WO2000043513A1 - Recepteur 17723 couple a la proteine g - Google Patents

Recepteur 17723 couple a la proteine g Download PDF

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Publication number
WO2000043513A1
WO2000043513A1 PCT/US2000/001592 US0001592W WO0043513A1 WO 2000043513 A1 WO2000043513 A1 WO 2000043513A1 US 0001592 W US0001592 W US 0001592W WO 0043513 A1 WO0043513 A1 WO 0043513A1
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receptor
amino acid
protein
polypeptide
nucleic acid
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PCT/US2000/001592
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English (en)
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Maria Alexandra Glucksmann
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Millennium Pharmaceuticals, Inc.
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Priority to AU27346/00A priority Critical patent/AU2734600A/en
Publication of WO2000043513A1 publication Critical patent/WO2000043513A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor

Definitions

  • the present invention relates to a newly identified receptor belonging to the superfamily of G-protein-coupled receptors
  • the invention also relates to polynucleotides encoding the receptor
  • the invention further relates to methods using the receptor polypeptides and polynucleotides as a target for diagnosis and treatment in receptor-mediated disorders
  • the invention further relates to drug-screening methods using the receptor polypeptides and polynucleotides to identify agonists and antagonists for diagnosis and treatment
  • the invention further encompasses agonists and antagonists based on the receptor polypeptides and polynucleotides
  • the invention further relates to procedures for producing the receptor polypeptides and polynucleotides
  • G-protein coupled receptors constitute a major class of proteins responsible for transducing a signal within a cell
  • 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
  • a signal is transduced within the cell that results in a change in a biological or physiological property of the cell
  • G-proteins and effectors intracellular enzymes and channels modulated by G-proteins
  • the GPCR protein superfamily can be divided into five families Family I, receptors typified by rhodopsin and the ⁇ 2-adrenergic receptor and currently represented by over 200 unique members (Dohlman et al , Annu. Rev. Biochem.
  • Drosophila expresses a photoreceptor-specific protein, bride of sevenless (boss), a seven-transmembrane-segment protein which has been extensively studied and does not show evidence of being a GPCR (Hart et al , Proc. Natl. Acad. Sci.
  • G proteins represent a family of heterotrimeric proteins composed of ⁇ , ⁇ and ⁇ subunits, that bind guanine nucleotides These proteins are usually linked to cell surface receptors, e g , receptors containing seven transmembrane segments Following ligand binding to the GPCR, a conformational change is transmitted to the G protein, which causes the ⁇ -subunit to exchange a bound GDP molecule for a GTP molecule and to dissociate from the ⁇ -subunits
  • the GTP-bound form of the ⁇ - subunit typically functions as an effector-modulating moiety, leading to the production of second messengers, such as cAMP (e g , by activation of adenyl)
  • G proteins include Gi, Go, Gq, Gs and Gt.
  • G proteins are described extensively in Lodish etal , Molecular Cell Biology, (Scientific American Books Inc., New York, N.Y., 1995), the contents of which are incorporated herein by reference.
  • GPCRs, G proteins and G protein-linked effector and second messenger systems have been reviewed in The G-Protein Linked Receptor Fact Book, Watson et al, eds., Academic Press (1994).
  • GPCRs are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown GPCRs.
  • the present invention advances the state of the art by providing a previously unidentified human GPCR.
  • a specific object of the invention is to identify compounds that act as agonists and antagonists and modulate the expression of the novel receptor.
  • a further specific object of the invention is to provide compounds that modulate expression of the receptor for treatment and diagnosis of GPCR- related disorders.
  • the invention is thus based on the identification of a novel GPCR, designated the 17723 receptor.
  • the invention provides isolated 17723 receptor polypeptides including a polypeptide having the amino acid sequence shown in SEQ ID NO 1.
  • the invention also provides isolated 17723 receptor nucleic acid molecules having the sequence shown in SEQ ID NO 2.
  • the invention also provides variant polypeptides having an amino acid sequence that is substantially homologous to the amino acid sequence shown in SEQ LD NO 1.
  • the invention also provides variant nucleic acid sequences that are substantially homologous to the nucleotide sequence shown in SEQ ID NO 2.
  • the invention also provides fragments of the polypeptide shown in SEQ ID NO 1 and nucleotide shown in SEQ ID NO 2, as well as substantially homologous fragments of the polypeptide or nucleic acid.
  • the invention also provides vectors and host cells for expressing the receptor nucleic acid molecules and polypeptides and particularly recombinant vectors and host cells.
  • the invention also provides methods of making the vectors and host cells and methods for using them to produce the receptor nucleic acid molecules and polypeptides.
  • the invention also provides antibodies that selectively bind the receptor polypeptides and fragments.
  • the invention also provides methods of screening for compounds that modulate the activity of the receptor polypeptides. Modulation can be at the level of the polypeptide receptor or at the level of controlling the expression of nucleic acid (RNA or DNA) expressing the receptor polypeptide.
  • Modulation can be at the level of the polypeptide receptor or at the level of controlling the expression of nucleic acid (RNA or DNA) expressing the receptor polypeptide.
  • the invention also provides a process for modulating receptor polypeptide activity, especially using the screened compounds, including to treat conditions related to expression of the receptor polypeptides.
  • the invention also provides diagnostic assays for determining the presence of and level of the receptor polypeptides or nucleic acid molecules in a biological sample.
  • the invention also provides diagnostic assays for determining the presence of a mutation in the receptor polypeptides or nucleic acid molecules.
  • Figure 1 shows a comparison of the 17723 receptor against the Prosite data base of protein patterns, specifically showing a high score against the seven transmembrane segment rhodopsin superfamily (SEQ LD NO 3)
  • the underlined area shows a GPCR signature, and specifically the position of an arginine residue, conserved in GPCRs
  • the most commonly conserved sequence is an aspartate, arginine, tyrosine (DRY) triplet DRY is implicated in signal transduction
  • Arginine is invariant Aspartate is conservatively placed in several GPCRs
  • the arginine is found in the sequence ERY, which matches the position of DRY or invariant arginine in GPCRs of the rhodopsin superfamily of receptors
  • Figure 2 shows an analysis of the 17723 amino acid sequence ⁇ turn and coil regions, hydrophilicity, amphipathic regions, flexible regions, antigenic index, and surface probability plot
  • Figure 3 shows a 17723 receptor hydrophobicity plot The amino acids correspond to 1-402 and show the seven transmembrane segments
  • Figure 4 shows an analysis of the 17723 open reading frame for amino acids corresponding to specific functional sites
  • Six glycosylation sites are found in the protein from about amino acids 5-8, 82-85, 150-153, 198-201, 367-370, and 390-393
  • a cAMP- and cGMP-dependent protein kinase phosphorylation site is found from about amino acids 362-365
  • Three protein kinase C phosphorylation sites are found from about amino acids 228-230, 353-355, and 386-388
  • Five casein kinase II phosphorylation sites are found at about amino acids 84-87, 353-356, 376-379, 382- 385, and 392-395
  • Six N-myristoylation sites are found from about amino acids 4-9, 15-20, 55-60, 94-99, 121-126, and 185-190
  • a visual pigments (opsins) retinal binding site is found from about amino acids 298-316
  • the 17723 receptor protein is a GPCR that participates in signaling pathways
  • a “signaling pathway” refers to the modulation (e g , stimulation or inhibition) of a cellular function/activity upon the binding of a ligand to the GPCR ( 17723 protein)
  • Examples of such functions include mobilization of intracellular molecules that participate in a signal transduction pathway, e g , phosphatidylinositol 4,5-bisphosphate (PLP 2 ), inositol 1,4,5-triphosphate (LP 3 ) and adenylate cyclase, polarization of the plasma membrane, production or secretion of molecules, alteration in the structure of a cellular component, cell proliferation, e g , synthesis of DNA, cell migration, cell differentiation, and cell survival Since the 17723 receptor protein is expressed in heart, brain, thymus, esophagus, and uterus, cells participating in a 17723 receptor protein signaling pathway include, but are not limited to
  • the response mediated by the receptor protein depends on the type of cell For example, in some cells, binding of a ligand to the receptor protein may stimulate an activity such as release of compounds, gating of a channel, cellular adhesion, migration, differentiation, etc , through phosphatidylinositol or cyclic AMP metabolism and turnover while in other cells, the binding of the ligand will produce a different result Regardless of the cellular activity/response modulated by the receptor protein, it is universal that the protein is a GPCR and interacts with G proteins to produce one or more secondary signals, in a variety of intracellular signal transduction pathways, e g , through phosphatidylinositol or cyclic AMP metabolism and turnover, in a cell
