JP2003504023A - 16405 receptor, a novel G protein-coupled receptor - Google Patents

Abstract

  (57) [Summary] The present invention relates to newly identified receptors belonging to the G protein-coupled receptor superfamily. The present invention also relates to a polynucleotide encoding the receptor. The invention further relates to methods of using receptor polypeptides and polynucleotides as targets for the diagnosis and treatment of receptor-mediated diseases. The present invention further relates to drug screening methods using receptor polypeptides and polynucleotides to identify agonists and antagonists for diagnosis and therapy. The invention further includes agonists and antagonists based on receptor polypeptides and polynucleotides. The invention further relates to methods for making receptor polypeptides and polynucleotides.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to a newly identified cyclic nucleotide receptor belonging to the superfamily of G protein coupled receptors. The present invention also relates to polynucleotides that encode the receptor. The invention further relates to methods of using the receptor polypeptides and polynucleotides as targets for the diagnosis and treatment of receptor-mediated diseases. The invention further relates to methods of drug screening that use receptor polypeptides and polynucleotides to identify agonists and antagonists for diagnosis and therapy. The invention further includes agonists and antagonists based on the receptor polypeptides and polynucleotides. The invention further comprises
Techniques for making receptor polypeptides and polynucleotides.

BACKGROUND ART G protein-coupled receptors G protein-coupled receptors (GPCR) constitute a major class of proteins responsible for signal transduction in cells. GPCRs have three structural domains: an amino-terminal extracellular domain, a transmembrane domain containing seven transmembrane segments, three extracellular loops and three intracellular loops and a carboxy-terminal intracellular domain. When a ligand binds to the extracellular portion of the GPCR, a signal is transduced into the cell, altering the biological or physiological properties of the cell. GPCRs are G proteins and effectors (intracellular enzymes and channels regulated by G proteins)
Together, it is a component of the modular signaling system that connects intracellular second messenger status to extracellular inputs.

GPCR genes and gene products appear to be causative agents of disease (Spiegel et.
al., J. Clin. Invest. 92: 1119-1125 (1993); McKusick et al., L. Med. Gen.
et. 30: 1-26 (1993)). Certain defects in the rhodopsin gene and V2 vasopressin receptor have led to various forms of retinitis pigmentosa (Nathans et al., Annu. Rev. Genet. 2
6: 403-424 (1992)) and renal diabetes insipidus (Holtzman et al., Hum. Mol. Genet. 2
: 1201-1204 (1993)). These receptors are of considerable importance to the central nervous system and peripheral physiological processes. Evolutionary analysis suggests that the ancestors of these proteins initially co-occur with complex body formation plans and the nervous system.

The GPCR protein superfamily can be divided into five families. Family I (Dohlman et al., Annu., Typed by rhodopsin and β2-adrenergic receptors and is now represented by more than 200 unique members.
Rev. Biochem. 60: 653-688 (1991)), Family II (Juppner et al., Science 254: 1024-1026 (19), which is a parathyroid hormone / calcitonin / secretory receptor family.
91); Lin et al., Science 254: 1022-1024 (1991)), a family of metabotropic glutamate receptors, family III (Nakanishi, Science 258 597: 603 (1992)).
, A family of cAMP receptor family IV (Klein et al., Science 241: 1467-1472 (1988)) and fungal mating pheromone receptors such as STE2 that are important for chemotaxis and development of D. discoideum V (Kurjan, Annu. Rev. Biochem.
61: 1097-1129 (1992)).

There are also a few other proteins that display seven putative hydrophobic segments and appear to be unrelated to GPCRs, which have not been shown to bind to G proteins. Drosophila is a photoreceptor-specific protein, bride of sevenless (bos
s), which expresses seven transmembrane segment proteins that have been extensively studied and have not been shown to be GPCRs (Hart et al., Proc. Natl. Acad. Sci. USA).
90: 5047-5051 (1993)). The Drosophila gene frizzled (fz) is also considered to be a protein having seven transmembrane segments. Like boss, fz is G
Not shown to bind to proteins (Vinson et al., Nature 338: 263-
264 (1989)).

The G protein represents a family of heterotrimeric proteins that contain α, β and γ subunits that bind guanine nucleotides. These proteins normally bind to cell surface receptors, such as those containing seven transmembrane segments. The binding of the ligand to the GPCR transfers a conformational change to the G protein, which causes the α-subunit to exchange bound GDP molecules for GTP molecules and dissociate them from the βγ-subunits. The GTP-bound form of the α-subunit typically functions as an effector regulatory moiety, producing a second messenger such as cAMP (eg, by activation of adenyl cyclase), diacylglycerol or inositol phosphate. Over 20 different types of α-subunits are known in humans. These subunits are smaller pools of α
, Β and γ subunits. Examples of mammalian G proteins include Gi, Go, G
q, Gs and Gt. G-protein is derived from Lodish et al., Molecular Cell.
Biology, (Scientific American Books Inc., New York, NY, 1995). This document is hereby incorporated by reference. GPCRs, G proteins and G protein-coupled effectors and second messenger systems are described in The G-protein Linked Receptor Fact Book, Watson et a
l., eds., Academic Press (1994).

GPCRs are major targets for drug action and development. Therefore, a previously unknown GP
Identifying and characterizing CR is useful in the field of drug development. The present invention advances the state of the art by providing hitherto unidentified human GPCRs.

SUMMARY OF THE INVENTION The object of the present invention is to identify novel GPCRs. Yet another object of the invention is to provide novel GPCR polypeptides useful as reagents or targets in receptor assays applicable in the treatment and diagnosis of GPCR-mediated diseases.

[0009] Yet another object of the present invention is useful as a receptor assay target and reagent applicable in the treatment and diagnosis of G protein-mediated diseases, in order to produce novel receptor polypeptides by recombinant methods. It is to provide a polynucleotide corresponding to a novel receptor polypeptide that is useful.

A specific object of the invention is to identify compounds that act as agonists and antagonists and modulate the expression of novel receptors.

Yet another specific object of the invention is to provide compounds that modulate receptor expression for treating and diagnosing receptor-related disorders.

The present invention is therefore based on the identification of a novel receptor designated as the 16405 receptor.

The present invention provides the amino acid sequence shown in SEQ ID NO: 1 or American Type Culture Colle
ction, 10801 University Blvd., Manassas, VA 20110-2209 US containing a polypeptide having an amino acid sequence encoded by a cDNA deposited under ATCC No. PTA-1653 on April 5, 2000 ("Deposited cDNA"). , An isolated receptor polypeptide is provided.

The present invention also provides an isolated 16405 receptor nucleic acid molecule having the sequence set forth in SEQ ID NO: 2 or the deposited cDNA.

The invention also provides a variant polypeptide having SEQ ID NO: 1, or an amino acid sequence that is substantially homologous to the amino acid sequence encoded by the deposited cDNA.

The present invention also provides a variant nucleic acid sequence that is substantially homologous to the nucleotide sequence set forth in SEQ ID NO: 2 or the deposited cDNA.

The invention also provides fragments of the polypeptide set forth in SEQ ID NO: 1 and nucleotides set forth in SEQ ID NO: 2, as well as substantially homologous fragments of the polypeptide or nucleic acid.

The present invention further provides a nucleic acid construct comprising the above nucleic acid. In a preferred embodiment, the nucleic acids of the invention are operably linked to regulatory sequences.

The present invention provides vectors and host cells for expressing receptor nucleic acid molecules and polypeptides, and in particular recombinant vectors and host cells.

The present invention also provides methods of making vectors and host cells and methods of using them to make receptor nucleic acid molecules and polypeptides.

The present invention also provides antibodies or antigen-binding fragments that selectively bind to receptor polypeptides and fragments.

The invention also provides methods of screening for compounds that modulate the expression or activity of a receptor polypeptide or nucleic acid (RNA or DNA).

The present invention also provides methods of modulating receptor polypeptide or nucleic acid expression or activity, particularly using the screened compounds. Modulation can be used to treat conditions associated with aberrant activity or expression of receptor polypeptides or nucleic acids, particularly heart disease and pain management.

The invention also provides assays, including for the diagnosis of disease, that determine the presence or level of receptor polypeptides or nucleic acid molecules in a biological sample.

The present invention also provides assays, including the diagnosis of disease, which measure the presence of a mutation in a receptor polypeptide or nucleic acid molecule.

In yet another embodiment, the invention provides a computer readable means containing the nucleotide and / or amino acid sequences of the nucleic acids and polypeptides of the invention, respectively.

DETAILED DESCRIPTION OF THE INVENTION Receptor Function / Signal Pathway 16405 Receptor protein is a GPCR involved in the signaling pathway. As used herein, "signaling pathway" refers to the regulation (eg, stimulation or inhibition) of cell function / activity as a result of ligand binding to a GPCR (16405 protein).
Examples of such functions involve signaling pathways such as, for example, phosphatidylinositol 4,5-bisphosphate (PIP ₂ ), inositol 1,4,5-triphosphate (IP ₃ ), and adenylate cyclase. This includes migration of intracellular molecules, polarization of the plasma membrane, production or secretion of molecules, changes in the structure of cellular components, eg cell proliferation such as DNA synthesis, cell migration, cell differentiation and cell survival. Since the 16405 receptor protein is expressed in the spleen, brain, glioma and sclerotic lesions (derived from atherosclerotic tissue), cells involved in the 16405 receptor protein signaling pathway include cells derived from these tissues. , But not limited to them.

The response mediated by the receptor protein depends on the cell type.
For example, in some cells, the binding of a ligand to a receptor protein may result in the release of compounds, channel gating, cell adhesion, migration, differentiation, etc. due to metabolism and turnover of phosphatidylinositol or cyclic AMP. It may stimulate, but in other cells, binding of the ligand produces different results. Despite cellular activities / responses mediated by receptor proteins, the proteins are GPCRs and GPCRs in various intracellular signaling pathways, for example, via phosphatidylinositol or cyclic AMP metabolism or turnover in cells.
It is invariant to interact with proteins to form one or more secondary signals.

As used herein, “phosphatidylinositol turnover and metabolism” refers to molecules involved in phosphatidylinositol 4,5-bisphosphate (PIP ₂ ) turnover and metabolism and the activity of these molecules. Say. PIP ₂ is a phospholipid found in the cytosolic leaflet of the plasma membrane. In some cells, when the ligand binds to the receptor it activates the plasma membrane enzyme phospholipase C,
This hydrolyzes PIP ₂ to give 1,2-diacylglycerol (DAG) and inositol 1
It is capable of producing 4,4,5-triphosphate (IP ₃ ). Once IP ₃ formed can diffuse to the endoplasmic reticulum surface where IP ₃ receptor, for example, can bind to the calcium channel protein containing an IP ₃ binding site. IP ₃ binding induces the opening of the channel, thereby releasing calcium ions into the cytoplasm. IP ₃ can also be phosphorylated by a specific kinase, is a molecule that can flow into the calcium from the extracellular medium into the cytoplasm, inositol 1,3,4,5 tetraphosphate
(IP ₄ ) can be formed. IP ₃ and IP ₄ then very rapidly proceed to the inactive products inositol 1,4-biphosphate (IP ₂ ) and inositol 1,3, respectively.
Can be hydrolyzed to 1,4-triphosphate. These inactive products can be recycled by the cells to synthesize PIP ₂ . Another second messenger produced by the hydrolysis of PIP ₂ , namely 1,2-diacylglycerol (DAG), remains in the cell membrane where it may serve to activate the enzyme protein kinase C. Protein kinase C is normally found in the cytoplasm of cells, but when intracellular calcium levels are elevated, this enzyme can move to the cell membrane where it can be activated by DAG. Activation of protein kinase C in different cells results in different cellular responses such as phosphorylation of glycogen synthase or phosphorylation of different transcription factors, eg NF-kB. As used herein
The term phosphatidylinositol activity "refers to one activity of PIP ₂ or its metabolites.

Another signaling pathway in which the receptor may be involved is the cAMP turnover pathway. As used herein, "cyclic AMP turnover and metabolism" refers to
It refers to molecules involved in the turnover and metabolism of cyclic AMP (cAMP) 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. In the cAMP signaling pathway, ligand binding to GPCR activates the enzyme adenylate cyclase, which catalyzes the synthesis of cAMP. The newly synthesized cAMP can activate a cAMP-dependent protein kinase. This activated kinase is voltage gated
It can phosphorylate (voltage-gated) potassium channel proteins or related proteins and not open potassium channels during the application of action potentials. If potassium channels are not opened, the outward flux of potassium is diminished, and neuronal membranes are usually repolarized, prolonging membrane depolarization.

Polypeptides The present invention is based on the discovery of novel G-coupled protein receptors. In particular,
An expressed sequence tag (EST) is selected based on its homology with the G protein coupled receptor sequence. This EST is used to design a primer based on the sequence it contains, and also EST is used to clone a cDNA from a human spleen cDNA library.
A was identified. Positive clones were sequenced and overlapping fragments assembled. Analysis of the assembled sequences revealed that the cloned cDNA molecule encodes a G protein-coupled receptor.

Accordingly, the present invention relates to a novel GPCR having the deduced amino acid sequence shown in Figure 1 (SEQ ID NO: 1) or having the amino acid sequence encoded by the deposited cDNA, ATCC No. PTA-1653.

The deposit refers to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms (the Budapest Trunk
eaty on the International Recognition of the Deposit of Microorganisms)
Be managed in accordance with. The deposit is provided as a convenience to those of ordinary skill in the art and is not a permit required under 35 U.SC § 112. The deposited sequences as well as the polypeptides encoded by the sequences are incorporated herein by reference and any discrepancies such as sequencing errors will be adjusted using the description herein.

“16405 Receptor Polypeptide” or “16405 Receptor Protein” refers to SEQ ID NO:
1 or refers to the polypeptide encoded by the deposited cDNA. However, the term "receptor protein" or "receptor polypeptide" further includes numerous variants described herein as well as full length polypeptides and fragments derived from variants.

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

The 16405 polypeptide is a 383-residue protein that exhibits three major structural domains, an amino-terminal extracellular domain, a transmembrane region and a carboxy-terminal intracellular domain. The transmembrane domain contains seven segments that span the membrane. There are three intracellular loops and three extracellular loops in the region spanning the transmembrane domain.

The transmembrane domain contains the GPCR signaling property, RYL, at residues 137-139. The sequence contains arginine, a conserved amino acid in the GPCR, at residue 138.

As used herein, a polypeptide, when it is isolated from recombinant and non-recombinant cells, is substantially free of cellular material, or is chemically synthesized, If it contains substantially no chemical precursors or other chemicals,
It is said to be “isolated” or “purified”. However, a polypeptide may be associated with another polypeptide that normally does not bind intracellularly, and is still considered "isolated" or "purified".

The receptor polypeptide can be purified to homogeneity. However, preparations where the polypeptide has not been purified to homogeneity are also useful and are believed to contain the polypeptide in isolated form. An important feature is that the preparation is capable of exerting the desired polypeptide function of the polypeptide in the presence of large amounts of other components. Thus, the present invention includes varying degrees of purity.

In one embodiment, the term “substantially free of cellular material” means about 30
% (Dry weight%) other proteins (ie contaminating proteins), less than about 20% other proteins, less than about 10% other proteins or less than about 5% other proteins. Preparations of. If the receptor polypeptide is made by recombinant techniques, the receptor polypeptide can also be substantially free of culture medium, i.e., the culture medium is less than about 20% of the protein preparation, Less than about 10% or less than about 5%.

Receptor polypeptides are also considered to be isolated if they are part of a membrane preparation or if purified and then reconstituted with membrane vesicles or liposomes.

The term “substantially free of chemical precursors or other chemicals” refers to a preparation of receptor polypeptides that is separated from the chemical precursors or other chemicals involved in its synthesis. including. In one embodiment, the term "substantially free of chemical precursors or other chemicals" means less than about 30% (dry weight%) chemical precursors or "other chemicals, about 20%. Less than about 10% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or preparations of polypeptides with less than about 5% chemical precursors or other chemicals. Including.

In one embodiment, the receptor polypeptide comprises the amino acids set forth in SEQ ID NO: 1. However, the invention also includes sequence variants. Variants include substantially homologous proteins encoded by the same locus in an organism, ie allelic variants. The 16405 receptor is mapped to chromosome 1 adjacent to the AFM297zg1 marker. Variants also include proteins from other loci in the organism,
1 includes a protein having substantial homology to the 16405 receptor protein. Variants also include proteins that are substantially homologous to the 16405 receptor protein, but that are derived from another organism, namely orthologs. Variants also include proteins that are substantially homologous to the 16405 receptor protein made by chemical synthesis. Variants also include proteins that are substantially homologous to the 16405 receptor protein produced by recombinant methods. However, it is understood that variants do not include any of the previously disclosed amino acid sequences of the invention.

As used herein, two proteins (or regions of proteins) are
Substantially if the amino acid sequence is at least about 55-60%, typically about 70-75%, more typically at least about 80-85%, and most typically about 90-95% or more homologous. Are homologous. A substantially homologous amino acid sequence according to the present invention is encoded by a nucleic acid sequence that hybridizes under stringent conditions as described in more detail below to the nucleic acid sequence of SEQ ID NO: 2 or a portion thereof. It

To determine the percent identity of two amino acid sequences or two nucleic acid sequences,
Arrange sequences so that optimal comparisons can be performed (for example, gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, ignoring heterologous sequences for comparison). can do). In a preferred embodiment, the length of the reference sequence arranged for comparison is at least 30%, preferably at least 40%, more preferably at least 50% of the length of the reference sequence,
Even more preferably at least 60%, even more preferably at least 70%, 80
% Or 90%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical in that position (the amino acid used herein or Nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology." Considering the number of gaps and the length of each gap that must be introduced to optimally align the two sequences, the percent identity between the two sequences is a function of the number of identical positions shared by the sequences.

