JP2003525571A - 7TM receptor GABAB-R2a - Google Patents

Abstract

  (57) [Summary] GABAB-R2a polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods of using GABAB-R2a polypeptides and polynucleotides for therapy, and diagnostic assays for such uses.

Description

Detailed Description of the Invention

This application is based on US Provisional Application Nos. 60 / 075,306 and 1998, filed February 20, 1998.
Claiming the benefit of priority of British Patent Application No. 9817907.0 filed August 17, 2010,
This is a continuation-in-part application of U.S. Patent Application No. 09 / 183,253, filed October 30, 1998.
Claims priority to US Partial Continuation Application filed February 19, 1999 (application number not yet given). The entire contents of these applications are incorporated herein by reference.

[0002] THE INVENTION The present invention relates to newly identified polypeptides, polynucleotides encoding such polypeptides, agonists that may be effective in as well as therapeutic treatment, a compound which is an antagonist and / or inhibitor It relates to the use of said polypeptides and polynucleotides in identifying, as well as methods of producing said polypeptides and polynucleotides.

Prior Art Drug discovery processes are currently undergoing major changes. Because it extends to "functional genomics," that is, high-throughput genome- or gene-based biology. This approach is rapidly replacing the relatively early approaches based on "positional cloning." A phenotype, i.e. a biological function or genetic disease, is identified,
Then, the etiological genes were located by using the position of the genetic map as a clue.

Genomic functioning relies heavily on various tools of bioinformatics to identify gene sequences that may be of interest from the many molecular biology databases currently available. Yet, there is a need to identify and characterize additional genes and their related polypeptides / proteins as targets for drug discovery.

Many medically important biological processes include proteins and / or second messengers (eg, cAM) involved in signal transduction pathways, including G proteins.
It is well known that it is mediated by (P) (Lefkowitz, Nature, 1991, 35.
1: 353-354). These proteins are referred to herein as PPG proteins, which are proteins involved in pathways involving G proteins. Some examples of these proteins include adrenergic agonists and receptors for dopamine (Kobilka, BK et al., Proc. Natl. Acad. Sci. USA, 1987, 84 : 46-50; Kobilka.
, BK et al., Science, 1987, 238: 650-656; Bunzow, JR et al., Nature, 1988, 336:
783-787), G proteins themselves, effector proteins (eg phospholipase C, adenyl cyclase, and phosphodiesterase), actuator proteins (eg protein kinase A and protein kinase C) (Simon,
MI et al., Science, 1991, 252: 802-8).

For example, in one form of signal transduction, the binding of hormones activates the enzyme adenylate cyclase intracellularly. Activation of this enzyme by the hormone depends on the presence of the nucleotide GTP. GTP also affects hormone binding. G-proteins link hormone receptors to adenylate cyclase. G protein was bound to GDP when activated by hormone receptor
Was exchanged for GTP. This GTP-bearing form then binds to activated adenylate cyclase. The hydrolysis of GTP to GDP is catalyzed by the G protein itself, returning it to its inactive ground state. Thus, the G protein functions as an intermediate that takes over the signal from the receptor to the effector, and as a clock that controls the duration of the signal, and plays two roles.

The membrane protein gene superfamily of G protein-coupled receptors has been characterized as having seven putative transmembrane domains. These domains are extracellular,
Alternatively, it is considered to correspond to a transmembrane α-helix connected by a cytoplasmic loop. G-protein coupled receptors include many biologically active receptors such as hormones, viruses, growth factors and neural receptors.

The G protein-coupled receptor (otherwise known as the 7TM receptor) is a conserved hydrophobic region of about 20-30 amino acids that connects at least eight different hydrophilic loops. Has been characterized as including. The family of G protein-coupled receptors includes the dopamine receptors that bind neuroleptic drugs used in the treatment of psychosis and neuropathy. Other examples of members of this family include calcitonin, adrenaline, endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, histamine, thrombin, quinine, follicle stimulating hormone, opsin, endothelial cell differentiation gene-1, rhodopsin, odorant, And cytomegalovirus receptors.

Most G-protein coupled receptors contain a conserved cysteine residue forming a disulfide bond in each of the first two extracellular loops that is thought to stabilize the structure of the functional protein. I have one. 7 transmembrane regions are TM1 and TM
2, referred to as TM3, TM4, TM5, TM6, and TM7. TM3 is involved in signal transduction.

Phosphorylation and lipidation (palmitoylation or farnesylation) of cysteine residues can influence the signaling of some G protein-coupled receptors. Most G protein-coupled receptors often contain a phosphorylation site within their third cytoplasmic loop and / or carboxy terminus. In some G protein-coupled receptors, such as the β-adrenergic receptor, phosphorylation by protein kinase A and / or specific receptor kinases mediates receptor desensitization.

In some receptors, the ligand binding site of a G protein-coupled receptor is a hydrophilic socket (soc) formed by several G protein-coupled receptor transmembrane domains.
ket), the socket is believed to be surrounded by hydrophobic residues of G protein-coupled receptors. It is hypothesized that the hydrophilic side of each G protein coupled receptor transmembrane helix faces inward and forms a polar ligand binding site. TM3 has a ligand binding site, such as the TM3 aspartic acid residue, and thus has been involved in ligand binding at some G-protein coupled receptors. TM5 serine, TM6 asparagine and TM6 or TM7 phenylalanine or tyrosine are also involved in ligand binding.

G protein-coupled receptors can bind intracellularly to a variety of intracellular enzymes, ion channels and transporters by heterotrimeric G proteins (Johnson
Endoc. Rev., 1989, 10: 317-331). The α subunit of each G protein preferentially stimulates specific effectors to modulate various biological functions within the cell. Phosphorylation of cytoplasmic residues of G protein-coupled receptors has been identified as an important mechanism for regulating G protein coupling of some G protein-coupled receptors. G-protein coupled receptors are found in numerous sites in the mammalian host. Over the last 15 years, nearly 350 therapeutic agents targeting the seven transmembrane (7TM) receptor have been successfully marketed.

SUMMARY OF THE INVENTION The present invention relates to GABAB-R2a, particularly GABAB-R2a polypeptides and GABAB-R2a polynucleotides, recombinant materials, and methods of producing the same. In a different embodiment, the present invention relates to infections such as bacterial infections, fungal infections, protist and viral infections, especially infections caused by HIV-1 or HIV-2; pain; cancer; diabetes; obesity;
Anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; urinary retention;
Osteoporosis; angina; myocardial infarction; stroke; ulcer; allergy; benign prostatic hypertrophy; migraine; vomiting; anxiety, schizophrenia, manic depression, depression, delirium, dementia, severe mental retardation, and other nervous system disorders Disease; and Huntington's disease or Jill de la
The present invention relates to a method for using the above-mentioned polypeptide and polynucleotide, including the treatment of movement disorders such as Tourette's syndrome (hereinafter collectively referred to as "the disease"). In another aspect, the present invention provides a method of identifying agonists and antagonists / inhibitors using the agents provided by the invention, and treating the conditions associated with GABAB-R2a imbalance with the identified compounds. Regarding In yet another aspect, the invention relates to a diagnostic assay for detecting a disease associated with inappropriate GABAB-R2a activity or GABAB-R2a levels.

[0014] In embodiments of the description one invention, the present invention relates to GABAB-R2a polypeptide. Peptides of this type have at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2. Includes an isolated polypeptide comprising an amino acid sequence having at least 95% identity, and most preferably at least 97-99% identity. Such polypeptides include the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.

For other peptides of the invention, the amino acid sequence is at least 70% identical, preferably at least 80% identical, more preferably to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2. Included are isolated polypeptides having at least 90% identity, more preferably at least 95% identity, and most preferably at least 97-99% identity. Such a polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.

Further peptides of the invention include an isolated polypeptide encoded by a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1.

The polypeptides of the present invention are considered to be members of the G protein-coupled receptor family of polypeptides. Therefore, they are interesting. Because G
Protein-coupled receptors are the targets of pharmaceutical intervention for more than any other gene family.