  • phosphatidylinositol turnover and metabolism refers to the molecules involved in the turnover and metabolism of phosphatidylinositol 4,5- bisphosphate (PLP 2 ) as well as to the activities of these molecules
  • PLP is a phospholipid found in the cytosolic leaflet of the plasma membrane Binding of ligand to the receptor activates, in some cells, the plasma-membrane enzyme phospholipase C that in turn can hydrolyze PIP 2 to produce 1,2-diacylglycerol (DAG) and inositol 1,4,5-triphosphate (LP 3 )
  • DAG 1,2-diacylglycerol
  • LP 3 inositol 1,4,5-triphosphate
  • IP 3 can diffuse to the endoplasmic reticulum surface where it can bind an IP 3 receptor, e g , a calcium channel protein containing an IP 3 binding site EP 3 binding can induce opening of the channel, allowing calcium ions to be released into the
  • cyclic AMP turnover and metabolism refers to the molecules involved in the turnover and metabolism of cyclic AMP (cAMP) as well as to the activities of these molecules
  • Cyclic AMP is a second messenger produced in response to ligand-induced stimulation of certain G protein coupled receptors
  • binding of a ligand to a GPCR can lead to the activation of the enzyme adenyl cyclase, which catalyzes the synthesis of cAMP
  • the newly synthesized cAMP can in turn activate a cAMP-dependent protein kinase
  • This activated kinase can phosphorylate a voltage-gated potassium channel protein, or an associated protein, and lead to the inability of the potassium channel to open during an action potential The inability of the potassium channel to open results in a decrease in the outward flow of potassium, which normally repolarizes the membrane of a neuron, leading to prolonged membrane depolarization
  • the invention is based on the discovery of a novel G-coupled protein receptor Specifically, an expressed sequence tag (EST) was selected based on homology to G-protein-coupled receptor sequences This EST was used to design primers based on sequences that it contains and used to identify a cDNA from a human heart cDNA library Positive clones were sequenced and the overlapping fragments were assembled Analysis of the assembled sequence revealed that the cloned cDNA molecule encodes a G-protein coupled receptor The invention thus relates to a novel GPCR having the deduced amino acid sequence shown in (SEQ ID NO 1)
  • the transmembrane domain contains seven transmembrane segments, three extracellular loops and three intracellular loops The transmembrane segments are found from about amino acid 44 to about amino acid 67, from about amino acid 78 to about amino acid 100, from about amino acid 119 to about amino acid 137, from about amino acid 157 to about amino acid 176, from about amino acid 202 to about amino acid 226, from about amino acid 256 to about amino acid 279, and from about amino acid 287 to about amino acid 307 Within the region spanning the entire transmembrane domain are three intracellular and three extracellular loops The three intracellular loops are found from about amino acid 68 to about amino acid 77, from about amino acid 138 to about amino acid 156, and from about amino acid 227 to about amino acid 255 The three extracellular loops are found at from about amino acid 44 to about amino acid 67, from about amino acid 78 to about amino acid 100, from about amino acid 119 to about amino acid 137, from about amino acid 157 to about amino acid 176, from
  • the transmembrane domain includes a GPCR signal transduction signature, ERY, at residues 138-140
  • the sequence includes an arginine at residue 139, an invariant amino acid in GPCRs
  • 17723 receptor polypeptide or "17723 receptor protein” refers to the polypeptide in SEQ LD NO 1
  • receptor protein or “receptor polypeptide”
  • receptor polypeptide further includes the numerous variants described herein, as well as fragments derived from the full length 17723 polypeptide and variants
  • the present invention thus provides an isolated or purified 17723 receptor polypeptide and variants and fragments thereof
  • the 17723 polypeptide is a 402 residue protein exhibiting three main structural domains
  • the amino terminal extracellular domain is identified to be within residues 1 to about 43 in SEQ ID NO 1
  • the transmembrane domain is identified to be within residues from about 44 to about 307 in SEQ ID NO 1
  • the carboxy terminal intracellular domain is identified to be within residues from about 308 to 402 in SEQ LD NO 1
  • the transmembrane domain contains seven segments that span the membrane The transmembrane segments are found from about amino acid 44 to about amino acid 67, from about amino acid 78 to about amino acid 100, from about amino acid 119 to about amino acid 137, from about amino acid 157 to about amino acid 176, from about amino acid 202 to about amino acid 226, from about amino acid 256 to about amino acid 279, and from about amino acid 287 to about amino acid 307
  • Within the region spanning the entire transmembrane domain are three intracellular and three extracellular loops The three intracellular loops are found from about amino
  • the transmembrane domain includes a GPCR signal transduction signature, ERY, at residues 138-140
  • the sequence includes an arginine at residue 139, an invariant amino acid in GPCRs
  • a polypeptide is said to be “isolated” or “purified” when it is substantially free of cellular material when it is isolated from recombinant and non- recombinant cells, or free of chemical precursors or other chemicals when it is chemically synthesized
  • a polypeptide can be joined to another polypeptide with which it is not normally associated in a cell and still be considered “isolated” or “purified "
  • the receptor polypeptides can be purified to homogeneity It is understood, however, that preparations in which the polypeptide is not purified to homogeneity are useful and considered to contain an isolated form of the polypeptide
  • the critical feature is that the preparation allows for the desired function of the polypeptide, even in the presence of considerable amounts of other components
  • the invention encompasses various degrees of purity
  • the language "substantially free of cellular material” includes preparations of the receptor polypeptide having less than about 30% (by dry weight) other proteins (i e , contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins
  • the receptor polypeptide When the receptor polypeptide is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20%, less than about 10%), or less than about 5% of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of the receptor polypeptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of the polypeptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10%> chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.
  • the receptor polypeptide comprises the amino acid sequence shown in SEQ ID NO 1.
  • the invention also encompasses sequence variants.
  • Variants include a substantially homologous protein encoded by the same genetic locus in an organism, i.e., an allelic variant.
  • the 17723 receptor has been mapped to chromosome 1 q42-44.
  • Variants also encompass proteins derived from other genetic loci in an organism, but having substantial homology to the 17723 receptor protein of SEQ LD NO 1.
  • Variants also include proteins substantially homologous to the 17723 receptor protein but derived from another organism, i.e., an ortholog.
  • Variants also include proteins that are substantially homologous to the 17723 receptor protein that are produced by chemical synthesis.
  • Variants also include proteins that are substantially homologous to the 17723 receptor protein that are produced by recombinant methods. It is understood, however, that variants exclude any amino acid sequences disclosed prior to the invention.
  • two proteins are substantially homologous when the amino acid sequences are at least about 50-55%, 55-60%, typically at least about 70-75%, more typically at least about 80-85%, and most typically at least about 90-95%> or more homologous.
  • a substantially homologous amino acid sequence, according to the present invention will be encoded by a nucleic acid sequence hybridizing to the nucleic acid sequence, or portion thereof, of the sequence shown in SEQ ID NO 2 under stringent conditions as more fully described below.
  • the sequences are aligned for optimal comparison purposes (e g , gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes)
  • the length of a reference sequence aligned for comparison purposes is at least 30%>, preferably at least 40%, more preferably at least 50%>, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence (e g , when aligning a second sequence to the amino acid sequences herein having 402 amino acid residues, at least 120, preferably at least 160, more preferably at least 200, even more preferably at least 240, and even more preferably at least 280, 320, and 360 amino acid residues are aligned)
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then
  • the comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm (Computational Molecular Biology, Lesk, A M , ed , Oxford University Press, New York, 1988, Bwcomputing: Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part 1, Griffin, A M , and Griffin, H G , 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 Stockton Press, New York, 1991)
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J.
  • Mol Biol. (48) 444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http //www gcg com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J , et al, Nucleic Acids Res.
  • nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences Such searches can be performed using the NBLAST and XBLAST programs (version 2 0) of Altschul, et al (1990) J.
  • Gapped BLAST can be utilized as described in Altschul et al , (1997) Nucleic Acids Res.