The invention also includes polypeptides that have a low degree of identity, but are sufficiently similar to perform one or more of the same functions performed by a 16405 polypeptide. Similarity is determined by conservative amino acid substitutions. Such a substitution is one in which a given amino acid in the polypeptide is replaced by another amino acid of similar characteristics. Conservative substitutions appear to be phenotypically silent. One-to-one substitution between the aliphatic amino acids Ala, Val, Leu and Ile, hydroxyl residue Ser
And Thr, exchange of acidic residues Asp and Glu, substitution of amide residues Asn and Gln, exchange of basic residues Lys and Arg and substitution of aromatic residues Phe, Tyr are typically conservative substitutions. it is conceivable that. Guidance on which amino acid changes are phenotypically silent can be found in Bowie et al., Science 247: 1306-1310 (1990).

[0047]

[Table 1]

Sequence comparisons and determination of percent identity and similarity between two sequences can be performed using a mathematical algorithm (Computational Molecular Biolo
gy, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomput
ing: Informatics and Genome Projects, Smith, DW, ed., Academic Press,
New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.
M., and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence
Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and
Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockt
on Press, New York, 1991).

A preferred, but non-limiting example of such a mathematical algorithm is Karlin et al.
(1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877. Altsch
ul et al. (1997) Nucleic Acids Res. 25: 3389-3402,
Such algorithms are incorporated into the NBLAST and XBLAST programs (version 2.0). When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. In one embodiment, the parameters for sequence comparison may be set to score = 100, wordlength = 12 or may be altered (eg W = 5 or W = 20).

In a preferred embodiment, the percent identity between the two amino acid sequences is Needlem
an et al. (1970) (J. Mol. Biol. 48: 444-453) algorithm was used by incorporating it into the GCG software package GAP program (available at http://www.gcg.com). 62 matrix or PAM250 matrix and gap weight 1
Determined using 6, 14, 12, 10, 8, 6 or 4 and length weights 1, 2, 3, 4, 5 or 6. In yet another preferred embodiment, the percentage of identity between the two nucleotide sequences is determined by the GCG program (Devereux et a
l. (1984) Nucleic Acids Res. 12 (1): 387) (available at http://www.gcg.com) using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70 or 8
Determined using 0 and length weights 1, 2, 3, 4, 5 or 6.

Another preferred non-limiting example of a mathematical algorithm used for sequence comparison is
The algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the CGC sequence alignment software package. When using the ALIGN program for comparing amino acid sequences, the PAM120 weight residue table, gap length penalty of 12 and gap penalty of 4 can be used. Alternative algorithms for sequence analysis are well known in the art, and Torellis et al. (1994) Comput. A
ADVANCE and ADAM described in ppl. Biosci. 10: 3-5 and Person et al.
1988) including FASTA as described in PNAS 85: 2444-8.

The protein sequences of the present invention may further be used as a “query sequence” to perform searches in public databases, for example to identify other family members or related sequences. it can. Such a search is Altschul et
al. (1990) J. Mol. Biol. 215: 403-10, using the NBLAST and XBLAST programs (version 2.0). To obtain nucleotide sequences homologous to the nucleic acid molecules of the invention, NBLAST program, score = 100, wordlength
= 12 can be used to perform a BLAST nucleotide search. BLAST protein searches can be performed using the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to proteins of the invention. To obtain a gapped alignment for comparison, Altschul et al. (1997) Nucleic Acids R
Gapped BLAST can be used as described in es. 25 (17): 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov .

Variant polypeptides may differ in amino acid sequence by one or more substitutions, deletions, insertions, transpositions, fusions and truncations or any combination thereof.

Variant polypeptides may be full-function or may lack one or more active functions. Thus, in the case of the present invention, mutations may affect, for example, the function of one or more of the regions corresponding to ligand binding, membrane binding, G protein binding and signal transduction.

Full-function mutants typically contain only conservative mutations or mutations of non-critical residues or non-critical regions. Functional variants also contain conservative amino acid substitutions that do not change or produce major changes in function. Alternatively, such substitutions may have some positive or negative effect on function.

Non-functional mutants typically have one or more non-conservative amino acid substitutions, deletions, insertions, transpositions or truncations or substitutions, insertions, transpositions or deletions of important residues or heavy regions. contains.

As indicated, the variants may be naturally occurring or made by recombinant means or chemical synthesis to provide useful and novel features of the receptor polypeptide. This involves rendering the formulation non-immunogenic by preventing protein aggregation.

Useful variants further include alteration of ligand binding properties. For example, one embodiment involves altering the binding site that binds a ligand but does not or slowly releases it. Yet another useful modification of the same site results in higher affinity for the ligand. Useful alterations also include alterations that provide an affinity for another ligand. Another useful modification includes those that allow binding but are not activated by the ligand. Another useful modification is the alteration of a transmembrane G protein binding / signaling region that provides for a decrease or increase in binding by a suitable G protein, or a binding by a G protein different from that to which the receptor normally binds. including. Another useful modification provides fusion proteins in which one or more regions or subregions are functionally fused to one or more regions or subregions of another G protein-coupled receptor.

Amino acids essential for function can be identified by methods well known in the art, such as site-directed mutations or alanine scanning mutations (Cunningham).
et al. Science 244: 1081-1085 (1989)). The latter method uses 1 for every residue in the molecule.
Introduce one alanine mutation. The resulting mutant molecules are then tested for biological activity such as receptor binding or in vitro, or in vitro proliferative activity. Sites important for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or optical affinity labeling (Smith et al. J.
Mol. Biol. 224: 899-904 (1992); de Vos et al. Science 225: 306-312 (1992).
).

Substantial homology may be entire nucleic acid or amino acid sequences or fragments of these sequences.

Accordingly, the present invention also includes polypeptide fragments of the 16405 receptor protein. The fragment can be derived from the amino acid sequence shown in SEQ ID NO: 1. However, the present invention also includes fragments of variants of the 16405 receptor protein described herein.

However, the fragments with which the invention is concerned should not be considered to include the previously disclosed fragments of the invention.

A fragment as used herein comprises at least 7 consecutive amino acids from amino acid 1 to about amino acid 356. A fragment is one or more of the biological activities of a protein, such as
It retains the ability to bind G proteins or ligands as well as fragments that can be used as immunogens to form receptor antibodies.

Biologically active fragments (eg chain lengths of eg 7, 10, 12, 15, 20, 30, 35)
, 36, 37, 38, 39, 40, 50, 100 or more amino acids) are domains or motifs, such as extracellular or intracellular domains or loops, one or more transmembrane segments, or their partial G-protein linkages. Sites or GPCR signatures, glycosylation sites, phosphorylation sites, amidation sites and N-myristoylation sites may be included. Such domains or motifs can be identified by conventional computerized homology search techniques.

Possible fragments extend to 1) a soluble peptide containing the entire amino-terminal extracellular domain or part thereof, 2) a peptide containing the entire carboxy-terminal intracellular domain or part thereof, 3) the entire transmembrane domain. Including, but not limited to, a region-comprising peptide, 4) any particular transmembrane segment or part thereof, 5) any three intracellular or three extracellular loops or any part thereof.

The fragment further combines the amino-terminal domain with one or more transmembrane segments and associated extra or intracellular loops or one or more transmembrane segments, and associated extra or extracellular loops plus a carboxy-terminal domain. Including combinations of the above fragments, such as Therefore, any of the above fragments may be combined. Other fragments include mature proteins. Other fragments contain various functional sites described herein. Fragments can include, for example, up to 5, 10, 15, 20, 30, 40, 50 or 100 amino acids extending from the functional site in one or both directions. In addition, fragments may include subfragments of the particular domains described above, which subfragments retain the function of the domain from which they are derived. These regions can be identified by well known methods including computerized homology analysis.

Fragments also include antigenic fragments, particularly those with a high antigenic index as shown in FIG.

Furthermore, possible fragments include, but are not limited to, those that define ligand binding sites, those that define membrane binding and those that define interactions between G proteins and signaling. By this is intended a discrete fragment that provides the relevant function or allows the identification of the relevant function. In a preferred embodiment, the fragment contains the ligand binding site.

The present invention also provides fragments that have immunogenic properties. These contain the epitope-forming portion of the 16405 receptor protein and variants. These epitope-forming peptides are useful in raising antibodies that specifically bind to the receptor polypeptide or region or fragment. These peptides can contain at least 7, 10, 12, at least 14 or at least about 15 to about 30 amino acids.

Non-limiting examples of antigenic polypeptides that can be used to form antibodies include peptides derived from either the amino-terminal extracellular domain or the extracellular loops. Regions with high antigenicity index are shown in Figure 3. However, intracellularly formed antibodies that appear to recognize intracellular receptor peptide regions (“intrab
odies ”) are also included.

Receptor polypeptides, including potentially previously disclosed variants and fragments of this invention, are useful in bioassays involving GPCRs. Such an assay may assay for any of the known GPCR functions or activities or properties useful in the diagnosis and treatment of conditions associated with GPCRs, particularly diseases involving the tissues in which the receptors are expressed as disclosed herein. Related to crab.

Epitope-forming receptor polypeptides can be produced by any conventional means (Houghten, RA Proc. Natl. Acad. Sci. USA 82: 5131-5135 (1985).
). Simultaneous multipeptide synthesis is described in US Pat. No. 4,631,211.

Fragments may be separate (not fused to other amino acids or polypeptides)
Or it may be within a larger polypeptide. In addition, some fragments 1
It may be contained within two larger polypeptides. In one embodiment, the fragment designed to be expressed in the host may include a heterologous prepolypeptide and propolypeptide region fused to the amino terminus of the receptor fragment and another region fused to the carboxyl terminus of the fragment. it can.

Accordingly, the present invention provides chimeric or fusion proteins. These include receptor proteins that functionally bind to heterologous proteins that have amino acid sequences that are not substantially homologous to the receptor protein. "Operably linked" indicates that the receptor protein and the heterologous protein are fused in frame. The heterologous protein may be fused to the N-terminus or C-terminus of the receptor protein.

In one embodiment, the fusion protein does not affect the receptor function itself. For example, the fusion protein may 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 enzyme fusion proteins,
For example, β-galactosidase fusion, yeast 2-hybrid GAL fusion, polyHis
Fusions and Ig fusions are included, but are not limited to. Such fusion proteins, especially polyHis fusions, can facilitate purification of recombinant receptor proteins. In certain host cells (eg, mammalian host cells), protein expression and / or secretion can be increased by using heterologous signal sequences. Therefore, in another embodiment, the fusion protein contains a heterologous signal sequence at the N-terminus.

European Patent Application No. EP-AO-464 533 discloses fusion proteins comprising various portions of immunoglobulin constant regions. Fc is useful in therapy and diagnosis and thus has improved pharmacokinetic properties (European Patent Application No. EP-A-0232 262). In drug discovery, for example, human proteins have been fused to Fc moieties for high throughput screening assays to identify antagonists (B
ennett et al. J. Mol. Recog. 8: 52-58 (1995) and Johanson et al. J. Biol.
Chem. 270: 9459-9471 (1995)). Thus, the invention also includes soluble fusion proteins containing the receptor polypeptide and various portions of the constant region of the heavy or light chain of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE). The constant region of the heavy chain of human IgG, in particular IgG1, in which the fusion occurs in the hinge region is preferred as immunoglobulin. For some applications, eg, where the fusion protein is to be used as an antigen for immunization, it is desirable to remove the Fc after the fusion protein has been used for its intended purpose. In certain embodiments, the Fc portion can be easily removed by a cleavage sequence that is integrated and can be cleaved with Factor Xa.

Chimeric or fusion proteins can be made by standard recombinant DNA techniques. For example, DNA fragments encoding different protein sequences are ligated in-frame by conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
Alternatively, PCR amplification of a gene fragment can be performed using anchor primers that produce complementary overhangs between two consecutive gene fragments, which are then annealed and reamplified to create a chimeric gene sequence. (Ausbel et al. C
urrent Protocols in Molecular Biology (1992)). In addition, a number of expression vectors can be purchased that already encode a fusion moiety (eg, GST protein). The nucleic acid encoding the receptor is labeled in-frame with the fusion moiety at the receptor.
frame) so that it can be ligated into such an expression vector.

Another form of fusion protein is one that directly affects receptor function. Thus, a receptor polypeptide in which one or more of the receptor region (or part thereof) is replaced by a homologous region (or part thereof) of another G protein-coupled receptor or other type of receptor is Included in the invention. Therefore, various substitutions are possible. The amino-terminal extracellular region or a subregion thereof (eg, ligand binding) can be replaced with a region or subregion of another ligand binding receptor protein. Alternatively, the entire transmembrane region, or any of the seven segments or loops, or portions thereof, such as G protein coupling / signalling, can be replaced. Finally, the carboxy-terminal intracellular region or subregion can be replaced. Therefore,
Chimeric receptors can be formed in which one or more of the natural regions or subregions have been replaced with another.

The isolated receptor protein can be purified from cells that naturally express it, such as spleen, glioma and sclerotic lesions, or modified to express it (recombinant. 2.) can be purified from cells or can be synthesized using known protein synthesis methods.

In one embodiment, the protein is produced by recombinant DNA technology. 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 purification techniques.

Peptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. In addition, many amino acids, including terminal amino acids, can be modified by natural processes such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. The usual naturally occurring modifications to polypeptides are described in basic textbooks, detailed research articles and literature, and are well known in the art.

Thus, a polypeptide also refers to a derivative or analog in which the substituted amino acid residue is not one encoded by the genetic code, a derivative or analog containing a substituent, a mature polypeptide is one-half of a polypeptide. Derivatives or analogs fused with another compound, such as a phase-extending compound (eg, polyethylene glycol), or another amino acid, a leader or secretory sequence, or a sequence for purifying a mature polypeptide, or a proprotein sequence, etc. Derivatives or analogs fused to the mature polypeptide of.

Known modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavins, covalent attachment of heme moieties, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, Phosphatidylinositol covalent bond, bridge, cyclization, disulfide bond formation, demethylation, covalent bridge formation,
Cystine formation, pyroglutamate formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic treatment, phosphorylation, prenylation, racemization, Includes, but is not limited to, transfer RNA-mediated addition of amino acids to proteins and ubiquitination such as selenoylation, sulfation, arginylation.

Such modifications are well known to those of skill in the art and have been described in considerable detail in the scientific literature. Some particularly common modifications such as glycosylation, lipid binding, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation are described in, for example, Proteins-Structure and Molecular Properties, 2nd ed., TE Creighton,
It is described in the most basic textbooks such as WH Freeman and Company, New York (1993). Wold, F., Posttranslational Covalent Modification of Protein
s, BC Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et a
l. (1990) Meth. Enzymol. 182: 626-646) and Rattan et al. (1992) Ann. N.
Many detailed reviews on this subject are available, such as Y. Acad. Sci. 663: 48-62).

As is well known, a polypeptide need not be entirely linear. For example, a polypeptide may diverge as a result of ubiquitination, and may or may not diverge as a result of post-translational events, which generally include natural processing events and non-naturally occurring artifactual events. May be Cyclic, branched and branched cyclic polypeptides can be synthesized by non-translation natural processes and synthetic methods.

Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxy termini. Blocking by covalent modification of the amino and / or carboxyl groups of a polypeptide is common in naturally occurring and synthetic polypeptides. For example, the amino terminal residue of a polypeptide produced by E. coli is almost always N-formylmethionine before undergoing the proteolytic process.

Modifications are a function of how the protein is made. For recombinant polypeptides, for example, the modification will be determined by the ability of the host cell to post-translationally modify and the modification signal of the polypeptide amino acid sequence. Thus, if glycosylation is desired, the polypeptide should be expressed in a glycosylation host, generally a eukaryotic cell. Insect cells often perform the same post-translational glycosylation as mammalian cells, and thus insect cell expression systems have been developed to efficiently express mammalian proteins with native patterns of glycosylation. Similar ideas apply to other modifications.

The same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain more than one type of modification.

Uses of Polypeptides Receptor polypeptides are useful for raising antibodies specific for 16405 receptor protein, region or fragment. Regions with high antigenicity scores are shown in Figure 3.

Receptor polypeptides, including potential variants and fragments previously disclosed herein, are useful in bioassays involving GPCRs. Such an assay involves any known GPCR function or activity or property useful in the diagnosis and treatment of GPCR-related conditions.

Receptor polypeptides are also useful in drug screening assays in cell-based or cell-free systems. The cell line may be a cell, such as a biopsy sample or a cultured cell that is native, ie normally expressing the receptor protein. However, in one embodiment, the cell line involves a recombinant host cell that expresses the receptor protein.

Determining the ability of a test compound to interact with a polypeptide determines that the test compound preferentially binds to the polypeptide as compared to the ability of the ligand or biologically active portion thereof to bind to the polypeptide. It may also include determining the ability to bind.

The polypeptides can be used to identify compounds that modulate receptor activity. Such compounds may, for example, increase or decrease binding affinity or rate with a known ligand, may compete with the ligand for binding to the receptor, or may displace the ligand bound to the receptor. May be. Both the 16405 protein and appropriate variants and fragments can be used in high throughput screens to assay candidate compounds for their ability to bind to the receptor. These compounds can be further screened for functional receptors to determine the effect of the compounds on receptor activity. Compounds that activate (agonist) or inactivate (antagonist) the receptor to the desired degree can be identified. The method of modulation can be performed in vitro (eg, by culturing cells with the drug) or in vivo (eg, by administering the drug to a subject).