These properties are hereafter referred to as “GABAB-R2a activity” or “GABAB-R2a polypeptide activity” or “GABAB-R2a biological activity”. These activities include GABAB-
It also includes the antigenic and immunogenic activities of R2a polypeptides, particularly the polypeptide of SEQ ID NO: 2. Preferably, the polypeptide of the invention comprises at least one GABAB-
5 shows the biological activity of R2a.

The polypeptide of the invention may be in the form of a "mature" protein or may be part of a larger protein, such as a fusion protein. It is often advantageous to include additional amino acid sequences, such as secretory or leader sequences, prosequences, sequences useful for purification such as multiple histidine residues, or during recombinant production. There are additional sequences etc. that ensure stability.

The present invention also includes variants of the above polypeptides, ie, polypeptides that differ from the reference polypeptide by conservative amino acid substitutions (where one residue is replaced by another residue of similar nature). include. Typical such substitutions are Ala, Val, Le
Between u and Ile; between Ser and Thr; between acidic residues Asp and Glu; between Asn and Gln; between basic residues Lys and Arg; or between aromatic residues Phe and Tyr. In particular, a variant in which several, 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1 amino acids are substituted, deleted or added in any combination is preferable.

The polypeptide of the present invention can be produced by any appropriate method. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Be done. Means for producing such polypeptides are well understood in the art.

In a further aspect of the invention, the invention relates to GABAB-R2a polynucleotides. Such a polynucleotide has at least 70% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2, preferably at least 80%.
An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having at least 90% identity, more preferably at least 90% identity, and even more preferably at least 95% identity. In this regard, at least 97
Polypeptides with% identity are more preferred, those with at least 98-99% identity are even more preferred, and polypeptides with at least 99% identity are most preferred. Examples of such a polynucleotide include a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1 which encodes the polypeptide of SEQ ID NO: 2.

A further polynucleotide of the present invention has at least 70% over its entire coding region, relative to the nucleotide sequence encoding the polypeptide of SEQ ID NO: 2.
Identity, preferably at least 80% identity, more preferably at least 9
Included is an isolated polynucleotide comprising a nucleotide sequence having 0% identity, more preferably at least 95% identity. In this regard, polynucleotides having at least 97% identity are more preferred, those with at least 98-99% identity are even more preferred, and those with at least 99% identity are most preferred. .

Further polynucleotides of the invention include SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.
A polynucleotide sequence having at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably at least 95% identity. Separated polynucleotides are included. In this regard, polynucleotides having at least 97% identity are more preferred, those with at least 98-99% identity are even more preferred, and those with at least 99% identity are most preferred. . Such polynucleotides include a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 and the polynucleotide of SEQ ID NO: 1.

The present invention also provides polynucleotides which are complementary to all the above polynucleotides.

The nucleotide sequence of SEQ ID NO: 1 is homologous to GABAB-R2 (K. Jones et al. (1998) Na
ture 396, 674-679; J. White et al. (1998) Nature, 396, 679-682; K. Kaupmann et al. (199
8) Nature 379, 683-686). The nucleotide sequence of SEQ ID NO: 1 is a cDNA sequence and includes a polypeptide coding sequence (nucleotide numbers 31-2652) encoding a polypeptide of 874 amino acids which is the polypeptide of SEQ ID NO: 2. Although the nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 is the same as the polypeptide coding sequence contained in SEQ ID NO: 1 because of the redundancy (degeneracy) of the genetic code,
It may be a sequence other than the sequence contained in SEQ ID NO: 1, which also encodes the polypeptide of SEQ ID NO: 2. The polypeptide of SEQ ID NO: 2 is structurally related to other proteins of the G protein-coupled receptor family that have homology and / or structural similarity to GABAB-R2 (K. Jones et al., Nature 396, 674-679 (1998); JW
hite et al., Nature, 396, 679-682 (1998); K. Kaupmann et al., Nature 379, 683-686 (19).
98)).

Suitable polypeptides and polynucleotides of the invention are expected to have, inter alia, similar biological functions / properties to the homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one GABAB-R2a activity.

The present invention also relates to partial or other polynucleotides and polypeptides initially identified prior to the determination of the corresponding full length sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

Accordingly, in a further aspect, the invention provides: (a) at least 70% identity, preferably at least 80% identity, more preferably to the nucleotide sequence of SEQ ID NO: 3 over the entire length of SEQ ID NO: 3. Is a nucleotide sequence having at least 90% identity, more preferably at least 95% identity, most preferably 97-99% identity, (b) to the nucleotide sequence of SEQ ID NO: 3 over the entire length of SEQ ID NO: 3. Having at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably at least 95% identity, most preferably 97-99% identity. Sequence, (c) polynucleotide of SEQ ID NO: 3, or (d) SEQ ID NO: 4 spanning the entire length of SEQ ID NO: 4 At least 70% identity to the amino acid sequence, preferably at least 80% identity, more preferably at least 90% identity, even more preferably at least 95% identity, most preferably 97-99% identity. A nucleotide sequence encoding a polypeptide consisting of an amino acid sequence having sex, an isolated polynucleotide comprising: and a polynucleotide of SEQ ID NO: 3.

Further, the invention provides (a) at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity to the amino acid sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4. A polypeptide comprising an amino acid sequence having identity, more preferably at least 95% identity, most preferably 97-99% identity, (b) to the amino acid sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4. Have at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably at least 95% identity, most preferably 97-99% identity. A polypeptide having an amino acid sequence, (c) a polypeptide comprising the amino acid of SEQ ID NO: 4, and (d) a polypeptide of SEQ ID NO: 4. Polypeptide is a peptide, and polypeptide encoded by a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 3, to provide.

The nucleotide sequence of SEQ ID NO: 3 and the peptide sequence encoded thereby are derived from the Expressed Sequence Tag (EST) sequence. One of ordinary skill in the art will appreciate that there will necessarily be some nucleotide sequence reading errors in the EST sequence (Adams, MD et al., Nature 37).
7 (supp) 3, 1995). Therefore, the nucleotide sequence of SEQ ID NO: 3 and the peptide sequence encoded thereby are subject to the same inherent limitations on sequence accuracy. Furthermore, the peptide sequence encoded by SEQ ID NO: 3 has the same region as the protein with the highest homology or structural similarity, or high homology and / or
Alternatively, it contains regions of structural similarity (eg, conservative amino acid differences).

The polynucleotides of the invention were analyzed by EST analysis from a cDNA library derived from mRNA in human hippocampal cells by standard cloning and screening techniques (Adams, MD et al., Science (1991) 252: 1651-. 1656; Adams, MD et al.
, Nature (1992) 355: 632-634; Adams, MD et al., Nature (1995) 377 Supp: 3-174.
). In addition, the polynucleotide of the present invention is a genomic DNA
It can be obtained from natural sources such as libraries or can be synthesized using well known commercially available techniques.

When the polynucleotide of the present invention is used for recombinant production of the polypeptide of the present invention, the polynucleotide may include the coding sequence of the mature polypeptide alone or other coding sequences (eg, leader or secretory sequences). Sequence, pre- or pro- or pre-pro protein sequence, or other fusion peptide portion coding sequence) in the same reading frame with the mature polypeptide coding sequence. For example, a marker sequence that facilitates purification of the fusion polypeptide can be encoded. In a preferred embodiment of this aspect of the invention, the marker sequence is provided by the pQE vector (Qiagen, Inc.) and is provided by Gentz et al., Proc. Natl. Acad. Sci. USA.
(1989) 86: 821-824, a hexa-histidine peptide, or HA tag. The polynucleotide may also include 5'and 3'non-coding sequences, such as transcribed but untranslated sequences, splicing and polyadenylation signals, ribosome binding sites, and mRNA stabilizing sequences.

Further specific examples of the present invention include several, for example, 5 to 10, 1 to 5, and 1 to 3.
There is a polynucleotide encoding a polypeptide variant comprising the amino acid sequence of SEQ ID NO: 2, in which one, one or two, or one amino acid residue is substituted, deleted or added in any combination. .

