JP2003530834A - Human G protein-coupled receptors belonging to the isolated proto-oncogene subfamily, nucleic acid molecules encoding human GPCR proteins, and uses thereof - Google Patents

Abstract

(57) Abstract The present invention provides the amino acid sequence of the GPCR peptide of the present invention, which is encoded in the human genome. The invention particularly provides isolated peptides and nucleic acid molecules, methods for identifying orthologs and paralogs of GPCR peptides, and methods for identifying modulators of GPCR peptides.

Description

Detailed Description of the Invention

[0001]

【Technical field】

The present invention belongs to the field of G-protein coupled receptors (GPCRs) and relates to the MAS proto-oncogene receptor subfamily, recombinant DNA molecules and protein production. The present invention is particularly directed to a novel GPC for use in developing human therapeutic methods.
The present invention provides R peptides, proteins, and nucleic acid molecules encoding these peptides and protein molecules.

[0002]

[Background technology]G protein coupled receptor   G-protein coupled receptor (GPCR) is a protein responsible for signal transduction in cells.
It constitutes the main part of quality. GPCR consists of an amino-terminal extracellular domain, 7
A transmembrane domain containing a transmembrane segment and three extracellular loops and three cells
Three structural domains, the inner loop and the carboxy-terminal intracellular domain.
Have an inn. Signaling inside the cell when the ligand binds to the extracellular part of the GPCR
Are transmitted, resulting in a change in the biological or physiological properties of the cell. GPCR,
And G protein, effector (intracellular enzyme, char that is modulated by G-protein
Is a module that links the state of intracellular second messengers to extracellular inputs.
Is a component of the Raler signal system.

GPCR genes and gene products can be causative agents that cause disease (S
piegel et al., J. Clin. Invest. 92: 1119-1125 (1993); McKusick et al., J.
Med. Genet. 30: 1-26 (1993)). Causes various types of retinitis pigmentosa as defects unique to the rhodopsin gene and V2 vasopressin receptor gene (Nathans e
t al., Annu. Rev. Genet. 26: 403-424 (1992)) and causing renal diabetes insipidus (Holtzman et al., Hum. Mol. Genet. 2: 1201-1204 (1993)). It is shown.
These receptors are very important in central nervous system and peripheral physiological processes. Evolutionary analysis suggests that these proteins originally developed with complex body design and nervous system.

The GPCR protein superfamily includes family I: rhodopsin and β
Receptors for which over 200 unique members have been shown to date, represented by 2-purinergic receptors (Dohlman et al., Annu. Rev. Biochem. 60: 653-688.
(1991)); Family II: Parathyroid hormone / calcitonin / secretin receptor family (Juppner et al., Science 254: 1024-1026 (1991); Lin et al., Scien.
ce 254: 1022-1024 (1991)); Family III: Metabolic glutamate receptor family (Nakanishi, Science 258 597: 603 (1992)); Family IV: chemotaxis, and
The cAMP receptor family important for the development of D. discoideum (Klein et al., Scienc
e 241: 1467-1472 (1988)); Family V: fungal mating pheromone receptors such as STE2 (Kurjan, Annu. Rev. Biochem. 61: 1097-1129 (1992)). Can

Also included here are a few other proteins that have a putative seven hydrophobic segment and appear to be GPCR-independent, and these did not show G protein binding. Drosophila is a photoreceptor-specific protein, brid
A protein that expresses e-of-sevenless (boss) and has this seven transmembrane segment has been extensively studied, but no evidence of a GPCR has been shown (Hart et al., Proc. Natl. Acad. Sci. USA 90: 5047-505
1 (1993)). A small gene (fz) in Drosophila is also considered to be a protein with seven transmembrane segments. Like boss, fz also showed no binding to G-proteins (Vinson et al., Nature 338: 263-264 (1
989)).

G-proteins are a family of heterotrimeric proteins composed of α, β, and γ subunits that bind guanine nucleotides. These proteins are
It is usually bound to a cell surface receptor, such as a receptor containing seven transmembrane segments. Following the binding of the ligand to the GPCR, a conformational change is transmitted to the G protein, which exchanges the GDP molecule bound to the α subunit with the GTP molecule, and separates it from the β and γ subunits. cause. The GTP binding mode of the α subunit typically functions as an effector modulator,
P (eg, by activation of adenyl cyclase), diacylglycerol,
Leads to the production of second messengers such as inositol phosphates. Over 20 different α subunits are known in humans. These subunits are associated with a smaller pool of β, γ subunits. Examples of mammalian G proteins include Gi, Go, Gq, Gs, and Gt. G proteins are described in Lodish et al., Molecular Cell Biology, (Scientific American Books In
c., New York, NY, 1995), the contents of which are incorporated herein by reference. About GPCR, G protein, G protein coupling effector, and second messenger system, The G-Protein Link
ed Receptor Fact Book, Watson et al., eds., Academic Press (1994).

[0007]MAS1 oncogene-like receptor   The human MAS1 oncogene was initially transformed by conversion into N1H3T3 cells.
Was found. The MAS1 oncogene is cotransfected, tumorigenic
It was isolated from the epidermoid oncogene cell line using E. coli (Young et al., 1984). Predicted
Based on the amino acid sequence shown, the MAS1 gene product is
It contains a transmembrane domain. Therefore, MAS1 protein probably
It is an intramembrane protein. The protein encoded by MAS1 is a receptor,
It can activate and modulate pathogenic components of the growth-regulatory pathway, leading to carcinogenic effects.
Jacson et al. (1988) found that the MAS oncogene was similar to the substance K receptor.
It was shown to be an array. Based on this, they found that inositol lipids
It encodes a peptide receptor with mitogenic activity that acts through a signaling pathway.
I expected it to be one. Xenopus egg cells, and transfected
Expression in a mammalian cell line that drives the MAS gene product into a functional angiotensin
It was demonstrated to be a receptor.

[0008] Ross et al. (1990) identified the RTA receptor, a GPCR associated with the MAS1 oncogene. RTA is abundantly expressed throughout the intestine, vas deferens, uterus, and aorta, but is only slightly detected in the liver, kidney, lung, and salivary gland. In the rat brain, RTA sequences are present in very high amounts in the cerebellum. RTA is most associated with the MAS oncogene, which is thought to become the forebrain angiotensin receptor (34% concordance). However, cDNA or mRNA, respectively,
After introduction into OS cells, Xenopus oocytes, binding of angiotensin to RTA receptors was not detected. Therefore, it was concluded that RTA is not an angiotensin receptor.

Young et al. (1988) et al. Cloned the MAS oncogene in rats, sequenced its DNA and investigated its expression in various rat tissues. Rat and human MAS
Comparison of the predicted sequences of the proteins showed that they were highly conserved except for the hydrophilic amino-terminal domain. In the hippocampus of the brain and the cerebral cortex, MA
High levels of SRNA transcription were detected, but not in neural tissue areas or other tissues. This expression pattern, and the similarities between MAS proteins and known receptor proteins, suggest that MAS encodes receptors that are associated with normal neurophysiology and / or development of specific neural tissues. (1. Ross P
C, et al., A candidate G protein-coupled receptor: cloning, sequencing,
and tissue distribution.Proc Natl Acad Sci USA 1990 Apr; 87 (8): 3052-6;
2. Young D, et al., Characterization of the rat mas oncogene and its hi
gh-level expression in the hippocampus and cerebral cortex of rat brain.
, Proc Natl Acad Sci USA 1988 Jul; 85 (14): 5339-42; 3) Young, D .; et al.
, Isolation and characterization of a new cellular oncogene encoding ap
rotein with multiple transmembrane domains. Cell 45: 711-719, 1984; and
4) Jackson, TR, et al., The mas oncogene encodes an angiotensin recep
tor. Nature 335: 437-440, 1988).

[0010] GPCRs, especially members of the MAS protooncogene receptor subfamily, are involved in drug behaviour, and development, particularly in the regulation of cells expressing this receptor (eg cancer therapy),
And regulation of cellular signals associated with expression of this receptor (eg modulation of thyroid activity)
Is an important target in. Therefore, identifying and characterizing previously unknown GPCRs is very useful in the field of pharmaceutical development. The present invention contributes to the progress of these technologies by providing a human GPCR that has not been confirmed so far.

[0011]

SUMMARY OF THE INVENTION

The present invention is based, in part, on the amino acid sequence identification of human GPCR peptides and proteins and relates to the MAS protooncogene receptor subfamily, their allelic variants, and their orthologs in other mammals. It is a thing. These unique peptide sequences and nucleic acid sequences encoding these peptides can be used as models for the development of human therapeutic targets, are useful for protein identification for therapeutic use, and are useful in human therapeutic agents. It can be a target for development.

The proteins according to the present invention include MAS in cells expressing these proteins.
GPCRs associated with signaling pathways mediated by protooncogene receptor subfamily
(See expression information in FIG. 1: screening of tissue-specific libraries shows that the GPCRs of the invention are expressed in at least brain, placenta, and thyroid).
. “Signal pathway” as used herein refers to the modulation (eg, promotion or inhibition) of cell function / activity in the binding of a ligand to a GPCR protein. Examples of these functions include, for example, phosphatidylinositol 4,5-
Bisphosphate (PIP ₂ ), inositol 1,4,5-triphosphate (I
P ₃ ), Mobilization of intracellular molecules involved in signal transduction pathways such as adenylate cyclase, polarization of plasma membrane, production or secretion of molecules, structural change of cellular components, cell proliferation such as DNA synthesis, cell migration , Cell differentiation, cell survival.

The response mediated by the receptor protein depends on the type of cell expressed. Information on cell types expressing other members of the GPCR subfamily of the present invention is already known in the art (background art, information on highly homologous proteins shown in Figure 2 and shown in Figure 1). Expression information: In the screening of a tissue-specific library, the GPCR of the present invention is
It has been shown to be expressed in placenta and thyroid gland). For example, in some cells, binding of a ligand to a receptor protein results in compound release, channel gating, cell adhesion, cell migration, cell differentiation, etc. through metabolism and turnover of phosphatidylinositol or cyclic AMP. Promote activity such as. On the other hand, in other cells, binding of the ligand produces different results. Specific G of the Invention
Regardless of the cellular activity / response modulated by PCR, a receptor protein that is a GPCR is introduced into a biological pathway in the cell or tissue in which the GPCR is expressed, eg, phosphatidylinositol, or cyclic AM.
It is well known in the art to interact with G proteins to generate one or more secondary signals in various intracellular signaling pathways through the metabolism and turnover of P.

“Phosphatidylinositol turnover and metabolism,” as used herein, refers to phosphatidylinositol 4,5-bisphosphate (PIP ₂ ) turnover and metabolism and molecules involved in these activities. Point to. PIP ₂ is a phospholipid found in intracytoplasmic leaflets of the plasma membrane. In some cells, binding of the ligand to the receptor activates the plasma membrane enzyme phospholipase C, leading to PI
P ₂ is subsequently hydrolyzed to produce 1,2-diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP ₃ ). I once generated
P ₃ is diffused onto the endoplasmic reticulum surface where it can bind to IP ₃ receptors such as calcium channel proteins that have IP ₃ binding sites. The IP ₃ binding opens the channel and allows the release of calcium ions into the cytoplasm. Also, IP ₃ can also be phosphorylated to the specific kinases 1,3,4,5 tetraphosphate (IP _4), this molecule is flowed calcium from the extracellular medium into the cytoplasm. IP ₃ and IP ₄ are hydrolyzed very rapidly in a chain to inactive products such as 1,4-biphosphate (IP ₂ ), inositol 1,3,4-triphosphate, respectively. These inactive products lead to PI in cells.
Reused for P ₂ synthesis. Another second messenger generated by the hydrolysis PIP _2, namely 1,2-diacylglycerol (DAG) can be left in the cell membrane, to activate the enzyme protein kinase C here. Although protein kinase C is normally dissolved in the cytoplasm of cells, it migrates to the plasma membrane when intracellular calcium concentration rises, where it is activated by DAG. Activation of protein kinase C in other cells results from various cellular responses such as, for example, phosphorylation of glycogen synthase or phosphorylation of various transcription factors such as NF-kB. The term “phosphatidylinositol activity” as used herein refers to the activity of PIP ₂ or one of its metabolites.

Another signaling pathway that may be associated with the receptor is the cAMP turnover pathway. As used herein, “cyclic AMP turnover and metabolism” refers to cyclic AMP (cAMP) turnover and metabolism and molecules involved in these activities. Cyclic AMP is a second messenger produced in response to ligand-derived stimuli of specific G protein-coupled receptors. In the cAMP signal pathway, binding of ligand to GPCR results in cAM
The enzyme adenyl cyclase, which catalyzes P synthesis, is activated. Newly synthesized c
AMP in turn activates cAMP-dependent protein kinases. This activated kinase phosphorylates voltage-gated potassium channel proteins and related proteins, which renders them unable to open potassium channels even at action potentials. The inability of the potassium channels to open leads to an increase in potassium in the external fluid, leading to sustained depolarization within the membrane of the normally repolarized nerve cell membrane.

By targeting agents that modulate GPCRs, signal activity and receptor-mediated biological processes can be agonized or antagonized in specific cells and tissues (of tissue-specific libraries). Screening has shown that the GPCRs of the invention are expressed in at least the brain, placenta and thyroid). Such agonism or antagonism is based on modulation of biological activity in a therapeutic setting (mammalian therapy) or a toxic setting (anti-cell therapy such as anti-cancer agents).

