JP2002517195A - G protein-coupled receptor named 2871 receptor - Google Patents

Abstract

  (57) [Summary] The present invention relates to newly identified G protein-coupled receptors. The present invention also relates to polynucleotides encoding such receptors. The invention further relates to methods of using receptor polypeptides and receptor polynucleotides for the diagnosis and treatment of receptor-mediated diseases. The invention further relates to methods of using such receptor polypeptides and polynucleotides to identify agonists and antagonists useful for diagnosis and treatment. The invention further includes agonists and antagonists based on such receptor polypeptides and polynucleotides. The invention further relates to procedures for producing such receptor polypeptides and polynucleotides by recombinant methods.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to newly identified receptors of the G protein-coupled receptor superfamily. The present invention also relates to polynucleotides encoding such receptors. The invention further relates to various methods of using the polypeptides and polynucleotides of the receptor, which can be applied in the diagnosis and treatment of diseases mediated by the receptor. The invention further relates to drug screening methods that use such receptor polypeptides and polynucleotides to identify agonists and antagonists, which can be applied in diagnosis and treatment. The invention further encompasses agonists and antagonists based on such receptor polypeptides and polynucleotides. The invention further relates to procedures for producing such receptor polypeptides and polynucleotides by recombinant methods.

BACKGROUND OF THE INVENTION G Protein-Coupled Receptors G-protein coupled receptors (GPCRs) constitute a major class of proteins responsible for intracellular signal transduction. GP
CR has seven transmembrane domains. When the ligand binds to the extracellular portion of the GPCR, a signal is transmitted within the cell that results in a change in the biological or physiological properties of the cell. GPCRs are composed of G proteins and effectors (
It is a component of a modular signal transduction system that links the state of a second messenger in a cell to extracellular stimuli together with intracellular enzymes and channels regulated by G proteins.

[0003] GPCR genes and gene products are potential causative agents of disease (Spie).
gel et al. Clin. Invest. 92; 1119 to 1125 (1993)
); McKusick et al. Med. Genet. 30: 1 to 26 (1993)
)). Certain defects in the rhodopsin gene and the V2 vasopressin receptor gene are associated with various forms of retinitis pigmentosa (Nathans et al., Annu. Rev.
Genet. 26: 403-424 (1992)), renal diabetes insipidus (Holtzm).
an, Hum. Mol. Genet. 2: 1201-1204 (1993))
It has been shown to cause These receptors are clinically important for both central nervous system and peripheral physiological processes. Evolutionary analysis suggests that the ancestors of these proteins originally evolved in concert with a complex system and nervous system.

[0004] The GPCR protein superfamily is represented by five families, namely the family I, rhodopsin and β-2-adrenergic receptors, and is currently represented by over 200 characteristic receptors (Dohlman et al., Annu Rev. Biochem. 60: 653-68.
8 (1991)); Family II, parathyroid hormone / calcitonin / secretin receptor family (Juppner et al., Science, 254: 10).
24-1026 (1991); Lin et al., Science, 254: 1022-
1024 (1991)); Family III, metabotropic glutamate receptor family (Nakanishi, Science, 258, 597: 603 (1)
992)); Family IV, D.C. The CAMP receptor family important in discoideum chemotaxis and development (Klein et al., Science,
241: 1467-1472 (1988)); and fungal conjugative pheromone receptors such as Family V, STE2 (Kurjan, Annu. Rev. B).
iochem. 61: 1097-1129 (1992)).

[0005] There are also a small number of other proteins that display seven hypothetical hydrophobic segments and are not thought to be related to GPCRs. However, they have been shown not to couple to G proteins. Drosophila is a protein specific to photoreceptors, such as blind of sevenless (boss),
Expresses a protein of seven transmembrane segments that has been extensively studied but has not been proven to be a GPCR (Hart et al., Proc. Natl.
. Acad. Sci. ScL USA, 90: 5047-5051 (1993)). Dr
The Osophila gene frizzled (fz) is also believed to be a protein with seven transmembrane segments. Like boss, fz is
It has been shown that it is not coupled to G proteins (Vinson et al., N
atature, 338: 263-264 (1989)).

[0006] G proteins represent a family of heterotrimers composed of α, β and γ subunits that bind guanine nucleotides.
These proteins usually bind to cell surface receptors, for example, receptors containing seven transmembrane domains. After the ligand binds to the GPCR, the conformational change is transmitted to the G protein, which causes the α subunit to displace the bound GDP molecule for a GTP molecule and dissociate from the βγ subunit. GTP-linked forms of the α subunit typically function as effector regulatory components that result in the production of second messengers such as cAMP (eg, by activation of adenyl cyclase), diacylglycerol or inositol phosphates. . More than 20 different types of α subunits are known in humans. These subunits associate with a smaller population of β and γ subunits. Examples of mammalian G proteins include:
Gi, Go, Gq, Gs and Gt are included. G protein is Lodish
Et al., Molecular Cell Biology (Scientific
American Books Inc. New York, N .; Y. , 19
95) (the contents of which are incorporated herein by reference).

[0007] GPCRs are major targets for drug action and drug development. Thus, identifying and characterizing previously unknown GPCRs is more valuable in the area of pharmaceutical development. The present invention advances the state of the art by providing a previously unknown human GPCR.

SUMMARY OF THE INVENTION It is an object of the present invention to identify a novel GPCR receptor.

[0009] It is a further object of the present invention to provide novel G proteins useful as reagents or targets in receptor assays that can be applied in the treatment and diagnosis of GPCR-mediated diseases.
It is to provide a PCR receptor polypeptide.

[0010] A further object of the present invention is to provide novel G proteins useful as reagents or targets in receptor assays that can be applied in the treatment and diagnosis of GPCR-mediated diseases.
An object of the present invention is to provide a polynucleotide corresponding to a PCR receptor polypeptide, which is useful for producing a novel receptor polypeptide by a recombinant method.

A particular object of the present invention is to identify compounds that act as agonists and antagonists and modulate receptor expression. It is a further particular object of the present invention to provide compounds that modulate receptor expression for the treatment and diagnosis of GPCR-related diseases.

[0012] Accordingly, the present invention is based on the identification of a novel GPCR called the 2871 receptor.

[0013] The present invention relates to an amino acid sequence represented by SEQ ID NO: 1, or Year Month ATC on the day
C Deposit No. No. 2871 comprising a polypeptide having the amino acid sequence encoded by the deposited cDNA ("deposited cDNA").
A receptor polypeptide is provided.

[0014] The invention also provides an isolated 2871 receptor nucleic acid molecule having the sequence set forth in SEQ ID NO: 2 or the sequence of the deposited cDNA.

[0015] The present invention also relates to the amino acid sequence shown in SEQ ID NO: 1 or the deposited cDNA.
A variant polypeptide having an amino acid sequence substantially homologous to the amino acid sequence encoded by

The present invention also relates to the nucleotide sequence shown in SEQ ID NO: 2 or the deposited cD
A variant nucleic acid sequence substantially homologous to the nucleotide sequence of NA is provided.

The invention also provides fragments of the polypeptide shown in SEQ ID NO: 1 and nucleotides shown in SEQ ID NO: 2, and substantially homologous fragments of such polypeptides or nucleic acids.

The present invention also provides vectors and host cells, particularly recombinant vectors and host cells, for expressing such receptor nucleic acid molecules and polypeptides.

The invention also provides methods for making such vectors and host cells, and methods for using them to produce such receptor nucleic acid molecules and polypeptides.

[0020] The invention also provides antibodies that selectively bind to such receptor polypeptides and fragments.

The present invention also provides a screening method for a compound that modulates the activity of such a receptor polypeptide. Regulation may be at the level of a polypeptide receptor, or may be at the level of controlling expression of a nucleic acid that expresses the receptor polypeptide.

The invention also provides a method for modulating the activity of a receptor polypeptide, comprising treating a condition associated with expression of the receptor polypeptide, particularly using the screened compound.

The invention also provides diagnostic assays that measure the presence and level of such receptor polypeptides or nucleic acid molecules in a biological sample.

The invention also provides diagnostic assays for determining the presence of a mutation in such a receptor polypeptide or nucleic acid molecule.

DETAILED DESCRIPTION OF THE INVENTION Receptor Function / Signal Pathway The 2871 receptor protein is a GPCR involved in the signal pathway.
As used herein, "signaling pathway" refers to the modulation (eg, stimulation or inhibition) of cell function / activity upon binding of a ligand to a GPCR (2871 protein). Examples of such functions include, for example, phosphatidylinositol 4,5-diphosphate (PIP
₂ ) recruitment of intracellular molecules involved in signal transduction pathways, such as inositol 1,4,5-triphosphate (IP ₃ ) or adenylate cyclase; polarization of plasma membrane; production or secretion of molecules; Changes in structure; cell proliferation such as DNA synthesis; cell migration; cell differentiation; and cell survival. Since the 2871 receptor protein is expressed in the prostate, uterus, placenta, and other tissues as disclosed herein,
Cells involved in the 71 receptor protein signaling pathway include, but are not limited to, cells obtained from these tissues.

Depending on the type of cell, the response mediated by the receptor protein may be different.
For example, in some cells, binding of a ligand to a receptor protein stimulates activities such as compound release, channel gating, cell adhesion, migration, differentiation, etc. through the metabolism and turnover of phosphatidylinositol or cyclic AMP. In other cells, ligand binding may produce different results. Regardless of the cellular activity / response regulated by the receptor protein, the protein is a GPCR,
Interacting with G proteins to generate one or more secondary signals in various intracellular signaling pathways in cells, for example, via metabolism and turnover of phosphatidylinositol or cyclic AMP, is constant It is a target.

“Phosphatidylinositol turnover and metabolism” as used herein refers to molecules involved in phosphatidylinositol 4,5-diphosphate (PIP ₂ ) turnover and metabolism and the activity of these molecules. I do. PIP ₂ is a phospholipid found in the cytoplasmic part of the plasma membrane. Binding of the ligand to the receptor activates the plasma membrane enzyme phospholipase C in some cells, which in turn
The P ₂ is hydrolyzed to the 1,2-diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP ₃₎ can be produced. Once formed IP ₃ can diffuse to the endoplasmic reticulum surface where IP ₃ receptor, e.g., can bind to the calcium channel protein containing an IP ₃ binding site. IP ₃ binding can induce opening of the channel, can release calcium ions in the cytoplasm. IP ₃ can also be phosphorylated by a specific kinase is Hikiokoseru molecule intrusion of calcium from the extracellular medium into the cytoplasm, it can form inositol 1,3,4,5-tetraphosphate (IP _4). IP ₃ and IP ₄ can subsequently very quickly inactive products inositol 1,4
It can hydrolyze to diphosphate (IP ₂ ) and inositol 1,3,4-triphosphate respectively. These inert product is recycled by the cell, it can be synthesized PIP _2. 1,2, another secondary messenger produced by the hydrolysis of PIP ₂
-Diacylglycerol (DAG) remains at the cell membrane where it can act to activate the enzyme protein kinase C. Protein kinase C is normally found soluble in the cytoplasm of cells, but upon increasing intracellular calcium concentration, the enzyme moves to the plasma membrane where it can be activated by DAG. Activation of protein kinase C in different cells results in glycogen synthase phosphorylation or NF-
Various cellular responses occur, such as phosphorylation of various transcription factors such as kB. The term “phosphatidylinositol activity” as used herein refers to the activity of PIP ₂ or its metabolites.

Another signaling pathway that may involve the receptor is the cAMP turnover pathway.
“Cyclic AMP turnover and metabolism” as used herein refers to molecules involved in cyclic AMP (cAMP) turnover and metabolism and the activity of these molecules. Cyclic AMP is a second messenger produced in response to ligand-induced stimulation of certain G protein-coupled receptors. cAMP
In the signaling pathway, binding of the ligand to the GPCR can activate the enzyme adenylate cyclase, which catalyzes cAMP synthesis. The newly synthesized cAMP is then c
AMP-dependent protein kinase can be activated. This activated kinase is able to phosphorylate voltage-gated potassium channel proteins, or associated proteins, disabling the opening of potassium channels during action potentials. The inability to open potassium channels reduces the outward flow of potassium, usually repolarizing the neural membrane and prolonging membrane depolarization.

Pharmacogenomics Pharmacogenomics deals with clinically significant genetic changes in response to drugs due to altered drug predisposition and abnormal effects in patients. For example, Eichelba
um, M .; (1996) Clin. Exp. Pharmacol. Physio
l. 23 (10-11): 983-985 and Linder, M .; W. (19
97) Clin. Chem. 43 (2): 254-266. From the clinical consequences of these changes, the metabolic changes in the individual may result in the therapeutic agent becoming severely toxic in some individuals, or ineffective in some individuals. Thus, the genotype of the individual can determine the manner in which the therapeutic compound acts on the organism or the manner in which the organism metabolizes the compound. In addition, the activity of drug metabolizing enzymes affects both the intensity and duration of drug action. Thus, the pharmacogenomics of an individual allows the selection of a compound and an effective amount of the compound that are effective for prophylactic or therapeutic treatment based on the genotype of the individual. The discovery of genetic polymorphisms for some drug-metabolizing enzymes may explain why patients do not achieve the expected drug effect, exhibit excessive drug effects, or cause severe toxicity from standard drug doses. Explain if there is Polymorphisms can be expressed in extensive metabolizer phenotypes and poor metabolizer phenotypes.

Disease / Cell Function The present invention relates to immune and respiratory diseases, in particular T helper (Th) cells and T
Methods and compositions for the modulation, diagnosis and treatment of h cell-like related diseases. The immune diseases include chronic inflammatory diseases and diseases such as Crohn's disease, reactive arthritis including Lyme disease, insulin-dependent diabetes mellitus, organ-specific autoimmunity including multiple sclerosis, Hashimoto's thyroiditis and Graves' disease, contact dermatitis Atopic conditions such as psoriasis, graft rejection, graft-versus-host disease, sarcoidosis, asthma and allergies including allergic rhinitis, gastrointestinal allergies including food allergies, eosinophilia, conjunctivitis, glomerulonephritis, parasitic infections ( Pathogen susceptibility, such as, for example, leishmaniasis), certain viral infections including HIV, and bacterial infections including tuberculosis and rheumatoid rye.