A polynucleotide which is identical or sufficiently identical to the nucleotide sequence contained in SEQ ID NO: 1 has been used for the isolation of full-length cDNA and genomic clones encoding the polypeptides of the invention and also in SEQ ID NO: 1. To isolate cDNAs and genomic clones of other genes with high sequence similarity to, including genes encoding homologues and orthologs from species other than human.
It can be used as a hybridization probe for DNA and genomic DNA or as a primer for nucleic acid amplification (PCR) reactions. Typically,
These nucleotide sequences are 70%, preferably 80% of the reference nucleotide sequence.
%, More preferably 90% and most preferably 95% identical. The probe or primer usually contains 15 or more nucleotides, preferably 30 or more nucleotides and may have 50 or more nucleotides. Particularly preferred probes are those having a range of 30-50 nucleotides.

A polynucleotide encoding the polypeptide of the present invention (including homologues and orthologs derived from species other than human) is a stringent probe using a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof. It is obtained by a method comprising the steps of screening a suitable library under hybridization conditions and isolating full-length cDNA and genomic clones containing the polynucleotide sequence. Such hybridization techniques are well known to those of ordinary skill in the art. Preferred stringent hybridization conditions are: 50% formamide, 5xSSC
(150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),
Incubate overnight at 42 ° C in a solution containing 5x Denhardt's solution, 10% dextran sulfate and 20 µg / ml denatured and sheared salmon sperm DNA, then wash the filters in 0.1x SSC at about 65 ° C. Thus, the present invention also includes a polynucleotide obtained by screening a suitable library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof.

As will be appreciated by those in the art, isolated cDNA sequences are often incomplete, as the region encoding the polypeptide is often truncated at the 5'end of the cDNA. Let's do it. It is due to reverse transcriptase, which originally has a low "processivity" (processivity: a measure of the enzyme's ability to remain bound to the template during the polymerization reaction), which causes mRNA template during first strand cDNA synthesis. Can not complete the DNA copy of.

There are several methods well known and available to those skilled in the art for obtaining full-length cDNAs or for extending short-chain cDNAs, for example, the cDNA terminal rapid amplification method (R
ACE) based methods (eg Frohman et al., PNAS USA 85, 8998-9002,
See 1988). Recent improvements in the above techniques, such as those demonstrated by Marathon ^™ technology (Clontech Laboratories Inc.), have greatly facilitated the search for longer cDNAs. With Marathon ^TM technology, mRNA extracted from a given tissue
CDNA is made from and the "adapter" sequences are ligated to each end. Subsequently, nucleic acid amplification (PCR) is performed using a combination of gene-specific and adapter-specific oligonucleotide primers to amplify the "deletion"5'end of the cDNA. next,
A "nested" primer, ie, a primer designed to anneal within the amplification product (typically an adapter-specific primer that anneals 3'to the adapter sequence and an additional 5'to the known gene sequence). PCR reaction is repeated using a gene-specific primer). After that, the product of this reaction is analyzed by DNA sequencing, and this product is directly bound to an existing cDNA to form a complete sequence, or a new sequence information for 5 ′ primer design is used to obtain another full-length sequence. A full-length cDNA can be constructed by performing PCR.

Recombinant polypeptides of the invention can be produced from genetically engineered host cells containing expression systems using methods well known in the art. Thus, in a further aspect, the invention relates to expression systems containing one or more polynucleotides of the invention, host cells genetically engineered by the expression systems, and the production of polypeptides of the invention by recombinant methods. Cell-free translation systems can also be used to produce proteins of this type using RNA derived from the DNA constructs of the invention.

For recombinant production, host cells are genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention. Introduction of polynucleotides into host cells is described by Davis et al., Basic Methods in Molecular Biology (1986) and Sambro.
ok et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbo
This can be done by the methods described in many standard laboratory manuals such as r Laboratory Press, Cold Spring Harbor, NY (1989). Suitable such methods include, for example, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape loading, bullet loading. Introduction (b
allistic introduction) or infection.

As typical examples of suitable hosts, bacterial cells (eg Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, Bacillus subtilis), fungal cells (eg yeast, Aspergillus), insect cells (eg Drosophila S2) , Spodoptera Sf9)
, Animal cells (eg, CHO, COS, HeLa, C 127, 3T3, BHK, HEK
293, Bowes melanoma cells) and plant cells.

A wide variety of expression systems can be used. As such an expression system, for example,
Chromosome-, episome- and virus-derived systems, such as bacterial plasmid-derived, bacteriophage-derived, transposon-derived, yeast episome-derived, insertion factor-derived, yeast chromosomal element-derived, viruses (eg baculovirus, papovavirus like SV40, vaccinia virus) , Adenovirus, fowlpox virus,
Pseudorabies virus, retrovirus) -derived vectors, and vectors derived from combinations thereof, such as those derived from genetic elements of plasmids and bacteriophage, such as cosmids and phagemids. These expression systems may include control regions that regulate not only expression but also expression. Typically,
Any system or vector capable of maintaining, amplifying and expressing a polynucleotide for production of a polypeptide in a host may be used. Sambrook et al, Molecu
The appropriate nucleotide sequence may be inserted into the expression system by any of the well-known and routine techniques, such as those described in lar Cloning: A Laboratory Manual (supra). Appropriate secretory signals may be incorporated into the polypeptide of interest in order to secrete the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular milieu. These signals may be endogenous or heterologous to the polypeptide of interest.

When attempting to express a polypeptide of the invention for use in a screening assay, it is generally preferred to produce the polypeptide on the surface of cells. In that case, cells are harvested prior to use in the screening assay. If the polypeptide is secreted into the medium, the medium is harvested to recover and purify the polypeptide. If produced intracellularly, the cells must first be lysed and then the polypeptide recovered.

To recover and purify the polypeptides of the present invention from recombinant cell culture, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity Well known methods can be used including chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably high performance liquid chromatography is used for purification. When the polypeptide is denatured during intracellular synthesis, isolation and / or purification, well known techniques for refolding proteins can be used to restore the active conformation.

The present invention also relates to the use of the polynucleotide of the present invention as a diagnostic agent. Detection of a mutant form of the gene characterized by the polynucleotide of SEQ ID NO: 1 associated with dysfunction can be used to diagnose a disease caused by underexpression, overexpression or altered expression of the gene or susceptibility to the disease. It would provide a diagnostic tool that could be added or made its diagnosis. Individuals with mutations in the gene can be found at the DNA level by various techniques.

Diagnostic nucleic acids can be obtained from cells of a subject, such as cells from blood, urine, saliva, tissue biopsy or autopsy material. The genomic DNA may be used directly for detection, or it may be enzymatically amplified using PCR or other amplification methods prior to analysis. RNA or cDNA can also be used in a similar manner. Deletions and insertions can be detected by a change in the size of the amplification product as compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled GABAB-R2a nucleotide sequences. Perfectly matched sequences and mismatched duplexes can be distinguished by RNase digestion or by differences in melting temperature.
Differences in DNA sequences can also be detected by changes in electrophoretic mobility of DNA fragments on gels with or without denaturing agents, or by direct DNA sequencing (eg, Myers et al., Science (1985)). 230: 1242). Sequence changes at specific positions can also be confirmed by nuclease protection assays (eg, RNase and S1 protection) or chemical cleavage methods (Cotton
Et al., Proc. Natl. Acad. Sci. USA (1985) 85: 4397-4401). In another embodiment, GABA is used, for example, for efficient screening of gene mutations.
An array of oligonucleotide probes containing the B-R2a nucleotide sequence or fragments thereof can be constructed. Array techniques are well known and have general applicability and have been used to solve various problems in molecular genetics, including gene expression, genetic linkage and genetic variability (e.g. Chee et al., Science
, Vol.274, pp.610-613 (1996)).

The diagnostic assay provides a method for diagnosing or determining susceptibility to the disease by detecting a mutation of the GABAB-R2a gene by the above method. Furthermore, the above-mentioned diseases can be diagnosed by a method of measuring an abnormal decrease or increase in the level of polypeptide or mRNA from a sample obtained from a subject. Decreased or increased expression can be achieved at the RNA level by any of the polynucleotide quantification methods known in the art, such as nucleic acid amplification (eg, PCR, RT-PCR), RNase protection, Northern blotting, and other hybridization methods. Can be measured. Those skilled in the art are familiar with assay methods that can be used to measure the level of a protein, such as a polypeptide of the invention, in a sample obtained from a host. Such assay methods include radioimmunoassays, competitive binding assays, Western Blot analysis, ELISA assays and the like.

