JP2003024068A - Human g-protein receptor hgber32 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain human G-protein receptor HGBER32 and techniques regarding the same. SOLUTION: Human G-protein coupled receptor polypeptides and DNA (RNA) encoding such polypeptides and a procedure for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing such polypeptides for identifying antagonists and agonists to such polypeptides and methods for using the agonists and antagonists therapeutically to treat conditions related to the underexpression and overexpression of the G-protein coupled receptor polypeptides, respectively. Also disclosed are diagnostic methods for detecting a mutation in the G-protein coupled receptor nucleic acid sequences and an altered level of the soluble form of the receptors.

Description

DETAILED DESCRIPTION OF THE INVENTION [0001] TECHNICAL FIELD The human G protein receptor
HGBER32 The present invention relates to a newly identified polynucleotide.
Otide, encoded by such a polynucleotide
Polypeptides, such polynucleotides and polypeptides
The use of repeptides, as well as such polynucleotides
And the production of polypeptides. More specifically,
The polypeptide of the present invention may comprise a human 7-transmembrane receptor.
It is. This transmembrane receptor is G-protein coupled
Type receptor. More specifically, this 7-transmembrane type
The receptor is a G protein chemokine receptor
It was putatively identified and is sometimes referred to hereinafter as “HGBER
32 ". The present invention also relates to such polypeptides.
It relates to inhibiting the action of tide. [0002] BACKGROUND OF THE INVENTION Many medically important biological processes
Is a G protein and / or a second messenger
-Involved in signal transduction pathways including (eg, cAMP)
Is well-established
(Lefowitz, Nature, 35
1: 353-354 (1991)). In this specification,
These proteins are involved in pathways using G proteins.
The protein or PPG protein to be given. this
Some examples of these proteins include the GPC receptor.
(Eg, adrenergic drugs and dopami
GPC receptor (Kobilka, B .;
K. Et al., PNAS, 84: 46-50 (1987); K.
obilka, B .; K. Et al., Science, 238:
650-656 (1987); R.
Et al., Nature, 336: 783-787 (198).
8))), G protein itself, effector protein
Proteins (eg, phospholipase C, adenyl cycler)
And phosphodiesterases) and actu
Actor protein
n) (eg, protein kinase A and protein
Kinase C) (Simon, MI, et al., Science)
e, 252: 802-8 (1991)).
You. [0003] For example, in one form of signaling,
The effect of hormone binding is the enzyme adenylate in cells
Activation of cyclase. Enzyme activity by hormones
Is dependent on the presence of the nucleotide GTP and GT
P also affects hormone binding. G protein
Links hormone receptors to adenylate cyclase
Let it. [0004] G protein is activated by hormone receptors.
When activated, it converts GTP to bound GDP.
Was shown. The form with GTP is then activated
To adenylate cyclase. GD of GTP
Hydrolysis to P is catalyzed by the G protein itself.
To return the G protein to its basal, inactive form. Follow
Thus, the G protein transmits signals from the receptor to the effector.
As an intermediate to relay to the
It plays two roles as a clock that controls time. [0005] G protein-coupled receptor membrane tamper
The protein superfamily consists of seven putative transmembrane
It is characterized as having a common domain. This
These domains are connected by extracellular or cytoplasmic loops.
It is believed to represent the combined transmembrane α helix. G
Protein-coupled receptors are hormones, viruses,
Extensive biology such as growth factors and neuroreceptors
Includes chemically active receptors. [0006] There are few G protein-coupled receptors.
From about 20 to about 8 which bind eight branched hydrophilic loops
These seven conserved hydrophobic ranges of 30 amino acids
It is characterized as including. Binding receptor
Dopamine receptor as a G protein family
-. This is a psychotic and neurological disorder
Binds to neuroleptic drugs used to treat
You. Other examples of members of this family include Cal
Cytonin, adrenergic, endothelin, cAM
P, adenosine, muscarinic, acetylcholine, cello
Tonin, histamine, thrombin, kinin, follicle stimulation
Lumon, opsin, endothelial differentiation gene-1 receptor and
Rhodopsin, odorant, cytomegalovirus receptor
And the like. Most G-protein coupled receptors
Is thought to stabilize functional protein structures
First two extracellular loops that form disulfide bonds
Each of the groups has a single conserved cysteine residue.
The seven transmembrane regions are TM1, TM2, TM3, TM4,
Named TM5, TM6, and TM7. TM
3 (the intracellular loop between TM3 and TM4) also
Associated with null transmission. [0008] Phosphorylation and lipidation of cysteine residues
(Palmitylated or farnesylated)
Influences G protein-coupled receptor signaling
Can give. Most G protein-coupled receptors are
Within the third cytoplasmic loop and / or carboxy terminus
Contains potential phosphorylation sites. Some G tampa
Protein-coupled receptors (eg, β-adrenoceptors)
-) For protein kinase A and / or
Phosphorylation by different receptor kinases
-Mediates desensitization. G protein-coupled receptor ligand
The binding site is located on several G protein-coupled receptors
A hydrophilic socket (sock) formed by the transmembrane domain
et), which is a G protein
It is surrounded by hydrophobic G residues of conjugated receptors.
Sparseness of each G protein-coupled receptor transmembrane helix
The aqueous side is inward and the polar ligand binding site
It is assumed to form a position. TM3 is a ligand binding
Combination site (eg, contains aspartic acid residue of TM3)
Some G protein-coupled receptors
Are associated with each other. In addition, TM5
Phosphorus, TM6 asparagine, and TM6 or T
The phenylalanine or tyrosine of M7 is also
Related to the bond. In addition, extracellular hydrophilic domains
Has also been shown to play a role in ligand binding
I have. [0010] G protein-coupled receptors are heterologous.
Various intracellular enzymes,
Intracellular conjugates to transport channels and transporters
(Johnson et al., Endoc., Rev.,
10: 317-331 (1989)). Different
Α subunit of G protein
Selective effectors to regulate biological function
Stimulate first. G protein-coupled receptor cells
Phosphorylation of protein residues is caused by some G protein-coupled
Key mechanisms for regulation of sceptor G-protein coupling
Has been identified as G protein-coupled receptor
Has been found at numerous sites within the mammalian host.
You. [0011] SUMMARY OF THE INVENTION The present invention provides a new identification method.
Polynucleotides, such polynucleotides
Polypeptides such as these
Uses of leotides and polypeptides and such
Production of various polynucleotides and polypeptides
You. [0012] According to the present invention, the following is provided.
Items 1 to 19 are provided to achieve the above object. (Item 1.) selected from the group consisting of:
An isolated polynucleotide comprising the member: (A) Polynucleic acid encoding the polypeptide of FIG.
Leotide; (B) ATCC accession number Expression by DNA contained in
A polynucleotide encoding a polypeptide to be obtained; (C) hives the polynucleotide of (a) or (b)
The polynuclease of (a) or (b)
Polynucleotides at least 70% identical to otide
Do; and (D) the polynucleotide of (a), (b) or (c)
Polynucleotide fragment. (Item 2.) The polypeptide described in FIG.
2. The polynucleotide according to item 1, which encodes. (Item 3.) The polynucleotide according to item 1.
The polynucleotide is an ATCC accession number
Matured polypeptide encoded by DNA contained in
A polynucleotide that encodes a code. (Item 4.) The polynucleotide according to item 1.
Vectors containing tides. (Item 5.) The vector described in Item 4
Genetically engineered host cells. (Item 6.) To produce a polypeptide
From the host cell of item 5
Generates a polypeptide encoded by a polynucleotide
A process that includes the steps of manifesting. (Item 7.) The vector according to item 4 is
Generating a polypeptide, including genetically engineering cells
A process for producing a reproducible cell. (Item 8.) Selected from the group consisting of:
Polypeptide: (I) a polypeptide having the deduced amino acid sequence of FIG. 1,
And fragments, analogs, and derivatives thereof;
And (ii) ATCC accession number Of cDNA
Polypeptides to be loaded, as well as polypeptide polypeptides
Fragments, analogs, and derivatives. (Item 9.) The polypeptide is a polypeptide of the type shown in FIG.
Item 9. The polypeptide according to Item 8, having a constant amino acid sequence.
De. (Item 10.) The polypeptide described in Item 8
Antibodies against (Item 11) The polypeptide described in Item 8
A compound that activates (Item 12.) The polypeptide described in Item 8
A compound that inhibits the activation of (Item 13.) It is necessary to activate the receptor.
A method of treating a patient having a need, as described in item 11
Administering to the patient a therapeutically effective amount of a compound of the formula
How. (Item 14.) Need to inhibit receptor
A method of treating a patient having sexual activity, comprising:
Administering to the patient a therapeutically effective amount of the compound.
How. (Item 15) The compound is a polypeptide
And a therapeutically effective amount of the compound is administered to the patient.
Providing DNA encoding the nyst, and in vivo
Item administered by expressing the agonist
14. The method according to 13. (Item 16.) The compound is a polypeptide
And a therapeutically effective amount of the compound is administered to the patient.
Provide DNA encoding the agonist, and
Administered by expressing the antagonist at
Item 15. The method according to Item 14. (Item 17) The polypeptide according to item 8
Compound that binds to and activates the polypeptide
A method for identifying a cell comprising the steps of:
Contacting the compound with a cell that expresses the described polypeptide.
Wherein the polypeptide is the polypeptide of the compound.
A detectable signal in response to the binding to the
Associated with a second component, which can be provided, wherein the contacting of the compound
Under conditions sufficient to permit binding to the polypeptide
A step; and a sig produced by the second component.
Detection of Null to Bind Polypeptide
Identifying the compound. (Item 18) The polypeptide described in Item 8
Binds and inhibits activation of the polypeptide
A method for identifying a compound comprising: a term on a cell surface
A host cell that expresses the polypeptide of item 8;
Analytically known to bind to the polypeptide
Contacting the ligand and compound that can be released.
Thus, the polypeptide allows binding to the polypeptide.