Respiratory diseases include apnea, asthma, especially bronchial asthma, beryllium disease, bronchiectasis, bronchitis, bronchial pneumonia, cystic fibrosis, diphtheria, dyspnea, emphysema, chronic obstructive pulmonary disease, allergic bronchi Including but not limited to pulmonary aspergillosis, pneumonia, acute pulmonary edema, pertussis, pharyngitis, atelectasis, Wegener's granuloma, Legionella disease, pleurisy, rheumatic fever, and sinusitis.

“2871 activity”, “biological activity of 2871” as used herein as synonyms
Or "functional activity of 2871" can be performed in vivo or in vivo according to standard techniques.
It refers to the activity elicited by the 2871 protein, polypeptide or nucleic acid molecule on the 2871 responsive cells as determined by tro. In one embodiment, 287
One activity is a direct activity, such as association with a target protein, preferably a 2871 target molecule (eg, a G protein α subunit or a 2871 ligand). In another embodiment, the 2871 activity is an indirect activity, such as inhibiting the synthesis or activity of a second protein (eg, a protein in a signaling pathway). In a preferred embodiment, the 2871 activity is at least one or more of the following activities. (I) a 2871 protein of the plasma membrane, a protein or other organic molecule secreted from the cell that expresses the 2871 protein molecule (eg, 2871 protein);
Ligand). (Ii) Interaction of the 2871 protein of the plasma membrane with proteins or other organic molecules (eg, 2871 ligand) secreted from cells different from the cell containing the 2871 protein molecule. (Iii) Complex formation between the 2871 protein and a secreted peptide, polypeptide or small molecule. (Iv) 28
71 protein interaction with target molecules in the extracellular environment (eg, soluble target molecules). (V) Interaction of 2871 protein with intracellular target molecules (eg, with internalized or phagocytosed ligands). (Vi) Complex formation with one, two or more intracellular target molecules.

[0033] In yet another preferred embodiment, the 2871 activity is at least one or more of the following activities. (1) modulates, eg, acts or antagonizes, a signaling pathway (eg, a 2871-dependent pathway). (2) regulate cytokine production and / or secretion (eg, proinflammatory cytokine production and / or secretion); (3
) Regulate lymphokine production and / or secretion; (4) Regulate brain function.
(5) regulate the production of adhesion molecules and / or cell adhesion; (6) regulate the expression or activity of nuclear transcription factors. (7) regulate the expression of IL-4, IL-5, or other cytokines involved in T cell function. (8) cell proliferation, development or differentiation,
For example, it regulates the differentiation of helper T cells into TH1 versus TH2 cells. (9) Regulates cell proliferation, development or differentiation of bone marrow and / or megakaryocyte precursor cells. (1
0) modulate the cellular immune response; (11) modulate cytokine-mediated pro-inflammatory effects (eg, inhibit acute phase protein synthesis, heat, and / or prostaglandin synthesis by hepatocytes, eg, PGE2 synthesis). (12) Promote and / or enhance wound healing.

[0034] Polypeptides The present invention is based on the discovery of novel G-coupled protein receptor. In particular, expressed sequence tags (ESTs) were selected based on homology to G protein-coupled receptor sequences. This EST is used to design primers based on the sequence it contains,
Used for identification of cDNA from prostate cDNA library. Positive clones were sequenced and overlapping fragments were assembled. Analysis of the assembled sequence revealed that the cloned cDNA molecule encodes a G protein-coupled receptor.

Accordingly, the present invention has the deduced amino acid sequence shown in FIG. 1 (SEQ ID NO: 1)
Alternatively, the present invention relates to a novel GPCR having an amino acid sequence encoded by a deposited cDNA, ATCC No._.

The deposit is maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms. Deposits are provided as a convenience to those skilled in the art and have a deposit of 35 U.S.M. S. C. §112
Not the approval required below. The deposited sequence, as well as the polypeptide encoded by the sequence, are hereby incorporated by reference and will be adjusted as described in the present application in case of conflict, such as sequence errors.

“2871 receptor polypeptide” or “2871 receptor protein” refers to a polypeptide encoded by SEQ ID NO: 1 or the deposited cDNA. However, the term "receptor protein" or "receptor polypeptide" further includes many variants described herein, as well as full length 2871 polypeptides and variants derived from the variants.

Accordingly, the present invention provides isolated or purified 2871 receptor polypeptides and variants and fragments thereof.

[0039] The 2871 polypeptide is a 359 residue protein that exhibits three major structural domains. The extracellular domain is identified within about 42 from residue 1 of SEQ ID NO: 1.
The transmembrane domain is identified as being within about residues 43 to about 318 of SEQ ID NO: 1. The intracellular domain is identified as being within about residues 319 to about 359 of SEQ ID NO: 1. The transmembrane domain binds the GPCR signaling signature DRY to 13
8th to 140th included.

As used herein, a polypeptide is substantially free of cellular material when isolated from recombinant and non-recombinant cells, or is substantially a chemical precursor or when chemically synthesized. If it is free of other chemicals, it is said to be "isolated" or "purified." However, the polypeptide may be linked to another polypeptide that is not normally associated with the cell, which is still considered "isolated" or "purified".

The receptor polypeptide can be purified to homogeneity. However, it will be understood that preparations in which the polypeptide has not been purified to homogeneity are also useful and are considered to include the polypeptide in isolated form. An important feature is that the preparation allows the desired function of the polypeptide, even in the presence of significant amounts of other components. Thus, the present invention encompasses varying degrees of purity.

In one embodiment, the term “substantially free of cellular material” comprises about 30
% Or less (by dry weight) of other proteins (ie contaminating proteins), about 20%
Or a preparation of a receptor polypeptide having about 10% or less of the other protein, or about 5% or less of the other protein. When the receptor polypeptide is produced recombinantly, it may be substantially free of cell medium, ie, it contains no more than about 20%, about 10%, or about 5% of the protein preparation.
The following capacities are shown.

The term “substantially free of chemical precursors or other chemicals” includes preparations of receptor polypeptides separated from chemical precursors or other chemicals involved in the synthesis. In one embodiment, the term "substantially free of chemical precursors or other chemicals" refers to about 30% or less (by dry weight) of chemical precursors or other chemicals, about 20% Preparation of a polypeptide having the following chemical precursors or other chemicals, up to about 10% chemical precursors or other chemicals, or up to about 5% chemical precursors or other chemicals: Including.

[0044] In one embodiment, the receptor polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. However, the invention also encompasses sequence variants. Mutants are
Includes substantially homologous proteins encoded by the same locus in the organism, ie, allelic variants. Variants are obtained from other loci of the organism,
And proteins having substantial homology to the 2871 receptor protein of US Pat. The variant is also substantially homologous to the 2871 receptor protein, but
Also includes proteins from other organisms, ie, orthologs. Variants also include proteins substantially homologous to the 2871 receptor protein produced by chemical synthesis. Variants also include proteins that are substantially homologous to the recombinantly produced 2871 receptor protein.

As used herein, two proteins (or regions of a protein) have an amino acid sequence of at least about 55-60%, typically at least about 70-75%, more typically at least about 80-80%. 85%, most typically at least about 90-9
Substantially homologous when 5% or more homologous. A substantially homologous amino acid sequence according to the present invention is encoded by a nucleic acid sequence that hybridizes to the nucleic acid sequence of SEQ ID NO: 2, or a portion thereof, under the stringent conditions described in more detail below.

To determine the percent homology of two amino acid sequences or two nucleic acids, the sequences are aligned for optimal comparison (eg, to optimally align one protein with another protein or nucleic acid). Or a gap can be introduced into the nucleic acid sequence)
. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in one sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the other sequence, then the molecules are homologous at that position. As used herein, "homology" of an amino acid or nucleic acid is equivalent to "identity" of the amino acid or nucleic acid. The percent homology between two sequences is a function of the number of identical positions shared by the sequences (ie, the percent homology is equal to the number of identical positions / total number of positions x 100).

The present invention also encompasses polypeptides of low identity but having sufficient similarity to perform one or more of the same functions performed by the 2871 polypeptide. Similarity is determined by conservative amino acid substitutions. The substitutions replace one particular amino acid of the polypeptide with another amino acid having similar properties. Conservative substitutions appear phenotypically silent. Typically seen as conservative substitutions are substitutions of one residue for another between the aliphatic amino acids Ala, Val, Leu, and Ile; exchange of hydroxyl residues Ser and Thr, acidic residues Exchange of Asp and Glu, substitution between amide residues Asn and Gln, basic residues Lys and Arg
And the substitution between the aromatic residues Phe and Tyr. Guidance on which amino acid changes appear phenotypically silent can be found in Bowie et al., Scie.
247: 1306-1310 (1990).

[0048]

[Table 1]

Both identity and similarity can be readily calculated (computational molecular biology,
Lesk, A .; M. Ed., Oxford University Press, New York, 1988;
Biocomputing: Information Science and Genome Project, Smith,
D. W. Ed., Academic Publishing, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A .; M. And Griffin,
H. G. FIG. Ed., Humana Publishing, New Jersey, 1994; Sequence analysis of molecular biology, von Heinje, G. et al. And Academic Publishing, 1987; and sequence analysis primers, Gribskov, M .; And Devereux, J
. Ed., M Stockton Publishing, New York, 1991).

Preferred computer programming methods for determining identity and similarity between two sequences include, but are not limited to, the GCG program package (Deve)
reux, J .; Et al., Nucleic Acids Res. 12 (1): 387
(1984)), BLASTP, BLASTN, FASTA (Atschul,
S. F. J. et al. Molec. Biol. 215: 403 (1990)).

[0051] Variant polypeptides may differ in amino acid by one or more substitutions, deletions, insertions, inversions, fusions and truncations, or any combination thereof.

[0052] A variant polypeptide may be fully functional or may lack one or more active functions. Thus, in this case, the variant can exert a function in one or more regions corresponding to, for example, ligand binding, transmembrane association, G protein binding and signal transduction.

[0053] Fully functional variants typically include only conservative changes, or changes in unimportant residues or unimportant regions. Functional variants may also include substitutions of similar amino acids that result in no change or insignificant change in function. Separately,
The substitutions may have some positive or negative effect on function.

Non-functional variants typically include one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncations, or deletions at important residues or regions. Substitutions, insertions, inversions, or deletions.

As indicated, variants may be natural, produced by recombinant means or by chemical synthesis, to provide useful and novel characteristics of the receptor polypeptide. This includes preventing immunogenicity from pharmaceutical preparations by preventing protein aggregation.

Useful changes further include changes in ligand binding characteristics. For example, one embodiment involves a change in the binding site that results in binding rather than release of the ligand. More useful changes at the same site can result in higher affinity for the ligand. Useful changes also include those that provide an affinity for another ligand. Other useful changes include those that allow binding to the ligand but prevent activation by the ligand. Another useful change is a decrease or increase in binding to the appropriate G protein,
Alternatively, it involves a change in the transmembrane G protein binding / signaling domain that results in binding to a G protein different from the G protein to which the receptor normally associates.
Another useful change provides for a fusion protein in which one or more domains are functionally fused to one or more domains from another G protein-coupled receptor.

Amino acids essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham et al., Science 244: 1081-1085 (1989)). The latter procedure introduces one alanine mutation at every residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as receptor binding, or for in vitro or in vivo growth activity. Important sites for ligand-receptor binding also include crystallization,
It can also be determined by structural analysis such as nuclear magnetic resonance or photoaffinity labeling (Smith et al.
J. Mol. Biol. 224: 899-904 (1992); de Vos et al., Science 255: 306-312 (1992)).

[0058] The invention also includes polypeptide fragments of the 2871 receptor protein. The fragment can be obtained from the amino acid sequence shown in SEQ ID NO: 1. However, the invention also encompasses fragments of the 2871 receptor protein variants described herein.

The fragments used herein contain at least 12 contiguous amino acids. Fragments retain one or more of the biological activities of the protein, such as, for example, the ability to bind to a G protein or ligand, as well as fragments that can be used as immunogens for making receptor antibodies.

A biologically active fragment (eg, 12, 15, 20, 30, 35, 36, 37, 38,
A peptide that is 39, 40, 50, 100 or more amino acids in length) comprises a domain or motif, such as an extracellular domain, one or more transmembrane domains, a G protein binding domain, or a GPCR signature. Can be.

Possible fragments include: 1) a soluble peptide comprising the entire extracellular domain from about amino acid 1 to about amino acid 42 of SEQ ID NO: 1, 2) the cell from about amino acid 319 to about amino acid 359 of SEQ ID NO: 1 3) Including, but not limited to, peptides containing the entire transmembrane domain from about amino acid 43 to amino acid 318.

The present invention also provides fragments having immunogenic properties. These include the epitope-containing portions of the 2871 receptor protein and variants. These epitope-containing peptides are useful for producing antibodies that specifically bind to a receptor polypeptide or region or fragment. These peptides can include at least 12, at least 14, or at least about 15 to about 30 amino acids.

[0063] Non-limiting examples of antigenic polypeptides that can be used for the production of antibodies include peptides from the extracellular domain.

[0064] Epitope-containing receptors and polypeptides can be produced by any conventional means (Houghten, RA, Proc. Natl. Acad. S.
ci. ScL USA 82: 5131-5135 (1985)). Multiple simultaneous peptide syntheses are described in U.S. Patent No. 4,631,211.

Even if the fragments are separate (not fused to other amino acids or polypeptides)
, May be within a larger polypeptide. In addition, several fragments can be included within one larger polypeptide. In one embodiment, a fragment designed for expression in a host may have a heterologous pre- and pro-polypeptide region fused to the amino terminus of the receptor fragment and an additional region fused to the carboxyl terminus of the fragment. .

