JP2001025391A - Human g protein chemokine receptor hdgnr10 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new polynucleotide which codes for a human G protein chemokine receptor polypeptide having a specific amino acid sequence, and is useful for treating and diagnosing many physiological and disease conditions including immunological diseases and so on. SOLUTION: This is a new polynucleotide which is selected from the group consisting of a polynucleotide coding for the (mature) polypeptide represented by formula I, a polynucleotide containing the base sequence represented by formula II coding for a (mature) human G protein chemokine receptor peptide, a polynucleotide having a homology at 90% or more, 95% or more, or 97% or more, respectively, to these polynucleotides, polynucleotides complimentary to these and so on, and is useful for producing a composition for treating and diagnosing many physiological and disease conditions including immunological diseases such as injury healing, blood formation regulation, allergy, asthma, arthritis and so on. This polynucleotide is obtained by screening a cDNA library derived from a human monocyte.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, and the use of such polynucleotides and polypeptides. For the production of peptides. More specifically, the polypeptide of the present invention is a human 7-transmembrane receptor that has been putatively identified as a chemokine receptor, and will be referred to herein as sometimes "G protein chemokine receptor" or "HDGNR1".
0 ". The invention also relates to inhibiting the action of such polypeptides.

[0002]

BACKGROUND OF THE INVENTION It is well established that a number of medically important biological processes are mediated by proteins involved in signal transduction pathways, including G proteins and / or second messengers (eg, cAMP). (Lefkowitz, Nature, 35
1: 353-354 (1991)). In this specification,
These proteins are referred to as proteins involved in pathways using G proteins or PPG proteins. Some examples of these proteins include GPC receptors (eg, GPC receptors for adrenergic drugs and dopamine (Kobilka, BK et al.
Et al., PNAS, 84: 46-50 (1987); Kob.
ilka, B .; K. Et al., Science, 238: 65.
0-656 (1987); R. Et al.
Nature, 336: 783-787 (198).
8))), the G protein itself, effector proteins (eg, phospholipase C, adenyl cyclase, and phosphodiesterase), and actuator proteins (actuator proteins)
n) (eg, protein kinase A and protein kinase C) (Simon, MI et al., Science)
e, 252: 802-8 (1991)).

For example, in one form of signaling,
The effect of hormone binding is the activation of the enzyme adenylate cyclase in the cell. Activation of enzymes by hormones depends on the presence of the nucleotide GTP, and GT
P also affects hormone binding. The G protein links the hormone receptor to adenylate cyclase. G proteins have been shown to convert GTP to bound GDP when activated by hormone receptors. The form with GTP then binds to activated adenylate cyclase. The hydrolysis of GTP to GDP is catalyzed by the G protein itself,
Return the protein to its basal, inactive form. Thus, the G protein plays two roles: as an intermediate that relays the signal from the receptor to the effector, and as a clock that controls the duration of the signal.

[0004] The membrane protein gene superfamily of G protein-coupled receptors has been characterized as having seven putative transmembrane domains. This domain is thought to represent a transmembrane α-helix connected by an extracellular or cytoplasmic loop. G protein-coupled receptors include a wide range of biologically active receptors such as hormones, viruses, growth factors, and neural receptors.

[0005] G protein-coupled receptors bind about 20 to 3 open-ended hydrophilic loops.
It has been characterized as including these seven conserved hydrophobic ranges of 0 amino acids. The G protein family of coupled receptors includes dopamine receptors that bind to neuroleptic drugs used in the treatment of psychiatric and neurological disorders. Other examples of this family include calcitonin, adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsin, endothelial differentiation gene 1 receptor and rhodopsin, odorants, Including cytomegalovirus receptor.

[0006] G protein-coupled receptors can bind intracellularly to various intracellular enzymes, ion channels and transporters via heterotrimeric G proteins (Johnson et al., Endoc., Rev., 1).
0: 317-331 (1989)). The α subunits of different G proteins preferably stimulate specific effectors to regulate various biological functions in the cell. Phosphorylation of cytoplasmic residues of G protein-coupled receptors has been identified as a key mechanism for the regulation of G protein coupling of some G protein-coupled receptors. G protein-coupled receptors are found at many sites in mammalian hosts.

[0007] Chemokines, also referred to as intercrine cytokines, are a subfamily of structurally and functionally related cytokines. These molecules are 8-10
kd size. Generally, chemokines are 20% ~
It shows 75% amino acid level homology and is characterized by four conserved cysteine residues forming two disulfide bonds. Based on the arrangement of the first two cysteine residues, chemokines can be
Into three subfamilies. In the α subfamily, the first two cysteines are separated by one amino acid and are hereafter referred to as the “CXC” subfamily. In the β subfamily; the two cysteines are in an articulated position and are therefore referred to as the “CC” subfamily. Thus, at least nine different members of this family have been identified in humans.

[0008] Intercrine cytokines exhibit a wide variety of functions. Salient features are monocytes, neutrophils,
The ability to induce chemotactic migration of different cell types, including T lymphocytes, basophils, and fibroblasts. Many cytokines have pro-inflammatory activity and participate in multiple stages during the inflammatory response. These activities include stimulation of histamine release, release of lysosomal enzymes and leukotrienes, increased adhesion of target immune cells to endothelial cells, enhanced complement protein binding, induction of expression of granule cell adhesion molecules and complement receptors, And breathing explosions. In addition to their association in inflammation, it has been suggested that certain chemokines exhibit other activities. For example, macrophage inflammatory protein 1 (MIP-1) can inhibit hematopoietic stem cell proliferation, platelet factor 4 (PF-4) is a potential inhibitor of endothelial cell proliferation, and interleukin 8 (IL-
8) promotes keratinocyte proliferation and GRO is an autocrine growth factor for melanoma cells.

[0009] In a wide range of biological activities, chemokines can be used to traffic lymphocytes,
It is not surprising that it is involved in a number of physiological and disease states, including wound healing, hematopoietic regulation and immunological diseases such as allergy, asthma and arthritis.

[0010]

SUMMARY OF THE INVENTION The present invention provides compositions and methods for diagnosing and / or treating a number of physiological and disease states, including immunological diseases.

[0011]

According to one aspect of the present invention, there is provided a novel mature receptor polypeptide and fragments, analogs and derivatives thereof that are biologically active and useful for diagnosis or therapy. . The receptor polypeptide of the invention is of human origin.

According to another aspect of the present invention there is provided an isolated nucleic acid molecule encoding a receptor polypeptide of the present invention, wherein the nucleic acid molecule comprises mRNA, DNA, cDNA,
Includes genomic DNA, and antisense analogs thereof, and fragments thereof that are biologically active and useful for diagnosis or therapy.

According to a further aspect of the present invention, a recombinant prokaryotic host cell comprising a nucleic acid sequence encoding a receptor polypeptide of the present invention under conditions which promote expression of said polypeptide and subsequent recovery of said polypeptide. Recombinant techniques, including culturing recombinant eukaryotic host cells, and / or provide a process for producing such receptor polypeptides.

[0014] According to yet a further aspect of the present invention, there are provided antibodies against such receptor polypeptides.

According to another aspect of the present invention, there is provided a screening method for a compound which binds to and activates or inhibits activation of the receptor polypeptide of the present invention.

In accordance with yet another embodiment of the present invention, there is provided a step of administering to a host a compound that binds and activates a receptor polypeptide of the present invention. This compound is useful for stimulating hematopoiesis, wound healing, coagulation, angiogenesis, treating solid tumors, chronic infections, leukemias, T-cell mediated autoimmune diseases, parasitic infections, psoriasis, and growth factor activity Stimulates.

According to another aspect of the present invention, a receptor polypeptide of the present invention is mediated by gene therapy to treat conditions associated with underexpression of the polypeptide or underexpression of the ligand of the receptor polypeptide. Methods of administration are provided.

In accordance with yet another embodiment of the present invention, there is provided a step of administering to a host a compound that binds to the receptor polypeptide of the present invention and inhibits its activation. This compound is useful for allergy, atherogenesis, hypersensitivity, malignancy, chronic and acute inflammation, histamine and IgE
Mediated allergic reactions, prostaglandin-independent fever, bone marrow failure, silicosis, sarcomas, rheumatoid arthritis,
Useful for prevention and / or treatment of shock and eosinophilia syndrome.

According to yet another aspect of the present invention there is provided a nucleic acid probe containing a nucleic acid molecule of sufficient length to specifically hybridize to a polynucleotide sequence of the present invention.

According to yet another aspect of the present invention, to detect a disease associated with a mutation in a nucleic acid sequence encoding such a polypeptide, and to detect altered levels of a soluble form of the receptor polypeptide. A diagnostic assay is provided for detecting.

According to a still further aspect of the present invention, such receptor polypeptides, or polynucleotides encoding such polypeptides, may be used for in vitro purposes related to scientific research, DNA synthesis and DNA vector production. A process is provided for utilizing for

These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.

The accompanying drawings are illustrative of embodiments of the invention, and are not meant to limit the scope of the invention, as encompassed by the claims.

[0024]

DETAILED DESCRIPTION OF THE INVENTION Disclosed are human G protein chemokine receptor polypeptides and DNA (RNA) encoding such polypeptides and procedures for producing such polypeptides by recombinant techniques. Methods of using such polypeptides to identify antagonists and agonists to such polypeptides and therapeutics of agonists and antagonists for the treatment of conditions associated with underexpression and overexpression of G protein chemokine receptor polypeptides The various uses are also disclosed. Also disclosed are diagnostic methods for detecting mutations in G protein chemokine receptor nucleic acid sequences and for detecting levels of soluble receptor in a sample derived from a host.

According to an aspect of the present invention, a mature polypeptide having the deduced amino acid sequence of FIGS. 1-3 (SEQ ID NO: 2), or ATCC Accession No. 9 on June 1, 1995.
American Type Cul as No. 7183
cure Collection, 12301 Pa
rkrawn Drive, Rockville, M
aryland, 20852, United St
Provided is an isolated nucleic acid (polynucleotide) encoding a mature polypeptide encoded by the cDNA of the clone deposited in the ates of America.