Receptor polypeptides can be used to screen compounds for their ability to stimulate or inhibit the interaction of a receptor protein with a target molecule that normally interacts with the receptor protein. The target is a ligand or component of the signaling pathway with which the receptor protein normally interacts (eg, a G protein or other interacting factor involved in cAMP or phosphatidylinositol turnover and / or adenylate cyclase or phospholipase C activation). It may be. The assay allows the receptor protein or fragment to interact with a target molecule and detect the formation of a protein-target complex or G protein phosphorylation, cyclic AMP or phosphatidylinositol turnover and adenylate cyclase or phospholipase C activation. Combining the receptor protein with a candidate compound under conditions that detect a biochemical outcome of the interaction of the receptor protein with the target, such as any of the related effects of.

Determining the ability of a protein to bind a target molecule can also be performed using techniques such as real-time Bimolecular Interaction Analysis (BIA) Sjolander et al. 1991) Anal. Chem. 63: 2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5: 699-705. As used herein, "BIA" refers to an interacting substance. All are techniques for studying biospecific interactions in real time, without labeling (eg, BIScore®).
Changes in Optical Phenomena Changes in surface plasmon resonance (SPR) can be used as an indicator of real-time reactions between biomolecules.

The test compounds of the present invention can be used in biological libraries, spatially processed in a spatially parallel manner.
Addressable parallel) solid phase or liquid phase libraries, synthetic library methods requiring reverse response, “1-bead 1-compound” library methods and synthetic library methods using affinity chromatography selection It can be obtained using any of a number of methods in the combinatorial library methods well known above. Biological library methods are limited to polypeptide libraries, but the other four methods are applicable to small molecule libraries of polypeptides, non-polypeptide oligomers or compounds (Lam, KS (1997) Anticancer Drug). Des.
12: 145).

Examples of methods for synthesizing molecular libraries are described, for example, in De Witt et al. (19
93) Proc. Natl. Acad. Sci. USA 90: 6909; Erb et al. (1994) Proc. Natl. Aca
d. Sci. USA 91: 11422; Zuckermann et al. (1994). J. Med. Chem. 37: 2678;
Cho et al. (1993) Science 261: 1303; Carell et al. (1994) Angew. Chem.
Int. Ed. Engl. 33: 2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl.
33: 2061; and Gallop et al. (1994) J. Med. Chem. 37: 1233.
Can be found in the art. A library of compounds is in the form of a solution (eg Ho
ughten (1992) Biotechniques 13: 412-421) or on beads (Lam (1991) Natur
e 354: 82-84), on chip (Fodor (1993) Nature 364: 555-556), bacteria (Ladner US
P 5,223,409), spores (Ladner USP '409), plasmids (Cull et al. (1992) Proc.
Natl. Acad. Sci. USA 89: 1865-1869) or phage (Scott and Smith (1990).
Science 249: 386-390); (Devlin (1990) Science 249: 404-406); (Cwirla et al.
(1990) Proc. Natl. Acad. Sci. USA 97: 6378-6382); (Felici (1991) J. Mol.
Biol. 222: 301-310); (Ladner supra).

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

One candidate compound is a soluble full-length receptor or fragment that competes for ligand binding. Other candidate compounds include mutant receptors or suitable fragments containing mutations that affect receptor function and compete with the ligand. Thus, for example, fragments that compete with the ligand for higher affinity or that bind but do not release the ligand are included in the invention.

The present invention provides other endpoints for identifying compounds that modulate (stimulate or inhibit) receptor activity. The assay typically involves assaying for events in the signaling pathway that are indicative of receptor activity. Thus, the expression of genes that are upregulated or downregulated in response to a receptor protein dependent signal cascade can be assayed. In one embodiment,
The regulatory regions of such genes can be operably linked to readily detectable markers such as luciferase. Alternatively, receptor phosphorylation or receptor targeting may be measured.

Either receptor-mediated biological or biochemical functions can be used as endpoint assays. These include all of the biochemical or biochemical / biological events described in the references cited herein for these endpoint assay targets and well known to those skilled in the art. Other features are included.

The binding and / or activating compound may also be an amino terminal extracellular region or part thereof, any of the seven transmembrane segments or an entire transmembrane region or subregion such as either an intracellular or extracellular loop. Alternatively, screening can be performed using a chimeric receptor protein in which the carboxy-terminal intracellular region or a part thereof is substituted with a heterologous region or subregion. For example, a G protein binding domain that interacts with a G protein different from that recognized by the natural receptor may be used. Therefore, different sets of signaling components are available as endpoint assays of activation. Alternatively, the entire transmembrane region or subregion (transmembrane segment or intracellular or extracellular loop) is specific for a host cell different from the host cell in which the amino-terminal extracellular region and / or G protein-binding region is derived. The entire transmembrane region or subregion may be replaced. This allows the assay to be performed in cells other than the particular host cell in which the receptor is derived. Alternatively, the amino terminal extracellular region (and / or other ligand binding region) binds to a different ligand (and / or other binding region)
May be exchanged for a heterologous amino terminal extracellular domain (or region)
Assays of test compounds that interact with but do not produce signal transduction are provided. Finally, activation can be detected by a reporter gene containing an easily detectable coding region operably linked to transcriptional regulatory sequences that are part of the natural signaling pathway.

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 to the polypeptide or interact with something other than binding. Soluble receptor polypeptide is also added to the mixture. When the test compound interacts with the soluble receptor polypeptide, the test compound reduces the amount of complex formed from the receptor target or the activity of the receptor target. This type of assay is particularly useful in searching for compounds that interact with specific regions of the receptor. Thus, soluble polypeptides that compete with the target receptor region are designed to contain peptide sequences corresponding to the region of interest.

In order to perform a cell-free drug screening assay, receptors are provided to facilitate separation of the complex from uncomplexed forms of one or both of the proteins, as well as to accommodate assay automation. It is desirable to immobilize the protein or fragment or its target molecule.

Techniques that immobilize proteins on substrates can be used in drug screening assays. In one embodiment, a fusion protein can be provided that adds a domain capable of binding the protein to a substrate. For example, the glutathione-S-transferase / 16405 receptor fusion protein was adsorbed to glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione-derivatized microtiter plates, followed by cell lysate (eg, ³⁵ S-labeled). ) And a candidate compound and under conditions that lead to complex formation (eg salt and pH).
The mixture can be incubated under physiological conditions). After incubation, the beads are washed to remove any unbound label, the substrate is fixed and the radioactivity is measured directly or in the supernatant after dissociation of the complex. Alternatively, the complexes are dissociated from the substrate, separated by SDS-PAGE, and standard electrophoretic techniques are used to quantify from the gel the level of receptor-binding protein found in the bead fraction. For example,
Coupling of biotin and streptavidin can be used to immobilize the polypeptide or its target molecule using techniques well known in the art. Alternatively, an antibody that is reactive with the protein but does not interfere with the binding of the protein to the target molecule can be incubated in the wells of the plate and the protein bound to the wells by antibody binding. A preparation of receptor binding protein and candidate compound can be incubated in the wells where the receptor protein is present to quantify the amount of complex captured in the wells. Methods for detecting such complexes other than those described above for GST-immobilized complexes include antibodies that are reactive with the receptor target molecule, or are reactive with the receptor protein and compete with the target molecule. And immunoassays of the complex using S. aureus as well as enzyme-linked assays using the detection of enzyme activity associated with the target molecule.

Modulators of receptor protein activity identified by these drug screening assays are used to treat cells expressing the 16405 receptor, such as those disclosed herein, by treating the receptor pathway. A subject having a disease mediated by can be treated. These methods of treatment include the step of administering to the subject in need of such treatment a modulator of protein activity in the pharmaceutical compositions described herein.

Since the gene is expressed in tissues including, but not limited to, the spleen, brain including glioma and sclerotic lesions, receptor expression and altered expression are diagnostic of diseases associated with these tissues. And is important for treatment.

Disorders involving the spleen include, but are not limited to, nonspecific acute splenitis, splenomegaly, including congestive spenomegaly and splenic infarction, malignancy, birth defects and rupture. Disorders associated with splenomegaly include nonspecific splenitis, infectious mononucleosis, tuberculosis, typhoid fever, brucellosis, cytomegalovirus, syphilis, malaria, histoplasmosis, toxoplasmosis, black fever, trypanosomiasis, Schistosomiasis, infections such as leishmaniasis and echinococcosis, congestion conditions associated with partial hypertension such as cirrhosis, portal vein or splenic vein thrombosis and heart failure, Hodgkin's disease, non-Hodgkin's lymphoma / leukemia, multiple myeloma , Myeloproliferative disorders, lymphatic circulation disorders such as hemolytic anemia and thrombocytopenic purpura, immune-inflammatory conditions such as rheumatoid arthritis and systemic lupus erythematosus, accumulation of Gaucher disease, Niemann-Pick disease and mucopolysaccharidosis And other conditions such as amyloidosis, primary and secondary cancers.

[0109]   Brain-related diseases include nerve-related diseases and astrocytes, oligodendrocytes.
Glial-related diseases such as transglial cells, ependymal cells and microglial cells
Tumor, increased cerebral pressure and herniation and hydrocephalus, neural tube malformation, forebrain abnormality, posterior skull
Dysplasia and dysplasia such as fossa and syringomyelia and hydromyelopathy, hypotension
, Hypoperfusion and hypoperfusion--global and focal cerebral ischemia--of local blood supply
Infarction due to occlusion, intracerebral (intraparenchymal) hemorrhage, subarachnoid hemorrhage and strawberry aneurysm rupture
, And hypertension including vascular malformations, hiatal infarction, slit hemorrhage and hypertensive encephalopathy
Associated with hypoxia such as intracranial hemorrhage, including cardiovascular disease, ischemia and infarction
Cerebral vascular diseases such as, purulent (bacterial) meningitis and acute aseptic (viral)
) Acute meningitis, including meningitis, cerebral abscess, subdural empyema, and epidural abscess
Localized purulent infection, tuberculosis and mycobacteriosis, neurosyphilis and neurob
Chronic bacterial meningoencephalitis, including orreliosis, arthropod-borne (Arbo) Will
Encephalitis, herpes simplex virus type 1, herpes simplex virus type 2, Varicalla-zoster
Herpes), cytomegalovirus, acute poliomyelitis, rabies and HIV-1 meningoencephalitis
Human immunodeficiency virus type 1, including acute encephalitis, vacuolar myelopathy, AIDS-related myopathy
Harm, peripheral neuropathy and childhood AIDS, progressive multifocal leukoencephalopathy, subacute sclerosing panencephalitis
Sensitivity, including viral meningoencephalitis, including fungal meningoencephalitis, other infectious diseases of the nervous system
Infectious disease, infectious spongiform encephalopathy (prion disease), multiple sclerosis, multiple sclerosis variant, acute
Disseminated encephalomyelitis and acute necrotizing hemorrhagic encephalomyelitis and other diseases associated with demyelination
Demyelinating diseases including, disorders affecting the cerebral cortex including Alzheimer's disease and Pick's disease
Sexual illness, Parkinsonism, idiopathic Parkinsonism (tremor paralysis), progression
Supranuclear paralysis, cortical basal degeneration, striatonigral degeneration, Shy-Drager syndrome and
Multiple system atrophy including olive pontine cerebellar atrophy and basal cerebrum including Huntington's disease
Degenerative diseases such as nuclear and brainstem degenerative diseases, Friedreich's ataxia and capillaries
Spinocerebellar ataxia including telangiectasia, amyotrophic lateral sclerosis (motor neuron disease
), Spinal cord atrophy (Kennedy syndrome) and spinal muscular atrophy
Cerebellar degeneration, including Klebbe's disease and metachromatic leukodystrophy, including degenerative diseases
, Adrenoleukodystrophy, Periceus-Merzbacher disease and Canavan disease
Mitochondria, including leukodystrophies, Leigh's disease and other mitochondrial encephalomyopathy
Inborn errors of metabolism such as brain muscle disorders, thiamine (vitamin B₁) Deficiency and vitamin B ₁₂ Acquired metabolic diseases, hypoglycemia, hyperglycemia due to toxicity including vitamin deficiency such as deficiency
Neurological sequelae of metabolic disorders including dementia and hepatic encephalopathy, carbon monoxide, methanol, ethanol
Toxic diseases involving irradiation, including combined radiation damage with knots and methotrexate
, Astrocytomas, including fibrillary (diffuse) astrocytomas and glioblastoma multiforme
Glioma including blastoma, pilocytic astrocytoma, pleomorphic yellow astrocytoma and
And brain stem glioma, oligodendroglioma and ependymoma and associated paraventricular mass disease
Malignant tumors with poor differentiation, including mass lesions, neuronal tumors, neuromedulloblastomas,
Other parenchymal tumors, including primary field brain lymphoma, germ cell tumors and pineal parenchymal tumors, meninges
Tumors, metastatic tumors, paraneoplastic syndromes, Schwannoma, neurofibroma and malignant peripheral
Peripheral nerve sheath tumors, including nerve sheath tumors (malignant Schwannoma), type 1 neurofibromatosis (NF
1) and neurocutaneous syndromes including neurofibromatosis including type 2 neurofibromatosis (NF2) (nevus
), Tuberous sclerosis and von Hippel-Lindau disease and other tumors
, But not limited to them.

Diseases related to the heart include, but are not limited to, cardiac hypertrophy, left and right heart failure, angina, myocardial infarction, chronic ischemic heart disease and sudden death. Including heart (left heart) hypertensive heart disease and lung (right) hypertensive heart disease,
Hypertensive heart disease, calcified aortic stenosis, congenital bicuspid aortic valve calcification and valvular degeneration caused by calcification such as annular calcification and mitral valve degenerative degeneration. (Mitral valve prolapse syndrome), rheumatic fever and rheumatic heart disease, infectious endocarditis and non-bacterial thrombotic endocarditis and systemic lupus erythematosus endocarditis (Livmann-Sachs endocarditis) Non-infectious hyperplasia, including carcinoid heart disease and complications of artificial valve, including but not limited to valvular heart disease, dilated cardiomyopathy, hypertrophic cardiomyopathy, constrictive cardiomyopathy and myocarditis. , Pericardial diseases, including but not limited to myocardial disease, epicardial exudate and pericardemia, and pericarditis, including acute pericarditis and cured pericarditis, and rheumatoid heart disease , Viscous Tumor, lipoma, papillary fibroelastosis (papillary fibroelasto
ma), rhabdomyomas and sarcomas, and primary heart tumors, including but not limited to cardiac effects of non-heart tumors, neoplastic heart disease, atrial septal defect, ventricular septal defect and atrioventricular septum. Left-right shunts such as defects: late cyanosis, Fallot 4 sign, translocation of the great arteries, common arteriosclerosis, leaflet atresia and abnormal pulmonary venous return-early cyanosis, aortic coarctation, Includes, but is not limited to, congenital heart disease, including but not limited to pulmonary stenosis and occlusion, and congenital obstructive abnormalities such as aortic stenosis and occlusion, and diseases associated with heart transplantation. .

Diseases involving blood vessels include responses to vascular cell wall insults such as endothelial dysfunction and endothelial activation and intimal thickening, hypertensive vascular disorders such as arteriovenous fistula, atherosclerosis and hypertension. Vascular disorders, including but not limited to congenital abnormalities such as, giant cell (transient) arteritis, Takayasu arteritis, polyarteritis nodosa (classical), Kawasaki disease (mucocutaneous lymph node syndrome) ), Microscopic polyanglitis (microscopic polyarteritis, hypersensitivity or leukocytoclastic anglitis), inflammatory diseases such as vasculitis and infectious arteritis associated with other diseases-vasculitis, Raynaud's disease, abdominal aortic aneurysm, syphilis (Syphilis) Malformations and dissections such as aneurysms and aortic detachments (dissociative hematomas), varicose veins, thrombophlebitis and venous thrombosis, superior vena cava obstruction (upper vena cava obstruction syndrome), inferior vena cava Benign tumors and tumor-like conditions such as obstruction (inferior vena cava syndrome) and lymphangiitis and lymphedema, hemangiomas, lymphangioma, glomus tumor (glomus tumor), vasodilatation and bacillary hemangiomatosis, Kaposi's sarcoma and Tumors including intermediate grade (low borderline grade malignant) tumors such as hemangloendothelioma and malignant tumors such as hemangiosarcoma and hemangiopericytoma, vascular replacement such as balloon angioplasty and related techniques and Includes, but is not limited to, venous and lymphatic disorders such as lesions of therapeutic interventions for vascular disorders such as coronary artery bypass graft surgery.

With regard to the expression patterns of the involved receptors and tissues, preferred treatments and diagnoses include cardiovascular diseases, in particular atherosclerosis and pain management.

Accordingly, receptor polypeptides are useful in treating receptor-related disorders characterized by aberrant expression or activity of receptor proteins. In one embodiment, the methods of the invention provide for an agent (eg, an agent identified by a screening assay described herein) or agent combination that modulates (eg, upregulates or downregulates) protein expression or activity. Is administered. In another embodiment, the invention comprises administering the protein as a therapeutic regimen that compensates for low or aberrant expression or activity of the protein.

Stimulation of protein activity is desirable in situations where the protein is abnormally downregulated and / or where high protein activity is likely to have a beneficial effect. Similarly, inhibition of protein activity is desirable in situations where the protein is abnormally upregulated and / or where low protein activity is likely to have a beneficial effect. In one example of this situation,
The subject has a disease characterized by abnormal development or cell differentiation. In another example of such a situation, the subject has a proliferative disorder (eg, cancer) or a disorder characterized by an abnormal hematopoietic response. In another example of such a situation, it may be desirable to regenerate tissue in a subject (eg, where the subject has brain or spinal cord injury and it is desirable to regenerate neural tissue in a controlled manner).

