EP1220914A1 - Human g-protein coupled receptor - Google Patents

Human g-protein coupled receptor

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Publication number
EP1220914A1
EP1220914A1 EP00967696A EP00967696A EP1220914A1 EP 1220914 A1 EP1220914 A1 EP 1220914A1 EP 00967696 A EP00967696 A EP 00967696A EP 00967696 A EP00967696 A EP 00967696A EP 1220914 A1 EP1220914 A1 EP 1220914A1
Authority
EP
European Patent Office
Prior art keywords
igs3
polypeptide
seq
nucleotide sequence
polynucleotide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00967696A
Other languages
German (de)
English (en)
French (fr)
Inventor
Willy Deleersnijder
Guy Nys
Nicole D'heuvaert
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Abbott Healthcare Products BV
Original Assignee
Solvay Pharmaceuticals BV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Solvay Pharmaceuticals BV filed Critical Solvay Pharmaceuticals BV
Priority to EP00967696A priority Critical patent/EP1220914A1/en
Publication of EP1220914A1 publication Critical patent/EP1220914A1/en
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • the present invention relates to novel identified polynucleotides, polypeptides encoded by them and to the use of such polynucleotides and polypeptides, and to their production More particularly, the polynucleotides and polypeptides of the present invention relate to a G-protein coupled receptor (GPCR), hereinafter referred to as IGS3
  • GPCR G-protein coupled receptor
  • the invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides, to a vector containing said polynucleotides, a host cell containing such vector and transgenic animals where the IGS3-gene is either overexpressed, misexpressed, underexpressed and/or suppressed (knock-out animals)
  • the invention further relates to a method for screening compounds capable to act as an agonist or an antagonist of said G-protein coupled receptor IGS3
  • proteins participating in signal transduction pathways that involve G-proteins and/or second messengers, e g , cAMP (Lefkowitz, Nature, 1991 , 351 353-354)
  • these proteins are referred to as proteins participating in pathways with G-proteins
  • Some examples of these proteins include the GPC receptors, such as those for adrenergic agents and dopamine (Kobilka, B K , et al , Proc Natl Acad Sci , USA, 1987, 84 46-50, Kobilka, B K , et al , Science, 1987, 238 650-656, Bunzow, J R , et al , Nature, 1988, 336 783-787), G-proteins themselves, effector proteins, e g , phospho pase C, adenylate cyclase, and phosphodiesterase, and actuator proteins, e g , protein kinase A and protein kinas
  • the receptor upon hormone binding to a GPCR the receptor interacts with the heterot ⁇ me ⁇ c G-protein and induces the dissociation of GDP from the guanine nucleotide-binding site
  • GTP fills the site immediately Binding of GTP to the ⁇ subunit of the G-protein causes the dissociation of the G-protein from the receptor and the dissociation of the G-protein into ⁇ and ⁇ subunits
  • the GTP-carrying form then binds to activated adenylate cyclase Hydrolysis of GTP to GDP, catalyzed by the G-protein itself ( ⁇ subunit possesses an intrinsic GTPase activity), returns the G-protein to its basal, inactive form
  • the GTPase activity of the ⁇ subunit is, in essence, an internal clock that controls an on/off switch
  • the GDP bound form of the ⁇ subunit has high affinity for ⁇ and subsequent reassociation of ⁇
  • G-protein coupled receptors The membrane bound superfamily of G-protein coupled receptors has been characterized as having seven putative transmembrane domains The domains are believed to represent transmembrane ⁇ -helices connected by extracellular or cytoplasmic loops G-protein coupled receptors include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors
  • the G-protein coupled receptor family includes dopamine receptors which bind to neuroleptic drugs used for treating CNS disorders
  • members of this family include, but are not limited to calcitonm, adrenergic, neuropeptideY, somastotatin, neurotensin, neurokinin, capsaicin, VIP, CGRP, CRF, CCK, bradykinin, galanm, motilin, nociceptin, endothelin, cAMP, adenosine, muscannic, acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsin, endothelial differentiation gene-1 , rhodopsin, odorant, and cytomegalovirus receptors
  • TM1 , TM2, TM3, TM4, TM5 The 7 transmembrane regions are designated as TM1 , TM2, TM3, TM4, TM5,
  • TM6 and TM7 The cytoplasmic loop which connects TM5 and TM6 may be a major component of the G-protein binding domain
  • G-protein coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxy terminus
  • G-protein coupled receptors such as the ⁇ -adrenoreceptor
  • phosphorylation by protein kinase A and/or specific receptor kinases mediates receptor desensitization
  • GPCRs like the calcitonin-receptor like receptor, might interact with small single pass membrane proteins called receptor activity modifying proteins (RAMP's)
  • RAMP's receptor activity modifying proteins
  • This interaction of the GPCR with a certain RAMP is determining which natural ligands have relevant affinity for the GPCR-RAMP combination and regulate the functional signaling activity of the complex (McLathie, L M et al , Nature (1998) 393 333-339)
  • the ligand binding sites of G-protein coupled receptors are believed to comprise hydrophilic sockets formed by several G-protein coupled receptor transmembrane domains, said sockets being surrounded by hydrophobic residues of the G-protein coupled receptors
  • the hydrophilic side of each G-protein coupled receptor transmembrane helix is postulated to face inward and form a polar hgand-binding site TM3 has been implicated in several G-protein coupled receptors as having a ligand-binding site, such as the TM3
  • G-protein coupled receptors can be mtracellularly coupled by heterotrimeric G-proteins to various intracellular enzymes, ion channels and transporters (see, Johnson et al , Endoc Rev , 1989, 10 317-331 )
  • Different G-protein ⁇ -subunits preferentially stimulate particular effectors to modulate various biological functions in a cell Phosphorylation of cytoplasmic residues of G- protein coupled receptors has been identified as an important mechanism for the regulation of G-protein coupling of some G-protein coupled receptors
  • G-protein coupled receptors are found in numerous sites within a mammalian host