Binding of the compound to the polypeptide under acceptable conditions
Second component capable of providing a detectable signal in response to
And the polypeptide and the polypeptide
That there are no signals generated from interaction with
By detecting that the ligand is
Deciding whether to bind to or not. (Item 19.) Described in Item 8 in patients
For diseases or disorders related to the under-expression of
A process for diagnosing susceptibility to
Mutations in the nucleic acid sequence encoding the peptide, or
Determining the amount of the polypeptide in a sample of origin
Process that encompasses the process. According to one aspect of the present invention, G novel
Repeptides, and their biologically active, and
Fragments and derivatives useful for diagnosis or therapy are provided.
Provided. The polypeptides of the present invention are of human origin. According to another aspect of the present invention, there is provided a polyhedral polymorph of the present invention.
Provided are isolated nucleic acid molecules encoding the peptide;
This nucleic acid molecule can be mRNA, DNA, cDNA, genomic
DNA, and their antisense analogs and
Biologically active and useful for diagnosis or treatment
Including these fragments. According to a further aspect of the present invention, the poly
Expression of the peptide and subsequent recovery of said polypeptide
Encoding a polypeptide of the invention under conditions that promote
A recombinant prokaryotic host cell containing the nucleic acid sequence and / or
Is a recombinant technology that involves culturing eukaryotic host cells.
To produce such polypeptides by surgery
A process is provided. According to a still further aspect of the present invention, this
Antibodies to such polypeptides are provided. According to another aspect of the present invention, the method of the present invention
Binds to and activates or activates the polypeptide
Compounds that inhibit activation and receptor ligands
A leaning method is provided. According to yet another embodiment of the present invention, the G
Treatment of conditions related to under-expression of protein-coupled receptors
Stimulate the receptor polypeptide of the invention for placement.
Processes using such active compounds to provide
Provided. According to another aspect of the present invention, the G protein
To treat conditions involving overexpression of coupled receptors
To this end, processes using such inhibitory compounds have been provided.
It is. According to yet another aspect of the invention, a receptor
Binds a G protein-coupled receptor ligand
Of the G protein-coupled receptor of the present invention
At least one transmembrane domain;
Consensus fragments and / or stored
A sequence having a amino acid substitution, or a G protein
Quantitative or qualitative ligand binding of coupled receptors
Non-natural, synthetic, isolated, and / or coupled
A recombinant G protein-coupled receptor is provided. According to yet another aspect of the present invention, a synthetic or
Is a recombinant G protein-coupled receptor polypeptide,
Conservative substitutions and derivatives thereof, antibodies, anti-idio
Type antibodies, due to their expected biological properties,
Modulates or regulates ligand binding
The potential of G protein-coupled receptor function
Compositions and compositions that may be useful as potential modulators
And methods that provide for diagnosis, treatment, and / or
Can be used for research applications. Still another object of the present invention is to provide a receptor type
Various G protein-coupled types
Inhibit or mimic receptors or their fragments
Synthetic, isolated or recombinant poly
Is to provide a peptide. According to a still further aspect of the present invention, the present invention
Sufficient to specifically hybridize to the
Diagnostic probes containing nucleic acid molecules of a length are also provided. According to yet another object of the present invention,
A disease or disease associated with a mutation in the nucleic acid sequence
Diagnostic assays are provided for detecting morbidity. [0044] These and other aspects of the invention are described in detail herein.
It should be apparent to those skilled in the art from the teachings herein. [0045] BRIEF DESCRIPTION OF THE DRAWINGS FIG.
1 (SEQ ID NO: 2)
Encodes a repeptide or dated June 1, 1995
ATCC accession number Of deposited clones like
Encodes mature polypeptide encoded by cDNA
An isolated nucleic acid (polynucleotide) is provided.
You. The polynucleotide of the present invention is a human gallbladder tissue
It was found in a cDNA library derived from a tissue. This is
Artificially related to the G protein-coupled receptor family
I do. This results in a mature protein of 355 amino acid residues.
Includes open reading frame to encode. this
The protein is a human mononuclear chemotactic protein
Up to about 40% identity to quality 1 receptor (MCP-1b)
And shows the highest degree of homology at about 64% similarity
You. The polynucleotide of the present invention may be in the form of RNA.
Or DNA (this DNA is cDNA, genomic DN
A, and synthetic DNA). DN
A can be double-stranded or single-stranded, and if single-stranded
Contains the coding or non-coding (antisense) strand.
You can. The coding sequence encoding the mature polypeptide is:
The coding sequence shown in FIG.
Can be identical to the loan coding sequence or
The coding sequence is the result of duplication or degeneracy of the genetic code
The DNA of FIG. 1 (SEQ ID NO: 1) or the deposited cDN
A different coding sequence encoding the same mature polypeptide as A
Can be a column. The mature polypeptide of FIG. 1 (SEQ ID NO: 2)
Or the mature polypeptide encoded by the deposited cDNA
The polynucleotide encoding the tide may include:
Only: coding sequence for mature polypeptide; mature polypeptide
Coding sequence of the tide (and additional coding if necessary
Sequences) and non-coding sequences (eg, introns or
Is 5 'of the coding sequence for the mature polypeptide and / or
3 'non-coding sequence). Thus, the term "encoding a polypeptide"
"Polynucleotide" refers to the polypeptide coding sequence only.
And a further coding sequence
And / or a polynucleotide comprising a non-coding sequence
Including. The present invention further relates to FIG. 1 (SEQ ID NO: 2).
A polypeptide having a constant amino acid sequence or a deposited
Of the polypeptide encoded by the loan cDNA.
The invention that codes for fragments, analogs, and derivatives
The present specification relates to variants of the above polynucleotide. Poly
Nucleotide variants are naturally occurring variants of the polynucleotide.
Naturally occurring allelic variants or polynucleotides
May be a variant that does not exist. Accordingly, the present invention provides a method as shown in FIG.
The same mature polypeptide as indicated, or
Mature polypeptide encoded by the cDNA of the
Polynucleotides encoding the same, as well as such
Includes polynucleotide variants. This variant is shown in FIG.
The polypeptide of (SEQ ID NO: 2) or the deposited clone
Fragmentation of the polypeptide encoded by the cDNA of
A derivative, derivative or analog. like this
Nucleotide variants include deletion variants, substitution variants, and
And addition or insertion variants. As indicated hereinabove, polynucleated
Leotide has the coding sequence shown in FIG. 1 (SEQ ID NO: 1) or
Is a naturally occurring allele of the coding sequence of the deposited clone
It may have a coding sequence that is a genetic variant. In the field
As is known, allelic variants can include one or more nucleic acids.
Polynucleotides that may have otide substitutions, deletions, or additions
Another form of the otide sequence, which is the encoded poly
Does not substantially alter the function of the peptide. The polynucleotide may also be a full-length polynucleotide of the present invention.
Cleaved from the TM and intracellular domains of the repeptide
The receptor polypeptide of the present invention, which is an extracellular portion of a polypeptide
It may encode a soluble form of the polypeptide. The polynucleotide of the present invention
To a marker sequence that allows for purification of the polypeptide
It may have the coding sequence fused in frame. marker
The sequence may be, in the case of a bacterial host, a sequence fused to a marker.
A pQE-9 vector that provides for purification of a mature polypeptide
Hex histidine tag supplied by Ah
Alternatively, for example, the marker sequence may be a mammalian host (eg,
If COS-7 cells are used, hemagglutination
Element (HA) tag. HA tag is Influence
Matches epitopes derived from the hemagglutinin protein
(Wilson, I., et al., Cell, 37: 767).
(1984)). The present invention further provides that at least 70
%, Preferably at least 90%, and more preferably
Here means that at least 95% identity is present
In the polynucleotide that hybridizes to the above sequence
Related. The invention particularly relates to the polynucleotides described herein above.
Hybridizes under stringent conditions
Related to polynucleotides. For use herein
The term "stringent conditions" is used for hybridization.
At least 95% between sequences, and preferably
Only occurs when at least 97% identity is present
Means that. In a preferred embodiment, the present specification provides
A polynucleotide hybridizing to the above polynucleotide
The nucleotide was obtained from the cDNA of FIG.
With the mature polypeptide encoded by the committed cDNA
Retains substantially the same biological function or activity
(Ie, for example, a second messenger response
Induces membrane-bound receptor polypeptides
For receptors mediated by polypeptides that do not function as
By retaining the binding capacity of the ligand
Function as receptor polypeptide)
Code. Alternatively, the polynucleotide may be
Hybridized to the polynucleotide described in
As noted above, it has identity and activity
Non-reactive, at least 20 bases, preferably
At least 30 bases, and more preferably at least 30 bases
It may have 50 bases. Such a polynucleotide is
For example, this polynucleotide for the polynucleotide of SEQ ID NO: 1
Probes or diagnostics for recovery of oligonucleotides
Can be used as a probe, or PCR primer
You. The deposit (single or plural) referred to in this specification
Number) relates to the international recognition of deposits of microorganisms in patent procedures.
Maintained under the Budapest Treaty. These deposits
Is provided only as a convenience to those skilled in the art, and
That a deposit is required under 35 U.S.C. 112
I did not admit that. Polynuclei contained in the deposit
Otide sequences and polypeptides encoded thereby
The amino acid sequence of the peptide is incorporated herein by reference.
And with any description of the sequences herein
It also suppresses contradictions. Manufacture, use, or deposit
A license may be required to sell and
Such a license is not granted by
No. The present invention further relates to the method of FIG. 1 (SEQ ID NO: 2).
CDNA having a defined amino acid sequence or deposited
G protein having an amino acid sequence encoded by
Conjugated receptor polypeptides, as well as such
For fragments, analogs and derivatives of
I do. The terms “fragment”, “derivative”, and
And "analog" are the polypeptides of FIG. 1 (SEQ ID NO: 2)
Or the polypeptide encoded by the deposited cDNA
Substantially the same as such a polypeptide when referring to
Biological function or activity (ie, G protein coupling
Function as a type receptor), or
The polypeptide is a G protein-coupled receptor (eg,
Does not function as a soluble form of this receptor)
Also retain the ability to bind ligand or receptor
Or a polypeptide that is either Anna
The log is activated by cleavage of the proprotein part
Proproteins that can produce active mature polypeptides
Including. The polypeptide of the present invention is a recombinant polypeptide.