In yet another embodiment of the invention, the proteins of the invention are used to identify other proteins (capture proteins) that bind or interact with the proteins of the invention to regulate their activity. It can be used as a “bait protein” in a hybrid assay or a 3-hybrid assay (eg, US Pat. No. 5,283,317; Zervos et al. (1993) Cell 72: 223-232; Madura e).
t al. (1993) J. Biol. Chem. 268: 12046-12054; Bartel et al. (1993) Biot.
echniques 14: 920-924; Iwabuchi et al. (1993) Oncogene 8: 1693-1696; and Brent WO 94/10300).

Receptor polypeptides are also useful in providing targets for diagnosing receptor protein mediated diseases or predisposition to diseases and diseases including sclerotic lesions, particularly in spleen, brain, glioblastoma. Is. Thus, methods of detecting the presence or level of receptor protein in a cell, tissue or organ are provided. The method of the present invention involves contacting a biological sample with a compound capable of interacting with a receptor protein so that the interaction can be detected.

Agents for detecting receptor proteins are antibodies that can selectively bind to the receptor protein. Biological samples include tissues, cells and fluids isolated from a subject as well as tissues, cells and fluids present in the subject.

Receptor proteins also provide targets for diagnosing active disease or predisposition to disease in patients with variant receptor proteins. Thus, receptor proteins can be isolated from biological samples and assayed for the presence of genetic mutations that result in aberrant receptor proteins. This includes amino acid substitutions, deletions, insertions, rearrangements (
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 binding or antibody binding patterns, changes in isoelectric point, direct amino acids. Included are any of the other known assay techniques useful for sequencing and detecting protein mutations.

In vitro techniques for detecting receptor proteins include enzyme-linked immunosorbent assay (ELISA), western blot, immunoprecipitation and immunofluorescence.
Alternatively, the protein can be detected in vivo in a subject by introducing into the subject a labeled anti-receptor antibody. 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 of detecting allelic variants of a receptor protein expressed in a subject and methods of detecting fragments of the receptor protein in a sample.

Receptor polypeptides are also useful for pharmacogenomic analysis. Pharmacogenomics deals with clinically significant genetic alterations in the response of drugs due to altered drug properties and abnormal effects in affected individuals. For example, Eichelbaum, M. Clin. Exp.
Pharmacol. Physiol. 23 (10-11): 983-985 (1996) and Linder, MW Clin. Ch.
See em. 43 (2): 254-266 (1997). The clinical consequences of these changes may lead to severe toxicity of the therapeutic agent in some patients and failure of drug treatment in some patients as a result of individual differences in metabolism. Thus, an individual's genotype can determine how a therapeutic compound acts on the organism or how the organism metabolizes the compound. Furthermore, the activity of drug-metabolizing enzymes affects the intensity and duration of drug action. Thus, an individual's pharmacogenomics can select compounds and doses of such compounds that are effective for prophylactic treatment or treatment based on the individual's genotype. Due to the discovery of genetic polymorphisms in several drug-metabolizing enzymes, some patients do not get the expected drug effect, some show an increased drug effect, or some patients receive standard drug doses. Have explained why they experience severe toxicity. Polymorphisms can be expressed in a wide range of metabolizer phenotypes and weak metabolizer phenotypes. Thus, genetic polymorphisms can result in allelic protein variants of a receptor protein in which one or more of the functions of the receptors in one population differs from that in another population. Thus, the polypeptide allows the target to identify genetic predispositions that may affect therapeutics. Thus, in ligand-based therapies, polymorphisms may result in activation of the amino-terminal extracellular domain and / or ligand binding domain and receptor activation, with stronger or weaker ligand binding activity. Thus, the dose of ligand will necessarily be adjusted to maximize the therapeutic effect within a given population containing the polymorphism. As an alternative to genotyping, specific polymorphic polypeptides are identified.

Receptor polypeptides are also useful for monitoring therapeutic efficacy during clinical trials and other treatments. Therefore, the therapeutic efficacy of agents designed to increase or decrease gene expression, protein levels or receptor activity should be monitored over the course of treatment using receptor polypeptides as endpoint targets. You can The monitoring can be performed as follows, for example. (i)
Obtaining a pre-dose sample from the subject prior to administering the drug, (ii) detecting the presence or activity level of a particular protein in the pre-dose sample, (iii) after one or more doses from the subject Obtaining the sample, (iv) detecting the expression or activity level of the protein in the sample after administration, (v) determining the expression or activity level of the protein in the sample before administration and the expression or activity level of the protein in the sample after administration Compare, and (vi) increase or decrease the administration of the drug to the subject accordingly.

Receptor polypeptides are also useful in treating receptor-related disorders. Accordingly, therapeutic methods include the use of soluble receptors or fragments of receptor proteins that compete for ligand binding. These receptors or fragments may have a higher affinity for the ligand so that competition is effective.

Antibodies The present invention also provides antibodies that selectively bind to the 16405 receptor protein and variants and fragments thereof. Antibodies are believed to selectively bind, even if they bind to other proteins that are not substantially homologous to the receptor protein. These other proteins have homology with fragments or regions of the receptor protein.
Such conservation of specific regions gives rise to antibodies that bind both proteins with respect to homologous sequences. In this case, it will be appreciated that the antibody that binds the receptor protein is still selective.

To form antibodies, the isolated receptor polypeptide is used as an immunogen to form antibodies using standard techniques for polyclonal and monoclonal antibody production. Full length proteins or antigenic peptide fragments can be used. Regions with high antigenicity index are shown in Figure 3.

Antibodies are preferably made from these regions or separate fragments of these regions. However, antibodies can be made from any region of the peptides described herein. Preferred fragments produce antibodies that reduce or completely interfere with ligand binding. An antibody can be a whole receptor or a portion of a receptor, such as an intracellular carboxy-terminal domain, an amino-terminal extracellular domain, an entire transmembrane domain or a particular segment, either intracellular or extracellular loop, or any of the above. It can be formed for any part. Antibodies can also be formed against the ligand binding site G protein binding site or specific functional sites such as glycosylation, phosphorylation, myristoylation or amidation sites.

An antigenic fragment typically comprises at least 7 consecutive amino acid residues. However, the antigenic peptide may comprise a contiguous sequence, at least 12, 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, eg hydrophilic regions. However, these fragments should not be considered to include any of the previously disclosed fragments of the invention.

The antibody may be polyclonal or monoclonal. An intact antibody or fragment thereof (eg, Fab or F (ab ') ₂ ) can be used.

Detection can be facilitated by coupling (ie, physically binding) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alcal phosphatase,
Examples include β-galactosidase or acetylcholinesterase, examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin, examples of suitable fluorophores are umbelliferone, fluorescein, fluorescein isothiocyanate. , Rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin, examples of luminescent materials include luminol, examples of bioluminescent materials include luciferase, luciferin and aequorin, examples of suitable radioactive materials Includes ¹²⁵ I, ¹³¹ I, ³⁵ S or ³ H.

Suitable immunogenic preparations can be derived from natural, recombinantly expressed or chemically synthesized peptides.

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

Antibodies are useful in detecting the presence of a receptor in a cell or tissue to measure the expression pattern of the receptor between various tissues of an organism over the normal developmental course.

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

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

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

In addition, the antibodies can be used to assess receptor expression in disease states such as individuals in the active phase of the disease or predisposed to the disease associated with receptor function. If the disease is caused by inappropriate tissue distribution, inappropriate developmental expression or inappropriate expression level of the receptor protein, antibodies can be raised against the normal receptor protein. If the disorder is characterized by a specific mutation in the receptor protein, antibodies specific for this mutant protein can be used to assay the presence of the specific mutant receptor protein. However, it also includes antibodies ("intrabodies") made in cells that are believed to recognize intracellular receptor peptide regions.

Antibodies can also be used to assess normal and abnormal subcellular localization of cells in various tissues of an organism. Antibodies can be made to the entire receptor or a portion of the receptor, eg, an amino acid extracellular region or a portion of an extracellular loop.

Diagnostic applications can be applied not only in genetic testing but also in monitoring therapeutics. Thus, where the ultimate aim of therapy is to correct the expression level of the receptor or the presence of aberrant receptors and the abnormal tissue distribution or aberrant developmental expression, the receptor or related fragment Antibodies can be used to monitor therapeutic efficacy.

Thus, antibodies can be used diagnostically, for example, to monitor protein levels in tissues as part of a clinical trial procedure to determine the efficacy of a given therapy.

Antibodies are also useful for pharmacogenomic analysis. Thus, antibodies raised against polymorphic receptor proteins can be used to identify individuals in need of treatment modalities.

Antibodies also serve as diagnostic tools as immunological markers of aberrant receptor proteins analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digestion and other physical assays well known to those of skill in the art. Is also useful.

Antibodies are also useful for tissue typing. Thus, if a particular receptor protein correlates with expression in a particular tissue, antibodies specific for this receptor protein can be used to identify tissue type.

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

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

These applications can be applied to therapeutic contexts in which treatment involves inhibition of receptor function. Antibodies can also be used to block ligand binding. Antibodies can be made to specific fragments that contain the site necessary for function, or to the intact receptor associated with the cell.

Fully human antibodies are particularly desirable for treating human patients. For a review of this technology for producing human antibodies, see Lonberg and Huszar (1995 Int. Rev. Immunol. 13: 65.
See -93). A detailed discussion of this technique for making human and human monoclonal antibodies, and protocols for making such antibodies, can be found in, for example:
See U.S. Patent No. 5,625,126, U.S. Patent No. 5,633,425, U.S. Patent No. 5,569,825, U.S. Patent No. 5,661,016 and U.S. Patent No. 5,545,806.

The invention also includes a kit that uses the antibody to detect the presence of a receptor protein in a biological sample. The kit comprises an antibody, such as a labeled or labelable antibody, a compound or agent for detecting a receptor protein in a biological sample, a means for measuring the amount of the receptor protein in the sample, and a sample in the sample. Means for comparing the amount of the receptor protein of E. coli with a standard. The compound or agent may be packaged in a suitable container. The kit may further include instructions for using the kit to detect the receptor protein.

Polynucleotide The nucleotide sequence of SEQ ID NO: 2 was obtained by sequencing the deposited human full length cDNA. Thus, the sequence of the deposited clone controls any discrepancies between the two, and any reference to the sequence of SEQ ID NO: 2 includes reference to the sequence of the deposited cDNA.

The specifically disclosed cDNAs include the coding region, 5 ′ and 3 ′ untranslated sequences (SEQ ID NO: 2).

The human 16405 receptor cDNA has a chain length of about 2040 nucleotides and encodes a full-length protein having a chain length of about 383 amino acid residues. Nucleic acid is expressed in spleen, brain, glioblastoma and sclerotic lesions. Structural analysis of the amino acid sequence of SEQ ID NO: 1 is shown in Figure 3, hydropathy plot. The figure shows the putative structures of the seven transmembrane segments, the amino-terminal extracellular region and the carboxy-terminal intracellular region.

The term “transmembrane” as used herein refers to a structural amino acid motif containing a hydrophobic helix that spans the plasma membrane.

The present invention provides an isolated polynucleotide encoding a 16405 receptor protein. The term "16405 polynucleotide" or "16405 nucleic acid" refers to the SEQ ID NO:
2 or the sequence shown in the deposited cDNA. The term "receptor polynucleotide" or "receptor nucleic acid" further includes variants and fragments of 16405 polynucleotides.

An “isolated” receptor nucleic acid refers to one that is separated from other nucleic acids that are present in naturally occurring receptor nucleic acids. Preferably, the "isolated" nucleic acid is the genomic DN of the organism from which the nucleic acid was derived.
It does not include sequences that naturally flank the nucleic acid in A (ie, sequences located at the 5'and 3'ends of the nucleic acid). However, some flanking nucleotide sequences, for example up to about 5 KB, may be present. Importantly, the nucleic acid is capable of performing the particular manipulations described herein, such as recombinant expression, probe and primer production, and other uses specific to receptor nucleic acid sequences. , Is isolated from the flanking sequences.

In addition, an "isolated" nucleic acid molecule, such as a cDNA or RNA molecule, can be substantially free of other cellular material or culture medium when made by recombinant techniques, or chemically It may contain no chemical precursors or other chemicals when synthesized. However, the nucleic acid molecule may be fused to other coding or regulatory sequences and still be considered isolated.

For example, the recombinant DNA molecule contained in the vector is considered isolated. Still other examples of isolated DNA molecules include recombinant DNA molecules maintained in a heterologous host cell or solution-state purified (partial or substantial) DNA molecules. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

In some cases, the isolated material forms part of a composition (eg, a crude extract containing other materials), buffer system or reagent mixture. In other cases, the substance is, for example,
It can be purified to the required homogeneity as measured by column chromatography such as PAGE or HPLC. Preferably, the isolated nucleic acid comprises at least about 50, 80 or 90% (based on the molecule) of all macromolecular species present.

The receptor polynucleotide encodes the mature protein plus additional amino or carboxy terminal amino acids, or (for example, if the mature form has one or more polypeptide chains) an amino acid within the mature polypeptide. You can Such sequences, among other things, play some role in processing the protein from precursor to mature form, facilitate protein trafficking, prolong or shorten protein half-life, or for assay or production. It may facilitate the manipulation of the protein. Since it is generally in situ in that case, additional amino acids may be processed from the mature protein by cellular enzymes.

Receptor polynucleotides include sequences that encode the mature polypeptide alone, the mature polypeptide and additional coding sequences such as leader or secretory sequences (eg, preproprotein or proprotein sequences). , Mature polypeptides with or without additional coding sequences plus additional non-coding sequences such as transcription, mRNA processing (including splicing and polyadenylation signals), transcription that plays a role in ribosome binding and mRNA stability. Introns and noncoding 5'and 3'such as sequences that are translated but not translated
Sequences that encode sequences are included, but are not limited to. The polynucleotide may also be fused to, for example, a marker sequence that encodes a peptide that facilitates purification.

Receptor polynucleotides may be in the form of RNA, such as mRNA, or in the form of DNA, including cDNA and genomic DNA made by cloning or chemical synthesis techniques or combinations thereof. The nucleic acid, especially DNA, may be double-stranded or single-stranded. Single-stranded nucleic acid may be the coding strand (sense strand) or the non-coding strand (antisense strand).

The receptor nucleic acid may comprise the nucleotide sequence shown in SEQ ID NO: 2, which corresponds to human spleen cDNA.

[0160]   In one embodiment, the acceptor nucleic acid comprises only the coding region.

The invention further provides a variant receptor polynucleotide encoding the same protein encoded by the nucleotide sequence set forth in SEQ ID NO: 2, which differs from the nucleotide sequence set forth in SEQ ID NO: 2 due to the degeneracy of the genetic code, and Provide a fragment of them.

The present invention also provides receptor nucleic acid molecules that encode the variant polypeptides described herein. Such polynucleotides can be present in their native form, including allelic variants (same locus) (map to chromosome 1 adjacent to AFM297zg1), homologues (different loci) and orthologs (different organisms). Even so,
Alternatively it may be constructed by recombinant DNA methods or by chemical synthesis. Such non-naturally occurring variants can be made by mutagenesis techniques including those applied to polynucleotides, cells or organisms. Therefore, as discussed above,
Variants may contain nucleotide substitutions, deletions, transpositions and insertions.

Mutations may occur in either or both the coding and non-coding regions. Mutations can result in conservative and non-conservative amino acid substitutions.

Typically, the variants have substantial homology with the nucleic acid molecule of SEQ ID NO: 2 and its complement.

Orthologs, homologues and allelic variants can be identified using methods well known in the art. These variants have at least about 55-60%, about 60-65%, 65-70%, and typically at least about 70-75%, and even more than the nucleotide sequence shown in SEQ ID NO: 2 or a fragment of this sequence Typically, it comprises a nucleotide sequence that encodes a receptor that is at least about 80-85% and most typically at least about 90-95% or more homologous. Such a nucleic acid molecule can be readily identified as capable of hybridizing under stringent conditions with the nucleotide sequence shown in SEQ ID NO: 2 or a fragment of this sequence. Stringent hybridization is substantially homologous if it is due to common homology, such as poly A sequences or sequences common to all or most proteins, all GPCRs, or all Family I GPCRs. It is understood that it does not exhibit sex. Furthermore, it is understood that variants do not include any of the nucleic acid sequences that may have been disclosed prior to the present invention.

As used herein, the term "hybridize under stringent conditions" typically refers to nucleotides encoding a receptor polypeptide that are at least about 50-55%, 55% homologous to each other. It is intended to describe the conditions of hybridization and washing that allow the sequences to hybridize to each other. Conditions are such that sequences that are at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95% or more identical to one another are capable of hybridizing to the other. May be. Such stringent conditions are well known to those of skill in the art and are incorporated herein by reference, Current Protocols in Molecular Biology, John Wiley & Sons, N.
It can be found in .Y. (1989), 6.3.1-6.3.6. An example of stringent hybridization conditions is hybridization in 6 × sodium chloride / sodium citrate (SSC) at about 45 ° C., followed by 0.2 × SSC at 50-65 ° C., 0 ° C.
.One or more washes in 1% SDS. Another non-limiting example is about 4 nucleic acid molecules.
Hybridize in 6 × sodium chloride / sodium citrate (SSC) at 5 ° C., followed by one or more low stringency washes in 0.2 × SSC / 0.1% SDS at room temperature or 0.2 at 42 ° C. × One or more moderate stringency washes with SSC / 0.1% SDS, or 65 ° C for high stringency
Perform a wash in 0.2 x SSC / 0.1% SDS in. In one embodiment, the isolated receptor nucleic acid molecule that hybridizes under stringent conditions with the sequence of SEQ ID NO: 2 corresponds to a naturally occurring nucleic acid molecule. As used herein, a "native" nucleic acid molecule refers to an RNA or DNA molecule having a naturally-occurring (eg, encoding a native protein) nucleotide sequence.