  • the new IGS3 GPCR described in this invention clearly satisfies a need in the art for identification and characterization of further receptors that can play a role in diagnosing, preventing, ameliorating or correcting dysfunctions, disorders, or diseases, hereafter generally referred to as "the Diseases"
  • the Diseases include, but are not limited to, psychiatric and CNS disorders, including schizophrenia, episodic paroxysmal anxiety (EPA) disorders such as obsessive compulsive disorder (OCD), post traumatic stress disorder (PTSD), phobia and panic, major depressive disorder, bipolar disorder, Parkinson's disease, general anxiety disorder, autism, delirium, multiple sclerosis, Alzheimer disease/dementia and other neurodegenerative diseases, severe mental retardation, dyskinesias, Huntington's disease, Tourett
  • the invention relates to IGS3 polypeptides, polynucleotides and recombinant materials and methods for their production
  • Another aspect of the invention relates to methods for using such IGS3 polypeptides, polynucleotides and recombinant materials
  • uses include, but are not limited to, use as a therapeutic target and for treatment of one of the Diseases as mentioned above
  • the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with IGS3 imbalance with the identified compounds
  • Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate IGS3 activity or levels
  • a further aspect of the invention relates to animal-based systems which act as models for disorders arising from aberrant expression or activity of IGS3
  • FIG. 1 Schematic representation of the relative positions of the different DNA clones that were isolated to generate the consensus IGS3 cDNA sequence HNT1370 represents the "founding" genomic clone ⁇ -IGS3 1A,B etc indicate separate (nearly) overlapping sequence contigs obtained from sequence analysis of DNA from lambda clone IGS3 1 PCR primers that have been described in this document are indicated (IP#)
  • CONSENSUS denotes the contig that was obtained after merging all obtained sequences
  • the part of the CONSENSUS contig that was fully validated by sequence analysis of at least three independent clones is represented by IGS3DNA (SEQ ID NO 1 )
  • the 330 ammo acids long open reading frame present in IGS3DNA is indicated with "**”
  • IGS3 is implied to play a role among other things in the Diseases mentioned above
  • IGS3 refers, among others, to a polypeptide comprising the ammo acid sequence set forth in SEQ ID NO 2, or a Variant thereof
  • Receptor Activity or "Biological Activity of the Receptor” refers to the metabolic or physiologic function of said IGS3 including similar activities or improved activities or these activities with decreased undesirable side effects Also included are antigenic and immunogenic activities of said IGS3
  • IGS3-gene refers to a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO 1 or Variants thereof and/or their complements
  • Antibodies as used herein includes polyclonal and monoclonal antibodies, chime ⁇ c, single chain, and humanized antibodies, as well as Fab fragments, including the products of a Fab or other immunoglobulin expression library
  • Isolated means altered “by the hand of man” from the natural state and/or separated from the natural environment Thus, if an "isolated” composition or substance that occurs in nature has been ' isolated”, it has been changed or removed from its original environment, or both For example, a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein
  • Polynucleotide generally refers to any polynbonucleotide or polydeoxnbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA
  • Polynucleotides include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single-and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single- stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions
  • polynucleotide may also include triple-stranded regions comprising RNA or DNA or both RNA and DNA
  • the term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons "Modified" bases include, for example, t ⁇ t
  • Polypeptide refers to any peptide or protein comprising two or more ammo acids joined to each other by peptide bonds or modified peptide bonds, i e , peptide isosteres
  • Polypeptide refers to short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins, and/or to combinations thereof Polypeptides may contain ammo acids other than the 20 gene-encoded ammo acids
  • Polypeptides include ammo acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art Such modifications are well-described in basic texts and in more detailed monographs, as well as in voluminous research literature Modifications can occur anywhere in a polypeptide, including the peptide backbone, the ammo acid side-chains and the ammo or carboxyl termini It will be appreciated that the same type of modification may be present in the same or
  • Variant is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties such as essential biological, structural, regulatory or biochemical properties
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the variant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical
  • a variant and reference polypeptide may differ in ammo acid sequence by one
  • Identity is a measure of the identity of nucleotide sequences or ammo acid sequences In general, the sequences are aligned so that the highest order match is obtained "Identity" per se has an art-recognized meaning and can be calculated using published techniques See, e g (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A M , ed , Oxford University Press, New York, 1988, BIOCOMPUTING INFORMATICS AND GENOME PROJECTS, Smith, D W , ed , Academic Press, New York, 1993, COMPUTER ANALYSIS OF SEQUENCE DATA, PART I Griffin, A M , and Griffin, H G , eds , Humana Press, New Jersey, 1994, SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Hemje, G , Academic Press, 1987, and SEQUENCE ANALYSIS PRIMER, Gnbskov, M and Devereux, J , eds , M Stockt
  • a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO 1 is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five nucleotide differences per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO 1
  • up to any 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to any 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence, or in a number of nucleotides of up to any 5% of the total nucleotides in the