Thus, in another embodiment, the invention provides: (a) a polynucleotide of the invention (preferably the nucleotide sequence of SEQ ID NO: 1) or a fragment thereof, (b) a nucleotide sequence complementary to (a) A nucleotide sequence, (c) the polypeptide of the present invention (preferably the polypeptide of SEQ ID NO: 2) or a fragment thereof, or (d) the antibody against the polypeptide of the present invention (preferably the polypeptide of SEQ ID NO: 2), Relates to a diagnostic kit comprising

It will be appreciated that in such a kit, (a), (b), (c) or (d) is a substantial constituent. Such kits can be used for diseases or susceptibility to diseases, especially bacterial, fungal, protist and viral infections, especially HIV-1 or HIV-2.
Infections such as those caused by; pain; cancer; diabetes, obesity; anorexia;
Bulimia; Asthma; Parkinson's disease; Acute heart failure; Hypotension; Hypertension; Urinary retention; Osteoporosis; Angina; Myocardial infarction; Stroke; Ulcer; Allergy; Benign prostatic hypertrophy; Migraine; Vomiting; Anxiety, schizophrenia, Manic It is useful for diagnosing psychosis and nervous system diseases such as depression, depression, delirium, dementia, and severe mental retardation; and movement disorders such as Huntington's disease or Gilles de la Tourette's syndrome.

The nucleotide sequences of the present invention are also useful for chromosome identification. This sequence can specifically target and hybridize with a particular location on an individual human chromosome. Generating a chromosomal map of related sequences according to the present invention is an important first step in correlating these sequences with gene-related diseases. Once a sequence is mapped to a precise chromosomal location, the physical location of that sequence on that chromosome can be correlated with genetic map data. This type of data can be found, for example, in V. McKusick, Mendelian Inheritance in Man (Johns Hopki
(available online at the ns University Welch Medical Library). Then, the relationship between the genes mapped to the same chromosomal region and the disease is confirmed by linkage analysis (co-inheritance of physically adjacent genes).

Differences in cDNA or genomic sequence between affected and unaffected individuals can also be examined. If a mutation is observed in some or all of the affected individuals, but not in any normal individual, then the mutation may be responsible for the disease.

The polypeptides of the invention, fragments or analogs thereof, or cells expressing them can also be used as immunogens to produce antibodies immunospecific for the polypeptides of the invention. By "immunospecific" is meant that the antibody exhibits a substantially higher affinity for a polypeptide of the invention than its affinity for other related polypeptides in the prior art.

Antibodies to the polypeptides of the present invention can be prepared by using conventional protocols in animals (
Preferably, it is obtained by administering to the nonhuman animal) a fragment, analog or cell containing the polypeptide or epitope. For preparation of monoclonal antibodies, any technique which produces antibodies from continuous cell line cultures can be used. For example, the hybridoma method (Kohler, G. and Milstein, C., Nature (
1975) 256: 495-497), trioma method, human B cell hybridoma method (Kozbor et al.,
Immunology Today (1983) 4:72) and the EBV-hybridoma method (Cole et al., Mo.
noclonal Antibodies and Cancer Therapy, pp.77-96, Alan R. Liss, Inc., 19
85) etc.

To produce single chain antibodies to the polypeptides of the invention, US Pat. No. 4,946,
The methods for preparing single chain antibodies as described in No. 778 can be adapted. Also, transgenic mice, or other organisms including other mammals, may be utilized to express humanized antibodies.

Using the above-mentioned antibody, a clone expressing the polypeptide can be isolated and identified, or the polypeptide can be purified by affinity chromatography.

Antibodies against the polypeptides of the invention may have potential use, inter alia, in the treatment of the above diseases.

In a further aspect of the invention, the invention provides a polypeptide of the invention, or a fragment thereof, as well as a variety of constant regions of H or L chains of immunoglobulins of different subclasses (IgG, IgM, IgA, IgE). And a soluble fusion protein produced by genetic engineering, which comprises: As the immunoglobulin, the constant region of the H chain of human IgG, particularly IgG1, is preferable, in which case the fusion occurs at the hinge region. In a particular example, the Fc portion can be easily removed by incorporating a cleavage sequence that can be cleaved by blood coagulation factor Xa. Furthermore, the invention relates to methods of genetically engineering these fusion proteins and their use in drug screening, diagnosis and therapy. Also, a further aspect of the present invention relates to a polynucleotide encoding such a fusion protein. An example of fusion protein technology is international patent application WO94 / 2945
8 and WO 94/22914.

A further aspect of the invention relates to a method of eliciting an immunological response in a mammal,
This method comprises inoculating a mammal with an antibody and / or a polypeptide of the invention sufficient to elicit an antibody and / or T cell immune response, particularly to protect said animal from said disease. Yet another aspect of the invention directs the expression of a polynucleotide encoding a polypeptide of the invention in vivo in order to elicit an immunological response which results in the production of antibodies which protect the mammal against said diseases. A method of eliciting an immunological response in a mammal comprising supplying the polypeptide via a vector.

A further aspect of the present invention relates to an immunological / vaccine formulation (composition) which, when introduced into a mammalian host, elicits an immunological response against the polypeptide of the present invention in the mammal. Contains a polypeptide or polynucleotide of the invention. The vaccine formulation may further include a suitable carrier. Since the polypeptide may be broken down in the stomach, it is preferably administered parenterally (eg by subcutaneous, intramuscular, intravenous or intradermal injection). Formulations suitable for parenteral administration include sterile aqueous and non-aqueous injection solutions that may contain antioxidants, buffers, bacteriostats and solutes that make this formulation isotonic with the blood of the recipient, and suspensions. There are aqueous and non-aqueous sterile suspensions which may include agents or thickeners. Such formulations may be presented in single or multi-dose containers (eg, sealed ampoules and vials) and may also be stored in lyophilized form with the addition of a sterile liquid carrier just prior to use. it can. The vaccine formulation may include an adjuvant system to enhance the immunogenicity of the formulation, such as an oil-in-water adjuvant system or any other adjuvant system known in the art. The dose varies depending on the specific activity of the vaccine, but can be easily determined by routine experimentation.

The polypeptides of the present invention are responsible for a variety of biological functions, including many pathological conditions, especially the diseases mentioned above. Therefore, it is desirable to develop screening methods to identify compounds that stimulate or inhibit the function of this polypeptide. Therefore, in a further aspect, the invention provides a method of screening compounds to identify compounds that stimulate or inhibit the polypeptide. In general,
Agonists or antagonists are used for the therapeutic and prophylactic purposes of said diseases. Compounds can be identified from a variety of sources, such as cells, cell-free preparations, chemical libraries and mixtures of natural products. The agonist, antagonist or inhibitor thus identified may optionally be a natural or modified substrate, ligand, receptor, enzyme, etc. of the polypeptide, and its structural or functional mimetics. (Coligan et al., C
urrent Protocols in Immunology 1 (2): Chapter 5 (1991)).

In the screening method, the binding of the candidate compound to the polypeptide of the present invention, a cell or membrane carrying the polypeptide, or a fusion protein thereof is carried out by using a label directly or indirectly bound to the candidate compound. And easy to measure. Alternatively, the screening method may use competition with a labeled competitor.
Furthermore, in such screening methods, whether a candidate compound results in a signal generated by activation or inhibition of the polypeptide can be tested using a detection system suitable for cells carrying the polypeptide. . In general, inhibitors of activation are assayed in the presence of known agonists to determine the effect of the presence of a candidate compound on agonist activation. Constitutively active polypeptides are used in methods of screening inverse agonists or inhibitors by examining whether a candidate compound inhibits activation of the polypeptide in the absence of agonist or inhibitor. In addition, these screening methods
Simply include the steps of combining the candidate compound with a solution containing the polypeptide of the invention to form a mixture, measuring the GABAB-R2a activity in the mixture, and comparing the GABAB-R2a activity of the mixture to a standard. Just enough. High throughput screening assays to identify antagonists of the polypeptides of the invention can also use fusion proteins such as those made from the Fc portion and GABAB-R2a polypeptides described above (D. Bennett et al., J. Mol. Recognition, 8: 52-5
8 (1995) and K. Johanson et al., J. Biol. Chem., 270 (16): 9459-9471 (1995)).