As will be appreciated by those in the art, the exact conditions can be determined empirically and depend on the ionic strength, temperature and concentration of destabilizing agents such as formamide or denaturing agents such as SDS. To do. Other factors considered in determining the desired hybridization conditions include the length of the nucleic acid sequences, the composition of the bases, the percentage of mismatches between the hybridizing sequences and the frequency of occurrence of sequence subsets within other non-identical sequences. Is included. Thus, equivalent conditions can be determined by altering one or more of these parameters but maintaining about the same degree of identity or similarity between the two nucleic acid molecules.

The present invention also provides an isolated nucleic acid containing a single-stranded or double-stranded fragment or portion that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 2 or the complementary strand of SEQ ID NO: 2. To do. In one embodiment, the nucleic acid consists of the nucleotide sequence of SEQ ID NO: 2 and part of the complementary strand of SEQ ID NO: 2. The nucleic acid fragments of the invention have a chain length of at least about 15, preferably at least about 18, 20, 23 or 25.
The nucleotides may be nucleotides having a chain length of 30, 40, 50, 100, 200 or more. Longer fragments encoding the antigenic proteins or polypeptides described herein are useful, eg, nucleotides with a chain length of 30 or greater.

The invention further provides polynucleotides that include fragments of the full length receptor polynucleotide. Fragments may be single or double stranded and may include DNA or RNA. Fragments may be derived from coding or non-coding sequences.

In one embodiment, the isolated receptor nucleic acid fragment has nucleotides 1 to about nucleotide 1237 and nucleotides 1754 to 2040 has a chain length of at least 5
Hybridizes under stringent conditions to a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO: 2. In one embodiment, the nucleic acid is a nucleotide with a chain length of 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 400 or 500 or more.

In another embodiment, the isolated receptor nucleic acid comprises about amino acid 1 to amino acid 3.
Code the entire 83 code regions. In another embodiment, the isolated receptor nucleic acid encodes a sequence corresponding to the mature protein from about amino acid 6 to amino acid 383.
Other fragments include nucleotide sequences that encode the amino acid fragments described herein. In addition, fragments may include subfragments of the particular regions or sites described herein. Fragments also include nucleic acid sequences corresponding to the particular amino acid sequences above or fragments thereof.

Receptor nucleic acid fragments further include sequences corresponding to the regions described herein, the subregions described herein and specific functional sites. Receptor nucleic acid fragments also include combinations of the regions, segments, loops and other functional sites described above. Thus, for example, the receptor nucleic acid may comprise a sequence corresponding to the amino-terminal extracellular domain and one transmembrane fragment. One of ordinary skill in the art will be aware of the many possible substitutions.

As long as the location of regions or sites is predicted by computer analysis, one of skill in the art will understand that the amino acid residues that make up these domains may vary depending on the criteria used to define the domains. I'm sure

However, it is understood that a receptor fragment includes any nucleic acid sequence that does not include the entire gene.

Accordingly, a receptor nucleic acid fragment is a polypeptide comprising an amino-terminal extracellular domain,
A polypeptide comprising a region spanning a transmembrane domain, a polypeptide comprising a carboxy-terminal subdomain, a nucleic acid molecule encoding a polypeptide having G protein receptor-identifying properties, seven transmembrane segments, an extracellular or intracellular loop, Nucleic acid molecules encoding any of glycosylation, phosphorylation, amidation and myristoylation are included. If the location of the region is predicted by computer analysis,
One of ordinary skill 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 an epitope harboring the region of the receptor protein described herein.

The isolated receptor polynucleotide sequences, particularly fragments, are useful as DNA probes and primers.

For example, the receptor coding region can be isolated using known nucleotide sequences for synthesizing oligonucleotide probes. The labeled probe can then be used to screen a cDNA library, genomic DNA library or mRNA to isolate the nucleic acid corresponding to the coding region. In addition, primers can be used in PCR reactions to clone specific regions of the receptor gene.

The probe / primer typically comprises substantially purified oligonucleotide. The oligonucleotide is typically at least about 5,10 of SEQ ID NO: 2.
, 12, typically about 25, more typically about 40, 50 or 75 contiguous nucleotides that hybridize to the sense or anti-sense strand or other receptor polynucleotide under stringent conditions. Contains the sequence region. The probe further comprises a label, such as a radioisotope, a fluorescent compound, an enzyme or an enzyme cofactor.

Uses of Polynucleotides Nucleic acid sequences of the invention can be used to search the public databases to identify, for example, other family members or related sequences.
uence) ”. Such a search is performed by Altshul et al. (19
90) J. Mol. Biol. 215: 403-10 NBLAST and XBLAST programs (version 2.
0) can be used. BLAST nucleotide searches are performed with the NBLAST program, score = 100, to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention.
, Word length = 12 can be implemented. Altshul et al. (1997) Nucle
Gapped BLAST can be used to insert and align gaps for comparison purposes, as described in ic Acids Res. 25 (17): 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

The nucleic acid fragments of the invention provide probes or primers for assays such as those described below. A "probe" is an oligonucleotide that base-specifically hybridizes to a complementary strand of nucleic acid. Such a probe is available from Nielsen
including polypeptide nucleic acids described in et al. (1991) Science 254: 1497-1500. Typically, at least about 15, and typically about 20-25 of the nucleic acid sequence of SEQ ID NO: 2.
, More typically about 40, 50 or 75 contiguous nucleotides and a region of nucleotide sequence that hybridizes to its complementary strand under highly stringent conditions. More typically, the probe further comprises a label such as, for example, a radioisotope, a fluorescent compound, an enzyme or an enzyme cofactor.

The term “primer” as used herein includes template-oriented using well-known methods (eg, PCR, LCR), including but not limited to those described herein. A single-stranded oligonucleotide that acts as a starting point for DNA synthesis. Suitable chain length primers will depend on the particular application, but typically range from about 15 to
It is in the range of 30 nucleotides. The term "primer site" refers to the region of target DNA with which a primer hybridizes. The term “primer pair” includes a 5 ′ (upstream) primer that hybridizes to the 5 ′ end of the nucleic acid sequence to be amplified and a 3 ′ (downstream) primer that hybridizes to the complementary strand of the sequence to be amplified. A primer set.

Receptor polynucleotides are useful for probes, primers and in bioassays.

When the polynucleotide is used to assess a GPCR property or function, such as the assays described herein, all of the full length cDNA or not all of the full length cDNA may be useful. In this case, the fragments known before the present invention are also included. Thus, assays specifically directed to GPCR function, such as assessing agonist or antagonist activity, involve the use of known fragments. Furthermore, diagnostic methods for assessing receptor function can be performed with any fragment, including those fragments that may have been known prior to the present invention. Similarly, methods related to the treatment of receptor dysfunction include all fragments, including those that may be well known in the art.

Receptor polynucleotides are variants or herein for the isolation of full-length cDNA and genomic clones encoding the polypeptide set forth in SEQ ID NO: 1, and producing the same polypeptide set forth in SEQ ID NO: 1. It is useful as a cDNA and genomic DNA hybridization probe to isolate cDNA and genomic clones corresponding to other variants described in the publication. Variants may be isolated from the same tissue and organism from which the polypeptide set forth in SEQ ID NO: 1 was isolated, from different tissues of the same organism, or from different organisms. . This method
Developmentally controlled is useful for isolating genes and cDNAs that may be expressed in the same or different tissues at different time points in the development of the organism.

The probe can correspond to any sequence of the entire chain length of the gene encoding the receptor. Thus, the probe may be derived from the 5'noncoding region, the coding region and the 3'noncoding region. However, as discussed herein, it is understood that the fragment corresponding to the probe does not include fragments that may have been previously disclosed.

Nucleic acid probes have, for example, a chain length of at least 5, 10, 12, 15, 30, 50, 100, 25
0 or 500 nucleotides, mRNA or DNA under stringent conditions
It may be a full-length cDNA of SEQ ID NO: 1, or a fragment thereof, such as an oligonucleotide sufficient to specifically hybridize with.

Fragments of the polynucleotides described herein are also useful for synthesizing larger fragments or full length polynucleotides described herein. For example,
The fragment can hybridize to any portion of the mRNA to produce a larger or full length cDNA.

Fragments are also useful in synthesizing antisense molecules of the desired chain length and sequence.

The antisense nucleic acids of the invention are designed using the nucleotide sequence of SEQ ID NO: 2 and can be constructed using chemical synthesis and enzymatic ligation reactions using techniques well known in the art. . For example, antisense nucleic acids (eg, antisense oligonucleotides) are used to increase the biological stability of naturally occurring nucleotides or molecules, or the physical stability of dimers formed between antisense and sense nucleic acids. It can be chemically synthesized using various modified nucleotides designed to increase sex. For example, thiophosphate ester derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides that can be used to make antisense nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminoethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylkeosine, inosine, N6-isopentenyladenine, 1-methyl Guanine, 1-methylinosine, 2,2
-Dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylkeosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-
N6-isopentenyl adenine, uracil-5-oxyacetic acid (v), wibutoxosine,
Pseudouracil, keosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-
Thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino3-N-2- Carboxypropyl) uracil, (acp3) w and 2,6-diaminopurine. Alternatively, antisense nucleic acids can be made biologically using expression vectors in which the nucleic acid is subcloned in the antisense orientation (
That is, the RNA transcribed from the inserted nucleic acid is in the antisense orientation of the target nucleic acid of interest).

The nucleic acid molecule of the present invention may also be modified at the base moiety, sugar moiety or phosphate backbone, for example to improve the stability, hybridization or solubility of the molecule. For example, the deoxyribose phosphate backbone of nucleic acids can be modified to create peptide nucleic acids (Hyrup et al. (1996) Bioorganic & Medicinal
Chemistry 4: 5). The term "peptide nucleic acid" or "PNA" as used herein refers to nucleic acid mimetics, for example, where the deoxyribose phosphate backbone is replaced with a pseudopeptide backbone and only the four natural nucleobases are retained. A DNA mimic that is present. PNAs
The neutral scaffold has been shown to be capable of specific hybridization with DNA and RNA under low ionic strength conditions. Synthesis of PNA oligomers is described by Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Aca.
d. Sci. USA 93: 14670 can be performed using the standard solid phase peptide synthesis protocol. PNAs can be synthesized, for example, by attaching a lipophilic group or other helper group to PNA to form a PNA-DNA chimera.
Alternatively, it can be further modified to increase stability, specificity or cellular uptake by using liposomes or other drug delivery techniques well known in the art. Synthesis of PNA-DNA chimeras is described in Hyrup (1996) supra, Finn et al. (1996) Nucleic.
Acids Res. 24 (17): 3357-63, Mag et al. (1989) Nucleic Acids Res. 17: 597.
3 and Peterser et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119.

Nucleic acid molecules and fragments of the present invention also include peptides (eg, for targeting host cell receptors in vivo) or agents that facilitate transport across cell membranes (
For example, Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86: 6553-6556;
Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84: 648-652; PCT Public
ation (see WO 88/0918) or other agents such as agents that facilitate transport across the blood-brain barrier (see, eg, PCT Publication WO 89/10134). . Oligonucleotides are also useful in hybridization-induced cleavage agents (eg, Krol et al. (1988) Bio-Techniques 6: 958-976).
) Or an intercalating agent (see, eg, Zon (1988) Pharm Res. 5: 539-549).

Receptor polynucleotides are also useful as primers for PCR to amplify any given region of the receptor polynucleotide.

Receptor polynucleotides are also useful in constructing recombinant vectors.
Such vectors include expression vectors that express some or all of the receptor polypeptide. The vector also includes the receptor gene and gene product in sit
u Includes an insert vector used to integrate into another polynucleotide sequence, such as the cell genome, to alter expression. For example, the endogenous receptor coding sequence is
All or part of the coding region containing one or more specifically introduced mutations may be replaced by homologous recombination.

Receptor polynucleotides are also useful for expressing the antigenic portion of the receptor protein.

Receptor polynucleotides can also be expressed in FISH (a review of this technique is provided by Verma et al. (1988
) Human Chromosomes: A Manual of Basic Techniques (Pergamon Press, New Yo
rk)) and as a probe for determining the chromosomal location of the receptor polynucleotide by in situ hybridization methods such as PCR mapping of somatic cell hybrids. The mapping of sequences to chromosomes is an important first step in linking these sequences with genes associated with disease.

Chromosome mapping reagents can be used individually to mark one chromosome or one site of that chromosome, and the reagent suite can be used for multiple sites and / or
Or it can be used to mark multiple chromosomes. Reagents corresponding to the non-coding regions of the gene are actually preferred for mapping purposes. The coating sequences are likely to be conserved within the gene family, thus increasing the probability of cross-hybridization during chromosome mapping.

Once the sequence has been mapped to a precise chromosomal location, the physical location of the sequence on the chromosome can be correlated to gene mapping data. (Such data is found, for example, in V. McKusick, Mendelian Inhetitance in Man, available online from Johns Hopkins University Welch Medical Library).
. Linkage analysis then identifies the association of the gene with a disease that maps to the same chromosomal region, as described, for example, in Egeland et al. ((1987) Nature 325: 783-787). Can (co-inheritance of physically adjacent genes).

In addition, DNA sequence differences between individuals affected and not affected by a disease associated with a particular gene can be determined. If a mutation is found in some or all of the affected patients but not in unaffected individuals, the mutation may be the causative agent of the particular disease. A comparison of affected and unaffected individuals can generally be seen from chromosome spreads, and of chromosomes such as defects or metastases that are detectable using PCR based on their DNA sequences. Related to seeing structural changes first. Finally, complete sequencing of genes in several individuals can be performed to confirm the presence of the mutation and distinguish the mutation from polymorphisms.

Receptor polynucleotide probes may also be used to identify receptors and their variants with respect to tissue distribution, eg, whether replication of the gene is occurring and whether replication is occurring in all or only a subset of tissues. It is useful for determining the presence pattern of the encoding gene. The gene may be naturally occurring or exogenously introduced into the cell, tissue or organism.

Receptor polynucleotides are also useful in designing ribozymes corresponding to all or part of the mRNA made from the genes encoding the polynucleotides described herein.

Receptor polynucleotides are also useful for constructing host cells expressing some or all of the receptor polynucleotides and polypeptides.

Receptor polynucleotides are also useful for constructing transgenic animals that express all or a portion of the receptor polynucleotides and polypeptides.

Receptor polynucleotides are also useful in making vectors that express some or all of the receptor polypeptide.

Receptor polynucleotides are also useful as hybridization probes for determining the level of receptor nucleic acid expression. Thus, the probe can be used to detect the presence of, or determine the level of, receptor nucleic acid in cells, tissues and organisms. The nucleic acid whose level is sought may be DNA or RNA. Accordingly, probes corresponding to the polypeptides described herein can be used to assess gene copy number in a given cell, tissue or organism. This is particularly relevant in cases where there is amplification of the receptor gene.

Alternatively, the probe is integrated into a chromosome in which the receptor gene is not normally found, such as on extrachromosomal elements or in regions that stain homogenously, for the location of the extra copy of the receptor gene. Can be used with respect to in situ hybridization to evaluate as.

These uses relate to the diagnosis of diseases associated with increased or decreased receptor expression relative to normal, such as proliferative diseases, differentiation or developmental diseases or hematopoietic diseases.

Accordingly, the present invention is a method for identifying a disease or disorder associated with aberrant expression or activity of a receptor nucleic acid, wherein a test sample is obtained from a subject and the nucleic acid (eg mRNA , Genomic DNA) and the presence of the nucleic acid is diagnostic of a subject having a disease or disorder associated with aberrant expression or activity of the nucleic acid or at risk of developing the disease or disorder. provide.

One aspect of the invention is whether an individual has a disease or disorder associated with aberrant nucleic acid expression or activity, or is at risk of developing a disease or disorder associated with aberrant nucleic acid expression or activity. It relates to a diagnostic assay for determining the expression and activity of nucleic acids in biological samples (eg blood, serum, cells, tissues) to determine whether. Such assays can be used for prognostic or predictive purposes of prophylactically treating an individual prior to the onset of a disorder characterized by or associated with the expression or activity of nucleic acid molecules.

In vitro techniques for detecting mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detecting DNA include Southern hybridizations and in situ hybridizations.

[0211] A probe is by measuring the level of a nucleic acid encoding a receptor, eg, mRNA or genomic DNA, in a cell sample of a subject, or by determining whether the receptor gene is mutated. Can be used as part of a diagnostic test kit to identify cells or tissues that express the receptor.

Nucleic acid expression assays are useful in drug screening to identify compounds that modulate receptor nucleic acid expression (eg, antisense, polypeptides, peptidomimetics, small molecules or other agents). Cells are contacted with a candidate compound and mRNA expression is measured. The expression level of mRNA in the presence of the candidate compound is compared to the expression level of mRNA in the absence of the candidate compound. Based on this comparison, the candidate compound can then be identified as a modulator of nucleic acid expression and can be used, for example, to treat disorders characterized by aberrant nucleic acid expression. The modulator may bind to the nucleic acid or may indirectly regulate expression, such as by interacting with other cellular components that affect the expression of the nucleic acid.