  • a polypeptide having an ammo acid sequence having at least, for example, 95% "identity" to a reference ammo acid sequence of SEQ ID NO 2 is intended that the ammo acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five ammo acid alterations per each 100 ammo acids of the reference ammo acid of SEQ ID NO 2
  • the polypeptide sequence may include up to five ammo acid alterations per each 100 ammo acids of the reference ammo acid of SEQ ID NO 2
  • up to any 5% of the ammo acid residues in the reference sequence may be deleted or substituted with another ammo acid, or a number of ammo acids up to any 5% of the total ammo acid residues in the reference sequence may be inserted into the reference sequence
  • These alterations of the reference sequence may occur at the ammo or carboxy terminal positions of the reference ammo acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one
  • the present invention relates to IGS3 polypeptides (including IGS3 proteins)
  • the IGS3 polypeptides include the polypeptide of SEQ ID NO 2 and the polypeptide having the am o acid sequence encoded by the DNA insert contained in the deposit no CBS 102196, deposited on September 15, 1999 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands), as well as polypeptides comprising the ammo acid sequence of SEQ ID NO 2 and the polypeptide having the ammo acid sequence encoded by the DNA insert contained in the deposit no CBS 102196 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands), and polypeptides comprising an ammo acid sequence having at least 80% identity to that of SEQ ID NO 2 and/or to the polypeptide having the am o acid sequence encoded by the DNA insert contained in the deposit no CBS 102196 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands) over its entire length
  • the IGS3 polypeptides may be a part of a larger protein such as a fusion protein It is often advantageous to include an additional am o acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidme residues, sequences which aid in detection such as antigenic peptide tags (such as the haemagglutinin (HA) tag), or an additional sequence for stability during recombinant production Fragments of the IGS3 polypeptides are also included in the invention A fragment is a polypeptide having an am o acid sequence that is the same as part of, but not all of, the ammo acid sequence of the aforementioned IGS3 polypeptides As with IGS3 polypeptides, fragments may be "free-standing," or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region Representative examples of polypeptide fragments of the invention, include, for example, fragment
  • Preferred fragments include, for example, truncation polypeptides having the ammo acid sequence of IGS3 polypeptides, except for deletion of a continuous series of residues that includes the ammo terminus, or a continuous series of residues that includes the carboxyl terminus or deletion of two continuous series of residues, one including the ammo terminus and one including the carboxyl terminus
  • fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil- formmg regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions
  • Other preferred fragments are biologically active fragments
  • Biologically active fragments are those that mediate receptor activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity Also included are those that are anti
  • polypeptides of the invention include polypeptides having an ammo acid sequence that is at least 80% identical to either that of SEQ ID NO 2 and/or the polypeptide having the ammo acid sequence encoded by the DNA insert contained in the deposit no CBS 102196 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands), or fragments thereof with at least 80% identity to the corresponding fragment
  • all of these polypeptide fragments retain the biological activity of the receptor, including antigenic activity
  • Variants of the defined sequence and fragments also form part of the present invention
  • Preferred variants are those that vary from the referents by conservative amino acid substitutions — i e , those that substitute a residue with another of like characteristics Typical such substitutions are among Ala, Val, Leu and He, among Ser and Thr, among the acidic residues Asp and Glu, among Asn and Gin, and among the basic residues Lys and Arg, or aromatic residues Phe and Tyr Particularly preferred are variants in which several, 5
  • the IGS3 polypeptides of the invention can be prepared in any suitable manner
  • Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods Methods for preparing such polypeptides are well known in the art
  • IGS3 polynucleotides include isolated polynucleotides which encode the IGS3 polypeptides and fragments, and polynucleotides closely related thereto More specifically, the IGS3 polynucleotide of the invention includes a polynucleotide comprising the nucleotide sequence contained in SEQ ID N0 1 , such as the one capable of encoding a IGS3 polypeptide of SEQ ID NO 2, polynucleotides having the particular sequence of SEQ ID NO 1 and polynucleotides which essentially correspond to the DNA insert contained in the deposit no CBS 102196 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands)
  • IGS3 polynucleotides further include polynucleotides comprising a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the IGS3 polypeptide of SEQ ID NO 2, polynucleotides comprising a nucleotide sequence that is at least 80% identical to that of SEQ ID NO 1 over its entire length and a polynucleotide which essentially corresponds to the DNA insert contained in the deposit no CBS 102196 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands)
  • polynucleotides with at least 90% identity are particularly preferred, and those with at least 95% are especially preferred Furthermore, those with at least 97% are highly preferred and those with at least 98-99% are most highly preferred, with at least 99% being the most preferred
  • the invention also provides polynucleotides which are complementary to such IGS3 polynucleotides
  • IGS3 of the invention is structurally related to other proteins of the G-protein coupled receptor family, as shown by the results of BLAST searches in the public databases
  • the ammo acid sequence of Table 2 (SEQ ID NO 2) has about 35 % identity (using BLAST, Altschul S F et al Nucleic Acids Res (1997) 25 3389-3402) over most of its length (ammo acid residues 2-306 ) with the protein encoded by the human mas oncogene (Sequence 1 in patent application WO 8707472)