[0017]

BEST MODE FOR CARRYING OUT THE INVENTIONOverview   The present invention is based on sequence analysis of the human genome. Sequence analysis of human genome and
By analyzing the sequence information in the construction, MAS cancer
It is a GPCR protein related to the original gene subfamily, or a part thereof.
Structural and / or sequence phase for the identified protein / peptide / domain
A previously unidentified fragment of the human genome encoding a homologous peptide.
Became clear. These sequences are used to construct additional genomic sequences and
And / or cDNA sequences were isolated and characterized. Based on this analysis, the present invention
, Amino acid sequences of human GPCR peptides and proteins, these GPCR peptides
Nucleic acid sequences, cDNA sequences and / or in transcribed form encoding tides and proteins
Is the genomic sequence, nucleic acid variation (allelic information), tissue distribution of expression, and G of the present invention.
Most related known tamper with structural or sequence homology to PCR
It provides information on proteins / peptides / domains.

The peptides provided in the present invention can be selected based on their utility in the development of commercially important products and services, in addition to being previously unknown. In particular, the peptides of the invention are selected on the basis of their homology and / or structural correlation to known GPCR proteins in the MAS protooncogene subfamily and their expression patterns ( Screening of tissue-specific libraries has shown that the GPCRs of the invention are expressed in at least the brain, placenta and thyroid). This technique establishes commercial importance in this family of proteins and peptides with expression patterns similar to the genes of the invention. More specific properties of the peptides of the invention and their use are well known in the present specification, especially in the background, drawings, and / or known MAS protooncogene family or GPCR protein subfamily, respectively. Is.

[0019]

[Details of Examples]Peptide molecule   The present invention provides a tag identified as a member of the GPCR family of proteins.
It provides a nucleic acid sequence that encodes a protein molecule (see Figure 2 for proteins).
Sequence, FIG. 1 shows the transcription / cDNA sequence, and FIG. 3 shows the genomic sequence). Provided in Figure 2
Peptide sequences, as well as obvious mutations such as allelic variants described herein.
The body is herein defined as a GPCR peptide of the invention, a GPCR peptide of the invention, or a peptide.
Called peptide / protein.

The present invention provides isolated peptide and protein molecules consisting of, consisting essentially of, or containing the amino acid sequence of the GPCR peptide shown in FIG. 2 (transcript / cDNA of FIG. 1, or FIG. 3). (Encoded by the nucleic acid molecule shown in the genomic sequence of E. coli), as well as obvious variants of these peptides prepared and used by the present technology. These variants are detailed below.

As used herein, a peptide is said to be “isolated” or “purified” if the peptide is substantially free of cellular material or free of chemical precursors or other chemicals. The peptides of the invention can be purified to homogeneity or other purity. The level of purification depends on the intended use. An important property is that it can exert the required peptide function even in the presence of large amounts of other components in the preparation (the properties of the isolated nucleic acid molecule are described below).

According to some examples, “substantially free of cellular material” means less than about 30% other proteins (ie, contaminating proteins) (dry weight), less than about 20% other proteins, Less than about 10% other proteins, or about 5% other proteins
Including peptide preparations having less than. If the peptide is recombinantly produced,
Substantially no medium can be present when the medium is less than 20% by volume of the protein preparation.

The term “substantially free of chemical precursors or other chemicals” refers to peptide preparations separated from the chemical precursors or other chemicals involved in the synthesis.
In one example, "substantially free of chemical precursors or other chemicals" means less than about 30% (dry weight) chemical precursors or other chemicals, chemical precursors or other chemicals. Of less than about 20%, less than about 10% of chemical precursors or other chemicals, or less than about 5% of chemical precursors or other chemicals.

The isolated GPCR peptides can be purified from naturally expressing cells, cells that have been modified for expression (recombinant), or can be synthesized using known protein synthesis methods (tissue-specific libraries. Of the GPCR of the present invention
Indicates that it is expressed in at least the brain, placenta, and thyroid gland). For example, GP
The nucleic acid molecule encoding the CR peptide is cloned into an expression vector, which is then introduced into a host cell so that the protein is expressed in the host cell. The protein can then be isolated from the cells by a suitable purification scheme using standard protein purification techniques. Many of these techniques are detailed below.

Therefore, the present invention provides the amino acid sequence (SEQ ID NO. 2) shown in FIG.
), For example, the transcription / cDNA nucleic acid sequence (SE
Q ID NO. 1) and the protein encoded by the genomic sequence (SEQ ID NO. 3) shown in FIG. The amino acid sequence of such a protein is shown in FIG. A protein consists of an amino acid sequence when the final amino acid sequence of the protein is this amino acid sequence.

The present invention further provides a protein consisting essentially of the amino acid sequence shown in Figure 2 (SEQ ID NO. 2), eg, the transcribed / cDNA nucleic acid sequence shown in Figure 1 (S
EQ ID NO. 1) and the genomic sequence shown in FIG. 3 (SEQ ID NO. 3)
It provides a protein encoded by: When such an amino acid sequence has a trace amount of additional amino acid residues, for example, about 1 to 100 additional residues, typically 1 to 20 additional residues, are present in the protein, the protein is It consists essentially of an array.

The present invention further includes a protein containing the amino acid sequence shown in FIG. 2 (SEQ ID NO. 2), such as the transcription / cDNA nucleic acid sequence shown in FIG. 1 (SEQ ID N.
O. 1) and the genomic sequence shown in FIG. 3 (SEQ ID NO. 3). A protein comprises an amino acid sequence when the amino acid sequence is at least part of the final amino acid sequence of the protein. In such cases, the protein may be a peptide only, or additional amino acids such as amino acid residues naturally adjacent to the protein (adjacent to the encoded sequence) or non-homologous amino acid residue / peptide sequences. Can have different amino acids. Such proteins may have small amounts of additional amino acid residues or may contain hundreds or more additional amino acids. A preferred species of protein that includes the GPCR peptides of the invention is the naturally occurring mature protein.
The method for preparing / isolating these various proteins will be briefly described below.

The GPCR peptides of the invention may be used to form chimeric or fusion proteins,
It can bind to heterologous sequences. Such chimeric and fusion proteins include a GPCR peptide that is operatively linked to a heterologous protein having an amino acid sequence that is substantially homologous to the GPCR peptide. "Effectively bound" indicates that the GPCR peptide and the heterologous protein are fused in frame. The heterologous protein can be fused to the N-terminus or C-terminus of the GPCR peptide.

In some examples, the fusion protein does not affect the activity of the GPCR peptide itself. Examples of fusion proteins include β-galactosidase fusion, yeast 2-hybrid GAL fusion, polyHis fusion, MYC labeling, HI labeling and Ig.
Enzyme fusion proteins such as fusions are included, but fusion proteins are not limited thereto. Such fusion proteins, especially polyHis fusions, are useful in the purification of recombinant GPCR peptides. In certain host cells (eg, mammalian host cells), protein expression and / or secretion can be increased by using heterologous signal sequences.

Chimeric or fusion proteins can be produced by standard recombinant DNA techniques. For example, DNA fragments encoding different protein sequences are placed together in frame according to conventional techniques. In another example, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, the chimeric gene sequence can be reamplified by using an anchor primer for PCR amplification of the gene fragment, forming a complementary overhang between two consecutive gene fragments and then annealing (Ausubel et al. al., Current
See Protocols in Molecular Biology, 1992). Furthermore, many expression vectors that already encode a fusion moiety (for example, GST protein) are commercially available. The nucleic acid encoding the GPCR peptide can be cloned into such an expression vector such that the fusion region is linked in frame to the GPCR peptide.

As explained above, the present invention also relates to proteins of the present invention, such as natural peptide mature forms, allelic / sequence variants of peptides, non-natural recombination-induced variants, orthologs and paralogs of peptides. It provides and enables feasible variants in the amino acid sequence. Such a mutant can be easily produced by using a technique known in the fields of nucleic acid recombination technology and protein biochemistry. However, it is understood that this variant does not include any of the amino acid sequences published prior to the present invention.

Such a mutant can be easily identified / manufactured by using the molecular technique and sequence information shown in the present invention. Moreover, such variants can be readily distinguished from other peptides based on sequence and / or structural homology to the GPCR peptides of the invention. The degree of homology / identity is based mainly on whether the peptides are functional or non-functional variants, the amount of differentiation present in the paralog family, the evolutionary differences between orthologs .

To determine the percent identity of two amino acid sequences, or two nucleic acid sequences, the sequences are aligned (eg, for optimal sequence comparison, gaps are separated by gaps) for the purpose of making optimal comparisons. Introduced into one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences can be ignored for comparison). Suitable examples include at least 30%, 40%, 50%, 60%, 70%, 80 of the length of the subject sequence.
%, 90% or more are aligned for comparison purposes. Then, amino acid residues or nucleotides on the corresponding amino acid positions or nucleotide positions are compared. When a configuration in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding configuration in the second sequence, the molecules are identical in that configuration (
As used herein, "identity" of amino acids or nucleic acids refers to "identity" of amino acids or nucleic acids.
Synonymous with "homology"). The percent identity between two sequences is a function of the number of identical configurations shared in the sequences, taking into account the number of gaps and each gap length,
Gaps need to be introduced so that the two sequences can be optimally compared.

Sequence comparison and determination of percent identity and similarity between two sequences is
Can be done using mathematical algorithms (Computational Molecular Biol
ogy, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomput
ing: Informatics and Genome Projects, Smith, DW, ed., Academic Press,
New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM
., and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Ana
lysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Se
quence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton
Press, New York, 1991). As a suitable example, the percent identity between two amino acid sequences is determined by the Needleman-Wunsch algorithm (J. Mol. J. Mol., J. Mol. Biol. (48): 444-453 (1970)) using Blossom 62 matrix or PAM250 matrix and gap weights 16, 14, 1
2, 10, 8, 6, 4, determined using length weights 1, 2, 3, 4, 5, 6.
In another preferred example, the percent identity between the two nucleotide sequences is GC
NWSgapdna.CMP using the GAP program (Devereux, J., et al., Nucleic Acids Res. 12 (1): 387 (1984)) of the G software package (available at http://www.gcg.com). Matrix, gap weight 40, 50, 60,
70, 80 and length weights 1, 2, 3, 4, 5, 6 determined. As yet another example, the percent identity between two amino acid or nucleotide sequences is
The E.I. program incorporated in the ALIGN program (version 2.0). Myers W
Using the Miller algorithm (CABIOS, 4: 11-17 (1989)), PAM120
It is determined using the weight residue table, the gap length penalty of 12, and the gap penalty of 4.

The nucleic acid and protein sequences of the present invention can be used as a “query sequence” to search a sequence database, for example to discover other families or related sequences. Such a search is available in NBLA by Altschul et al.
ST and XBLAST programs (version 2.0) (J. Mol. Biol. 215: 4)
03-10 (1990)). BLAST nucleotide search
To obtain a nucleotide sequence homologous to the nucleic acid molecule of the present invention, NBLAST
It can be performed by using a program, score = 100, wordlength = 12. The BLAST protein search uses the XBLAST program to obtain amino acid sequences homologous to the protein of the present invention, and score = 50.
, Wordlength = 3. To obtain gap sequences for comparison purposes, Altschul et al. Gapped BLAST (Nucleic Acids Res.
25 (17): 3389-3402 (1997)) can be used. BLAST and Gappe
When using the dBLAST program, each program (for example, XBLAST
Or NBLAST) default parameters can be used.

The full-length morphology of the protein comprising one of the peptides of the invention before and after undergoing maturation processing has perfect sequence identity to one of the GPCR peptides of the invention, It can be easily identified as being encoded by the same gene position as the GPCR peptide of the present invention. The RH panel map shows that the GPCR of the present invention is encoded by the gene shown on chromosome 11 in the vicinity of markers SHGC-32486 and SHGC-20653 (LOD score 12.02).

An allelic variant of a GPCR peptide is a human protein that has a high degree of (significant) sequence homology / identity to at least a portion of the GPCR peptide,
Similarly, it can be easily identified as being encoded at the same gene position as the GPCR peptide of the invention. The gene location can be readily determined based on the genomic information shown in Figure 3, such as the genomic sequence mapped to the human of interest (in the RH panel map, the GPCR of the invention is:
Markers SHGC-32486 and SHGC-20653 (LO on chromosome 11
It is shown that it is encoded by the gene shown in the vicinity of D score 12.02). ). As used herein, in the amino acid sequence, typically at least about 70-80%, 80-90%, more typically at least about 90-95.
Two proteins (or regions of a protein) are significantly homologous if they have a% or greater homology. According to the present invention, an amino acid sequence having significant homology is encoded by a nucleic acid sequence which hybridizes with a nucleic acid molecule encoding a GPCR peptide under stringent conditions as described in more detail below. To be done.

FIG. 3 provides SNP information discovered from the gene encoding the GPCR protein of the present invention. Here, mutations of C1435G, T1262C, and A532G were found.

A paralog of a GPCR peptide has some significant sequence homology / identity to at least a portion of the GPCR peptide, is encoded by a human gene, and has similar activity or function. Can be easily identified. Two proteins are considered to be typical paralogs if the amino acid sequences typically have at least about 60% or more, and more typically 70% or more homology through a given region or domain. Paralogs like this
More specifically, it is encoded by a nucleic acid sequence that hybridizes to a nucleic acid molecule encoding a GPCR peptide under moderate to stringent conditions, as described below.

GPCR peptide orthologs have some significant sequence homology / identity to at least a portion of the GPCR peptide and are readily identified as being encoded by genes of other organisms. You can Orthologs are preferably isolated from mammals, more preferably primates, and used for the development of human therapeutic targets and therapeutic agents. Such orthologs can be isolated from GPC under moderate to stringent conditions, as described in more detail below.
Encoded by a nucleic acid sequence that hybridizes to a nucleic acid molecule that encodes an R peptide, which depends on the association of the two organisms that produce the protein.