Tide, a natural polypeptide or a synthetic polypeptide.
And preferably a recombinant polypeptide. The polypeptide of FIG. 1 (SEQ ID NO: 2)
Phage of the polypeptide encoded by the deposited cDNA
A fragment, derivative or analog is (i) one or more
Amino acid residues above are conserved amino acids or non-conserved amino acids
A non-acid residue (preferably a conserved amino acid residue)
And such a substituted amino acid residue is the genetic code
May be an amino acid residue that can be encoded by
Or (ii) one or more
The amino acid residue above contains a substituent, or
(Iii) the mature polypeptide has a half-life of the polypeptide;
Another compound such as polyethylene (eg, polyethylene
Glycol) or (iv)
Additional amino acids are needed for purification of the mature polypeptide
Fused to the mature polypeptide used, or
Or (v) the fragment of the polypeptide is soluble
That is, it does not bind to membranes, but
Gand). Such a fragment
Are derivatives, analogs and derivatives of the teachings herein.
Are considered to be within the purview of those skilled in the art. Polypeptides and Polynucleotides of the Invention
The tide is preferably provided in an isolated form, and
And preferably homogeneously purified. The polypeptide of the present invention corresponds to the polypeptide of SEQ ID NO: 2.
Ripeptides (especially mature polypeptides) and at least
Also 70% similarity (preferably at least 70% identity)
G) to the polypeptide of SEQ ID NO: 2, and
More preferably at least 90% similarity (more preferred
Or at least 90% identity) with the polypeptide of SEQ ID NO: 2.
Have against the peptide, and even more preferably less
95% similarity (even more preferably at least 9
5% identity) to the polypeptide of SEQ ID NO: 2.
And generally also at least
30% amino acids and more preferably at least 50
Having portions of such polypeptides containing amino acids
Includes portions of such polypeptides. As is known in the art, two polypep
"Similarity" between tides refers to the amino acids of one polypeptide
The sequence and its conserved amino acid substitutions into a second polypeptide.
It is determined by comparing with the sequence of the peptide. A fragment of the polypeptide of the present invention or
Is the corresponding full-length polypeptide by peptide synthesis.
Can be used to produce Therefore, this flag
Is an intermediate for producing the full-length polypeptide.
Can be used. Fragmentation of the polynucleotide of the present invention
Part or portion combines the full-length polynucleotide of the invention.
Can be used to generate The term “isolated” means that a substance is
Environment (for example, the natural environment, if it occurs naturally)
Means that it has been removed from For example, alive
Naturally occurring polynucleotides present in animals
Alternatively, the polypeptide is not isolated, but is a naturally occurring system.
Separated from some or all of the coexisting materials in
The same polynucleotide or polypeptide that is
Has been isolated. Such a polynucleotide is a vector
And / or such polynucleic acid
The leotide or polypeptide is part of the composition
Obtained, and such a vector or composition is
Because it is not part of the environment, it can still be isolated. The term “gene” refers to the production of a polypeptide chain.
Means a segment of DNA involved in
This is before and after the code area (reader and tray).
Region) and individual code segments (Exo
Intervening sequence (intron). The present invention also relates to the polynucleotide of the present invention.
Using the vector of the present invention
Host cells to be used
For the production of peptides. The host cell is a vector of the present invention, which comprises
For example, a cloning vector or an expression vector.
Genetically engineered (transduced)
Or transformed or transfected
Is done). Vectors include, for example, plasmids, viruses
It can be in the form of particles, phages, and the like. Manipulated host
Cells activate the promoter or select transformants.
Or G protein-coupled receptor gene
In a conventional nutrient medium modified appropriately to amplify
And cultured. Culture conditions (eg, temperature, pH
Are previously used in the host cell selected for expression.
Conditions, and will be apparent to those skilled in the art. The polynucleotide of the present invention can be produced by a recombinant technique.
Can be used to produce a polypeptide. Obedience
Thus, for example, a polynucleotide emits a polypeptide
Included in any one of a variety of expression vectors for expression
Can be Such vectors contain chromosomal DNA sequences,
Includes chromosomal and synthetic DNA sequences.
Such vectors include, for example, derivatives of SV40;
Bacterial plasmid; phage DNA; baculovirus;
Yeast plasmid; a set of plasmid and phage DNA
Vectors derived from the combination, viral DNA (eg,
Vaccinia, adenovirus, fowlpox virus, and
And pseudorabies). But can replicate in the host
Any other vector can be used as long as it is
Can be used. The appropriate DNA sequence may be obtained by various procedures.
Can be inserted into the reactor. Generally, the DNA sequence
Appropriate restriction endonuclease sites by procedures known in the art
It is inserted in the place. These and other procedures are
It is considered within the scope of the trader. The DNA sequence in the expression vector is
Operably linked to a current control sequence (promoter);
Directs RNA synthesis. Such promoters
Tabular examples may include: LTR or
SV40 promoter,E. FIG. coli lacOrt
rp, Phage P_LPromoters and prokaryotic cells
Genes in vesicles or eukaryotic cells or their viruses
Other promoters known to control expression of
- The expression vector also contains ribosomes for translation initiation.
Contains a binding site and a transcription terminator. Vector
May also contain appropriate sequences for amplifying expression.
You. Furthermore, the expression vector is preferably
Phenotypic traits for selection of transformed host cells (eg,
For eukaryotic cell cultures, dihydrofolate reductor
Ze or neomycin resistance, or for exampleE. FIG. col
iResistance to tetracycline or ampicillin in rats
Containing one or more selectable marker genes that provide
You. A suitable DNA sequence as described herein above
Contains appropriate promoter or control sequences
The appropriate vector is transformed into an appropriate host and
It can be used to express protein. Representative examples of suitable hosts include:
Can include: bacterial cells (eg,E. FIG. coli,St
reptomyces,Salmonella type
himuriumFungal cells (eg, yeast); insect cells
Vesicles (for example,Drosophila S2andSpo
doptera Sf9); Animal cells (eg, CH
O, COS or Bowes melanoma); adenovirus
And plant cells. Selection of an appropriate host is described herein.
It is considered within the skill of the art from the teachings. More specifically, the present invention also relates to
Includes a recombinant construct containing one or more of the exemplified sequences
I do. The construct is a vector (eg, a plasmid vector).
Or viral vectors).
In the sequence of the present invention is inserted in the forward or reverse direction
I have. In a preferred aspect of this embodiment, the construct comprises
In addition, a regulatory sequence operably linked to the sequence (eg,
If it contains a promoter). Very large number
Clear vectors and promoters are known to those of skill in the art.
And can be purchased. The following vectors are examples
Provided. Bacterial: pQE70, pQE60, pQE
-9 (Qiagen), pbs, pD10, phage
script, psiX174, pbluescript
t SK, pbsks, pNH8A, pNH16a, p
NH18A, pNH46A (Stratagene);
pTRC99a, pKK223-3, pKK233-
3, pDR540, pRIT5 (Pharmaci
a). Eukaryotic: pWLNEO, pSV2CAT, pOG
44, pXT1, pSG (Stratagene); p
SVK3, pBPV, pMSG, pSVL (Pharm
acia). However, any other plasmid or vector
Are also replicable and viable in the host.
It can be used as long as it can. The promoter region is CAT (chloramph
Enicol transferase) vector or selection
Any other desired vector using other vectors with
It can be selected from genes. Two suitable vectors are P
KK232-8 and pCM7. Especially well-known
The bacterial promoters are lacI, lacZ, T3,
T7, gpt, λP_R, P_LAnd trp. true
Nuclear promoters include CMV immediate, HSV thymidine
Kinases, early and late SV40, retro
LTR derived from irs and mouse metallothionein I
Is included. Choosing the right vector and promoter
Is well within the level of ordinary skill in the art. In a further embodiment, the invention relates to the above-described structure.
The present invention relates to host cells containing structures. The host cell is higher
Eukaryotic cells (eg, mammalian cells) or lower eukaryotes
Cells (eg, yeast cells) or
The primary cell can be a prokaryotic cell (eg, a bacterial cell)
You. Introduction of the construct into the host
Transfection, DEAE-dextran mediated tiger
By transfection or electroporation
(Davis, L., Dibner,
M. Battey, I .; , Basic Method
s in Molecular Biology, (1
986)). Conventional methods using constructs in host cells
To produce the gene product encoded by the recombinant sequence.
You. Alternatively, the polypeptides of the present invention can
It can be produced synthetically by a synthesizer. A fragment of the polypeptide of the present invention comprises
Peptide synthesis produces the corresponding full-length polypeptide
Can be used to So this fragment
Is used as an intermediate to produce a full-length polypeptide.
Can be used. Fragments of the polynucleotide of the invention
Is used to synthesize the full-length polypeptide of the present invention.
Can be used. The mature protein can be a mammalian cell, yeast,
Under control of an appropriate promoter in bacteria or other cells
Can be expressed. The cell-free translation system is also a DNA of the present invention.
Using RNA from the construct,
It can be used to produce proteins. Prokaryotic hosts and
Cloning vectors for use in eukaryotic hosts
And expression vectors are described in Sambrook et al., Mol.
eco Cloning: A Laborat
ory Manual, 2nd edition, Cold Spring
g Harbor, N .; Y. , (1989) (this disclosure
Is incorporated herein by reference).
I have. DNA encoding the polypeptide of the present invention
Transcription by higher eukaryotes enhances vector
Augmented by inserting sequences. Enhancers
Cis-acting element of DNA, usually about 10 to about
300 bp, which act on the promoter and
Increase transcription. As an example, bp100 to
270 late-stage SV40 enhancer, cytomegalo
Virus early promoter enhancer, replication origin
Late-stage polyoma enhancers and adenovirus
Suenhancer is mentioned. In general, the recombinant expression vector is a host cell
Origins and selectable markers that allow transformation of yeast
(For example,E. FIG. coliAmpicillin resistance gene and
AndS. cerevisiaeTRP1 gene)
From highly expressed genes that direct transcription of structural sequences downstream
Contains a promoter. Such a promoter
Is a glycolytic enzyme (eg, 3-phosphoglycerate)
Kinase (PGK)), α-factor, acid phosphatase,
Or an operon encoding a heat shock protein, etc.
