence obtained is shown in SEQ ID NO: 7. This sequence contains an open reading frame of 1,260 bases (the first sequence of SEQ ID NO: 7).
The open reading frame contains the predicted sequence.
The predicted amino acid sequence (419 amino acids) is shown in SEQ ID NO: 3.
The sequence contains a sparse sequence that appears to contain seven transmembrane domains characteristic of G protein-coupled receptors.
Since this gene contains an aqueous domain, it is believed to encode a G protein-coupled receptor.
It was found that amplification of the novel G protein-coupled receptor GPRv21 involves human fetal-derived Marat
hon Ready cDNA (Clontech) was used as the template cDNA.
5'-ATGGAGACCACCATGGGGTTCA as word primer
TGGATG-3' (SEQ ID NO: 15), and 5'-TT as a reverse primer
ATTTTAGTCTGATGCAGTCCACCTCTTC-3' (SEQ ID NO:
PCR was performed using Pyrobest DNA polymerase.
(Takara Shuzo) in the presence of 5% formamide, and then heated at 94°C (2.5 minutes).
5 cycles of 94°C (5 seconds)/72°C (4 minutes), 94°C (5 seconds)/70°C (4 minutes)
) cycle five times, and 94°C (5 seconds)/68°C (4 minutes) cycle 25 times.
As a result, a DNA fragment of about 1.2 kbp was amplified.
Cloning was performed using the R2.1 plasmid (Invitrogen).
The nucleotide sequence of the obtained clone was determined to be ABI3 by the dideoxy terminator method.
77 DNA Sequencer (Applied Biosystems)
The sequence identified is shown in SEQ ID NO: 8. This sequence has an open reading frame of 1,182 bases (SEQ ID NO: 8).
The amino acid sequence predicted from the open reading frame (393
The predicted amino acid sequence is shown in SEQ ID NO: 4.
It has seven hydrophobic regions that are thought to be transmembrane domains, which are characteristic of
It was found that this gene encodes a G protein-coupled receptor. Amplification of the novel G protein-coupled receptor GPRv40 was performed using human fetal Marat
hon Ready cDNA (Clontech) was used as the template cDNA.
5'-ATGGAGGAGGATCTCTTTAGCCCCT as word primer
CAATTC-3' (SEQ ID NO: 27), 5'-CT as a reverse primer
AGAAGGCACTTTCGCAGGAGCAAGGC-3' (SEQ ID NO: 2
PCR was performed using Pyrobest DNA polymerase (Takara).
In the presence of 5% formamide, the reaction mixture was heated at 98°C for 2.5 minutes, then heated at 98°C for 2.5 minutes.
5 cycles of 98°C (5 seconds)/72°C (4 minutes), 98°C (5 seconds)/70°C
(4 min) cycle five times, 98°C (5 sec) / 68°C (4 min) cycle
This was repeated 25 times. As a result, a DNA fragment of approximately 1.3 kbp was amplified.
The fragment was cloned using pCR2.1 plasmid (Invitrogen).
The nucleotide sequence of the obtained clone was determined by the dideoxy terminator method.
ABI377 DNA Sequencer (Applied Biosys)
The sequence identified is shown in SEQ ID NO: 22. This sequence contains an open reading frame of 1,305 bases (SEQ ID NO: 22).
The amino acid sequence predicted from the open reading frame (43
The predicted amino acid sequence is shown in SEQ ID NO: 17.
It has seven hydrophobic regions that are thought to be transmembrane domains, which are characteristic of the receptor.
It was found that this gene encodes a G protein-coupled receptor. Amplification of the novel G protein-coupled receptor GPRv47 involved the Mara gene from human fetal brain.
The template cDNA was Thon Ready cDNA (Clontech).
Forward primer: 5'-ATGGAGTCCTCACCCATCCCC
CAGTCATC-3' (SEQ ID NO: 29), and 5'- as a reverse primer
TCATGACTCCAGCCGGGGTGAGGCGGCAG-3' (SEQ ID NO:
PCR was performed using Pyrobest DNA polymerase (No. 30).
e (Takara Shuzo) in the presence of 5% formamide, and then heated at 94°C (2 minutes).
35 cycles of 50°C (30 seconds) / 50°C (30 seconds) / 72°C (1.5 minutes)
As a result, a DNA fragment of about 1.4 kbp was amplified.
was cloned using pCR2.1 plasmid (Invitrogen).
The nucleotide sequence of the obtained clone was determined by the dideoxy terminator method.
BI377 DNA Sequencer (Applied Biosystem)
The sequence identified is shown in SEQ ID NO: 23. This sequence contains an open reading frame of 1,356 bases (SEQ ID NO: 23).
The amino acid sequence predicted from the open reading frame (45
The predicted amino acid sequence is shown in SEQ ID NO: 18.
It has seven hydrophobic regions that are thought to be transmembrane domains, which are characteristic of the receptor.
It was revealed that this gene encodes a G protein-coupled receptor. Amplification of the novel G protein-coupled receptor GPRv51 requires the use of Marat
hon Ready cDNA (Clontech) was used as the template cDNA.
5'-ATGAACCAGACTTTGAATAGCA as word primer
GTGG-3' (SEQ ID NO: 31), 5'-TCAA as a reverse primer
GCCCCCATCTCATTGGTGCCCACG-3' (SEQ ID NO: 32)
PCR was performed using Pyrobest DNA polymerase (Takara Shuzo).
) was used, followed by 98°C (2.5 minutes), 98°C (30 seconds) / 50°C (30 seconds)
The cycle of 68°C (4 min)/68°C (4 min) was repeated 35 times.
A DNA fragment of 100 bp was amplified. This fragment was inserted into pCR2.1 plasmid (I
The nucleotide sequence of the obtained clone was
The sequence is ABI377 DNA Sequencing by the dideoxy terminator method.
The analysis was carried out using a .RTM. RT-PCR software (Applied Biosystems).
The resulting sequence is shown in SEQ ID NO: 24. This sequence has an open reading frame of 966 bases (SEQ ID NO: 24).
The amino acid sequence predicted from the open reading frame (321
The predicted amino acid sequence is shown in SEQ ID NO: 19.
It has seven hydrophobic regions that are thought to be transmembrane domains, which are characteristic of the body.
It was found that this gene encodes a G protein-coupled receptor. Amplification of the novel G protein-coupled receptor GPRv71 involved the use of Marat
hon Ready cDNA (Clontech) was used as the template cDNA.