Non-naturally occurring variants of the GPCR peptides of this invention can be readily produced using recombinant techniques. Such variants include, but are not limited to, deletions, additions, and substitutions in the amino acid sequence of the GPCR peptide. For example, one type of substitution includes conservative amino acid substitutions. This substitution is such that a particular amino acid in the GPCR peptide is replaced by another amino acid with similar characteristics. In the conservative substitution, one of the aliphatic amino acids Ala, Val, Leu, and Ile is substituted with the other, exchange of a hydroxyl residue Ser with Thr, exchange of an acidic residue Asp with Glu, amide, Residue As
There are substitutions between n and Gln, exchange of basic residues Lys and Arg, and substitution of aromatic residues Phe and Tyl. Guidelines for amino acid substitutions that do not appear phenotypically are set forth in Bowie et al., Science 247: 1306-1310 (1990).

The mutant GPCR peptide may be fully functional or may be deficient in one or more of its activities such as ligand binding capacity, G protein binding capacity, signal transduction mediating capacity, etc. . Fully functional variants typically include only conservative mutations or variants within non-lethal residues or regions. The results of the protein analysis are shown in Figure 2 and can be used to identify lethal domains / regions. Functional variants also include substitutions of similar amino acids that do not change function or have no significant change in function. On the other hand, such substitutions may have some positive or negative impact on function.

Non-functional variants typically include one or more non-conservative amino acid substitutions, deletions, introductions, inversions, truncations, or substitutions, deletions at lethal residues or regions. Lost, introduced,
Includes inversion.

Amino acids essential for function are, for example, site-directed mutagenesis or alanine scanning mutagenesis (Cunningham et al., Science 244: 1081-1085 (1
989)) and the like, which can be confirmed by a method known in the art, particularly using the results shown in FIG. Alanine scanning mutagenesis involves single alanine mutations at every residue in the molecule. The resulting mutant molecule is
It is then tested for biological activity such as ligand / effector molecule binding, or assays such as in vitro proliferative activity assays. The important site for ligand / receptor binding is determined by structural analysis such as crystallization, nuclear magnetic resonance, and optical affinity labeling (Smith et al., J. Mol. Biol. 224: 899-904 (1992). de Vos et al. Scien
ce 255: 306-312 (1992)).

The present invention provides fragments of GPCR peptides, and additionally provides proteins and peptides comprising and consisting of said fragments, especially the fragments identified in FIG. However, fragments related to the present invention are not considered to include fragments published prior to the present invention.

Fragments, as used herein, contain at least 8, 10, 12, 14, 16 or more amino acid residues flanking the GPCR peptide. Such fragments may be selected for their ability to retain the biological activity of one or more GPCR peptides, or for their ability to perform functions such as binding to ligands, effector molecules, or behaving as antigens. Particularly important fragments are bioactive fragments, which are, for example, peptides of 8 or more amino acids. Such fragments typically include domains or motifs of GPCR peptides, such as G protein binding sites, transmembrane domains, or ligand binding domains. Furthermore, possible fragments include, but are not limited to, domain or motif containing fragments, lytic peptide fragments, antigenic structure containing fragments. The predicted domains and functional sites can be easily confirmed by known computer programs (eg, PROSITE analysis) that are easily available to those skilled in the art. One of the results of such analysis is shown in FIG.

Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. In addition, many amino acids, including terminal amino acids, can be modified by natural processes such as processing and post-translational modification, or by chemical modification techniques known in the art. General modifications that occur naturally in GPCR peptides are described in the basic text, detailed research papers and literature, which are well known to those skilled in the art (some of these properties are shown in FIG. 2). It is confirmed).

Known modifications include acetylation, acylation, ADP ribosylation, amidation,
Flavin covalent addition, heme moiety covalent addition, nucleotide or nucleotide derivative covalent addition, lipid or lipid derivative covalent addition, phosphatidylinositol covalent addition, cross-linking, cyclization, disulfide bond formation, demethylation. , Formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing , Phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation, etc., including transfer RNA-mediated addition of amino acids to proteins, ubiquitination,
The modification is not limited to this.

Thus, a GPCR peptide of the invention is a mature GPCR peptide in which the substituted amino acid residue is not encoded by the genetic code, contains a substituent, eg a longer half-life of the GPCR peptide. Is fused to another compound, such as a compound (eg, polyethylene glycol), or an additional amino acid is, for example, the main, subsequence, or purified sequence of the mature GPCR peptide, or the mature GP.
It also includes derivatives or analogs such as fused to a mature GPCR peptide, such as the CR protein preprotein sequence.

Such modifications are well known to those of skill in the art and have been described in great detail in the scientific literature. Particularly common modifications such as glycosylation, lipidation, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, ADP-ribosylation are Prot.
eins-Structure and Molecular Properties, 2nd Ed., TE Creighton, W. H
It is described in most basic texts, such as Freeman and Company, New York (1993). For detailed literature on this point, see Wold, F., Posttransl.
ational Covalent Modification of Proteins, BC Johnson, Ed., Academic P
ress, New York 1-12 (1983); Seifter et al. (Meth. Enzymol. 182: 626-646.
(1990)) and Rattan et al. (Ann. NY Acad. Sci. 663: 48-62 (1992)).

[0051]Use of protein / peptide   The protein of the present invention is associated with the functional information shown in the drawings and background art.
In qualitative and specific assays, for example, raising antibodies or other immune reactions
(Or its binding target or receptor) in biological fluid for inducing
As reagents (including labeling reagents) used in the assay for level quantification,
Or, a tissue marker that selectively expresses the corresponding protein (tissue differentiation or
Can be used as a particular stage of development, or as a disease state). Ta
If the protein binds or is capable of binding another protein (eg, receptor-
Ligand-ligand interaction), use this protein to identify the binding partner and
A system can be developed to identify inhibitors for use. Some or all of these
Can be used as a reagent grade or as a commercial product in a kit format.
It becomes possible to develop to

The manner of making the uses listed above are well known to those skilled in the art. References disclosing such methods include "Molecular Cloning: A Labor
atory Manual ”2d ed, Cold Spring Harbor Laboratory Press, Sambrook, J.,
EF Fritsch and T. Maniatis eds., (1989) “Methods in Enzymology: Guid
e to Molecular Cloning Techniques ”, Academic Press, Berger, SL and A
There is R. Kimmel eds., (1987).

Potential uses of the peptides of the invention are based primarily on protein origin and protein class / action. For example, GPCRs isolated from humans, and their human / mammalian orthologs, may be used as therapeutic pharmaceuticals for mammals, eg, human pharmaceuticals,
In particular, it is useful as a target for discovering a drug used for modulation of cells or tissue expressing a receptor (in the screening of a tissue-specific library, the GPCR of the present invention is expressed in at least the brain, placenta, and thyroid. Shown). About 70% of all pharmaceutical formulations modulate the activity of GPCRs. The combination of invertebrate and mammalian orthologs can be used in a selective screening method to discover invertebrate-specific drugs. The structural and functional information provided in the background and figures provides specific and essential uses of the molecules of the invention. Such use can be readily determined using the information provided in the present invention, as well as information known to those of skill in the art, and routine experimentation.

This receptor polypeptide, including the variants and fragments disclosed before this invention, is useful in biological assays related to GPCRs. Such an assay is useful for diagnosing and treating GPCR-related conditions that are specifically related to the function of a known GPCR, or in particular cells and tissues that express this receptor, to the GPCR subfamily to which one of the present invention belongs. , Either activity or property (screening of tissue-specific libraries shows that the GPCRs of the invention are expressed at least in brain, placenta, and thyroid).

The receptor polypeptide is also useful in drug screening assays in cell-based or cell-free systems. In a cell line, a native type, ie, a cell that normally expresses a receptor protein, that grows in a biopsy or cell culture medium (
Screening of tissue-specific libraries has shown that the GPCRs of the invention are expressed in at least the brain, placenta and thyroid). However, in one example, the cell-based assay involves recombinant host cells expressing the receptor protein.

The polypeptides can be used to identify compounds that naturally modulate the receptor activity of a protein, or variants that cause a particular disease or condition associated with the receptor. Both the GPCRs of the invention and the appropriate mutants and fragments can be used in a high efficiency screen to assay candidate compounds capable of binding to the receptor. These compounds can be screened against functional receptors to further determine their effect on receptor activity. Furthermore, these compounds are active / active in animal or invertebrate systems.
It can be tested to determine the effect. The compound is determined to activate (agonist) or inactivate (antagonist) the receptor to the desired extent.

In addition, the receptor polypeptide may interact with a receptor protein and a molecule that normally interacts with the receptor protein, eg, a ligand or component of a signaling pathway with which the receptor protein normally interacts. Use to screen for compounds with the ability to enhance or inhibit action (eg, G proteins, or other interacting substances related to cAMP, phosphatidylinositol turnover and / or adenylate cyclase or phospholipase C activation) You can Such assays generally involve the receptor protein or fragment interacting with a target molecule, detecting a complex of protein and target, or G protein phosphorylation, cAMP or phosphatidylinositol turnover, Under conditions that allow the detection of the biochemical consequences of the interaction between the receptor protein and the target, such as signaling-related effects such as adenylate cyclase, phospholipase C activation, etc. The step of combining with a candidate compound is included.

Candidate compounds include, for example, 1) a fusion peptide in which the last part is Ig, and a soluble peptide containing a random peptide library (eg, Lam et al., Nature 354).
: 82-84 (1991); Houghten et al., Nature 354: 84-86 (1991)), and D-
And / or peptides containing chemically derived molecular libraries made of combinations of L-type amino acids, 2) phosphopeptides (eg, random or partially modified phosphopeptide libraries, eg Songyang et al., Cell 72: 767-778 (19
93)), 3) antibody (for example, a polyclonal antibody, a monoclonal antibody, a humanized antibody, an anti-idiotype antibody, a chimeric antibody, Fab, F (ab ') 2, a single-chain antibody containing a Fab expression library fragment, And epitope-binding fragments of antibodies), 4) small organic and inorganic molecules (eg, molecules derived from combinatorial and natural product libraries).

One candidate compound is a soluble fragment of the receptor that competes for ligand binding. Other candidate compounds include mutant receptors, or suitable fragments containing mutants that affect receptor function, which result in competition with the ligand. Thus, for example, fragments that compete with the ligand are included in the invention such that they have a high affinity or that the fragment binds to the ligand and does not dissociate.

The present invention further includes other endpoint assays for identifying compounds that modulate (enhance or inhibit) receptor activity. This assay is generally associated with assaying behavior in signal transduction pathways that are indicative of receptor activity. Thus, assays are performed for cellular processes such as cell proliferation and gene expression that is regulated to promote or suppress response to receptor protein dependent signal cascades. In one example, the regulatory regions of these genes can be linked to a readily detectable marker such as luciferase.

Any biological or biochemical function mediated by the receptor can be used as an endpoint assay. These include all biochemical or biochemical / biological behaviours described herein, the references cited therein are folded into the targets of these endpoint assays, and others The function of is known to those skilled in the art, or with the help of the drawings, in particular FIG.
It can be easily confirmed. In particular, assays can be performed for the biological function of cells or tissues that express the receptor (screening of tissue-specific libraries has shown that the GPCRs of the invention are expressed in at least the brain, placenta, and thyroid. Exist).

Bound and / or active compounds can also be screened by using chimeric receptor proteins, such as amino terminal extracellular domains or portions thereof, or any of the seven transmembrane segments or intracellular or One of the extracellular loops,
And the entire transmembrane domain, such as the carboxy-terminal intracellular domain or a subregion or part thereof, may be replaced by a heterologous domain or subregion. For example, G protein-binding regions can be used as those that interact with different G proteins and are recognized by natural receptors. Thus, a different set of signaling components can be utilized as an endpoint assay for activation. Alternatively, the entire transmembrane region or subregion (transmembrane segment, or intracellular or extracellular loop) is different from the host cell from which the amino-terminal extracellular region and / or G protein-binding region is derived, and is a transmembrane region specific to the host cell. It can be replaced with the whole part or subregion. This allows the assay to be performed in something other than the particular host cell from which the receptor is derived. Alternatively, the amino-terminal extracellular domain (and / or ligand binding region) can be replaced with a domain that binds other ligands (and / or other binding regions), and thus a heterologous amino-terminal extracellular domain. An assay for a test compound that interacts with (or a region) but results in signal transduction is provided. Finally, activation is detected by a reporter gene containing a detectable coding region that can be linked to transcriptional regulatory sequences that are part of the natural signaling pathway.

The receptor polypeptides are also useful in competitive binding assays, a method designed to discover compounds that interact with the receptor. To this end, the compound is contacted with the receptor polypeptide under conditions which allow the compound to bind or interact with the polypeptide. Soluble receptor polypeptide is also added to the mixture. When the test compound interacts with the soluble receptor polypeptide, the amount or activity of the complex formed from the receptor target is reduced. This type of assay is particularly useful when seeking compounds that interact with specific regions of the receptor. Thus, a soluble polypeptide that competes with the target receptor region is designed to include a peptide sequence corresponding to the target region.

To perform a cell-free drug screening assay, a receptor protein or fragment, in order to streamline the separation of the complexed form from the uncomplexed form of one or both of the proteins and to automate the assay, It may be desirable to immobilize any of the target molecules.