The modulation method can be performed in vitro (eg, by culturing cells with the drug) or in vivo in a patient or transgenic animal (eg, by administering the drug to a subject).

Accordingly, the invention provides methods for identifying compounds that can be used to treat disorders associated with nucleic acid expression of a receptor gene. The methods of the invention are typically compounds that can be used to assay the ability of a compound to modulate expression of a receptor nucleic acid, thereby treating a disorder characterized by unwanted receptor nucleic acid expression. Identifying.

The assay can be performed in cell-based and cell-free systems. Cell-based assays include cells that naturally express the receptor nucleic acid and recombinant cells that have been genetically engineered to express a particular nucleic acid sequence.

Alternatively, candidate compounds can be assayed in vivo in patients or transgenic animals.

Assays for receptor nucleic acid expression may involve direct assays at the nucleic acid level, such as mRNA levels, or may involve secondary compounds involved in signal pathways (such as cyclic AMP turnover). . In addition, the expression of genes that are upregulated or downregulated in response to the receptor signaling pathway can also be assayed. In this embodiment, the regulatory regions of these genes can be operably linked to a reporter gene such as luciferase.

Therefore, in a method of contacting cells with a candidate compound and measuring the expression of mRNA,
Modulators of receptor gene expression can be identified. The expression level of the receptor mRNA in the presence of the candidate compound is compared to the expression level of the receptor mRNA in the absence of the candidate compound. Based on this comparison, the candidate compound can then be identified as a modulator of nucleic acid expression and can be used, for example, to treat disorders characterized by aberrant nucleic acid expression. A candidate compound is identified as a stimulator of nucleic acid expression if mRNA expression is statistically significantly greater in the presence of the candidate compound than in the absence thereof. A candidate compound is identified as an inhibitor of nucleic acid expression if nucleic acid expression is statistically significantly less in the presence of the candidate compound than in the absence thereof.

Accordingly, the present invention provides therapeutic methods using compounds identified by drug screening as gene modulators to regulate receptor nucleic acid expression and using nucleic acids as targets. Modulation includes upregulation (ie, activation or activity) or downregulation (eg, suppression or antagonism) or effect on the activity of nucleic acids (eg, when nucleic acids are mutated or inappropriately modified) . Treatment is treatment for disorders characterized by aberrant expression or activity of nucleic acids.

Alternatively, the modulator of receptor nucleic acid expression may be a small molecule or drug identified using the screens described herein, as long as the drug or small molecule inhibits receptor nucleic acid expression. Good.

Receptor polynucleotides are also useful for monitoring the effectiveness of modulatory compounds on receptor gene expression or activity in clinical trials or therapeutic methods. Thus, the expression pattern of a gene can serve as a barometer of the long-term efficacy of treatment with compounds, especially those with which patients may develop resistance. The expression pattern of a gene can also serve as a marker of the physiological response of diseased cells to the compound. Thus, such monitoring allows for increased dosing of the compound or administration of another compound for which the patient has not developed resistance. Similarly, if the expression level of the nucleic acid falls below the desired level, the administration of the compound may be correspondingly reduced.

Monitoring can be performed, for example, as follows. (i) obtaining a pre-dose sample from the subject prior to administering the drug, (ii) a particular mRNA of the invention in the pre-dose sample
Or detecting the expression level of genomic DNA, (iii) obtaining one or more post-administration samples from a subject, (iv) detecting the expression or activity level of mRNA or genomic DNA in the post-administration sample, ( v) comparing the expression or activity level of mRNA or genomic DNA in the pre-dose sample with the expression or activity level of mRNA or genomic DNA in the post-dose sample, and (vi) correspondingly administering the drug to the subject. Increase or decrease.

Receptor polynucleotides are also useful in diagnostic assays for quantitative alterations of receptor nucleic acids, particularly for abnormal alterations. The polynucleotide is mR
It can be used to detect mutations in receptor genes such as NA and gene expression products. Use of the Polynucleotides as Hybridization Probes to Detect Naturally Occurring Mutations in Receptor Genes and thereby Determine Whether a Mutated Subject Is at Risk of Disorders Caused by the Mutations You can Mutations include alterations in gene copy number, such as deletions, additions or substitutions of one or more nucleotides in a gene, chromosomal modifications such as inversions or transpositions, modifications or amplification of genomic DNA such as aberrant methylation patterns. included. Detection of mutant forms of the receptor gene associated with dysfunction is a diagnostic tool for active disease or susceptibility of the disease if the disease is caused by overexpression, underexpression or altered expression of the receptor.

Mutations in the receptor gene can be detected at the nucleic acid level by a variety of techniques. Genomic DNA may be analyzed directly or may be amplified using PCR prior to analysis. RNA or cDNA can be used as well.

[0225] In certain embodiments, mutation detection is performed by anchor PCR or RACE PC.
R polymerase chain reaction (PCR) (see, eg, US Pat. Nos. 4,683,195 and 4,6
83,202) or ligation chain reaction (LCR) (see, eg, Landegran et a
l. (1988) Science 241: 1077-1080 and Nakazawa et al. (1994) PNAS 91: 360-3.
64), the latter being particularly useful for detecting point mutations in genes (Abravaya et al. (1995). Nucleic Ac
ids Res. 23: 675-682). This method comprises the steps of recovering a cell sample from a patient, isolating nucleic acid (eg, genomic, mRNA or both) from the cells of the sample, and hybridizing the gene (if present) to the nucleic acid sample. And contacting one or more primers that specifically hybridize to the gene under conditions in which amplification occurs, detecting the presence or absence of an amplification product or detecting the size of the amplification product, and the chain length as a control sample. And comparing with. Deletions and insertions can be detected by a change in the size of the amplification product as compared to the normal genotype. Point mutation is normal RN
It can be identified by hybridizing amplified DNA to A or antisense DNA sequences.

It is contemplated that PCR and / or LCR may be desirable for use as a preliminary amplification step in combination with any of the techniques used to detect the mutations described herein. .

[0227] Another amplification method is self sustained sequence replication.
) (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcription amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173-1177), Q
-Beta Replicase (Lizardi et al. (1988) Bio / Technology 6: 1197) or any other nucleic acid amplification method, followed by detection of the amplified molecule using techniques well known to those skilled in the art. These detection schemes are particularly useful for detecting nucleic acid molecules when such molecules are present in very low numbers.

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

In addition, sequence-specific ribozymes (US Pat. No. 5,498,531) can be used for scoring the presence of specific mutations by the formation or loss of ribozyme cleavage sites.

Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or melting temperature differences.

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

[0232] Furthermore, the sequence difference between the mutant receptor gene and the wild-type gene is a direct DN
A can be determined by sequencing. Mass spectrometry (eg WO 9
4/16101; Cohen et al. Adv. Chromatogr. 36: 127-162 (1996); and Griffin.
38. 147-159 (1993)), et al. Appl. Biochem. Biotechnol. 38: 147-159 (1993)), using various automated sequencing techniques when performing diagnostic assays ((1995) Biotechniques 19: 448). You can

Another method of detecting gene mutations is to use protection from cleaving agents to detect mismatched bases in RNA / RNA or RNA / DNA duplexes (Myers et al.
Science230: 1242 (1985)); Cotton et al. PNAS 85: 4397 (1988); Saleeba et al.
Meth. Enzymol. 217: 286-295 (1992)), a method of comparing electrophoretic mobilities of mutant and wild-type nucleic acids (Orita et al. PNAS 86: 2766 (1989); Cotton et al. Mut.
at. Res. 285: 125-144 (1993); and Hayashi et al. Genet. Anal. Tech. Appl.
.9: 73-79 (1992)) and a method for assaying migration of mutant or wild-type fragments in a polyacrylamide gel containing gradient denaturants using denaturing gradient gel electrophoresis ( Myers et al. Nature 313: 495 (1985)). The sensitivity of the assay can be enhanced using RNA (rather than DNA) whose secondary structure is more sensitive to sequence changes. In one embodiment, the method of the invention utilizes heteroduplex analysis to separate double-stranded heteroduplex molecules based on alterations in electrophoretic mobility (Keen et al. (1991). Trends Genet. 7: 5). Examples of other techniques for detecting point mutations include selective oligonucleotide hybridization, selective amplification and selective primer extension.

In another embodiment, gene mutations are performed by hybridizing a sample and control nucleic acid, eg, DNA or RNA, to a high density array containing hundreds or thousands of oligonucleotide probes. Can be identified (Cronin et al. (1996) Human Mutation 7: 244-255; Kozal et al. (1996) N
ature Medicine 2: 753-759). For example, mutations in genes contain light-generated DNA probes as described by Cronin et al. 2
Can be identified in a directional array. Briefly, the first hybridization probe array was designed to scan long DNA in samples and controls to identify base changes between sequences by creating a linear array of overlapping probes. Can be used for This step allows the identification of point mutations. This step is followed by a second, in which specific mutations can be characterized by using a smaller, specialized probe array complementary to all detected variants or mutations. The hybridization array of is used. Each mutation array contains parallel probe sets, one complementary to the wild-type gene and one complementary to the mutant gene.

Receptor polynucleotides are also useful in testing individuals for genotypes that affect treatment, although not necessarily causing disease. Thus, the polynucleotides can be used to study the relationship between an individual's genotype and the individual's response to compounds used in therapy (pharmacogenomic relationship). In the case of the present invention, for example, mutations in the receptor gene that alter its affinity for cAMP would lead to excess or diminished drug effects at standard concentrations of cAMP that activate the receptor. Thus, the receptor polynucleotides described herein can be used to assess mutation content of a gene in an individual to select appropriate compounds or regimens for treatment.

Accordingly, polynucleotides that exhibit genetic alterations that affect their treatment represent diagnostic targets that can be used to form a treatment for an individual. Thus, by producing recombinant cells and animals containing these polymorphisms, therapeutic compounds and regimens can be effectively clinically designed.

The method of the present invention involves obtaining a control biological sample from a control subject and determining the mRNA or genomic DN.
The control sample is contacted with a compound or agent capable of detecting mRNA or genomic DNA such that the presence of A is detected in the biological sample, and the presence of mRNA or genomic DNA in the control sample is tested for the presence of mRNA in the test sample. Or it may relate to comparison with the presence of genomic DNA.

Receptor polynucleotides are also useful for chromosome identification, where the sequence is identified using individual chromosomes at specific locations on the chromosome. First, set the DNA sequence in si
Chromosomes are matched by tu or chromosome-specific hybridization.
Sequences can also be correlated to particular chromosomes by making PCR primers that can be used in PCR screening of somatic cell hybrids containing individual chromosomes from the desired species. Only hybrids containing a chromosome containing the gene homologous to the primer will produce an amplified fragment. Sublocalization (sublo
calization) can be performed using a chromosome fragment. Other methods include prescreening with labeled flow-sorted chromosomes and preselection by hybridization with a chromosome-specific library. Yet another mapping method involves fluorescent in situ hybridization, which allows hybridization with shorter probes than previously used. Chromosome mapping reagents can be used individually to mark one chromosome or one site of a chromosome, and reagent pools can be used for multiple sites and / or
Or it can be used to mark multiple chromosomes. Reagents corresponding to the non-coding regions of the gene are indeed preferred for mapping purposes. The code sequence is
It is likely to be conserved within the gene family, thus increasing the probability of cross-hybridization during chromosome mapping.

Receptor polynucleotides can also be used to identify individuals from small biological samples. This is a restriction fragment length polymorphism used to identify individuals, for example.
(RFLP) can be used. Accordingly, the polynucleotides described herein are used as DNA markers for RFLP (see US Pat. No. 5,272,057).

In addition, receptor sequences can be used to provide another technique for determining the actual DNA sequence of selected fragments of an individual's genome. Therefore, the receptor sequences described herein can be used to make two PCR primers from the 5'and 3'ends of the sequence. Then for subsequent sequencing,
These primers can be used to amplify DNA from an individual.

Since each individual has a unique set of such DNA sequences, the corresponding set of DNA sequences from the individuals thus created can provide unique identification of the individual. It is estimated that human allelic variants occur approximately once every 500 bases. Allelic variants occur to some extent in the coding regions of these sequences and to a large extent in non-coding regions. Receptor sequences can be used to obtain such identifying sequences from individuals and tissues. The sequence is a unique fragment of the human genome. Each of the sequences described herein are to some extent derived from an individual.
The DNA can be used as a standard against which it can be compared for identification purposes.

When a sequence-derived reagent group is used to create a unique identification database for an individual, such same reagents can later be used to identify tissue from that individual. A unique identification database can be used to perform positive identification of living or dying individuals from very small tissue samples.

Receptor polynucleotides can also be used in forensic discrimination procedures.
PCR techniques can be used to amplify DNA sequences taken from very small biological samples such as single hair follicles, body fluids (eg blood, saliva or semen).
The amplified sequence can then be compared to a standard to identify the origin of the sample.

[0244] Thus, a receptor polynucleotide may be used to improve the reliability of DNA-based forensic identification, for example, by providing another "discriminating marker" (ie, another DNA sequence unique to a particular individual). It can be used to provide polynucleotide reagents such as PCR primers that target specific loci in the human genome that can be expanded. As mentioned above, the actual nucleotide sequence information can be used for identification as an accurate alternative to the pattern formed by the fragments produced by the restriction enzymes. Sequences that target non-coding regions are particularly useful because larger polymorphisms occur in non-coding regions that make it easier to identify individuals using this technique.

Receptor polynucleotides are used to identify specific tissues, eg, in situ.
It can be used to provide polynucleotide reagents such as labeled or labelable probes that can be used in hybridization techniques.
This is also useful if the forensic pathologist is provided with tissue of unknown origin. Receptor probe groups can be used to identify tissues by species and / or organ type.

[0246] Similarly, these primers and probes can be used to screen tissue cultures for contamination (ie, for the presence of a mixture of different cell types in culture).

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

Accordingly, receptor polynucleotides are useful as antisense constructs for controlling the expression of receptor genes in cells, tissues and organisms. DN
A antisense polynucleotides are designed to be “complementary to the gene regions involved in transcription and prevent transcription, and thus production of the receptor protein. Antisense RNA or DNA polynucleotides hybridize to mRNA, Therefore
Blocks translation of mRNA into receptor protein.

Examples of antisense molecules useful in inhibiting the expression of nucleic acids include those antisense molecules of SEQ ID NO: 2 complementary to a fragment of the 5 ′ untranslated region of SEQ ID NO: 2, including the initiation codon. An antisense molecule complementary to the 3'untranslated region fragment is included.

Alternatively, a class of antisense molecules may be used to reduce expression of receptor nucleic acid,
It can be used to inactivate mRNA. Thus, these molecules can treat diseases characterized by abnormal or unwanted expression of receptor nucleic acids. This technique involves cleavage by ribozymes that contain nucleotide sequences complementary to one or more regions of the mRNA, which render the mRNA less translatable. Possible regions include coding regions, particularly those that correspond to the catalytic and other functional activities of the receptor protein. It is understood that these regions include any of the specific regions, sites, segments, loops, etc. disclosed as specific regions or sites herein.

Receptor polynucleotides also provide a vector for gene therapy for patients containing cells with aberrant expression of the receptor gene. Thus, recombinant cells, including patient cells that have been engineered in exo vivo and returned to the patient, are introduced into the individual, where the cells produce the desired receptor protein for treating the individual.

The present invention also includes a kit for detecting the presence of receptor nucleic acid in a biological sample. For example, the kit may include reagents such as labeled or labelable nucleic acids or agents capable of detecting receptor nucleic acid in a biological sample, means for measuring the amount of receptor nucleic acid in the sample, and Means for comparing the amount of the receptor nucleic acid of The compound or agent may be packaged in a suitable container. Kit is a receptor
It may further include instructions for using the kit to detect mRNA or DNA.

Computer Readable Means The nucleotide or amino acid sequences of the invention are also provided in a variety of media to facilitate their use. As used herein, "provided" refers to a product other than an isolated nucleic acid or amino acid molecule that contains a nucleotide or amino acid sequence of the invention. Such products are available to those of skill in the art using methods that are not directly applicable to investigating nucleotide or amino acid sequences or subsets thereof when they occur in their naturally occurring or purified form. A possible form of a nucleotide or amino acid sequence or a subset thereof (eg, a subset of the open reading frame (ORF)) is provided.

In one application of this embodiment, the nucleotide or amino acid sequences of the invention can be recorded on a computer readable medium. As used herein, "computer-readable medium" is read directly by a computer,
Refers to any medium that can be evaluated. Such media include magnetic storage media such as floppy disks, hard disk storage media and magnetic tapes, optical storage media such as CD-ROMs, electrical storage media such as RAM and ROM, and magnetic / engineering media. Includes, but is not limited to, hybrids of such categories, such as physical storage media. Those of skill in the art will readily assess which of the currently known computer readable media can be used to make a product comprising the computer readable media recording the nucleotide or amino acid sequences of the invention. be able to.

As used herein, "recorded" refers to a process for storing information on a computer-readable medium. One of skill in the art can readily employ any of the presently known methods for recording information on computer readable media to produce products containing the nucleotide or amino acid sequence information of the present invention.

A variety of data storage structures are available to those of skill in the art to make computer readable media having recorded the nucleotide or amino acid sequences of the invention. The choice of data storage structure is generally based on the means chosen to utilize the information stored. Also, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable media. Sequence information is WordPerfect and Microsoft
It may be provided in a word processing text file formatted in a commercial software such as Word or in the form of an ASCII file and can be stored in a database application such as DB2, Sybase, Oracle, etc. One of skill in the art can readily adapt any number of data processor structural formats (eg, text files or databases) to obtain a computer readable medium having the nucleotide sequence information of the present invention recorded thereon.