One of the screening techniques is to express cells expressing the receptor of the present invention in a system for measuring changes in extracellular pH or intracellular calcium caused by activation of the receptor (
(Eg, transfected CHO cells). In this technique, the compound may be contacted with cells expressing the receptor polypeptide of the invention. Then
Second messenger responses such as signal transduction, changes in pH, or changes in calcium levels are measured to determine if the candidate compound activates or inhibits the receptor.

Another method involves screening for receptor inhibitors by measuring inhibition or stimulation of receptor-mediated cAMP and / or adenylate cyclase accumulation. Such a method comprises transfecting a eukaryotic cell with the receptor of the present invention to express the receptor on the cell surface. The cells are then exposed to candidate antagonists in the presence of the receptor of the invention. After that, the amount of accumulated cAMP is measured. When a candidate antagonist binds to the receptor, receptor binding is inhibited and receptor-mediated cA
Reduced or increased levels of MP or adenylate cyclase. Another method for detecting agonists or antagonists of the receptors of the present invention is described in US Pat.
This is a yeast-based technique described in 5,482,835.

Further, a screening method for detecting the effect of an added compound on intracellular mRNA or polypeptide production can be constructed using the polynucleotide, polypeptide or antibody against the polypeptide of the present invention. it can. For example, an EL for measuring the secretion level or cell binding level of a polypeptide using a monoclonal or polyclonal antibody by a standard method known in the art.
An ISA assay can be constructed that can be used to search for substances (also referred to as antagonists or agonists) that suppress or enhance the production of the polypeptide from appropriately engineered cells or tissues.

If a membrane-bound or soluble receptor is present, the polypeptide of the invention may be used to identify this type of receptor by standard receptor binding methods known in the art. Can be used. Such receptor binding methods include, but are not limited to, ligand binding assays and cross-linking assays in which the polypeptide is labeled with a radioactive isotope (eg, ¹²⁵ I) or chemically modified (eg. Eg biotinylated) or fused to peptide sequences suitable for detection and purification and incubated with putative receptor sources (cells, cell membranes, cell supernatants, tissue extracts, body fluids etc.). Other methods include biophysical methods such as surface plasmon resonance and spectroscopy. These screening methods can also be used to identify agonists or antagonists of the polypeptide that compete with the binding of the polypeptide or its receptor (if present). Standard methods for conducting screening assays are well understood in the art.

Examples of potential antagonists of the polypeptides of the present invention include antibodies, in some cases oligonucleotides or proteins (eg, oligonucleotides) that are intimately associated with the polypeptide's ligands, substrates, receptors, enzymes, etc. , Ligand, substrate, receptor,
Fragments, such as enzymes), or small molecules that bind the polypeptide of the invention but do not induce a response (and thus interfere with the activity of the polypeptide).

Thus, in another aspect, the invention identifies an agonist, antagonist, ligand, receptor, substrate, enzyme, etc. of a polypeptide of the invention, or a compound that reduces or increases the production of this type of polypeptide. The present invention relates to a screening kit for: (a) the polypeptide of the present invention; (b) the recombinant cell expressing the polypeptide of the present invention; (c) the polypeptide of the present invention. A cell membrane, or (d) an antibody against the polypeptide of the present invention, wherein said polypeptide is preferably the polypeptide of SEQ ID NO: 2.

It will be appreciated that in such a kit, (a), (b), (c) or (d) is a substantial constituent.

Those skilled in the art will readily appreciate that the polypeptides of the present invention can also be used in methods of designing agonists, antagonists or inhibitors of the polypeptides based on their structure. This method comprises (a) first analyzing the three-dimensional structure of the polypeptide, (b) assuming a three-dimensional structure of a likely reaction site or binding site of an agonist, antagonist or inhibitor, and (c) assuming Synthesizing a candidate compound that is predicted to bind or react with a given reactive or binding site, and (d) determine whether the candidate compound is in fact an agonist, antagonist or inhibitor. It will be further understood that this is a normal interaction process.

In a further embodiment, the present invention relates to either over- or under-expression of GABAB-R2a polypeptide activity, such as bacterial infections, fungal infections, protist and viral infections, in particular HIV-1 or HIV. Infections such as those caused by -2; pain;
Cancer; diabetes, obesity; anorexia nervosa; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina; myocardial infarction; stroke; ulcer; allergy;
Benign prostatic hypertrophy; migraine; vomiting; anxiety, schizophrenia, manic depression, depression, delirium, dementia, psychotic and nervous system disorders such as severe mental retardation; and Huntington's disease or Jill de la Tourette syndrome To provide a method for treating abnormal conditions such as movement disorders.

If the activity of the polypeptide is in excess, several approaches are available. One approach is to inhibit the function of the polypeptide, for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc., or by reducing an abnormal condition by inhibiting a second signal. Comprising administering to the patient an inhibitor compound (antagonist) as described above, together with a pharmaceutically acceptable carrier. In another approach, soluble forms of the polypeptide that are still capable of binding ligands, substrates, enzymes, receptors, etc. in competition with the endogenous polypeptide can be administered. A typical example of such a competitor is a fragment of GABAB-R2a polypeptide.

In yet another approach, expression inhibition methods can be used to repress expression of the gene encoding the endogenous GABAB-R2a polypeptide. Such known techniques require the use of antisense sequences produced in the body or administered separately (e.g., Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression), C
O'Connor, J. Neurochem (1991) 56: 560 in RC Press, Boca Raton, FL (1988).
checking). Alternatively, an oligonucleotide that forms a triple helix with this gene can be supplied (eg, Lee et al., Nucleic Acids Res (19
79) 6: 3073; Cooney et al., Science (1988) 241: 456; Dervan et al., Science (1991) 2
51: 1360). These oligomers can be administered per se or the relevant oligomers can be expressed in vivo.

Several approaches can also be taken to treat abnormal conditions associated with underexpression of GABAB-R2a and its activity. One approach involves administering to a patient a therapeutically effective amount of a compound that activates a polypeptide of the invention (ie, the agonist) together with a pharmaceutically acceptable carrier to alleviate the abnormal condition. It consists of Alternatively, gene therapy can be used to endogenously produce GABAB-R2a in the patient's relevant cells. For example, the polynucleotides of the invention are genetically engineered for expression by a replication defective retroviral vector as described above. The retroviral expression construct is then isolated and introduced into packaging cells transduced with a retroviral plasmid vector containing RNA encoding the polypeptides of the invention. As a result, the packaging cells will produce infectious viral particles containing the gene of interest. These producer cells are administered to the patient for in vivo cell engineering and in vivo polypeptide expression. For an overview of gene therapy, see Human Molecular Genetics, T Strach.
Chapter 20, Ge in an and AP Read, BIOS Scientific Publishers Ltd (1996).
See ne Therapy and other Molecular Genetic-based Therapeutic Approaches (and references therein). Another approach is to administer a therapeutic amount of a polypeptide of the invention together with a suitable pharmaceutical carrier.

In a further aspect, the invention provides a therapeutically effective amount of a polypeptide (eg, a soluble form of the polypeptide of the invention), an agonist or antagonist peptide, or a small molecule compound in a pharmaceutically acceptable carrier or Provided is a pharmaceutical composition containing together with an excipient. Carriers of this type include, but are not limited to, saline, saline, dextrose, water, glycerol, ethanol, and combinations thereof. The present invention further comprises 1 filled with one or more components of the composition of the present invention described above.
The present invention relates to a medicine pack and kit containing the above-mentioned containers. The polypeptides of the invention and the other compounds may be used alone or in combination with other compounds, eg therapeutic compounds.

The pharmaceutical composition may be adapted to the route of administration, eg the systemic or oral route of administration. Suitable forms for systemic administration are infusion, typically intravenous.
Other injection routes such as subcutaneous, intramuscular or intraperitoneal can also be used. Another means of systemic administration is transmucosal and transdermal administration using penetrants such as bile salts, fusidic acids and other surfactants. Furthermore, oral administration is possible provided that the polypeptide of the present invention or other compound can be formulated as an enteric solution or capsule. These compounds may be topically administered and / or localized in the form of ointments, pastes, gels and the like.