Can be derived from The heterologous structural sequence includes a translation initiation sequence and a translation sequence.
Assembled in proper phase with the translation stop array. As needed
Thus, the heterologous sequence has the desired characteristics (eg, expressed recombinant
N-terminus to provide stable or simplified purification of the product
It may encode a fusion protein comprising a terminal identification peptide. Expression vectors useful for bacterial use are functional
Proper promoter and operable reading phase
Along with the translation start and translation termination signals,
Inserting a structural DNA sequence that encodes a protein
More built. Vectors contain one or more phenotype selection
And the maintenance of the vector
If necessary, provide an origin of replication to provide amplification in the host.
contains. Suitable prokaryotic hosts for transformation are
E. FIG. coli,Bacillus subtilis,
Salmonella typhimurium,
Genus Pseudomonas, Streptomyc
Species within the genus es and Staphylococcus
Includes various species, but other species are also used for selection
Can be As a representative, but not limiting, example,
Expression vectors useful for the use of bacteria are well-known cloning
Inheritance of vector pBR322 (ATCC 37017)
Selection plasmids derived from commercially available plasmids containing
And a bacterial origin of replication. like this
A commercially available vector is, for example, pKK223-3 (Ph
armasia Fine Chemicals, Up
psala, Sweden, and GEM1 (Prom
ega Biotec, Madison, WI, US
A). These pBR322 "backbone" portions are suitable
Smart promoters and structural sequences to be expressed
Are combined. Transformation of a suitable host strain and suitable cells
Following growth of the host strain to density, the selected promoter
Appropriate means (eg, temperature shift or chemical induction).
And the cells are cultured for a further period of time.
It is. The cells are typically collected by centrifugation.
Crushed by physical or chemical means, and
The crude extract obtained is retained for further purification.
You. Microbes used in protein expression
Cells are subjected to freeze-thaw cycles, sonication, mechanical disruption
Any convenient method, including disruption or use of cell lysing agents
Can be crushed by the method. Such methods are well known to those skilled in the art.
It is. Various mammalian cell culture systems are also recombinant.
And can be used to express proteins. Mammal
Examples of expression systems include Gluzman, Cell, 23:
175 (1981).
COS-7 strain, and others capable of expressing compatible vectors
Cell lines (eg, C127, 3T3, CHO, HeL
a, and BHK cell lines). Mammalian expression vector
The originator, the appropriate promoter and the enhancer
Sensors and any necessary ribosome binding sites,
Liadenylation sites, splice donor sites and splice
Is acceptor site, transcription termination sequence, and 5 '
It also contains a non-transcribed non-transcribed sequence. SV40 splice
Site derived from the DNA site and the polyadenylation site
Sequence to provide the necessary non-transcribed genetic elements
Can be used. G protein-coupled receptor of the present invention
Repeptides can be obtained from recombinant cell culture by methods including:
And can be purified. Ammonium sulfate precipitation or
Tanol precipitation, acid extraction, anion or cation exchange
Chromatography, phosphocellulose chromatography
-, Hydrophobic interaction chromatography, affinity
-Chromatography, hydroxylapatite chroma
Chromatography and lectin chromatography. Must
If necessary, refold the protein (refold
ing) process completes the placement of the mature protein
Can be used for Finally, high-performance liquid chromatography
(HPLC) can be used for the final purification step
You. [0091] The polypeptides of the present invention may be naturally occurring, purified.
Or is the product of a chemical synthesis procedure
Or prokaryotic or eukaryotic host (eg, culture
Bacteria, yeast, higher plants, insects, and mammalian cells
Vesicles) can be produced by recombinant techniques. Recombinant producer
Depending on the host used in turn, the polypeptide of the invention
Can be glycosylated or glycosylated
Maybe not. The polypeptides of the present invention also
Methionine amino acid residue. Full length G protein-coupled receptor gene
Fragment isolates the full-length gene and
High sequence similarity or similar biological activity to the gene
CDNA clones to isolate other genes
Used as a hybridization probe for Bally
Can be done. This type of probe has at least 20 salts
Group, preferably 30 bases, and most preferably 50 salts
Having more than one group. Probes also have full-length transcription
CDNA clone and genomic clone corresponding to the product,
Or regulatory and promoter regions, exons, and
G-protein coupled receptor containing introns
Can be used to identify clones containing the gene.
You. Using a known DNA sequence as an example of screening
To synthesize oligonucleotide probes.
G protein-coupled receptor gene coding region
Is isolated. Sequence and sequence phase of the gene of the present invention
The complementary labeled oligonucleotide is human c
Libraries of DNA, genomic DNA, or mRNA
Used to screen for
Determine which members will hybridize to the probe
I do. G protein-coupled receptor of the present invention
Is a compound that activates the receptor polypeptide of the present invention.
(Agonist) or compound that inhibits activation (an
TAGONIST) In the process for screening
Can be used. Generally, such screening procedures
Expresses the receptor polypeptide of the present invention on its surface
Providing suitable cells. Such cells
Is a mammal, yeast, Drosophila, or
E. FIG. E. coli-derived cells. In particular, the receptor of the present invention
The polynucleotide encoding the promoter
The G protein-coupled receptor
It is used to express Next, the expression
The prober is contacted with a test compound to allow binding, functional response
Observe irritation or inhibition. One such screening procedure is:
To express the G protein-coupled receptor of the present invention
Including the use of melanophore cells transfected into
No. Such a screening technique is described in PCT WO
92/01810 (released February 6, 1992)
Have been. Thus, for example, such an assay
Receptor ligands and agents to be screened
Both the compound and the melanin-retaining cell encoding the receptor.
By contacting the cells with the cells of the present invention.
Screen for compounds that inhibit peptide activation
Can be used to Signal generated by ligand
Inhibition of the compound is a potential
Be a nest, i.e.
Indicates harm. This screening was performed with such cells.
Contacting with the compound to be screened, and
Such a compound produces a signal, ie
Determine whether such compounds activate the receptor
To determine the compound that activates the receptor
Can be used to [0098] Other screening techniques include, for example, Sc
issue, 246, 181-296 (1989)
(October 1998)
To measure extracellular pH changes caused by
Expressing G protein-coupled receptor
(Eg, using transfected CHO cells).
No. For example, if the compound is a receptor polypeptide of the invention
Can be contacted with cells expressing the
Anger response (eg, signal transduction or pH change)
Is a potential compound that activates or blocks the receptor
It can be measured to determine what harms. Another such screening technique is G
Xe encoding RNA encoding protein-coupled receptor
Introduced into nopus oocytes to temporarily generate receptors
Including manifestation. Then the receptor oocyte
Is the receptor ligand and the
Contact with compounds, and subsequently inhibit receptor activation
When screening for compounds that are likely to
Inhibition or activation of the lucium signal can be detected. Another screening technique involves using the receptor
G-protein coupled to phospholipase C or D
Includes expression of protein-coupled receptors. Such fine
Representative examples of endothelial cells, smooth muscle cells, fetal renal cells
Vesicles and the like. Screening is described herein.
Activation of the receptor, or phospholipa
Of receptor activation derived from the secondary signal of the enzyme
This can be achieved by detecting harm. Another approach is to have the receptor on the cell surface.
Determine the inhibition of labeled ligand binding to cells
The receptor polypeptide of the present invention
Screening for compounds that inhibit activation of
You. Such a method allows cells to place receptors on their surface.
Encodes a G protein-coupled receptor to be expressed
Transfecting eukaryotic cells with the DNA
And compounds in the presence of known ligands in labeled and labeled form
And the step of contacting The ligand is, for example,
It can be labeled by radioactivity. Label binding to receptor
The amount of ligand can be used, for example, to measure receptor radioactivity.
Is measured. Of labeled ligand that binds to the receptor
Compound binds to receptor as determined by reduction
The binding of the labeled ligand to the receptor is inhibited.
It is. G protein-coupled receptors are mammalian
Many ubiquitous in the host and encompass many medical conditions
Responsible for biological functions. Therefore, on the one hand G protein coupling
To stimulate G-type receptors, while
Find compounds and drugs that can interfere with the function of
Is desirable. For example, a G protein-coupled receptor
Activating compounds include asthma, Parkinson's disease, acute heart disease
Therapeutic objectives, such as treating illness, hypotension, urinary retention, osteoporosis
Can be used for Generally, G protein-coupled receptors
Compounds that inhibit activation include, for example, hypertension, angina,
Myocardial infarction, ulcer, asthma, allergy, benign prostatic hyperplasia,
And psychotic and neurological disorders (schizophrenia, manic
Morbid excitement, depression, delirium, dementia or severe mental retardation
Stagnation, Huntington's disease or Jill de la Tourette
(Gilles dila Tourette's)
Various treatments for the treatment of group-like dyskinesia)
Can be used for purpose. G protein coupled receptor
A compound that inhibits teratogen also reverses endogenous anorexia
And control of pathological hunger. The antibody is a G protein-coupled receptor of the present invention.
Antagonized putter or, if present, oligopeptide
Can be This is linked to G protein-coupled receptors.
But does not trigger a second messenger response,
Therefore, activation of G protein-coupled receptor is prevented.
You. Antibodies are unique, generally associated with the antigen-binding site of an antibody
And anti-idiotype antibodies that recognize the determinant of Latent
Antagonist compounds are also G-protein coupled
A protein closely related to the ligand of the receptor
That is, it includes a fragment of the ligand. It is a living thing
Lacking biological functions, and
It does not elicit any response upon binding to the scepter. Prepared through the use of antisense technology
Antisense constructs have triple helix formation or
Control gene expression via sense DNA or RNA
Can be used to Both of these methods are polynucleated
Based on the binding of otide to DNA or RNA. example
For example, the 5 'coding portion of the polynucleotide sequence (of the present invention)
Encodes the mature polypeptide) is about 10-40 bases
Set a pair-length antisense RNA oligonucleotide
Used to measure. DNA oligonucleotides
Complementary to the region of the gene involved in transcription (triple overlap)
Lee et al., Nucl. Acids Res. , 6:
3073 (1979); Cooney et al., Science.
e, 241: 456 (1988); and Dervan
Et al., Science, 251: 1360 (1991).