5'-ATGGAGAAGGTGGACATGAATA as word primer
CATCAC-3' (SEQ ID NO: 33), 5'-TT as a reverse primer
ACCCAGATCTGTTCAACCCTGGGCATC-3' (SEQ ID NO:
PCR was performed using Pyrobest DNA polymerase (
Takara Shuzo) was used, and the temperature was 94°C (2.5 minutes), followed by 98°C (5 seconds)/72°C (4
5 cycles of 98°C (5 seconds) / 70°C (4 minutes)
The cycle of 98°C (5 seconds)/68°C (4 minutes) was repeated 25 times.
As a result, a DNA fragment of approximately 1.0 kbp was amplified.
Cloning was performed using asmid (Invitrogen).
The base sequence of the clone was determined by the dideoxy terminator method.
Analysis using Sequencer (Applied Biosystems)
The sequence identified is shown in SEQ ID NO: 25. This sequence contains an open reading frame of 1002 bases (SEQ ID NO: 25).
The amino acid sequence predicted from the open reading frame (33
The predicted amino acid sequence is shown in SEQ ID NO: 20.
It has seven hydrophobic regions that are thought to be transmembrane domains, which are characteristic of the receptor.
It was found that this gene encodes a G protein-coupled receptor. Amplification of the novel G protein-coupled receptor GPRv72 required human genomic DNA (Clo
The template DNA was 5'-ATGAC
GTCCACCTGCACCAACAGCACGC-3' (SEQ ID NO: 35),
5'-TCAAGGAAAGTAGCAGAATC as the reverse primer
GTAGGAAG-3' (SEQ ID NO: 36) was used. PCR was performed using Pyrobes
After using tDNA polymerase (Takara Shuzo) at 94°C (2 minutes),
30 cycles of 94°C (30 seconds) / 55°C (30 seconds) / 68°C (4 minutes)
As a result, a DNA fragment of about 1.5 kbp was amplified.
was cloned using pCR2.1 plasmid (Invitrogen).
The nucleotide sequence of the obtained clone was determined by the dideoxy terminator method.
BI377 DNA Sequencer (Applied Biosystem)
The sequence identified is shown in SEQ ID NO: 26. This sequence contains an open reading frame of 1,527 bases (SEQ ID NO: 26).
The amino acid sequence predicted from the open reading frame (50
The predicted amino acid sequence is shown in SEQ ID NO: 21.
It has seven hydrophobic regions that are thought to be transmembrane domains, which are characteristic of the receptor.
It was revealed that this gene encodes a G protein-coupled receptor. [Example 2] SWISS-PR in the amino acid sequence of a novel G protein-coupled receptor
BLAST search against OT BLAST against SWISS-PROT with the amino acid sequence of "GPRv8"
The search results are shown in Figure 1. "GPRv8" is the only known G protein-coupled receptor
HUMAN VASOPRESSIN V1B RECEPTOR (P4790
The highest homology was 36% with the nucleotide sequence (1,424 aa).
"GPRv8" was found to be a novel G protein-coupled receptor. BLAS against SWISS-PROT for the amino acid sequence of "GPRv12"
The search results are shown in Figure 2. "GPRv12" is one of the known G protein-coupled receptors.
RAT 5-HYDROXYTRYPTAMINE 6 RECEPTOR
(P31388, 436 aa) showed the highest homology of 27%.
This revealed that "GPRv12" is a novel G protein-coupled receptor.
. BLAS against SWISS-PROT for the amino acid sequence of "GPRv16"
The search results are shown in Figure 3. "GPRv16" is one of the known G protein-coupled receptors.
So, MOUSE GALANIN RECEPTOR TYPE 1 (P564
The highest homology was 28% with the nucleotide sequence (79,348 aa).
"GPRv16" was found to be a novel G protein-coupled receptor. BLAS against SWISS-PROT for the amino acid sequence of "GPRv21"
The search results are shown in Figure 4. "GPRv21" is one of the known G protein-coupled receptors.
So, "GPRv21" is one of the known G protein-coupled receptors.
ROPEPTIDE Y RECEPTOR TYPE 2 (P79113,3
The highest homology was 30% with GPRv (84 aa).
21" was found to be a novel G protein-coupled receptor. BLAS against SWISS-PROT for the amino acid sequence of "GPRv40"
The search results are shown in Figure 5. "GPRv40" is one of the known G protein-coupled receptors.
There is no other product that is the same as OXYTOCIN RECEPTOR (P9792
The highest homology was 34% with the nucleotide sequence of 6,388 aa.
"GPRv40" was found to be a novel G protein-coupled receptor. BLAS against SWISS-PROT for the amino acid sequence of "GPRv47"
The search results are shown in Figure 6. "GPRv47" is one of the known G protein-coupled receptors.
There is no other product that is the same as GPRX_ORYLA PROBABLE G P
ROTEIN-COUPLED RECEPTOR (Q91178,428aa
) showed the highest homology of 43%.
It was found to be a novel G protein-coupled receptor. BLAS against SWISS-PROT for the amino acid sequence of "GPRv51"
The search results are shown in Figure 7. "GPRv51" is the most abundant G protein-coupled receptor.
There is no identical one, PROBABLE G PROTEIN-COUP
37 for LED RECEPTOR RTA (P23749, 343aa)
This indicates that "GPRv51" is a novel G protein cofactor.
It was found to be a phospholipase C receptor. BLAS against SWISS-PROT for the amino acid sequence of "GPRv71"
The search results are shown in Figure 8. "GPRv71" is one of the known G protein-coupled receptors.
There is no identical product, Chicken P2Y PURINOCEPTO
The highest affinity was 45% for R3 (P2Y3) (Q98907, 328 aa).
This indicates that "GPRv71" is a novel G protein-coupled receptor.
It was found that the amino acid sequence of "GPRv72" was BLAS against SWISS-PROT.
The search results are shown in Figure 9. "GPRv72" is one of the known G protein-coupled receptors.
There is no identical one, ALPHA-1A ADRENNERGIC REC
It shows the highest homology of 30% to EPTOR (O02824, 466 aa).
This indicates that "GPRv72" is a novel G protein-coupled receptor.
It was found. [Example 3] Expression Tissue Analysis 1. Reagents 1.1 Quantitative Polymerase Chain Reaction (PCR)
) Primers and TaqMan probes: sense primer, antisense primer
The markers and TaqMan probes were from PE Biosystems.