The polynucleotide of the present invention has been found in a cDNA library derived from human monocytes. It is structurally related to the G protein-coupled receptor family. It contains an open reading frame encoding a protein of 352 amino acid residues. This protein spans a range of 347 amino acids and has 70.1% identity and 82.9% to the human MCP-1 receptor.
Shows the highest degree of homology with similarities.

The polynucleotide of the present invention may be in the form of RNA or DNA (this DNA may be cDNA, genomic DN).
A, and synthetic DNA). DN
A can be double-stranded or single-stranded, and if single-stranded, can be the coding strand or the non-coding (antisense) strand. The coding sequence encoding the mature polypeptide is:
The coding sequence shown in Figures 1-3 (SEQ ID NO: 1) or the coding sequence of the deposited clone may be identical, or the coding sequence may be the result of duplication or degeneracy of the genetic code. It may be a different coding sequence encoding the same mature polypeptide as the DNA of SEQ ID NO: 1) or the deposited cDNA.

The polynucleotide encoding the mature polypeptide of FIGS. 1-3 or the mature polypeptide encoded by the deposited cDNA may include: the coding sequence of the mature polypeptide only; the coding sequence of the mature polypeptide; And additional coding sequences, such as transmembrane (TM) or intracellular domains; the coding sequence of the mature polypeptide (and any additional coding sequences) and non-coding sequences (eg, 5 'and / Or 3 'non-coding sequence).

Thus, the term "polynucleotide encoding a polypeptide" encompasses polynucleotides that include only the coding sequence of the polypeptide, as well as polynucleotides that include additional coding and / or non-coding sequences.

The present invention further provides a polynucleotide as described herein above which encodes a polypeptide having the deduced amino acid sequence of FIGS. 1-3, or fragments, analogs and derivatives of the polypeptide encoded by the cDNA of the deposited clone. To a variant thereof. A variant of a polynucleotide can be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.

Accordingly, the present invention relates to FIGS.
Includes polynucleotides encoding the same mature polypeptide as shown in 2) or the same mature polypeptide encoded by the cDNA of the deposited clone, as well as variants of such polynucleotides. This variant encodes a fragment, derivative or analog of the polypeptide of Figures 1-3 (SEQ ID NO: 2) or the polypeptide encoded by the cDNA of the deposited clone. Such nucleotide variants are deletion variants,
Includes substitution variants and addition or insertion variants.

As indicated herein above, a polynucleotide is a naturally occurring allelic variant of the coding sequence shown in FIGS. 1-3 (SEQ ID NO: 1) or the coding sequence of the deposited clone. It can have a coding sequence. As is known in the art, an allelic variant is another form of a polynucleotide sequence that may have one or more nucleotide substitutions, deletions or additions, which alter the function of the encoded polypeptide. Not substantially changed.

The polynucleotide may also encode a soluble form of the G protein chemokine receptor polypeptide which is the extracellular portion of the polypeptide cleaved from the TM and intracellular domains of the full length polypeptides of the invention.

The polynucleotide of the present invention may also have the coding sequence fused in frame to a marker sequence that allows for purification of the polypeptide of the present invention. The marker sequence may be, in the case of a bacterial host, a hexahistidine tag provided by the pQE-9 vector, which provides for purification of the mature polypeptide fused to the marker. Alternatively, for example, the marker sequence can be a hemagglutinin (HA) tag when a mammalian host (eg, COS-7 cells) is used. The HA tag is consistent with an epitope derived from the influenza hemagglutinin protein (Wilson, I. et al., Cell, 37: 767 (1.
984)).

The term "gene" means a DNA segment involved in the production of a polypeptide chain; this includes regions preceding and following the coding region (leaders and trailers) as well as individual coding segments (exons). Includes intervening sequences (introns).

The fragments of the full-length gene of the present invention can be used to isolate full-length cDNAs and to isolate other cDNAs having high sequence similarity or similar biological activity to this gene. Can be used as a hybridization probe for a cDNA library. Probes of this type preferably have at least 30 bases and may include, for example, 50 or more bases. The probe also provides a cDNA clone corresponding to the full-length transcript, and a genomic clone (s) containing the complete gene including regulatory and promoter regions, exons, and introns.
Can be used to identify Examples of screening include isolating the coding region of a gene by using a known DNA sequence to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to the sequence of the gene of the present invention may be used to determine which member of the library the probe hybridizes with, a human cDNA library,
Used to screen genomic DNA or mRNA.

The present invention further provides that at least 70
%, Preferably at least 90%, and more preferably at least 95% of identity refers to polynucleotides that hybridize to the sequences herein above. The invention particularly relates to polynucleotides that hybridize under stringent conditions to the herein above-described polynucleotides. The term "stringent conditions" as used herein means that hybridization occurs only when there is at least 95%, and preferably at least 97%, identity between the sequences. In a preferred embodiment, a polynucleotide that hybridizes to a polynucleotide as described herein above is substantially the same organism as the mature polypeptide encoded by the cDNA of Figures 1-3 (SEQ ID NO: 1) or the deposited cDNA. Encodes a polypeptide that retains either a biological function or activity.

[0038] Alternatively, the polynucleotide may have at least 20 bases, preferably 30 bases, and more preferably 50 bases. These hybridize to the polynucleotides of the present invention, and have identity thereto, and may or may not retain activity, as described herein above. For example,
Such a polynucleotide may be used, for example, as a probe for the polynucleotide of SEQ ID NO: 1 for recovery of the polynucleotide, or as a diagnostic probe or PC.
Can be used as an R primer.

Thus, the present invention relates to polynucleotides having at least 70%, preferably at least 90%, and more preferably at least 95% identity. This polynucleotide encodes the polynucleotide of SEQ ID NO: 2 as well as fragments thereof, which fragment is at least 30 bases, and preferably 5
0 bases. And the present invention relates to a polynucleotide encoded by such a polynucleotide.

The deposit (s) referred to herein are maintained under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Patent Procedure. These deposits are provided only as a convenience to those of skill in the art and are not an admission that a deposit is required under 35 USC 112. The sequence of the polynucleotide contained in the deposit, as well as the amino acid sequence of the polypeptide encoded thereby, is incorporated herein by reference and controls any inconsistencies with the sequence description herein. . A license may be required to make or sell the deposit, and no such license is granted by this.

The present invention further relates to FIGS.
2) G protein chemokine receptor polypeptides having the deduced amino acid sequence or having the amino acid sequence encoded by the deposited cDNA, and analogs and derivatives of such polypeptides.

The terms "fragment,""derivative," and "analog" when referring to the polypeptides of FIGS. 1-3 or the polypeptide encoded by the deposited cDNA are substantially the same as those polypeptides. Binds a ligand or receptor, even though it retains a functional function or activity (ie, functions as a G protein chemokine receptor) or the polypeptide does not function as a G protein chemokine receptor (eg, a soluble form of this receptor) Or a polypeptide that either retains the ability to do so. Analogs include proproteins that can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.

Fragments, derivatives or analogs of the polypeptides of FIGS. 1-3 (SEQ ID NO: 2) or of the polypeptide encoded by the deposited cDNA comprise (i) 1
One or more amino acid residues are replaced with conserved or non-conserved amino acid residues (preferably conserved amino acid residues), and such substituted amino acid residues are those encoded by the genetic code. May be
Or not, or (ii) one or more amino acid residues contain a substituent, or (iii) a compound wherein the mature polypeptide increases the half-life of the polypeptide (eg, polyethylene Glycol)), or (iv) an additional amino acid is fused to the mature polypeptide for purification of the polypeptide, or (v) a fragment of the polypeptide. It can be soluble (ie, not membrane bound) and still further bind the ligand to a membrane bound receptor. Such fragments, derivatives and analogs are deemed to be within the skill of the art in light of the teachings herein.

The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and are preferably purified to homogeneity.

The polypeptide of the present invention has SEQ ID NO: 2
(Especially the mature polypeptide) and at least 70% similarity (preferably 70%) to the polypeptide of SEQ ID NO: 2.
% Identity), and more preferably 90% similarity (more preferably 90% identity) to the polypeptide of SEQ ID NO: 2, and even more preferably SEQ ID NO: 2
A polypeptide having 95% similarity (even more preferably 90% identity) to a polypeptide of the invention, and generally having such a portion of a polypeptide comprising at least 30 amino acids and more preferably at least 50 amino acids. Such polypeptides include portions of the polypeptides.

As is known in the art, "similarity" between two polypeptides is to compare the amino acid sequence of the polypeptide and conserved amino acid substitutions thereof with the sequence of a second polypeptide. Is determined by

[0048] Fragments or portions of a polypeptide of the present invention can be used to produce the corresponding full-length polypeptide by peptide synthesis. Thus, this fragment can be used as an intermediate to produce a full-length polypeptide. Fragments or portions of a polynucleotide of the invention can be used to synthesize a full-length polynucleotide of the invention.

The term "gene" means a DNA segment involved in the production of a polypeptide chain; the regions preceding and following the coding region ("leaders" and "trailers") and the individual coding segments. Sequences intervening between (exons) (introns)
including.

The term "isolated" means that the material has been removed from its original environment (eg, the natural environment, if it occurs naturally). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is the same polynucleotide that has not been isolated, but has been separated from some or all of the coexisting materials in the natural system. Or the polypeptide
Has been isolated. Such a polynucleotide may be part of a vector, and / or such a polynucleotide or polypeptide may be part of a composition, and such a vector or composition may be part of its natural environment. Because it is not part, it can still be isolated.

The polypeptides of the present invention may comprise the polypeptide of SEQ ID NO: 2 (particularly the mature polypeptide) as well as at least 70% similarity (preferably at least 70% identity) to the polypeptide of SEQ ID NO: 2 More preferably at least 90% of the polypeptide of SEQ ID NO: 2
(More preferably at least 90% identity), and even more preferably a polypeptide having at least 95% similarity (even more preferably at least 95% identity) to the polypeptide of SEQ ID NO: 2 And also generally comprises a portion of such a polypeptide having such a portion of the polypeptide comprising at least 30 amino acids and more preferably at least 50 amino acids.