See also G-protein coupled receptors
Designed to prevent transcription and production of Anti
Sense RNA oligonucleotides are mRNA in vivo
And G protein-coupled receptor
Blocking translation of the mRNA molecule into the
, Okano, J .; Neurochem. , 56:
560 (1991); antisense inhibition of gene expression.
Oligodeoxynucleotides as CRC, CRC
Press, Boca Raton, FL (198
8)). The above oligonucleotides are also delivered to cells.
As a result, the antisense RNA or DNA
In order to inhibit the production of protein-coupled receptors,
It can be expressed in vivo. Binds to G protein-coupled receptor
Small molecules use G protein-coupled receptors as ligands
Inaccessibility, which hinders normal biological activity
It is. For example, small peptides or peptide-like molecules
Inhibiting the activation of the receptor polypeptide of the present invention.
Can be used to Soluble forms of G protein-coupled receptors
The form (eg, a fragment of a receptor) of the invention
By binding to a ligand for the polypeptide
And ligand are membrane-bound G protein-coupled receptors
Of the receptor by preventing it from interacting with
Can be used to inhibit activation. The present invention further relates to excessive G protein coupling.
To treat abnormal conditions related to type 1 receptor activity
To provide a way. This method uses G protein-coupled
Blocking binding of the ligand to the sceptor or
Inhibits activation by inhibiting a second signal
An effective amount of a pharmaceutically acceptable carrier and
The above-mentioned inhibitor compound is administered to the subject.
And thereby mitigating abnormal conditions
Is included. The present invention also relates to a G protein-coupled receptor.
-To treat abnormal conditions associated with under-expression of activity
To provide a way. This method uses the receptor poi of the present invention.
The therapeutically effective amount of a compound that activates a
In combination with a pharmaceutically acceptable carrier such as
Administer to the specimen to thereby alleviate the abnormal condition
Process. Soluble form of G protein-coupled receptor
To activate or block such receptors
Harmful compounds may be combined with a suitable pharmaceutical carrier
Can be used. Such compositions may contain a therapeutically effective amount of
A peptide or compound, and a pharmaceutically acceptable key.
Carrier or excipient. As such a carrier
Is saline, buffered saline, dextrose,
Water, glycerol, ethanol, and combinations thereof
But not limited thereto. The prescription is
It should suit the mode of administration. The present invention also relates to one of the pharmaceutical compositions of the present invention.
Pharmaceutical comprising one or more containers filled with one or more ingredients
Provide packs or kits. Such containers (single
Or more than one) regarding the manufacture of drugs or biological products
Specified by governmental agencies governing the manufacture, use, or sale of
Product labeling, which can be used in human
Institutions in the manufacture, use or sale of dosing
Represents authorization by In addition, the pharmaceutical composition may have other therapeutic
It can be used in combination with the compound. Pharmaceutical compositions can be administered, for example, topically, intravenously,
By intraperitoneal, intramuscular, subcutaneous, intranasal, or intradermal route
It can be administered in a convenient manner. Pharmaceutical compositions may have certain suitability.
Administered in an amount effective to treat and / or prevent
You. Generally, the pharmaceutical composition will have at least about 10 μg / k
g of body weight, and often they
It is administered in an amount not to exceed about 8 mg / kg body weight daily. Many
In most cases, the dosage is from about 10 μg / kg body weight to about
1 mg / kg body weight, taking into account administration route, symptoms, etc.
It is. G protein-coupled receptor polypeptide
And compounds that also activate or inhibit the compound
In accordance with the present invention, such a polypeptide in vivo
It can be used by expression of a tide. This is often called "hereditary
Called child treatment. Thus, for example, cells from a patient
A polynucleotide encoding a peptide (DNA or
RNA) can be manipulated ex vivo and then manipulated
The cells produced are the patients to be treated with this polypeptide
Provided to Such methods are well known in the art.
You. For example, a cell encodes a polypeptide of the invention.
The use of retroviral particles containing RNA
It can be operated by procedures known in the art. Similarly, cells can be used to produce polypeptides in vivo.
For expression of the peptide, for example, by procedures known in the art.
Can be manipulated in vivo. As known in the art,
Les containing RNA encoding the polypeptide of the present invention.
Producer cells for producing torovirus particles
To manipulate cells in vivo and in vivo
May be administered to a patient for expression of the polypeptide. This
The method of administering the polypeptide of the present invention by a method such as
These and other methods for making
It should be apparent to one skilled in the art. For example, manipulate cells
Expression vehicles other than retroviruses
(Eg, adenovirus). This is appropriate
Cells in vivo after combination with a suitable delivery vehicle
Can be used to make. The retroviral plasmid described above in the present specification.
Retroviruses from which the vector can be derived include Moro
Knee mouse leukemia virus, spleen necrosis virus, Rous
Retrovirus like sarcoma virus, Harvey sarcoma
Ils, chicken leukemia virus, gibbon monkey leukemia
Ills, human immunodeficiency virus, adenovirus, bone
Myeloproliferative sarcoma virus, and breast cancer virus
But not limited to these. In one embodiment
The retroviral plasmid vector is Molonema
Derived from the Usus leukemia virus. [0118] Vectors may contain one or more promoters.
No. Suitable promoters that can be used include Mil.
ler et al.Biotechniques, Volume 7, 9
No. 980-990 (1989)
LTR; SV40 promoter;
Gallovirus (CMV) promoter, or any other
Promoters (eg, eukaryotic cell promoters)
(Histone, pol III, and β-actin pro
Motors (including but not limited to motors)
Vesicle promoter), but are not limited thereto.
Absent. As other viral promoters that can be used
Is an adenovirus promoter, thymidine kinase
(TK) promoter and B19 parvovirus pump
Motor, but is not limited thereto. Suitable
The selection of a sharp promoter is based on the teachings contained herein.
Will be apparent to those skilled in the art. Nucleic acid sequence encoding the polypeptide of the present invention
The rows are under the control of a suitable promoter. Can be used
Suitable promoters include adenovirus promoters.
(Eg, the adenovirus major late promoter)
-); Or a heterologous promoter (eg, cytomegalo
Virus (CMV) promoter); RS virus (R
SV) promoter; an inducible promoter (eg, M
MT promoter, metallothionein promoter);
Heat shock promoter; albumin promoter; A
poAI promoter; human globin promoter;
Ilstymidine kinase promoter (eg,
Rupesthymidine kinase promoter); retrovirus
LTR (modified retroviral LTR as described herein above)
Β-actin promoter; and human growth factor
Remon promoter, but not limited to
Absent. Promoters also encode polypeptides
It can be a natural promoter that controls the gene. [0120] The retroviral plasmid vector
Transducing the packaging cell line into producer cells
Used to form strains. Transfected
Examples of packaging cells obtained include Miller,
Human Gene Therapy, Volume 1, 5-
14 (1990) (in its entirety as a reference herein)
PE501, PA31 as described in US Pat.
7, ψ-2, ψ-AM, PA12, T19-14X, V
T-19-17-H2, $ CRE, $ CRIP, GP +
E-86, GP + envAm12, and DAN cell lines
But not limited thereto. The vector is
Packaging cells by any means known in the art
Can be transduced. Such means include elect
Loporation, use of liposomes, and CaPO_Four
But not limited to precipitation. One generation
Alternatively, the retroviral plasmid vector is
Can be encapsulated in a liposome or bound to a lipid
And can then be administered to a host. The producer cell line contains the polypeptide
An infectious agent containing the encoding nucleic acid sequence (s)
Produce torovirus vector particles. Then this
Such retroviral vector particles can be used in vitro or
For transducing eukaryotic cells, either in vivo
Can be used for The transduced eukaryotic cells are
Nucleic acid sequence (s) encoding the repeptide
Express. Eukaryotic cells that can be transduced include embryos
Blast stem cells, embryonal carcinoma cells and hematopoietic stem cells, hepatocytes,
Fibroblasts, myoblasts, keratinocytes, endothelial cells,
And bronchial epithelial cells, including but not limited to
Absent. The present invention also provides the G protein conjugate of the present invention.
Ligands that are not known to be able to bind to type receptors
Determine if it can bind to such receptors
Provide a way to This method uses G protein
Conditions that permit binding of ligand to conjugated receptor
And a mammal expressing a G protein-coupled receptor
Contacting cells with ligand, binding to receptor
Detecting the presence of the ligand, and thereby
Whether Gand binds to G protein-coupled receptors
Deciding whether or not to use The present invention further provides a human G
Specifically interacts with protein-coupled receptors,
To identify drugs that bind to
Provide a method for training. This is G protein coupling
Contains an isolated DNA molecule encoding a type 1 receptor
Contacting a plurality of drugs with a mammalian cell to be treated.
Determining which drugs bind to mammalian cells, and
And thereby the human G protein-coupled receptor of the present invention
Drugs that specifically interact with and bind to pter
Identifying the object. Then such drugs
Activates or activates the receptor of the present invention.
It can be used therapeutically either to inhibit. The present invention also relates to a G protein-coupled receptor.
By detecting the presence of mRNA encoding the
G protein-coupled receptor on cell surface
Methods for detecting the expression of This method works on cells
Obtaining total mRNA, and the thus obtained mRNA.
RNA encoding human G protein-coupled receptor
Specifically hybridizes with sequences contained within the sequence of the nucleic acid molecule
A hybridizable nucleic acid probe of the present invention which can be hybridized
Contacting under probe conditions, hybridizing probe
Detecting the presence of the amplified mRNA, and
Therefore, expression of G protein-coupled receptor by cells
The step of detecting The present invention also relates to the receptor polypeptide of the present invention.