Using the software Primer Express version 1.0
The conventional primers were designed by Amersham Pharmacia Biotech (Tokyo).
TaqMan probes are manufactured by PE Biosystems Japan.
The TaqMan probe has a reporter dye, FAM, at the 5' end.
The quencher tamra was attached to the 3' end.
The base sequence of the n probe is shown below. 1.2 Disease-derived cDNA cDNA derived from tumor and normal tissues of the same patient was obtained from Clontech's Mat
The tissues used were lung, stomach, colon, ovary, and prostate.
, uterus, and kidneys. The brain, pancreas, and testes of tumor patients and normal adults, and the liver of cirrhotic patients and normal adults.
, kidneys of lupus patients, hippocampus of Alzheimer's disease (AD) patients and normal adults
and cDNA derived from the frontal lobe was purchased from the BioChain Institute.
The following reagents were purchased and used: 1.3 Reagents for quantitative PCR reaction: TaqMan Universal PCR Master Mix (PE
TaqMan β-
actin Control Reagents (PE Biosystems)
) was used. 2. Quantitative PCR Reaction: 1) Dilution of Template cDNA BioChain cDNA was diluted 50-fold with water, and Clontech cDNA was used.
NA was diluted 5-fold with water before use. 2) Preparation of master mix A reaction solution with the following composition was prepared. 3) Preparation of PCR reaction solution Add 6 μl of template cDNA to 54 μl of master mix solution, then perform quantitative PCR.
Place 25 μl of duplicate sample wells on the PCR plate for the device.
The two non-template control wells were filled with the above
The master mix was dispensed in 25 μl portions.
The cDNA subcloned into the target was diluted starting from 100 pg/μl and then diluted to 1/10.
54 μl of the master mix of 2) and Stan
Add 6 μl of standard solution to each well and add 25 μl of the solution to each well.
That is, the highest concentration of 250p was dispensed into the standard well.
g, low concentration 25ag (a: atto, 10^-18) plasmid DNA is inserted
After attaching the 8-strip cap, the plate was briefly centrifuged to remove any air bubbles. 4) PCR Reaction The plate was placed in a quantitative PCR device (GeneAmp 5700 Sequence
Detection System (PE Biosystems)
The reaction was performed using the following operating program: 50°C, 2 minutes: 1 cycle 95°C, 10 minutes: 1 cycle 5) Quantitative Analysis Quantitative analysis was performed and output according to the GeneAmp 5700 operating manual.
3. Results and Summary: GPCR expression profiles using cDNA derived from normal and diseased human organs
The relative ratio was calculated using the expression level of the actin gene as an internal standard, and the results were compared in duplicate.
The average results are summarized in Table 1. Reproducible changes in expression of 3-fold or greater are considered significant.¹⁾From marked organs
cDNA was purchased from BioChain, and unmarked organ-derived cDNA
A was purchased from Clontech.
The following summarizes the changes in expression due to cancer. GPRv8 expression was not detected in normal pancreas or uterus, but was moderately elevated during cancer
It was expressed equally in the colon, but was expressed even more strongly in colon cancer. GPRv12 was weakly expressed overall. Expression was detected in normal ovaries and testes.
In Alzheimer's disease, expression was detected in the hippocampus.
was decreased. GPRv16 is expressed in the colon, but its expression became undetectable after canceration.
In the brain, expression increased with cancer. In the liver, expression became undetectable with cirrhosis.
In Alzheimer's disease brains, expression was increased in the hippocampus. GPRv21 expression was low, but expression was detected in the colon and testis due to cancer.
GPRv40 expression increased in the brain and testis due to cancer.
GPRv47 expression increased in the brain and kidneys and decreased in the testes due to canceration.
Expression in the liver became undetectable in cirrhosis. GPRv51 is strongly expressed in the colon, but expression is attenuated by cancer. Testis
In liver cirrhosis, expression was decreased compared to normal liver.
Although expression was weak in the brain, expression increased in the hippocampus in Alzheimer's disease. GPRv71 expression decreased in the colon and kidney due to canceration. In liver cirrhosis
Expression in the frontal lobe is reduced in Alzheimer's disease.
GPRv72 is strongly expressed in the colon, but its expression becomes undetectable after canceration.
Although expression was weak in the brain, expression increased in the hippocampus of patients with Alzheimer's disease. [Example 4] Bioinformatics Analysis of GPRv8 1. GPRv8 Homology Search The amino acid sequence of GPRv8 was compared with known sequences (known sequence databases include the EMBL
(Release 64, http://www.ebi.ac.uk/), G
ENBANK (Release 120.0, http://www.ncbi
．． nlm. nih. gov/), PIR (Release 66.00, http
p://www-nbrf.georgetown.edu/pir/)
) using the analysis program (BLAST 2.0) (Altschul, Step
hen F. et al. (1997) Nucleic Acids Res.
25:3389-3402.) was used to search for the sequence shown in Table 2.
It has been revealed that GPRv8 has homology with GPCRs.
The amino acid sequence of GPRv8 was found to be a novel clone.
The results of a search using the analysis program (blast 2.0) for
(those with lue less than e-39) are shown in Table 2. 2. Transmembrane site prediction The amino acid sequence of GPRv8 was used to predict the transmembrane site using the Kyte-Doolittle method (J
．． Kyte and R. F. Doolittle, (1982), J. Mol
. Biol., 157, 105-132.) to create a hydropathy plot.
As a result, GPRv8 has seven transmembrane domains (
It was found that the GPRv8 amino acid sequence was involved in the HMMPfam search (Figure 10). 3. HMMPfam search PF was performed using a hidden Markov model with the GPRv8 amino acid sequence as a query.
AM search (HMMPFAM (Sonnhammer EL, et al., Nu
Cleic Acids Res’1998 Jan 1;26(1):320
The hidden Markov model was performed using HMMER version 2.
1 (http://hmmer.wustl.edu/), PFAM database
The software is Pfam Version 5.5 (http://www.sanger
.ac.uk/Software/Pfam/index.shtml)
As a result, GPRv8 was found to be tm7_1 (Rhodopsin family)
It was found to have the following. Table 3 shows the results of the HMMPfam search. Hit: Name of the predicted domain resulting from the search. Score: The higher this value, the higher the confidence. Expect: The closer this value is to 0, the higher the confidence. Q from: Start position of the predicted domain Q to: End position of the predicted domain Description: Description of the predicted domain 4. Amino Acid Sequence Alignment Clustalw 1.7 was used to align the amino acid sequences of GPRv8 and the proteins in Table 2.