  • a variant polypeptide can differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations or a combination of any of these
  • Variant polypeptides can be fully functional or can lack function in one or more activities Thus, in the present case, variations can affect the function, for example, of one or more of the regions corresponding to ligand binding, membrane association, G-protein binding and signal transduction
  • Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions Functional variants can also contain substitution of similar amino acids which result in no change or an insignificant change in function Alternatively, such substitutions may positively or negatively affect function to some degree
  • Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region
  • variants can be naturally-occurring or can be made by recombinant means or chemical synthesis to provide useful and novel characteristics for the receptor polypeptide This includes preventing immunogenicity from pharmaceutical formulations by preventing protein aggregation Useful variations further include alteration of ligand binding characteristics
  • one embodiment involves a variation at the binding site that results in binding but not release, or slower release, of ligand
  • a further useful variation at the same sites can result in a higher affinity for ligand
  • Useful variations also include changes that provide for affinity for another ligand
  • Another useful variation includes one that allows binding but which prevents activation by the ligand
  • Another useful variation includes variation in the transmembrane G-protein-binding/signal transduction domain that provides for reduced or increased binding by the appropriate G-protein or for binding by a different G-protein than the one with which the receptor is normally associated
  • Another useful variation provides a fusion protein in which one or more domains or subregions is operationally fused to one or more domains or subregions from another G-protein coupled receptor
  • Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al. , Science 244 1081 - 1085 ( 1989)) The latter procedure introduces single alanine mutations at every residue in the molecule The resulting mutant molecules are then tested for biological activity such as receptor binding or in vitro, or in vitro proliferative activity Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al, J. Mol. Biol. 224 899-904 (1992), de Vos et al. Science 255 306-312 (1992))
  • Substantial homology can be to the entire nucleic acid or amino acid sequence or to fragments of these sequences
  • the invention thus also includes polypeptide fragments of the 17723 receptor protein Fragments can be derived from the amino acid sequence shown in SEQ ID NO 1
  • the invention also encompasses fragments of the variants of the 17723 receptor protein as described herein
  • the fragments to which the invention pertains, however, are not to be construed as encompassing fragments that may be disclosed prior to the present invention
  • a fragment comprises at least 9, 12 contiguous amino acids Fragments retain one or more of the biological activities of the protein, for example the ability to bind to a G-protein or ligand, as well as fragments that can be used as an immunogen to generate receptor antibodies
  • Biologically active fragments can comprise a domain or motif, e g , an extracellular or intracellular domain or loop, one or more transmembrane segments, or parts thereof, G-protein binding site, or GPCR signature, glycosylation sites, cAMP- and cGMP-dependent phosphorylation sites, protein kinase C phosphorylation sites, and casein kinase II phosphorylation sites, and N- myristoylation sites
  • Possible fragments include, but are not limited to 1) soluble peptides comprising the entire amino terminal extracellular domain amino acid 1 to about amino acid 43 of SEQ LD NO 1 or parts thereof, 2) peptides comprising the entire carboxy terminal intracellular domain from about amino acid 308 to amino acid 402 of SEQ ID NO 1 or parts thereof, 3) peptides comprising the region spanning the entire transmembrane domain from about amino acid 44 to amino acid 307, 4) any of the specific transmembrane segments, or parts thereof, 5) any of the three intracellular or three extracellular loops, or parts thereof
  • Possible fragments include, but are not limited to 1) soluble peptides comprising the entire amino terminal extracellular domain about amino acid 1 to about amino acid 43 of SEQ ID NO 1, or parts thereof, 2) peptides comprising the entire carboxy terminal intracellular domain from about amino acid 308 to amino acid 402 of SEQ LD NO 1, or parts thereof, 3) peptides comprising the region spanning the entire transmembrane domain from about amino acid 44 to about amino acid 307, or parts thereof, 4) any of the specific transmembrane segments, or parts thereof, from about amino acid 44 to about amino acid 67, from about amino acid 78 to about amino acid 100, from about amino acid 119 to about amino acid 137, from about amino acid 157 to about amino acid 176, from about amino acid 202 to about amino acid 226, from about amino acid 256 to about amino acid 279, and from about amino acid 287 to about amino acid 307, 5) any of the three intracellular or three extracellular loops, or parts thereof, from about amino acid 68 to about amino acid
  • Fragments also include antigenic fragments and specifically those shown to have a high antigenic index in Figure 2
  • fragments include fragments defining a ligand-binding site, fragments defining a phosphorylation site, fragments defining membrane association, fragments defining myristoylation sites, fragments defining interaction with G proteins and signal transduction, and fragments defining glycosylation sites
  • fragments defining a ligand-binding site fragments defining a phosphorylation site
  • fragments defining membrane association fragments defining myristoylation sites
  • fragments defining interaction with G proteins and signal transduction fragments defining glycosylation sites
  • the invention also provides fragments with immunogenic properties These contain an epitope-bearing portion of the 17723 receptor protein and variants These epitope-bearing peptides are useful to raise antibodies that bind specifically to a receptor polypeptide or region or fragment These peptides can contain at least 9, 12, at least 14, or between at least about 15 to about 30 amino acids
  • Non-limiting examples of antigenic polypeptides that can be used to generate antibodies include peptides derived from the amino terminal extracellular domain or any of the extracellular loops Regions having a high antigenicity index are shown in Figure
  • intracellularly-made antibodies are also encompassed, which would recognize intracellular receptor peptide regions
  • the receptor polypeptides are useful for biological assays related to GPCRs
  • Such assays involve any of the known GPCR functions or activities or properties useful for diagnosis and treatment of GPCR-related conditions
  • the epitope-bearing receptor and polypeptides may be produced by any conventional means (Houghten, R A , Proc. Natl. Acad. Sci. USA 82 5131-5135 (1985)) Simultaneous multiple peptide synthesis is described in U S Patent No 4,631,211
  • Fragments can be discrete (not fused to other amino acids or polypeptides) or can be within a larger polypeptide Further, several fragments can be comprised within a single larger polypeptide In one embodiment a fragment designed for expression in a host can have heterologous pre- and pro-polypeptide regions fused to the amino terminus of the receptor fragment and an additional region fused to the carboxyl terminus of the fragment
  • the invention thus provides chimeric or fusion proteins These comprise a receptor protein operatively linked to a heterologous protein having an amino acid sequence not substantially homologous to the receptor protein "Operatively linked" indicates that the receptor protein and the heterologous protein are fused in-frame
  • the heterologous protein can be fused to the N-terminus or C-terminus of the receptor protein
  • the fusion protein does not affect receptor function per se
  • the fusion protein can be a GST-fusion protein in which the receptor sequences are fused to the C-terminus of the GST sequences
  • Other types of fusion proteins include, but are not limited to, enzymatic fusion proteins, for example beta- galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions and Ig fusions
  • Such fusion proteins, particularly poly-His fusions can facilitate the purification of recombinant receptor protein In certain host cells (e g , mammalian host cells), expression and/or secret
  • EP-A-O 464 533 discloses fusion proteins comprising various portions of immunoglobulin constant regions The Fc is useful in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 262)
  • human proteins have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists Bennett et al (J. Mol. Recog. 8 52-58 (1995)) and Johanson et al (J. Biol. Chem.