  • the sequence is 37% identical (ammo acid residues 35-315) with the G-protein coupled receptor published in patent application WO 9616087 (GENESEQ 96P-R97222 )
  • the nucleotide sequence of Table 1 (SEQ ID NO 1 ) has 52 % and 54 % identity over most of its length to the two receptors above (GENESEQ 87N-70685 and 96N-T28807 respectively) Also there is 48% identity to the human Somato
  • Polynucleotides of the invention can be obtained from natural sources such as genomic DNA
  • degenerated PCR primers can be designed that encode conserved regions within a particular GPCR gene subfamily PCR amplification reactions on genomic DNA or cDNA using the degenerate primers will result in the amplification of several members (both known and novel) of the gene family under consideration (the degenerated primers must be located within the same exon, when a genomic template is used) (Libert et al , Science, 1989, 244 569-572)
  • Polynucleotides of the invention can also be synthesized using well-known and commercially available techniques
  • the nucleotide sequence encoding the IGS3 polypeptide of SEQ ID NO 2 may be identical to the polypeptide encoding sequence contained in SEQ ID NO 1 (nucleotide number 149 to 1138), or it may be a different nucleotide sequence, which as a result of the redundancy (degeneracy) of the genetic code might also show alterations compared to the polypeptide encoding sequence contained in SEQ ID NO 1 , but also encodes the polypeptide of SEQ ID NO 2
  • the polynucleotide may include the coding sequence for the mature polypeptide or a fragment thereof, by itself, the coding sequence for the mature polypeptide or fragment in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions
  • a marker sequence which facilitates purification of the fused polypeptide can be encoded
  • the marker sequence is a hexa-histidme peptide, as provided in the pQE vector (Qiagen, Inc ) and described in Gentz et al , Proc Natl Acad Sci USA (1989) 86 821-824, or is an HA tag
  • the polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, nb
  • IGS3 variants comprising the ammo acid sequence of the IGS3 polypeptide of SEQ ID NO 2 in which several, 5-10, 1 -5, 1 - 3, 1-2 or 1 ammo acid residues are substituted, deleted or added, in any combination
  • polynucleotides of the invention can be engineered using methods generally known in the art in order to alter IGS3-encod ⁇ ng sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences
  • oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create am o acid substitutions, create new restriction sites, alter modification (e g glycosylation or phosphorylation) patterns, change codon preference, produce splice variants, and so forth
  • the present invention further relates to polynucleotides that hybridize to the herein above- described sequences
  • the present invention especially relates to polynucleotides which hybridize under stringent conditions to the polynucleotides described above
  • stringent conditions means hybridization will occur only if there is at least 80%, and preferably at least 90%, and more preferably at least 95%, yet even more preferably at least 97%, in particular at least 99% identity between the sequences
  • Polynucleotides of the invention which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO 1 or a fragment thereof, may be used as hybridization probes for cDNA and genomic DNA, to isolate full-length cDNAs and genomic clones encoding IGS3 and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than human) that have a high sequence similarity to the IGS3 gene
  • these nucleotide sequences are 80% identical, preferably 90% identical, more preferably 95% identical to that of the referent
  • the probes generally will comprise at least 5 nucleotides, and preferably at least 8 nucleotides, and more preferably at least 10 nucleotides, yet even more preferably at least 12 nucleotides, in particular at least 15 nucleotides Most preferred, such probes will have at least 30 nucleotides and may have at least 50 nucle
  • a polynucleotide encoding the IGS3 polypeptide, including homologs and orthologs from species other than human comprises the steps of screening an appropriate library under stringent hybridization conditions with a labeled probe having the SEQ ID NO 1 or a fragment thereof, and isolating full-length cDNA and genomic clones containing said polynucleotide sequence
  • Stringent hybridization conditions are as defined above or alternatively conditions under overnight incubation at 42 °C in a solution comprising 50% formamide, 5xSSC (150mM NaCI, 15mM t ⁇ sodium citrate), 50 mM sodium phosphate (pH7 6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0 1 xSSC at about 65°C
  • the present invention also relates to vectors which comprise a polynucleotide or polynucleotides of the present invention, and host cells which are genetically engineered with vectors of the invention and to the production of polypeptides of the invention by recombinant techniques Cell-free translation systems can also be used to produce such proteins using RNAs derived from the DNA constructs of the present invention
  • host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention
  • Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al , BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y (1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid- mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection
  • Representative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, E coli, Streptomyces and Bacillus subtihs cells, fungal cells, such as yeast cells and Aspergillus cells, insect cells such as
  • a great variety of expression systems can be used such systems include, among others, chromosomal, episomal and virus-derived systems, e g , vectors derived from bacterial plasmids, from bacte ⁇ ophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacte ⁇ ophage genetic elements, such as cosmids and phagemids
  • the expression systems may contain control regions that regulate as well as engender expression Generally, any system or vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide in a host may be used The appropriate nucleotide sequence may be inserted into an expression system by any of a