The sequences were aligned (Figs. 11 and 12). GPRv8 has seven transmembrane domains.
C, which has positions (#### ###) and is thought to perform SS binding specific to GPCRs.
It was found to contain a Cys (Cys with an @). [Example 5] Bioinformatics Analysis of GPRv12 1. Homology Search of GPRv12 The amino acid sequence of GPRv12 was compared with known sequences (known sequence databases include EMB
L (Release 64, http://www.ebi.ac.uk/),
GENBANK (Release 120.0, http://www.ncb
i. nlm. nih. gov/), PIR (Release 66.00, ht
tp://www-nbrf.georgetown.edu/pir/)
The analysis program (BLAST 2.0) (Altschul, Ste
phen F. et al. , (1997) Nucleic Acids Re
s. 25:3389-3402.), the sequence shown in Table 4 was obtained.
It was revealed that GPRv12 has homology with GPCR.
It was found to be a novel clone having the amino acid sequence of GPRv12.
The results of a search using an analysis program (blast 2.0) for known sequences (
E-values less than e-15) are shown in Table 4. 2. Transmembrane site prediction The amino acid sequence of GPRv12 was used to predict the transmembrane site using the Kyte-Doolittle method (
J. Kyte and R. F. Doolittle, (1982), J. Mo
1. Biol., 157, 105-132.)
As a result, GPRv12 has seven transmembrane regions.
It was found to have TM1-TM7 positions (Figure 13). 3. HMMPfam Search Using the amino acid sequence of GPRv12 as a query, a hidden Markov model was used to perform P
FAM search (HMMPFAM (Sonnhammer EL, et al., N
ucleic Acids Res 1998 Jan 1;26(1):32
The hidden Markov model was HMMER version 2.
．． 1 (http://hmmer.wustl.edu/), PFAM database
The source is Pfam Version 5.5 (http://www.sang
r.ac.uk/Software/Pfam/index.shtml)
As a result, GPRv12 was found to be tm7_1 (Rhodopsin family
) was found to have the following structure. Table 5 shows the results of the HMMPfam search. Hit: Name of the predicted domain resulting from the search. Score: The higher this value, the higher the confidence. Expect: The closer this value is to 0, the higher the confidence. Q from: Start position of the predicted domain Q to: End position of the predicted domain Description: Description of the predicted domain 4. Amino acid sequence alignment Clustalw 1.7 was used to align GPRv12 and orphan G pro
tein-coupled receptor GPR26-Rattus n
The amino acid sequence was aligned with that of C. orvegicus (AF208288).
This was achieved (Figure 14). GPRv12 has seven transmembrane domains (####).
Cys (with @) is thought to form a SS bond specific to GPCRs.
It was found that the amino acid sequence of GPRv16 is a homologous sequence of GPRv16. [Example 6] Bioinformatics Analysis of GPRv16 1. Homology Search of GPRv16 The amino acid sequence of GPRv16 was compared with known sequences (known sequence databases, such as EMB
L (Release 64, http://www.ebi.ac.uk/),
GENBANK (Release 120.0, http://www.ncb
i. nlm. nih. gov/), PIR (Release 66.00, ht
tp://www-nbrf.georgetown.edu/pir/)
The analysis program (BLAST 2.0) (Altschul, Ste
phen F. et al. (1997) Nucleic Acids Res
25:3389-3402.) was used to search, and the sequences shown in Table 6 were found.
It was revealed that GPRv16 has homology with GPCR.
It was found to be a novel clone that has the amino acid sequence of GPRv16.
The results of a search using an analysis program (blast 2.0) for known sequences (E
-value less than e-18) are shown in Table 6. 2. Transmembrane site prediction The amino acid sequence of GPRv16 was used to predict the transmembrane site using the Kyte-Doolittle method (
J. Kyte and R. F. Doolittle, (1982), J. Mo
1. Biol., 157, 105-132.)
As a result, GPRv16 has seven transmembrane regions.
It was found to have TM1-TM7 positions (Figure 15). 3. HMMPfam Search Using the amino acid sequence of GPRv16 as a query, a hidden Markov model was used to perform P
FAM search (HMMPFAM (Sonnhammer EL, et al., N
ucleic Acids Res 1998 Jan 1;26(1):32
The hidden Markov model was HMMER version 2.
．． 1 (http://hmmer.wustl.edu/), PFAM database
The source is Pfam Version 5.5 (http://www.sang
r.ac.uk/Software/Pfam/index.shtml)
As a result, GPRv16 was found to be tm7_1 (Rhodopsin family
) was found to have the following structure: Table 7 shows the results of the HMMPfam search. Hit: The name of the predicted domain as a result of the search. Score: The higher this value, the higher the confidence level. Expect: The closer this value is to 0, the higher the confidence level. Q from: The start position of the predicted domain. Q to: The end position of the predicted domain. Description: A description of the predicted domain. 4. The results of 3. and 4. are summarized in Figure 16. GPRv16 contains the GPCR-specific nucleotide sequence.
It was found to contain a Cys(@) that forms an S-S bond. [Example 7] Bioinformatics Analysis of GPRv21 1. Homology Search of GPRv21 The amino acid sequence of GPRv21 was compared with known sequences (known sequence databases include EMB
L (Release 64, http://www.ebi.ac.uk/),
GENBANK (Release 120.0, http://www.ncb
i. nlm. nih. gov/), PIR (Release 66.00, ht
tp://www-nbrf.georgetown.edu/pir/)
The analysis program (BLAST 2.0) (Altschul, Ste
phen F. et al. , (1997) Nucleic Acids Re
s. 25:3389-3402.), the sequence shown in Table 8 was obtained.
It was revealed that GPRv21 has homology with GPCR.
The amino acid sequence of GPRv21 was found to be a novel clone.
The results of a search using an analysis program (blast 2.0) for known sequences (
E-values less than e-35) are shown in Table 8. 2. Transmembrane site prediction The amino acid sequence of GPRv21 was used to predict the transmembrane site using the Kyte-Doolittle method (
J. Kyte and R. F. Doolittle, (1982), J. Mo
1. Biol., 157, 105-132.)
As a result, GPRv21 has seven transmembrane regions.