As is known in the art, "similarity" between two polypeptides is to compare the amino acid sequence of one polypeptide and its conserved amino acid substitutions to the sequence of a second polypeptide. To be determined.

[0053] Fragments or portions of the polypeptides of the present invention can be used to produce the corresponding full-length polypeptide by peptide synthesis. Thus, this fragment can be used as an intermediate to produce a full-length polypeptide. Fragments or portions of a polynucleotide of the invention can be used to synthesize a full-length polynucleotide of the invention.

The present invention also relates to vectors containing the polynucleotides of the present invention, host cells genetically engineered with the vectors of the present invention, and the production of polypeptides of the present invention by recombinant techniques.

The host cell is a vector of the present invention, which comprises
For example, it may be genetically engineered (transduced or transformed or transfected) with a cloning vector or expression vector. Vectors can be, for example, in the form of plasmids, viral particles, phages, and the like. The engineered host cells can be cultured in a conventional nutrient medium suitably activated to activate the promoter, select transformants, or amplify the gene of the invention. Culture conditions (eg, temperature, pH, etc.) are those previously used for the host cell selected for expression, and will be apparent to those skilled in the art.

[0056] The polynucleotides of the present invention can be used to produce polypeptides by recombinant techniques. Thus, for example, a polynucleotide can be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, non-chromosomal and synthetic DNA sequences.
Such vectors include, for example, derivatives of SV40; bacterial plasmids; phage DNA; baculovirus;
Yeast plasmids; vectors derived from combinations of plasmid and phage DNA, viral DNA (eg, vaccinia, adenovirus, fowlpox virus, and pseudorabies). However, any other vector can be used as long as it is replicable and viable in the host.

The appropriate DNA sequence can be inserted into the vector by various procedures. Generally, the DNA sequence is inserted into an appropriate restriction endonuclease site by procedures known in the art. Such and other procedures are deemed to be within the purview of those skilled in the art.

[0058] The DNA sequence in the expression vector is operably linked to an appropriate expression control sequence (promoter).
Directs RNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, E. coli lac or t
rp , lambda phage P _L promoter, and other promoters known to regulate gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. Vectors may also contain appropriate sequences for amplifying expression.

In addition, the expression vector is preferably a phenotypic trait for selection of transformed host cells (eg, dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or E. coli , for example ).
i ) containing one or more selectable marker genes that provide for resistance to tetracycline or ampicillin.

A vector containing a suitable DNA sequence as described herein above and a suitable promoter or control sequence can be used to transform a suitable host to express the protein in the host.

Representative examples of suitable hosts may include: bacterial cells (eg, E. coli , St.
reptomyces, Salmonella typ
Himurium); fungal cells (such as yeast); insect cells (e.g., Drosophila S2 and Spo
doptera Sf9 ); animal cells (eg, CH
O, COS or Bowes melanoma); adenovirus; plant cells and the like. Selection of an appropriate host is considered to be within the skill of the art in light of the teachings herein.

More particularly, the present invention also includes a recombinant construct comprising one or more sequences as broadly described above. Constructs include a vector (eg, a plasmid vector or a viral vector) into which a sequence of the invention is inserted in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises a regulatory sequence operably linked to the sequence, including, for example, a promoter. Numerous suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided as examples. 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 can be used as long as they are replicable and viable in the host.

The promoter region can be selected from any desired gene using a CAT (chloramphenicol transferase) vector or other vector having a selectable marker. Two suitable vectors are P
KK232-8 and PCM7. Particularly well-known bacterial promoters include lacI, lacZ, T3, T3.
7, including gpt, λP _R , P _L and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell (eg, a mammalian cell) or a lower eukaryotic cell (eg, a yeast cell), or the host cell can be a prokaryotic cell (eg, a bacterial cell). . Introduction of the construct into host cells can be accomplished by calcium phosphate transfection, DEAE-dextran mediated transfection, or electroporation (Davis, L., Dibner,
M. Battey, I .; , Basic Method
s in Molecular Biology, 19
86).

The construct in a host cell can be used to produce the gene product encoded by the recombinant sequence in a conventional manner. Alternatively, the polypeptide of the present invention can be produced synthetically by a conventional peptide synthesizer.

Mature proteins include mammalian cells, yeast,
It can be expressed in bacteria, or other cells, under the control of a suitable promoter. The cell-free translation system is also a DNA of the present invention.
RNA from the construct can be used to produce such proteins. Suitable cloning and expression vectors for use in prokaryotic and eukaryotic hosts are described in Sambrook et al., Mol.
eco Cloning: A Laborato
ry Manual, 2nd edition, Cold Spring
Harbor, N .; Y. , (1989), the disclosure of which is incorporated herein by reference.

DNA encoding the polypeptide of the present invention
Is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to about 300 bp, which act on a promoter to increase its transcription. As an example, bp100 to
270, the SV40 enhancer on the late side, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

In general, recombinant expression vectors contain an origin of replication and a selectable marker (eg, the ampicillin resistance gene of E. coli and the TRP1 gene of S. cerevisiae ) that allow transformation of the host cell, as well as downstream structural sequences. Contains a promoter from a highly expressed gene that directs transcription. Such a promoter may be derived from an operon encoding a glycolytic enzyme (eg, 3-phosphoglycerate kinase (PGK)), α-factor, acid phosphatase, or heat shock protein. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably a leader sequence capable of directing secretion of the translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein that includes an N-terminal identification peptide that provides the desired characteristics (eg, stabilization or simplified purification of the expressed recombinant product).

Expression vectors useful for bacterial use are constructed by inserting a structural DNA sequence encoding the desired protein, with an appropriate translation initiation and termination signals, with a functional promoter and operable reading phase. Is done. The vector contains one or more phenotypic selectable markers, and an origin of replication to ensure maintenance of the vector and, if desired, provide for amplification in the host. Suitable prokaryotic hosts for transformation, E. co
li , Bacillus subtilis , Salm
onella typhimurium and Ps
genus Eudomonas, Streptomyces,
And various species within the genus Staphylococcus, but other species may also be used as selections.

As a representative, but non-limiting example, expression vectors useful for bacterial use include selectable markers derived from commercially available plasmids containing the genetic elements of the well-known cloning vector pBR322 (ATCC 37017) and bacterial replication. An origin may be included. Such commercially available vectors include, for example, pKK223-3 (Ph
armasia Fine Chemicals, U
ppsala, Sweden) and GEM1 (Pr
omega Biotec, Madison, W
I, USA). These pBR322 "backbone" portions are combined with an appropriate promoter and the structural sequence to be expressed.

Following transformation of the appropriate host strain and propagation of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (eg, temperature shift or chemical induction), and the cells are transformed. Incubate for an additional period.

The cells are typically collected by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification.

The microbial cells used in protein expression can be disrupted by any convenient method, including freeze-thaw cycles, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well-known to those skilled in the art.

[0074] A variety of mammalian cell culture systems can also be used to express the recombinant protein. Examples of mammalian expression systems include Gluzman, Cell, 23: 1.
75 (1981), C of monkey kidney fibroblasts.
The OS-7 strain and other cell lines capable of expressing compatible vectors (eg, C127, 3T3, CHO, HeL
a, and BHK cell lines). Mammalian expression vectors contain an origin of replication, a suitable promoter and enhancer, and any necessary ribosome binding, polyadenylation, splice donor and splice acceptor sites, transcription termination sequences, and 5 'flanking non-transcribed sequences. contains. DNA sequences derived from the SV40 splice site, and polyadenylation site, can be used to provide the necessary non-transcribed genetic elements.

The G protein chemokine receptor polypeptide can be recovered from the recombinant cell culture and purified by methods including the following. Ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. If necessary, refold the protein (refold
ing) step can be used to complete the configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be used for the final purification step.

The polypeptides of the present invention can be natural, purified products, or products of chemical synthesis procedures, or can be prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants in culture). , Insects, and mammalian cells). Depending on the host used for the recombinant production procedure, the polypeptides of the invention may or may not be glycosylated. The polypeptides of the present invention may also include an initial methionine amino acid residue.

The polynucleotides and polypeptides of the present invention can be used as research reagents and materials for the discovery of treatment and diagnosis for human disease.

The G protein chemokine receptor of the present invention can be used in a process for screening an activating compound (agonist) or an inhibitory compound (antagonist) of the receptor polypeptide of the present invention.

In general, such screening procedures involve the provision of suitable cells that express the receptor polypeptide of the present invention on the cell surface. Such cells can be mammalian, yeast, Drosophila, or E. coli. E. coli-derived cells. In particular, cells are transfected with a polynucleotide encoding a receptor of the invention, thereby expressing a G protein chemokine receptor. The expressed receptor is then bound,
Contact with a test compound to observe stimulation or inhibition of a functional response.

One such screening procedure is:
It involves the use of transfected melanophore cells to express the G protein chemokine receptor of the present invention. Such a screening technique is described in PCT W
O 92/01810 (published February 6, 1992).

Thus, for example, such an assay
It can be used to screen for compounds that inhibit the activation of the receptor polypeptide of the invention by contacting both the receptor ligand and compound to be screened with the melanophore cells encoding the receptor. Inhibition of the signal produced by the ligand indicates that the compound is a potential antagonist of this receptor, ie, inhibits activation of the receptor.

This screen can be used to determine compounds that activate the receptor by contacting such cells with the compound to be screened, and to determine whether such compound produces a signal. Can be used to determine, ie, determine whether such a compound activates the receptor.

Other screening techniques include, for example, Sc
246, pp. 181-296 (October 1989), in a system for measuring extracellular pH changes caused by receptor activation, cells expressing the G protein chemokine receptor (for example, , Transfected CHO cells). For example, a compound can be contacted with a cell that expresses a receptor polypeptide of the invention, and a second messenger response (eg, signal transduction or pH change) determines whether the potential compound activates or inhibits the receptor. It can be measured to determine.

Another such screening technique is G
Introducing RNA encoding the protein chemokine receptor into Xenopus oocytes and transiently expressing the receptor. When screening for a compound that appears to inhibit receptor activation, the receptor oocyte can then be contacted with the receptor ligand and the compound being screened, followed by detection of inhibition or activation of the calcium signal. .