Techniques for immobilizing proteins on matrices can be used in drug screening assays. In one example, the fusion protein can add a domain capable of binding the protein to a matrix. For example, glutathione S-transferase fusion protein is adsorbed onto glutathione Sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione-induced microtiter plates and cell lysates (eg, ³⁵ S-label).
ed) and the candidate compound are bound and the mixture is incubated under complex formation conditions (eg, physiological conditions of salt and pH). After incubation, the beads are washed for removal of unbound label and quantified by immobilizing the matrix and measuring the radiolabel directly or after separating the complex and measuring the supernatant. Alternatively, the complexes can be separated from the matrix by SDS-PAGE and standard electrophoresis techniques can be used to quantify the level of receptor binding protein in the bead fraction from the gel. For example, either the polypeptide or its target molecule is immobilized using conjugation of biotin and streptavidin utilizing techniques well known to those of skill in the art. Alternatively, an antibody that reacts with the protein and does not interfere with the binding of the protein to the target molecule is derivatized to the well of the plate, and the protein is captured in the well by binding to the antibody. The composition of the receptor binding protein and the candidate compound are incubated in the receptor protein present wells and the amount of complex trapped in the wells is measured. As a method for detecting such a complex, in addition to the above-mentioned method using a GST-immobilized complex, an antibody reactive with a receptor protein target molecule, or a receptor protein reactive with a target molecule competes with the target molecule. Immunodetection of complexes with antibodies and enzyme-linked assays based on detection of enzyme activity associated with the target molecule are included.

Agents that modulate one of the GPCRs of the present invention can be identified using the above analytical methods alone or in combination. In general, it is preferable to use cell-based or cell-free systems first and last to confirm activity in animal or other model systems. Such model systems are well known to those of skill in the art and can be readily used in this description.

Modulators of receptor protein activity identified by these drug screening assays can be obtained by treating cells or tissues expressing a GPCR by
It can be used to treat patients with diseases mediated by the receptor pathway (screening of tissue-specific libraries shows that the GPCRs of the invention are expressed in at least brain, placenta, and thyroid). These methods of treatment include administering a modulator of GPCR activity in a pharmaceutical composition in an amount required to treat the patient, and the modulator is identified as described herein.

In another aspect of the invention, a 2-hybrid assay or 3-hybrid assay (US Patent No. 5,283,317; Zervos et al. (19
93) Cell 72: 223-232; Madura et al. (1993) J. Biol. Chem. 268: 12046-12054.
Bartel et al. (1993) Biotechniques 14: 920-924; Iwabuchi et al. (1993)
Oncogene 8: 1693-1696; and Brent WO94 / 10300), GPCR proteins can be used as "bait proteins". GP like this
The CR binding protein is, for example, a GPCR protein as a downstream factor, or G
It is involved in signal transduction by PCR targets. Alternatively, such GPC
The R-binding protein may be a GPCR inhibitor.

The two-hybrid system is based on the modular nature of most transcriptional regulators, which consist of separable DNA-binding and activation domains. Briefly, this assay utilizes two different DNA structures. In one structure, the gene encoding the GPCR protein is fused to the gene encoding the DNA binding domain of a known transcription factor (eg GAL-4). In the other structure, a DNA sequence obtained from a DNA sequence library and encoding an unknown protein (“prey” or “sample”) is transferred to a gene encoding the activation domain of a known transcription factor. Be fused. "B
When the "ait protein" and the "prey protein" can interact in vivo and form a GPCR-dependent complex, the DNA binding domain and the activation domain of the transcription factor are in close proximity. This proximity allows transcription of a reporter gene (eg LacZ) capable of binding to a transcriptional regulatory site that responds to a transcriptional regulatory factor. Expression of the reporter gene is detectable and cell colonies containing functional transcriptional regulators can be isolated and used to obtain a cloned gene encoding a protein that interacts with the GPCR protein. .

The present invention further relates to novel agents identified by the screening assays described above. Therefore, it is within the scope of the invention to use the agents identified as described herein in suitable animal models. For example, agents identified in the present invention (eg GPCR modulating agents, antisense GPCR nucleic acid molecules, GPCRs
Specific antibodies, or GPCR binding targets), can be used in animals or other models to determine efficacy, toxicity, side effects in the formulation of these agents. Alternatively, the agents identified in the present invention can be used in animals or other models to determine the mechanism of action of such agents. Further, the present invention provides
It relates to the use of novel agents identified by the screening assays for treatment described herein.

The GPCR proteins of the present invention are useful to provide targets for the diagnosis of peptide mediated diseases or predispositions for diseases. Therefore, the present invention
The present invention provides a method for detecting the presence of a protein (or encoded mRNA) in a cell, tissue, or living body, or its level (in the screening of a tissue-specific library, the GPCR of the present invention comprises at least the brain, It has been shown to be expressed in placenta and thyroid gland). This method involves contacting a biological sample with a compound capable of interacting with a receptor protein, where the interaction is detected. Such an assay is provided in a single detection format or a multi-detection format such as an antibody chip array.

One agent that detects proteins in a sample is an antibody that can selectively bind to the protein. Biological samples include tissues, cells, biological fluids isolated from or present within a subject.

The peptides of the present invention target the activity of proteins in patients with peptide variants, their targets for use in the diagnosis of disease or predisposition to disease, in particular those known as other than the existing protein family, and symptom targets. It is provided. Therefore, the peptide can be isolated from a biological sample, and
Assays can be performed for the presence of genetic mutations that result in aberrant peptides. This includes amino acid substitutions, deletions, insertions, rearrangements (resulting from aberrant splicing behavior), and inappropriate post-translational modifications. Analytical methods include altered electrophoretic mobility, altered tryptic peptide digestion, altered GPCR activity by cell-based or cell-free assays, altered binding patterns of ligands or antibodies, altered isoelectric points, direct amino acid sequences, and protein mutations. Other known assay techniques useful for detection are included. Such an assay is provided in a single detection format or a multi-detection format such as an antibody chip array.

As a technique for detecting peptides in vitro, an enzyme-linked immunosorbent assay (ELI) is used.
SAs) Western blots, immunoprecipitation with detection reagents such as antibodies or protein binders, immunofluorescence. Alternatively, by introducing a labeled anti-peptide antibody, or other type of detection reagent, into the subject, in vivo.
o Detection can be done. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Methods of detecting allelic variants of peptides expressed in a subject, and methods of detecting fragments of peptides in a sample are particularly useful.

Peptides are also useful in genomic pharmacological analysis. Genomic pharmacology clinically deals with significant genetic variations in the response to drugs, according to the trends in drug changes and the abnormal behavior of affected humans. For example, Eichelbaum
, M. (Clin. Exp. Pharmacol. Physiol. 23 (10-11): 983-985 (1996)), and Lin.
der, MW (Clin. Chem. 43 (2): 254-266 (1997)). The clinical consequences of these mutations result in severe toxicity of the therapeutic agent to some individuals or treatment failure to some individuals as a result of metabolic variations in the individual. Thus, the genotype of an individual can determine how the therapeutic compound acts in the body or how the body metabolizes the compound. In addition, the activity of drugs that metabolize enzymes affects the intensity and duration of drug action. Thus, the genomic pharmacology of an individual allows for the selection of effective compounds, or effective doses of such compounds, in prophylactic or therapeutic treatments based on the individual's genotype. With the discovery of genetic polymorphism,
The reasons may be explained for the enzyme-metabolizing drug such that the patient does not have the expected drug effect, exhibits an unnatural drug effect, or suffers significant toxicity from the standard dosage. Polymorphism can be represented by a strong metabolic system phenotype and a weak metabolic system phenotype. Thus, polymorphisms in a gene may lead to allelic variants of the receptor protein such that one or more of the receptor's functions in one population differs from that in another population. As such, peptides can be targets for confirming the predisposition of genes that affect therapeutics. Thus, in ligand-based therapy, polymorphism may cause higher or lower ligand binding and receptor activity in the extracellular domain of the terminal amino groups and / or other ligand binding regions. Thus, in populations containing polymorphisms, the ligand dosage will necessarily be modified to maximize therapeutic effect. As an alternative to genotype, specific polymorphic peptides can be identified.

Peptides are also useful in treating disorders characterized by lack of expression, inadequate expression, or unnecessary expression of proteins (in tissue-specific library screening, GPCR is at least in the brain, placenta,
It has been shown to be expressed in the thyroid gland). Therefore, as a treatment method, GPC
The use of R proteins or fragments is included.

[0077]antibody   The present invention provides the peptides of the present invention, proteins containing such peptides,
An antibody that selectively binds to one of the variants and fragments of
. As used herein, the antibody binds to the target peptide and is
Antibodies selectively bind target peptides when they do not bind strongly to proteins
is doing. The antibody is associated with another protein that is not substantially homologous to the target peptide.
Even if it binds, the peptide does not bind to the target peptide of the antibody.
Antibodies selectively bind peptides as long as they have homology in the fragment or domain.
It is thought to tie the do. In this case, the antibody bound to the peptide is
It is understood to be selective despite having some degree of cross-reactivity.

As used herein, antibodies are defined in the same terms as are accepted in the art, which are multi-subunit proteins produced by the mammalian organism and which respond to antigenic challenge. Antibodies of the invention include, but are not limited to, polyclonal antibodies, monoclonal antibodies, and fragments of antibodies such as Fab or F (ab ') 2, and Fv.

Many methods are known for producing and / or identifying antibodies to obtain target peptides. Some of these methods are available from Harlow, Antibodies, Co.
ld Spring Harbor Press, (1989).

In general, to produce antibodies, the isolated peptide is used as an immunogen and administered to a mammalian organism such as a rat, rabbit or mouse. Full length proteins, antigenic peptide fragments or fusion proteins are used. Fragments of particular interest are those that cover functional domains as specified in Figure 2 and also have sequence homology or differences with the family that can be readily identified using protein placement methods. It is a domain with sex.

Antibodies are suitably prepared from regions of the GPCR protein, or isolated fragments. Antibodies can be prepared from any of the peptides described herein. However, preferred areas include function / activity and / or GPC
Regions involved in R-binding interactions are included. Figure 2 can be used to identify regions of particular interest, and sequence alignment can be used to identify retained unique sequence fragments.

Antigenic fragments generally consist of at least 8 contiguous amino acid residues. An antigenic peptide can consist of at least 10, 12, 14, 16 or more amino acid residues. Such fragments should be selected based on, for example, physical properties such as a fragment corresponding to a region located on the surface of a protein, for example, a hydrophilic region, or sequence specificity (see FIG. 2). You can

Detection of the antibody of the present invention can be facilitated by coupling (ie, physical association) between the detectable substance and the antibody. Examples of detectable substances include, for example, various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase, and examples of suitable prosthetic groups include streptavidin /
Examples of suitable fluorescent substances include biotin, avidin / biotin, and examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. Examples of substances include luminol, examples of bioluminescent substances include luciferase, luciferin and aequorin, and examples of suitable radioactive substances are ¹²⁵ I, ¹³¹ I, ³⁵ S,
Or ³ H is included.

[0084]Use of antibodies   Antibodies can be applied to standard techniques such as affinity chromatography or immunoprecipitation.
Thus, it can be used to isolate one of the proteins of the invention. antibody
Is produced by the native protein from the cell and the recombinant expressed in the host cell
The purified protein can be easily purified. Moreover, such antibodies
In order to determine the expression pattern of proteins in tissues and organisms,
It can be used for detecting the presence of the protein of the present invention without going through a normal development stage.
it can. In addition, such antibodies can be evaluated by assessing the amount and pattern of expression.
For the detection of proteins in situ, in vitro, in cell lysates and in the supernatant
Can be used for. In addition, such antibodies are also useful for the development or progression of biological conditions.
Can be used to assess abnormal tissue distribution, or abnormal expression between
(For screening tissue-specific libraries, the GPCR of the present invention
Has also been shown to be expressed in the brain, placenta, and thyroid). Full-length protein circulation
Antibody detection in pigments can be used to confirm turnover.

In addition, the antibodies may be used to assess the onset of a disease, such as an active stage of a disease associated with protein function, particularly protein function in cells and tissues that express the receptor, or an individual predisposed to the disease. It can be used (screening of tissue-specific libraries has shown that the GPCRs of the invention are expressed in at least the brain, placenta, and thyroid). If the disorder is due to inappropriate tissue distribution, developmental expression, protein expression level, or expression / progression status, antibodies are prepared against the normal protein. If the disorder is characterized by a particular mutation in the protein, then antibodies specific for this mutant protein can be used to assay for the presence of the particular mutant protein.

Antibodies can also be used to assess the location of normal or abnormal organelles of cells in various tissues in vivo (in a tissue-specific library screen, the GPCR of the invention is at least the brain). , Placenta and thyroid gland). Its use as a diagnostic can be applied not only to genetic testing, but also to monitoring therapy. Thus, if the treatment ultimately aims to correct the expression level, or the presence of aberrant sequences and aberrant tissue distribution, or the developmental expression, treat the antibody directly against the protein or related fragment with Can be used to monitor the effectiveness of

In addition, the antibodies are useful in genomic pharmacological analysis. Thus, antibodies prepared against polymorphic proteins can be used to identify individuals in need of treatment modification. Antibodies are also useful as diagnostic tools such as immunological markers of abnormal proteins analyzed by electrophoresis, isoelectric point, tryptic peptide digestion, and other physical assays well known in the art. .

Antibodies are also useful for tissue typing (screening of tissue-specific libraries has shown that the GPCRs of the invention are expressed in at least brain, placenta, and thyroid). Thus, if a particular protein was correlated with expression in a particular tissue, antibodies specific for this protein can be used to identify the tissue type.

Antibodies are also useful for inhibiting protein function, for example blocking the binding of GPCR peptides to their binding partners such as ligands. These uses
It can be applied in a therapeutic environment in therapeutics involving the inhibition of protein function. Antibodies can be used to inhibit modulation of peptide activity (agonizing or antagonizing), for example by blocking binding. Antibodies are prepared against specific fragments containing the sites necessary for function, or against intact proteins associated with the cell or cell membrane. FIG. 2 shows structural information regarding the protein of the present invention.

The present invention includes kits that use the antibodies to detect the presence of proteins in biological samples. The kit comprises a labeled or labelable antibody and a compound or reagent for detecting a protein in a biological sample,
Means for determining the amount of protein in a sample; means for comparing with the amount of protein in a standard sample; and instructions for use. Such a kit can be provided to detect a single protein, or epitope, or can be configured to detect one of multiple epitopes, such as an antibody detection array. .
As the array, a nucleic acid array is described below, and similar methods for antibody arrays have been developed.

