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WO1999015551A1 - Nouveau recepteur - Google Patents

Nouveau recepteur Download PDF

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Publication number
WO1999015551A1
WO1999015551A1 PCT/AU1998/000805 AU9800805W WO9915551A1 WO 1999015551 A1 WO1999015551 A1 WO 1999015551A1 AU 9800805 W AU9800805 W AU 9800805W WO 9915551 A1 WO9915551 A1 WO 9915551A1
Authority
WO
WIPO (PCT)
Prior art keywords
receptor
seqvience
leu
protein
seq
Prior art date
Application number
PCT/AU1998/000805
Other languages
English (en)
Inventor
Herbert Herzog
Original Assignee
Garvan Institute Of Medical Research
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 Garvan Institute Of Medical Research filed Critical Garvan Institute Of Medical Research
Priority to CA002271700A priority Critical patent/CA2271700A1/fr
Priority to JP51834899A priority patent/JP2001513653A/ja
Priority to AU93300/98A priority patent/AU737148B2/en
Priority to EP98946142A priority patent/EP0973800A4/fr
Publication of WO1999015551A1 publication Critical patent/WO1999015551A1/fr
Priority to US11/218,716 priority patent/US20060137029A1/en

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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
    • 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 isolated polynucleotide molecules which encode a novel seven-transmembrane G-protein-coupled receptor designated TSR32.
  • the present invention relates to the use of these molecules in the production of TSR32 receptors using recombinant technology and to methods of screening compounds for agonists and/or antagonists of the TSR32 receptor.
  • Proteins with seven-transmembrane (7TM) segments define a superfamily of receptors with a common stmcture comprising an extracellular N-terminus, three extramembranous loops on either side of the plasma membrane, and a cytoplasmic C-terminus.
  • the majority of such proteins f inction as cell-surface receptors for a variety of ligands, including small molecules, peptides, hormones, ions, and external sensory stimuli such as odorants (Watson, S. and Arkinstall, S., The G-protein Linked Receptor Facts Book, Academic Press, London, 1994).
  • secretin receptor family One family within the 7TM superfamily is known as the secretin receptor family and comprises member receptors with specificity for secretin, calcitonin, glucagon, glucagon-like peptide 1.
  • parathyroid hormone parathyroid-related peptide
  • corticotropin- releasing factor CRF
  • growth hormone-releasing hormone GHRH
  • gastric inhibitory polypeptide pituitary adenylate-cyclase-activating polypeptide (PACAP), vasoactive intestinal peptides (VIP) and insect diuretic hormone
  • TSR32 a further member receptor of the secretin receptor family.
  • This receptor designated TSR32
  • TSR32 appears to be expressed in a large variety of tissue types including heart, kidney, lung and brain with highest levels in the thyroid gland. Since agonists and/or antagonists for this receptor may have commercial value, for example as regulators of important thyroid functions in growth, development and metabolic activity, the ability to produce TSR32 receptors by recombinant DNA technology would be advantageous. Disclosure of the Invention:-
  • the present invention provides an isolated polynucleotide molecule encoding a G-protein-coupled receptor, designated TSR32, which is characterised by the N-terminal amino acid sequence: MTPQSLLQTT (SEQ ID NO: 1), or a functionally equivalent fragment of said receptor.
  • the polynucleotide molecule encodes a human TSR32 receptor of about 693 amino acids.
  • the isolated polynucleotide molecule encodes a human TSR32 receptor comprising an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 2 or a functionally equivalent fragment thereof.
  • the nucleotide sequence of a polynucleotide molecule in accordance with the first aspect may comprise a nucleotide sequence substantially corresponding to or, showing at least 75% (preferably, at least 90% or. even more preferably, at least 95%) sequence identity to that shown as SEQ ID NO: 3 or SEQ ID NO: 4 or any portion thereof encoding a functionally equivalent TSR32 receptor fragment.
  • the polynucleotide molecvile may be incorporated into plasmids or expression vectors (including viral vectors), which may then be introduced into S iitable host cells (e.g. bacterial, yeast, insect and mammalian host cells). Such host cells may be used to express the TSR32 receptor encoded by the isolated polynucleotide molecule.
  • S iitable host cells e.g. bacterial, yeast, insect and mammalian host cells.
  • the present invention provides a host cell transformed with the polynucleotide molecule of the first aspect.
  • the present invention provides a method of producing TSR32 receptors or functionally equivalent fragments thereof, comprising culturing the host cell of the second aspect under conditions enabling the expression of the polynucleotide molecule and, optionally, recovering the TSR32 receptor or fragments thereof.
  • the host cell is mammalian or of insect origin.
  • the cell is mammalian, it is presently preferred that it be a Chinese hamster ovary (CHO) cell or a human embryonic kidney (HEK) 293 cell.
  • the cell is of insect origin, it is presently preferred that it be an insect Sf9 cell.
  • the TSR32 receptor or fragments thereof are expressed onto the surface of the host cells.
  • the polynucleotide molecules of the present invention represent a new subfamily of the secretin receptor family which may be of interest both clinically and commercially as it is expressed in many tissue types and may therefore be involved in a wide variety of systems. By using the polynucleotide molecules of the present invention it is possible to obtain TSR32 receptor protein or functionally equivalent fragments thereof in a substantially pure form.
  • the present invention provides a G- protein-coupled receptor, designated TSR32, which is characterised by the N- terminal amino acid sequence:
  • the purified TSR32 receptor has an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 2.
  • the present invention provides an antibody or antibody fragment capable of specifically binding to a TSR32 receptor according to the fourth aspect.