  • secretion signals may be incorporated into the desired polypeptide
  • These signals may be endogenous to the polypeptide or they may be heterologous signals, i e derived from a different species
  • the IGS3 polypeptide is to be expressed for use in screening assays, generally, it is preferred that the polypeptide be produced at the surface of the cell In this event, the cells may be harvested prior to use in the screening assay In case the affinity or functional activity of the IGS3 polypeptide is modified by receptor activity modifying proteins (RAMP), coexpression of the relevant RAMP most likely at the surface of the cell is preferred and often required Also in this event harvesting of cells expressing the IGS3 polypeptide and the relevant RAMP prior to use in screening assays is required If the IGS3 polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide, if produced intracellularly, the cells must first be lysed before the polypeptide is recovered Membranes expressing the IGS3 polypeptide can be recovered by methods that are well known to a person skilled in the art In general, such methods include harvesting of the cells expressing the IGS3 polypeptide and homogenization of the cells by a method such as, but not limited
  • IGS3 polypeptides can be recovered and purified from recombinant cell cultures by well- known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectm chromatography Most preferably, high performance liquid chromatography is employed for purification Well-known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification
  • This invention also relates to the use of IGS3 polynucleotides for use as diagnostic reagents Detection of a mutated form of the IGS3 gene associated with a dysfunction will provide a diagnostic tool that can add to or define a diagnosis of a disease or susceptibility to a disease which results from under-expression, over-expression or altered expression of IGS3 Also in this event co-expression of relevant receptor activity modifying proteins can be required to obtain diagnostic assays of desired quality Individuals carrying mutations in the IGS3 gene may be detected at the DNA level by a variety of techniques
  • Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis RNA or cDNA may also be used in similar fashion
  • Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype
  • Point mutations can be identified by hybridizing amplified DNA to labeled IGS3 nucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures
  • DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing See, e g , Myers et al , Science (1985) 230 1242 Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and
  • the diagnostic assays offer a process for diagnosing or determining a susceptibility to among other things the Diseases as mentioned above, through detection of mutation in the IGS3 gene by the methods described
  • the Diseases as mentioned above can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of the IGS3 polypeptide or IGS3 mRNA
  • RNA level can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods
  • Assay techniques that can be used to determine levels of a protein, such as an IGS3, in a sample derived from a host are well known to those of skill in the art
  • Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays
  • the present invention relates to a diagnostic kit for among other things the Diseases or suspectability to one of the Diseases as mentioned above
  • the kit may comprise
  • an IGS3 polynucleotide preferably the nucleotide sequence of SEQ ID NO 1 , or a fragment thereof, and/or
  • an antibody to an IGS3 polypeptide preferably to the polypeptide of SEQ ID NO 2, and/or
  • the nucleotide sequences of the present invention are also valuable for chromosome identification
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome
  • the mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease
  • genetic map data are found, for example, in V McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library)
  • the relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinhe ⁇ tance of physically adjacent genes)
  • the differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease
  • polypeptides of the invention or their fragments or analogs thereof, or cells expressing them if required together with relevant RAMP's, may also be used as immunogens to produce antibodies immunospecific for the IGS3 polypeptides
  • immunospecific means that the antibodies have substantial! greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art
  • Antibodies generated against the IGS3 polypeptides may be obtained by administering the polypeptides or epitope-bea ⁇ ng fragments, analogs or cells to an animal, preferably a nonhuman, using routine protocols
  • any technique which provides antibodies produced by continuous cell line cultures, may be used Examples include the hyb ⁇ doma technique (Kohler, G and Milstem, C , Nature (1975) 256 495-497), the t ⁇ oma technique, the human B-cell hyb ⁇ doma technique (Kozbor et al , Immunology Today (1983) 4 72) and the EBV-hyb ⁇ doma technique (Cole et al , MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp 77-96, Alan R Liss, Inc , 1985)
  • antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography
  • Antibodies against IGS3 polypeptides as such, or against IGS3 polypeptide-RAMP complexes, may also be employed to treat among other things the Diseases as mentioned above
  • Non-human animal-based model systems may also be used to further characterize the activity of the IGS3 gene Such systems may be utilized as part of screening strategies designed to identify compounds which are capable to treat IGS3 based disorders such as among other things the Diseases as mentioned above
  • animal-based models may be used to identify pharmaceutical compounds, therapies and interventions which may be effective in treating disorders of aberrant expression or activity of IGS3
  • animal models may be used to determine the LD 50 and the
  • Animal-based model systems of IGS3 based disorders may include both non-recombmant animals as well as recombinantly engineered transgenic animals
  • Animal models for IGS3 disorders may include, for example, genetic models Animal models exhibiting IGS3 based disorder-like symptoms may be engineered by utilizing, for example, IGS3 sequences such as those described, above, in conjunction with techniques for producing transgenic animals that are well known to persons skilled in the art