It was found to have TM1-TM7 positions (Figure 17). 3. HMMPfam Search Using the amino acid sequence of GPRv21 as a query, a hidden Markov model was used to perform P
FAM search (HMMPFAM (Sonnhammer EL, et al., N
ucleic Acids Res 1998 Jan 1;26(1):32
The hidden Markov model was HMMER version 2.
．． 1 (http://hmmer.wustl.edu/), PFAM database
The source is Pfam Version 5.5 (http://www.sang
r.ac.uk/Software/Pfam/index.shtml)
As a result, GPRv21 was found to be tm7_1 (Rhodopsin family
) was found to have the following structure: Table 9 shows the results of the HMMPfam search. Hit: The name of the predicted domain resulting from the search. Score: The higher this value, the higher the confidence. Expect: The closer this value is to 0, the higher the confidence. Q from: The start position of the predicted domain. Q to: The end position of the predicted domain. Description: A description of the predicted domain. 4. Amino Acid Sequence Alignment Clustalw 1.7 was used to align the amino acid sequences of GPRv21 with the proteins in Table 8.
The sequences were aligned (Figs. 18 and 19). GPRv8 contains seven transmembrane domains.
It has a binding site (######) and is thought to perform disulfide binding specific to GPCRs.
It was found that the amino acid sequence of GPRv40 contains a Cys (Cys with an @). [Example 8] Bioinformatics Analysis of GPRv40 1. Homology Search of GPRv40 The amino acid sequence of GPRv40 was compared with known sequences (known sequence databases include EMB
L (Release 64, http://www.ebi.ac.uk/),
GENBANK (Release 120.0, http://www.ncb
i. nlm. nih. gov/), PIR (Release 66.00, ht
tp://www-nbrf.georgetown.edu/pir/)
The analysis program (BLAST 2.0) (Altschul, Ste
phen F. et al. , (1997) Nucleic Acids Re
s. 25:3389-3402.), the sequence shown in Table 10 was obtained.
It was revealed that GPRv40 has homology with GPCR.
The amino acid sequence of GPRv40 was found to be a novel clone having the following structure:
The results of searching for known sequences using an analysis program (blast 2.0)
(E-value less than e-11) are shown in Table 10. 2. Transmembrane site prediction The amino acid sequence of GPRv40 was used to predict the transmembrane site using the Kyte-Doolittle method (
J. Kyte and R. F. Doolittle, (1982), J. Mo
1. Biol., 157, 105-132.)
As a result, GPRv40 has seven transmembrane regions.
It was found to have TM1-TM7 positions (Figure 20). 3. HMMPfam Search Using the amino acid sequence of GPRv40 as a query, a hidden Markov model was used to perform P
FAM search (HMMPFAM (Sonnhammer EL, et al., N
ucleic Acids Res 1998 Jan 1;26(1):32
The hidden Markov model was HMMER version 2.
．． 1 (http://hmmer.wustl.edu/), PFAM database
The source is Pfam Version 5.5 (http://www.sang
r.ac.uk/Software/Pfam/index.shtml)
As a result, GPRv40 was found to be tm7_1 (Rhodopsin family
) was found to have the following structure: Table 11 shows the results of the HMMPfam search. Hit: The name of the predicted domain as a result of the search. Score: The higher this value, the higher the confidence. Expect: The closer this value is to 0, the higher the confidence. Q from: The start position of the predicted domain. Q to: The end position of the predicted domain. Description: A description of the predicted domain. 4. The results of 3. and 4. are summarized in Figure 21. GPRv40 has the S domain characteristic of GPCRs.
It was found to contain a Cys(@) that forms an -S bond. [Example 9] Bioinformatics Analysis of GPRv47 1. Homology Search of GPRv47 The amino acid sequence of GPRv47 was compared with known sequences (known sequence databases include EMB
L (Release 64, http://www.ebi.ac.uk/),
GENBANK (Release 120.0, http://www.ncb
i. nlm. nih. gov/), PIR (Release 66.00, ht
tp://www-nbrf.georgetown.edu/pir/)
The analysis program (BLAST 2.0) (Altschul, Ste
phen F. et al. , (1997) Nucleic Acids Re
s. 25:3389-3402.), the sequence shown in Table 12 was obtained.
It was revealed that GPRv47 has homology with GPCR.
The amino acid sequence of GPRv47 was found to be:
The results of searching for known sequences using an analysis program (blast 2.0)
(E-value less than e-11) is shown in Table 12. 2. Transmembrane site prediction The amino acid sequence of GPRv47 was used to predict the transmembrane site using the Kyte-Doolittle method (
J. Kyte and R. F. Doolittle, (1982), J. Mo
1. Biol., 157, 105-132.)
As a result, GPRv47 has seven transmembrane regions.
It was found to have TM1-TM7 positions (Figure 22). 3. HMMPfam Search Using the amino acid sequence of GPRv47 as a query, a hidden Markov model was used to perform P
FAM search (HMMPFAM (Sonnhammer EL, et al., N
ucleic Acids Res 1998 Jan 1;26(1):32
The hidden Markov model was HMMER version 2.
．． 1 (http://hmmer.wustl.edu/), PFAM database
The source is Pfam Version 5.5 (http://www.sang
r.ac.uk/Software/Pfam/index.shtml)
As a result, GPRv47 was found to be tm7_1 (Rhodopsin family
) was found to have the following structure: Table 13 shows the results of the HMMPfam search. Hit: Name of the predicted domain resulting from the search. Score: The higher this value, the higher the confidence. Expect: The closer this value is to 0, the higher the confidence. Q from: Start position of the predicted domain Q to: End position of the predicted domain Description: Description of the predicted domain 4. Amino Acid Sequence Alignment Amino acid sequences of GPRv47 and similar proteins were aligned using Clustalw 1.7.
The sequences were aligned (Figures 23-25). GPRv8 has seven transmembrane domains.
It has a site (#### ###) and is thought to perform disulfide binding specific to GPCRs.
It was found to contain Cys (Cys with an @). [Example 10] Bioinformatics Analysis of GPRv51 1. Homology Search of GPRv51 The amino acid sequence of GPRv51 was compared with known sequences (known sequence databases include EMB
L (Release 64, http://www.ebi.ac.uk/),
GENBANK (Release 120.0, http://www.ncb
i. nlm. nih. gov/), PIR (Release 66.00, ht
tp://www-nbrf.georgetown.edu/pir/)
The analysis program (BLAST 2.0) (Altschul, Ste
phen F. et al. (1997) Nucleic Acids Res
25:3389-3402.) was used to search, the sequence shown in Table 14 was obtained.