[0091]Nucleic acid molecule   The invention further provides a core encoding a GPCR peptide or protein of the invention.
It provides acid molecules (cDNA, transcription and genomic sequences). Such a nucleus
The acid molecule is a nucleic acid molecule encoding one of the GPCR peptides of the invention, or
Consisting essentially of an allelic variant or an ortholog or paralog thereof
, Or includes.

As used herein, an "isolated" nucleic acid molecule is one that is separated from the presence of other nucleic acids of natural origin. Preferably, an "isolated" nucleic acid does not include the flanking sequences of the nucleic acid (ie, the sequences located at the 5'and 3'ends) in the genomic DNA of the organism from which the nucleic acid was derived. However, there are several flanking nucleotide sequences, such as, for example, adjacent peptides encoded by sequences of about 5 KB, 4 KB, 3 KB, 2 KB or especially 1 KB or more, or less, especially sequences, but by the sequence of the same gene The encoded peptides are separated by introns in the genomic sequence. The important point is that the nucleic acid can be handled in a specific manner as described herein, such as recombinant expression, preparation of probes and primers, other uses for nucleic acid sequences, etc. It is separated from the unimportant flanking sequences.

Furthermore, “isolated” nucleic acid molecules, such as, for example, cDNA molecules, can
It is substantially free of media when produced by recombinant techniques and precursors or other chemicals when chemically synthesized. However, this nucleic acid molecule
It can be fused to other coding or regulatory sequences, but is considered isolated.

For example, a recombinant DNA molecule contained in a vector is considered isolated. Further, examples of isolated DNA molecules include recombinant DNA molecules retained in a heterologous host cell or purified (partially or substantially) DNA molecules in solution. The isolated RNA molecule includes isolated DN of the present invention.
In vivo or in vitro RNA transcripts of the A molecule are included. Nucleic acid molecules isolated according to the present invention further include synthetically produced molecules.

Therefore, the present invention is based on FIG. 1 or 3 [SEQ ID NO. 1 (transcribed sequence), and SEQ ID NO. 3 (genomic sequence)], or a nucleic acid molecule encoding the protein shown in FIG. 2 (SEQ ID NO. 2). A nucleic acid molecule consists of a nucleotide sequence when the nucleotide sequence is the complete nucleotide sequence of this nucleic acid molecule.

The present invention further includes the configuration of FIG. 1 or 3 [SEQ ID NO. 1 (transcribed sequence), and SE
Q ID NO. 3 (genomic sequence)], or a nucleic acid molecule encoding the protein shown in FIG. 2 (SEQ ID NO. 2). In the nucleic acid molecule, when such a nucleotide sequence is finally present with only a few additional nucleic acid residues, eg 1 to 300 additional nucleotides, the nucleic acid molecule consists essentially of the nucleotide sequence.

The present invention further includes the configuration of FIG. 1 or 3 [SEQ ID NO. 1 (transcribed sequence), and SE
Q ID NO. 3 (genomic sequence)], or a nucleic acid molecule encoding the protein shown in FIG. 2 (SEQ ID NO. 2). A nucleic acid molecule comprises a nucleotide sequence when at least a portion of the final nucleic acid sequence of the nucleic acid molecule is this nucleic acid sequence. According to this, the nucleic acid molecule may be only its nucleotide sequence or additional nucleic acid residues,
For example, it may have a nucleic acid sequence related to nature or a heterologous nucleic acid sequence. Such nucleic acid molecules have very few additional nucleotides or can include hundreds or more of additional nucleotides. A brief description of how to easily generate / isolate these various types of nucleic acid molecules is provided below.

Both coded and non-coded sequences are shown in FIGS. 1 and 3. From the human genome sequence of the present invention (FIG. 3) and the cDNA / transcription sequence (FIG. 1), the nucleic acid molecule in the figure is a genomic intron sequence, 5 ′ and 3 ′ non-coding sequences, gene regulatory regions, and non-coding genes. Includes an array. In general, the features of such sequences described in both FIGS. 1 and 3 can be readily ascertained using computational tools known in the art. As discussed below, some non-coding sites, particularly regulatory elements of genes such as promoters, can be used to control a variety of non-homologous gene expressions, targets for identifying compounds that modulate gene activity, etc. It is useful for the purpose and is particularly claimed as a fragment of the genomic sequence provided herein.

The isolated nucleic acid molecule encodes the mature protein and additional amino or carboxy terminal amino groups, or amino groups within the mature peptide (eg, when the mature form has one or more peptide chains). be able to. An array like this
In processing the protein from its precursor to its mature form, it may play a role in facilitating protein delivery, extending or shortening the protein half-life, or streamlining operations in the assay or production of the protein, or in other events. In general,
In situ, additional amino acids are processed by cellular enzymes into the mature protein.

As described above, the isolated nucleic acid molecule may include a sequence encoding the GPCR peptide alone, a sequence encoding the mature peptide, and a major or minor sequence (eg, pre-pro, (including pro-protein sequences), but is not limited to additional coding sequences, plus additional non-coding sequences such as introns and non-coding 5'and 3 'With or without those that play a role in transcription, mRNA processing (including splicing and polyadenylation), ribosome binding, and mRNA stability, such as the' sequence that is not translated. Is also good. In addition, the nucleic acid molecule can be fused to a marker of the sequence encoding the peptide, for example to facilitate purification.

The isolated nucleic acid molecule may be in the form of RNA such as mRNA, or cDNA and genomic DNA produced by cloning, chemical synthesis techniques or a combination thereof.
Can take the form of DNA containing. The nucleic acid, especially DNA, can be double-stranded or single-stranded. Single-stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (antisense strand).

The present invention further provides nucleic acid molecules that encode obvious variants of the GPCR proteins of the present invention as described above, as well as nucleic acid molecules that encode fragments of the peptides of the present invention. . Such nucleic acid molecules can occur naturally, such as allelic variants (same position), paralogs (different positions) and orthologs (different organisms), or can be produced by recombinant DNA methods or chemical synthesis. Such non-naturally occurring variants can be produced by mutagenesis techniques applied to nucleic acid molecules, cells or organisms. Thus, as mentioned above, variants include nucleotide substitutions, deletions, inversions and insertions. Mutations can occur in either the coding or non-coding regions, or both. Mutations can occur in both conservative and non-conservative amino acid substitutions.

The present invention further provides non-coding fragments of the nucleic acid molecules shown in Figures 1 and 3. Suitable non-coding fragments include, but are not limited to, promoter sequences, enhancer sequences, gene modulation sequences, gene termination sequences. Such fragments are useful in the control of heterologous gene expression, and in the development of screens for the identification of gene modulating agents. It is confirmed.

Fragments include 12 or more flanking nucleotide sequences. Further, the fragment is at least 30, 40, 50, 100, 250, or 500.
Nucleotide length. The length of the fragment depends on the intended use. For example, the fragment can encode an epitope result region of a peptide, or is useful as a DNA probe and DNA primer. Such fragments can be isolated using known nucleotide sequences for synthesizing oligonucleotide probes. The labeled probe can be used for screening a cDNA library, a genomic DNA library, or mRNA to isolate the nucleic acid corresponding to the coding region. In addition, the primers can be used in PCR reactions to clone specific regions of the gene.

A typical probe / primer comprises a substantially purified oligonucleotide or oligonucleotide pair. Oligonucleotides generally include a nucleotide sequence region hybridized under stringent conditions to at least 12, 20, 25, 40, 50 or more contiguous nucleotides.

Orthologs, homologs, and allelic variants can be identified using methods well known in the art. As mentioned in the peptide section, these variants comprise a nucleotide sequence encoding the peptide and are typically 60-70% relative to the nucleotide sequence shown in the figure, or a fragment of this sequence. ,
70-80%, 80-90%, and more typically at least 90-95% or more homology. Such a nucleic acid molecule can be easily identified as capable of hybridizing under moderate to stringent conditions to the nucleotide sequence shown in the drawing, or a fragment of this sequence. Allelic variants can be readily determined at the genetic location of the encoding gene (in the RH panel map, the GPCRs of the invention show that chromosome 11
Above markers SHGC-32486 and SHGC-20653 (LOD score 1
It is shown to be encoded by the gene shown in the proximity of 2.02)).

FIG. 3 shows SNP information found in the gene encoding the GPCR protein of the present invention. Here, mutations of C1435G, T1262C and A532G are found.

The term “hybridize under stringent conditions”, as used herein, is such that the peptide-encoding nucleotide sequences remaining hybridized to each other are at least 60-70% homologous to each other. It means the conditions under which hybridization and washing are carried out to the extent that they have properties. Under this condition, the remaining hybridized sequence will be at least about 60%, at least about 70%, at least about 80%, or more. Such stringent conditions are well known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John W.
iley & Sons, NY (1989), 6.3.1-6.3.6. One example of stringent hybridizing conditions is hybridizing in 6X sodium chloride / sodium citrate (SSC) at about 45 ° C followed by 0.2X SSC0.1.
Wash at 50-65 ° C in% SDS. Examples of low stringency, moderated hybridization conditions are well known to those of skill in the art.

[0109]Use of nucleic acid molecules   The nucleic acid molecule of the present invention includes a probe, a primer, a chemical synthesis intermediate, and a biological reagent.
Useful in essays. The nucleic acid molecule encodes the peptide shown in Figure 2.
For the isolation of full-length cDNA and genomic clones
Mutants that produce peptides that are identical or related to steroids (alleles, orthologs, etc.)
Messenger RNA, transcription / c for isolating a genomic clone corresponding to
Useful as a hybridization probe for DNA and genomic DNA
. In FIG. 3, found in the gene encoding the GPCR protein of the invention
Indicates SNP information. Here, the C1435G, T1262C and A532G
Differences can be seen.

The probe can correspond to any sequence in the entire length of the nucleic acid molecule shown in the figure. Therefore, it has 5'non-coding regions, coding regions, and 3 '
'Can be derived from non-coding regions. However, as already mentioned, fragments are considered to be free of fragments published prior to the present invention.

Nucleic acid molecules are also useful as primers in PCR to amplify any region of the nucleic acid molecule and in the synthesis of antisense molecules of the required length and sequence.

Nucleic acid molecules are also useful in the production of recombinant vectors. Such a vector includes an expression vector expressing part or all of the peptide sequence. Vectors also include insertion vectors, which are integrated into other nucleic acid molecules and used, for example, to alter the in situ expression of genes and / or gene products in the cell genome. For example, an endogenous coding sequence may be replaced via homologous recombination with some or all of the coding region containing one or more specifically introduced mutations.

[0113]   Nucleic acid molecules are also useful for expressing the antigenic portion of proteins.

Nucleic acid molecules are also useful as probes for determining the chromosomal location of nucleic acid molecules by in situ hybridization methods (in the RH panel map, the GPCR of the invention uses the marker SHGC on chromosome 11). -32486 and SH
(It has been shown to be encoded by the gene shown in the proximity of GC-20653 (LOD score 12.02)). This is particularly useful in determining whether a particular protein is an allelic variant of the proteins described herein.

Nucleic acid molecules are also useful in the production of vectors containing the gene regulatory region of the nucleic acid molecules of the invention as described below.

Nucleic acid molecules are also useful in the design of ribozymes that correspond to some or all of the mRNA produced from the nucleic acid molecules described herein.

Nucleic acid molecules are also useful in making host cells expressing some or all of the nucleic acid molecules and peptides.

Nucleic acid molecules are also useful in the production of transgenic animals expressing some or all of the nucleic acid molecules and peptides.

Nucleic acid molecules are also useful in the production of vectors expressing part or all of a peptide.

Nucleic acid molecules are also useful as hybridization probes to determine the presence, level, morphology, distribution of nucleic acid expression. Thus, the probe can be used to detect the presence or to determine the level of a nucleic acid molecule in cells, tissues, or organisms (for screening tissue-specific libraries, the GPCRs of the invention are used in at least the brain, placenta, and thyroid). It has been shown to express). The level of nucleic acid is DNA
Or it is determined as RNA. Therefore, probes corresponding to the peptides described herein can be used to assess expression and / or gene copy number in a given cell, tissue or organism. These uses, compared to the normal case, GP
It is suitable for the diagnosis of disorders associated with increased or decreased CR protein.

In vitro detection techniques for mRNA include Northern hybridizations, and in
In situ hybridization is included. In vitro detection techniques for DNA include Southern hybridizations and in situ hybridizations.

The probe can be used as part of a diagnostic test kit to determine cells or tissues that express a GPCR protein. This is, for example, to measure the level of a GPCR-encoding nucleic acid molecule such as mRNA or genomic DNA in a cell sample of a subject or to determine whether the GPCR gene is mutated (tissue In the screening of specific libraries, the GPCR of the present invention
It has been shown to be expressed in at least the brain, placenta, and thyroid).

Nucleic acid expression assays are useful in drug screening to identify compounds that modulate GPCR nucleic acid expression, particularly nucleic acid expression in cells and tissues that express the receptor. The GPCR of the invention is
It has been shown to be expressed in at least the brain, placenta, and thyroid).

Accordingly, the present invention provides methods for identifying compounds for use in treating disorders associated with nucleic acid expression of the GPCR gene. This method typically uses G
The compounds are assayed for their ability to modulate the expression of PCR nucleic acids, and then compounds that can be used to treat disorders characterized by unwanted GPCR nucleic acid expression are identified. This assay can be performed in cell and cell free systems. For cell-based assays, cells that naturally express GPCR nucleic acids (screening of tissue-specific libraries have shown that the GPCRs of the invention are expressed in at least brain, placenta, thyroid), or specific cells. Included are recombinant cells designed to express a nucleic acid sequence.