Another screening technique involves the expression of a G protein chemokine receptor whose receptor is linked to phospholipase C or D. Representative examples of such cells can include endothelial cells, smooth muscle cells, fetal kidney cells, and the like. Screening can be accomplished by detection of receptor activation or inhibition of receptor activation by a secondary phospholipase signal, as described herein above.

Another method is to screen for compounds that inhibit the activation of the receptor polypeptide of the antagonist of the present invention by determining the inhibition of the binding of the labeled ligand to cells having the receptor on the cell surface of the labeled ligand. Include. Such methods include transfecting a eukaryotic cell with DNA encoding a G protein chemokine receptor such that the cell expresses the receptor on its surface, and combining the cell with the compound in the presence of a labeled form of a known ligand. Is contacted. The ligand can be labeled, for example, by radioactivity. The amount of the labeled ligand bound to the receptor is measured, for example, by measuring the radioactivity of the receptor. If the compound binds to the receptor as determined by a decrease in labeled ligand that binds to the receptor, the binding of the labeled ligand to the receptor is inhibited.

The antibody may bind to the G protein chemokine receptor of the invention, or in some cases, an oligonucleotide that binds to the G protein chemokine receptor but does not elicit a second messenger response that inhibits the activity of the G protein chemokine receptor. Can antagonize. Antibodies include anti-idiotypic antibodies that generally recognize unique determinants associated with the antigen-binding site of the antibody. Potential antagonist compounds also include proteins closely related to the ligand of the G protein chemokine receptor, ie, fragments of the ligand,
It lacks biological function and does not elicit a response when binding to the G protein chemokine receptor.

[0088] Antisense constructs prepared by use of antisense technology can be used to control triple helix formation or gene expression via antisense DNA or RNA, and both of these methods involve the use of polynucleotides. Based on binding to DNA or RNA. For example, the 5 'coding portion of a polynucleotide sequence encoding a mature polypeptide of the present invention can be used to design an antisense RNA oligonucleotide about 10 to 40 base pairs in length. DNA oligonucleotides are designed to be complementary to the region of the gene involved in transcription (triple helix-Lee et al., Nucl. Acids).
Res. , 6: 3073 (1979); Cooney.
Et al., Science, 241: 456 (1988); and Dervan et al., Science, 251: 136.
0 (1991)), thereby inhibiting transcription and production of G protein chemokine receptors. Antisense RNA oligonucleotides hybridize to mRNA in vivo and block translation of mRNA molecules into G protein-coupled receptors (antisense-Okano, J. Neuroche).
m. , 56: 560 (1991); Oligodeox.
nucleides as Antisense
Inhibitors of Gene Express
session, CRCPress, Boca Rato
n, FL (1988)). The oligonucleotides described above can also be delivered to cells so that antisense RNA or DNA can be expressed in vivo to inhibit the production of G protein chemokine receptors.

Small molecules that bind to G-protein Chemokine Receptors that make it impossible to gain access to ligands that inhibit normal biological activity, such as, for example, small peptides or peptide-like molecules, are also of the present invention. It can be used to inhibit activation of a receptor polypeptide.

[0090] Soluble forms of the G protein chemokine receptor (eg, fragments of the receptor) bind the ligand to a polypeptide of the invention and protect the ligand from interaction with the membrane-bound G protein chemokine receptor to thereby protect the ligand. Can be used to inhibit activation.

The compound that binds to and activates the G protein chemokine receptor of the present invention may be a hematopoietic (h
aematopoiesis), solid tumors, chronic infections, leukemias, to stimulate wound healing, coagulation, angiogenesis
Treating T cell mediated autoimmune diseases, parasitic infections, psoriasis,
And can be used to stimulate the activity of growth factors.

Compounds that bind to and inhibit the G protein chemokine receptors of the present invention include allergies, atherogenesis, hypersensitivity, malignancy, chronic and acute inflammation, histamine and IgE-mediated allergic reactions, prostaglandin-independent It can be used to treat fever, bone marrow failure, silicosis, sarcoidosis, rheumatoid arthritis, shock and eosinophilia syndrome.

The compounds can be used in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the compound, and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.

The present invention also relates to one of the pharmaceutical compositions of the present invention.
There is provided a pharmaceutical pack or kit comprising one or more containers filled with one or more components. For such container (s), a product label may be provided in a form prescribed by a governmental agency that controls the manufacture, use, or sale of the drug or biological product,
Represents an institutional approval for manufacture, use, or sale for human administration. Further, the compounds of the present invention
It can be used in combination with other therapeutic compounds.

Pharmaceutical compositions include, for example, topical, intravenous,
It can be administered in a convenient manner by the intraperitoneal, intramuscular, subcutaneous, intranasal, or intradermal route. The pharmaceutical composition can be administered in an amount effective to treat and / or prevent the specific condition. 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. In most cases, the dosage is from about 10 μg / kg to about 1 mg / kg body weight daily, taking into account the route of administration, symptoms, etc. (CONFIRM DOSAGES).

G-protein Chemokine Receptor polypeptides and antagonists or agonists, which are polypeptides, can also be used in accordance with the present invention by expression of such polypeptides in vivo. This is often called "gene therapy".

Thus, for example, cells from a patient can be engineered ex vivo with a polynucleotide (DNA or RNA) encoding a polypeptide, and the engineered cells can then be treated with the polypeptide to be treated with the polypeptide. Provided to Such methods are well-known in the art. For example, cells can be prepared by using retroviral particles containing RNA encoding a polypeptide of the invention.
It can be operated by procedures known in the art.

Similarly, cells can be engineered in vivo for expression of the polypeptide in vivo, for example, by procedures known in the art. As known in the art,
Producing cells for producing retroviral particles containing RNA encoding a polypeptide of the invention can be administered to a patient for engineering cells in vivo and for expression of the polypeptide in vivo. These and other methods for administering a polypeptide of the invention by such methods will be apparent to those skilled in the art from the teachings of the present invention. For example, an expression vehicle for manipulating cells can be other than a retrovirus (eg, an adenovirus). This can be used to manipulate cells in vivo after being combined with a suitable delivery vehicle.

The retrovirus derived from the retroviral plasmid vector which can be derived as described above includes Moloney murine leukemia virus, spleen necrosis virus, retrovirus (for example, Rous sarcoma virus), Harvey sarcoma virus, chicken leukemia virus, and gibbon. Includes, but is not limited to, leukemia virus, human immunodeficiency virus, adenovirus, spinal cord sarcoma virus, and mammalian tumor virus. In one embodiment, the retroviral plasmid vector is derived from Moloney murine leukemia virus.

[0100] Vectors contain one or more promoters. Suitable promoters that can be used are
Retroviral LTR; SV40 promoter; and Miller et al., Biotechniques, No. 7.
Vol. 9, No. 9, 980-990 (1989), or any other promoter (e.g., histone, pol)
Cell promoters such as, but not limited to, eukaryotic promoters, including, but not limited to, III and β-actin promoters. Other viral promoters that can be used include, but are not limited to, the adenovirus promoter, the thymidine kinase (TK) promoter, and the B19 parvovirus promoter. The selection of an appropriate promoter will be apparent to the skilled artisan from the techniques included herein.

The nucleic acid sequence encoding the polypeptide of the present invention is under the control of a suitable promoter. Suitable promoters that can be used include, but are not limited to, adenovirus promoters, such as the adenovirus major late promoter; also heterologous promoters, such as the cytomegalovirus (CMV) promoter; respiratory syn.
cytial virus (RSV) promoter;
Inducible promoters such as the MMT promoter; metallothionein promoter; heat shock promoter; albumin promoter; ApoAI promoter; human globulin promoter; viral thymidine kinase promoter such as herpes simplex thymidine kinase promoter; Retroviral LTR); β
An actin promoter; and a human growth hormone promoter. The promoter can also be a natural promoter that controls the gene encoding the polypeptide.

[0102] Retroviral plasmid vectors are used to transduce packaging cell lines to form producer cell lines. Examples of packaging cells that can be transfected are PE501, PA31
7, ψ-2, ψ-AM, PA12, T19-14X, V
T-19-17-H2, $ CRE, $ CRIP, GP +
E-86, GP + envAm12, and Miller, Human, which is incorporated herein by reference in its entirety.
Gene Therapy, Vol. 1, pp. 5-14 (1
990), but is not limited thereto. Vectors can transduce packaging cells through any technique known in the art.
Such techniques include, but are not limited to, electroporation, the use of liposomes, and CaPO ₄ precipitation. In one alternative, the retroviral plasmid vector can be encapsulated in liposomes or associated with a lipid and then administered to a host.

The producer cell line produces infectious retroviral vector particles that contain the nucleic acid sequence (s) encoding the polypeptide. Such retroviral vector particles can then be used to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence (s) encoding the polypeptide. Eukaryotic cells that can be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, and hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells. .

The present invention also provides a method for determining whether a ligand that is not known to be capable of binding a G protein chemokine receptor is capable of binding to such a receptor, the method comprising: Contacting a mammalian cell expressing a G protein chemokine receptor with the ligand under conditions that permit binding of the ligand to the G protein chemokine receptor, detecting the presence of the ligand bound to the receptor, and Determining whether it binds to the G protein chemokine receptor. The systems for determining agonists and / or antagonists described herein above can also be used to determine ligand binding to a receptor.

The present invention also relates to the receptor encoding m.
Provided is a method for detecting the expression of a G protein chemokine receptor polypeptide of the present invention on the surface of a cell by detecting the presence of RNA, comprising the steps of obtaining total mRNA from a cell, and obtaining the obtained mRN.
A is at least 10 capable of specifically hybridizing to a sequence contained in the sequence of the nucleic acid molecule encoding the receptor.
Contacting with a nucleic acid probe containing a nucleotide nucleic acid molecule under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of the receptor by the cell.