Thus, the present invention provides chimeric or fusion proteins. These include receptor proteins operably linked to heterologous proteins having amino acid sequences that are not substantially homologous to the receptor proteins. "Operably linked" indicates that the receptor protein and the heterologous protein are fused in-frame. Heterologous proteins can be fused to the N-terminus or C-terminus of the receptor protein.

In one embodiment, the fusion protein does not itself affect receptor function. For example, the fusion protein can be a GST fusion protein in which the receptor sequence is fused to the C-terminus of the GST sequence. Other types of fusion proteins include, but are not limited to, enzyme fusion proteins, such as β-galactosidase fusions, yeast double hybrid GAL fusions, poly-His fusions and Ig fusions. The fusion protein, especially a poly-His fusion, can facilitate purification of the recombinant receptor protein. In certain host cells (eg, mammalian host cells), expression and / or secretion of a protein can be increased through the use of a heterologous signal sequence. Thus, in another embodiment, the fusion protein includes a heterologous signal sequence at its N-terminus.

EP-A-O 464 533 discloses a fusion protein comprising various parts of an immunoglobulin constant region. Fc is useful for therapy and diagnosis, and therefore has improved pharmacokinetic properties (EP-A 0232 262). In drug discovery, for example, human proteins are fused with an Fc portion for high-throughput screening assays to identify antagonists. Bennett et al., Journal of
Molecular Recognition 8: 52-58 (1995)
And Johanson et al., The Journal of Biological
al Chemistry 270, 16: 9449-9471 (1995).
Accordingly, the present invention also includes receptor polypeptides as well as soluble fusion proteins comprising various portions of the heavy or light chain constant region of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE). Preferred as immunoglobulins are the constant parts of the heavy chains of human IgG, especially IgG1, in which the fusion takes place in the hinge region. In some uses, it may be desirable to remove Fc after using the fusion protein for its intended purpose, for example when using the fusion protein as an antigen for immunization. In certain embodiments, the Fc portion is easily removed by a cleavage sequence, which is also incorporated and can be cleaved using factor Xa.

[0069] Chimeric or fusion proteins can be produced by standard recombinant DNA techniques. For example, DNA fragments encoding different protein sequences are ligated together in-frame according to conventional techniques. In another embodiment, the fusion gene is
It can be synthesized by conventional techniques including automatic DNA synthesizers. Alternatively, amplification of a gene fragment by PCR can be performed using anchor primers, resulting in a complementary overhang between two consecutive gene fragments, followed by annealing and re-amplification to produce a chimeric gene sequence (Asuhel Et al., Current Protocols in Molecular Biology, 1992). In addition, many expression vectors are commercially available that already encode a fusion moiety (eg, a GST protein). The nucleic acid encoding the receptor protein can be cloned into an expression vector such that the fusion moiety is linked in-frame to the receptor protein.

Another form of the fusion protein is one that directly affects receptor function.
Thus, a receptor polypeptide in which one or more of the receptor domains have been replaced by homologous domains from another G protein-coupled receptor or another type of receptor is encompassed by the invention. Therefore, various permutations are possible. The extracellular domain, or a small region thereof (eg, ligand binding) can be replaced with a domain or small region from another ligand binding receptor protein. Alternatively, transmembrane regions, eg, G protein binding / signaling, may be replaced. Finally, the intracellular domain may be replaced. Thus, chimeric receptors can be formed in which one or more of the natural domains or small regions have been replaced.

The isolated receptor protein can be purified from cells that naturally express it, such as the prostate, placenta, uterus, and cells that have been altered to express it (recombinant), as shown in FIG. ) Or can be synthesized using known protein synthesis methods.

[0072] In one embodiment, the protein is produced by recombinant DNA techniques.
For example, a nucleic acid molecule encoding a receptor polypeptide is cloned into an expression vector, the expression vector is introduced into a host cell, and the protein is expressed in the host cell. The protein can then be isolated from the cells by a suitable purification scheme using standard protein purification techniques.

[0073] Polypeptides often contain amino acids other than 20 amino acids, commonly referred to as the 20 natural amino acids. In addition, many amino acids, including terminal amino acids, can be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques known in the art. Common modifications that occur naturally in polypeptides are described in basic textbooks, detailed research texts, and research literature, which are known to those skilled in the art.

Thus, the polypeptide may also be a non-substituted amino acid residue that is not a residue encoded by the genetic code, contains a substituent, or has a mature polypeptide, such as a compound that increases the half-life of the polypeptide. (Eg, polyethylene glycol)
Derivatives or analogs that are fused to or to an additional amino acid fused to a mature polypeptide, such as a leader or secretory sequence or a sequence for purifying a mature polypeptide or a proprotein sequence.

Known modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of heme moieties, covalent attachment of nucleotides or nucleotide derivatives, lipids or lipids Covalent attachment of derivatives, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, dimethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamic acid, formylation, gamma carboxylation, Glycosylation, GPI anchoring, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenization, sulphation, arginine, etc. Transfer RNA-mediated addition and ubiquitination Including, but not limited to.

The modifications are known to those skilled in the art and have been described in great detail in the scientific literature. Several particularly common modifications, such as glycosylation, lipid attachment, sulfation, γ-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, have been described in most basic textbooks, such as protein-structure. And molecular properties, 2nd edition, T.M. E. FIG. Creighton, W.C. H. Freeman and Compa
ny, New York (1993). On this subject, Wold,
F, post-translational covalent modification of the protein; C. Edited by Johnson, Acad
emic Publishing, New York 1-12 (1983); Seifter et al., Met.
h. Enzymol. 182: 626-646 (1990) and Rattan.
Et al., Ann. N. Y. Acad. Sci. A number of detailed commentaries are available, such as 663: 4862 (1992).

As is also known, polypeptides are not necessarily completely linear. For example, polypeptides can be branched as a result of ubiquitin binding, and they are generally cyclic, with or without branches, as a result of natural process events and non-naturally occurring events resulting from human manipulation. Can be Cyclic, branched and branched cyclic polypeptides can be synthesized by non-translated natural processes and by synthetic methods.

Modifications can occur anywhere in the polypeptide, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. Blockage of the amino or carboxyl group or both of the polypeptides by covalent modifications is common with natural and synthetic polypeptides. For example, before proteolytic processing,
The amino-terminal residue of polypeptides made in E. coli is almost invariably N-formylmethionine.

The modification can be a function of how the protein is made. For example, in a recombinant polypeptide, the modification is determined by the ability of the host cell to post-translate and to modify the amino acid sequence of the polypeptide. Thus, where glycosylation is desired, the polypeptide is expressed in a glycosylation host, generally a eukaryotic cell. Insect cells often perform the same post-translational glycosylation as mammalian cells, and for this reason, insect cell expression systems are developed to efficiently express mammalian proteins having a native glycosylation pattern. Similar considerations apply to other modifications.

The same type of modification may be present, to an equal or different extent, at several sites in a particular polypeptide. Also, certain polypeptides may contain one or more types of modifications.

Polypeptide Use Receptor polypeptides are useful for producing antibodies specific to the 2871 receptor protein, region or fragment.

Receptor polypeptides are also useful in drug screening assays, cell-based or cell-free systems. A cell-based system can be a cell that normally expresses a receptor protein, either native, that is, grown as a biopsy or in a cell culture, such as in the cells disclosed herein. However, in one embodiment, the cell-based assay involves a recombinant host cell expressing the receptor protein.

The polypeptides can be used to identify compounds that modulate receptor activity. 2
Both the 871 protein and appropriate variants and fragments can be used in high-throughput screening systems to assay candidate compounds for receptor binding ability. These compounds can be further screened against a functional receptor to determine the effect of the compound on receptor activity. Compounds that activate (agonist) or inactivate (antagonist) the receptor to a desired extent can be identified.

The receptor polypeptide can be used to screen compounds for the ability to stimulate or inhibit the interaction between the receptor protein and a target molecule that normally interacts with the receptor protein. A target can be a component of a signaling pathway with which a ligand or receptor protein normally interacts (eg, cAMP).
Or G proteins or other interactors involved in phosphatidylinositol turnover and / or adenylate cyclase, or phospholipase C activation). The assay can detect whether the receptor protein or fragment can interact with the target molecule and detect the formation of a complex between the protein and the target, or the biochemical consequences of the interaction between the receptor protein and the target, eg, Oxidation,
Combining the receptor protein with the candidate compound under conditions that can detect any of the cyclic AMP or phosphatidylinositol turnover and the associated effects of signaling such as adenylate cyclase or phospholipase C activation.

Candidate compounds include, for example, 1) soluble peptide containing Ig tail fusion peptides and random peptide libraries (eg, Lam et al., Nature 354:
82-84 (1991); Houghten et al., Nature 354: 84-.
86 (1991)) and peptides such as members of a combinatorial chemistry derived molecular library consisting of D and / or L configuration amino acids, 2) phosphopeptides (eg, members of random and partially denatured indicating phosphopeptide libraries, eg, Cell 72: 767-778 (1993).
3) Antibodies (eg, polyclonal, monoclonal, humanized, anti-idiotype, chimeric, and single-chain antibodies and Fab, F (ab ') ₂ , Fab expression library fragments, and epitope-binding fragments of antibodies) And 4) small organic and inorganic molecules (eg, molecules obtained from combinatorial and natural product libraries)
including.

One candidate compound is a soluble full-length receptor or fragment that competes for ligand binding. Other candidate compounds include mutant receptors or appropriate fragments that contain mutations that affect receptor function and thus compete with the ligand. Thus, for example, fragments that compete with the ligand with high affinity, or that bind but do not release the ligand, are contemplated by the present invention.

The present invention provides other indicators for identifying compounds that modulate (stimulate or inhibit) receptor activity. Assays typically include assays for signaling pathway events indicative of receptor activity. Thus, the expression of genes that are up-regulated or down-regulated in response to a receptor protein-dependent signal cascade can be assayed. In one embodiment, the regulatory region of the gene can be operably linked to a readily detectable marker such as luciferase. Alternatively, phosphorylation of the receptor protein or receptor protein target can be measured.

The binding and / or activation of a compound can be screened by the use of a chimeric receptor protein in which the extracellular domain, transmembrane domain or small region, and intracellular domain can be replaced by a heterologous domain. For example, a G-protein binding region that interacts with a different G-protein and thus is recognized by a natural receptor can be used. Thus, different sets of signaling components can be used as end-point assays for activation. Alternatively, the transmembrane portion can be replaced by a transmembrane portion specific to a host cell different from the host cell from which the extracellular domain and / or G protein binding region was obtained. This allows the assay to be performed on a host other than the specific host cell from which the receptor was obtained. Alternatively, the extracellular domain can be replaced with a domain that binds a different ligand, thus allowing assays for test compounds that interact with the heterologous extracellular domain but still cause signal transduction. Finally, activation can be detected by a reporter gene that contains an easily detectable coding region operably linked to a transcriptional regulatory sequence that is part of the natural signaling pathway.

[0089] Receptor polypeptides are also useful in competitive binding assays in methods designed to discover compounds that interact with the receptor. Thus, the compound is exposed to the receptor polypeptide under conditions that allow the compound to bind or otherwise interact with the polypeptide. A soluble receptor polypeptide is also added to the mixture. When the test compound interacts with the soluble receptor polypeptide, it reduces the amount of complex formed or the activity of the receptor target. This type of assay is particularly useful when searching for compounds that interact with specific regions of the receptor. Therefore, a soluble polypeptide is designed that competes with the target receptor region, including a peptide sequence corresponding to the region of interest.

To perform a cell-free drug screening assay, immobilize the receptor protein, or fragment, or target molecule thereof, to facilitate separation of the complex from one or both proteins in uncomplexed form; It would be desirable to adapt to the automation of the assay.

[0091] Techniques for immobilizing proteins on matrices can be used in drug screening assays. In one embodiment, a fusion protein can be provided that adds a domain that can bind the protein to a matrix. For example, glutathione-S-
The transferase / flh385 fusion protein can be adsorbed to glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione-derivatized microtiter plates, which are then combined with a cell lysate (eg, ³⁵ S-labeled) and a candidate compound and mixed with the mixture. Is incubated under conditions that favor complex formation (eg, salts and pH under physiological conditions). After incubation, the beads are washed to remove any unbound label, the matrix is fixed, and the radiolabel is determined directly or in the supernatant after dissociating the complex. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the levels of receptor binding protein found in the bead fraction can be quantified from the gel using standard electrophoresis techniques. For example, a polypeptide or its target molecule can be immobilized using biotin and streptavidin conjugates using techniques known in the art. Alternatively, antibodies that are reactive with the protein but do not interfere with the binding of the protein to its target molecule can be derivatized into wells of the plate and the protein captured by the antibody conjugate in the well. Preparations of the receptor binding protein and the candidate compound are incubated in the receptor protein display well and the amount of complex captured in the well can be quantified. Methods for detecting the complex include those described above for GST-immobilized complexes, as well as complexes using antibodies that are reactive with the receptor protein target molecule or that are reactive with the receptor protein and compete with the target molecule. Includes immunodetection, as well as enzyme binding assays that rely on detecting enzyme activity associated with the target molecule.

[0092] Using modulators of receptor protein activity identified by these drug screening assays, a subject suffering from a disease mediated by the receptor pathway, including but not limited to those disclosed herein. Can be treated. These methods of treatment include administering a modulator of the protein activity of a pharmaceutical composition described herein to a subject in need of the treatment.

[0093] Receptor polypeptides are also useful for providing targets for diagnosing a disease or predisposition mediated by a receptor protein, as disclosed herein. Accordingly, a method is provided for detecting the presence or level of a receptor protein in a cell, tissue or organism. The method comprises contacting a biological sample with a compound capable of interacting with the receptor protein, such that the interaction can be detected.

One substance that detects a receptor protein is an antibody that can selectively bind to the receptor protein. Biological samples include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present in a subject.