It was revealed that GPRv51 has homology with GPCR.
The amino acid sequence of GPRv51 was found to be a novel clone.
The results of a search using an analysis program (blast 2.0) for known sequences (
E-values less than e-18) are shown in Table 14. 2. Transmembrane site prediction The amino acid sequence of GPRv51 was used to predict the transmembrane site using the Kyte-Doolittle method (
J. Kyte and R. F. Doolittle, (1982), J. Mo
1. Biol., 157, 105-132.)
As a result, GPRv51 has seven transmembrane regions.
It was found to have TM1-TM7 positions (Figure 26). 3. HMMPfam Search Using the amino acid sequence of GPRv51 as a query, a hidden Markov model was used to perform P
FAM search (HMMPFAM (Sonnhammer EL, et al., N
ucleic Acids Res 1998 Jan 1;26(1):32
The hidden Markov model was HMMER version 2.
．． 1 (http://hmmer.wustl.edu/), PFAM database
The source is Pfam Version 5.5 (http://www.sang
r.ac.uk/Software/Pfam/index.shtml)
As a result, GPRv51 was found to be tm7_1 (Rhodopsin family
) was found to have the following structure. Table 15 shows the results of the HMMPfam search. Hit: Name of the predicted domain resulting from the search. Score: The higher this value, the higher the confidence. Expect: The closer this value is to 0, the higher the confidence. Q from: Start position of the predicted domain Q to: End position of the predicted domain Description: Description of the predicted domain 4. Amino acid sequence alignment Clustalw 1.7 was used to align GPRv51 and G-protein cou
Amino acid sequence alignment with the pluripotent receptor-Rat (M35297)
GPRv51 has seven transmembrane domains (###
It was found that the GPRv71 amino acid sequence has the following structure: [Example 11] Bioinformatics Analysis of GPRv71 1. GPRv71 Homology Search The amino acid sequence of GPRv71 was compared with known sequences (known sequence databases include EMB
L (Release 64, http://www.ebi.ac.uk/),
GENBANK (Release 120.0, http://www.ncb
i. nlm. nih. gov/), PIR (Release 66.00, ht
tp://www-nbrf.georgetown.edu/pir/)
The analysis program (BLAST 2.0) (Altschul, Ste
phen F. et al. , (1997) Nucleic Acids Re
s. 25:3389-3402.), the sequence shown in Table 16 was obtained.
It was revealed that GPRv71 has homology with GPCR.
The amino acid sequence of GPRv71 was found to be:
The results of searching for known sequences using an analysis program (blast 2.0)
(E-value less than e-35) are shown in Table 16. 2. Transmembrane site prediction The amino acid sequence of GPRv71 was used to predict the transmembrane site using the Kyte-Doolittle method (
J. Kyte and R. F. Doolittle, (1982), J. Mo
1. Biol., 157, 105-132.)
As a result, GPRv71 has seven transmembrane regions.
It was found to have TM1-TM7 positions (Figure 28). 3. HMMPfam Search Using the amino acid sequence of GPRv71 as a query, a P2P search was performed using a hidden Markov model.
FAM search (HMMPFAM (Sonnhammer EL, et al., N
ucleic Acids Res 1998 Jan 1;26(1):32
The hidden Markov model was HMMER version 2.
．． 1 (http://hmmer.wustl.edu/), PFAM database
The source is Pfam Version 5.5 (http://www.sang
r.ac.uk/Software/Pfam/index.shtml)
As a result, GPRv71 was found to be tm7_1 (Rhodopsin family
) was found to have the following structure: Table 17 shows the results of the HMMPfam search. Hit: The name of the predicted domain resulting from the search. Score: The higher this value, the higher the confidence. Expect: The closer this value is to 0, the higher the confidence. Q from: The start position of the predicted domain. Q to: The end position of the predicted domain. Description: A description of the predicted domain. 4. Amino Acid Sequence Alignment Clustalw 1.7 was used to align the amino acid sequences of GPRv71 and its related proteins.
The sequences were aligned (Figs. 29 and 30). GPRv71 has seven membrane-binding domains.
It was found to have a transmembrane site (####). [Example 12] Bioinformatics Analysis of GPRv72 1. Homology Search of GPRv72 The amino acid sequence of GPRv72 was compared with known sequences (known sequence databases, such as EMB
L (Release 64, http://www.ebi.ac.uk/),
GENBANK (Release 120.0, http://www.ncb
i. nlm. nih. gov/), PIR (Release 66.00, ht
tp://www-nbrf.georgetown.edu/pir/)
The analysis program (BLAST 2.0) (Altschul, Ste
phen F. et al. , (1997) Nucleic Acids Re
s. 25:3389-3402.), the sequence shown in Table 18 was obtained.
It was revealed that GPRv72 has homology with GPCR.
The amino acid sequence of GPRv72 was found to be:
The results of searching for known sequences using an analysis program (blast 2.0)
(E-value less than e-24) are shown in Table 18. 2. Transmembrane site prediction The amino acid sequence of GPRv72 was used to predict the transmembrane site using the Kyte-Doolittle method (
J. Kyte and R. F. Doolittle, (1982), J. Mo
1. Biol., 157, 105-132.)
As a result, GPRv72 has seven transmembrane regions.
It was found to have TM1 to TM7 (Figure 31). 3. HMMPfam Search Using the amino acid sequence of GPRv72 as a query, a hidden Markov model was used to perform P
FAM search (HMMPFAM (Sonnhammer EL, et al., N
ucleic Acids Res 1998 Jan 1;26(1):32
The hidden Markov model was HMMER version 2.
．． 1 (http://hmmer.wustl.edu/), PFAM database
The source is Pfam Version 5.5 (http://www.sang
r.ac.uk/Software/Pfam/index.shtml)
As a result, GPRv72 was found to be tm7_1 (Rhodopsin family
) was found to have the following structure: Table 19 shows the results of the HMMPfam search. Hit: The name of the predicted domain resulting from the search. Score: The higher this value, the higher the confidence. Expect: The closer this value is to 0, the higher the confidence. Q from: The start position of the predicted domain. Q to: The end position of the predicted domain. Description: A description of the predicted domain. 4. Amino Acid Sequence Alignment Clustalw 1.7 was used to align the amino acid sequences of GPRv72 with those of the proteins in Table 18.
The sequences were aligned (Figures 32-34). GPRv72 has seven membrane-binding domains.