By providing a computer readable form of the nucleotide or amino acid sequences of the invention, one of ordinary skill in the art can commonly utilize the sequence information for a variety of purposes. For example, one of skill in the art can use the computer readable form of the nucleotide or amino acid sequences of the invention to compare a target sequence or target structural motif with sequence information stored in a data storage means. Search means can be used to identify fragments or regions of the sequences of the invention that match a particular target sequence or target motif.

As used herein, a “target sequence” may be any DNA or amino acid sequence of 6 or more nucleotides or 2 or more amino acids. One of skill in the art can readily recognize that the longer the target sequence, the less likely it is to occur in the database as a random occurrence. The most preferred sequence length of the target sequence is about 10-100 amino acids or about 30-300 nucleotide residues. However, it is well recognized that commercially important fragments such as sequence fragments involved in gene expression and protein processing may have shorter chain lengths.

As used herein, a “target structural motif” or “target motif” is a rationally selected sequence that is selected based on the three-dimensional arrangement formed as a result of the folding of the protein motif. Refers to any sequence or combination of sequences. There are various target motifs known in the art. Protein target motifs include, but are not limited to, enzyme active sites and signal sequences. Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).

Computer software is publicly available to those skilled in the art for sequence information provided in the form of computer readable media for analysis and comparison with other sequences. Various known algorithms have been publicly disclosed, and various commercially available software for implementing the search means have been and can be used in the computer-based system of the present invention. Examples of such software include MacPattern (EMBL), BLASTN and BLASTX (NCBIA
), But is not limited thereto.

For example, BLAST (Altshul et al. (1990) J. Mol. Biol. 215: 403-410) and BL.
AZE (Brutlag et al. (1993) Comp. Chem. 17: 203-207) Search algorithm Sybas
Software running on the e-system can be used to identify open reading frames (ORFs) of the sequences of the invention that have homology to other library ORFs or proteins. Such ORFs are protein-encoding fragments and are useful in producing commercially important proteins such as enzymes used in various reactions and in producing commercially useful metabolites.

Vectors / Host Cells The invention also provides vectors containing the receptor polynucleotides. The term “vector” refers to a nucleic acid molecule capable of transporting a vehicle, preferably a receptor polynucleotide. When the vector is a nucleic acid molecule, the acceptor polynucleotide is covalently linked to the vector nucleic acid. In this aspect of the invention, the vector is a plasmid, single-stranded or double-stranded phage, single-stranded or double-stranded RNA or DNA.
Contains viral vectors or artificial chromosomes such as BAC, PAC, YAC OR MAC.

The vector may be maintained in the host cell as an extrachromosomal element when it replicates and makes additional copies of the receptor polynucleotide. Alternatively, the vector can be integrated into the host cell genome, making additional copies of the receptor polynucleotide as the host cell replicates.

The present invention provides a vector for maintenance of the receptor polynucleotide (cloning vector) or a vector for expression (expression vector). The vector can act in prokaryotic cells or eukaryotic cells or both (shuttle vectors).

Expression vectors are engineered to allow transcription of the polynucleotide in a host cell.
It contains a cis-acting regulatory region operably linked to the receptor polynucleotide vector. The polynucleotide may be introduced into the host cell along with a separate polynucleotide capable of affecting transcription. Therefore, the second polynucleotide is a cis-nucleotide in order to allow transcription of the receptor polynucleotide from the vector.
A trans-acting factor that interacts with the regulatory control region can be provided. Alternatively, the trans-acting factor may be supplied by the host cell. Finally, the trans-acting factor may be produced from the vector itself.

However, it is understood that in certain embodiments transcription and / or translation of the receptor polynucleotide may occur in a cell-free system.

Regulatory sequences to which the polynucleotides described herein are operably linked include promoters that direct transcription of mRNA. These include the left promoter of bacteriophage λ, E. coli lac, TRP and TAC promoters, SV40 early and late protein kinases, CMV immediate early promoter, adenovirus early and late promoters and retroviral terminal repeat sequences. Included, but not limited to.

In addition to control regions that promote transcription, expression vectors may include regions that regulate transcription, such as repressor binding sites and enhancers. For example, SV
40 enhancers, cytomegalovirus immediate-type enhancers, polyoma enhancers, adenovirus enhancers and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation and control, expression vectors also contain sequences necessary for transcription termination and a ribozyme binding site for translation in the transcribed region. Other regulatory control elements for expression include start and stop codons and polyadenylation signals. One of ordinary skill in the art will be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook e
t al. (1989) Molecular Cloning: A Laboratory Manual 2nd.ed., Cold Sprin
g Harbor Laboratory Press, Cold Spring Harbor, NY).

A variety of expression vectors can be used to express the receptor polynucleotide. Such vectors include chromosomes, episome- and virus-derived vectors, such as bacterial plasmids, bacteriophage, yeast episomes, yeast chromosomal elements including yeast artificial chromosomes, baculovirus, papovaviruses such as SV40, vaccinia virus, adenovirus, poxvirus, Vectors derived from viruses such as pseudorabies virus and retroviruses are included. Vectors may be derived from combinations of these origins, such as those derived from plasmids and bacteriophage genetic elements, eg, cosmids and phagemids. Suitable cloning and expression vectors for prokaryotic and eukaryotic hosts are found in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual 2nd. E.
d., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

A regulatory sequence may provide for constitutive expression in one or more host cells (eg, tissue-specific), but also one such as temperature, nutrient addition, or exogenous factors such as hormones or other ligands. Inducible expression may be provided in the above cell types. Various vectors that provide constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of skill in the art.

Receptor polynucleotides can be inserted into vector nucleic acids by well known methods. Generally, the final expressed DNA sequence is ligated to the expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Techniques for restriction enzyme digestion and ligation are well known to those of skill in the art.

Vectors containing the appropriate polynucleotides can be introduced into an appropriate host cell for propagation or expression using well known techniques. Bacterial cells include E. coli, Streptpmyces, and Salmonella typhi.
murium), but is not limited to them. 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.

As described herein, it may be desirable to express the polypeptide as a fusion protein. Accordingly, the present invention provides fusion vectors that allow the production of receptor polynucleotides. The fusion vector can increase the expression of the recombinant protein, increase the solubility of the recombinant protein, and help the purification of the protein, for example, by acting as a ligand for affinity purification. Proteolytic cleavage sites can be introduced at the junction of the fusion moiety so that the desired protein can ultimately be separated from the fusion moiety. Proteolytic enzymes include, but are not limited to, Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include glutathione S-transferase (GST), pGEX (Smith et al. Gene 67: 31-40 (1988)), which fuses maltose E binding protein or protein A to the target recombinant protein, respectively, pMAL (N
ew England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ). An example of a suitable inducible unfused E. coli expression vector is pTrc (Amann
et al. Gene 69: 301-315 (1988)) and pET 11d (Studier et al. Gene Express
ion Technology: Methods in Enzymology 185: 60-89 (1990)).

Recombinant protein expression is achieved by providing a genetic background in which the host cell has an impaired ability to proteolytically cleave the recombinant protein.
Can be maximized without host bacteria. (Gottesman, S. Gene Expression Tec
hnology: Methods in Enzymology 185, Academic Press, San Diedo, Californi
a (1990) 119-128). Alternatively, the sequence of the polynucleotide of interest can be modified to provide preferential codon usage for certain host cells, such as E. coli (wada et al. Nucleic Acids Res. 20: 2111-2118 (1992)).

Receptor polynucleotides can also be expressed by expression vectors that are functional in yeast. An example of a vector for expression in yeast such as S. cerevisiae is pYepSec1 (Baldari et al. EMBO J. 6: 229-234 (198).
7)), pMFa (Kurjan et al. Cell 30: 933-943 (1982)), pJRY88 (Schultz et al. G).
ene 54: 113-123 (1987)) and pYES2 (Invitorogen Corporation, San Diego, CA.
) Is mentioned.

Receptor polynucleotides can also be expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors available for expression of proteins in insect cells in culture (eg, Sf9 cells) include pAc series (
Smith et al. Mol. Cell Biol. 3: 2156-2165 (1983)) and pVL series (Lucklow
et al. Virology 170: 31-39 (1989)).

In certain embodiments of the invention, the polynucleotides described herein are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329: 840) and pMT2PC (Kaufman
et al. EMBO J. 6: 187-195 (1987)).

The expression vectors provided herein are provided solely to exemplify well-known vectors available to those of skill in the art that may be useful for expressing receptor polynucleotides. One of ordinary skill in the art will be aware of other vectors suitable for maintenance growth or expression of the polynucleotides described herein. These are, for example:
Sambrook et al. Molecular Cloning: A Laboratory Manual 2nd, ed., Cold Sp
ring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Found in Harbor, NY 1989.

The present invention also includes vectors in which the nucleic acid sequences described herein are cloned into the vector in the opposite orientation, but operably linked to regulatory sequences that allow transcription of the antisense RNA. Accordingly, antisense transcripts can be generated for all or part of the polynucleotide sequence described herein, including coding and non-coding regions. The expression of this antisense RNA is subjected to each of the parameters described above in relation to the expression of sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).

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

Recombinant host cells are made by introducing into a cell the vector construct described herein by techniques readily available to those of skill in the art. These include calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, lipofection and Sambrook et al.
al. (Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbo
r Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY, 1989) and other techniques, including but not limited to.

Host cells may contain more than one vector. Therefore, different nucleotide sequences may be introduced into different vectors in the same cell. Similarly, the receptor polynucleotide may be introduced alone or with other polynucleotides unrelated to the receptor polynucleotide, such as those that provide trans-acting factors for the expression vector. If more than one vector is introduced into the cell, the vectors may be introduced independently, simultaneously or may be linked to the acceptor polynucleotide vector.

In the case of bacteriophage and viral vectors, these can be introduced into cells as packaged or encapsulated virus by standard techniques for infection and transduction. Viral vectors may be replication competent or replication defective. When viral replication is defective, replication occurs in host cells that provided the function of compensating for the defect.

Vectors generally include a selectable marker that allows for selection of the subpopulation of cells that contain the recombinant vector construct. The marker may be contained on the same vector containing the polynucleotides described herein or on a separate vector. Markers include tetracycline or ampicillin resistance genes in prokaryotic host cells and dihydrofolate reductase or neomycin resistance in eukaryotic host cells. However, any marker that selects for a phenotypic trait is effective.

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

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

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

Depending on the host cell in the recombinant production of the polynucleotides described herein, the polypeptides may have different glycosylation patterns depending on the cell and when produced in bacteria. May not be glycosylated. Also, the polypeptide may be subject to initial modification if it is a result of host-mediated processes.
ied) Methionine may be included.

Host cells of particular interest include cells that are derived from the tissue in which the receptor is expressed and are involved in the brain, spleen, glioblastoma and sclerosing lesions. Including.

Uses of Vectors and Host Cells It is understood that "host cell" and "recombinant host cell" refer not only to the particular subject cell, but to the progeny or possible progeny of such a cell. Although some modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not be identical to the parent cell, but are nevertheless used herein. Included within the scope of the term.

Host cells expressing the polypeptides described herein, particularly recombinant host cells, have a variety of uses. First, the cells are useful to produce a receptor protein or polypeptide that can be further purified to produce the desired amount of receptor protein or fragment. Therefore, host cells containing the expression vector are useful for producing the polypeptide.

Host cells are also useful for performing cell-based assays involving receptors or receptor fragments. Thus, recombinant host cells expressing the native receptor are useful in assaying compounds that stimulate or inhibit receptor function. This includes ligand binding, gene expression at the transcriptional or translational level, G protein interactions and components of signal transduction pathways.

Particularly relevant cells are cells in which the receptor is expressed as disclosed herein.

Host cells are also useful in identifying receptor mutants in which these functions are affected. When the mutant occurs naturally and causes an abnormality, the host cell containing the mutation has a desired effect on the mutant receptor that is not shown by its effect on the natural receptor (eg, stimulates or inhibits function). Is useful for assaying compounds having

Recombinant host cells also express the chimeric polypeptides described herein in order to evaluate compounds that activate or repress activity by the heterologous amino-terminal extracellular region (or other binding region). Useful for. Alternatively, the transmembrane region (
Or a portion thereof) can be used to assess the effect of the desired amino-terminal extracellular region (or other binding region) of any given host cell. In this embodiment, a region spanning the entire transmembrane region (or a portion thereof) compatible with the particular host cell is used to make the chimeric vector. Alternatively, a heterologous carboxy-terminal cell, eg, a signal transduction region, can be introduced into a host cell.

In addition, mutant receptors can be designed that are engineered to increase or decrease one or more of various functions (eg, cAMP binding or kinase A binding),
It can be used to increase or replace individual receptor proteins. Thus, the host cell may provide a therapeutic benefit by exchanging aberrant receptors or by providing aberrant receptors that provide a therapeutic result. In one embodiment, the cells provide a receptor that is abnormally active.

In another embodiment, the cell provides a receptor that is abnormally inactive. These receptors can compete with endogenous receptors in the individual.

In another embodiment, cells expressing a receptor that cannot be activated in order to compete with an endogenous receptor for a ligand are introduced into an individual. For example, if excess ligand is part of the treatment regimen, it may be necessary to inactivate this molecule at some point in the treatment. It would be useful to provide cells that compete for this ligand but are unaffected by receptor activation.

It is also possible to produce homologous recombinant host cells which allow in situ modification of the endogenous receptor polynucleotide sequence of the host cell genome. This technique is disclosed in WO 93
Further details are described in WO / 09222, WO 91/12650 and US Pat. No. 5,641,670. Briefly, a homologous recombination that can affect the expression of a gene introduces a specific polynucleotide sequence corresponding to the receptor polynucleotide or the proximal or distal sequence of the receptor gene into the host cell genome. It is possible. In one embodiment, regulatory sequences that increase or decrease expression of endogenous sequences are introduced. Thus, the receptor protein can be made in cells that normally do not produce the receptor protein, or increased expression of the receptor protein can result in cells that normally produce a particular level of the protein. Alternatively, the entire gene may be deleted. Additional specific mutations can be introduced into any desired region of the gene to create a mutant receptor protein. Such mutations may be introduced, for example, in the particular regions disclosed herein.

In one embodiment, the host cell may be a fertilized oocyte or embryonic stem cell that can be used to produce a transgenic animal containing a modified receptor gene. Alternatively, the host cell may be a stem cell or other early tissue precursor that is capable of giving rise to a particular subset of cells and forming transgenic tissue in the animal. For a description of homologous recombination vectors, see Thomas et al.,
See also Cell 51: 503 (1987). The vector is introduced (eg, by electroporation) into an embryonic stem cell line and selects for cells in which the introduced gene has homologous recombination with the endogenous receptor gene (eg, Li. E. et al. Cell 69: 915). (1992)). The selected cells are then transferred to an animal (eg, mouse) to form a chimeric population.
Inserted into blastocysts (eg Bradley, A. in Tetracarcinomas and Embryonic
Stem Cells: A Practical Approach, EJ Robertoson, ed. (IRL, Oxford, 19
87) pp. 113-152). The chimeric embryo can then be implanted in a suitable pseudopregnant female foster animal and the embryo allowed to grow to term. Recombinant DNA homologous to germ cells
Progeny harboring S. cerevisiae can be used to breed animals in which all cells of the animal contain homologous recombinant DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombination animals are described in Bradley, A. (1991) Curr.
ent Opinion in Biotechnology 2: 823-829 and WO 90/11354,
Further description is made in WO 91/01140 and WO 93/04169.

Genetically engineered host cells can be used to produce nonhuman transgenic animals. The transgenic animal is preferably a rodent, such as a mammal, eg, rat or mouse, in which one or more of the cells of the animal contains the transgene. A transgene is exogenous DNA that is introduced into the genome of a cell in which a transgenic animal develops and is held in the genome of a mature animal in one or more cell types or tissues of the transgenic animal. These animals are useful for studying the function of the receptor protein and for identifying and evaluating modulators of receptor protein activity.

Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens and amphibians.

[0304] In one embodiment, the host cell is a fertilized oocyte or embryonic stem cell into which the receptor polynucleotide sequence has been introduced.

Transgenic animals are produced, for example, by introducing nucleic acids into the male pronucleus of fertilized oocytes by microinjection, retroviral infection, and generating the oocytes in pseudopregnant female foster relatives. be able to. Any of the receptor nucleotide sequences can be introduced as a transgene into the genome of a non-human animal such as a mouse.

Any of the regulatory or other sequences useful in expression vectors may form part of the transgene sequence. These include intron sequences and polyadenylation signals, if not already included. Tissue-specific regulatory sequences may be operably linked to the transgene to express the receptor protein in specific cells.

Methods for producing transgenic animals, especially animals such as mice, by embryo manipulation and microinjection are conventional in the art, eg, both US Pat. Nos. 4,736,866 and 4,870,009 by Leder et al. , Wagner et
al., U.S. Pat. No. 4,873,191 and Hogan, B., Manipulating the Mouse E.
mbyo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY., 198
It is described in 6). Similar methods are used to generate other transgenic animals. Transgenic founders can be identified based on the presence of the transgene within the genome and / or expression of the transgenic mRNA in animal tissues or cells. The transgenic founder animal can then be used to breed another animal carrying the transgene. In addition, transgenic animals carrying the transgene can be further bred to other transgenic animals carrying other transgenes. Transgenic animals also include animals in which whole animals or animal tissues have been produced using the homologous recombinant host cells described herein.