[0095] The receptor protein also provides a target for diagnosing an active disease or predisposition in a patient having a mutant receptor protein. Thus, receptor proteins can be isolated from biological samples and assayed for the presence of genetic mutations that result in abnormal receptor proteins. This includes amino acid substitutions, deletions,
Includes insertions, transpositions (as a result of aberrant splicing events), and inappropriate post-translational modifications. Analytical methods include changes in electrophoretic mobility, changes in tryptic peptide digestion, changes in receptor activity in cell-based or cell-free assays, changes in ligand or antibody binding patterns, changes in isoelectric point, direct amino acid sequence Determination and any other known assay technique useful for detecting mutations in a protein. In vitro techniques for receptor protein detection include enzyme linked immunosorbent assays (ELISA), western blots, immunoprecipitations, and immunofluorescence. Alternatively, the protein may be introduced into a subject in vivo by introducing a labeled anti-receptor antibody into the subject.
Can be detected. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Particularly useful are methods for detecting allelic variants of a receptor protein expressed in a subject, and methods for detecting a receptor protein fragment in a sample.

[0096] Receptor polypeptides are also useful for pharmacogenomic analysis. Thus, a genetic polymorphism can result in an allelic variant of a receptor protein, wherein one or more receptor functions in one population differ from those of another population. Thus, the polypeptide allows the target to ascertain a genetic predisposition that can affect the mode of treatment. Thus, in ligand-based treatments, polymorphisms can result in a somewhat active extracellular domain of ligand binding and receptor activation.
Therefore, the ligand dose needs to be modified to maximize the therapeutic effect within a particular population, including the polymorphism. Apart from genotype, specific polymorphic polypeptides can be identified.

[0097] Receptor polypeptides are also useful for monitoring therapeutic effects during clinical trials and other treatments. Thus, the therapeutic effect of a substance designed to increase or decrease gene expression, protein levels or receptor activity can be monitored during a course of treatment using the receptor polypeptide as an endpoint target.

[0098] Receptor polypeptides are also useful for treating receptor-related disorders, as described herein. Thus, treatment methods involve the use of soluble receptors or receptor protein fragments that compete for ligand binding. These receptors or fragments have a high affinity for the ligand and can provide an effective composition.

Antibodies The present invention also provides antibodies that selectively bind to the 2871 receptor protein and variants and fragments thereof. Antibodies are believed to selectively bind, even when bound to other proteins that are not substantially homologous to the receptor protein. These other proteins share homology with fragments or domains of the receptor protein. This conservation in specific regions results in antibodies that bind to both proteins by homologous sequences. In this case, it is understood that the antibodies binding to the receptor protein are still selective.

[0100] Antibodies can be polyclonal or monoclonal. An intact antibody, or a fragment thereof (eg, Fab or F (ab ') ₂ ) can also be used.

Detection can be facilitated by binding (ie, physical linkage) of the antibody to a detectable substance. Examples of detectable substances include various enzymes, conjugates, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, α-galactosidase, or acetylcholinesterase, examples of suitable conjugates include streptavidin / biotin and avidin / biotin, and examples of suitable fluorescent materials include Umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include
Examples of suitable radioactive materials include luciferase, luciferin, and aequorin, include ¹²⁵ I, ¹³¹ I, ³⁵ S or ³ H.

[0102] To produce antibodies, the isolated receptor polypeptide is used as an immunogen,
Antibodies are produced using standard techniques for polyclonal and monoclonal antibody preparation. Full length proteins or antigenic peptide fragments can be used. Antigenic fragments typically contain at least 12 contiguous amino acid residues. However, an antigenic peptide can include at least 14 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, or at least 30 amino acid residues. In one embodiment, the fragments correspond to regions located on the surface of the protein, for example, hydrophilic regions.

[0103] Suitable immunogenic preparations can be obtained from naturally occurring, recombinantly expressed proteins, or from chemically synthesized peptides.

Antibody Use Antibodies can be used to isolate receptor proteins by standard techniques, such as affinity chromatography or immunoprecipitation. Antibodies can facilitate the purification of natural receptor proteins from cells and recombinantly produced receptor proteins expressed in host cells.

Antibodies detect the presence of the receptor protein in cells or tissues and are useful in determining the pattern of receptor expression between organisms and various tissues in the course of normal development.

Antibodies can be used in situ, in vitro or in the detection of receptor proteins in cell lysates or supernatants to assess the amount and pattern of expression.

The antibodies can be used to assess abnormal tissue distribution or expression during development.

[0108] Antibody detection of circulating fragments of the full-length receptor protein can be used to identify receptor turnover.

In addition, antibodies can be used to assess receptor expression in a disease state, such as an active disease stage, or in an individual predisposed to a disease associated with receptor function. If the disease is caused by inappropriate tissue distribution, developmental expression, or expression levels of the receptor protein, antibodies can be prepared against the normal receptor protein. If the disease is characterized by a specific mutation in the receptor protein, antibodies specific for the mutant protein can be used to assay for the presence of the specific mutant receptor protein.

Antibodies can also be used to assess the subcellular localization of normal and abnormal cells in various tissues of an organism. Antibodies can be raised against the entire receptor or a portion of the receptor, for example, a portion of the extracellular domain.

The diagnostic use is applicable not only to genetic testing, but also to monitoring of treatment modalities. Thus, where treatment ultimately corrects for the level of receptor expression or presence of abnormal receptors and the presence of abnormal tissue distribution or developmental expression, antibodies directed against the receptor or related fragments can be used to monitor therapeutic efficacy.

In addition, antibodies are useful for pharmacogenomic analysis. Accordingly, antibodies prepared against the polymorphic receptor protein can be used to identify individuals in need of a modified treatment modality.

Antibodies are also useful as diagnostic tools as immunological markers for abnormal receptor proteins analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digestion, and other physical assays known in the art. is there.

The antibodies are also useful for tissue genotypes. Thus, where a specific receptor protein is associated with the expression of a specific tissue, a tissue type can be identified using an antibody specific for the receptor protein.

Antibodies are also useful for forensic identification. Thus, if an individual is associated with a specific genetic polymorphism that results in a specific polymorphic protein, antibodies specific to the polymorphic protein can be used as an aid in identification.

Antibodies are also useful for inhibiting receptor function, for example, blocking ligand binding.

[0117] These uses are also applicable to therapeutic situations where treatment involves inhibition of receptor function. Antibodies can be used, for example, to block ligand binding. Antibodies can be prepared against specific fragments that contain sites necessary for function, or against intact receptors associated with the cells. The invention also includes kits that use the antibodies to detect the presence of a receptor protein in a biological sample. The kit comprises an antibody, such as a labeled or labelable antibody, and a compound or substance that detects the receptor protein in the biological sample, a means for determining the amount of the receptor protein in the sample, and the amount of the receptor protein in the sample. Means for comparing with a standard may be included. The compound or substance can be packaged in a suitable container. The kit further comprises:
It may include instructions for using the receptor protein detection kit.

The nucleotide sequence of polynucleotide SEQ ID NO: 2 was obtained by sequencing the deposited human full-length cDNA. Therefore, the sequence of the deposited clone is:
Any reference to the sequence of SEQ ID NO: 2 controls for any mismatch between the two, including a reference to the sequence of the deposited cDNA.

[0119] The specifically disclosed cDNA consists of the coding region, the 5 'and 3' untranslated sequences (SEQ ID NO: 2). In one embodiment, the receptor nucleic acid consists only of the coding region.

[0120] The human 2871 receptor cDNA is approximately 1489 nucleotides in length and encodes a full-length protein that is 359 amino acid residues in length. Nucleic acids are expressed in the prostate, uterus, and placenta. A structural analysis of the amino acid sequence of SEQ ID NO: 1 is presented in FIG. 3 and is a hydropathy concept. The figure shows the putative structure of the seven transmembrane, extracellular and intracellular domains. As used in this application, the term "transmembrane domain" refers to a structural amino acid motif that includes a hydrophobic helix that spans the cell membrane.

The present invention provides isolated polynucleotides encoding the 2871 receptor protein. The term "2871 polynucleotide" or "2871 nucleic acid" refers to the sequence set forth in SEQ ID NO: 2 or the deposited cDNA. "Receptor polynucleotide" or "receptor nucleic acid" further includes variants and fragments of 2871 polynucleotide.

A “isolated” receptor nucleic acid is one that is separated from other nucleic acids that are present in the natural source of the receptor nucleic acid. Preferably, an “isolated” nucleic acid is free from sequences that naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (ie, sequences located at the 5 ′ and 3 ′ ends of the nucleic acid). are doing. However, there may be any flanking nucleotide sequence, for example, up to about 5 KB. Importantly, nucleic acids may be subject to specific manipulations described in this application, such as recombinant expression, generation of probes and primers, and other uses specific to the receptor nucleic acid sequence. Second, the nucleic acid is separated from the flanking sequences.

In addition, “isolated” nucleic acid molecules, such as cDNA molecules, can be produced from other cellular materials or culture media when produced by recombinant techniques, or chemical precursors or other when chemically synthesized. It may be substantially free of chemicals.
However, the nucleic acid molecule can be fused to other coding or regulatory sequences, but still considered to be separate.

For example, a recombinant DNA molecule contained in a vector is considered to be separate. Further, examples of isolated DNA molecules include recombinant DNA molecules that are maintained in solution within heterologous host cells or (partially or substantially) purified DNA molecules. The isolated RNA molecule includes the isolated DNA of the present invention.
Includes in vivo or in vitro RNA transcription of the molecule. The isolated nucleic acid molecule according to the present invention further includes a molecule produced synthetically and the like.

A receptor polynucleotide may comprise, in addition to the mature protein, additional amino acids or carboxyl terminal amino acids or amino acids internal to the mature polypeptide (eg, whose mature form has more than one polypeptide chain). If you can). Such sequences, among other things, play a role in the process of proteins from precursors to one mature form, facilitating protein trafficking, extending or shortening protein half-life, or measuring or Manipulation of the protein for production can be facilitated. Generally, in situ, but the additional amino acids are
It may be processed away from the mature protein by cellular enzymes.

Receptor polynucleotides include sequences encoding only the mature polypeptide,
A sequence encoding a mature polypeptide and additional coding sequences such as a leader or secretory sequence (eg, prepro or proprotein), additional non-coding sequences,
For example, add and attach introns and noncoding 5 'and 3' sequences, such as transcription, mRNA processes (including splicing and polyadenylation signals), and transcribed and untranslated sequences that play a role in mRNA ribosome binding and stability. But not limited to, but not limited to, the sequence encoding the mature polypeptide. In addition, the polynucleotide may be fused to a marker sequence that facilitates purification, for example, encoding the peptide.

[0127] Receptor polynucleotides can be obtained by cloning, by chemical synthesis techniques, or by combinations thereof, such as mRNAs.
Or in the form of DNA including cDNA and genomic DNA.
The nucleic acid is in particular DNA, but can be double-stranded or single-stranded. A single-stranded nucleic acid can be a coding strand (sense strand) or a non-coding strand (antisense strand).

One receptor nucleic acid corresponds to human prostate cDNA and consists of the nucleic acid sequence shown in SEQ ID NO: 2.

The present invention further provides variant receptor polynucleotides and fragments thereof that differ from the nucleotide sequence set forth in SEQ ID NO: 2 due to the degeneracy of the genetic code, and thus Encodes the same protein as the protein encoded by the given nucleotide sequence.

[0130] The present invention also provides receptor nucleic acid molecules that encode the variant polypeptides described in the present application. Such polynucleotides, such as allelic variants (same locus), homologous chromosomes (different loci), orthologs (different organisms), may occur naturally, or may be constructed by recombinant DNA methods or by chemical synthesis. May be done. These non-naturally occurring variants are
It may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms. Thus, as discussed above, variants can include nucleotide substitutions, deletions, inversions, and insertions.

Variants can be generated in either or both the coding and non-coding regions. Mutations can produce both conservative and non-conservative amino acid substitutions.

Orthologs, homologous chromosomes, allelic variants can be identified using methods well known to those skilled in the art. These variants are at least about 55% homologous to the nucleotide sequence set forth in SEQ ID NO: 2 or a fragment of this sequence;
Typically at least about 70-75% homologous, more typically at least about 80-85% homologous, and most typically at least about 90-95% homologous.
Consists of a nucleotide sequence that encodes a homologous or more homologous receptor. Such nucleic acid molecules can be readily identified as being capable of hybridizing under stringent conditions to the nucleotide sequence set forth in SEQ ID NO: 2 or a fragment thereof. Stringent hybridization involves substantial homology due to common homologies such as polyA sequences or sequences common to all or most proteins, all GPCRs, or all family I GPCRs. It turns out that it does not mean.

As used in this application, the phrase “hybridizes under stringent conditions” refers to nucleotide sequences encoding receptors that are at least 55% homologous to each other, typically with each other. It is intended to describe conditions for hybridization and washing that remain hybridized. Sequences that are at least about 65% homologous to each other, sequences that are at least about 70% homologous to each other, or at least about 7
It is possible that sequences that are 5% homologous to each other, or more homologous to each other, will typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and are described in "Current Protocols in Molecular B".
ioology, ”6.3.1 to 6.3.6. , John Wiley & Son
See New York, 1989. One example of stringent hybridization conditions is hybridization with 6 × sodium chloride / sodium acetate (SSC) at about 45 ° C. followed by 5 ×
One or more washes are performed with 0.2 × SSC, 0.1% SDS at 0-65 ° C. In one embodiment, the isolated receptor nucleic acid molecule that hybridizes under stringent conditions to the sequence of SEQ ID NO: 2 corresponds to a naturally occurring nucleic acid molecule. As used in this application, "natural" nucleic acid molecule refers to an RNA or DNA molecule having a naturally occurring nucleotide sequence (eg, encoding a naturally occurring protein).

Further, the present invention provides a polynucleotide consisting of a fragment of the full-length receptor polynucleotide. The fragment can be single-stranded or double-stranded,
It may also consist of DNA or RNA. The fragments can be derived from either coding or non-coding sequences.