It has a transmembrane site (######) and is thought to perform SS binding specific to GPCRs.
It was found that the amino acid sequence of the amino acid sequence of the present invention is Cys (Cys with @).Possibility of industrial applicability The present invention provides novel G protein-coupled receptors (GPRv8, GPRv12 ...
Rv16, GPRv21, GPRv40, GPRv47, GPRv51, GPR
v71, GPRv72), genes encoding the proteins, and vectors containing the genes
Furthermore, the present invention provides a host cell containing the vector and a method for producing the protein.
A method for screening compounds that modify the activity of a protein is provided.
Compounds that modify the activity of proteins, their genes, or proteins of the present invention are also useful in the treatment of cancer.
To develop new preventive and therapeutic drugs for diseases involving G protein-coupled receptor proteins
It is expected to be used.

[Sequence List]

[Brief explanation of the drawings]

FIG. 1 shows the amino acid sequence of "GPRv8" as a "Query" and the SWISS-
1 shows the results of a BLAST search performed on the entire PROT sequence.
The amino acid sequence of GPRv12 was Query-based and showed 36% homology to the N VASOPRESSIN V1B RECEPTOR.
1 shows the results of a BLAST search for the entire RAT 5-HYDROXYTRYPTAMINE 6 RECEPTOR sequence.
% homology.
1 shows the results of a BLAST search performed on the entire MOU-PROT sequence.
It showed 28% homology to SE GALANIN RECEPTOR TYPE 1. Figure 4 shows the amino acid sequence of "GPRv21" as a "Query" and the results of SWIS
FIG. 1 shows the results of a BLAST search performed on the entire S-PROT sequence.
It showed 30% homology to VIN NEUROPEPTIDE YRECEPTOR TYPE 2. Figure 5 shows the results of the "GPRv40" amino acid sequence query performed on the SWISS
1 shows the results of a BLAST search performed on the entire OXY-PROT sequence.
The amino acid sequence of "GPRv47" was queried using the SWISS
1 shows the results of a BLAST search performed on the entire GPR-PROT sequence.
X_ORYLA PROBABLE G PROTEIN-COUPLED R
The amino acid sequence of "GPRv51" was queried using the SWISS
1 shows the results of a BLAST search performed on the entire PROT sequence.
BABLE G PROTEIN-COUPLED RECEPTOR RTA
The amino acid sequence of "GPRv71" was queried using the SWISS
45% to P2Y PURINOCEPTOR 3 (P2Y3) (Q98907).
FIG. 9 shows the homology of the "GPRv72" amino acid sequence to the SWISS
FIG. 1 shows the results of a BLAST search performed on the entire ALP-PROT sequence.
It showed 30% homology to HA-1A adrenaline receptor (O02824). Figure 10 shows the hydropathy plot of GPRv8. Figure 11 shows the alignment of GPRv8 with similar families. '*' means that the position is completely conserved in all sequences. ':' means that one of the following groups is conserved at that position: {STA}, {NEQK}, {NHQK}, {NDBQ}, {QH
{RK}, {MILV}, {MILF}, {HY}, {FYW} '.' means that one of the following groups is stored at that position: {CSA}, {ATV}, {SAG}, {STNK}, {STPA
}, {SGND}, {SNDEQK}, {NDEQHK}, {NEQHRK} Figure 12 is a continuation of Figure 11. Figure 13 shows the hydropathy plot of GPRv12. Figure 14 shows the alignment of GPRv12 and AF208288. '*' means that the position is completely conserved in all sequences. ':' means that one of the following groups is conserved at the position: {STA}, {NEQK}, {NHQK}, {NDBQ}, {QH
{RK}, {MILV}, {MILF}, {HY}, {FYW} '.' means that one of the following groups is stored at that position: {CSA}, {ATV}, {SAG}, {STNK}, {STPA
}, {SGND}, {SNDEQK}, {NDEQHK}, {NEQHRK} Figure 15 shows the hydropathy plot of GPRv16. Figure 16 is a diagram summarizing the HMMPFAM, transmembrane region, and S-S bonds of GPRv16. *** indicates the region assigned as 7tm_1 as a result of HMMPFAM. ### indicates the transmembrane region. @ indicates Cys that forms an S-S bond. Figure 17 shows the hydropathy plot of GPRv21. Figure 18 shows an alignment of GPRv21 and its similar proteins. '*' means that the position is completely conserved in all sequences. ':' means that one of the following groups is conserved at that position: {STA}, {NEQK}, {NHQK}, {NDBQ}, {QH
{RK}, {MILV}, {MILF}, {HY}, {FYW} '.' means that one of the following groups is stored at that position: {CSA}, {ATV}, {SAG}, {STNK}, {STPA
}, {SGND}, {SNDEQK}, {NDEQHK}, {NEQHRK} Figure 19 is a continuation of Figure 18. Figure 20 is a diagram showing the hydropathy plot of GPRv40. Figure 21 is a diagram summarizing the HMMPFAM, transmembrane region, and S-S bonds of GPRv40. *** indicates the region assigned as 7tm_1 as a result of HMMPFAM. ### indicates the transmembrane region. @ indicates a Cys that forms an S-S bond. Figure 22 is a diagram showing the hydropathy plot of GPRv47. Figure 23 is a diagram showing an alignment of GPRv47 and its similar proteins. '*' means that the position is completely conserved in all sequences. ':' means that one of the following groups is conserved at that position. {STA}, {NEQK}, {NHQK}, {NDBQ}, {QH
{RK}, {MILV}, {MILF}, {HY}, {FYW} '.' means that one of the following groups is stored at that position: {CSA}, {ATV}, {SAG}, {STNK}, {STPA
}, {SGND}, {SNDEQK}, {NDEQHK}, {NEQHRK} Figure 24 is a continuation of Figure 23. Figure 25 is a continuation of Figure 24. Figure 26 is a diagram showing the hydropathy plot of GPRv51. Figure 27 is a diagram showing the alignment of GPRv51 with similar proteins. '*' means that the position is completely conserved in all sequences. ':' means that one of the following groups is conserved at the position. {STA}, {NEQK}, {NHQK}, {NDBQ}, {QH
{RK}, {MILV}, {MILF}, {HY}, {FYW} '.' means that one of the following groups is stored at that position: {CSA}, {ATV}, {SAG}, {STNK}, {STPA
}, {SGND}, {SNDEQK}, {NDEQHK}, {NEQHRK} Figure 28 shows the hydropathy plot of GPRv71. Figure 29 shows the alignment of GPRv71 and its similar proteins. '*' means that the position is completely conserved in all sequences. ':' means that one of the following groups is conserved at that position: {STA}, {NEQK}, {NHQK}, {NDBQ}, {QH
{RK}, {MILV}, {MILF}, {HY}, {FYW} '.' means that one of the following groups is stored at that position: {CSA}, {ATV}, {SAG}, {STNK}, {STPA
}, {SGND}, {SNDEQK}, {NDEQHK}, {NEQHRK} Figure 30 is a continuation of Figure 29. Figure 31 shows the hydropathy plot of GPRv72. Figure 32 shows the alignment of GPRv72 and its similar proteins. '*' means that the position is completely conserved in all sequences. ':' means that one of the following groups is conserved at that position. {STA}, {NEQK}, {NHQK}, {NDBQ}, {QH
{RK}, {MILV}, {MILF}, {HY}, {FYW} '.' means that one of the following groups is stored at that position: {CSA}, {ATV}, {SAG}, {STNK}, {STPA
}, {SGND}, {SNDEQK}, {NDEQHK}, {NEQHRK} Figure 33 is a continuation of Figure 32. Figure 34 is a continuation of Figure 33.