The dosage range required will depend on the choice of peptide or other compound of the invention, the route of administration,
It will depend on the nature of the formulation, the condition of the patient, and the judgment of the physician. However, suitable dosages are in the range of 0.1-100 μg / kg of patient weight. Wide variations in the required dosage are to be expected in view of the wide variety of compounds available and the differing efficiencies of administration routes. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Such variation in dosage levels can be adjusted using standard empirical optimization procedures, as is well understood in the art.

Polypeptides used in treatment can also be generated endogenously in the patient, in treatment modalities often referred to as "gene therapy" as described above. For example, cells from a patient
It is engineered ex vivo by a polynucleotide such as a DNA or RNA encoding a polypeptide, eg using a retroviral plasmid vector. Thereafter, these cells are introduced into the patient.

Polynucleotide and polypeptide sequences provide a valuable information resource with which to identify other sequences of similar homology. This is maximally facilitated by storing such sequences in a computer readable medium and then using the stored data to search a sequence database with known search tools such as GCG.
Accordingly, in a further aspect, the present invention provides a computer readable medium having stored thereon a polynucleotide comprising the sequence of SEQ ID NO: 1 and / or the polypeptide encoded thereby.

The following definitions are provided to aid understanding of terms often used in the above description.

As used herein, “antibody” includes polyclonal and monoclonal antibodies, chimeric antibodies, single chain antibodies, humanized antibodies, as well as Fab fragments that include the products of Fab or other immunoglobulin expression libraries. Be done.

“Isolated” means altered “by the hand of man” from the natural state. If an "isolated" composition or substance is naturally-occurring, it has been altered or separated from its original environment, or both.
For example, a polynucleotide or polypeptide naturally occurring in the body of a living animal is not "isolated", but a polynucleotide or polypeptide separated from its naturally occurring coexisting material is As used in the text, it is "isolated".

“Polynucleotide” generally refers to any polyribonucleotide or polydeoxyribonucleotide, which is unmodified RNA or DNA.
, Or modified RNA or DNA. “Polynucleotide” includes, but is not limited to, single-stranded and double-stranded DNA, DNA in which single-stranded region and double-stranded region are mixed, single-stranded and double-stranded RNA, single-stranded Hybrid molecule containing RNA, DNA and RNA in which region and double-stranded region are mixed (single-stranded or, more typically, double-stranded, single-stranded region and double-stranded region are mixed Can be included). In addition, "polynucleotide" refers to RNA or D
It means a triple-stranded region consisting of both NA or RNA and DNA. The term "polynucleotide" also refers to DNA or RNA containing one or more modified bases.
, And DNA or R having a backbone modified for stability or other reasons
Including NA. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA. Thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms of polynucleotides commonly found in nature, as well as chemical forms of DNA and RNA characteristic of viruses and cells. . "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

By “polypeptide” is meant a peptide or protein containing two or more amino acids linked by peptide bonds or modified peptide bonds (ie, peptide isosteres). "Polypeptide" refers to short chains (usually referred to as peptides, oligopeptides or oligomers) and long chains (commonly referred to as proteins).
Both. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. A "polypeptide" comprises an amino acid sequence modified either by natural processes, such as post-translational processing, or by chemical modification methods known in the art. Such modifications are detailed in basic textbooks, more detailed academic and research literature. Modifications can be made anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. The polypeptide may be branched for ubiquitination, or cyclic with or without branching. Cyclic, branched, or branched cyclic polypeptides may result from post-translational natural processes or may be produced synthetically. Modifications include acetylation, acylation, ADP-ribosylation, amidation, flavin covalent bond, heme moiety covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, phosphatidylinositol covalent bond. , Cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-link formation, cystine formation, pyroglutamate formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination , Methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation, transfer RNA-mediated addition of amino acids to proteins, ubiquitination, etc. (
For example, Proteins-Structure and Molecular Properties 2nd Ed., TE Cre
ighton, WH Freeman and Company, New York, 1993; Posttranslational Cova
lent Modification of Proteins, BC Johnson, Academic Press, New York,
Wold, F., Post-translational Protein Modifications: Perspective in 1983
s and Prospects, pgs. 1-12; Seifter et al., “Analysis for protein modificati.
ons and nonprotein cofactors ", Meth Enzymol (1990) 182: 626-646; and R
attan et al., “Protein Synthesis: Post-translational Modifications and Aging
", Ann NY Acad Sci (1992) 663: 48-62).

As used herein, a “variant” is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide but retains essential properties. Typical polynucleotide variants differ in nucleotide sequence from the reference polynucleotide. The nucleotide sequence changes in this variant are
The amino acid sequence of the polypeptide encoded by the reference polynucleotide may or may not be altered. Nucleotide changes may result in amino acid substitutions, deletions, additions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. Typical polypeptide variants differ in amino acid sequence from a reference polypeptide. Generally, the sequence of the reference polypeptide and the sequence of the variant are generally similar and limited to differences that are identical in many regions. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, deletions, additions in any combination. The substituted or added amino acid residue may or may not be one encoded by the genetic code. Variants of polynucleotides or polypeptides may be naturally occurring variants, such as allelic variants, or variants not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides can be made by mutagenesis or direct synthesis.

"Identity," as known in the art, is an affinity between two or more such sequences, as determined by comparing the polypeptide or polynucleotide sequences. In the art, "identity" also means the degree of sequence similarity between such sequences, as determined by the match between the strands of the polypeptide or polynucleotide sequences. "Identity" and "homology" can be easily calculated by known methods, and as such a method, for example, Computational Molecular Biolo
gy, Lesk, AM, Oxford University Press, New York, 1988; Biocomputing:
Informatics and Genome Projects, Smith, DW Edition, Academic Press, New Yo
rk, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and
Griffin, HG, Humana Press, New Jersey, 1994; Sequence Analysis in M
olecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysi
s Primer, Gribskov, M. and Devereux, J. Ed., M Stockton Press, New York,
1991; and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48: 1073.
(1988), but is not limited to these. Preferred methods to determine identity are designed to give the largest match between the sequences under consideration. Methods to determine identity and homology have been compiled into publicly available computer programs. A preferred computer program method for determining identity and homology between two sequences is the GCG program package (Deve
reux, J. et al., Nucleic Acids Research 12 (1): 387 (1984)), BLASTP, BL
ASTN and FASTA (Atschul, SF et al., J. Molec. Biol. 215: 403-410 (
1990)), but not limited to these. The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI
NLM NIH Bethesda, MD 20894; Altschul, S. et al., J. Mol. Biol. 215: 403-410.
(1990)). The well known Smith Waterman algorithm can also be used to determine identity.

Preferred parameters for comparing polypeptide sequences include:
1) Algorithm: Needleman & Wunsch, J. Mol. Biol. 48: 443-453 (1970)
Comparison matrix: BLOSSUM62, Hentikoff and Hentikoff, Proc. Natl. Acad.
Sci. USA, 89: 10915-10919 (1992) Gap Penalty: 12 Gap Length Penalty: 4 A useful program with these parameters is the Genetics Computer Group.
(Madison WI), generally available as the "gap" program. The above parameters are the default parameters for peptide comparison (default paramet
er) (no end gap penalty).

Preferred parameters for comparing polynucleotide sequences include: 1) Algorithm: Needleman & Wunsch, J. Mol. Biol. 48: 443-453 (1970); Comparison matrix: Match = + 10, Mismatch = 0 Gap penalty: 50 Gap length penalty: 3 A useful program with these parameters is Genetics Computer Group (M
It is available as a "gap" program from adison WI). These are the default parameters for nucleic acid comparisons.