  • IGS3 sequences may be introduced into, and overexpressed and/or misexpressed in, the genome of the animal of interest, or, if endogenous IGS3 sequences are present, they may either be overexpressed, misexpressed, or, alternatively, may be disrupted in order to underexpress or inactivate IGS3 gene expression
  • IGS3 gene sequence may be ligated to a regulatory sequence which is capable of driving high level gene expression or expression in a cell type in which the gene is not normally expressed in the animal type of interest Such regulatory regions will be well known to those skilled in the art, and may be utilized in the absence of undue experimentation
  • a regulatory sequence which is capable of driving high level gene expression or expression in a cell type in which the gene is not normally expressed in the animal type of interest
  • Such regulatory regions will be well known to those skilled in the art, and may be utilized in the absence of undue experimentation
  • an endogenous IGS3 gene sequence such a sequence may be isolated and engineered such that when remtroduced into the genome of the animal of interest, the endogenous IGS3 gene alleles will be inactivated, or "knocked-out"
  • the engineered IGS3 gene sequence is introduced via gene targeting such that the endogenous IGS3 sequence is disrupted upon integration of the engineered IGS3 gene sequence into the animal's genome
  • mice Animals of any species, including, but not limited to, mice, rats, rabbits, squirrels, guinea- pigs, pigs, micro-pigs, goats, and non-human primates, e_g_, baboons, monkeys, and chimpanzees may be used to generate animal models of IGS3 related disorders
  • any technique known in the art may be used to introduce a IGS3 transgene into animals to produce the founder lines of transgenic animals
  • Such techniques include, but are not limited to pronuclear micromjection (Hoppe, P C and Wagner, T E , 1989, U S Pat No 4,873,191 ), retrovirus mediated gene transfer into germ lines (van der Putten et al , Proc Natl Acad Sci , USA 82 6148-6152, 1985), gene targeting in embryonic stem cells (Thompson et al .
  • the present invention provides for transgenic animals that carry the IGS3 transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, J L, mosaic animals (See, for example, techniques described by Jakobovits, Curr Biol 4 761-763, 1994)
  • the transgene may be integrated as a single transgene or in concatamers, e g , head-to- head tandems or head-to-tail tandems
  • the transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al (Lasko, M et al . Proc Natl Acad Sci USA 89 6232-6236, 1992)
  • the IGS3 transgene When it is desired that the IGS3 transgene be integrated into the chromosomal site of the endogenous IGS3 gene, gene targeting is preferred Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous IGS3 gene of interest (e g , nucleotide sequences of the mouse IGS3 gene) are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of, the nucleotide sequence of the endogenous IGS3 gene or gene allele
  • the transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene of interest in only that cell type, by following, for example, the teaching of Gu et al (Gu, H et al -, Science 265 103-106, 1994)
  • the regulatory sequences required for such a cell-type specific mactivation will depend upon the particular cell
  • the expression of the recombinant IGS3 gene and protein may be assayed utilizing standard techniques
  • Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to assay whether integration of the transgene has taken place
  • the level of mRNA expression of the IGS3 transgene in the tissues of the transgenic animals may also be assessed using techniques which include but are not limited to Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and RT-PCR Samples of target gene-expressing tissue, may also be evaluated immunocytochemically using antibodies specific for the target gene transgene product of interest
  • the IGS3 transgenic animals that express IGS3 gene mRNA or IGS3 transgene peptide (detected immunocytochemically, using antibodies directed against target gene product epitopes) at easily detectable levels may then be further evaluated to identify those animals which display characteristic IGS3 based disorder symptoms
  • IGS3 transgenic founder animals are produced (i e . those animals which express
  • IGS3 proteins in cells or tissues of interest may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal
  • breeding strategies include but are not limited to outbreedmg of founder animals with more than one integration site in order to establish separate lines, inbreeding of separate lines in order to produce compound IGS3 transgenics that express the IGS3 transgene of interest at higher levels because of the effects of additive expression of each IGS3 transgene, crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the possible need for screening of animals by DNA analysis, crossing of separate homozygous lines to produce compound heterozygous or homozygous lines, breeding animals to different inbred genetic backgrounds so as to examine effects of modifying alleles on expression of the IGS3 transgene and the development of IGS3-l ⁇ ke symptoms
  • One such approach is to cross the IGS3 transgenic founder animals with
  • Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises administering to (for example by inoculation) the mammal the IGS3 polypeptide, or a fragment thereof, if required together with a RAMP polypeptide, adequate to produce antibody and/or T cell immune response to protect said animal from among other things one of the Diseases as mentioned above
  • Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises delivering the IGS3 polypeptide via a vector directing expression of the IGS3 polynucleotide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.
  • a further aspect of the invention relates to an immunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to an IGS3 polypeptide wherein the composition comprises an IGS3 polypeptide or IGS3 gene
  • immunological/vaccine formulations may be either therapeutic immunological/vaccine formulations or prophylactic immunological/vaccine formulations
  • the vaccine formulation may further comprise a suitable carrier.