  • this invention also encompasses soluble fusion proteins containing a receptor polypeptide and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclass (IgG, IgM, IgA, IgE)
  • immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hinge region
  • the Fc part can be removed in a simple way by a cleavage sequence which is also incorporated and can be cleaved with factor Xa
  • a chimeric or fusion protein can be produced by standard recombinant DNA techniques For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see Ausubel et al , Current Protocols in Molecular Biology, 1992)
  • many expression vectors are commercially available that already encode a fusion moiety (e g , a GST protein)
  • a receptor protein-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the receptor protein
  • Another form of fusion protein is one that directly affects receptor functions
  • a receptor polypeptide is encompassed by the present invention in which one or more of the receptor domains (or parts thereof) has been replaced by homologous domains (or parts thereof) from another G-protein coupled receptor or other type of receptor Accordingly, various permutations are possible
  • the amino terminal extracellular domain, or subregion thereof, (for example, ligand-binding) can be replaced with the domain or subregion from another ligand-binding receptor protein
  • the entire transmembrane domain, or any of the seven segments or loops, or parts thereof, for example, G-protein-binding/signal transduction can be replaced
  • the carboxy terminal intracellular domain or subregion can be replaced
  • chimeric receptors can be formed in which one or more of the native domains or subregions has been replaced
  • the isolated receptor protein can be purified from cells that naturally express it, such as from heart, brain, thymus, esophagus, and uterus, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods
  • the protein is produced by recombinant DNA techniques
  • a nucleic acid molecule encoding the receptor polypeptide is cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell
  • the protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques
  • Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally-occurring amino acids
  • many amino acids, including the terminal amino acids may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art Common modifications that occur naturally in polypeptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art Accordingly, the polypeptides also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature polypeptide is fused with another compound, such as a compound to
  • Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination
  • polypeptides are not always entirely linear
  • polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of post-translation events, including natural processing event and events brought about by human manipulation which do not occur naturally Circular
  • branched and branched circular polypeptides may be synthesized by non-translational natural processes and by synthetic methods Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini Blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, is common in naturally-occurring and synthetic polypeptides
  • the amino terminal residue of polypeptides made in E. coh, prior to proteolytic processing almost invariably will be N-formylmethionine
  • the modifications can be a function of how the protein is made
  • the modifications will be determined by the host cell posttranslational modification capacity and the modification signals in the polypeptide amino acid sequence Accordingly, when glycosylation is desired, a polypeptide should be expressed in a glycosylating host, generally a eukaryotic cell Insect cells often carry out the same posttranslational glycosylations as mammalian cells and, for this reason, insect cell expression systems have been developed to efficiently express mammalian proteins having native patterns of glycosylation Similar considerations apply to other modifications
  • a given polypeptide may contain more than one type of modification
  • the receptor polypeptides are useful for producing antibodies specific for the 17723 receptor protein, regions, or fragments Regions having a high antigenicity index score are shown in Figure 2
  • the receptor polypeptides are useful for biological assays related to GPCRs
  • Such assays involve any of the known GPCR functions or activities or properties useful for diagnosis and treatment of GPCR-related conditions
  • the receptor polypeptides are also useful in drug screening assays, in cell- based or cell-free systems
  • Cell-based systems can be native, i e , cells that normally express the receptor protein, as a biopsy or expanded in cell culture In one embodiment, however, cell-based assays involve recombinant host cells expressing the receptor protein
  • the polypeptides can be used to identify compounds that modulate receptor activity
  • Both 17723 protein and appropriate variants and fragments can be used in high-throughput screens to assay candidate compounds for the ability to bind to the receptor
  • These compounds can be further screened against a functional receptor to determine the effect of the compound on the receptor activity
  • Compounds can be identified that activate (agonist) or inactivate (antagonist) the receptor to a desired degree
  • the receptor polypeptides can be used to screen a compound for the ability to stimulate or inhibit interaction between the receptor protein and a target molecule that normally interacts with the receptor protein
  • the target can be ligand or a component of the signal pathway with which the receptor protein normally interacts (for example, a G-protein or other interactor involved in cAMP or phosphatidylinositol turnover and/or adenylate cyclase, or phospholipase C activation)
  • the assay includes the steps of combining the receptor protein with a candidate compound under conditions that allow the receptor protein or fragment to interact with the target molecule, and to detect the formation of a complex between the protein and the target or to detect the biochemical consequence of the interaction with the receptor protein and the target, such as any of the associated effects of signal transduction such as G- protein phosphorylation, cyclic AMP or phosphatidylinositol turnover, and adenylate cyclase or phospholipase C activation
  • Candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e g , Lam et al , Nature 354 82-84 (1991), Houghten et al , Nature 354 84-86 (1991)) and combinatorial chemistry-derived molecular libraries made of D- and/or L- configuration amino acids, 2) phosphopeptides (e g , members of random and partially degenerate, directed phosphopeptide libraries, see, e g , Songyang et al , Cell 72 767-778 (1993)), 3) antibodies (e g , polyclonal, monoclonal, humanized, anti- idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab') , Fab expression library fragments, and epitope-binding fragments of antibodies), and 4) small organic and inorganic molecules (e g
  • One candidate compound is a soluble full-length receptor or fragment that competes for ligand binding
  • Other candidate compounds include mutant receptors or appropriate fragments containing mutations that affect receptor function and thus compete for ligand Accordingly, a fragment that competes for ligand, for example with a higher affinity, or a fragment that binds ligand but does not allow release, is encompassed by the invention
  • the invention provides other end points to identify compounds that modulate
  • the assays typically involve an assay of events in the signal transduction pathway that indicate receptor activity
  • the expression of genes that are up- or down-regulated in response to the receptor protein dependent signal cascade can be assayed
  • the regulatory region of such genes can be operably linked to a marker that is easily detectable, such as luciferase
  • phosphorylation of the receptor protein, or a receptor protein target could also be measured
  • any of the biological or biochemical functions mediated by the receptor can be used as an endpoint assay These include all of the biochemical or biochemical/biological events described herein, in the references cited herein, incorporated by reference for these endpoint assay targets, and other functions known to those of ordinary skill in the art
  • Binding and/or activating compounds can also be screened by using chimeric receptor proteins in which the amino terminal extracellular domain, or parts thereof, the entire transmembrane domain or subregions, such as any of the seven transmembrane segments or any of the intracellular or extracellular loops and the carboxy terminal intracellular domain, or parts thereof, can be replaced by heterologous domains or subregions
  • a G-protein-binding region can be used that interacts with a different G-protein than that which is recognized by the native receptor Accordingly, a different set of signal transduction components is available as an end-point assay for activation
  • the entire transmembrane portion or subregions (such as transmembrane segments or intracellular or extracellular loops) can be replaced with the entire transmembrane portion or subregions specific to a host cell that is different from the host cell from which the amino terminal extracellular domain and/or the G-protein-binding region are derived This allows for assays to be performed in other than the specific host cell from
  • the receptor polypeptides are also useful in competition binding assays in methods designed to discover compounds that interact with the receptor Thus, a compound is exposed to a receptor polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide. Soluble receptor polypeptide is also added to the mixture If the test compound interacts with the soluble receptor polypeptide, it decreases the amount of complex formed or activity from the receptor target This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the receptor Thus, the soluble polypeptide that competes with the target receptor region is designed to contain peptide sequences corresponding to the region of interest
  • a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix
  • glutathione-S- transferase/17723 fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the cell lysates (e g , 35 S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e g , at physiological conditions for salt and pH) Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of receptor-binding protein found in the bead fraction quantitate
  • Modulators of receptor protein activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the receptor pathway, by treating cells that express the 17723 protein, such as in heart, brain, thymus, esophagus, and uterus
  • These methods of treatment include the steps of administering the modulators of protein activity in a pharmaceutical composition as described herein, to a subject in need of such treatment
  • the receptor polypeptides also are useful to provide a target for diagnosing a disease or predisposition to disease mediated by the receptor protein, especially in heart, brain, thymus, esophagus, and uterus Accordingly, methods are provided for detecting the presence, or levels of, the receptor protein in a cell, tissue, or organism The method involves contacting a biological sample with a compound capable of interacting with the receptor protein such that the interaction can be detected
  • receptor protein can be isolated from a biological sample, assayed for the presence of a genetic mutation that results in aberrant receptor protein This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification Analytic methods include altered electrophoretic mobility, altered tryptic peptide digest, altered receptor activity in cell-based or cell-free assay, alteration in ligand or antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein
  • the protein can be detected in vivo in a subject by introducing into the subject a labeled anti-receptor antibody
  • 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 which detect the allelic variant of a receptor protein expressed in a subject and methods which detect fragments of a receptor protein in a sample
  • the receptor polypeptides are also useful in pharmacogenomic analysis
  • the genotype of the individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound Further, the activity of drug metabolizing enzymes effects both the intensity and duration of drug action Thus, the pharmacogenomics of the individual permit the selection of effective compounds and effective dosages of such compounds for prophylactic or therapeutic treatment based on the individual's genotype
  • the discovery of genetic polymorphisms in some drug metabolizing enzymes has explained why some patients do not obtain the expected drug effects, show an exaggerated drug effect, or experience serious toxicity from standard drug dosages Polymorphisms can be expressed in the phenotype of the extensive metabolizer and the phenotype of the poor metabolizer Accordingly, genetic polymorphism may lead to allelic protein variants of the receptor protein in which one or more of the receptor functions in one population is different from those in another population
  • the polymorphism may lead to allelic protein variants of the receptor protein in which one or more of the receptor functions in one population is different from those in another population.