The present invention also provides a method for identifying a receptor associated with the receptor polypeptide of the present invention. These related receptors may interact with homologous G protein chemokine receptor polypeptides of the invention, by low stringency cross-hybridization, or with related natural or synthetic ligands, and / or with the chemokine receptor of the invention. By identifying a receptor that elicits a similar behavior after genetic or pharmacological blocking of the polypeptide, it can be identified by homology to the G protein chemokine receptor polypeptide of the present invention.

Fragments of a gene may be used as hybridization probes in a cDNA library to isolate other genes having high sequence similarity to the gene of the present invention or having similar biological activity. obtain. Probes of this type are at least 20 bases, preferably at least 30 bases, and most preferably at least 50 bases or more. Probes are also used to identify cDNA clones corresponding to the full-length transcript, and genomic clone (s) containing the complete gene of the invention, including regulatory and promoter regions, exons and introns. obtain. An example of this type of screen is the known DNA for synthesizing oligonucleotide probes.
Isolating the coding region of the gene using the sequence. Labeled oligonucleotides having a sequence complementary to the sequence of the gene of the present invention may be used to determine which members of the library will hybridize with the probe.
A human cDNA, genomic cDNA, or mRNA library is screened and used.

The present invention also contemplates the use of the gene of the present invention as a diagnostic. For example, some diseases are caused by inherited defective genes. These genes can be detected by comparing the sequence of the defective gene with the sequence of a normal gene. Consequently, a "mutated" gene may prove to be associated with abnormal receptor activity. Further, as yet another means for demonstrating and identifying mutations, mutant receptor genes can be expressed using functional assay systems (eg, colorimetric assays in receptor-deficient strains of HEK293 cells, expression on MacConkey plates, complementation). Test) can be inserted into a suitable vector. Once the "mutant" gene has been identified, a population of carriers of the "mutant" receptor gene can then be screened.

An individual having a mutation in the gene of the present invention can be detected at the DNA level by various techniques. Nucleic acids for diagnosis can be obtained from patient cells, including but not limited to blood, urine, saliva, tissue biopsy, and autopsy material. Genomic DNA can be used directly for detection or can be enzymatically amplified by using PCR before analysis (Saiki et al., Nature,
324: 163-166 (1986)). RNA or cDNA can also be used for the same purpose. By way of example, PCR primers complementary to the nucleic acids of the invention can be used to identify and analyze mutations in the gene of the invention. For example, deletions and insertions can be detected by a change in the size of the amplified product in comparison to the normal genotype. Point mutations can be detected by hybridizing the amplified DNA to a radiolabeled RNA of the invention or, alternatively, a radiolabeled antisense DNA sequence of the invention. Perfectly matched sequences can be distinguished from mismatched duplexes by ribonuclease A digestion or by differences in melting temperatures. Such a diagnosis is particularly useful for prenatal testing or neonatal testing.

[0110] Sequence differences between the related gene and the "mutant" gene can be revealed by direct DNA sequencing. In addition, the cloned DNA segment
It can be used as a probe to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For example, sequence primers are used with double-stranded PCR products or single-stranded template molecules generated by modified PCR. Sequencing is performed by conventional methods using radiolabeled nucleotides or by automated sequencing methods using fluorescent tags.

Genetic testing based on DNA sequence differences can be performed on DNA in gels with or without denaturing agents.
This can be achieved by detecting a change in the electrophoretic mobility of the fragment. Sequence changes at specific positions can also be caused by nuclease protection assays (eg, RNase protection and S1 protection or chemical cleavage methods (eg, Cotton)
Et al., PNAS, USA, 85: 4397-4401 (1
985))).

In addition, some diseases are the result of or characterized by changes in gene expression that can be detected by changes in mRNA. Alternatively, the gene of the present invention can be used as a reference to identify individuals who express reduced function associated with this type of receptor.

The present invention also relates to diagnostic assays for detecting altered levels of a soluble form of a G protein chemokine receptor polypeptide of the present invention in various tissues. Assays used to detect levels of soluble receptor polypeptide in a sample derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and preferably ELISA assays. I do.

The ELISA assay involves first preparing an antibody (preferably a monoclonal antibody) specific for the antigen of the G protein chemokine receptor polypeptide. In addition, receptor antibodies are prepared against monoclonal antibodies. Attached to the receptor antibody is a detectable agent, such as, for example, radioactivity, fluorescence, or, in this example, horseradish peroxidase enzyme. The sample is then removed from the host and incubated on a solid support (eg, a polystyrene dish) that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as, for example, bovine serum albumin. The monoclonal antibody is then incubated in the dish, during which time the monoclonal antibody binds to any G protein chemokine receptor protein attached to the polystyrene dish.
Wash any unbound monoclonal antibody with buffer.
The receptor antibody linked to horseradish peroxidase is then placed in the dish, resulting in the binding of the receptor antibody to any monoclonal antibody bound to the G protein chemokine receptor protein. The unbound receptor antibody is then washed. The peroxidase substrate is then added to the dish, and the amount of color that appeared at a given time interval is compared to a standard curve:
It is a measure of the amount of G protein chemokine receptor protein present in a given volume of patient sample.

The sequences of the present invention are also valuable for chromosome identification. This sequence can specifically target a particular location on an individual human chromosome and hybridize to that location. In addition, it is now necessary to identify specific sites on the chromosome. Currently, there are few chromosome labeling reagents based on actual sequence data (repetitive polymorphisms) available for labeling chromosomal locations. The mapping of DNA to chromosomes according to the present invention is an important first step in correlating these sequences with genes for disease.

[0117] Briefly, the sequence was converted from cDNA to P
The chromosome can be mapped by preparing a CR primer (preferably 15-25 bp). Computer analysis of the cDNA is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. Then
These primers are used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.

[0117] PCR mapping of somatic cell hybrids is a rapid procedure for assigning specific DNA to specific chromosomes. Using the present invention with the same oligonucleotide primers, sublocalization is determined using a panel of fragments from a particular chromosome or a pool of large genomic clones in a similar fashion (subloca).
lization) can be achieved. Other mapping schemes that can also be used to map to chromosomes include in situ hybridization, pre-screening on labeled flow-sorted chromosomes, and high-level techniques for constructing chromosome-specific cDNA libraries. Preselection by hybridization is included.

Fluorescence in situ hybridization (FIS) of a cDNA clone to a metaphase chromosome spread
H) can be used to provide a precise chromosomal location in one step. This technique can be used with cDNAs as short as 50 or 60 bases. For a review of this technology,
Verma et al., Human Chromosomes:
a Manual of Basic Techniq
us, Pergamon Press, New Yo
rk (1988).

Once a sequence has been mapped to a precise chromosomal location, the physical location of the sequence on the chromosome can be correlated with genetic map data. Such data is described, for example, in V.A. McKusick, Mendelian In
found in heritance in Man (Joh
ns Hopkins University Wel
ch Online available from the Medical Library). Then, the relationship between the gene mapped to the same chromosomal region and the disease is identified by linkage analysis (simultaneous inheritance of physically adjacent genes).

Next, the cD between affected and unaffected individuals
It is necessary to determine NA or genomic sequence differences. If the mutation is observed in some or all affected individuals but not in normal individuals, then the mutation appears to be a causative agent of the disease.

At the current resolution of physical and genetic mapping techniques, a cDNA correctly located in a chromosomal region associated with a disease may be one of between 50 and 500 potential causative genes . (this is,
Assuming 1 megabase mapping resolution and 1 gene per 20 kb. 2.) The polypeptides, fragments or other derivatives thereof, or analogs thereof, or cells expressing them, can be used as immunogens to raise antibodies against them. These antibodies can be, for example, polyclonal or monoclonal antibodies. The invention also encompasses chimeric, single chain and humanized antibodies, as well as Fab fragments, or the products of a Fab expression library. Various procedures known in the art can be used for the production of such antibodies and fragments.

Antibodies raised against polypeptides corresponding to the sequences of the present invention can be obtained by direct injection of the polypeptide into an animal or by administration of the polypeptide to an animal. The animal is preferably non-human. The antibody thus obtained then binds to the polypeptide itself. In this way, even sequences encoding only fragments of the polypeptide can be used to generate antibodies that bind to the entire native polypeptide.
Such antibodies can then be used to isolate the polypeptide from a tissue expressing the polypeptide.

For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the 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: 7
2), and EBV hybridoma technology for producing human monoclonal antibodies (Cole et al., 1985, M
onclonal Antibodies and
Cancer Therapy, Alan R.A. Lis
s, Inc. , Pp. 77-96).

The techniques described for generating single-chain antibodies (US Pat. No. 4,946,778) can be adapted to generate single-chain antibodies to the immunogenic polypeptide products of the invention. Also, transgenic mice
It can be used to express humanized antibodies against the immunogenic polypeptide products of the invention.

The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts are by weight unless otherwise specified.

To facilitate understanding of the following examples,
Certain methods and / or terms that frequently occur are described.

“Plasmids” are designated by the lower case p preceded and / or followed by capital letters and / or numbers. The starting plasmid herein is
It is commercially available, publicly available without limitation, or can be constructed from available plasmids according to published procedures. In addition, plasmids equivalent to the described plasmids are known in the art, and will usually be apparent to those skilled in the art.

“Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions,
Cofactors and other requirements used were known to those skilled in the art. For analytical purposes, typically for 1 μg of plasmid or DNA fragment, about 2 units of enzyme are used in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5-50 μg of DNA is digested with 20-250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37 ° C. are usually used, but this can vary according to the supplier's instructions. After digestion, the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.

The size separation of the cleaved fragments was performed according to Goe
ddel, D.E. Et al., Nucleic Acids Re
s. , 8: 4057 (1980).
This is performed using a polyacrylamide gel.

“Oligonucleotide” refers to either a single-stranded polydeoxynucleotide or two complementary polydeoxynucleotide chains, which can be chemically synthesized. Such synthetic oligonucleotides are
It has no 5 'phosphate and therefore will not ligate to another oligonucleotide without the addition of phosphate and ATP in the presence of the kinase. Synthetic oligonucleotides ligate to fragments that have not been dephosphorylated.

“Ligation” refers to the process of forming a phosphodiester bond between two double-stranded nucleic acid fragments (Maniatis, T et al., Supra, p. 146). Unless otherwise provided, ligation can be accomplished with known buffers and conditions using approximately equimolar amounts of 10 units of T4 DNA ligase per 0.5 μg of DNA fragment to be ligated (“ligase”). .