Assays for GPCR nucleic acid expression can be performed at the nucleic acid level, such as the mRNA level,
Alternatively, it is associated with the direct assay of accessory compounds associated with the signal pathway. In addition, the expression of genes that enhance or decrease responsiveness in the GPCR protein signaling pathway is also assayed. As an example of this, the regulatory regions of these genes are:
It can be effectively linked to a reporter gene such as luciferase.

Therefore, modulators of GPCR gene expression can be identified by the method of contacting cells with a candidate compound and determining the expression of mRNA. The expression level of GPCR mRNA in the presence of the candidate compound was
It is compared to the expression level of PCR mRNA. Based on this comparison, the candidate compound is identified as a modulator of nucleic acid expression and can be used, for example, in the treatment of disorders characterized by aberrant nucleic acid expression. A candidate compound is identified as a promoter of nucleic acid expression if the expression of mRNA in the presence of the candidate compound is statistically significantly greater than that in the absence. A candidate compound is identified as an inhibitor of nucleic acid expression if the expression of mRNA in the presence of the candidate compound is statistically significantly less than in the absence.

The invention further targets compounds identified through drug screening as gene modulators for modulating GPCR nucleic acid expression, particularly gene modulators for modulating activity in cells and tissues that express the protein. To provide a therapeutic method to be used (screening of a tissue-specific library shows that the GPCR of the present invention is expressed in at least the brain, placenta and thyroid). Modulation includes both up-regulation (eg activation or agonisation), down-regulation (suppression or antagonisation) or nucleic acid expression.

Alternatively, GP in cells or tissues in which the drug or small molecule expresses the protein
Modulators of GPCR nucleic acid expression, so long as they inhibit CR expression, can be agents or small molecules identified using the screening assays described herein (for screening tissue-specific libraries, the GPCR of the invention). Indicates that it is expressed in at least the brain, placenta, and thyroid gland).

Nucleic acid molecules are also useful in monitoring the effects of modulatory compounds on GPCR gene expression and activity in clinical trials or therapeutic methods. Thus, gene expression patterns can be a barometer of continued efficacy in treatment with compounds, especially those that improve patient tolerance. The gene expression pattern can also be a marker for the physiological response of cells to the compound. Thus, such monitoring may allow for increased doses of the compound or administration of alternative compounds that the patient does not tolerate. Similarly, if the level of nucleic acid expression is reduced to the desired level, administration of the compound can be proportionally reduced.

Nucleic acid molecules are also useful in diagnostic assays for qualitative changes in GPCR nucleic acid expression, particularly those leading to disease. The nucleic acid molecule can be used to detect mutations in GPCR genes, such as mRNA, and gene expression products. The nucleic acid molecule can be used as a hybridization probe to detect naturally occurring gene mutations in the GPCR gene and determine if the subject with the mutation is at risk for the disorder caused by the mutation. Mutations include
Includes deletions, additions, or substitutions of one or more nucleotides in a gene, chromosomal recombination such as inversions or transfers, modification of genomic DNA such as aberrant methylation patterns, or changes in gene copy number such as amplification. Be done. Detection of mutants of the GPCR gene associated with dysfunction provides a diagnostic tool for activity or sensitivity when the disease results from overexpression, underexpression, or mutant expression of the GPCR protein.

Individuals carrying mutations in the GPCR gene can be detected at the nucleic acid level by a variety of techniques (in the RH panel map, the GPCRs of the invention show that the markers SHGC-32486 and SHGC on chromosome 11). -2
0653 (LOD score 12.02) indicating that it is encoded by the gene shown in the proximity). Genomic DNA can be analyzed directly or after prior amplification using PCR. FIG. 3 shows SNP information found in the gene encoding the GPCR protein of the present invention. Here, C1435G, T
Mutations of 1262C and A532G are seen. RNA or cDNA can be used in the same manner. In one use, the detection of mutations can be accomplished by, for example, polymerase chain reaction (PCR), such as anchor PCR, RACE PCR (see, eg, US Patent Nos. 4,683,195, and 4,683,202), or else by ligation Reaction (LCR) (eg, Landegran et al., Science 241: 1
077-1080 (1988); and Nakazawa et al., PNAS 91: 360-364 (1994)), which relates to the use of probes / primers, the latter being particularly useful for identifying mutational positions in genes ( See Abravaya et al., Nucleic Acids Res. 23: 675-682 (1995)). This method involves the steps of collecting a cell sample from a patient, isolating nucleic acid (eg, genome, mRNA, or both) from the cells of the sample, and hybridizing and amplifying the gene (if present). A step of contacting the nucleic acid with one or more primers specifically hybridized to a gene under possible conditions, detecting the presence of an amplification product, or detecting the size of the amplification product, a control sample Including the step of comparing with the length of. Deletions and insertions can be detected by comparing the change in size of the amplification product with the normal genotype. Point mutations can be confirmed by hybridizing amplified DNA with normal RNA or antisense DNA sequences.

Alternatively, mutations in the GPCR gene can be directly confirmed by, for example, alteration of the restriction enzyme digestion pattern determined by gel electrophoresis.

In addition, sequence specific ribozymes (US Patent No. 5,498,531) can be used to score the presence of specific mutations by the growth or reduction of ribozyme cleavage sites. Perfectly matched sequences can be separated from mismatched sequences by nuclease cleavage digestion assay evaluation or by different melting points.

Sequence changes at specific positions can be assessed by RNase and S1 protection, or nuclease protection assays such as chemical cleavage methods. Furthermore, mutant GP
The sequence difference between the CR gene and the wild type gene can be determined by direct DNA sequence analysis. A variety of automated sequence analysis tools are available for diagnostic assays (Naeve,
CW, (1995) Biotechniques 19: 448), which include sequence analysis by mass spectroscopy (eg, PCT International Publication No.
WO 94/16101; Cohen et al., Adv. Chromatogr. 36: 127-162 (1996), and Gri
ffin et al., Appl. Biochem. Biotechnol. 38: 147-159 (1993)).

[0135] Examples of other techniques for detecting mutations in genes include protection from cleavage reagents to detect mismatched bases from RNA / RNA or RNA / DNA duplexes (Myers et al. al., Science 230: 1242 (1985), Cotton et al., PNAS 85:
4397 (1988), Saleeba et al., Meth. Enzymol. 217: 286-295 (1992)), a method for comparing electrophoretic mobilities of mutant and wild type nucleic acids (Orita et al., PNAS 86: 2766).
(1989); Cotton et al., Mutat. Res. 285: 125-144 (1993); and Hayashi et a
l., Genet. Anal. Tech. Appl. 9: 73-79 (1992)), and the movement of the mutant or wild-type fragment in a polyacrylamide gel containing a gradient of denaturing agents was determined by gradient gel electrophoresis. (Myers et al., Nature 313: 495 (1985)
))including. Examples of other techniques for detecting point mutations include selective oligonucleotide hybridization, selective amplification, and selective primer extension.

Nucleic acid molecules are also useful in personal testing for genotypes that, despite having therapeutic efficacy, do not necessarily cause disease. Thus, nucleic acid molecules can be used in studies of the correlation between an individual's genotype and the individual's response to compounds used in therapy (pharmacogenomic correlation). Thus, the nucleic acid molecules described herein can be used to assess mutations in an individual's GPCR gene to select appropriate compounds and dosages for treatment. FIG. 3 shows SNP information found in the gene encoding the GPCR protein of the present invention. Here, C1435G, T1262C and A532
A G mutation is seen.

Thus, nucleic acid molecules exhibiting therapeutically-affected gene mutations provide diagnostic targets that can be used for Taylor therapy in an individual. Thus, the production of recombinant cells and animals containing these polymorphisms allows for effective clinical design of therapeutic compounds and drug administration.

Nucleic acid molecules are useful as antisense constructs for controlling GPCR gene expression in cells, tissues and organisms. DNA antisense nucleic acid molecules
Designed to be complementary to a site of a gene involved in transcription and, therefore, to G
Generation and transcription of PCR proteins is prevented. Antisense RNA or DNA nucleic acid molecules are hybridized to mRNA, which blocks translation of the mRNA in the GPCR protein.

Alternatively, certain antisense molecules can be used for inactivated mRNAs that reduce expression of GPCR nucleic acids. Thus, these molecules can be used in the treatment of disorders characterized by abnormal or unwanted GPCR nucleic acid expression. This technique involves cleavage by ribozymes that contain nucleotide sequences complementary to one or more regions of the mRNA that reduce the translational capacity of the mRNA.
Possible regions include coding regions, particularly those that correspond to catalytic and other functional activities of GPCR proteins, such as ligand binding.

The nucleic acid molecule also provides a vector for gene therapy in patients with cells that have abnormal GPCR gene expression. Recombinant cells include cells that are prepared ex vivo and returned to the patient where they are introduced into the body of an individual where they produce the GPCR proteins required for treatment of the individual.

The present invention also includes kits that use the antibodies to detect the presence of GPCR nucleic acids in biological samples, especially cells and tissues that normally express this receptor (for screening tissue-specific libraries, The GPCR of the present invention has been shown to be expressed in at least the brain, placenta and thyroid gland). For example, the kit is labeled,
Or a labelable nucleic acid, or a reagent capable of detecting GPCR nucleic acid in a biological sample; means for determining the amount of GPCR nucleic acid in a sample; means for comparing to the amount of GPCR nucleic acid in a standard sample; Can be included. The compound or reagent can be packaged in a suitable container. The kit can further include instructions for use as a detection kit for GPCR protein mRNA or DNA.

[0142]Nucleic acid array   The present invention further provides a nucleic acid detection kit, which includes, for example,
Nucleic acid molecule based on the sequence information shown in FIGS. 1 and 3 (SEQ ID NO. 1, 3)
Array or microarray.

As used herein, an “array” or “microarray” is synthesized on a substrate such as paper, nylon or other membrane, filter, chip, glass slide, or other suitable solid support. Array of discrete polynucleotides or oligonucleotides that have been created. As one example, microarrays are described in US Patent 5,837,
832, Chee et al., PCT application W095 / 11995 (Chee et al.), Lockhart, D.
J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1
996; Proc. Natl. Acad. Sci. 93: 10614-10619, all of which are incorporated herein by reference. Alternatively, such arrays are manufactured by the method described in Brown et al., US Patent No. 5,807,522.

The microarray or detection kit preferably comprises a number of specific single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or cDNA fragments, on a solid support. Fixed. Oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably 20-25 nucleotides in length. For certain types of microarrays or detection kits it may be preferable to use only oligonucleotides of 7-20 nucleotides in length. Microarrays or detection kits are known
It may contain an oligonucleotide containing a'or 3'sequence, an oligonucleotide containing a full-length sequence, or a specific oligonucleotide selected from a specific region of sequence length. The polynucleotide used in the microarray or detection kit can be a gene or an oligonucleotide specific for the gene of interest.

In a microarray or detection kit, the gene of interest (or the ORF identified according to the invention) is typically labeled with a nucleic acid using a computer algorithm to produce an oligonucleotide of known sequence. Start 5'of the sequence,
Or end with 3 '. Typical algorithms identify gene-specific length defined oligomers, have GC components in the preferred range for hybridization, and no secondary structure that would be expected to interfere with hybridization. Under certain conditions, it may be preferable to use a pair of oligonucleotides in a microarray or detection kit. A "pair" of oligonucleotides is preferably identical, except for one nucleotide, which is located in the middle of the sequence. The second pair of oligonucleotides (mismatched with one) are used as controls. The number of oligonucleotide pairs is between 2 and 1 million. Oligomers are synthesized at designated regions on a substrate using a photoinduced chemical process. The base is paper,
Nylon or other membrane, filter, chip, glass slide, or other suitable solid support.

On the other hand, oligonucleotides were used for PCT application W095 / 251116 (Baldeschweile
(R et al.) and synthesized on the surface of the substrate using chemical coupling means and inkjet application equipment, all of which are folded in here as a reference. In another aspect, a vacuum system, heating, UV, mechanical, or chemical bonding steps are used to deposit the cDNA or oligonucleotide onto the surface of the substrate, such as a dot (or slot) blot in a "grid" array. Can be combined. Arrays such as those described above are manufactured by hand or using available equipment (slot blot or dot blot equipment), materials (suitable solid support), and machinery (including robotic equipment). , 24, 96, 384, 1
It may also contain 536, 6144 or more, or other numbers between 2 and 1 million oligonucleotides that are effectively used in commercially used devices.

RNA or DNA obtained from a biological sample is prepared in a hybridization probe for analysis of the sample using a microarray or detection kit. mRNA is isolated and cDNA is prepared and used as a template to prepare antisense RNA (aRNA). The aRNA is amplified in the presence of fluorescent nucleotides, the labeled probe is incubated with the microarray or detection kit, and the sequence of the probe is hybridized with the complementary oligonucleotide in the microarray or detection kit. Incubation conditions are adjusted to be exactly complementary, or to the extent that hybridization can occur with varying degrees of complementarity. After removing unhybridized probe, a scanner is used to determine the level and pattern of fluorescence. The scanned images are examined to determine the degree of complementarity and the relative amount of each oligonucleotide sequence on the microarray or detection kit. Biological samples include body fluids (eg blood, urine, saliva, sputum,
Gastric juice, etc.), cultured cells, biopsy, or other tissue preparation.
The detection system is used to simultaneously measure the presence, absence and amount of hybridization in all different sequences. This data is used for large-scale correlation studies of sequences, expression patterns, mutations, variants, or polymorphisms in a sample.