In another embodiment, transgenic non-human animals can be produced that contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre / loxP recombinase system of bacteriophage P1. cre / loxP
A description of recombinase systems can be found, for example, in Lakso et al. PNAS 89: 6232-6236 (1992).
See. Another example of a recombinase system is the S. cerevisiae FLP recombinase system (O'Gorman et al. Science 251: 1351-1335 (1991). Cre / loxP.
Cre when the recombinase system is used to regulate transgene expression.
There is a need for animals containing transgenes that encode both recombinases and selected proteins. Such animals can be transformed into, for example, a "double" transgenic animal by crossing two transgenic animals, one containing the transgene encoding the selected protein and the other containing the transgene encoding the recombinase. It can be provided by constructing a transgenic animal.

The non-human transgenic animal clones described herein also include
It can be prepared by the method described in Wilmut et al. Nature 385: 810-813 (1997) and WO 97/07668 and WO 97/07669. Briefly, cells of transgenic animals, eg, somatic cells, can be isolated and induced to exit the proliferative phase and enter the G ₀ phase. The quiescent cells can be fused to the enucleated oocytes of the same species of animal from which the quiescent cells were isolated, for example, by using electrical pulses. Then, the reconstructed oocyte is cultured so as to develop to morula or blastocyst, and transferred to pseudopregnant female foster animal. The offspring born from this female foster animal are clones of the animal from which the cells, eg somatic cells, are isolated.

Transgenic animals containing recombinant cells expressing the polypeptides described herein are useful for performing the assays described herein in vivo. Thus, the various physiological factors that are present in vivo and appear to influence cAMP binding, receptor activation and signaling may not be revealed from in vitro cell-free or cell-based assays. . Thus, in vivo assay of receptor function, including ligand interaction, receptor function and the effect of specific mutant receptors on ligand interaction, as well as the effect of chimeric receptors, provides non-human transgenic animals. Useful for. It is also possible to assess the effect of null mutations, ie mutations that substantially or completely eliminate one or more receptor functions.

Pharmaceutical Compositions Receptor nucleic acid molecules, proteins (particularly fragments such as the amino terminal extracellular region), protein modulators and antibodies (referred to herein as "agonists").
Can be incorporated into a pharmaceutical composition suitable for administration to a subject, eg, a human. Such compositions typically include a nucleic acid molecule, protein, modulator or antibody and a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically acceptable carrier” refers to solvents, dispersion media, coatings, antibacterial and antifungal agents that are compatible with the administration of the formulation.
It is intended to include any and all isotonic and absorption delaying agents and the like.
The use of such media and agents for pharmaceutically active substances is well known in the art. Such vehicles can be used in the compositions of the present invention, unless any conventional vehicle or agent is compatible with the active compound. Additional active compounds can also be incorporated into the compositions. The pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (topical), transmucosal and rectal administration. Solutions or suspensions for parenteral, intradermal or subcutaneous application may contain the following ingredients: sterile diluents such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerine, propylene glycol or Other synthetic solvents, antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, buffering agents such as acetates, citrates or phosphates and chlorides Drugs to regulate the osmotic pressure, such as sodium or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions 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 saline, bacteriostatic water, Cremophor EL (B
ASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be easy to syringe.
must be liquid enough to exist. The composition 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 may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg glycerol, polyethylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of microbial activity can be carried out by various antibacterial and antifungal agents such as, for example, paraben, chlorobutanol, phenol, ascorbic acid, thimerosal and the like.
In many cases, it will be preferable to include, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions include the required amount of the active compound (eg, receptor protein or anti-receptor antibody) in a suitable solvent, optionally with one or a combination of the above-listed components, followed by filter sterilization. It can be prepared by Generally, dispersions are prepared by incorporating the active compound 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 preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying to obtain the active ingredient plus any additional desired ingredients from the previously sterile filtered solution.

Oral compositions generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For oral administration, the drug may be contained in an enteric form such that it is not degraded in the stomach or it may be coated or mixed by known methods for release in specific areas of the digestive tract. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a liquid carrier for use as a mouthwash in which the compound in a liquid carrier is orally applied and swished and exhaled or swallowed. Pharmaceutically compatible binders and / or auxiliary substances can be included as part of the composition. Tablets, pills, capsules, troches and the like may contain any of the following ingredients or compounds of similar nature. Microcrystalline cellulose, binders such as tragacanth gum or gelatin, excipients such as starch or lactose, alginic acid, disintegrants such as Primogel or corn starch, lubricants such as magnesium stearate or Sterotes, slip agents such as colloidal silicon dioxide ( glidant
), Sweeteners such as sucrose or saccharin or flavoring agents such as peppermint, methyl salicylate or orange flavors.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, such as a gas such as carbon dioxide, or a nebulizer.

Systemic administration may be by transmucosal or transdermal routes. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and for transmucosal administration, for example, surfactants,
Includes bile acid and fusidic acid derivatives. Transmucosal administration can be carried out by nasal spray or the use of suppositories. For transdermal administration, the active compound is
They are generally formulated into ointments, salves, gels or creams well known in the art.

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

In one embodiment, the active compounds are prepared with carriers that do not provide rapid elimination of the compound from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. Methods for preparing such formulations will be apparent to those of skill in the art. Materials are Alza Corporation and Nova Pha
It can also be purchased from rmaceuticals, Inc. Liposomal suspensions, including liposomes that target infected cells and monoclonal antibodies to viral antigens, can also be used as pharmaceutically acceptable carriers. These can be prepared by methods well known to those skilled in the art, for example, as described in US Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, "dosage unit form" refers to physically discrete units suitable as unit dosages for the subject to be treated, each unit being associated with the required pharmaceutical carrier. Containing a predetermined amount of active compound calculated to produce the desired therapeutic effect. The specification of the dosage unit form of the present invention is
Required and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect achieved, as well as the essential limitations in the art of combining such active compounds to treat an individual.

The nucleic acid molecule of the present invention can be inserted into a vector and used as a gene therapy vector. Gene therapy vectors are, for example, intravenously injected, topically applied (US Pat. No. 5,328,470) or stereotactically injected (eg Chen et al. PNAS 91: 3054-305).
7 (see 1994)). The pharmaceutical preparation of the gene therapy vector may include the gene therapy vector in an acceptable diluent or may include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be made intact from recombinant cells, eg, retroviral vectors, the pharmaceutical preparation creates a gene delivery system.
It may include one or more cells.

The pharmaceutical composition may be included in a container, pack or dispenser with instructions for administration.

The present invention may be embodied in a number of different forms and should not be construed as limited to the embodiments set forth herein, but rather these embodiments are the disclosures of the present invention. The contents are provided to convey those skilled in the art to the invention in detail.
Many modifications and other embodiments of the invention will be apparent to those skilled in the art to which the invention pertains and benefit from the teachings provided in the above specification. Specific terms are used, but unless otherwise indicated, they are used as in the art.

[Sequence list]

[Brief description of drawings]

1 shows the 16405 nucleotide sequence (SEQ ID NO: 2) and the deduced 16405 amino acid sequence (SEQ ID NO: 1).

FIG. 2 shows a comparison of 16405 receptors against the Prosite database of protein patterns, specifically the seven transmembrane segment rhodopsin superfamily (SEQ ID NO: 3
) Shows a high score. The underlined portion indicates the GPCR discrimination characteristic, specifically the position of the arginine residue. The most commonly conserved sequences are aspartate, arginine and tyrosine (DRY) triplets. DRY is involved in signal transduction. Arginine is unchanged. Aspartate is conservedly located in some GPCRs. In the case of the invention, arginine is found in the sequence RYL, which corresponds to DRY or the invariant arginine of the GPCR of the rhodopsin superfamily of receptors.

FIG. 3 shows an analysis of the 16405 receptor amino acid sequence, αβ turn and coil regions, hydrophilic, amphipathic regions, flexible regions, antigenic index and surface probability plots.

FIG. 4 shows a hydrophobicity plot for the 16405 receptor. The amino acids correspond to positions 1-383 and represent 7 transmembrane segments.

FIG. 5 shows an analysis of the 16405 open reading frame of amino acids corresponding to a specific functional site (SEQ ID NO: 1). The glycosylation site is from about amino acid 3 to about amino acid 6. The cAMP and cGMP-dependent protein kinase phosphorylation sites are found at about amino acids 155 to about amino acids 158, about amino acids 224 to about amino acids 227 and about amino acids 279 to 282. Protein kinase C phosphorylation sites are found at about amino acids 58-60, 111-113, 222-224, 337-339 and 346-348. N-myristoylation sites are amino acids 40-45, 92-97, 107-112, 117-122.
, 123-128, 239-244, 316-321, 353-358 and 376-381. The amidation site is found at about amino acid 28 to about amino acid 31. The leucine zipper pattern is found from about amino acid 115 to about amino acid 136. Amino acids 1-31 constitute the amino-terminal extracellular domain, amino acids 32-338 constitute the region spanning the transmembrane domain, and amino acids 339-383 are predicted to constitute the carboxy-terminal intracellular domain. . The transmembrane domain contains 7 transmembrane segments, 3 extracellular loops and 3 intracellular loops. The transmembrane segment includes about amino acid 32 to about amino acid 56, amino acid about 68 to amino acid 85, amino acid about 118 to amino acid 1136, amino acid about 159 to amino acid 176, amino acid about 194 to amino acid 216, amino acid about 281 to amino acid. About 305 and about 319 amino acids to about amino acids
Found at the 338th. The region spanning the transmembrane domain has three intracellular loops and three extracellular loops. The three intracellular loops are about 57 to about 67 amino acids, about 137 to about 158 amino acids and about 217 to about 2 amino acids.
Found in 80. Three extracellular loops are found at about amino acid 86 to about amino acid 117, amino acid about 177 to about amino acid 193, and about amino acid 304 to about amino acid 318. The transmembrane region contains the GPCR signaling property, RYL, at residues 137-139. The sequence contains arginine at residue 138, the constant amino acid of the GPCR.

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Claims (26)

[Claims] 1. A) Nucleotide sequence according to SEQ ID NO: 2 or Patent Deposi
a nucleic acid molecule containing a nucleotide sequence that is at least 45% identical to the cDNA insert of any of the plasmids deposited in the ATCC as t Number PTA-1653 or its complementary strand; and b) the nucleotide sequence of SEQ ID NO: 2 or Patent Deposit. A nucleic acid molecule containing a cDNA insert of any of the plasmids deposited at the ATCC as Number PTA-1653 or a fragment of at least 15 nucleotides of the complementary strand thereof, and c) the amino acid sequence of SEQ ID NO: 1 or Patent Deposit Number PTA- ATC as 1653
A nucleic acid molecule encoding a polypeptide comprising an amino acid sequence encoded by the cDNA insert of any of the plasmids deposited in C, and d) the amino acid sequence of SEQ ID NO: 1 or AT as Patent Deposit Number PTA-1653.
A fragment of a polypeptide comprising an amino acid sequence encoded by the cDNA insert of any of the plasmids deposited with CC, which is SEQ ID NO: 1 or Patent Dep
osit Number cDNA of one of the plasmids deposited with ATCC as PTA-1653
A nucleic acid molecule encoding a fragment containing at least 12 contiguous amino acids of the amino acid sequence encoded by the insert, and e) the amino acid sequence of SEQ ID NO: 1 or ATC as Patent Deposit Number PTA-1653.
SEQ ID NO: encodes a natural allelic variant of a polypeptide containing the amino acid sequence encoded by the cDNA insert of one of the plasmids deposited in C.
An isolated nucleic acid molecule selected from the group consisting of a nucleic acid molecule containing 2 or its complementary strand and a nucleic acid molecule that hybridizes under stringent conditions. 2. A) a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO: 2 or a cDNA insert of any of the plasmids deposited at the ATCC as Patent Deposit Number PTA-1653 or its complementary strand, and b) a sequence. AT as number 1 amino acid sequence or Patent Deposit Number PTA-1653
A nucleic acid molecule according to claim 1, selected from the group consisting of: a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence encoded by the cDNA insert of any of the plasmids deposited with the CC. 3. The nucleic acid molecule of claim 1, further comprising a vector nucleic acid sequence. 4. The nucleic acid sequence further encoding a heterologous polypeptide.
1 nucleic acid molecule. 5. A host cell containing the nucleic acid molecule of claim 1. 6. The host cell according to claim 5, which is a mammalian host cell. 7. A non-human mammalian host cell containing the nucleic acid molecule of claim 1. 8. A) Amino acid sequence of SEQ ID NO: 1 or Patent Deposit Number P
A fragment of a polypeptide comprising an amino acid sequence encoded by the cDNA insert of any of the plasmids deposited with the ATCC as TA-1653, SEQ ID NO: 1
Or a fragment of a polypeptide containing at least 15 contiguous amino acids of the amino acid sequence encoded by the cDNA insert of any of the plasmids deposited with the ATCC as Patent Deposit Number PTA-1653, and b) of SEQ ID NO: 1. AT as amino acid sequence or Patent Deposit Number PTA-1653
A naturally occurring allelic variant polypeptide comprising an amino acid sequence encoded by the cDNA insert of any of the plasmids deposited with CC, SEQ ID NO: 2
Or a naturally occurring allelic variant polypeptide encoded by a nucleic acid molecule that hybridizes to a nucleic acid molecule containing its complementary strand under stringent conditions; and c) a nucleic acid containing SEQ ID NO: 2 or the nucleotide sequence of its complementary strand. At least 45%
An isolated polypeptide selected from the group consisting of a polypeptide encoded by a nucleic acid molecule comprising nucleotide sequences that are identical. 9. Amino acid sequence of SEQ ID NO: 1 or Patent Deposit Number PTA
An isolated polypeptide according to claim 8 comprising the amino acid sequence encoded by the cDNA insert of any of the plasmids deposited with the ATCC as -1653. 10. The polypeptide of claim 8, further comprising a heterologous amino acid sequence. 11. An antibody that selectively binds to the polypeptide of claim 8. 12. A) Amino acid sequence of SEQ ID NO: 1 or Patent Deposit Numbe
A polypeptide containing an amino acid sequence encoded by one of the cDNA inserts of the plasmid deposited with ATCC as r PTA-1653, and b) the amino acid sequence of SEQ ID NO: 1, or as Patent Deposit Number PTA-1653.
A polypeptide comprising a fragment of the amino acid sequence encoded by the cDNA insert of any of the plasmids deposited with the ATCC, which is SEQ ID NO: 1 or Patent Defect
cDN of one of the plasmids deposited with ATCC as posit number PTA-1653
A polypeptide comprising a fragment containing at least 12 contiguous amino acids of the amino acid sequence encoded by the A insert, and c) the amino acid sequence of SEQ ID NO: 1 or AT as Patent Deposit Number PTA-1653.
A naturally occurring allelic variant polypeptide comprising an amino acid sequence encoded by the cDNA insert of any of the plasmids deposited with CC, SEQ ID NO: 2
Alternatively, a method for producing a polypeptide selected from the group consisting of a naturally occurring allelic variant polypeptide encoded by a nucleic acid molecule that hybridizes to a nucleic acid molecule containing its complementary strand under stringent conditions. And culturing the host cell of claim 5 under conditions in which the nucleic acid molecule is expressed. 13. The method of claim 10, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO: 1. 14. A) contacting a sample with a compound that selectively binds to the polypeptide of claim 8, and b) determining whether the compound binds to the polypeptide in the sample. A method for detecting the presence of the polypeptide of claim 8 in a sample comprising. 15. The method of claim 14, wherein the compound that binds to the polypeptide is an antibody. 16. A kit comprising a compound that selectively binds to the polypeptide of claim 8 and instructions for use. 17. A) contacting a sample with a nucleic acid probe or primer that selectively hybridizes to a nucleic acid molecule; and b) determining whether the nucleic acid probe or primer binds to the nucleic acid molecule in the sample. A method for detecting the presence of a nucleic acid molecule according to claim 1 in a sample comprising. 18. The sample comprises mRNA molecules and is contacted with a nucleic acid probe,
The method according to claim 17. 19. A kit comprising a compound that selectively hybridizes to the nucleic acid molecule of claim 1 and instructions for use. 20) a) contacting a test compound with cells of the polypeptide of claim 8 or of the polypeptide of claim 8, and b) determining whether the polypeptide binds to the test compound. A method for identifying a compound that binds to the polypeptide of claim 8 comprising the step of determining. 21. Binding of a test compound to a polypeptide comprises the steps of:   a) detection of binding by direct detection of binding between the test compound and the polypeptide,   b) detection of binding using a competitive binding assay,   c) detection of binding using an assay for GPCR-like mediated signaling and 21. The method of claim 20, detected by a method selected from the group consisting of: 22. A method comprising contacting a compound that binds to the polypeptide of claim 8 with the polypeptide or cells expressing the polypeptide at a concentration sufficient to modulate the activity of the polypeptide. A method for modulating the activity of the polypeptide according to 8. 23) a) contacting a polypeptide according to claim 8 with a test compound, and b) determining the effect of the test compound on the activity of the polypeptide and thereby determining a compound which modulates the activity of the polypeptide. 9. A method for identifying a compound that modulates the activity of the polypeptide of claim 8 comprising the step of identifying. 24. A method for identifying an agent that regulates the expression level of a nucleic acid molecule according to claim 1 in a cell, wherein said expression level of said nucleic acid molecule is regulated in said cell by said agent. Contacting a cell expressing said nucleic acid molecule with said agent, and measuring said expression level of said nucleic acid molecule. 25. A method for regulating the expression level of a nucleic acid molecule according to claim 1, wherein the agent is contacted with the nucleic acid molecule under conditions capable of regulating the expression level of the nucleic acid molecule. Including the method. 26. A pharmaceutical composition comprising any of the polypeptides of claim 8 in a pharmaceutically acceptable carrier.