Transformations were performed by Graha unless otherwise noted.
m, F. And Van der Eb, A. et al. , Vir
, 52: 456-457 (1973).

[0133]

EXAMPLES Example 1 Bacterial Expression and Purification of HDGNR10 A DNA sequence encoding HDGNR10 (AT
CC Accession No. 97183) to the processed HD
It is first amplified using PCR oligonucleotide primers corresponding to the 5 ′ sequence of the GNR10 protein (excluding the signal peptide sequence) and the vector sequence 3 ′ to the HDGNR10 gene. Additional nucleotides corresponding to HDGNR10 were replaced with 5 ′ and 3 ′, respectively.
Added to the sequence. The 5 'oligonucleotide primer has the sequence 5'CGGAATTCCTCCATGGATT
An 18 nucleotide HDG with ATCAAGTGTCA 3 ', starting from the putative terminal amino acid of the EcoRI restriction enzyme site followed by the processed protein codon
Contains the NR10 coding sequence. 3 'sequence, 5' CGGAA
GCTTTCGTCACAAGCCCCACAGATAT
3 'contains the sequence complementary to the HindIII site, and subsequently contains 18 nucleotides of the coding sequence of HDGNR10. The restriction enzyme site is located on the bacterial expression vector pQE.
-9 (Qiagen, Inc. 9259 Eton
Avenue, Chatsworth, CA, 9131
1) Corresponds to the above restriction enzyme site. pQE-9 is characterized by antibiotic resistance (Amp ^r ), bacterial origin of replication (ori), I
PTG regulatable promoter operator (P / O),
It encodes a ribosome binding site (RBS), a 6-His tag, and a restriction enzyme site. Then, pQE-9 was converted to E
digested with coRI and HindIII. The amplified sequence was ligated into pQE-9 and a histidine tag and RB
Inserted into frame with sequence encoding S. The ligation mixture is then used to, E. coli strain M15 /
rep4 (Qiagen, Inc.) to Sambrook
k, J .; Et al., Molecular Cloning: A
Laboratory Manual, Cold Sp
ring Laboratory Press, (19
89). M15 / re
p4 expresses the lacI repressor and also confers the plasmid pR which confers kanamycin resistance (Kan ^r ).
Includes multiple copies of EP4. Transformants were identified by their ability to grow on LB plates and ampicillin / kanamycin resistant colonies were selected. Plasmid DNA was isolated and confirmed by restriction enzyme analysis.
Clones containing the desired constructs were transferred to Amp (100 μg /
ml) and Kan (25 μg / ml) in liquid culture in LB medium supplemented with both (O / N) overnight. The O / N culture is used to inoculate a large culture at a ratio of 1: 100 to 1: 250. Cells were grown until the optical density (OD ⁶⁰⁰ ) of ⁶⁰⁰ was between 0.4 and 0.6. Then, IPTG ("Isopropyl-BD"
-Thiogalactopyranoside ") to a final concentration of 1 mM. IPTG clears P / O and induces increased gene expression by inactivating the lacI repressor. Cells were grown for an additional 3-4 hours. The cells were then harvested by centrifugation. The cell pellet is treated with the chaotropic agent 6M guanidine HCl.
Solubilized in water. After clearing, solubilized HDGNR10
Was purified from this solution by chromatography on a nickel-chelate column under conditions that bind more tightly to the protein containing the 6-histtag. Hochu
li, E et al. Chromatography 41
1: 177-184 (1984). HDGNR10 to 6
The column was eluted with M guanidine HCl (pH 5.0) and, for regeneration purposes, 3M guanidine HCl, 10 M
It was adjusted to 0 mM sodium phosphate, 10 mM glutathione (reduced form), and 2 mM glutathione (oxidized form). After a 12 hour incubation in this solution, the protein was dialyzed against 10 mM sodium phosphate.

Example 2 Recombinant H in COS Cells
Expression of DGNR10) Expression of plasmid, HDGNR1
0 HA was transferred to the vector pcDNAI / Amp (Invi
trogen). Vector pcDNAI /
Amp contains: 1) SV40 origin of replication, 2).
Ampicillin resistance gene, 3) E. coli. coli origin of replication,
4) CMV promoter followed by a polylinker region, an SV40 intron and a polyadenylation site. Full HD
The DNA fragment encoding the GNR10 precursor and the HA tag fused in-frame to its 3 'end was cloned into the polylinker region of the vector. Therefore, recombinant protein expression is controlled under the CMV promoter. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson, H. Niman, RH
Eighten, A Cherenson, M .; Co
nnoly, and R.M. Lerner, 1984, C
ell 37,767). Fusion of the HA tag to the target protein allows for easy detection of the recombinant protein using an antibody that recognizes the HA epitope.

The plasmid construction strategy is described below: The DNA sequence encoding HDGNR10 (ATC
C. Accession No. 97183) was constructed by PCR using two primers: 5 ′ primer 5 ′ GTC
CAAGCTTGCCACCCATGGATTATCAA
GTGTCA3 'is a HindIII site followed by 18 of the HDGNR10 coding sequence starting at the start codon.
3 'sequence 5' CTAGCTCGA
GTCAAGCGTAGTCTGGGACGTCGTA
TGGGTAGCACAAGCCCCACAGATTATT
TC3 'contains an XhoI site, a translation stop codon, an HA tag, and a sequence complementary to the last 18 nucleotides (excluding the stop codon) of the HDGNR10 coding sequence. Therefore, the PCR product contains a HindIII site, an HDGNR10 coding sequence followed by an in-frame fused HA tag, a translation stop codon adjacent to the HA tag, and an XhoI site. The PCR amplified DNA fragment and the vector (pcDNAI / Amp) were
Digested with III and XhoI restriction enzymes and ligated. The ligation mixture is coli SURE strain (S
Tratagene Cloning System
s, 11099 North Torrey Pines
(Rod, La Jolla, CA 92037), transformed cultures were plated on ampicillin medium plates, and resistant colonies were selected. Plasmid DNA was isolated from transformants and tested for the presence of the correct fragment by restriction analysis. For expression of recombinant HDGNR10, COS cells were transformed into DEA
Transfected with the expression vector by the E-DEXTRAN method (J. Sambrook, E. Fritsc)
h. Maniatis, Molecular Cl
oning: A Laboratory Manua
1, Cold Spring Laboratory
Press, (1989)). HDGNR10 HA protein expression was detected by radiolabeling and immunoprecipitation. (E. Harlow, D. Lane, Ant
ibodies: A Laboratory Manu
al, Cold Spring Harbor Lab
laboratory Press, (1988)). Cells were labeled with ³⁵ S-cysteine for 8 hours two days after transfection. The culture medium is then harvested and the cells are washed with detergent (RIPA buffer (150 mM NaC
1, 1% NP-40, 0.1% SDS, 1% NP-
40, 0.5% DOC, 50 mM Tris (pH
7.5)) (Wilson, I. et al., Ibid.)
37: 767 (1984)). Both cell lysate and culture medium were sedimented with an HA-specific monoclonal antibody. Precipitated protein was purified by 15% SDS-PAG.
Analyzed on E-gel.

Example 3 Cloning and Expression of HDGNR10 Using Baculovirus Expression System The DNA sequence encoding the full-length HDGNR10 protein (A
TCC Accession No. 97183) was amplified using PCR oligonucleotide primers corresponding to the 5 'and 3' sequences of the gene.

The 5 'primer has the sequence 5'CGGGAT
CCCTCCATGGATTATCAAGTGTCA
4 nucleotides with a 3 'and similar signal to the BamHI restriction enzyme site followed by an efficient signal for initiation of translation in eukaryotic cells (J. Mol. Biol. 198).
7, 196, 947-950, Kozak, M .; ), And immediately after it, the first 18 nucleotides of the HDGNR10 gene (the translation initiation codon is "ATG").

The 3 'primer has the sequence 5' CGGGAT
CCCGCTCACAAGCCCCACAGATAT3 '
And comprises a cleavage site for the restriction endonuclease BamHI and 18 nucleotides complementary to the 3 'untranslated sequence of the HDGNR10 gene. The amplified sequence was prepared using a commercially available kit (“Geneclean” BIO 101 In).
c. , La Jolla, Ca. ) Was used to isolate from a 1% agarose gel. The fragment was then digested with the endonuclease BamHI and purified as described above. This fragment is called F2.

The vector pRG1 (a modified version of the pVL941 vector, described below) was transformed into H using the baculovirus expression system
Used for expression of the DGNR10 protein (for a review, see Summers, MD and Smith,
G. FIG. E. FIG. 1987, A manual of meth
ods for baculovirus vector
rs and insect cell culture
e procedures, Texas Agric
ultra ExperimentalStati
on Bulletin No. 1555). This expression vector is Autographac.
Alifornica nuclear polyhedrosis virus (AcMNP)
V) a strong polyhedrin promoter followed by a recognition site for the restriction endonuclease BamHI. The simian virus (SV) 40 polyadenylation site is used for efficient polyadenylation. For easy selection of recombinant viruses, E. coli was used. The β-galactosidase gene from E. coli is inserted in the same direction as the polyhedrin promoter, followed by a polyadenylation signal for the polyhedrin gene. The polyhedrin sequence is flanked at both ends by viral sequences for cell-mediated homologous recombination of co-transfected wild-type viral DNA. Many other baculovirus vectors can be used in place of pRG1. For example, pAc373, pVL941, and p
AcIM1 (Luckow, VA and Su)
mmers, M .; D. , Virology, 170: 3.
1-39).

The plasmid was digested with the restriction enzyme BamHI and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. The DNA was then isolated from a 1% agarose gel as described above. This vector DNA is called V2.

The fragment F2 and the dephosphorylated plasmid V2 were ligated using T4 DNA ligase.
Then, E. E. coli HB101 cells,
Then, a bacterium containing a plasmid having the HDGNR10 gene (pBacHDGNR10) was identified using the enzyme BamHI. The sequence of the cloned fragment is
Confirmed by NA sequencing.