The present invention provides methods for identifying the expression of GPCR proteins / peptides of the present invention, and allelic variants of this gene / protein, using such arrays. In particular, such methods involve incubating a test sample with one or more nucleic acid molecules and assaying for binding of the components in the test sample to the nucleic acid molecules. Such an assay involves an array of many genes or alleles, at least one of which is a gene of the invention and / or an allelic variant of the GPCR gene of the invention (FIG. 3, GPC of the present invention
The SNP information found in the gene encoding the R protein is shown. Here, mutations of C1435G, T1262C, and A532G are found).

Conditions for incubating the test sample with the nucleic acid molecule are varied. Incubation conditions depend on the assay format used, the detection method used, and the type and nature of the nucleic acid molecule used in the assay. Those skilled in the art who are aware of commonly available hybridization, amplification, or array assay formats can readily make applications using the novel fragments of the human genome described herein. An example of such an assay is given by Chard, T, An Introduction to
Radioimmunoassay and Related Techniques, Elsevier Science Publishers, A
msterdam, The Netherlands (1986); Bullock, GR et al., Techniques in I
mmunocytochemistry, Academic Press, Orlando, FL Vol. 1 (1 982), Vol. 2 (
1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of Enzyme Immunoa
ssays: Laboratory Techniques in Biochemistry and Molecular Biology, Else
vier Science Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, proteins, membrane extracts from cells.
The test sample used in the above method should have the format of the assay, the nature of the detection method,
And based on the tissue, cells, or extract thereof used as the sample for the assay. The preparation of nucleic acid extracts or cell extracts is well known to those of skill in the art,
It can be easily applied by obtaining a sample that is compatible with the system used.

As another example of the present invention, a kit containing the reagents necessary for performing the assay of the present invention is provided.

In particular, the invention provides (a) a first container containing one or more nucleic acid molecules capable of binding to a fragment of a GPCR described herein, (b) one or more wash reagents, detecting bound nucleic acids. The present invention provides a kit, which is divided into one or more containers containing one or more other containers containing the reagents that can be used and sealed.

In detail, segmented kits include kits in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers, strips of plastic, glass or paper, or array materials such as silicon dioxide. Such containers can efficiently transfer reagents from one compartment to another so that the sample and reagents do not mix and contaminate, and the reagents or solutions in each container are in the other container. Can be quantitatively added to. Such containers include test container, nucleic acid probe-containing container, washing reagent (eg, phosphate buffer, Tris-buffer, etc.), and reagent used for detecting bound probe. Including a container containing. A person skilled in the art can recognize a conventionally unknown GPCR gene according to the present invention, and can routinely confirm it using the sequence information disclosed herein, and further confirm this by an established kit form well known to those skilled in the art. In particular, it can be used by incorporating it into an expression array.

[0154]Vector / host cell   The invention also provides a vector comprising a nucleic acid molecule described herein.
is there. The term "vector" refers to a vehicle, preferably a nucleic acid molecule.
Yes, it is capable of transporting nucleic acid molecules. Vector is nucleic acid
When it is a child, the nucleic acid molecule is covalently linked to the nucleic acid of the vector. This view of the invention
In terms of vectors, vectors include plasmids, single-stranded or double-stranded RNA or DNA
Di-, single-chain or double-stranded viral vector, or BAC, PAC, YAC, OR
Artificial chromosomes such as MAC are included.

The vector is retained in the host cell as an extrachromosomal component in which it replicates and produces additional copies of the nucleic acid molecule. Alternatively, the vector integrates into the genome of the host cell, producing additional copies of the nucleic acid molecule during replication of the host cell.

The present invention provides a vector for holding a nucleic acid molecule (cloning vector) or a vector for expressing a nucleic acid molecule (expression vector). This vector can function in prokaryotic cells or eukaryotic cells, or both (shuttle vectors).

Expression vectors contain a cis-acting regulatory region capable of binding to a nucleic acid molecule in the vector, which allows transcription of the nucleic acid molecule in a host cell. The nucleic acid molecule can be separated from the nucleic acid molecule that affects transcription and introduced into the host cell. Therefore, the second nucleic acid molecule is a ci that effects transcription of the nucleic acid molecule from the vector.
It provides a trans-acting factor that interacts with the s regulatory control region. Alternatively, the trans-acting factor is provided by the host cell. Finally, trans-acting factors can be produced from the vector itself. However, in some cases transcription and / or translation of the nucleic acid molecule may occur in a cell-free system.

The regulatory sequences of the nucleic acid molecules described herein can be operably linked to include a promoter for transcription of the mRNA of interest. These include the left promoter from bacteriophage λ, E. lac, TRP and T obtained from E. coli
AC promoter, early and late promoter derived from SV40, CMV
And the adenovirus early and late promoters, and retrovirus LTRs, but are not limited thereto.

In addition to control regions that promote transcription, expression vectors may also include regions that regulate transcription, such as repressor binding sites and enhancers. Examples include the SV40 enhancer, the cytomegalovirus immediate-early enhancer, the polyoma enhancer, the adenovirus enhancer, the retrovirus LTR enhancer.

In addition to containing transcription initiation and control regions, expression vectors can also include sequences necessary for transcription termination in the transcription region, a ribosome binding site for transcription. Other expression regulation control components include start and stop codons as well as polyadenylation signals. One of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al., Molec.
ular Cloning: A Laboratory Manual. 2nd.ed., Cold Spring Harbor Laborato
ry Press, Cold Spring Harbor, NY, (1989).

Various expression vectors can be used to express a nucleic acid molecule. Such vectors include vectors derived from chromosomes, episomes, viruses, such as bacterial plasmids, bacteriophages, yeast episomes, yeast chromosome components such as artificial yeast chromosomes, baculoviruses, papovaviruses such as SV40. , Vaccinia virus, adenovirus, poxvirus, pseudorabis virus, and retrovirus. Vectors can also be derived from combinations of these sources, for example from plasmids such as cosmids and phagemids and the genetic components of bacteriophage. Suitable cloning and expression vectors for prokaryotic and eukaryotic host cells are described in Sambrook et al., Molecular Cloning: A Labo.
ratory Manual.2nd.ed., Cold Spring Harbor Laboratory Press, Cold Sprin
g Harbor, NY, (1989).

In regulatory sequences, expression as a constituent of one or more host cells (ie tissue specificity)
Or provide directional expression in one or more cell types due to temperature, nutrient addition, or exogenous factors such as hormones and other ligands. A variety of vectors that are constitutively and directedly expressed in prokaryotic and eukaryotic host cells are well known to those of skill in the art.

Nucleic acid molecules can be introduced into vector nucleic acids by well known methods.
Usually, the finally expressed DNA sequence is ligated to the expression vector by cleaving the DNA sequence with the expression vector and one or more restriction enzymes and ligating the fragments together. Restriction enzyme digestion and ligation procedures are well known to those of skill in the art.

The vector containing the appropriate nucleic acid molecule can be introduced into an appropriate host cell for propagation or expression using known techniques. For bacterial cells, E.
coli, Streptomyces, and Salmonella typhi
murium, but is not limited thereto. For eukaryotic cells,
It includes but is not limited to yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.

The expression of the peptide as a fusion protein is preferred, as described herein. Therefore, the present invention provides a fusion vector capable of producing a peptide. Fusion vectors can improve the expression and solubility of recombinant proteins, and can also improve protein purification, eg, by the action of a ligand for affinity purification. The proteolytic cleavage site is introduced at the point of attachment to the fusion moiety, which ultimately separates the peptide of interest from the fusion moiety. Proteolytic enzymes include, but are not limited to, Factor Xa, thrombin, enterokinase. Typical fusion expression vectors include glutathione S-transferase (GST), maltose E.
PGEX (Smith et al., Gene 67: 31-40 (1988)), pMAL (Ne, in which each of the binding protein or protein A was fused to a target recombinant protein.
w England Biolabs, Beverly, MA), pRIT5 (Pharmacia, Piscataway, NJ
), But is not limited thereto. Suitable indicator non-fusion E. co
Examples of li expression vectors include pTrc (Amann et al., Gene 69: 301-315 (1
988), pET11d (Studier et al., Gene Expression Technology: Methods
in Enzymology 185: 60-89 (1990)).

Recombinant protein expression can be maximized in host bacteria by providing a background for genes in the host cell that are capable of proteolytic cleavage deficiency of the recombinant protein (Gottesman, S., Gene Expression. T
echnology: Methods in Enzymology 185, Academic Press, San Diego, Califor
nia (1990) 119-128). Alternatively, the sequence of the nucleic acid molecule of interest may be, for example, E.
E. coli can be modified to be the preferentially used codon for certain host cells (Wada et al., Nucleic Acids Res. 20: 2111-2118 (
1992)).

Nucleic acid molecules can also be expressed by expression vectors that act in yeast. S. Examples of vectors that are expressed in yeast such as cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6: 229-234 (1987)), p
MFa (Kurjan et al., Cell 30: 933-943 (1982)), pJRY88 (Schultz et.
al., Gene 54: 113-123 (1987)), pYES2 (Invitrogen Corporation, San
Diego, CA) is included.

Nucleic acid molecules can also be expressed in insect cells, for example using baculovirus expression vectors. Vectors used to express proteins in cultured insect cells (eg, Sf9 cells) include pAc series (Smith et al., Mol. Cel.
Biol. 3: 2156-2165 (1983)), and pVL series (Lucklow et al., Virolo
gy 170: 31-39 (1989)).

In one embodiment of the invention, the nucleic acid molecules described herein are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, B. Nature 329: 840 (1987)), and pMT2PC (Kau.
fman et al., EMBO J. 6: 187-195 (1987)).

As the expression vectors listed here, only those known as vectors that are useful for expressing a nucleic acid molecule and are available to those skilled in the art are shown. Other suitable vectors for maintaining or expressing the nucleic acid molecules described herein are well known to those of skill in the art. These include, for example, Sambrook, J., Fritsh,
EF, and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed.
, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Co
ld Spring Harbor, NY, 1989.

Vectors in which the nucleic acid sequences described herein are cloned into the vector in the reverse orientation are capable of binding regulatory sequences which allow transcription of the antisense RNA, but the invention also includes such vectors. It also includes. Thus, antisense transcription can produce all or part of a nucleic acid molecule sequence described herein, including both coding and non-coding regions. The expression of this antisense RNA corresponds to each of the above-mentioned parameters regarding the expression of the sense RNA (regulatory sequence, constitutive or directive expression, tissue-specific expression).

The present invention also relates to recombinant host cells containing the vectors described herein. Thus, host cells include prokaryotic cells, low eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and high eukaryotic cells such as mammalian cells.

Recombinant host cells can be prepared by introducing into a cell a vector constructed as described herein by techniques readily available to those of skill in the art. These include calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection,
Electroporation, transduction, infection, lipofection, and Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2n
d, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Pre
ss, Cold Spring Harbor, NY, 1989), including but not limited to other techniques.

Host cells can include one or more vectors. This allows different nucleotide sequences to be introduced into different vectors in the same cell. Similarly, the nucleic acid molecule can be introduced alone or with other unrelated nucleic acid molecules, such as those providing the trans-acting factor of the expression vector. When more than one vector is introduced into a cell, the vectors can be introduced alone, co-introduced, or linked to a nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, they can be introduced intracellularly as encapsulated or encapsulated virus by standard infection and transduction procedures. Viral vectors are replicable,
Or it may be a replication defect. If the replication of the virus is defective, replication can occur in host cells where the function of complementing the defect is provided.

Vectors generally contain a selectable marker that allows for selection of a subpopulation of cells that contain the recombinant vector constructs. The marker can be included in the same vector containing the nucleic acid molecule described herein or in another vector. Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, markers that provide selectivity for the phenotypic trait are effective in each case.

Although mature proteins can be produced in bacteria, yeast, mammalian cells, and other cells under the control of appropriate regulatory sequences, cell-free transcription and translation systems are also described herein. RNA derived from the described DNA constructs can be used to produce these proteins.

If secretion of the peptide is required, this is difficult to achieve within a multi-transmembrane domain containing protein such as a GPCR and an appropriate secretion signal is incorporated into the vector. The signal sequence can be endogenous to these peptides or heterologous to the peptides.

If the peptide is not secreted in the medium, typically in the case of GPCRs, the protein is isolated from the host cell by standard disruption procedures such as freeze-thaw, sonication, mechanical disruption, degradation reagents. Can be Peptides are recovered by known purification methods, including ammonium sulfate precipitation, acid extraction, or anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography. , Can be purified.

Also, depending on the host cell of the recombinant production of the peptides described herein,
It is understood that peptides have different glycosylation patterns and, depending on the cell, may not be glycosylated when produced in bacteria. In addition, the peptide may include a methionine that was first modified in some cases as a result of a host-mediated process.

[0181]Use of vectors and host cells   Recombinant host cells expressing the peptides described herein include a variety of
There are uses. First, these cells are involved in the production of GPCR proteins or peptides.
For producing the required amount of GPCR protein or fragment.
Can be purified. For this reason, host cells containing expression vectors are
This is useful for generating code.

Host cells are useful in performing cell-based assays involving GPCR proteins or GPCR protein fragments, such as those described above, as well as other forms well known to those of skill in the art. As such, recombinant host cells expressing the native GPCR protein are useful in assaying compounds that promote or inhibit GPCR protein function.

Host cells are also useful for identifying functionally affected GPCR protein variants. When the mutation naturally occurs and causes pathology, the host cell containing the mutation does not exhibit the effects of the native GPCR protein, but the effect (eg, promoting or inhibiting function) required of the GPCR protein variant. ) Is useful in the assay of compounds.

The genetically engineered host cells can be further used to produce transgenic animals other than humans. The transgenic animal is preferably a mammal, eg a rodent such as a rat or mouse in which one or more cells contain the recombinant gene. A recombinant gene is an exogenous DNA that integrates into the genome of a cell of a growing transgenic animal and remains in the genome of the mature animal in one or more cell types or tissues. These animals are useful for studying GPCR protein function and identifying and evaluating modulators of GPCR protein activity. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.