  • IGS3 polypeptide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, mtradermal etc injection)
  • parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacte ⁇ ostats and solutes which render the formulation isotonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-d ⁇ ed condition requiring only the addition of the sterile liquid carrier immediately prior to use
  • the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-m water systems and other systems known in the art
  • the dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation
  • the IGS3 polypeptide of the present invention may be employed in a screening process for compounds which bind the receptor and which activate (agonists) or inhibit activation of (antagonists) the receptor polypeptide of the present invention
  • polypeptides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures
  • substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics
  • IGS3 polypeptides are responsible for biological functions, including pathologies
  • agonists are employed for therapeutic and prophylactic purposes for such conditions as among other things the Diseases as mentioned above
  • Antagonists may be employed for a variety of therapeutic and prophylactic purposes for such conditions as among other things the Diseases as mentioned above
  • such screening procedures involve producing appropriate cells, which express the receptor polypeptide of the present invention on the surface thereof and, if essential co- expression of RAMP's at the surface thereof
  • Such cells include cells from mammals, yeast, Drosophila or E coli Cells expressing the receptor (or cell membrane containing the expressed receptor) are then contacted with a test compound to observe binding, or stimulation or inhibition of a functional response
  • One screening technique includes the use of cells which express the receptor of this invention (for example, transfected CHO cells) in a system which measures extracellular pH, mtracellular pH, or mtracellular calcium changes caused by receptor activation
  • compounds may be contacted with cells expressing the receptor polypeptide of the present invention
  • a second messenger response, e g , signal transduction, pH changes, or changes in calcium level, is then measured to determine whether the potential compound activates or inhibits the receptor
  • Another method involves screening for receptor inhibitors by determining modulation of a receptor-mediated signal, such as cAMP accumulation and/or adenylate cyclase activity
  • a receptor-mediated signal such as cAMP accumulation and/or adenylate cyclase activity
  • transfectmg an eukaryotic cell with the receptor of this invention to express the receptor on the cell surface
  • the cell is then exposed to an agonist to the receptor of this invention in the presence of a potential antagonist If the potential antagonist binds the receptor, and thus inhibits receptor binding, the agonist-mediated signal will be modulated
  • Another method for detecting agonists or antagonists for the receptor of the present invention is the yeast-based technology as described in U S Patent 5,482,835
  • the assays may simply test binding of a candidate compound wherein adherence to the cells bearing the receptor is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor Further, these assays may test whether the candidate compound results in a signal generated by activation of the receptor, using detection systems appropriate to the cells bearing the receptor at their surfaces Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed
  • the assays may simply comprise the steps of mixing a candidate compound with a solution containing an IGS3 polypeptide to form a mixture, measuring the IGS3 activity in the mixture, and comparing the IGS3 activity of the mixture to a standard
  • the IGS3 cDNA, protein and antibodies to the protein may also be used to configure assays for detecting the effect of added compounds on the production of IGS3 mRNA and protein in cells
  • an ELISA may be constructed for measuring secreted or cell associated levels of IGS3 protein using monoclonal and polydonal antibodies by standard methods known in the art, and this can be used to discover agents which may inhibit or enhance the production of IGS3 (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues Standard methods for conducting screening assays are well known in the art
  • IGS3 antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligand of the IGS3, e g , a fragment of the ligand, or small molecules which bind to the receptor but do not elicit a response, so that the activity of the receptor is prevented
  • the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc for IGS3 polypeptides, or compounds which decrease, increase and/or otherwise enhance the production of IGS3 polypeptides, which comprises
  • This invention provides methods of treating abnormal conditions related to both an excess of and insufficient amounts of IGS3 activity
  • IGS3 activity of IGS3 is in excess.
  • an inhibitor compound (antagonist) as heremabove described administered to a subject an inhibitor compound (antagonist) as heremabove described along with a pharmaceutically acceptable carrier in an amount effective to inhibit activation by blocking binding of ligands to the IGS3, or by inhibiting interaction with a RAMP polypeptide or a second signal, and thereby alleviating the abnormal condition
  • soluble forms of IGS3 polypeptides still capable of binding the ligand in competition with endogenous IGS3 may be administered Typical embodiments of such competitors comprise fragments of the IGS3 polypeptide
  • oligonucleotides which form triple helices with the gene, can be supplied See, for example, Lee et al , Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, Dervan et al, Science (1991 ) 251 1360
  • These oligomers can be administered per se or the relevant oligomers can be expressed in vivo Synthetic antisense or triplex oligonucleotides may comprise modified bases or modified backbones Examples of the latter include methylphosphonate, phosphorothioate or
  • Ribozymes are catalytically active RNAs that can be natural or synthetic (see for example Usman, N, et al , Curr Opin Struct Biol (1996) 6(4), 527- 33 )
  • Synthetic ribozymes can be designed to specifically cleave IGS3 mRNAs at selected positions thereby preventing translation of the IGS3 mRNAs into functional polypeptide
  • Ribozymes may be synthesized with a natural ⁇ bose phosphate backbone and natural bases, as normally found in RNA molecules Alternatively the ⁇ bosymes may be synthesized with non- natural backbones to provide protection from ⁇ bonuclease degradation, for example, 2'-0- methyl RNA, and may contain modified bases
  • a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above.