In one embodiment, the isolated receptor nucleic acid is at least 36 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO: 2. In other embodiments, the nucleic acids are at least 40, 50, 100, 250, 500 nucleotides in length.

[0136] However, receptor fragments have been found to include any nucleic acid sequence that does not include the entire gene.

Receptor nucleic acid fragments include polypeptides consisting of an extracellular domain containing from 1 to about 42 amino acid residues, polypeptides consisting of a transmembrane domain (about 43 to about 318 amino acid residues), intracellular Domain (amino acid residues from about 318 to about 359), a polypeptide encoding the G protein receptor signature (about 1
Nucleic acid molecules encoding DRY or surrounding amino acid residues from 27 to about 143). Where the positions of the domains are predicted by computer analysis, those skilled in the art will appreciate that the amino acid residues that make up these domains may vary depending on the criteria used to define the domains.

The present invention also provides a receptor nucleic acid fragment encoding a region bearing the epitope of the receptor protein described in the present application.

[0139] Isolated receptor polynucleotide sequences, and particularly fragments, are useful as DNA probes and primers.

For example, the coding region of a receptor gene can be isolated using a known nucleotide sequence to synthesize an oligonucleotide probe. The labeled probe is then used to separate the cDNA corresponding to the coding region from the cDNA.
Used to screen libraries, genomic DNA libraries, or mRNA. In addition, primers can be used in PCR reactions to clone specific regions of the receptor gene.

The probes / primers typically consist essentially of purified oligonucleotides. Oligonucleotides typically have at least about 12 contiguous nucleotides, typically about 25 contiguous nucleotides, more typically about 40, 50, 7 of the SEQ ID NO: 2 sense or antisense strand or other receptor polynucleotide.
It contains one region of the nucleotide sequence that hybridizes under stringent conditions to 5 contiguous nucleotides. One probe also:
Labels include, for example, radioisotopes, fluorescent components, enzymes or enzyme cofactors.

Uses of the Polynucleotides The receptor polynucleotides were used to separate full-length cDNA and genomic clones encoding the polypeptides set forth in SEQ ID NO: 1, and to the same polypeptides set forth in SEQ ID NO: 1. It is useful as a hybridization probe for cDNA and genomic DNA in order to separate genomic coulomb from cDNA corresponding to a mutant that produces E. coli or other mutants described in the present application. A variant can be isolated from the same tissue and from an organism from which the polypeptide of SEQ ID NO: 1 has been isolated, and can separate different tissues from the same organism or different tissues from different organisms. . This method is useful for separating genes and cDNAs that are developmentally regulated and therefore may be expressed in the same tissue at different points in the development of the organism.

A probe can correspond to any sequence along the entire length of the gene encoding the receptor. Therefore, it can be derived from a 5 'non-coding region, a coding region, a 3' coding region, and a 3 'non-coding region.

Nucleic acid probes may be, for example, at least 12, 15, 30, 50, 100,
SEQ ID NO: 1 such as an oligonucleotide of 250 or 500 nucleotides
Or a fragment thereof, but is sufficient to specifically hybridize to mRNA or DNA under stringent conditions.

The fragments of the polynucleotides described in this application are also useful for use in synthesizing larger fragments or full-length polynucleotides described in this application. For example, fragments can hybridize to any part of the mRNA and produce larger or full-length cDNAs.

The fragments are also useful for synthesizing antisense molecules of desired length and sequence.

[0147] Receptor polynucleotides are also useful as primers in PCR methods to propagate any region of the receptor polynucleotide.

[0148] Receptor polynucleotides are also useful for constructing recombinant vectors. Such vectors include expression vectors that express part or all of the receptor polypeptide. Vectors also include insertion vectors that are used to modify the in situ expression of the receptor gene and gene product for integration into another polypeptide sequence, such as into the genome of the cell. For example, the endogenous receptor coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations.

[0149] Receptor polypeptides are also useful as probes for determining the chromosomal location of a receptor polynucleotide by in situ hybridization techniques.

Receptor polynucleotide probes also relate to their receptors and their variants with respect to tissue distribution, eg, whether gene replication has occurred and whether that replication occurs in all or only a subset of tissues. It is useful to determine the pattern of presence of the body-encoding gene. The gene could be naturally occurring or could be introduced into the organism in a cell, tissue or exogenously. Receptor polynucleotides are also useful for designing ribozymes corresponding to all or a portion of mRNA produced from a gene encoding a polynucleotide described in the present application.

[0151] Receptor polynucleotides are also useful for constructing transgenic animals that express all or part of the receptor nucleotides and polypeptides.

[0152] Receptor polynucleotides are also useful in creating vectors that express some or all of the receptor polypeptide.

[0153] Receptor polynucleotides are also useful as hybridization probes to determine the level of receptor nucleic acid expression. Thus, the probe can be used to detect or determine the level of a receptor nucleic acid in a cell, tissue, or organism. The nucleic acid whose level is to be determined can be DNA or RNA. Accordingly, probes corresponding to the polypeptides described in the present application can be used to assess gene copy number in any cell, tissue, or organism. This is especially true in the case where the receptor gene has been amplified.

Alternatively, the probe may be integrated on an extrachromosomal factor or, for example, in a chromosome where the receptor gene is not normally found, such as in a region that has been stained homologously. Can be used in the context of in situ hybridization to assess the location of extra copies of a receptor gene.

The use of these is appropriate for the diagnosis of disorders involving increased or decreased receptor expression for normal outcome.

In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vi for DNA detection
The tro technology includes Southern and in situ hybridization methods.

The probe may express a receptor protein, such as by measuring the level of a receptor-encoding nucleic acid in a sample of cells derived from the subject, eg, mRNA or genomic DNA, or by determining whether the receptor gene has been mutated. It can be used as a part of a diagnostic test kit for identifying cells or tissues to be made.

Nucleic acid expression measurements are useful for drug screening to identify components that regulate receptor nucleic acid expression.

The present invention is thus a method for identifying components that can be used to treat nucleic acid expression of a receptor gene, eg, a disorder associated with the nucleic acid expression disclosed in the present application. I will provide a. The method typically involves determining the ability of the component to modulate the expression of the receptor nucleic acid, and thus, the component that can be used to treat a disorder characterized by unwanted receptor nucleic acid expression. An identifying step is included.

The measurements can be made in cell-based and cell-free systems. Cell-based assays include cells that naturally express receptor nucleic acids such as those disclosed in the present application, or recombinant cells that have been genetically engineered to express specific nucleic acid sequences. included.

[0161] Alternatively, the candidate component can be measured in a patient or in a transgenic animal.

Assays for receptor nucleic acid expression can include direct measurement of mRNA levels or nucleic acid levels such as incidental components involved in signal pathways (such as cyclic AMP or phosphatidylinositol rotational displacement). In addition, the expression of genes that are up- or down-regulated in response to the receptor protein signaling pathway can also be measured. In this embodiment, the regulatory regions of these genes can be operably linked to a reporter gene such as luciferase. Thus, modulators of receptor gene expression can be identified in a method in which a cell is contacted with a candidate component and mRNA expression is measured. The level of receptor mRNA expression in the presence of the candidate component is compared to the level of receptor mRNA expression in the absence of the candidate component. Candidate components can then be identified as modulators of nucleic acid expression based on this comparison, and
It can be used to treat disorders characterized by abnormal nucleic acid expression. If the presence of a candidate component for mRNA expression is statistically significantly greater than the absence of the candidate component, the candidate component is identified as a stimulator of nucleic acid expression. A candidate component is identified as an inhibitor of nucleic acid expression when nucleic acid expression is statistically significantly less in the presence of the candidate component than in the absence thereof.

Accordingly, the present invention provides a method of treating with a nucleic acid as a target, using components identified by drug screening as gene regulators that regulate receptor nucleic acid expression, such as in the disorders disclosed in the present application. I will provide a. Regulation includes both up-regulation (ie, activation and activation) or down-regulation (suppression or antagonism) or nucleic acid expression.

[0164] Alternatively, modulators for receptor nucleic acid expression are identified using the screening assays described in this application while the drug or small molecule inhibits receptor nucleic acid expression. , Small molecules or drugs.

[0165] Receptor polynucleotides are also useful in clinical trials or in therapeutic regimens to monitor the effects of modulating components on the expression or activity of the receptor gene. Therefore, the gene expression pattern depends on the components, in particular,
It can serve as a barometer for continuing the effects of treatment with components that the patient can resist. Gene expression patterns can also serve as markers that indicate the physiological response of diseased cells to that component. Thus, such monitoring is possible either with escalating doses of the component or with alternative components that do not render the patient resistant. Similarly, if the level of nucleic acid expression falls below the desired level, administration of that component can be reduced proportionately.

[0166] Receptor polynucleotides are also useful in diagnostic assays for quantitative changes in receptor nucleic acids, and are qualitatively linked to pathology, particularly in the disorders disclosed in the present application. It is useful in various changes. The polynucleotide can be used to detect mutations in receptor genes such as mRNA and gene expression products. The polynucleotide can be used as a hybridization probe to detect that a genetic mutation in the receptor gene occurs naturally, and thereby subject the subject to a risk for a disorder caused by the mutation with respect to the mutation. Determine whether Mutations include genes, ie, genomic DNs, such as abnormal methylation patterns or changes in gene copy number, such as amplification.
Inversion or transposition of A, deletion, addition or substitution of one or more nucleotides in the modified chromosomal rearrangement is included. Detection of mutant forms of a receptor gene associated with deficiency is a diagnostic tool for disease activity or susceptibility to the disease when the disease results from overexpression, low expression, or altered expression of the receptor protein. I will provide a.

[0167] Individuals with mutations in the receptor gene can be detected at the nucleic acid level by a variety of procedures. Genomic DNA can be analyzed directly or can be amplified using PCR methods prior to analysis. RNA or cDNA can be used as well.

In certain embodiments, methods for detecting mutations include the use of probes / primers in a polymerase chain reaction (PCR) method (eg, US Pat.
683,195 and 4,683,202), anchor PCR, or RACE PCR, or alternatively, a binding chain reaction (LCR).
) Method (Landgeran et al., Science 241: 1077-1080 (
1988); Nakazawa et al., PNAS 91: 360-364 (19).
94)), the latter of which may be particularly useful for detecting point mutations in genes (Abrabaya et al., Nucleic Acids Res. 2).
3: 675-682 (1995)). The method includes the steps of collecting a sample of cells from the patient, isolating nucleic acids (eg, genomic DNA, mRNA or both) from the cells of the sample, hybridization of genes (if present) and Contacting the nucleic acid sample with one or more primers that specifically hybridize the gene under conditions such that amplification occurs, and detecting the presence or absence of the amplification product, or the amplification product And measuring its length against a control sample. Deletions and insertions can be detected by a change in the size of the amplified product relative to the normal genotype. Point mutation is performed by converting amplified DNA to normal RNA or antisense DNA.
It can be identified by hybridizing to the sequence.

Alternatively, mutations in the receptor gene can be identified directly, for example, by alterations in restriction enzyme hydrolysis patterns measured by gel electrophoresis.

In addition, sequence-specific ribozymes (US Pat. No. 5,498,531) can be used to score for the presence of specific mutations due to the occurrence or deletion of a ribozyme cleavage site.

Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage hydrolysis assays or by differences in melting temperatures.

Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and S1 protection, or by chemical cleavage methods.

In addition, sequence differences between a mutant receptor gene and a wild-type gene can be determined by direct DNA sequencing. Various automated sequencing methods are described by mass spectrometry sequencing (eg, PCT International Publication No. WO 94/16101; C
ohen et al., Adv. Chromatogr. 36: 127-162 (1996)
); And Griffin et al., Appl. Biochem. Biotechno
l. 38: 147-159 (1993)) can be used when performing diagnostic assays ((1995) Biotechniques 19: 448).

Other methods for detecting mutations in a gene include methods in which a protection agent from a cleavage agent is used to detect incompatible bases in RNA / RNA or RNA / DNA duplexes (Myers et al., Science). 230: 1242 (1
985); Cotton et al., PNAS 85: 4397 (1988); Sal.
eeba et al., Meth. Enzymol. 217: 286-295 (19
92)), a method for comparing the electrophoretic mobilities of a mutant and a wild-type nucleic acid (Ori
ta et al., PNAS 86: 2766 (1989); Cotton et al., Mut.
at. Res. 285: 125-144 (1993); Hayashi et al.,
Genet. Anal. Tech. Appl. 9: 73-79 (1992)
)), A method in which the migration of a mutant or wild-type fragment in a polyacrylamide gel containing a gradient of denaturants is measured using denaturing gradient gel electrophoresis (Myers
Nature 313: 495 (1985)). Examples of other techniques for detecting point mutations include selective oligonucleotide hybridization, selective amplification, and selective primer extension.

[0175] Receptor polynucleotides are also useful for testing individuals for genotypes that do not necessarily cause the disease, but do not affect the method of treatment. Thus, polynucleotides can be used to study the relationship between an individual's genotype and the individual's response to components used for therapy (pharmacogenomic relationship). In this case, for example, a mutation in the receptor gene that results in an altered affinity for the ligand will result in excess or decreased drug effect at the standard density of the ligand activating the receptor. Thus, the receptor polynucleotides described in this application can be used to assess the mutation content of a receptor gene in an individual to select the appropriate component or dosage regimen for treatment.

[0176] Thus, polynucleotides displaying genetic mutations that effect a treatment provide a diagnostic target that can be used to tailor treatment in an individual. Thus, the production of recombinant cells and animals containing these polymorphisms allows for effective clinical design of therapeutic components and dosage regimes.