  • the receptor polypeptides are also useful for monitoring therapeutic effects during clinical trials and other treatment
  • the therapeutic effectiveness of an agent that is designed to increase or decrease gene expression, protein levels or receptor activity can be monitored over the course of treatment using the receptor polypeptides as an end-point target
  • the receptor polypeptides are also useful for treating a receptor-associated disorder Accordingly, methods for treatment include the use of soluble receptor or fragments of the receptor protein that compete for ligand binding These receptors or fragments can have a higher affinity for the ligand so as to provide effective competition
  • the invention also provides antibodies that selectively bind to the 17723 receptor protein and its variants and fragments An antibody is considered to selectively bind, even if it also binds to other proteins that are not substantially homologous with the receptor protein These other proteins share homology with a fragment or domain of the receptor protein This conservation in specific regions gives rise to antibodies that bind to both proteins by virtue of the homologous sequence In this case, it would be understood that antibody binding to the receptor protein is still selective
  • an isolated receptor polypeptide is used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation
  • Antibodies are preferably prepared from these regions or from discrete fragments in these regions However, antibodies can be prepared from any region of the peptide as described herein
  • a preferred fragment produces an antibody that diminishes or completely prevents ligand-binding
  • Antibodies can be developed against the entire receptor or portions of the receptor, for example, the intracellular carboxy terminal domain, the amino terminal extracellular domain, the entire transmembrane domain or specific segments, any of the intra or extracellular loops, or any portions of the above Antibodies may also be developed against specific functional sites, such as the site of ligand-binding, the site of G protein coupling, or sites that are glycosylated, phosphorylated, myristoylated, or otherwise modified as described herein
  • an antigenic fragment will typically comprise at least 9 contiguous amino acid residues
  • the antigenic peptide can comprise, however, 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
  • fragments correspond to regions that are located on the surface of the protein, e g , hydrophilic regions
  • Antibodies can be polyclonal or monoclonal An intact antibody, or a fragment thereof (e g Fab or F(ab') 2 ) can be used
  • Detection can be facilitated by coupling (i e , physically linking) the antibody to a detectable substance
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase
  • suitable prosthetic group complexes include streptavidin/biotin and avidin biotin
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin
  • an example of a luminescent material includes luminol
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin
  • suitable radioactive material include 125 I, 131 I, 35 S
  • the antibodies can be used to isolate a receptor protein by standard techniques, such as affinity chromatography or immunoprecipitation
  • the antibodies can facilitate the purification of the natural receptor protein from cells and recombinantly produced receptor protein expressed in host cells
  • the antibodies are useful to detect the presence of receptor protein in cells or tissues to determine the pattern of expression of the receptor among various tissues in an organism and over the course of normal development
  • the antibodies can be used to detect receptor protein in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression
  • the antibodies can be used to assess abnormal tissue distribution or abnormal expression during development
  • Antibody detection of circulating fragments of the full length receptor protein can be used to identify receptor turnover
  • the antibodies can be used to assess receptor expression in disease states such as in active stages of the disease or in an individual with a predisposition toward disease related to receptor function
  • the antibody can be prepared against the normal receptor protein
  • antibodies specific for this mutant protein can be used to assay for the presence of the specific mutant receptor protein
  • intracellularly-made antibodies (“intrabodies”) are also encompassed, which would recognize intracellular receptor peptide regions
  • the antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tissues in an organism
  • Antibodies can be developed against the whole receptor or portions of the receptor, for example, portions of the amino terminal extracellular domain or extracellular loops
  • the diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality Accordingly, where treatment is ultimately aimed at correcting receptor expression level or the presence of aberrant receptors and aberrant tissue distribution or developmental expression, antibodies directed against the receptor or relevant fragments can be used to monitor therapeutic efficacy
  • antibodies are useful in pharmacogenomic analysis
  • antibodies prepared against polymo ⁇ hic receptor proteins can be used to identify individuals that require modified treatment modalities
  • the antibodies are also useful as diagnostic tools as an immunological marker for aberrant receptor protein analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digest, and other physical assays known to those in the art
  • the antibodies are also useful for tissue typing
  • tissue typing where a specific receptor protein has been correlated with expression in a specific tissue, antibodies that are specific for this receptor protein can be used to identify a tissue type
  • the antibodies are also useful in forensic identification Accordingly, where an individual has been correlated with a specific genetic polymo ⁇ hism resulting in a specific polymorphic protein, an antibody specific for the polymorphic protein can be used as an aid in identification
  • the antibodies are also useful for inhibiting receptor function, for example, blocking ligand binding
  • Antibodies can be prepared against specific fragments containing sites required for function or against intact receptor associated with a cell
  • kits for using antibodies to detect the presence of a receptor protein in a biological sample can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting receptor protein in a biological sample, means for determining the amount of receptor protein in the sample, and means for comparing the amount of receptor protein in the sample with a standard
  • the compound or agent can be packaged in a suitable container
  • the kit can further comprise instructions for using the kit to detect receptor protein Polynucleotides
  • the specifically disclosed cDNA comprises the coding region and 5' and 3' untranslated sequences (SEQ LD NO 2)
  • the human 17723 receptor cDNA is approximately 2144 nucleotides in length and encodes a full length protein that is approximately 402 amino acid residues in length
  • the nucleic acid is expressed in heart, thymus, brain, esophagus, and uterus Structural analysis of the amino acid sequence of SEQ ID NO 1 is provided in Figure 2, a hydropathy plot The figure shows the putative structure of the seven transmembrane segments, the amino terminal extracellular domain and the carboxy terminal intracellular domain
  • transmembrane segment refers to a structural amino acid motif which includes a hydrophobic helix that spans the plasma membrane The entire transmembrane domain spans from about amino acid 44 to about amino acid 307 Seven segments span the membrane and there are three intracellular and three extracellular loops in this domain
  • the invention provides isolated polynucleotides encoding a 17723 receptor protein
  • the term “17723 polynucleotide” or “17723 nucleic acid” refers to the sequence shown in SEQ ID NO 2
  • the term “receptor polynucleotide” or “receptor nucleic acid” further includes variants and fragments of the 17723 polynucleotide
  • An "isolated" receptor nucleic acid is one that is separated from other nucleic acid present in the natural source of the receptor nucleic acid
  • an "isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i e , sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived
  • flanking nucleotide sequences for example up to about 5KB The important point is that the nucleic acid is isolated from flanking sequences such that it can be subjected to the specific manipulations described here
  • recombinant DNA molecules contained in a vector are considered isolated Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution 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
  • the receptor polynucleotides can encode the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance) Such sequences may play a role in processing of a protein from precursor to a mature form, facilitate protein trafficking, prolong or shorten protein half-life or facilitate manipulation of a protein for assay or production, among other things As generally is the case in situ, the additional amino acids may be processed away from the mature protein by cellular enzymes
  • the receptor polynucleotides include, but are not limited to, the sequence encoding the mature polypeptide alone, the sequence encoding the mature polypeptide and additional coding sequences, such as a leader or secretory sequence (e g , a pre-pro or pro-protein sequence), the sequence encoding the mature polypeptide, with or without the additional coding sequences, plus additional non- coding sequences, for example introns and non-coding 5' and 3' sequences such as transcribed but non-translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA
  • the polynucleotide may be fused to a marker sequence encoding, for example, a peptide that facilitates purification
  • Receptor polynucleotides can be in the form of RNA, such as mRNA, or in the form DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof
  • the nucleic acid, especially DNA can be double-stranded or single-stranded Single-stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (anti-sense strand)
  • One receptor nucleic acid comprises the nucleotide sequence shown in SEQ ID NO 2, corresponding to human heart cDNA
  • the receptor nucleic acid comprises only the coding region
  • the invention further provides variant receptor polynucleotides, and fragments thereof, that differ from the nucleotide sequence shown in SEQ ID NO 2 due to degeneracy of the genetic code and thus encode the same protein as that encoded by the nucleotide sequence shown in SEQ LD NO 2
  • the invention also provides receptor nucleic acid molecules encoding the variant polypeptides described herein
  • polynucleotides may be naturally occurring, such as allelic variants (same locus) (maps to chromosome 1 q42-44), homologs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis
  • allelic variants allelic locus
  • homologs different locus
  • orthologs different organism
  • Such non-naturally occurring variants may be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions
  • Variation can occur in either or both the coding and non-coding regions
  • the variations can produce both conservative and non-conservative amino acid substitutions
  • Orthologs, homologs, and allelic variants can be identified using methods well known in the art These variants comprise a nucleotide sequence encoding a receptor that is 50%, at least about 55%>, typically at least about 70-75%, more typically at least about 80-85%, and most typically at least about 90-95% or more homologous to the nucleotide sequence shown in SEQ LD NO 2 or a fragment of this sequence
  • Such nucleic acid molecules can readily be identified as being able to hybridize under stringent conditions, to the nucleotide sequence shown in SEQ LD NO 2 or a fragment of the sequence It is understood that stringent hybridization does not indicate substantial homology where it is due to general homology, such as poly A sequences, or sequences common to all or most proteins, all GPCRs, or all family I GPCRs Moreover, it is understood that variants do not include any
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a receptor at least 50%, 55% homologous to each other typically remain hybridized to each other
  • the conditions can be such that sequences at least about 65%), at least about 70%, or at least about 75% or more homologous to each other typically remain hybridized to each other
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N Y (1989), 6 3 1-6 3 6
  • stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0 2 X SSC, 0 1% SDS at 50-65°C
  • an isolated receptor nucleic acid molecule that hybridizes under stringent conditions to the sequence of SEQ LD NO 2 corresponds to a naturally- occurring nucleic acid molecule
  • the invention provides polynucleotides that comprise a fragment of the full length receptor polynucleotides
  • the fragment can be single or double stranded and can comprise DNA or RNA
  • the fragment can be derived from either the coding or the non-coding sequence
  • an isolated receptor nucleic acid fragment is at least 27 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO 2
  • the nucleic acid is at least 40, 50, 100, 250 or 500 nucleotides in length
  • an isolated receptor nucleic acid encodes the entire coding region from amino acid 1 to amino acid 402
  • the isolated receptor nucleic acid encodes a sequence corresponding to the mature protein from about amino acid 6 to amino acid 402
  • Receptor nucleic acid fragments further include sequences corresponding to the domains and segments described herein, subregions also described, and specific functional sites Receptor nucleic acid fragments also include combinations of the domains, segments, loops, and other functional sites described above
  • a receptor nucleic acid could include sequences corresponding to the amino terminal extracellular domain and one transmembrane fragment
  • a receptor fragment includes any nucleic acid sequence that does not include the entire gene
  • Other fragments include nucleotide sequences encoding the amino acid fragments described herein
  • Further fragments can include subfragments of the specific domains or sites described herein Fragments also include nucleic acid sequences corresponding to specific amino acid sequence
  • the invention also provides receptor nucleic acid fragments that encode epitope bearing regions of the receptor proteins described herein
  • the isolated receptor polynucleotide sequences, and especially fragments, are useful as DNA probes and primers
  • the coding region of a receptor gene can be isolated using the known nucleotide sequence to synthesize an oligonucleotide probe
  • a labeled probe can then be used to screen a cDNA library, genomic DNA library, or mRNA to isolate nucleic acid corresponding to the coding region
  • primers can be used in PCR reactions to clone specific regions of receptor genes
  • a probe/primer typically comprises substantially purified oligonucleotide
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, typically about 25, more typically about 40, 50 or 75 consecutive nucleotides of SEQ ID NO 2 sense or anti- sense strand or other receptor polynucleotides
  • a probe further comprises a label, e g , radioisotope, fluorescent compound, enzyme, or enzyme co-factor
  • the receptor polynucleotides are useful for probes, primers, and in biological assays Where the polynucleotides are used to assess GPCR properties or functions, such as in the assays described herein, all or less than all of the entire cDNA can be useful In this case, even fragments that may have been known prior to the invention are encompassed Thus, for example, assays specifically directed to GPCR functions, such as assessing agonist or antagonist activity, encompass the use of known fragments Further, diagnostic methods for assessing receptor function can also be practiced with any fragment, including those fragments that may have been known prior to the invention Similarly, in methods involving treatment of receptor dysfunction, all fragments are encompassed including those which may have been known in the art
  • the receptor polynucleotides are useful as a hybridization probe for cDNA and genomic DNA to isolate a full-length cDNA and genomic clones encoding the polypeptide described in SEQ ID NO 1 and to isolate cDNA and genomic clones that correspond to variants producing the same polypeptide shown in SEQ ID NO 1 or the other variants described herein
  • Variants can be isolated from the same tissue and organism from which the polypeptide shown in SEQ LD NO 1 was isolated, different tissues from the same organism, or from different organisms This method is useful for isolating genes and cDNA that are developmentally-controlled and therefore may be expressed in the same tissue or different tissues at different points in the development of an organism
  • the probe can correspond to any sequence along the entire length of the gene encoding the receptor Accordingly, it could be derived from 5' noncoding regions, the coding region, and 3 ' noncoding regions
  • the nucleic acid probe can be, for example, the full-length cDNA of SEQ LD
  • a fragment thereof such as an oligonucleotide of at least 12, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to mRNA or DNA Fragments of the polynucleotides described herein are also useful to synthesize larger fragments or full-length polynucleotides described herein For example, a fragment can be hybridized to any portion of an mRNA and a larger or full-length cDNA can be produced The fragments are also useful to synthesize antisense molecules of desired length and sequence
  • the receptor polynucleotides are also useful as primers for PCR to amplify any given region of a receptor polynucleotide.
  • the receptor polynucleotides are also useful for constructing recombinant vectors
  • Such vectors include expression vectors that express a portion of, or all of, the receptor polypeptides
  • Vectors also include insertion vectors, used to integrate into another polynucleotide sequence, such as into the cellular genome, to alter in situ expression of receptor genes and gene products
  • an endogenous receptor coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations
  • the receptor polynucleotides are also useful for expressing antigenic portions of the receptor proteins
  • the receptor polynucleotides are also useful as probes for determining the chromosomal positions of the receptor polynucleotides by means of in situ hybridization methods
  • the receptor polynucleotide probes are also useful to determine patterns of the presence of the gene encoding the receptors and their variants with respect to tissue distribution, for example, whether gene duplication has occurred and whether the duplication occurs in all or only a subset of tissues
  • the genes can be naturally occurring or can have been introduced into a cell, tissue, or organism exogenously
  • the receptor polynucleotides are also useful for designing ribozymes corresponding to all, or a part, of the mRNA produced from genes encoding the polynucleotides described herein
  • the receptor polynucleotides are also useful for constructing host cells expressing a part, or all, of the receptor polynucleotides and polypeptides
  • the receptor polynucleotides are also useful for constructing transgenic animals expressing all, or a part, of the receptor polynucleotides and polypeptides
  • the receptor polynucleotides are also useful for making vectors that express part, or all, of the receptor polypeptides
  • the receptor polynucleotides are also useful as hybridization probes for determining the level of receptor nucleic acid expression Accordingly, the probes can be used to detect the presence of, or to determine levels of, receptor nucleic acid in cells, tissues, and in organisms
  • the nucleic acid whose level is determined can be DNA or RNA
  • 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 in which there has been an amplification of the receptor genes
  • the probe can be used in an in situ hybridization context to assess the position of extra copies of the receptor genes, as on extrachromosomal elements or as integrated into chromosomes in which the receptor gene is not normally found, for example as a homogeneously staining region
  • In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations
  • In vitro techniques for detecting DNA includes Southern hybridizations and in situ hybridization Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express a receptor protein, such as by measuring a level of a receptor- encoding nucleic acid in a sample of cells from a subject e g , mRNA or genomic DNA or determining if a receptor gene has been mutated
  • Nucleic acid expression assays are useful for drug screening to identify compounds that modulate receptor nucleic acid expression
  • the assays can be performed in cell-based and cell-free systems
  • Cell-based assays include cells naturally expressing the receptor nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences
  • candidate compounds can be assayed in vivo in patients or in transgenic animals
  • the assay for receptor nucleic acid expression can involve direct assay of nucleic acid levels, such as mRNA levels, or on collateral compounds involved in the signal pathway (such as cyclic AMP or phosphatidylinositol turnover) Further, the expression of genes that are up- or down-regulated in response to the receptor protein signal 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
  • modulators of receptor gene expression can be identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA determined The level of expression of receptor mRNA in the presence of the candidate compound is compared to the level of expression of receptor mRNA in the absence of the candidate compound The candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression When expression of mRNA is statistically significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid expression When nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression
  • the invention provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate receptor nucleic acid expression Modulation includes both up-regulation (i e activation or agonization) or down-regulation (suppression or antagonization) or nucleic acid expression
  • a modulator for receptor nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drug or small molecule inhibits the receptor nucleic acid expression
  • the receptor polynucleotides are also useful for monitoring the effectiveness of modulating compounds on the expression or activity of the receptor gene in clinical trials or in a treatment regimen
  • the gene expression pattern can serve as a barometer for the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance
  • the gene expression pattern can also serve as a marker indicative of a physiological response of the affected cells to the compound Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant Similarly, if the level of nucleic acid expression falls below a desirable level, administration of the compound could be commensurately decreased
  • the receptor polynucleotides are also useful in diagnostic assays for qualitative changes in receptor nucleic acid, and particularly in qualitative changes that lead to pathology
  • the polynucleotides can be used to detect mutations in receptor genes and gene expression products such as mRNA
  • the polynucleotides can be used as hybridization probes to detect naturally-occurring genetic mutations in the receptor gene and thereby to determine whether a subject with the mutation is at risk for a disorder caused by the mutation Mutations include deletion, addition, or substitution of one or more nucleotides in the gene, chromosomal rearrangement, such as inversion or transposition, modification of genomic DNA, such as aberrant methylation patterns or changes in gene copy number, such as amplification Detection of a mutated form of the receptor gene associated with a dysfunction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of a receptor protein
  • RNA or cDNA can be used in the same way
  • detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e g U S Patent Nos 4,683, 195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e g , Landegran et al , Science 241 1077-1080 (1988), and Nakazawa et al , PNAS 91 360-364 (1994)), the latter of which can be particularly useful for detecting point mutations in the gene (see Abravaya et al , Nucleic Acids Res.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e g , genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences
  • mutations in a receptor gene can be directly identified, for example, by alterations in restriction enzyme digestion patterns determined by gel electrophoresis
  • sequence-specific ribozymes (U S Patent No 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site
  • Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and SI protection or the chemical cleavage method
  • sequence differences between a mutant receptor gene and a wild-type gene can be determined by direct DNA sequencing