  • the antibody may be monoclonal or polyclonal, however, it is presently preferred that the antibody is a monoclonal antibody.
  • Suitable antibody fragments include Fab, (F(ab') 2 and scFv.
  • the present invention provides a non-human animal transformed with a polynucleotide molecule according to the first aspect of the present invention.
  • the present invention provides a method for detecting agonist and/or antagonist compounds of the TSR32 receptor, comprising contacting a TSR32 receptor, functionally equivalent fragment thereof or a host cell transformed with and expressing the polynucleotide molecule of the first aspect, with a test compound under conditions enabling the activation of the TSR32 receptor or functionally equivalent fragment thereof, and detecting an increase or decrease in the activity of the TSR32 receptor or functionally equivalent fragment thereof.
  • an increase or decrease in activity of the receptor or functionally eq iivalent fragment thereof may be detected by measuring changes in CAMP production, CA 2+ levels or IP3 turnover after activating the receptor or fragment thereof with specific agonists or antagonists.
  • the present invention provides an oligonucleotide or polynucleotide probe comprising a icleotide seqLience of 10 or more nucleotides, the probe comprising a nucleotide sequence such that the probe specifically hybridises to the polynucleotide molecule of the first aspect under high stringency conditions (Sambrook et ah, Molecular Cloning: a laboratory manual, Second Edition, Cold Spring Harbor Laboratory Press).
  • the probe is labelled.
  • the present invention provides an antisense polynucleotide molecule comprising a nucleotide sequence capable of specifically hybridising to an mRNA molecule which encodes a TSR32 receptor so as to prevent translation of the mRNA molecule.
  • Such antisense polynucleotide molecules may include a ribozyme region to catalytically inactivate mRNA to which it is hybridized.
  • the polynucleotide molecule of the first aspect of the invention may be a dominant negative mutant which encodes a gene product ca ising an altered phenotype by, for example, reducing or eliminating the activity of endogenous TSR32 receptors.
  • substantially corresponding as vised herein in relation to amino acid sequences is intended to encompass minor variations in the amino acid sequences which do not result in a decrease in the biological activity of the TSR32 receptor. These variations may include conservative amino acids substitutions.
  • the substitutions envisaged are:-
  • G, A, V, I, L, M D, E; N. Q; S, T; K, R, H; F. Y, W, H; and
  • nucleotide sequences are intended to encompass minor variations in the nucleotide seqLience which due to degeneracy in the DNA code do not result in a change in the encoded protein. Further, this term is intended to encompass other minor variations in the sequence which may be reqLiired to enhance expression in a particular system but in which the variations do not result in a decrease and biological activity of the encoded protein.
  • fragments of the TSR32 receptor that exhibit binding specificity and activity that is substantially equivalent to the full length receptor from which it/they is/are derived.
  • the terms "comprise”, “comprises” and “comprising” as used throughout the specification are intended to refer to the inclusion of a stated step, component or feature or group of steps, components or features with or without the inclusion of a further step, component or feature or group of steps, components or features.
  • Figure 1 provides the nucleotide sequence of a cDNA encoding the human TSR32 receptor and includes the predicted amino acid sequence.
  • Figure 2 provides a sequence alignment of the amino acid sequences of the human TSR32 receptor and the human epididymis-specific HE6 receptor (Osterhoff, C, DNA and Cell Biology, Vol. 16 No. 4. 1997).
  • Glucagon-like peptide receptor GLPl
  • Library DNA from human hypothalamus and heart yielded fragments of the expected size. These fragments were subcloned and sequenced. One fragment, 456bp long, revealed a novel 7TM receptor sequence distantly related to the secretin receptor family. Screening a human heart cDNA library (Stratagene) using this fragment under high stringency conditions identified two positive hybridising clones, 1 and 2.9kb in size, respectively.
  • the larger clone contains an open reading frame of 2079 nucleotides encoding a 693 amino acid long protein (designated TSR32).
  • TSR32 693 amino acid long protein
  • the nucleotide and pvitative amino acid seqvience is provided at Figure 1.
  • EMR1 (27% identity), the insect DHR (26% identity) and the rat latrophilin- related protein 1 precursor (35% identity) all of which share a large extracellular domain with numerous putative O- and N-glycosylation sites. Except for DHR, in all of the receptors there is a cysteine motif preceding the first transmembrane domain. Further, in contrast to the CD97 and EMR1 receptors, TSR32 does not contain any EGF-domains or calcium binding sites in the extracellular domain.
  • Northern analysis indicates a low level of expression of the receptor mRNA in a large variety of tissues including kidney, lung, cerebellum and heart. The highest level of mRNA however, was found in the thyroid gland. This expression pattern of the receptor mRNA indicates that TSR32 has important functions in metabolic regulations throughout the body via the thyroid gland.
  • TSR32 The gene for TSR32 has been mapped to human chromosome 16q31 by in situ hybridisation and this localisation was confirmed by radiation hybrid analysis.
  • An avitosomal recessive disorder (Bardet Biedl Syndrome; Kwitek- Black, A.E. et al, Nature Genetics 5 : 392-396, 1993) has also been linked to this region.
  • This syndrome include obesity, retinal degeneration, hypogonadism and mental retardation.
  • Organism Homo sapiens
  • Organism Homo sapiens
  • Ser Pro Gin Asn lie Ser Leu Pro Ser Ala Ala Ser Phe Thr Phe Ser 145 150 155 160
  • Glu Glu Gin Ser Glu lie Met Glu Tyr Ser Val Leu Leu Pro Arg Thr 260 265 270
  • Val Val Gin lie Leu Arg Leu Arg Pro His Thr Gin Lys Trp Ser His 595 600 605
  • Trp Tyr Trp Ser Met Arg Leu Gin Ala Arg Gly Gly Pro Ser Pro Leu 660 665 670
  • Ser Ser Ser Ser Ser Arg lie SEQ ID NO: 3
  • Organism Homo sapiens
  • Organism Homo sapiens