[0177] Receptor polynucleotides are also useful for chromosome identification if the sequence is identified by the individual's chromosome and to a particular location on the chromosome. First,
The DNA sequence is adapted to the chromosome by in situ or other chromosome-specific hybridization methods. Sequences can also be correlated to a particular chromosome by creating PCR primers that can be used for PCR screening of somatic cell hybrids containing individual chromosomes from the desired species.
Only those hybrids containing a chromosome containing the gene homologous to the primer will yield an amplified fragment. Sublocalization can be achieved using chromosomal fragments. Other strategies include prescreening with labeled and flow sorted chromosomes and preselection by hybridization to a chromosome-specific library. In addition, mapping strategies include fluorescence in situ hybridization methods that allow hybridization with shorter probes than traditionally used. Chromosome mapping reagents can be used individually to label a single chromosome or a single site on that chromosome, and a panel of reagents can label multiple sites and / or multiple chromosomes. Can be used to Reagents corresponding to non-coding regions of the gene are in fact suitable for cartographic purposes. The coding sequence may become conserved within the gene family, thereby increasing the chance of cross-hybridization during chromosome mapping.

[0178] Receptor polynucleotides can also be used to identify individuals from small biological samples. This is due, for example, to restriction fragment length polymorphism (R
FLP) can be used to identify an individual. Thus, the polynucleotide described in this application is a DNA for RFLP
Useful as markers (see US Pat. No. 5,272,057).

In addition, receptor sequences can provide an alternative technique for determining the actual DNA sequence of a selected fragment in an individual's genome. Thus, the receptor sequences described in this application can be used to generate two PCR primers from the 5N and 3N ends of the sequence. These primers can then be used to amplify DNA from an individual for subsequent sequencing.

[0180] Corresponding panels of DNA sequences from individuals generated in this manner can provide a unique method of identifying individuals, as each individual has a unique set of such DNA sequences. . Allelic variation in humans is approximately 500
Occurs once per base. Allelic variation occurs to some extent in the coding regions of these sequences, and more often in non-coding regions.
Receptor sequences can be used to obtain such identified sequences from individuals and from tissues. Such sequences represent unique fragments of the human genome. Each of such sequences described in the present application can, to some extent, be used as a basis by which DNA from an individual can be compared for identification purposes.

If a panel of reagents from a sequence is used to generate a unique identification database for an individual, these same reagents can be used later to identify tissue from that individual. Using a unique identification database, positive identification of the individual, whether alive or dead, can be made from extremely small tissue samples.

[0182] Receptor polynucleotides can also be used in forensic identification procedures. PCR technology can be used to amplify DNA sequences from very small biological samples, such as a single hair follicle, bodily fluids (eg, blood, saliva, or semen). The amplified sequence can then be compared to a standard that allows identification of the source of the sample.

Receptor polynucleotides may thus be DNA-based forensic, for example, by providing another "identifying marker" (ie, another DNA sequence that is unique to a particular individual). It can be used to provide polynucleotide reagents, eg, PCR primers, that are targeted to specific loci in the human genome that can increase the confidence in identification. As described above, the actual nucleotide sequence information can be used for identification as an accurate alternative to the pattern formed by restriction enzyme generated fragments. Sequences targeted to non-coding regions are particularly useful because more polymorphisms occur in non-coding regions, making individual identification easier using this technique.
Fragments are at least 12 bases.

[0184] Receptor polynucleotides can also be used to identify specific tissues, for example,
It can be used to provide polynucleotides, eg, labeled or labelable probes, that can be used in in situ hybridization techniques. This is useful in cases where a forensic pathologist is presented with tissue of unknown origin. Panels of receptor probes can be used to identify tissues by species and / or organ type. In a similar manner, these primers and probes can be used to screen tissue culture media for contamination (ie, screen for the presence of a mixture of different types of cells in the culture media).

Alternatively, receptor polynucleotides can be used directly to block transcription or translation of receptor gene expression by means of antisense or ribozyme constructs. Thus, in disorders characterized by abnormally high or undesirable receptor gene expression, the nucleic acids can be used directly for therapy.

[0186] Receptor polynucleotides are therefore useful as antisense constructs for controlling receptor gene expression in cells, tissues, and organisms. DN
A antisense polynucleotides are designed to be complementary to one region of the gene involved in transcription, preventing transcription and, thus, production of the receptor protein. The antisense RNA or DNA polynucleotide will hybridize to the mRNA and thus prevent translation of the mRNA into a receptor protein.

Examples of antisense molecules useful for inhibiting nucleic acid expression include a start codon that is complementary to a fragment of the 3 ′ untranslated region of SEQ ID NO: 2 and a SEQ ID NO: Antisense molecules complementary to fragments of the 2 5 'untranslated region are included.

[0188] Alternatively, one class of antisense molecules can be used to inactivate mRNA to reduce the expression of receptor nucleic acids. Thus, these molecules can treat disorders characterized by abnormal or unwanted receptor nucleic acid expression. This technology includes the mR to be translated
Includes cleavage by ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of NA. Potential regions include coding regions, especially those coding for receptor protein catalysis and other functional activities.

[0189] Receptor polynucleotides also provide vectors for gene therapy of patients having cells that are abnormal in receptor gene expression. Therefore, ex v
Recombinant cells, including the patient's cells that have been genetically engineered in vivo and returned to the patient, are introduced into the individual whose cells produce the desired receptor protein to treat the individual. Is done.

[0190] The present invention also includes kits for detecting the presence of a receptor nucleic acid in a biological sample. For example, the kit may be a labeled or labelable nucleic acid or drug capable of detecting a receptor nucleic acid in a biological sample, a means for measuring the amount of the receptor nucleic acid in the sample, a standard and the sample. It can comprise a means for comparing the amount of the receptor nucleic acid therein. The component or agent can be packaged in a suitable container. The kit further comprises instructions for using the kit to detect receptor mRNA or DNA.

Vectors / Host Cells The invention also provides vectors that include a receptor polynucleotide.
The term "vector" refers to a vehicle, and preferably refers to a nucleic acid molecule capable of transporting a receptor polynucleotide. If the vector is a nucleic acid molecule,
The receptor polynucleotide is covalently linked to the vector nucleic acid. According to this aspect of the invention, the vector comprises a plasmid, a single-stranded or double-stranded phage,
Single- or double-stranded RNA or DNA virus vector, or BAC
, PAC, YAC, OR MAC and the like.

The vector can be maintained in a host cell as an extrachromosomal element with which the vector replicates and produces additional copies of the receptor polynucleotide. Alternatively, the vector may integrate into the genome of the host cell and produce additional copies of the receptor polynucleotide when the host cell replicates.

The present invention provides a vector for maintaining a receptor polynucleotide (cloning vector) and a vector for expression (expression vector). Vectors can function in prokaryotic or eukaryotic cells, or both (shuttle vectors).

The expression vector includes a cis-acting regulatory region operably linked in the vector to the receptor polynucleotide such that transcription of the polynucleotide is permitted in the host cell. The polynucleotide can be introduced into the host cell by another polynucleotide that can affect transcription. Thus, this second polynucleotide may provide a trans-acting factor that interacts with the cis-regulatory control region to allow transcription of the receptor polynucleotide from the vector. Alternatively, the trans-acting factor may be supplied by the host cell. Finally, the trans-acting factor can be produced from the vector itself.

However, in some embodiments, it has been found that transcription and / or translation of the receptor polynucleotide can occur in a cell-free system.

[0196] Regulatory sequences capable of operably linking the polynucleotides described in the present application include promoters to direct mRNA transcription.
A left promoter from bacteriophage λ, lac from E. coli (E. coli),
TRP, TAC promoter, early and late promoters from SV40, C
Included but not limited to the MV immediate promoter, adenovirus early and late promoter, retroviral long terminal repeat (LTR).

In addition to control regions that promote transcription and expression, vectors also include regions that regulate transcription, such as repressor binding sites and enhancers. Examples include SV40
Includes enhancers, cytomegalovirus immediate-type enhancers, polyoma enhancers, adenovirus enhancers, and retroviral LTR enhancers.

In addition to including sites for transcription initiation and control, expression vectors also include sequences necessary for transcription termination, and the transcribed region can include a ribosome binding site for translation. Other regulatory controls for expression include start and stop codons, as well as polyadenylation signals. If you are skilled in the art,
You will know a number of regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook, "Molecular Cloni".
ng: A Laboratory Manual ", 2nd edition, Cold Spr
ing Harbor Laboratory Press, Cold S
Spring Harbor, New York, 1989.

A variety of expression vectors can be used to express the receptor polynucleotide. Such vectors include chromosome-inducing betacu, episomal-inducing vectors, virus-inducing vectors, for example, vectors derived from bacterial plasmids, vectors derived from bacteriophages, vectors derived from yeast episomes, yeasts containing yeast artificial chromosomes. Vectors derived from chromosomal factors, vectors derived from viruses such as baculovirus, papovavirus such as SV40, vaccinia virus, adenovirus, poxvirus, pseudorabies virus, and retrovirus are included. Vectors may also be derived from combinations of these sources, such as those derived from plasmids and bacteriophage genetic elements, for example, cosmids and phagemids. Suitable cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook, Ed., Molecular Cloning: A La.
bounty Manual, 2nd edition, Cold Spring Har
Bor Laboratory Press, Cold Spring H
Arbor, New York, 1989.

Regulatory sequences may provide for constitutive expression in one or more host cells (ie, tissue-specific), or may be exogenous factors such as temperature, nutrient additives, or hormones or other ligands. Preparations for inducible expression in one or more cell types may be made, for example. A variety of vectors that prepare for construction and inducible expression in prokaryotic and eukaryotic hosts are well known to those of skill in the art.

[0201] The receptor polynucleotide can be inserted into the vector nucleic acid by well-known methods. Generally, the final expressed DNA sequence is ligated into an expression vector by cleaving the DNA sequence, and the expression vector is accompanied by one or more restriction enzymes and then ligated together with the fragment. did. Procedures for restriction enzyme hydrolysis and conjugation are well known to those skilled in the art.

[0202] Vectors containing the appropriate polynucleotides can be introduced into appropriate host cells for propagation or expression using well known techniques. For bacterial cells,
Includes, but is not limited to, E. coli, Streptomyceds, Salmonella typhimurium. Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.

[0203] As described in the present application, it is considered desirable to express the polypeptide as a fusion protein. Accordingly, the present invention provides fusion vectors that allow for the production of a receptor polypeptide. Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of that protein, for example, by acting as a ligand for affinity purification. A proteolytic cleavage site may be introduced at the junction of the fusion half so that the desired polypeptide can be eventually separated from the fusion half. Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, enterokinase. A typical fusion expression vector includes pGEX (Smith et al., (1)
988) Gene 67: 31-40), pMAL (New England)
Biolabs, Beverly, Mass., PRIT5 (
Pharmacia, Piscataway City, NJ). Examples of suitable inducible non-fused E. coli, expression vectors include pTrc (Ama
nn et al., Gene 69: 301-315 (1988)) and pET 11d (
Studier et al., Gene Expression Technology.
: Methods in Enzymology 185: 60-89 (199
0)) are included.

[0204] Recombinant protein expression can be maximized in host bacteria by providing a genetic background with the damaging ability of the host cell to proteolytically cleave the recombinant protein (Gottesman). ,
S, Gene Expression Technology: Method
s in Enzymology 185, Academic Press,
City of San Diego, California (1990) 119-128). Alternatively, the sequence of the polynucleotide of interest can be altered to provide preferential use of codons for a specific host cell, eg, E. coli (Wada et al., Nucleic Acids Res. 20). : 2111-21
18 (1992)).

[0205] Receptor polynucleotides can also be expressed by yeast-operable expression vectors. Yeast, for example, Saccharomyces cerev
Examples of vectors for expression in Isiae include pYepSec1 (B
aldar et al., EMBO J .; 6: 229-234 (1987)), pMF
a (Kurjan et al., Cell 30: 933-943 (1982)), pJ
RY88 (Schultz et al., Gene 54: 113-123 (1987)
)), PYES2 (Invitrogen Corporation, San
Diego City, CA).

[0206] Receptor polynucleotides can also be expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors useful for expressing proteins in cultured insect cells (eg, Sf9 cells) include the pAC series (Smith et al., Mol. Cell Bi).
ol. 3: 2156-2165 (1983)) and the pVL series (Luckl
ow et al., Virology 170: 31-39 (1989)).

In certain embodiments of the present invention, the polynucleotides described in the present application are expressed in mammalian cells using a mammalian expression vector.
Examples of mammalian expression vectors include pCDM8 (Seed, B. Natu
re 329: 840 (1987)) and pMT2PC (Kaufman et al., E
MBOJ. 6: 187-195 (1987)).

The expression vectors listed in this application are presented only as examples of well-known vectors available to those of skill in the art that are useful for expressing the receptor polynucleotide. One of skill in the art would know other vectors suitable for maintaining, propagating or expressing the polynucleotides described in this application. These are described, for example, in Sambrook, J .; Fritsh, E .;
F. Manjatis, T .; Edited by Molecular Cloning:
A Laboratory Manual, 2nd edition, Cold Spring
Published by Harbor Laboratory Press, Cold Spri
ng Harbor, New York, 1989.

The invention also relates to a vector wherein the nucleic acid sequences described in this application are cloned into a vector in the reverse orientation, but operably linked to regulatory sequences that permit transcription of the antisense RNA. Is also included. Thus, antisense transcripts can be produced for all or a portion of the polynucleotide sequences described in this application, including both coding and non-coding regions. This example of an antisense RNA requires each of the parameters described above in relation to the expression of the sense RNA (regulatory sequence, constitutive or inducible expression, tissue-specific expression).

[0210] The present invention also relates to recombinant host cells comprising the vectors described in this application. Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammals.

[0211] Recombinant host cells are created by introducing the vector constructs described in this application into cells by techniques that are readily available to those skilled in the art. These include calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection,
Electroporation, transduction, infection, lipofection, and Sambro
ok "Molecular Cloning: Laboratory Manual (Molecular Cloni)
ng: A Laboratory Manual), 2nd edition, Cold Sp
ring Harbor Laboratory Press, Cold
Spring Harbor, New York, 1989, but not limited to other technologies.

[0212] A host cell can include one or more vectors. Thus, different nucleotide sequences can be introduced on different vectors of the same cell. Similarly, the receptor polynucleotide can be introduced either alone or with other polynucleotides not related to the receptor polynucleotide, such as those that provide a trans-acting factor for the expression vector. . When one or more vectors are introduced into a cell, the vectors can be introduced alone, co-introduced, or linked to a receptor polynucleotide vector.