  • diagnostic assays ((1995) Biotechniques 19 448), including sequencing by mass spectrometry (see, e g , PCT International Publication No WO 94/16101, Cohen et al , Adv. Chromatogr. 36 127-162 (1996), and Griffin etal , Appl. Biochem Biotechnol 35 147-159 (1993))
  • RNA/RNA or RNA/DNA duplexes Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al , Science 230 1242 (1985)), Cotton et al , PNAS
  • the receptor polynucleotides are also useful for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality
  • the polynucleotides can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment (pharmacogenomic relationship)
  • a mutation in the receptor gene that results in altered affinity for ligand could result
  • polynucleotides displaying genetic variations that affect treatment provide a diagnostic target that can be used to tailor treatment in an individual Accordingly, the production of recombinant cells and animals containing these polymo ⁇ hisms allow effective clinical design of treatment compounds and dosage regimens
  • the receptor polynucleotides are also useful for chromosome identification when the sequence is identified with an individual chromosome and to a particular location on the chromosome
  • the DNA sequence is matched to the chromosome by in situ or other chromosome-specific hybridization
  • Sequences can also be correlated to specific chromosomes by preparing PCR primers that can be used for PCR screening of somatic cell hybrids containing individual chromosomes from the desired species Only hybrids containing the chromosome containing the gene homologous to the primer will yield an amplified fragment
  • Sublocalization can be achieved using chromosomal fragments
  • Other strategies include prescreening with labeled flow-sorted chromosomes and preselection by hybridization to chromosome-specific libraries
  • Further mapping strategies include fluorescence in situ hybridization which allows hybridization with probes shorter than those traditionally used Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on the chromos
  • the receptor sequence can be used to provide an alternative technique which determines the actual DNA sequence of selected fragments in the genome of an individual
  • the receptor sequences described herein can be used to prepare two PCR primers from the 5' and 3 ' ends of the sequences These primers can then be used to amplify DNA from an individual for subsequent sequencing Panels of corresponding DNA sequences from individuals prepared in this manner can provide unique individual identifications, as each individual will have a unique set of such DNA sequences It is estimated that allelic variation in humans occurs with a frequency of about once per each 500 bases Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions
  • the receptor sequences can be used to obtain such identification sequences from individuals and from tissue
  • the sequences represent unique fragments of the human genome
  • Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification pu ⁇ oses
  • a panel of reagents from the sequences is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples
  • the receptor polynucleotides can also be used in forensic identification procedures PCR technology can be used to amplify DNA sequences taken from very small biological samples, such as a single hair follicle, body fluids (eg blood, saliva, or semen) The amplified sequence can then be compared to a standard allowing identification of the origin of the sample
  • the receptor polynucleotides can thus be used to provide polynucleotide reagents, e g , PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i e another DNA sequence that is unique to a particular individual)
  • an identification marker i e another DNA sequence that is unique to a particular individual
  • actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments
  • Sequences targeted to the noncoding region are particularly useful since greater polymo ⁇ hism occurs in the noncoding regions, making it easier
  • the receptor polynucleotides can be used directly to block transcription or translation of receptor gene sequences by means of antisense or ribozyme constructs
  • nucleic acids can be directly used for treatment
  • the receptor polynucleotides are thus useful as antisense constructs to control receptor gene expression in cells, tissues, and organisms
  • a DNA antisense polynucleotide is designed to be complementary to a region of the gene involved in transcription, preventing transcription and hence production of receptor protein
  • An antisense RNA or DNA polynucleotide would hybridize to the mRNA and thus block translation of mRNA into receptor protein
  • antisense molecules useful to inhibit nucleic acid expression include antisense molecules complementary to a fragment of the 5' untranslated region of SEQ ID NO 2 which also includes the start codon and antisense molecules which are complementary to a fragment of the 3 ' untranslated region of SEQ LD NO 2
  • a class of antisense molecules can be used to inactivate mRNA in order to decrease expression of receptor nucleic acid Accordingly, these molecules can treat a disorder characterized by abnormal or undesired receptor nucleic acid expression
  • This technique involves cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated Possible regions include coding regions and particularly coding regions corresponding to the catalytic and other functional activities of the receptor protein, such as ligand binding
  • the receptor polynucleotides also provide vectors for gene therapy in patients containing cells that are aberrant in receptor gene expression
  • recombinant cells which include the patient's cells that have been engineered ex vivo and returned to the patient, are introduced into an individual where the cells produce the desired receptor protein to treat the individual
  • kits for detecting the presence of a receptor nucleic acid in a biological sample can comprise reagents such as a labeled or labelable nucleic acid or agent capable of detecting receptor nucleic acid in a biological sample, means for determining the amount of receptor nucleic acid in the sample, and means for comparing the amount of receptor nucleic acid in the sample with a standard
  • the compound or agent can be packaged in a suitable container
  • the kit can further comprise instructions for using the kit to detect receptor mRNA or DNA
  • the invention also provides vectors containing the receptor polynucleotides
  • vehicle refers to a vehicle, preferably a nucleic acid molecule, that can transport the receptor polynucleotides
  • the receptor polynucleotides are covalently linked to the vector nucleic acid
  • the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC
  • a vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the receptor polynucleotides Alternatively, the vector may integrate into the host cell genome and produce additional copies of the receptor polynucleotides when the host cell replicates
  • the invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of the receptor polynucleotides
  • the vectors can function in procaryotic or eukaryotic cells or in both (shuttle vectors)
  • Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the receptor polynucleotides such that transcription of the polynucleotides is allowed in a host cell
  • the polynucleotides can be introduced into the host cell with a separate polynucleotide capable of affecting transcription
  • the second polynucleotide may provide a trans-acting factor interacting with the cis- regulatory control region to allow transcription of the receptor polynucleotides from the vector
  • a trans-acting factor may be supplied by the host cell
  • a trans-acting factor can be produced from the vector itself
  • transcription and/or translation of the receptor polynucleotides can occur in a cell-free system
  • the regulatory sequence to which the polynucleotides described herein can be operably linked include promoters for directing mRNA transcription These include, but are not limited to, the left promoter from bacteriophage ⁇ , the lac, TRP, and TAC promoters from E. coh, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats
  • expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers Examples include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers
  • expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation
  • Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals
  • initiation and termination codons as well as polyadenylation signals
  • a variety of expression vectors can be used to express a receptor polynucleotide
  • Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses
  • Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, eg cosmids and phagemids Appropriate cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al , Molecular
  • the regulatory sequence may provide constitutive expression in one or more host cells (i e tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand
  • host cells i e tissue specific
  • inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand
  • the receptor polynucleotides can be inserted into the vector nucleic acid by well-known methodology Generally, the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art
  • Bacterial cells include, but are not limited to, E. coh, Streptomyces, and Salmonella typhimunum
  • 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
  • the invention provides fusion vectors that allow for the production of the receptor polypeptides
  • Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification
  • a proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired polypeptide 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 pGEX (Smith et al , Gene 6731-40 (1988)), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or
  • coh expression vectors include pTrc ( Amann etal , Gene 69 301 -315 ( 1988)) and pET 11 d (Studier et al , Gene Expression Technology: Methods inEnzymology 185 60-89 (1990))
  • Recombinant protein expression can be maximized in a host bacteria by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S , Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128)
  • the sequence of the polynucleotide of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coh (Wada etal , Nucleic Acids Res. 202111-2118 (1992))
  • the receptor polynucleotides can also be expressed by expression vectors that are operative in yeast Examples of vectors for expression in yeast e g , S.
  • the receptor polynucleotides can also be expressed in insect cells using, for example, baculovirus expression vectors
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al , Mol. Cell Biol. 3 2156-2165 ( 1983)) and the p VL series (Lucklow et al. , Virology 170 31-39 (1989))
  • the polynucleotides described herein are expressed in mammalian cells using mammalian expression vectors
  • mammalian expression vectors include pCDM8 (Seed, B Nature 329 840(1987)) and pMT2PC (Kaufman et al , EMBO J. 6 187- 195 ( 1987))
  • the invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA
  • an antisense transcript can be produced to all, or to a portion, of the polynucleotide sequences described herein, including both coding and non-coding regions Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of the sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression)
  • the invention also relates to recombinant host cells containing the vectors described herein Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells
  • the recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook, etal (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989)
  • Host cells can contain more than one vector
  • the receptor polynucleotides can be introduced either alone or with other polynucleotides that are not related to the receptor polynucleotides such as those providing transacting factors for expression vectors
  • the vectors can be introduced independently, co-introduced or joined to the receptor polynucleotide vector
  • these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction
  • Viral vectors can be replication-competent or replication-defective In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects
  • Vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs The marker can be contained in the same vector that contains the polynucleotides described herein or may be on a
  • secretion of the polypeptide is desired, appropriate secretion signals are inco ⁇ orated into the vector
  • the signal sequence can be endogenous to the receptor polypeptides or heterologous to these polypeptides
  • the protein can be isolated from the host cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like.