Associated with the presence of a mutation in the nucleic acid sequence encoding the peptide
Diagnostics to detect the disease or its susceptibility to the disease
G protein-coupled receptor as part of the assay
Concerning the use of genes. As an example, such diseases are
For vesicle transformation (eg, tumors and cancers). Human G protein-coupled receptor gene
Individuals with mutations in them can be DNA-leveled by various techniques.
Can be detected by Nucleic acids for diagnosis are located in patient cells
(Eg, blood, urine, saliva, tissue biopsy, and autopsy material)
Quality). Genomic DNA is used directly for detection.
Can be used directly or use PCR before analysis
Can be amplified enzymatically (Saiki et al., Natu).
re, 324: 163-166 (1986)). RNA
Alternatively, cDNA can also be used for the same purpose.
You. As an example, a G protein-coupled receptor protein
PCR primers complementary to the nucleic acid encoding the quality
Identify and analyze protein-coupled receptor mutations
Can be used for For example, deletions and insertions are normal
Of amplified product size in comparison to different genotypes
It can be detected by a change. Point mutation was radiolabeled
G protein-coupled receptor RNA or radiation
-Labeled G protein-coupled receptor antisense
The amplified DNA is hybridized to the DNA sequence.
Can be identified. Perfectly matched sequence
Is due to the difference in RNase A digestion or melting temperature.
It can be distinguished from the matched duplex. Between the control gene and the gene having the mutation
Sequence differences revealed by direct DNA sequencing
Can be done. In addition, the cloned DNA segment
Is a probe for detecting a specific DNA segment
Can be used as The sensitivity of this method is
If given, it will be greatly enhanced. For example, sequencing
The primer is a double-stranded PCR product or a modified PC
With the single-stranded template molecule created by R
used. Sequencing uses radiolabeled nucleotides
Automated procedures using conventional procedures or fluorescent tags
Implemented in order. Genetic Testing Based on DNA Sequence Differences
Is the DNA in gels with or without denaturing agents
For detecting changes in electrophoretic mobility of fragments
More can be achieved. Small sequence deletions and insertions
It can be visualized by resolution gel electrophoresis. Different arrays
DNA fragments on denaturing formamide gradient gels
Can be distinguished. Where different DNA fragments
The mobility of a polymer depends on its specific melting temperature or partial melting
Delayed to different locations in the gel according to temperature (e.g.,
See, Myers et al., Science, 230: 1242.
(1985)). A sequence change at a particular position may also
Ase protection assays (eg, RNase protection and S
1 Protection or chemical cleavage methods (eg, cotton et al.,
PNAS, USA, 85: 4397-4401 (198
5))). Therefore, the detection of a specific DNA sequence is high.
Hybridization, RNase protection, chemical cleavage, direct
Direct DNA sequencing or the use of restriction enzymes (eg,
For example, restriction fragment length polymorphism (RFLP)) and genomic DNA
Achieved by methods such as Southern blotting
obtain. More conventional gel electrophoresis and DNA sequencing
In addition to sequencing, mutations are also detected by in situ analysis.
Can be issued. The present invention also relates to the present invention in various tissues.
Levels of soluble forms of human receptor polypeptides
A diagnostic assay for the detection of Derived from the host
The level of soluble receptor polypeptide in a sample
Assays used to detect are well known to those of skill in the art.
Yes, and radioimmunoassays, competitive binding assays
B) Western blot analysis, and preferably ELI
Includes SA assay. The ELISA assay uses a receptor polypeptide.
Antibodies specific for peptide antigens, preferably monoclonal
First preparing the antibody. Furthermore, this thing
A reporter antibody to the clonal antibody is prepared.
Radioactivity, fluorescence, or horseradish in this example
Detectable reagents such as the enzyme oxidase reporter
-Attached to antibodies. Here the sample is taken from the host.
And a solid support (eg, tantalum in the sample).
Ink on polystyrene dish)
It is private. Then any free on the dish
A protein binding site is a non-specific protein (eg,
By incubating with bovine serum albumin)
Coated. The monoclonal antibody is then
Incubate in the
The body is attached to any polystyrene dish according to the invention
To the receptor polypeptide. Do not combine all
Monoclonal antibodies are washed away with buffer.
You. Here, the horseradish peroxidase-linked
The porter antibody is placed in the dish, which
Any monoclonal antibody conjugated to the scepter protein
The reporter antibody binds to the body. Then does not adhere
The reporter antibody is washed away. Then, peroxy
Diase substrate is added to the dish and
The amount of color development between
Determination of receptor protein present in large amounts of patient samples
It is a fixed value. The sequences of the present invention are also useful for chromosome identification.
It is. This sequence is located at a specific location on individual human chromosomes.
And specifically hybridize to that position
I can do it. In addition, we now identify specific sites on the chromosome
Need to be Currently available for labeling chromosome locations
Chromosome labeling based on accurate actual sequence data (repeated polymorphism)
There are almost no chemical reagents. The DNA of the present invention
Papping matches these sequences with disease-related genes.
This is an important first step in the relation. [0135] Briefly, the sequence was converted from cDNA to P
Prepare CR primer (preferably 15-25 bp)
Can be mapped to chromosomes. 3 'untranslated region
Computer analysis of more than one in genomic DNA
Does not span exons, thus complicating the amplification process
Used to quickly select the primer to be used. Next
These primers contain individual human chromosomes
Used for PCR screening of somatic cell hybrids
You. Hybrid containing the human gene corresponding to the primer
Only yield amplified fragments. PCR mapping of somatic cell hybrids
To assign specific DNA to specific chromosomes
This is a quick procedure. The same oligonucleotide primer
For use with the present invention, derived from specific chromosomes in a similar manner
Panel of fragments or large genomic clones
Pooling is used to determine partial localization (subloca).
lization can be achieved. Mappin to chromosome
Other mapping strategies that can also be used to
Teggie, in situ hybridization, labeling
Flow-sorted chromosomes
Prescreening used and chromosome-specific cDN
Hybridization for constructing A library
Preselection by Metaphase chromosome spread of cDNA clones
In situ hybridization (FIS)
H) is used to provide accurate chromosomal locations in one step.
Can be used. This technique can be as short as 50 or 60 bases.
Can be used with new cDNAs. A review of this technology
And Verma et al., Human Chromoso.
mes: a Manual of Basic Te
chniques, Pergamon Press, N
ew York (1988). Once the sequence maps to the exact chromosomal location
The location of the sequence on the chromosome
It can be correlated with the data in the figure. Such data, for example,
If V. McKusick, Mendelian In
heritance inMan (Johns Hop
kins University Welch Med
available online from ical Library
A) is found in Then, on the same chromosome region
The relationship between the gene being mapped and the disease
Analysis (simultaneous inheritance of physically adjacent genes)
It is. Next, the cD between affected and unaffected individuals
Need to determine differences in NA or genomic sequence
There is. Mutations are observed in some or all affected individuals
If this is not observed in any normal individuals,
Mutations appear to be causative factors of the disease. Physical Mapping Techniques and Gene Mapping
At the current resolution of imaging technology, chromosomal regions associated with disease
The cDNAs located exactly in the region are 50 and 500
May be one of the potential causative genes. (this is,
1 megabase mapping resolution and 20kb warm
One gene). The polypeptide, its fragments and
Is another derivative, or their analog, or it
Cells expressing them produce antibodies against them
Can be used as an immunogen for These antibodies are
For example, polyclonal antibodies or monoclonal antibodies
Can be The present invention also relates to chimeric, single chain,
And humanized antibodies, and Fab fragments, or
Includes the products of the Fab expression library. In the field
Various procedures known in the art may be used to make such antibodies and fragments.
Can be used for the production of The polypeptide corresponding to the sequence of the present invention
Antibodies produced by direct injection of the polypeptide into animals.
Or by administration of the polypeptide to the animal.
Can be done. The animal is preferably a human
Absent. Subsequently, the antibody thus obtained was
Binds to the peptide itself. In this way, this polypep
Even sequences encoding only fragments of
To generate antibodies that bind to the entire natural polypeptide
Can be used. Such antibodies are then
To isolate polypeptides from tissues expressing the peptide
Can be used for For preparation of monoclonal antibodies, cells
Any technique for providing antibodies produced by continuous culture of a strain
Surgery may be used. Examples include hybridoma technology
(Kohler and Milstein, 1975, N
ature, 256: 495-497), trioma
Technology, human B cell hybridoma technology (Kozbor)
Et al., 1983, Immunology Today.
4:72), and produces human monoclonal antibodies
EBV hybridoma technology (Cole et al., 19
85, Monoclonal Antibodies
and CancerTherapy, Alan R.A.
Liss, Inc. , Pp. 77-96). Techniques Described for Producing Single Chain Antibodies
(U.S. Pat. No. 4,946,778) shows the immunity of the invention.
To generate a single-chain antibody to a non-genic polypeptide product
Can be adapted. Also, transgenic mice
Humanized antibodies to immunogenic polypeptide products of the invention
Can be used to express The present invention will be further described with reference to the following examples.
However, the invention is limited to such examples.
It should be understood that not. All parts also
The amounts are by weight unless otherwise specified. To facilitate understanding of the following examples,
Certain methods and / or terms that appear frequently are described.
You. “Plasmids” are preceded by a lower case p and
Indicates capital letters and / or numbers that follow
Is specified by The starting plasmid herein is
It is commercially available, publicly available without restriction, or
Or from available plasmids according to published procedures.
Can be built. In addition, an equivalent of the described plasmid
Plasmids are known in the art and
It is clear to the person. “Digestion” of DNA refers to specific arrangements in the DNA.
Catalytically catalyzes the DNA with a restriction enzyme that acts only in the
Refers to cutting. Various controls used in this specification
Restriction enzymes are commercially available and their reaction conditions,
Cofactors and other requirements are those known to those skilled in the art.