───────────────────────────────────────────────────────── Continued from the front page (51) Int.Cl. ⁷ Identification Symbol FI C07K 14/705 C07K 14/705 16/28 16/28 C12N 1/15 C12N 1/15 1/19 1/19 1/21 1/21 5/10 C12P 21/02 C C12P 21/02 C12Q 1/02 C12Q 1/02 1/68 A 1/68 G01N 33/15 Z G01N 33/15 33/50 Z 33/50 33/53 D 33/53 M 33/566 33/566 C12N 5/00 A (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF) , BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), UA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, B Z, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, J P, KE, KG, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Makiko Suwa 1-24-4 Kamata, Ota-ku, Tokyo (72) Inventor: Tomoyasu Sugiyama 2-6-23-102 Kiyomidai, Kisarazu, Chiba (72) Inventor: Toshimitsu Kishimoto 2-2-6 Nihonbashi-Honcho, Chuo-ku, Tokyo Mitsubishi Pharma Corporation Tokyo Headquarters (72) Inventor: Koji Kanzaki 2-2-6 Nihonbashi-Honcho, Chuo-ku, Tokyo Mitsubishi Pharma Corporation Tokyo Headquarters (72) Inventor: Shinichiro Yasuda 2-2-6 Nihonbashi-Honcho, Chuo-ku, Tokyo Mitsubishi Pharma Corporation Tokyo Headquarters (72) Inventor: Yoshihisa Inoue 2-2-6 Nihonbashi-Honcho, Chuo-ku, Tokyo Mitsubishi Pharma Corporation Tokyo Headquarters (Note) This publication is based on the gazette published internationally by the International Bureau of WIPO. The effect of the international publication of the Japanese patent application (Japanese utility model registration application) related to this publication arises pursuant to Article 184-10, Paragraph 1 of the Patent Act (Article 48-13, Paragraph 2 of the Utility Model Act), and is unrelated to this publication.

Claims (17)

[Claims] [Claim 1] DNA encoding a guanosine triphosphate-binding protein-coupled receptor, as set forth in any one of (a) to (d) below: (a) DNA encoding a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4, 17 to 21. (b) DNA comprising a coding region for the base sequence set forth in any one of SEQ ID NOs: 5 to 8, 22 to 26. (c) DNA encoding a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4, 17 to 21 in which one or more amino acids have been substituted, deleted, added, and/or inserted. (d) DNA that hybridizes under stringent conditions to DNA consisting of the base sequence set forth in any one of SEQ ID NOs: 5 to 8, 22 to 26. 2. A DNA encoding a partial peptide of a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4 and 17 to 21. 3. A vector containing the DNA of claim 1 or 2. 4. A transformant harboring the DNA according to claim 1 or 2 or the vector according to claim 3. 5. A protein or peptide encoded by the DNA of claim 1 or 2. 6. The method according to claim 5, further comprising the steps of culturing the transformant according to claim 4 and recovering the expressed protein or peptide from the transformant or its culture supernatant.
A method for producing the protein or peptide described in . [Claim 7] A method for screening for a ligand that binds to the protein described in claim 5, comprising the steps of: (a) contacting a test sample with the protein or peptide described in claim 5; and (b) selecting a compound that binds to the protein or peptide. [Claim 8] A method for screening for a compound having activity that inhibits the binding between the protein of claim 5 and its ligand, comprising: (a) a step of contacting a ligand with the protein of claim 5 or a partial peptide thereof in the presence of a test sample, and detecting the binding activity between the protein or partial peptide thereof and the ligand; and (b) a step of selecting a compound that reduces the binding activity detected in step (a) by comparing it with the binding activity in the absence of the test sample. [Claim 9] A method for screening for a compound that inhibits or promotes the activity of the protein described in claim 5, comprising the steps of: (a) contacting a ligand of the protein with cells that express the protein in the presence of a test sample; (b) detecting changes in the cells due to the binding of the ligand to the protein; and (c) selecting a compound that suppresses or enhances the changes in the cells detected in step (b) compared to the changes in the cells in the absence of the test sample. 10. The method according to claim 8 or 9, wherein the change in the cell is a change in cAMP concentration or a change in calcium concentration. 11. An antibody that binds to the protein of claim 5. 12. A compound isolated by the screening method according to any one of claims 7 to 10. 13. A pharmaceutical composition comprising the compound according to claim 12 as an active ingredient. 14. The pharmaceutical composition according to claim 13, for the treatment of a disease selected from the group consisting of cancer, cirrhosis, and Alzheimer's disease. 15. A nucleotide having a chain length of at least 15 nucleotides, which is complementary to a DNA consisting of a base sequence set forth in any one of SEQ ID NOs: 5 to 8 and 22 to 26, or a complementary strand thereof. [Claim 16] A method for diagnosing a disease selected from the group consisting of cancer, cirrhosis, and Alzheimer's disease, comprising detecting expression of the DNA described in claim 1 in tissue associated with the disease from a subject, or detecting a mutation in the DNA described in claim 1 in the subject. 17. A diagnostic agent for a disease selected from the group consisting of cancer, liver cirrhosis, and Alzheimer's disease, comprising the antibody according to claim 11 or the nucleotide according to claim 15.