By way of example, the polynucleotide sequence of the invention is identical to the reference sequence of SEQ ID NO: 1, ie has 100% identity to the reference sequence or is constant relative to said reference sequence. It may contain up to an integer number of nucleotide variations. Such mutations are selected from the group consisting of deletions, substitutions (including transitions and transversions) or insertions of at least one nucleotide, such mutations being the 5'or 3'terminal position of the reference nucleotide sequence, or these It may be anywhere between the terminal positions, interspersed between the nucleotides in the reference sequence individually or as one or more contiguous groups within the reference sequence. The number of nucleotide mutations is
By multiplying the total number of nucleotides of SEQ ID NO: 1 by the absolute ratio of the respective% identity values (divided by 100) and subtracting the product from the total number of nucleotides of SEQ ID NO: ₁ It can be determined by ≦ x _n − (x _n · y). Where n _n is the number of nucleotide variations, x _n is the total number of nucleotides of SEQ ID NO: 1, y is, for example, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 90% for 85% Is 0.90 and 95% is 0.95
Etc., and the product of non-integers of x _n and y is rounded down to the nearest integer before subtracting the product from x _n . Modification of the polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 results in nonsense, missense or frameshift mutations in the coding sequence, thereby altering the polypeptide encoded by the polynucleotide after such mutations. be able to.

Similarly, the polypeptide sequence of the present invention is identical to the reference sequence of SEQ ID NO: 2,
That is, it may have 100% identity to the reference sequence, or may contain up to a certain integer number of amino acid mutations to the reference sequence such that the percent identity is less than 100%. Such mutations are selected from the group consisting of deletions, substitutions (including conservative and non-conservative amino acid substitutions) or insertions of at least one amino acid, and these mutations are amino or carboxy terminal positions of the reference polypeptide sequence. , Or anywhere between these terminal positions, interspersed individually between amino acids in the reference sequence or as one or more contiguous groups within the reference sequence. The number of amino acid mutations is the percent identity (divided by 100) for each of the total number of amino acids in SEQ ID NO: 2.
Is calculated by subtracting the product from the total number of amino acids in SEQ ID NO: 2, that is, by the following formula.

N _a ≦ x _a − (x _a · y) In the formula, n _a is the number of amino acid mutations, x _a is the total number of amino acids in SEQ ID NO: 2, and y is 0.70 for 70%, for example. 0.80 for 80%, 0.85 for 85%
And the non-integer product of x _a and y is rounded down to the nearest integer before subtracting the product from x _a .

A “fusion protein” is a protein encoded by two, often unrelated, fused genes or fragments thereof. As an example, EP-A-0 4
64 describes fusion proteins comprising various parts of the constant region of immunoglobulin molecules and other human proteins or parts thereof. Often for use in therapy and diagnostics, the immunoglobulin Fc as part of a fusion protein
It is advantageous to use regions, which improve eg the pharmacokinetic properties (see eg EP-A-0232 262). On the other hand, for some uses it may be desirable to remove the Fc portion after expressing, detecting and purifying the fusion protein.

All publications, including patents and patent application publications, cited herein are incorporated by reference in their entirety as if each publication was explicitly and individually indicated. Incorporated here.

Examples Example 1: Cloning Bioinformatic searches identified potential genomic sequences for 7TM receptors from public databases. 5'end of clone (Nature 396, 674-687, 199
The whole cDNA clone derived from human hippocampus was used for PCR using the primers corresponding to 8) and 3'end as templates. The PCR fragment was subcloned into pCR2.1 and sequenced. The cloning operation was performed twice to confirm the amino acid sequence. By comparing the nucleotide sequence of GABAB-R2a to the published GABAB-R2 sequence (Nature 396, 674-687 (1998)), there is an in-frame deletion of 66 amino acids near the 5'region of the clone. It has been found.

Example 2 Expression in Mammalian Cells Receptors of the invention were expressed in either human embryonic kidney 293 (HEK293) cells or adherent dhfr CHO cells. In order to maximize the expression of the receptor, all 5'and 3'untranslated regions (U) are typically labeled prior to insertion into the pCDN or pCDNA3 vector.
TRs) was removed from the receptor cDNA. These cells were transfected with individual receptor cDNAs using lipofectin and selected in the presence of 400 mg / ml G418. After 3 weeks of selection, individual clones were picked and expanded for further analysis. HEK293 or CHO cells transfected with vector alone were used as negative controls. In order to isolate cell lines stably expressing individual receptors,
Generally about 24 clones were selected and analyzed by Northern blot analysis. Receptor mRNA was usually detectable in about 50% of the G418 resistant clones analyzed.

Example 3 Ligand Banks for Binding and Functional Assays A bank of over 200 putative receptor ligands was assembled for screening. This bank includes: That is, the human 7-transmembrane (7
TM) transmitters, hormones and chemokines known to act via receptors; naturally occurring compounds that may be putative agonists of the human 7TM receptor; non-mammalian bioactive peptides (mammalian (The animal counterpart has not yet been identified.)
And a compound that activates the 7TM receptor with a natural ligand that is not found in nature but is unknown. This bank provides binding assays and functions (ie calcium, cAM
P, microphysiometer, oocyte electrophysiology, etc.
Both assays were used to initially screen the receptor for known ligands.

Example 4: Ligand binding assay The ligand binding assay provides a direct method for locating receptor pharmacology and is also applicable to high throughput formats. The purified ligand of the receptor was radiolabeled with a high specific activity (50-2000 Ci / mmol) for binding experiments. Next, it was determined that the process of radiolabelling did not reduce the activity of the ligand for its receptor. Assay conditions for buffers, ions, pH and other modulators such as nucleotides were optimized to establish workable possible signal-to-noise ratios for both membrane and whole cell receptor sources. For these assays, specific receptor binding was defined as total bound radioactivity minus radioactivity measured in the presence of excess unlabeled competing ligand. If possible, use two or more competing ligands to establish residual non-specific binding.

Example 5: Functional Assay in Xenopus Oocytes Capped from template of linearized plasmid encoding the receptor cDNA of the invention.
RNA transcripts were synthesized in vitro using RNA polymerase according to standard procedures. The in vitro transcript was suspended in water at a final concentration of 0.2 mg / ml. Ovary lobe (ova
rian lobes) were removed from adult female frogs to obtain stage V defolliculated oocytes and RNA transcripts (10 ng / oocyte) were injected once at 50 nl using a microinjector. Two
Currents from individual Xenopus oocytes in response to agonist exposure were measured using a single electrode potential clamp. Bar (Ca ²⁺ free bar at room temperature
th's) recordings were made in medium. This Xenopus system can be used to screen known ligands and tissue / cell extracts for activating ligands.

Example 6: Microphysiological Assay Activation of various second messenger systems releases small amounts of acid from cells. This acid is produced primarily as a result of enhanced metabolic activity required to fuel intracellular signaling processes. PH in the medium surrounding this cell
Change is very small, but can be detected by a cytosensor (CYTOSENSOR) microphysiometer (Molecular Devices Ltd., Menlo Park, CA). Therefore, this cytosensor can detect activation of a receptor that is coupled to energy utilizing an intracellular signal transduction pathway, such as the G protein-coupled receptor of the present invention.

Example 7: Extract / Cell Supernatant Screen There are many mammalian receptors, but so far no activating ligand (agonist) of the same animal species (cognate) has been found. Therefore, the ligands that activate these receptors may not be included in the ligand banks identified to date. Thus, the 7TM receptor of the present invention is also functionally functional to tissue extracts to identify natural ligands (using functional screens such as calcium, cAMP, microphysiometer, oocyte electrophysiology, etc. ) Was screened. Extracts exhibiting a positive functional response can be sequentially subfractionated until the activating ligand is isolated and identified.

Example 8 Calcium and cAMP Functional Assays 7TM receptors expressed in HEK293 cells were shown to be functionally coupled to PLC and calcium mobilization activation and / or cAMP stimulation or inhibition. Basal calcium levels in HEK293 cells in receptor-transfected cells or vector control cells were observed in the normal range of 100 nM to 200 nM. HEK293 cells expressing recombinant receptor were spiked with fura2 and> 150 selected ligands or tissue / cell extracts per day were evaluated for agonist-induced calcium mobilization. Similarly, HEK293 cells expressing recombinant receptor were evaluated for stimulation or inhibition of cAMP production using a standard cAMP quantitation assay. Agonists that transiently mobilize calcium or exhibit cAMP fluctuations were tested in vector control cells to determine if the response was characteristic of the receptor-expressing transfected cells.