Was used. For analytical purposes, typically 1 μg
Rasmid or DNA fragment contains about 2 units of enzyme
Together with about 20 μl of buffer solution. Plasmi
For the purpose of isolating DNA fragments for constructing
For this purpose, typically 5 to 50 μg of DNA are
Digested in a larger volume with 50 units of enzyme
You. Appropriate buffers and substrate for specific restriction enzymes
Is specified by the manufacturer. About 1 hour at 37 ° C
The incubation time is usually used, but this depends on the supplier
May vary according to instructions. After digestion, the reaction is
Direct electrophoresis on a acrylamide gel to obtain the desired fragment
Isolate. The size separation of the cleavage fragments was performed according to Goe
ddel, D.E. Et al., Nucleic Acids Re
s. , 8: 4057 (1980).
% Polyacrylamide gel. "Oligonucleotide" refers to a single-stranded polynucleotide.
Oxynucleotide or two complementary polydeoxy
Any of the nucleotide chains, which are chemically combined
Can be achieved. Such synthetic oligonucleotides are
No 5 'phosphate and therefore ATP in the presence of kinase
If no phosphoric acid is added with the
Do not connect with Synthetic oligonucleotides are dephosphorylated
Ligate to unfragmented fragments. "Ligation" refers to two double-stranded nucleic acid fragments.
Process to form a phosphodiester bond between
(Maniatis, T et al., Supra, p. 146). other
If not provided, ligation uses known buffers and conditions
And an approximately equimolar amount of the DNA fragment to be ligated.
10 units of T4 DNA ligase per 0.5 μg
("Ligases"). Unless otherwise stated, Graham, F. et al.
And Van der Eb, A .; , Virlog
y, 52: 456-457 (1973).
Transformations were performed as described. [0153] 【Example】Example 1 Expression of recombinant HGBER32 in COS7 cells The expression of the plasmid, HGBER32HA, was
Derived from cDNA3 (Invitrogen). Be
Vector pcDNA3 contains the following: 1) SV40 copy
Origin of origin, 2) ampicillin resistance gene, 3) E. coli. col
i replication origin, 4) polylinker region, SV40 intro
CMV promoter followed by an adenylation and polyadenylation site
- The entire HGBER32 precursor and its 3 'end
DNA encoding the HA tag fused in frame
Fragments into the polylinker region of the vector
It has become. Therefore, recombinant protein expression is
Dominated by the motor. The HA tag was previously described
(Wilson et al., Cell 37:67, 1984).
Such as influenza hemagglutinin protein
Corresponds to pitope. Fusion of HA tag to target protein
Optionally, recombination using an antibody that recognizes the HA epitope
Protein can be easily detected. The plasmid construction strategy is described below.
The DNA sequence encoding HGBER32 (ATC
C accession number No.) using the following two primers
Genomic lambda clones were constructed by PCR:
Immer (5'-ACCAGGATCCGCTGCCTT
GATGGATTAT) (SEQ ID NO: 4) is a BAMHI
Site followed by HGBER32 starting from the start codon
Contains 9 nucleotides of coding sequence; 3 'primer
(SEQ ID NO: 5) 5'-CTGCTTCTAGAATG
CCATTCAAGAAAATGTT is the XbaI part
Position, translation stop codon, and HGBER32 coding sequence
Complementary to 10 nucleotides (not including the stop codon)
Including sequences. Therefore, the PCR product is in the BAMHI section
HGBE followed by a position, a translation stop codon and an XbaI site
Contains the R32 coding sequence. PCR amplified DNA fragment
And the vector (pcDNAI / Amp)
Digest with HI and Xbal restriction enzymes and
Tied. The ligation mixture is coli SURE strain (S
Tratagene Cloning System
s, 11099 North Torrey Pine
s Road, La Jolla, CA 92037
To obtain the transformed culture.
Inoculate phosphorus medium plates and select resistant colonies
did. Plasmid DNA is isolated from the transformant and
The presence of the correct fragment was checked by restriction enzyme analysis.
For expression of the recombinant HGBER32 receptor, CO
S7 cells were subjected to the DEAE-DEXTRAN method (Sambro
Ok et al., Molecular Cloning: A
Laboratory Manual, 2nd edition, Col
d Spring Laboratory Pres
1989).
did. HGBER32 HA protein expression was radiated
(Harlow)
And Lane, Antibodies: A Lab
Oratory Manual, Cold Spring
g Harbor Laboratory Press,
(1988)). Cells were transferred 2 days after transfection
rear,³⁵Labeled with S-cysteine for 8 hours. Then culture
The ground is collected and the cells are washed with detergent (RIPA buffer).
(150 mM NaCl, 1% NP-40, 0.1%
  SDS, 1% NP-40, 0.5% DOC, 50
mM Tris (pH 7.5)).
on et al., ibid., 37: 767 (1984)). Cell lysis
Both product and culture medium are HA specific monoclonal
Precipitated with antibody. 15% of precipitated protein
  Analyzed on SDS-PAGE gel. [0155]Example 2 Cloning of HGBER32 using baculovirus expression system
Learning and expression Copy full length HGBER32 protein
DNA sequence (ATCC Accession No.)
PCR orientations corresponding to the 5 'and 3' sequences of the gene
Amplified using oligonucleotide primers: [0156] Embedded image Having TCAGTG (SEQ ID NO: 6) and BAMHI
Restriction enzyme sites (in bold) followed by eukaryotic cells
18 similar signals that are efficient at initiating translation in
Nucleotides (Kozak, J. Mol. Biol.1
96, 947-950, 1987), and immediately behind
The first 25 nucleotides of this HGBER32 gene
(The translation start codon "ATG" is underlined)
No. [0157] Embedded image And restriction endonuclease XbaI cleavage
Position and the 3 'untranslated sequence of this HGBER32 gene.
Includes four complementary nucleotides. Amplified sequence is commercially available
Kit ("Geneclean" BIO 101
Inc. , LaJolla, Ca. ) Using 1%
Isolated from a galose gel. Then this fragment
Digested with endonucleases BAMHI and XbaI
And then purified as described in Example 1. This hula
This fragment is referred to as F2. The vector pA2 (of the PUL941 vector)
Variant, described below), using H.
Used for GBER32 protein expression (reviewed
In summary, Summers and Smith, 1987,
A Manual of Methods for B
aculovirus Vectors and In
sec Cell Culture Procedure
res, TexasAgricultural Exp
ermental Station Bulleti
n No. 1555, 1987). This departure
The current vector is Autograph Califor
Nica nuclear polyhedrosis virus (AcMNPV)
Polyhedrin promoter followed by restriction endonuclei
Includes recognition sites for the reases BAMHI and XbaI.
Simian virus (SV) 40 polyadenylation site
Is used for efficient polyadenylation. Recombinant c
For easy selection of ills, E. E. coli β
-The galactosidase gene to a polyhedrin promoter
Followed by polyadenylation of the polyhedrin gene.
Insert in the same direction as the signal. Polyhedrin sequence
Cell-mediated nature of transfected wild-type viral DNA
It is flanked on both ends by viral sequences for homologous recombination.
Many other baculovirus vectors replace pA2.
Can be used. For example, pRG1, pAc373,
pVL941 and pAcIM1 (Lucko
w and Summers. Virology, 170:
31-39). The plasmid was replaced with the restriction enzymes BAMHI and X
digest with baI, then pups by procedures known in the art.
Dephosphorylated using bovine intestinal phosphatase. Next
Then, the DNA was replaced with 1% agarose as described in Example 1.
Isolated from sgel. This vector DNA is called V2.
You. The fragment F2 and the dephosphorylated
Rasmid V2 was ligated using T4 DNA ligase
Was. Then, E. E. coli HB101 cells
And a plasmid having the HGBER32 gene (pBa
Bacteria containing c-HGBER32) were identified. clone
The sequence of the fragment was confirmed by DNA sequencing.
Was. 5 μg of plasmid pBac-HGPCR
By the lipofection method (Felgner et al. Pro
c. Natl. Acad. Sci. USA, 84:74
13-7417 (1987)).
A commercially available linearized baculovirus ("BaculoG
old^TM  baculovirus DNA ", Pha
rmingen, San Diego, CA. With
Was co-transfected. 1 μg of BaculoGold^TMVirus
DNA and 5 μg of plasmid pBac-HGBER
32 was added to 50 μl of serum-free Grace's medium (Life
Technologies Inc. , Gaither
sburg, MD) containing microtiter plates
Mix in sterile well. Thereafter, 10 μl of lipofer
Add Kutin and 90 μl Grace medium and mix
And incubated for 15 minutes at room temperature. Next
The transfection mixture without serum.
35 mm tissue culture plate containing 1 ml of Grace's medium
Sf9 insect cells (ATCC CR
L 171l). New plate
The solution was shaken back and forth to mix.
The plate was then incubated for 5 hours at 27 ° C.
Was. After 5 hours, place the transfection solution on a plate
1 ml supplemented with 10% fetal bovine serum
Grace insect medium was added. Incubate plate
Return to beta and continue culturing at 27 ° C. for 4 days. After 4 days, the supernatant was collected and the Summe
rs and Smith (supra)
Workup assay was performed. As a modification, stained blue
"Blue Ga", which allows easy isolation of plaques
l ”(Life Technologies In
c. , Agarose with Gaithersburg)
A gel was used. (Detailed description of “plaque assay”
In addition, Life Technologies In
c. Insect cell culture distributed by Gaithersburg
Nursing and baculovirology user guides (9-10
P.)). Four days after serial dilution, the virus was added to the cells.
And plaque stained blue with Eppendorf
Picked up with pet chips. Next, the recombinant virus
Mug agar in an eppen containing 200 μl of Grace's medium
Resuspended in Dorf tube. Centrifuge the agar briefly
Removed by isolation and containing recombinant baculovirus
Sf9 cells seeded on a 35 mm dish
Used for infection. Four days later, these cultures
Wash supernatants were collected and then stored at 4 ° C. Sf9 cells were supplemented with 10% heat-inactivated FBS.