5 μg of plasmid pBacHDGNR1
0 by the lipofection method (Felgner et al., Pro
c. Natl. Acad. Sci. USA, 84:74
13-7417 (1987)), using 1.0 μg of a commercially available linearized baculovirus (“BaculoG”).
old ^TM baculovirus DNA ", Pha
rmingen, San Diego, CA. ) And co-transfected.

1 μg of BaculoGold ^™ viral DNA and 5 μg of plasmid pBacHDGNR1
0 with 50 μl of serum-free Grace's medium (Life
Technologies Inc. , Gaither
sburg, MD) in sterile wells of a microtiter plate. Thereafter, 10 μl Lipofectin and 90 μl Grace's medium were added and mixed,
And incubated at room temperature for 15 minutes. Then
The transfection mixture was transferred to Sf9 insect cells (ATCC CRL 171) seeded in 35 mm tissue culture plates with 1 ml of serum-free Grace's medium.
1) was dropped. The plate was shaken back and forth to mix the newly added solution. The plate was then incubated at 27 ° C. for 5 hours. After 5 hours, the transfection solution was removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum was added. The plate was returned to the incubator and the culture continued at 27 ° C. for 4 days.

After 4 days, the supernatant was collected and the Summe
Plaque assays were performed as described by rs and Smith (supra). As a modification, "Blue Ga" allows easy isolation of blue-stained plaques.
l ”(Life Technologies In
c. Agarose gel with Gaithersburg) was used. (A detailed description of the “plaque assay” is also available from Life Technologies Incorporated.
c. User's Guide for Insect Cell Culture Methods and Baculovirology distributed at Gaithersburg, Inc. (9
10 to 10).

Four days after the serial dilutions of virus were added to the cells, the blue-stained plaques were picked with an Eppendorf pipette tip. The agar containing the recombinant virus was then resuspended in an Eppendorf tube containing 200 μl Grace's medium. The agar was removed by brief centrifugation and the supernatant containing the recombinant baculovirus was added to
It was used to infect Sf9 cells seeded on a 5 mm dish. Four days later, the supernatants of these culture dishes were collected and then stored at 4 ° C.

Sf9 cells were cultured in Grace's medium supplemented with 10% heat-inactivated FBS. Cells were grown at a multiplicity of infection (MOI) of 2 by recombinant baculovirus V-HDGNR.
Infected at 10. After 6 hours, the medium was removed and SF900 I without methionine and cysteine was removed.
I Medium (Life Technologies In
c. , Gaithersburg). 4
After 2 hours, 5 μCi of ³⁵ S-methionine and 5 μCi
³⁵ was added to the S cysteine (Amersham) of. Cells were incubated for an additional 16 hours, after which cells were harvested by centrifugation and labeled proteins were visualized by SDS-PAGE and autoradiography.

Example 4 Expression Through Gene Therapy Fibroblasts were obtained from a subject by skin biopsy. The resulting tissue was placed in tissue culture medium and cut into small sections. Small sections of tissue are placed on the wet surface of a tissue culture flask, and approximately 10 sections are placed in each flask. Rotate the flask up and down, seal tightly and leave at room temperature overnight. After 24 hours at room temperature, the flask is inverted and the tissue section is kept fixed at the bottom of the flask and fresh medium (eg, Ham's F with 10% FBS, penicillin, streptomycin).
12 medium). It is then incubated at 37 ° C. for about one week. At this time, fresh medium is added and subsequently changed every few days. After another two weeks of culture, a monolayer of fibroblasts results. Trypsinize the monolayer and add to a larger flask (scal
e).

PMV-7 (Kirschmeier, F.) adjacent to the long terminal repeat of Moloney murine sarcoma virus.
P. T. DNA, 7: 219-25 (1988)).
Is digested with EcoRI and HindIII and then treated with calf intestinal phosphatase. The linear vector is separated on an agarose gel and purified using glass beads.

CDN encoding the polypeptide of the present invention
A is the corresponding PR of the 5 'and 3' terminal sequences, respectively.
Amplify using C primer. The 5 'primer is E
contains a coRI site and the 3 'primer is Hi
Contains an ndIII site. Equal amounts of Moloney mouse sarcoma virus linear scaffold, and EcoRI and Hin
The dIII fragments are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The concatenated mixture,
Used for transformation of the bacterium HB101, which is then plated on an agar plate containing kanamycin for the purpose of confirming that the vector has the gene of interest properly inserted.

Amphoteric pA317 or GP + am12
The packaging cells are grown to confluent density in tissue culture in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the medium, and the packaging cells are transduced with the vector. At this point, the packaging cells produce infectious viral particles that contain the gene (at this point, the packaging cells are called producer cells).

Fresh medium was added to the transduced producer cells, followed by 10c of confluent producer cells.
Collect medium from plate. The spent medium containing the infectious virus particles is then transferred to Millipore
e) Filter through a filter to remove separated producer cells and then infect fibroblasts with this medium. The medium is removed from the subconfluent plate of fibroblasts and quickly replaced with the medium from the producer cells. Remove this medium,
Then replace with fresh medium. If the virus titer is high, virtually all fibroblasts are infected and selection is not required. If the titer is very low, it is necessary to use a retroviral vector with a selectable marker (eg, neo , or his ).

Next, the engineered fibroblasts were used alone,
Alternatively, they are injected into the host either after confluent growth on Cytodex 3 microcarrier beads. Thereafter, the fibroblasts produce a protein product.

Many modifications and variations of the present invention are possible in light of the above teachings, and, unless otherwise indicated, the invention may be practiced within the scope of the appended claims.

[0154]

In accordance with the present invention, there is a wide range of biological activities involving chemokines, lymphocyte trafficking, wound healing, hematopoietic regulation and immunological diseases such as allergy, asthma and arthritis. Treats a number of physiological and disease states, including
Or compositions and methods for diagnosing are provided.

[0155]