  • the retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest
  • producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo
  • Chapter 20 Gene Therapy and other Molecular Genetic-based Therapeutic
  • any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans
  • Peptides such as the soluble form of IGS3 polypeptides, and agonists and antagonist peptides or small molecules, may be formulated in combination with a suitable pharmaceutical carrier
  • a suitable pharmaceutical carrier Such formulations comprise a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable carrier or excipient Formulation should suit the mode of administration, and is well within the skill of the art
  • the invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention
  • Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds
  • systemic administration of the pharmaceutical compositions include injection, typically by intravenous injection
  • Other injection routes such as subcutaneous, intramuscular, or intrape ⁇ toneal
  • Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents
  • penetrants such as bile salts or fusidic acids or other detergents
  • oral administration may also be possible
  • Suitable dosages are in the range of 0 1-100 ⁇ g/kg of subject Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration For example, oral administration would be expected to require higher dosages than administration by intravenous injection Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art
  • Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above
  • cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector The cells are then introduced into the subject
  • Example 1a Homology PCR cloning of a genomic fragment encoding a novel G-protein coupled receptor (GPCR).
  • GPCR G-protein coupled receptors
  • F20 forward
  • R42, R43 reverse
  • PCR primers were designed in conserved areas of the neurotensm receptor gene family (Vita N et al [1993] Febs Lett 317 139-142, Vita N et al [1998] Eur J Pharmacol 360 265-272) at the boundary of mtracellular loop n°1 (11 ) with transmembrane domain 2 (TM2) and at the boundary of transmembrane domain 3 with mtracellular loop n°2 (TM3/I2) respectively
  • primers R42 and R43 were chosen in such a way that it was either not complementary to the corresponding position of the human NTR1 cDNA (R42) or to the corresponding position of both NTR1 and NTR2 cDNA (R43)
  • the primary PCR reaction was carried out in a 60 ⁇ l volume and contained 100 ng human genomic DNA (Clontech), 6 ⁇ l GeneAmpTM 10 x PCR buffer II (100mM T ⁇ s-HCI pH 8 3, 500 mM KCI Perkin Elmer), 3 6 ⁇ l 25 mM MgCI 2 0 36 ⁇ l dNTPs (25mM of each dNTP), 1 5 units
  • reaction tubes were heated at 95°C for 10 mm and then subjected to 35 cycles of denaturation (95°C, 1 mm), annealing (55°C, 2 mm) and extension (72°C, 3m ⁇ n) Finally reaction tubes were heated for 10 mm at 72°C
  • Example 1b Cloning of genomic DNA fragments containing the complete IGS3 coding sequence.
  • the complete coding sequence of IGS3 was obtained via hybridization screening of a human genomic library
  • a human genomic DNA library (Clontech #HL1067j), constructed in the lambda EMBL3 SP6 T7 phage vector was screened by hybridization using an IGS3 specific probe
  • This probe was derived from a 130 bp PCR fragment amplified from the HNT1355 plasmid (which contained an identical insert as HNT1370) using IGS3 specific primers IP11969 (SEQ ID NO 6) and IP12008 (SEQ ID NO 7) (Fig 1 )
  • the 130 bp fragment was purified from gel using the Qiaex-llTM purification kit (Qiagen) and radiolabelled via random primed incorporation of [ ⁇ - 32 P]dCTP to a specific activity of > 10 s cpm/ ⁇ g using the Prime-It II kitTM (Stratagene) according to the instructions provided by the supplier Aproximately 550,000 plaques were screened with the 130 bp probe according to
  • the IGS3 coding sequence was subcloned into a plasmid vector after PCR amplification PCR reactions were carried out on the isolated ⁇ -IGS3 1 , ⁇ -IGS3.3 and ⁇ -IGS3 5 phage DNA (500 ng) with the IP12936 (SEQ ID NO 8) and IP12937 (SEQ ID NO 9) ohgonucleotide primers using the ExpandTM High Fidelity PCR system (Boeh ⁇ nger) PCR reaction tubes were heated at 95°C for 2 mm and then subjected to 35 cycles of denaturation (95°C, 30 sec), annealing (58°C, 30 sec) and extension (72°C, 1 mm) There was a final elongation step at 72°C (10 mm) A ⁇ 1 ,200 bp PCR product was purified from gel and ligated into the pGEM-T vector The recombinant DNA was then used to transform E coli bacterial strain DH5 ⁇ F' This yield
  • the bacterial strain harboring plasmid HNT4971 (containing the 1GS3DNA sequence) was recloned after replatmg on LB agar plates containing 100 ⁇ g ampicillin/ml and deposited both in the Innogenetics N V strain list (ICCG4319) and at the "Centraalbureau voor Schimmelculturen (CBS)" in Baarn, The Netherlands (accession n° 102196) Plasmid DNA was prepared from the recloned isolate and the insert was resequenced and found to be identical to the IGS3DNA sequence

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US7510845B2 (en) 2000-05-04 2009-03-31 California Institute Of Technology Assay employing G protein-coupled receptor expressed in dorsal root ganglia
US20030092035A1 (en) 2000-05-04 2003-05-15 Anderson David J. Pain signaling molecules
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WO2002004641A1 (fr) * 2000-07-07 2002-01-17 Takeda Chemical Industries, Ltd. Nouvelle proteine receptrice couplee a la proteine g et son adn
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