Grow in filled Grace's medium. Infect cells
Recombinant baculovirus V-HG with severe (MOI) 2
Infected with BER32. After 6 hours, the medium is removed and
SF900 excluding methionine and cysteine
II Medium (Life Technologies In
c. , Gaithersburg). 42
After hours, 5 μCi³⁵S-methionine and 5 μCi
³⁵S-cysteine (Amersham) was added. cell
For an additional 16 hours, after which the cells are centrifuged.
Separated and labeled proteins are collected by SD
Visible by S-PAGE and autoradiography
It has become. [0166]Example 3 Expression pattern of HGBER32 in human tissues Northern blot analysis was performed using HGBER in human tissues.
Performed to determine 32 expression levels. Total cell RNA
Sample, RNAzol^TMB system (Biotecx)
  Laboratories, Inc. 6023 So
uth Loop East, Houston, TX
77033). Each identified chick
Approximately 10 μg of total RNA isolated from
Separate on a loin gel and blot on a nylon filter.
Lot. (Sambrook et al., Supra). Labeling reaction
To the Stratagene Prime-It kit
Therefore, it is performed using 50 ng of the DNA fragment. Mark
Using the Select-G-50 column
(5 Prime-3 Prime, In
c. 5603 Arapahoe Road, Bouul
der, CO 80303). Then, filter
Full length radioactively labeled at 1,000,000 cpm / ml
HGBER32 gene and 0.5M NaPO_Four(PH
7.4) and hive overnight at 65 ° C in 7% SDS
Let it redisize. 2 times at room temperature and 2 times at 60 ° C
Washed with 0.5 × SSC, 0.1% SDS
Afterwards, the filters were then removed using an intensifying screen at -70.
Expose overnight at <RTIgt; Messe for HGBER32
Tanger RNA is abundant in cerebellar tissue. [0167]Example 4 Expression via gene therapy Fibroblasts are obtained from the subject by skin biopsy. Obtained
Tissue is placed in tissue culture medium and divided into small pieces.
You. Small chunks of tissue are removed from tissue culture
Place on a moist surface of a culture flask and add approximately 10 pieces each
Place in a flask. Turn the flask upside down and seal
And leave overnight at room temperature. After 24 hours at room temperature,
Invert the Lasco and secure the tissue mass to the bottom of the flask.
Leave on fresh medium (eg, 10% F
Has BS, penicillin, and streptomycin
Ham's F12 medium) is added. This is then
Incubate for approximately 1 week at ° C. at this point,
Add fresh medium and replace every few days thereafter. Further
After two weeks of culture, a monolayer of fibroblasts appears. Single layer
Trypsinize and scale to larger flask
Together. Moloney murine sarcoma virus long terminal repeat
PMV-7 (Kirschmeier, P .;
T. Et al., DNA, 7: 219-25 (1988)).
Was digested with EcoRI and HindIII and then
Treat with calf intestinal phosphatase. Linear vector
Fractions on an agarose gel and use glass beads.
And purify. CDN encoding the polypeptide of the present invention
A corresponds to the 5 'terminal sequence and the 3' terminal sequence, respectively
Amplification using PCR primers. 5 'primer
Contains an EcoRI site and the 3 'primer is
And a HindIII site. T4 DNA ligase
Of an equivalent amount of Moloney mouse sarcoma virus in the presence of
Skeleton and EcoRI and HindIII fragment
Add the components together. The resulting mixture is divided into two flags
Maintain conditions appropriate for ligation of the components. The concatenated mixture
To transform bacteria HB101, which is then
Vector has the gene of interest inserted properly.
For identification purposes, on agar containing kanamycin
Plating. Amphotropic pA317 or GP
+ Am12 packaging cells with 10% calf serum
(CS), penicillin, and streptomycin
Dulbecco's Modified Eagle's Medium (DMEM)
In tissue culture to a confluent density in
Let them breed. Next, the MSV vector containing the gene was added to the medium.
And packaging cells using the vector
To transduce. At this time, the packaging cells
Produce infectious virions containing the offspring (this
Sometimes, packaging cells are called producer cells
Is). The fresh medium was transformed with the transduced producer.
-Add cells to the cells and then remove the medium
Collect from 10 cm plate of reducer cells. Feeling
Spent media containing infectious virus particles is
Produced after filtration through a pore filter
Sir cells are removed and fibroblasts are then
Infect. Culture medium with subconfluent fibroblasts
Cells from the vesicle plate, and quickly producer
Replace with medium from cells. Remove the medium and remove
And replace with fresh medium. High virus titer
If substantially all fibroblasts are infected, and the selection is
unnecessary. If the titer is very low, select a marker (eg,
IfneoOrhis) Having a retroviral vector
It is necessary to use a monitor. Next, the engineered fibroblasts were isolated alone.
Or cytodex3 microcarrier beads
Any after growing to confluence on
And inject into the host. At this time, fibroblasts
Produce the product. Numerous modifications and variations of the present invention are described above.
It is possible with reference to the teachings and therefore the scope of the appended claims.
Within the bounds, the invention may be practiced other than as specifically described.
obtain. The following drawings are illustrative of embodiments of the present invention.
The present invention as present and as encompassed by the claims
Does not mean limiting the range. [0175] Since the present invention has the above-described structure,
The stated task is achieved.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A shows the cDNA sequence of the HGBER32 G protein-coupled receptor of the present invention (SEQ ID NO: 1)
And the corresponding deduced amino acid sequence (SEQ ID NO: 2). Use the standard one-letter abbreviations for amino acids. FIG. 1B is a cDNA sequence of the HGBER32 G protein-coupled receptor of the present invention (SEQ ID NO: 1).
And the corresponding deduced amino acid sequence (SEQ ID NO: 2). Use the standard one-letter abbreviations for amino acids. FIG. 1C shows the cDNA sequence of the HGBER32 G protein-coupled receptor of the present invention (SEQ ID NO: 1).
And the corresponding deduced amino acid sequence (SEQ ID NO: 2). Use the standard one-letter abbreviations for amino acids. FIG. 1D shows the cDNA sequence of the HGBER32 G protein-coupled receptor of the present invention (SEQ ID NO: 1).
And the corresponding deduced amino acid sequence (SEQ ID NO: 2). Use the standard one-letter abbreviations for amino acids. FIG. 2A shows the secondary structure characteristics of HGBER332 G protein-coupled receptor. The first seven figures describe regions of the amino acid sequence that are α-helices, β-sheets, turn regions, or coil regions. The range of the black square is a region corresponding to the indicated region. The second series shows the range of amino acid sequences that are exposed to the intracellular, cytoplasmic, or transmembrane areas. The hydrophilicity plot shows the region of the protein sequence that is the lipid bilayer of the membrane (and is therefore hydrophobic) and the region outside the lipid bilayer that is hydrophilic. The antigenic index corresponds to the hydrophilicity plot since the antigenic region is the region outside the lipid bilayer membrane and is the area to which antigen can bind. The surface probability plot further corresponds to the antigenic index and the hydrophilicity plot. The amphipathic plot shows regions of the protein sequence that are polar and non-polar. The flexible region corresponding to the second series of figures means that the outside of the membrane is a flexible region and the transmembrane region is a non-flexible region. FIG. 2B shows the secondary structure features of HGBER332 G protein-coupled receptor. The first seven figures describe regions of the amino acid sequence that are α-helices, β-sheets, turn regions, or coil regions. The range of the black square is a region corresponding to the indicated region. The second series shows the range of amino acid sequences that are exposed to the intracellular, cytoplasmic, or transmembrane areas. The hydrophilicity plot shows the region of the protein sequence that is the lipid bilayer of the membrane (and is therefore hydrophobic) and the region outside the lipid bilayer that is hydrophilic. The antigenic index corresponds to the hydrophilicity plot since the antigenic region is the region outside the lipid bilayer membrane and is the area to which antigen can bind. The surface probability plot further corresponds to the antigenic index and the hydrophilicity plot. The amphipathic plot shows regions of the protein sequence that are polar and non-polar. The flexible region corresponding to the second series of figures means that the outside of the membrane is a flexible region and the transmembrane region is a non-flexible region. FIG. 3A shows a G protein-coupled receptor of the present invention starting at amino acid 6 (HGBER32) and a human mononuclear chemotactic tractant starting at amino acid 40 (ch).
2 shows an amino acid alignment with the E. tractattractant) protein 1 receptor (MCP-1b) (SEQ ID NO: 3). Line symbols indicate identical amino acids (4
0.401%), and the dot symbol indicates a similar amino acid (64.470%). FIG. 3B shows a G protein-coupled receptor of the invention starting at amino acid 6 (HGBER32) and a human mononuclear chemotactic tractant starting at amino acid 40 (ch).
2 shows an amino acid alignment with the E. tractattractant) protein 1 receptor (MCP-1b) (SEQ ID NO: 3). Line symbols indicate identical amino acids (4
0.401%), and the dot symbol indicates a similar amino acid (64.470%). FIG. 4 shows the predicted transmembrane domain of HGBER32.

   ────────────────────────────────────────────────── ─── Continuation of front page    (71) Applicant 597018381             9410 Key West Avenue,               Rockville, Marylan             d 20850, United State             s of America (72) Inventor Daniel Earl. Sopet             United States Virginia 22020,             Centerville, Stillfield             Wraith 15050 (72) Inventor Li             United States Maryland 20878,               Geysersburg, Howard Lande             Driving 16125 (72) Inventor Craig A. Rosen             United States Maryland 20878,               Geysersburg, Rolling Hill               Road 22400 (72) Inventor Stephen M. Leuven             United States Maryland 20832,               Olney, Heritage Hills Dora             Eve 18528 F term (reference) 4B024 AA11 BA63 CA04 DA02 EA02                       EA04 FA02 GA11 GA18 HA03                       HA11

Claims (1)

Claims 1. An isolated polynucleotide comprising a member selected from the group consisting of: (a) a polynucleotide encoding the polypeptide of Figure 1; (b) ATCC deposit number (C) capable of hybridizing to the polynucleotide of (a) or (b), and at least 70% capable of hybridizing to the polynucleotide of (a) or (b). A polynucleotide that is the same; and (d) a polynucleotide fragment of the polynucleotide of (a), (b), or (c).