Genetically modified animals introduce nucleic acid molecules into male prokaryotic cells of fertilized oocytes by, for example, microinjection or retroviral infection, and the oocytes are bred in pseudopregnant female rearing animals. It is produced by Which GPCR
Protein nucleotide sequences can also be introduced as recombinant genes into the genome of non-human animals such as mice.

Any of the regulatory or other sequences useful in expression vectors can form part of the recombinant gene sequence. The intron sequence and the polyadenylation signal
This is also included if it is not already included. Tissue-specific regulatory sequences can be effectively linked to recombinant genes due to the direct expression of GPCR proteins in specific cells.

Methods for producing genetically modified animals through conception operations and microinjection, especially methods using animals such as mice, are generalized in the art, for example, US Patent Nos. 4,736,866, and 4,870,009, by. Leder et
al., US Patent No. 4,873,191 by Wagner et al. and in Hogan, B., Manipu
lating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spri
ng Harbor, NY, 1986). Similar methods have also been used for the production of other transgenic animals. The first transgenic animal can be identified based on the presence of the recombinant gene in the genome and / or the expression of the transgenic mRNA in the tissues or cells of the animal. The first transgenic animal can then be used to breed animals with additional recombinant genes. Moreover, a transgenic animal having a recombinant gene can be bred to another transgenic animal having another recombinant gene. Transgenic animals also include any animal or animal tissue produced using the homologous recombinant host cells described herein.

In another example, non-human transgenic animals can be produced that contain a selection system that provides for regulated expression of the recombinant gene. One example of such a system is the cre / loxP recombinase system of bacteriophage P1. For a description of the cre / loxP recombinase system, see, eg, Lakso et al. PNAS 89: 6232-6236 (1992). Another example of a recombinase system is the S. cerevisiae FLP recombinase system (O'Gorman et al. Science 251: 1351-1355 (1991). Cre / lo.
If the xP recombinase system is used to regulate the expression of recombinant genes, it is necessary for the animal to contain a recombinant gene encoding Cre recombinase and a selected protein. Such animals can be "doubled", for example, by crossing two recombinant gene animals, one carrying a recombinant gene encoding a selected protein and the other carrying a recombinant gene encoding a recombinase. It is provided by constructing a transgenic animal.

Non-human transgenic animal clones described herein were also cloned by Wilmut.
, I. et al. Nature 385: 810-813 (1997), and PCT International Publication.
It can be produced according to the method described in Nos. WO 97/07668 and WO 97/07669. Briefly, cells from transgenic animals, such as somatic cells, can be isolated and induced to exit the growth cycle and enter G ₀ phase. The quiescent cells can be fused to, for example, the use of electric pulses to denuclearized oocytes of animals of the same species as the isolated quiescent cells. The reconstituted oocyte is cultured so as to develop into a morula or a blast cell, and then,
Transferred into pseudopregnant female breeders. The offspring born from this female breeding animal are
It becomes a clone of an animal in which cells, for example, somatic cells are isolated.

Transgenic animals containing recombinant cells expressing the peptides described herein are useful for performing assays as described herein in an in vivo environment. Therefore, various physiological factors that are present in vivo and that influence ligand binding, GPCR protein activation, and signal transduction may not be revealed by in vitro cell-free or cell-based assays. As such, they are non-human for assaying in vivo GPCR protein function, including ligand interaction, GPCR protein function and the effect of certain variant GPCR proteins on ligand interaction, and the effect of chimeric GPCR proteins. It is useful for providing transgenic animals. It is also possible to assess the effect of null mutations, which are mutations that substantially or completely eliminate one or more GPCR protein functions.

All publications and patents mentioned above are incorporated herein by reference. Various modifications and variations of the method and system described in this invention will be apparent to those skilled in the art without departing from the scope and spirit of this invention. Although the present invention has been described in relation to particular preferred embodiments, it is to be understood that the invention as claimed is not unduly limited to such particular embodiments. . Indeed, various modifications of the above-described methods for carrying out the present invention will be apparent to those skilled in molecular biology or related fields, and such are also within the scope of the following claims.

[Brief description of drawings]

FIG. 1 shows the nucleotide sequence of a cDNA molecule or a transcribed sequence encoding the GPCR of the present invention (SEQ ID NO. 1). Furthermore, structural and functional information such as ATG initiation, termination, and tissue distribution is presented here, which can be used to easily determine a particular application of the invention based on this molecular sequence. Screening of tissue-specific libraries has shown that the GPCRs of the invention are expressed in at least brain, placenta and thyroid.

FIG. 2 shows the predicted amino acid sequence of the GPCR of the present invention (SEQ ID NO. 2).
). In addition, structural and functional information such as protein family, function, alteration sites is presented here, which can be used to easily determine specific applications of the invention based on this molecular sequence and important fragments of the proteins of the invention. can do.

FIG. 3 shows the genomic sequence of the gene region encoding the GPCR protein of the present invention (SEQ ID NO.3). Further, here, structural and functional information such as intron / exon structure, promoter position, etc. is shown, which is important for use in specific applications of the invention based on this molecular sequence, designing probes and primers, and controlling heterologous gene expression. Different fragments can be easily determined. Also shown in FIG. 3 is the SNP information found in the gene encoding the GPCR protein of the invention, where C1435G, T1262C, and A532G.
Mutations are seen.

[Sequence list]

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (Reference) C07K 16/28 C12N 1/15 4B065 C12M 1/00 1/19 4C084 C12N 1/15 1/21 4H045 1 / 19 C12P 21/02 C 1/21 C12Q 1/02 5/10 1/68 A C12P 21/02 G01N 33/15 Z C12Q 1/02 33/50 Z 1/68 33/53 D G01N 33/15 M 33 / 50 33/566 33/53 C12N 15/00 ZNAA F 33/566 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, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE , GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ , VN, YU, ZA, ZW (72) Inventor Kravchik, Anivar United States Merri Land 20850 Rockville, West Goode Drive 45, Serera (72) Inventor DI, Francesco, Valentina United States Maryland 20850 Rockville, West Goode Drive 45, Serera (72) Inventor Beasley, Ellen, Em 20850 Rockville, MD, USA, West Goode Drive 45, F term in cerera (reference) 2G045 AA34 AA40 BA11 BB50 DA12 DA13 DA36 FB02 4B024 AA01 BA63 BA80 CA02 CA09 CA12 DA01 DA02 DA05 DA11 DA12 FA02 GA11 HA11 HA14 4B029 AA07 AA07 A21 BB20 CC01 CC03 CC08 FA12 FA15 4B063 QA01 QA05 QQ21 QQ41 QQ43 QQ53 QQ61 QQ89 QR32 QR35 QR40 QR55 QR77 QR80 QR84 QS34 QS36 QX01 QX02 4B064 AG20 CA01 CA20 CC01 CC24 CC01 DA24 DAB ZB262 ZC021 ZC022 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA50 DA75 EA20 EA51 FA71 FA74

Claims (23)

[Claims] 1. An isolated peptide comprising an amino acid sequence selected from the following group. (A) SEQ ID NO. Amino acid sequence shown in FIG. 2; (b) SEQ ID NO. The amino acid sequence of an allelic variant of the amino acid sequence set forth in 2, wherein said allelic variant is SEQ ID NO. 1 (transcription) or 3 (genome)
An amino acid sequence characterized by being encoded by a nucleic acid molecule that hybridizes to the opposite strand of the nucleic acid molecule shown in (1) under stringent conditions; (c) SEQ ID NO. The amino acid sequence of the ortholog of the amino acid sequence shown in 2, wherein the ortholog is SEQ ID NO. 1 (transcription) or 3 (genome)
An amino acid sequence characterized by being encoded by a nucleic acid molecule that hybridizes to the opposite strand of the nucleic acid molecule shown in Figure 4 under stringent conditions; and (d) SEQ ID NO. A fragment of the amino acid sequence shown in 2,
An amino acid sequence characterized in that the fragment comprises at least 10 contiguous amino acids. 2. An isolated peptide comprising an amino acid sequence selected from the following group. (A) SEQ ID NO. Amino acid sequence shown in FIG. 2; (b) SEQ ID NO. The amino acid sequence of an allelic variant of the amino acid sequence set forth in 2, wherein said allelic variant is SEQ ID NO. 1 (transcription) or 3 (genome)
An amino acid sequence characterized by being encoded by a nucleic acid molecule that hybridizes to the opposite strand of the nucleic acid molecule shown in (1) under stringent conditions; (c) SEQ ID NO. The amino acid sequence of the ortholog of the amino acid sequence shown in 2, wherein the ortholog is SEQ ID NO. 1 (transcription) or 3 (genome)
An amino acid sequence characterized by being encoded by a nucleic acid molecule that hybridizes to the opposite strand of the nucleic acid molecule shown in Figure 4 under stringent conditions; and (d) SEQ ID NO. A fragment of the amino acid sequence shown in 2,
An amino acid sequence characterized in that the fragment comprises at least 10 contiguous amino acids. 3. An isolated antibody that selectively binds to the peptide of claim 2. 4. An isolated nucleic acid molecule consisting of a nucleotide sequence selected from the following group: (A) SEQ ID NO. A nucleotide sequence encoding the amino acid sequence shown in 2; (b) SEQ ID NO. A nucleotide sequence encoding an allelic variant of the amino acid sequence shown in SEQ ID NO: 2. 1 (
(Transcription) or 3 (genome) to the opposite strand of the nucleic acid molecule hybridized under stringent conditions; (c) SEQ ID NO. A nucleotide sequence encoding an ortholog of the amino acid sequence shown in 2, which has the nucleotide sequence of SEQ ID NO. 1 (
(Transcription) or 3 (genome) to the opposite strand of the nucleic acid molecule hybridized under stringent conditions; and (d) SEQ ID NO. A nucleotide sequence encoding a fragment of the amino acid sequence shown in 2, wherein said fragment comprises at least 10 contiguous amino acids. (E) A nucleotide sequence that is complementary to the nucleotide sequences of (a)-(d). 5. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group below. (A) SEQ ID NO. A nucleotide sequence encoding the amino acid sequence shown in 2; (b) SEQ ID NO. A nucleotide sequence encoding an allelic variant of the amino acid sequence shown in SEQ ID NO: 2. 1 (
(Transcription) or 3 (genome) to the opposite strand of the nucleic acid molecule hybridized under stringent conditions; (c) SEQ ID NO. A nucleotide sequence encoding an ortholog of the amino acid sequence shown in 2, which has the nucleotide sequence of SEQ ID NO. 1 (
(Transcription) or 3 (genome) to the opposite strand of the nucleic acid molecule hybridized under stringent conditions; and (d) SEQ ID NO. A nucleotide sequence encoding a fragment of the amino acid sequence shown in 2, wherein said fragment comprises at least 10 contiguous amino acids. (E) A nucleotide sequence that is complementary to the nucleotide sequences of (a)-(d). 6. A gene chip containing the nucleic acid molecule according to claim 5. 7. A non-human transgenic animal comprising the nucleic acid molecule of claim 5. 8. A nucleic acid vector comprising the nucleic acid molecule according to claim 5. 9. A host cell containing the vector according to claim 8. 10. A method for producing the peptide according to claim 1, which comprises introducing a nucleotide sequence encoding the amino acid sequence of any one of (a) to (d) into a host cell to obtain the peptide. A method of culturing a host cell under conditions in which is expressed from a nucleotide sequence. 11. A method for producing the peptide according to claim 2, which comprises introducing a nucleotide sequence encoding the amino acid sequence according to any one of (a) to (d) into a host cell to obtain the peptide. A method of culturing a host cell under conditions in which is expressed from a nucleotide sequence. 12. A method for detecting the presence of any of the peptides according to claim 2 in a sample, comprising contacting the sample with a reagent for specifically detecting the presence of the peptide in the sample, To detect the presence of. 13. A method for detecting the presence of the nucleic acid molecule according to claim 5 in a sample, which comprises contacting an oligonucleotide hybridizing with the nucleic acid molecule under stringent conditions with the sample, A method for determining whether or not the nucleic acid molecule and the oligonucleotide bind. 14. A method for identifying a modulator of the peptide according to claim 2, wherein the peptide is contacted with a reagent and it is determined whether the reagent modulates the function or activity of the peptide. 15. The method according to claim 14, wherein the reagent is provided to a host cell containing an expression vector expressing the peptide. 16. A method for identifying a reagent that binds to any of the peptides according to claim 2, wherein the peptide and the reagent are contacted to form a complex in which the peptide and the reagent are bound in a contact mixture. A method of assaying whether or not. 17. A pharmaceutical composition comprising a reagent identified by the method of claim 16 and pharmaceutically acceptable carriers thereof. 18. A method of treating a disease or condition mediated by the human G protein-coupled receptor, which comprises administering to a patient a pharmaceutically effective amount of the reagent identified by the method of claim 16. . 19. A method for identifying a modulator of the expression of the peptide according to claim 2, comprising contacting a cell expressing the peptide with a reagent, and determining whether the reagent modulates the expression of the peptide. How to measure. 20. The SEQ ID NO. An isolated human G protein-coupled receptor peptide having an amino acid sequence having at least 70% homology with the amino acid sequence shown in 2. 21. The peptide according to claim 20, wherein SEQ ID NO. A peptide having an amino acid sequence having at least 90% homology to the amino acid sequence shown in 2. 22. An isolated nucleic acid molecule encoding a human G protein-coupled receptor peptide comprising SEQ ID NO. A nucleic acid molecule having at least 80% homology with the nucleic acid molecule shown in 1 (transcription) or 3 (genome). 23. The nucleic acid molecule of claim 22, wherein SEQ ID NO. 1 (
Nucleic acid molecule having at least 90% homology with the nucleic acid molecule shown in (transcription) or 3 (genome).