Sequence listing free text sequence information SEQ ID NO : 1 Length: 3039 Sequence number 2 Sequence number 3 Sequence number 4

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 1/04 A61P 1/14 4C084 1/08 3/04 4C086 1/14 3/10 4H045 3/04 9 / 02 3/10 9/10 9/02 9/12 9/10 11/06 9/12 13/02 11/06 13/08 13/02 25/00 13/08 25/06 25/00 25/14 25/06 25/16 25/14 25/18 25/16 25/22 25/18 25/24 25/22 25/28 25/24 31/00 25/28 35/00 31/00 37/08 35 / 00 C07K 14/705 37/08 16/28 C07K 14/705 C12P 21/02 C 16/28 C12Q 1/68 Z C12N 5/10 G01N 33/15 Z C12P 21/02 33/50 Z C12Q 1/68 33 / 68 G01N 33/15 C12N 15/00 ZNAA 33/50 A61K 37/02 33/68 C12N 5/00 B (31) Priority claim number 09/183, 253 (32) Priority date October 30, 1998 (October 30, 1998) (33) United States of America claiming priority (US) (31) Priority claim number 09 / 253,216 (32) Priority date February 19, 1999 (February 19, 1999) (33) Priority claiming country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), JP (72) Inventor Elshore buggy, Neville United States 19380 West Chester, Bowtree Drive, Pennsylvania 1380 (72) Inventor Shevon, Uzman United States 19426 Pennsylvania, Collegeville, Hamilton Road 926 (72) Inventor Boutor, Lisa United States 18036 Coopersburg, Ebert, Pennsylvania Lord 1075 (72) Inventor Groger, Israel London, East Finkley, London England Summerly Avenue 13 (72) Inventor Starmers, Melanie UK CB 16 Ezzet Cambridge Shire, Balsham, Fox Road 33 F term (reference) 2G045 AA34 AA35 AA40 BB20 CB01 DA12 DA13 DA14 DA36 DA77 FB02 FB03 4B024 AA01 AA11 BA63 CA04 CA09 GA13 GA04 DA20 FA02 DA02 FA02 DA02 HA11 HA13 HA14 4B063 QA01 QA05 QA08 QQ43 QQ53 QQ61 QR56 QR77 QR80 QS34 QX05 QX10 4B064 AG20 AG27 CA10 CA19 CA20 CC01 CC24 DA01 DA13 4B065 AA90X AA93X AA93Y AB01 AC14 BA02 BA05 BA25 BD50 CA24 CA44 CA46 4C084 AA02 AA07 AA13 AA17 BA01 BA22 NA14 ZA022 ZA052 ZA082 ZA122 ZA182 ZA222 ZA362 ZA372 ZA402 ZA422 ZA432 ZA592 ZA702 ZA812 ZA842 ZA892 ZA972 ZB132 ZB262 ZB332 ZB352 ZC352 ZC552 4C086 AA01 EA16 MA01 MA04 NA14 ZA02 ZA05 ZA08 ZA12 ZA18 ZA22 ZA36 ZA37 ZA40 ZA42 ZA43 ZA59 ZA70 ZA81 ZA84 ZA89 ZB13 ZB26 ZB33 ZB35 ZC35 ZC55 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA50 DA75 EA20 EA50 FA71 FA74

Claims (14)

[Claims] 1. An isolated polypeptide selected from the group consisting of (i) to (iii) below: (i) at least (a) with respect to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2. An isolated amino acid sequence selected from the group having 70% identity, (b) 80% identity, (c) 90% identity, or (d) 95% identity. Polypeptide,
(ii) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or
(iii) An isolated polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. 2. An isolated polynucleotide selected from the group consisting of (i) to (vi) below, or a nucleotide sequence complementary to the isolated polynucleotide: (i) SEQ ID NO: 2 At least (a) 70% identity, (b) 80% identity, (c) 90% identity, or (d) 95% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having (ii) at least (a) 70% identity to the nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 over its entire length An isolated polynucleotide comprising (b) 80% identity, (c) 90% identity, or (d) 95% identity, (iii) SEQ ID NO: 1 Sequence number over the entire length of A nucleotide sequence having at least (a) 70% identity, (b) 80% identity, (c) 90% identity, or (d) 95% identity to one nucleotide sequence. An isolated polynucleotide comprising (iv) an isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO: 2, (v) an isolated polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 1 A polynucleotide,
And (vi) an isolated polynucleotide obtained by screening a suitable library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof. 3. An antibody immunospecific for the polypeptide of claim 1. 4. A method of treating a subject, comprising: (i) if the subject requires increased activity or expression of the polypeptide of claim 1, (a) treatment against the polypeptide. Administering an effective amount of an agonist to the subject, and / or (b) causing an isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide to produce the polypeptide activity in vivo. Administering to a subject in such a form, (ii) when the subject requires suppression of the activity or expression of the polypeptide according to claim 1, (a) for the polypeptide Administering to the subject a therapeutically effective amount of the antagonist, and / or (b) administering to the subject a nucleic acid molecule that suppresses expression of the nucleotide sequence encoding the polypeptide. And / or (c) administering to the subject a therapeutically effective amount of the polypeptide that competes with the polypeptide for its ligand, substrate, or receptor. . 5. A method for diagnosing a disease or susceptibility to the disease of a subject associated with expression or activity of the polypeptide according to claim 1 in the subject, comprising: (a) within the genome of the subject. Determining whether there is a mutation in the nucleotide sequence encoding the polypeptide of, and / or (b) analyzing the presence or amount of expression of the polypeptide in a sample obtained from the subject. A method comprising:,. 6. A screening method for identifying a compound that stimulates or suppresses the function of the polypeptide according to claim 1, comprising: (a) a candidate compound and the polypeptide (or a protein carrying the polypeptide). Cell or its membrane) or its fusion protein is measured by a label directly or indirectly bound to the candidate compound, (b) the candidate compound and the polypeptide (or the polypeptide) Binding to a cell or its membrane (or its membrane) or its fusion protein is measured in the presence of a labeled competitor, (c) the candidate compound results in a signal generated by activation or inhibition of the polypeptide. The use of a detection system suitable for cells or cell membranes carrying the polypeptide, (d) the candidate compound, and the method according to claim 1. Preparing a mixture with a solution containing the polypeptide and measuring the activity of the polypeptide in the mixture and comparing the activity of the mixture to a standard, or (e) the candidate compound in a cell A screening method, comprising a method selected from the group consisting of detecting the mRNA encoding the polypeptide and the effect on the production of the polypeptide using, for example, an ELISA assay. 7. An agonist or antagonist of the polypeptide according to claim 1. 8. An expression system containing a polynucleotide capable of producing the polypeptide of claim 1 when said expression system is present in a compatible host cell. 9. A host cell which, under suitable culture conditions, produces a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 2 over its entire length. 9. A method for producing a recombinant host cell, comprising transforming or transfecting a cell with the expression system according to claim 8. 10. A recombinant host cell obtained by the method of claim 9. 11. A recombinant host according to claim 10, which expresses a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2. Cell membrane. 12. A method according to claim 10, comprising culturing the host cell of claim 10 under conditions sufficient to produce the polypeptide and recovering the polypeptide from its culture medium. Production method. 13. An isolated polynucleotide selected from the group consisting of (a) to (d) below: (a) a minority relative to the nucleotide sequence of SEQ ID NO: 3 over the entire length of SEQ ID NO: 3.
An isolated polynucleotide comprising a nucleotide sequence having 70%, 80%, 90%, 95%, 97% identity, (b) an isolated polynucleotide comprising the polynucleotide of SEQ ID NO: 3 A polynucleotide, (c) the polynucleotide of SEQ ID NO: 3, or (d) at least 70%, 80%, 90%, 95%, 97-99% of the amino acid sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4. An isolated polynucleotide comprising a nucleotide sequence that encodes a polypeptide having identity. 14. A polypeptide selected from the group consisting of (a) to (e) below: (a) at least 70%, 80%, 90 relative to the amino acid sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4. A polypeptide comprising an amino acid sequence having%, 95%, 97-99% identity, (b) at least 70%, 80%, 90% relative to the amino acid sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4. A polypeptide having an amino acid sequence having 95%, 97-99% identity, (c) a polypeptide comprising the amino acid of SEQ ID NO: 4, (d) a polypeptide consisting of the polypeptide of SEQ ID NO: 4, e) A polypeptide encoded by a polynucleotide comprising the sequence contained in SEQ ID NO: 3.