In the case of bacteriophage and viral vectors, these can be introduced into cells as packaged or encapsulated viruses by standard procedures for infection and transduction. Viral vectors can be replication responsive or replication deficient. When viral replication is defective, replication occurs in a host cell that provides a function that complements the defect.

[0214] Vectors generally include a selectable marker that allows the selection of a lineage of cells containing the recombinant vector construct. Markers can be included in the same vector containing the polynucleotide described in this application, or can be on a separate vector. Markers include tetracycline or ampicillin resistance genes for prokaryotic host cells, and dihydrofolate reductase or neomycin resistance genes for eukaryotic host cells. But,
Any marker that provides a selection for one phenotypic trait is effective.

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

If secretion of the polypeptide is desired, an appropriate secretion signal is incorporated into the vector. The signal sequence may be endogenous to the receptor polypeptide or may be heterologous to these polypeptides.

If the polypeptide is not secreted into the medium, the protein can be isolated from host cells by standard disruption methods, including freeze-thawing, sonication, mechanical disruption, use of lysing agents, and the like. . The polypeptide may then be subjected to ammonium sulfate precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or It can be recovered and purified by well-known purification methods including high performance liquid chromatography.

[0218] Depending on the host cell in the recombinant production of the polypeptides described in this application, the polypeptides may have different glycosylation patterns depending on the cell, or may have non-glycosylation when produced in bacteria. Glycosylated. In addition, the polypeptide may in some cases include an initial modified methionine as a result of a host-mediated process.

Methods for Using Vectors and Host Cells Host cells for expressing the polypeptides described in this application, and especially recombinant host cells, have a variety of uses. First, the cells are useful for producing a receptor protein or polypeptide that can be further purified to produce a desired quantity of the receptor protein or fragment. Therefore, host cells containing the expression vector are useful for polypeptide production.

[0220] Host cells are also useful for performing cell-based assays that include a receptor or receptor fragment. Therefore, recombinant host cells that express native receptors are useful for measuring components that stimulate or inhibit receptor function. This includes components of ligand binding, gene expression at the level of transcription or translation, G protein interactions, and signal transduction pathways.

[0221] Host cells are also useful for identifying receptor variants in which these functions are affected. If the variant occurs naturally and causes pathology, the host cell containing the mutation is directed against the mutant receptor, which appears not to be effective at the native receptor It is useful for evaluating a component having a desired effect (for example, a stimulating or inhibiting function).

[0222] Recombinant host cells are also useful for expressing the chimeric polypeptides described in this application to evaluate components that activate or repress activity by a heterologous extracellular domain. Alternatively, the heterologous transmembrane domain can be used to assess the effect of the desired extracellular domain on any any host cell. In this embodiment, a transmembrane domain that is compatible with a particular host cell is used to create a chimeric vector. Alternatively, a heterologous cell, eg, signal transduction, a domain can be introduced into its host cell.

In addition, mutant receptors can be designed, in which one or more of the various functions are engineered to enhance or attenuate (ie, ligand binding, G -Protein binding) and also used to increase or replace the receptor protein in an individual. Thus, a host cell can provide therapeutic benefit by replacing or supplying an abnormal receptor to provide a therapeutic result. In one embodiment, the cells provide a receptor that is abnormally active.

[0224] In another embodiment, the cells provide a receptor that is abnormally inactive. These receptors can antagonize endogenous receptors in an individual. In another embodiment, cells expressing a non-activatable receptor are introduced into an individual to antagonize the endogenous receptor for the ligand. For example, if excess ligand is part of the method of treatment, it may be necessary to inactivate the ligand at some point in the treatment. Providing cells that antagonize the ligand but are not likely to be affected by receptor activation is a benefit.

[0225] Homogeneous recombinant host cells can also be produced to allow for in situ modification of the endogenous receptor polynucleotide sequence in the host cell genome. This technology is more fully described in PCT International Publication Nos. WO 93/09222, WO 9
1/126650, and in U.S. Pat. No. 5,641,670. Briefly, a particular polynucleotide sequence corresponding to a receptor polynucleotide or sequence proximal or distal to a receptor gene is:
Homologous recombination, whose expression of the gene can be affected, can be integrated into the host cell genome. In one embodiment, regulatory sequences are introduced to either increase or decrease expression of the endogenous sequence. Thus, the receptor protein can be produced in cells that do not normally produce it, or increased expression of the receptor protein results in cells that normally produce that protein at certain levels. It is possible that Alternatively, the entire gene can be deleted. Still further, certain mutations can be introduced into any desired region of the gene to produce a mutant receptor protein. Such mutations, for example,
It can be introduced into a specific functional region such as a ligand binding site or a G protein binding site.

In one embodiment, the host cell can be a fertilized oocyte or embryonic stem cell that can be used to produce a transgenic animal containing the modified receptor gene. Alternatively, host cells can be stem cells or other early tissue precursors that give rise to a particular subset of cells, and can be used to produce transgenic tissue in animals. For a description of homologous recombination vectors, see Thomas et al., Cell 51: 503 (19
87). The vector is introduced into an embryonic stem cell line (for example, by electroporation), and the introduced gene is selected from cells that have homologously recombined with the endogenous receptor gene (for example, Li, E et al.). Cell 69: 9
15 (1992)). The selected cells are then injected into undifferentiated germ cells of an animal (eg, a mouse) to form an aggregate chimera (eg, “Teratocarcinoma and embryonic stem cells: a practical approach ( Teratocarc
inomas and Embryonic Stem Cells: A Pr
physical Approach) ", E.T. J. Robertson (
Bradley, A., IRL, Oxford, 1987). See the writing section, pages 113-152). The chimeric germ layer is then implanted in a suitable pseudopregnant female foster animal and the embryo is grown to term. The offspring harboring the homologously recombined DNA in the germ cell can be used to raise an animal that contains the homologously recombined DNA by germline transmission of the transforming gene to all cells of the animal. Can be used. Methods for constructing homologous recombination vectors and homologous recombinant animals are described in more detail in Bradley, A .; (1991) "Cu
rent Opinion in Biotechnology "2: 823
-829 and PCT International Publication No. WO 90/11354, WO 91/011
No. 40, WO 93/04169.

Genetically engineered host cells can be used to produce non-human transgenic animals. The transgenic animal is preferably a mammal, for example, a rodent such as a rat or mouse, wherein one or more of the animal's cells contains a transforming gene. A transforming gene is exogenous DNA that is integrated into the genome of one cell from which the transgenic animal grows, and the mature gene in one or more cell types or tissues of the transgenic animal. Exogenous DNA that remains in the genome. These animals are useful for studying receptor protein function and for identifying and assessing modulators of receptor protein activity.

Other examples of transgenic animals include non-human apes, sheep, dogs,
Includes cattle, goats, chickens and amphibians.

In one embodiment, the host cell is a fertilized oocyte or embryonic stem cell into which the receptor polynucleotide sequence has been introduced.

A transgenic animal can be produced by introducing a nucleic acid into the male pronucleus of a fertilized oocyte, for example, by microinjection, retroviral infection, and Can be developed in pseudopregnant female foster animals. The receptor nucleotide sequence can be introduced as a transforming gene into the genome of a non-human animal such as a mouse.

Any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intron sequences and polyadenylation signals if they are not already contained. Tissue-specific regulatory sequences can be operably linked to the transforming gene to direct the expression of the receptor protein to a particular cell.

Methods for producing transgenic animals, particularly animals such as mice, via embryo manipulation and microinjection have become routine to those skilled in the art and are described, for example, in US Pat. No. 866, 4,870,009 (both Leder et al.); U.S. Pat. No. 4,873,191 to Wagner et al.
Also, Hogan, B,. "Manipulating the mouse embryo"
e Mouse Embryo) ", (Cold Spring Harbo)
r Laboratory Press, Cold Spring Harb
or, N. Y. , 1986). Similar methods have been used for the production of other transgenic animals. The transgenic founder is a transgenic mRNA of the animal tissue or cell.
Can be identified based on the presence of the transformed gene in its genome and / or expression. The transgenic founder can then be used to breed additional animals carrying the transformed gene. Furthermore, a transgenic animal carrying a transforming gene can be further bred to another transgenic animal carrying another transforming gene. Transgenic animals also include animals produced using the host cells in which the whole animal or tissue is recombined into the homology described in this application.

In another embodiment, a transgenic non-human animal can be produced that includes a selection system that allows for regulated expression of the transformed gene. One example of such a system is the cre / loxP recombinase system of bacteriophage P1. For a description of the cre / loxP recombinase system, see, eg, Lakso et al., PNAS 89: 6232-6236 (1.
992). Another example of a recombinase system is the S. recombinase system.
cerevisiae FLP recombinase system (O'Gorma
n et al., Science 251: 1351-1355 (1991)). cre
If the / loxP recombinase system is used to regulate the expression of the transforming gene, an animal containing the transforming gene encoding both the Cre recombinase and the selected protein is required. Such animals can be "double" transfected by, for example, crossing two transgenic animals, one containing a transforming gene encoding a selection protein and the other containing a transforming gene encoding a recombinase. It can be provided by construction of a transgenic animal.

The non-human transgenic animals described in the present application also include Wil
mut, I .; Et al., Nature 385: 810-813 (1997) and PC
It can be produced by the methods described in International Publication Nos. WO 97/07668 and WO 97/07669. Briefly, cells from transgenic animals, eg, somatic cells, can be separated and induced to leave the growth cycle and enter the Go phase. Quiescent cells can be fused to enucleated oocytes from the same species from which the quiescent cells are subsequently separated, for example, by using an electrical pulse. The reconstructed oocyte is then cultured so that it develops into a morula or an undifferentiated germ cell and subsequently transfers to a pseudopregnant female foster animal. The offspring born from this female foster animal will be a clone of the animal from which cells, for example, somatic cells, will be isolated.

Transgenic animals containing recombinant cells that express the polypeptides described in this application are useful for performing the measurements described in this application in an in vivo context. Therefore, the various physiological factors that exist in vivo and affect ligand binding, receptor activation, and signal transduction may not be evident from cell-free or cell-based measurements in vitro. Conceivable. Therefore, it would be useful to provide non-human transgenic animals for measuring receptor function in vivo, including ligand interaction, receptor function and the effects of specific mutant receptors on ligand interaction, and the effects of chimeric receptors. It is. Although a null mutation is a mutation that substantially or completely eliminates one or more receptor functions, it is also possible to evaluate the effects of the null mutation.

Pharmaceutical component receptor nucleic acid molecules, proteins (especially fragments such as extracellular domains), modulators of the protein, and antibodies (referred to in the present application as "active ingredients") can be administered to a subject, eg, a human. It can be incorporated into pharmaceutical ingredients suitable for administration. Such components typically comprise nucleic acid molecules, proteins, modulators, or antibodies, as well as pharmaceutically acceptable carriers.

As used in this application, the term “pharmaceutically acceptable carrier” refers to any and all solvents, compatible with pharmaceutical administration,
It is intended to include dispersing media, coatings, antibacterial and antifungal agents, isotonic solutions, absorption delaying agents, and the like. Such vehicles and agents for pharmaceutically active substances are well known to those skilled in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, such media can be used in the components of the present invention. Supplementary active ingredients are also incorporated into the ingredients. A pharmaceutical component of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include, for example, parenteral, such as intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (topical), transmucosal, rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous applications can include the following ingredients. That is, sterile diluents such as water for injection, physiological saline, fixed oil, polyethylene glycol, glycerin, and propylene glycol, or other synthetic solvents; benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite Agents; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetic acid, citric acid or phosphoric acid and agents for regulating tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be sealed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical ingredients suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. . For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELJ (BASF, Parsi
ppany, New Jersey) or phosphate buffered saline (PBS)
Is included. In all cases, the components must be sterile and, to some extent, must be fluids that can be easily syringed. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), or a suitable mixture thereof. Suitable fluidity, for example, by the use of a coating such as lecithin,
In the case of a dispersion, it can be maintained by maintaining the required particle size and by using a surfactant. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, components include isotonic agents,
For example, it is desirable to include saccharides such as mannitol, sorbitol, and sodium chloride, and polyhydric alcohols. Long-acting absorption of the injectable component can be brought about by including in the component, for example, aluminum monostearate and an agent that delays the absorption of gelatin.

Sterile injectable solutions are prepared by incorporating the active ingredient (eg, receptor protein or anti-receptor antibody) in the required amount in an appropriate solvent with one or a combination of the above-listed components. After that, necessary filtration sterilization is performed. Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the production of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization, whereby the powder of the active ingredient and, in addition to it, previously sterile filtered Produce any desired additional components from the solution.

[0240] Oral ingredients generally include an inert diluent or an edible carrier. They can be sealed in gelatin capsules or compressed into tablets. For oral administration, the drug can be included in the form of an enteric coating to save the stomach and can be further coated or mixed for release in specific areas of the gastrointestinal tract by well known methods. You. For the purpose of oral therapeutic administration, the active ingredient can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral components can also be made using a fluid carrier for use as a mouthwash, in which case the components in the fluid carrier are applied orally and whisper inhaled. And then expectorant, or swallowed. Pharmaceutically compatible binding agents, and / or adjuvants can be included as part of the ingredient. Tablets, pills, capsules, troches and the like can contain any of the following components or a component of a similar nature: Binders such as microcrystalline cellulose, tragacanth gum or gelatin; excipients such as starch or lactose; decomposing agents such as alginic acid, primogel or corn starch; lubricants such as magnesium stearate or Sterates; colloidal silicon dioxide Glidants, sweeteners such as sucrose or saccharin; or flavors such as peppermint, methyl salicylate, or orange flavors.

For administration by inhalation, the components are delivered in the form of a pressure container or dispenser containing a suitable propellant, eg, a gas such as carbon dioxide, or an aerosol spray from a nebulizer.