  • the polypeptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography
  • polypeptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria
  • polypeptides may include an initial modified methionine in some cases as a result of a host-mediated process
  • the host cells expressing the polypeptides described herein, and particularly recombinant host cells have a variety of uses First, the cells are useful for producing receptor proteins or polypeptides that can be further purified to produce desired amounts of receptor protein or fragments Thus, host cells containing expression vectors are useful for polypeptide production
  • Host cells are also useful for conducting cell-based assays involving the receptor or receptor fragments
  • a recombinant host cell expressing a native receptor is useful to assay for compounds that stimulate or inhibit receptor function This includes ligand binding, gene expression at the level of transcription or translation, G-protein interaction, and components of the signal transduction pathway
  • Host cells are also useful for identifying receptor mutants in which these functions are affected If the mutants naturally occur and give rise to a pathology, host cells containing the mutations are useful to assay compounds that have a desired effect on the mutant receptor (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native receptor
  • Recombinant host cells are also useful for expressing the chimeric polypeptides described herein to assess compounds that activate or suppress activation by means of a heterologous amino terminal extracellular domain (or other binding region)
  • a heterologous region spanning the entire transmembrane domain (or parts thereof) can be used to assess the effect of a desired amino terminal extracellular domain (or other binding region) on any given host cell
  • a region spanning the entire transmembrane domain (or parts thereof) compatible with the specific host cell is used to make the chimeric vector
  • a heterologous carboxy terminal intracellular, e g , signal transduction, domain can be introduced into the host cell
  • mutant receptors can be designed in which one or more of the various functions is engineered to be increased or decreased (e g , ligand binding or G-protein binding) and used to augment or replace receptor proteins in an individual
  • host cells can provide a therapeutic benefit by replacing an aberrant receptor or providing an aberrant receptor that provides a therapeutic result
  • the cells provide receptors that are abnormally active
  • the cells provide receptors that are abnormally inactive
  • These receptors can compete with endogenous receptors in the individual
  • cells expressing receptors that cannot be activated are introduced into an individual in order to compete with endogenous receptors for ligand For example, in the case in which excessive ligand is part of a treatment modality, it may be necessary to inactivate this ligand at a specific point in treatment
  • Homologously recombinant host cells can also be produced that allow the in situ alteration of endogenous receptor polynucleotide sequences in a host cell genome
  • This technology is more fully described in WO 93/09222, WO 91/12650 and U S 5,641,670 Briefly, specific polynucleotide sequences corresponding to the receptor polynucleotides or sequences proximal or distal to a receptor gene are allowed to integrate into a host cell genome by homologous recombination where expression of the gene can be affected
  • regulatory sequences are introduced that either increase or decrease expression of an endogenous sequence
  • a receptor protein can be produced in a cell not normally producing it, or increased expression of receptor protein can result in a cell normally producing the protein at a specific level Alternatively, the entire gene can be deleted Still further, specific mutations can be introduced into any desired region of the gene to produce mutant receptor proteins Such mutations could be introduced, for example, into the specific functional regions such as the ligand-binding site or the G-protein binding site
  • the host cell can be a fertilized oocyte or embryonic stem cell that can be used to produce a transgenic animal containing the altered receptor gene
  • the host cell can be a stem cell or other early tissue precursor that gives rise to a specific subset of cells and can be used to produce transgenic tissues in an animal See also Thomas et al , Cell 51 503 (1987) for a description of homologous recombination vectors
  • the vector is introduced into an embryonic stem cell line (e g , by electroporation) and cells in which the introduced gene has homologously recombined with the endogenous
  • the genetically engineered host cells can be used to produce non-human transgenic animals
  • a transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal in one or more cell types or tissues of the transgenic animal
  • transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians
  • a host cell is a fertilized oocyte or an embryonic stem cell into which receptor polynucleotide sequences have been introduced
  • a transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e g , by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal 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 can form part of the transgenic sequence This includes intronic sequences and polyadenylation signals, if not already included
  • a tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the receptor protein to particular cells
  • transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U S Patent Nos 4,736,866 and 4,870,009, both by Leder et al , U S Patent No 4, 873 , 191 by Wagner etal and in Hogan, B , Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y , 1986) Similar methods are used for production of other transgenic animals A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of transgenic mRNA in tissues or cells of the animals A transgenic founder animal can then be used to breed additional animals carrying the transgene Moreover, transgenic animals carrying a transgene can further be bred to other transgenic animals carrying other transgenes A transgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein In another embodiment, transgenic non-human animals
  • Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I et al Nature 385 810-813 (1997) and PCT International Publication Nos WO 97/07668 and WO 97/07669
  • a cell, e g a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G 0 phase
  • the quiescent cell can then be fused, e g , through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated
  • the reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to a pseudopregnant female foster animal
  • the offspring born of this female foster animal will be a clone of the animal from which the cell, e g , the somatic cell, is isolated
  • compositions suitable for administration to a subject typically comprise the nucleic acid molecule, protein, modulator, or antibody and a pharmaceutically acceptable carrier
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents, and the like, compatible with pharmaceutical administration
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents, and the like, compatible with pharmaceutical administration
  • the use of such media and agents for pharmaceutically active substances is well known in the art Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the invention
  • Supplementary active compounds can also be inco ⁇ orated into the compositions
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration Examples of routes of administration include parenteral, e g ,
  • 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
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS)
  • the composition must be sterile and should be fluid to the extent that easy syringability exists It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the composition
  • Oral compositions generally include an inert diluent or an edible carrier They can be enclosed in gelatin capsules or compressed into tablets
  • the agent can be contained in enteric forms to survive the stomach or further coated or mixed to be released in a particular region of the GI tract by known methods
  • the active compound can be inco ⁇ orated with excipients and used in the form of tablets, troches, or capsules
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature a binder such as microcrystalline cellulose, gum tragacanth or gelatin, an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primo
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e g , a gas such as carbon dioxide, or a nebulizer
  • Systemic administration can also be by transmucosal or transdermal means
  • penetrants appropriate to the barrier to be permeated are used in the formulation
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art
  • the compounds can also be prepared in the form of suppositories (e g , with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery
  • suppositories e g , with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems
  • 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 preparation of such formulations will be apparent to those skilled in the art
  • the materials can also be obtained commercially from Alza Co ⁇ oration and Nova Pharmaceuticals, Inc
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers
  • These can be prepared according to methods known to those skilled in the art, for example, as described in U S Patent No 4,522,811
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U S 5,328,470) or by stereotactic injection (see e g , Chen etal , PNAS 91 3054-3057 (1994))
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e g retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system
  • compositions can be included in a container, pack, or dispenser together with instructions for administration

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Abstract

Cette invention concerne un récepteur faisant partie de la superfamille des récepteurs couplés à la protéine G. Elle concerne également des polynucléotides codant pour ce récepteur. De plus, elle porte sur des procédés dans lesquels les polypeptides et polynucléotides du récepteur interviennent comme cible pour le diagnostic et le traitement de troubles provoqués par ledit récepteur. Par ailleurs, l'invention concerne des méthodes de criblage de médicaments dans lesquelles les polypeptides et polynucléotides du récepteur servent à identifier des agonistes et des antagonistes à des fins de diagnostic et de traitement. Elle porte également sur des agonistes et des antagonistes par rapport aux polypeptides et polynucléotides du récepteur ainsi que sur des méthodes de production des polypeptides et polynucléotides du récepteur.
PCT/US2000/001592 1999-01-21 2000-01-21 Recepteur 17723 couple a la proteine g WO2000043513A1 (fr)

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WO2001072773A2 (fr) * 2000-03-29 2001-10-04 Novartis Ag Composes organiques
WO2001072773A3 (fr) * 2000-03-29 2003-04-17 Novartis Ag Composes organiques
GB2371303A (en) * 2000-10-05 2002-07-24 Glaxo Group Ltd Receptor polypeptides with immunomodulatory activity

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