[Sequence List] SEQUENCE LISTING <110> HUMAN GENOME SCIENCES, INC. <120> HUMAN G-PROTEIN CHEMOKINE RECEPTOR HDGNR10 <130> F100959C05 <140> <141> <160> 9 <170> PatentIn Ver. 2.1 <210> 1 <211> 1414 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (259) .. (1314) <400> gtagacatct atgtaggcaa ttaaaaacct attgatgtat aaaacagttt 180 gcattcatgg agggcaacta aatacattct aggactttat aaaagatcac tttttattta 240 tgcacagggt ggaacaag atg gat tat caa gtg tca agt cca atc Tat Asc tc gat tat gac tc gat tat gac tc gat tat gac tat gac tat gac tc gat tat gac tc gat tat gac gt g tat gac tc gat tat gac rt cca aaa atc aat gtg aag caa 339 Ile Asn Tyr Tyr Thr Ser Glu Pro Cys Pro Lys Ile Asn Val Lys Gln 15 20 25 atc gca gcc cgc ctc ctg cct ccg ctc tac tca ctg gtg ttc atc ttt 387 Ile Ala Ala Arg Pro Pro Leu Tyr Ser Leu Val Phe Ile Phe 30 35 40 ggt ttt gtg ggc aac atg ctg gtc atc ctc atc ctg at a aac tgc caa 435 Gly Phe Val Gly Asn Met Leu Val Ile Leu Ile Leu Ile Asn Cys Gln 45 50 55 agg ctg gag agc atg act gac atc tac ctg ctc aac ctg gcc atc tct 483 Arg Leu Glu Ser Met Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile Ser 60 65 70 75 gac ctg ttt ttc ctt ctt act gtc ccc ttc tgg gct cac tat gct gcc 531 Asp Leu Phe Phe Leu Leu Thr Val Pro Phe Trp Ala His Tyr Ala Ala 80 85 90 gcc cag tgg gac ttt gga aat aca atg tgt caa ctc ttg aca ggg ctc 579 Ala Gln Trp Asp Phe Gly Asn Thr Met Cys Gln Leu Leu Thr Gly Leu 95 100 105 tat ttt ata ggc ttc ttc tct gga atc ttc ttc atc atc atc atc atc atc atc atc atc atc atc atc atc atc atc atc atc atg atc Tyr Phe Ile Gly Phe Phe Ser Gly Ile Phe Phe Ile Ile Leu Leu Thr 110 115 120 atc gat agg tac ctg gct atc gtc cat gct gtg ttt gct tta aaa gcc 675 Ile Asp Arg Tyr Leu Ala Ile Val His Ala Val Phe Ala Leu Lys Ala 125 130 135 agg acg gtc acc ttt ggg gtg gtg aca agt gtg atc act tgg gtg gtg 723 Arg Thr Val Thr Phe Gly Val Val Thr Ser Val Ile Thr Trp Val Val 140 145 150 155 gct gtg ttt gcg tct ctc cca gga atc atc ttt a cc aga tct caa aaa 771 Ala Val Phe Ala Ser Leu Pro Gly Ile Ile Phe Thr Arg Ser Gln Lys 160 165 170 gaa ggt ctt cat tac acc tgc agc tct cat ttt cca tac agt cag tat 819 Glu Gly Leu His Tyr Thr Cys Ser Ser His Phe Pro Tyr Ser Gln Tyr 175 180 185 caa ttc tgg aag aat ttc cag aca tta aag ata gtc atc ttg ggg ctg 867 Gln Phe Trp Lys Asn Phe Gln Thr Leu Lys Ile Val Ile Leu Gly Leu 190 195 200 gtc ctg ctg ctt gtc atg gtc atc tgc tac tcg gga atc cta aaa 915 Val Leu Pro Leu Leu Val Met Val Ile Cys Tyr Ser Gly Ile Leu Lys 205 210 215 act ctg ctt cgg tgt cga aat gag aag aag agg cac agg gct gtg agg 96 Thr Leu Leu Arg Cys Arg Asn Glu Lys Lys Arg His Arg Ala Val Arg 220 225 230 235 ctt atc ttc acc atc atg att gtt tat ttt ctc ttc tgg gct ccc tac 1011 Leu Ile Phe Thr Ile Met Ile Val Tyr Phe Leu Phe Trp Ala Pro Tyr 240 245 250 aac att gtc ctt ctc ctg aac acc ttc cag gaa ttc ttt ggc ctg aat 1059 Asn Ile Val Leu Leu Leu Asn Thr Phe Gln Glu Phe Phe Gly Leu Asn 255 260 265 aat tgc agt agcttc gac caa gct atg cag gtg aca gag 1107 Asn Cys Ser Ser Ser Asn Arg Leu Asp Gln Ala Met Gln Val Thr Glu 270 275 280 act ctt ggg atg acg cac tgc tgc atc aac ccc atc atc tat gcc ttt 1155 Thr Leu Gly Met Thr His Cys Cys Ile Asn Pro Ile Ile Tyr Ala Phe 285 290 295 gtc ggg gag aag ttc aga aac tac ctc tta gtc ttc ttc caa aag cac 1203 Val Gly Glu Lys Phe Arg Asn Tyr Leu Leu Val Phe Phe Gln Lys His 300 300 315 att gcc aaa cgc ttc tgc aaa tgc tgt tct att ttc cag caa gag gct 1251 Ile Ala Lys Arg Phe Cys Lys Cys Cys Ser Ile Phe Gln Gln Glu Ala 320 325 330 ccc gag cga gca agc tca gtt tac acc cga tcc gag cag gaa 1299 Pro Glu Arg Ala Ser Ser Val Tyr Thr Arg Ser Thr Gly Glu Gln Glu 335 340 345 ata tct gtg ggc ttg tgacacggac tcaagtgggc tggtgaccca gtcagagttg 1354 Ile Ser Val Gly Leu 350 tgcacatgggtggtggtggtgg14gg <211> 352 <212> PRT <213> Homo sapiens <400> 2 Met Asp Tyr Gln Val Ser Ser Pro Ile Tyr Asp Ile Asn Tyr Tyr Thr 1 5 10 15 Ser Glu P ro Cys Pro Lys Ile Asn Val Lys Gln Ile Ala Ala Arg Leu 20 25 30 Leu Pro Pro Leu Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gly Asn 35 40 45 Met Leu Val Ile Leu Ile Leu Ile Asn Cys Gln Arg Leu Glu Ser Met 50 55 60 Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile Ser Asp Leu Phe Phe Leu 65 70 75 80 Leu Thr Val Pro Phe Trp Ala His Tyr Ala Ala Ala Gln Trp Asp Phe 85 90 95 Gly Asn Thr Met Cys Gln Leu Leu Thr Gly Leu Tyr Phe Ile Gly Phe 100 105 110 Phe Ser Gly Ile Phe Phe Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu 115 120 125 Ala Ile Val His Ala Val Phe Ala Leu Lys Ala Arg Thr Val Thr Phe 130 135 140 Gly Val Val Thr Ser Val Ile Thr Trp Val Val Ala Val Phe Ala Ser 145 150 155 160 Leu Pro Gly Ile Ile Phe Thr Arg Ser Gln Lys Glu Gly Leu His Tyr 165 170 175 Thr Cys Ser Ser His Phe Pro Tyr Ser Gln Tyr Gln Phe Trp Lys Asn 180 185 190 Phe Gln Thr Leu Lys Ile Val Ile Leu Gly Leu Val Leu Pro Leu Leu 195 200 205 Val Met Val Ile Cys Tyr Ser Gly Ile Leu Lys Thr Leu Leu Arg Cys 210 215 220 Arg Asn Glu Lys Lys Arg His Arg Ala Val Arg Leu Ile Phe Thr Ile 225 230 235 240 Met Ile Val Tyr Phe Leu Phe Trp Ala Pro Tyr Asn Ile Val Leu Leu 245 250 255 Leu Asn Thr Phe Gln Glu Phe Phe Gly Leu Asn Asn Cys Ser Ser Ser 260 265 270 Asn Arg Leu Asp Gln Ala Met Gln Val Thr Glu Thr Leu Gly Met Thr 275 280 285 His Cys Cys Ile Asn Pro Ile Ile Tyr Ala Phe Val Gly Glu Lys Phe 290 295 300 Arg Asn Tyr Leu Leu Val Phe Phe Gln Lys His Ile Ala Lys Arg Phe 305 310 315 320 Cys Lys Cys Cys Ser Ile Phe Gln Gln Glu Ala Pro Glu Arg Ala Ser 325 330 335 Ser Val Tyr Thr Arg Ser Thr Gly Glu Gln Glu Ile Ser Val Gly Leu 340 345 350 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer sequence <400> 3 cggaattcct ccatggatta tcaagtgtca 30 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer sequence <400> 4 cggaagcttc gtcacaagcc cacagatat 29 <210> 5 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Se quence: primer sequence <400> 5 gtccaagctt gccaccatgg attatcaagt gtca 34 <210> 6 <211> 61 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer sequence <400> 6 ctagctcgag tcaagcgtag tctgggacgt cgtatgggta gcacaagccc acagatattt 60 c 61 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer sequence <400> 7 cgggatccct ccatggatta tcaagtgtca 30 <210> 8 <211 > 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer sequence <400> 8 cgggatcccg ctcacaagcc cacagatat 29 <210> 9 <211> 344 <212> PRT <213> Homo sapiens < 400> 9 Glu Glu Val Thr Thr Phe Phe Asp Tyr Asp Tyr Gly Ala Pro Cys His 1 5 10 15 Lys Phe Asp Val Lys Gln Ile Gly Ala Gln Leu Leu Pro Pro Leu Tyr 20 25 30 Ser Leu Val Phe Ile Phe Gly Phe Val Gly Asn Met Leu Val Val Leu 35 40 45 Ile Leu Ile Asn Cys Lys Lys Leu Lys Cys Leu Thr Asp Ile Tyr Leu 50 55 60 Leu Asn Leu Ala Ile Ser Asp Leu Leu Phe Leu Ile Thr Leu Pro Le u 65 70 75 80 Trp Ala His Ser Ala Ala Asn Glu Trp Val Phe Gly Asn Ala Met Cys 85 90 95 Lys Leu Phe Thr Gly Leu Tyr His Ile Gly Tyr Phe Gly Gly Ile Phe 100 105 110 Phe Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu Ala Ile Val His Ala 115 120 125 Val Phe Ala Leu Lys Ala Arg Thr Val Thr Phe Gly Val Val Thr Ser 130 135 140 Val Ile Thr Trp Leu Val Ala Val Phe Ala Ser Val Pro Gly Ile Ile 145 150 155 160 Phe Thr Lys Cys Gln Lys Glu Asp Ser Val Tyr Val Cys Gly Pro Tyr 165 170 175 Phe Pro Arg Gly Trp Asn Asn Phe His Thr Ile Met Arg Asn Ile Leu 180 185 190 Gly Leu Val Leu Pro Leu Leu Ile Met Val Ile Cys Tyr Ser Gly Ile 195 200 205 Leu Lys Thr Leu Leu Arg Cys Arg Asn Glu Lys Lys Arg His Arg Ala 210 215 220 Val Arg Val Ile Phe Thr Ile Met Ile Val Tyr Phe Leu Phe Trp Thr 225 230 235 240 Pro Tyr Asn Ile Val Ile Leu Leu Asn Thr Phe Gln Glu Phe Phe Gly 245 250 255 Leu Ser Asn Cys Glu Ser Thr Ser Gln Leu Asp Gln Ala Thr Gln Val 260 265 270 Thr Glu Thr Leu Gly Met Thr His Cys Cys Ile Asn Pro Ile Ile Tyr 275 280 285 Ala Phe Val Gly Glu Lys Phe Arg Ser Leu Phe His Ile Ala Leu Gly 290 295 300 Cys Arg Ile Ala Pro Leu Gln Lys Pro Val Cys Gly Gly Pro Gly Val 305 310 315 320 Arg Pro Gly Lys Asn Val Lys Val Thr Thr Gln Gly Leu Leu Asp Gly 325 330 335 Arg Gly Lys Gly Lys Ser Ile Gly 340

[Brief description of the drawings]

FIG. 1 cDN of G protein-coupled receptor of the present invention
FIG. 2 shows the A sequence and the corresponding deduced amino acid sequence. Use the standard one-letter abbreviations for amino acids. Sequencing was performed using a 373 automated DNA sequencer (Applied).
Biosystems, Inc. ).

FIG. 2 is a continuation of FIG. 1;

FIG. 3 is a continuation of FIG. 2;

FIG. 4 is a diagram illustrating an alignment of amino acid sequences between a G protein chemokine receptor of the present invention and a human MCP-1 receptor.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) A61P 17/02 A61P 17/02 19/02 19/02 37/08 37/08 C12P 21/02 C12P 21 / 02 C (C12N 15/09 ZNA C12R 1:91) (71) Applicant 597018381 9410 Key West Avenue, Rockville, Maryland 20850, United States of America (72) Inventor Ili United States of America Maryland 20878, Guy , Howard Landing Drive 16125 (72) Inventor Stephen M. Leuven United States Maryland 20832, Olney, Heritage Hills Drive 18528

Claims (1)

[Claims] 1. A polynucleotide comprising a member selected from the group consisting of: (a) a polynucleotide encoding the polypeptide shown in SEQ ID NO: 2; (b) encoding a mature polypeptide shown in SEQ ID NO: 2 (C) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1, encoding a human G protein chemokine receptor polypeptide; (d) SEQ ID NO: encoding a mature human G protein chemokine receptor polypeptide (E) a polynucleotide comprising a nucleotide sequence that is at least 90% identical to the encoded amino acid sequence of (a) or (b); (f) (a) or At least 95% identical to the encoded amino acid sequence of (b) (G) a polynucleotide that is at least 90% identical to the polynucleotide of (a), (b), (c), or (d); (h) (a), (b) A) a polynucleotide that is at least 95% identical to the polynucleotide of (c) or (d); (i) at least 97% of the polynucleotide of (a), (b), (c), or (d) (J) a polynucleotide that encodes at least 50 contiguous amino acids of SEQ ID NO: 2; and (k) (a), (b), (c), (d), (e) ,
A polynucleotide which is the complement of (f), (g), (h), (i), or (j).