[0242] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known to those of skill in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active ingredients are formulated into ointments, salves, gels, or creams as generally known to those skilled in the art.

The components can also be made in the form of suppositories (eg, the usual suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active ingredient is prepared with carriers that will protect the ingredient against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. You. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and the like. Methods for preparing such formulations will be apparent to those skilled in the art. The material is also A
lza Corporation and Nova Pharmaceuticals
Inc. It is commercially available from. Liposomal suspensions (including liposomes targeted to infected cells by monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, in US Pat. No. 4,522,81.
Prepared as described in No. 1.

It is especially advantageous to formulate oral or parenteral ingredients in dosage unit form for ease of administration and uniformity of dosage. As used in this application, dosage unit form refers to the appropriate number of physically discrete units as a unit dose for a subject to be treated. That is, each unit contains a predetermined quantity of active ingredient which has been calculated to produce the desired therapeutic effect associated with the required pharmaceutical formulation carrier. The specifications for the dosage unit forms of the present invention dictate and are directly dependent on the unique characteristics of the active ingredient, and may achieve a particular therapeutic effect, but will also There are inherent technical limitations in the preparation of such active ingredients for therapy.

[0246] The nucleic acid molecules of the present invention can be inserted into a vector and can be used as a gene therapy vector. Gene therapy vectors can be administered, for example, by intravenous injection, local administration (US Pat. No. 5,328,470), or by stereotactic injection (see, for example, Chen et al., PNAS 91: 3054-3057 (1994)). Can be delivered to a subject. Pharmaceutical formulations of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or the formulation can consist of a sustained release matrix in which the gene delivery vehicle is embedded. Alternatively, a complete gene delivery vector can be produced directly from a recombinant cell, eg, a retroviral vector, and the pharmaceutical formulation can include one or more cells that create the gene delivery system. it can.

The pharmaceutical component can include a container, package, or dispenser together with instructions for administration.

The present invention may be embodied in many different forms, but should not be construed as limiting any of the embodiments set forth in the present application. Rather, these embodiments are provided so that this disclosure will fully convey the invention to those skilled in the art. Many modifications and other embodiments of the present invention have
The invention will be apparent to those skilled in the art to which the invention pertains and which will benefit from the teachings provided in the foregoing description. Certain terms are used,
These terms are used in the art and have been briefly outlined otherwise.

Example Gene Expression Studies by TaqMan Quantitative Polymerase Chain Reaction Total RNA from various tissues was extracted using a single step method according to the manufacturer's instructions (RNA STAT-60, TelTest, Inc.).
). Each RNA preparation was treated with DNase I (Ambion) at 37 ° C. for 1 hour. After phenol extraction, the samples were prepared according to the manufacturer's instructions (GibcoBRL).
Were subjected to reverse transcription using a Superscript kit. A negative control sample containing RNA but no reverse transcription was run simultaneously. The simulated reverse transcription sample generated in the case where no reverse transcription is present is DN
Each RNA / cDNA sample was tested to ensure that the Case I treatment was complete. The integrity of the RNA samples after DNase I treatment was examined by agarose gel electrophoresis and ethidium bromide staining. Internal reference amp
If at least 38 amplification cycles are required to reach the fluorescence threshold level using licon β-2 microglobulin, the sample is required to receive complete DNase I treatment.

The probe uses the consensus sequence to obtain Pri from PE Biosystems.
Designed by merExpress software. RNA of gene 2871
The primer and probe sequences for expression analysis are as follows.

[0251]

[Table 2]

The target probe gene 2871 is labeled using 6-carboxyfluorescein (FAM). The internal reference amplicon for β2-microglobulin is labeled using VIC. Thus, the levels of the target gene and the internal reference can be measured in the same test tube by multiplex PCR. Forward and reverse primers and probes for both the internal reference gene and the target gene are TaqMan U
universal PCR Mater Mix (PE Applied B
iosystems). The final densities of the primers and probes vary, but are internally consistent within any experiment. In one typical experiment, 200 nM forward and reverse primers for β2-microglobulin,
In addition, a 100 nM probe is included, and for the target gene, 600 nM forward and reverse primers, plus a 200 nM probe. TaqMan
Matrix experiments were performed using the ABI PRPSM 7700 Sequence Detection System (PE
Applied Biosystems). The thermal cycler conditions are as follows. That is, hold at 50 ° C. for 2 minutes and at 95 ° C. for 10 minutes,
This is followed by a two step PCR 40 cycle, melting at 95 ° C for 15 seconds and annealing / extension at 60 ° C for 1 minute.

[0253] RNA from various tissues and cell types was purified and converted to cDNA using reverse transcriptase. Cells and tissues used to analyze 2871 include:
Includes the following: Various organs including lymph nodes, spleen, thymus, heart, brain, liver, fetal liver, fibrotic liver; 0 or 24 hours stimulated by antibodies against CD3 subunit of T cell receptor (_CD3 stimulation) in
In vitro differentiated helper T cell population; resting and _CD3 stimulation exv from peripheral blood
Ivo purified CD3 T cells; granulocytes, CD4, CD8 positive cells, B cells purified by anti-CD19 antibody and stimulated by LPS for 24 hours, resting and peripheral blood mononuclear cells (PBMC) stimulated by phytoagglutinin And other cells purified from peripheral blood. Other cells analyzed in this experiment include CD34 positive and negative (CD34 + and CD34-) cells or leukocytes purified from peripheral blood (mPB) or bone marrow (mBM) of patients treated with G-CSF. It is. CD34 + and CD34- cells were also purified from normal adult bone marrow (ABM) or spinal cord blood (CB). Nuclear peripheral blood (mPB) or bone marrow (mBM) of patients treated with G-CSF was also examined. Erythroblasts from normal bone marrow were also examined. Transformed cell lines include erythroleukemia cell line K562, acute promyelocytic leukemia cell line HL-60, and Hep3b hepatocellular carcinoma cells cultured under normal or reduced oxygen pressure.

The comparative Ct method is used for relative quantification of gene expression. The threshold cycle or Ct value is the cycle at which a statistically significant increase in ΔRn is detected. A low Ct value indicates a higher density of mRNA of the gene corresponding to the target probe sequence. The Ct value of the target gene is normalized relative to the internal reference gene Ct value to purify the delta Ct value using the following formula. That is, ΔCt = Ct target−Ct reference A cDNA sample with a relatively low expression level in the matrix is selected as a calibrator sample to generate a value for relative expression. The ΔCt value of the calibrator tissue is then subtracted from ΔCt for each, according to the following formula: That is, the value to be used for [Delta] [Delta] Ct = ACt sample -ΔCt calibrator relative expression 2 ^- is calculated using the arithmetic formula given by .DELTA..delta ^Ct. This value is then used to graph the relative expression of the target gene in multiple tissues in this study.

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows a 2871 nucleotide sequence (SEQ ID NO: 2) and a deduced 2871 amino acid sequence (SEQ ID NO: 1). Amino acids 1-42 are predicted to constitute an extracellular domain, amino acids 43 to 318 constitute a transmembrane domain, and amino acids 319 to 359 are predicted to constitute an intracellular domain.

FIG. 2 shows 2871 for the protein pattern in the Prosite database.
Figure 3 shows a comparison of the receptors, especially showing a high score for the seven transmembrane domain rhodopsin family. The underlined area indicates the GPCR signature. The most common conserved intracellular sequences are aspartate, arginine, tyrosine (DRY) adjacent to TM3.
It is a triplet. Arginine is unchanged. Aspartate has several GPs
Placed conservatively in CR. DRY is involved in signal transduction.

FIG. 3 shows analysis of 2871 amino acid sequence: αβ turn and coil region; hydrophilicity; amphipathic region; movable part; antigenicity coefficient; and surface probability.

FIG. 4 shows a 2871 receptor hydrophobicity plot. 43-318 amino acids
Correspondingly, seven transmembrane segments are shown.

FIG. 5 shows 2871 RNA expression in various tissues.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/15 C12N 1/19 4C084 1/19 1/21 4H045 1/21 C12P 21/02 C 5/10 C12Q 1/68 A C12P 21/02 G01N 33/68 C12Q 1/68 C12N 15/00 ZNAA G01N 33/68 5/00 A (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES , FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE) , SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, R U, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB , GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ , VN, YU, ZA, ZWF terms (reference) 2G045 AA25 AA34 AA35 AA40 BB20 CB01 DA12 DA13 DA14 DA36 DA77 FB02 4B024 AA01 AA11 BA44 BA63 DA02 DA06 EA04 GA11 HA01 4B063 QA01 QQ43 4 AA26X AA90X AA93Y AA99Y AB01 BA02 CA24 CA44 4C084 AA17 NA14 ZB111 ZB112 ZC022 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA50 DA76 EA20 EA50 FA74

Claims (20)

[Claims] 1. an amino acid sequence represented by SEQ ID NO: 1; (C) the amino acid sequence of an allelic variant of the amino acid sequence shown in SEQ ID NO: 1, (d) the ATCC deposit number (E) an amino acid sequence of a sequence variant of the amino acid sequence shown in SEQ ID NO: 1, which is a nucleic acid shown in SEQ ID NO: 2; An amino acid sequence encoded by a nucleic acid molecule that hybridizes under stringent conditions to the molecule; (f) ATCC Deposit No. The amino acid sequence of a sequence variant of the amino acid sequence encoded by the cDNA clone contained in SEQ ID NO. An amino acid sequence encoded by a nucleic acid molecule that hybridizes under stringent conditions to the cDNA contained in the amino acid sequence; (g) a fragment of the amino acid sequence set forth in SEQ ID NO: 1 comprising at least 12 contiguous fragments; A fragment containing an amino acid; (h) ATCC Deposit No. A fragment of the amino acid sequence encoded by the cDNA contained in SEQ ID NO: 1, comprising at least 12 contiguous amino acids; (J) the amino acid sequence of the mature receptor polypeptide of (K) the amino acid sequence of the mature polypeptide from about the 6th amino acid to about the 359th amino acid encoded by the cDNA clone contained in SEQ ID NO: 1; About 31
The amino acid sequence of the transmembrane domain up to the eighth amino acid, (l) ATCC Deposit No. Amino acid sequence of the transmembrane domain from about amino acid number 43 to about amino acid number 318 in the polypeptide encoded by the cDNA contained in SEQ ID NO: 1; and (m) any of the polypeptides (a) to (l) An isolated polypeptide having an amino acid sequence selected from the group consisting of the amino acid sequence of one of the epitope producing regions. 2. An isolated antibody which selectively binds to the polypeptides (a) to (m) of claim 1. (3) a nucleotide sequence represented by SEQ ID NO: 2, (b) an ATCC accession number (C) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1; (d) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1; (E) a nucleotide sequence that is complementary to any of the nucleotide sequences of (a), (b), (c) or (d); An isolated nucleic acid molecule having a nucleotide sequence selected from the group consisting of: (A) a nucleotide sequence encoding an amino acid sequence of a sequence variant of the amino acid sequence shown in SEQ ID NO: 1 which is higher than the nucleotide sequence shown in SEQ ID NO: 2 under stringent conditions; (B) ATCC Deposit No. A nucleotide sequence encoding an amino acid sequence of a sequence variant of the amino acid sequence encoded by the cDNA contained in the sequence number, wherein the nucleic acid sequence of the sequence variant is ATCC Deposit No. Contained in the code
A nucleotide sequence that hybridizes to the NA under stringent conditions; and (c) a nucleotide sequence that is complementary to any of the nucleotide sequences of (a) or (b). An isolated nucleic acid molecule having the formula: 5. (a) a fragment of the amino acid sequence shown in SEQ ID NO: 1,
A nucleotide sequence encoding a fragment comprising at least 12 contiguous amino acid sequences, (b) ATCC Deposit No. (C) a nucleotide sequence encoding a fragment of the amino acid sequence encoded by the cDNA contained in the sequence, the fragment comprising at least 12 contiguous amino acid sequences; An isolated nucleic acid molecule having a nucleotide sequence selected from the group consisting of: 6. A nucleic acid vector comprising the nucleic acid sequence according to claim 3. 7. A host cell containing the vector according to claim 6. 8. A method for producing any of the polypeptides according to claim 1, wherein a nucleotide sequence encoding any of the polypeptide sequences (a) to (m) is introduced into a host cell. And culturing said host cell under conditions in which a protein is expressed from said nucleic acid. 9. A method for detecting the presence of any of the polypeptides of claim 1 in a sample, wherein the sample is contacted with an agent capable of specifically detecting the presence of the polypeptide in the sample. And then detecting the presence of said polypeptide. 10. The method of claim 9, wherein said agent allows selective physical association with said polypeptide. 11. The method of claim 10, wherein said agent binds to said polypeptide. 12. The method according to claim 11, wherein said agent is an antibody. 13. The method of claim 11, wherein said agent is a ligand. 14. A kit comprising a reagent used in the method of claim 9, wherein the reagent comprises an agent that specifically binds to the polypeptide. 15. A method for detecting the presence of any of the nucleic acid sequences according to any one of claims 3 to 5 in a sample, the method comprising detecting the presence of the nucleic acid sequence under stringent conditions. Contacting a sample with the oligonucleotide to be hybridized; and detecting whether the oligonucleotide binds to the nucleic acid sequence in the sample. 16. The method of claim 15, wherein said nucleic acid whose presence is detected is an mRNA. 17. A kit comprising reagents used in the method of claim 15, wherein said reagents comprise a compound that hybridizes under stringent conditions to any of said nucleic acid molecules. 18. A method for identifying an agent that binds to any of the polypeptides of claim 1, comprising contacting the polypeptide with an agent that binds to the polypeptide; Assaying the complex formed by said agent bound to the agent. 19. A method for modulating the activity of any of the polypeptides of claim 1, wherein any of the polypeptides of claim 1 comprises:
Contacting under conditions whereby the activity of the polypeptide can be modulated by the agent. 20. The method of claim 19, wherein the activity is modulated in a subject having an inflammatory disease.