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Toxicology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Endocrinology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne des molécules de polynucléotides isolées qui codent pour un nouveau récepteur couplé aux protéines G (dénommé TSR32). On peut utiliser ces molécules pour exprimer ledit récepteur dans des cellules pouvant servir à cribler des composés afin de détecter une activité agoniste ou antagoniste.
PCT/AU1998/000805 1997-09-24 1998-09-24 Nouveau recepteur WO1999015551A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
CA002271700A CA2271700A1 (fr) 1997-09-24 1998-09-24 Recepteur couple aux proteines g, tsr32
JP51834899A JP2001513653A (ja) 1997-09-24 1998-09-24 新規な受容体
AU93300/98A AU737148B2 (en) 1997-09-24 1998-09-24 Novel receptor
EP98946142A EP0973800A4 (fr) 1997-09-24 1998-09-24 Nouveau recepteur
US11/218,716 US20060137029A1 (en) 1997-09-24 2005-09-06 Novel G protein-coupled receptor encoding gene and diagnostic uses therefor

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPO9386A AUPO938697A0 (en) 1997-09-24 1997-09-24 Novel receptor
AUPO9386 1997-09-24

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US09308696 A-371-Of-International 1999-06-11
US10/073,054 Continuation-In-Part US20030167485A1 (en) 1997-09-24 2002-02-12 Novel G protein-coupled receptor encoding gene and diagnostic uses therefor

Publications (1)

Publication Number Publication Date
WO1999015551A1 true WO1999015551A1 (fr) 1999-04-01

Family

ID=3803668

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU1998/000805 WO1999015551A1 (fr) 1997-09-24 1998-09-24 Nouveau recepteur

Country Status (5)

Country Link
EP (1) EP0973800A4 (fr)
JP (1) JP2001513653A (fr)
AU (1) AUPO938697A0 (fr)
CA (1) CA2271700A1 (fr)
WO (1) WO1999015551A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000020590A3 (fr) * 1998-10-06 2000-07-20 Incyte Pharma Inc Proteines de recepteurs couples a des proteines g
WO2000034473A3 (fr) * 1998-12-10 2000-08-17 Zymogenetics Inc Domaine transmembranaire 7 zsig56
WO2001044281A3 (fr) * 1999-12-15 2001-10-11 Zymogenetics Inc Recepteur humain de type secretine
WO2001025430A3 (fr) * 1999-10-01 2001-10-18 Lexicon Genetics Inc Nouvelles proteines membranaires humaines
WO2001083558A1 (fr) * 2000-05-03 2001-11-08 Astrazeneca Ab Agonistes/antagonistes du recepteur gpr56 utilises comme agents de regulation de l'appetit
WO2002034781A1 (fr) * 2000-10-20 2002-05-02 Solvay Pharmaceuticals Gmbh Recepteur de diadenosine tetraphosphate (ap4ar) couple a la proteine g humaine
US7029874B2 (en) * 1998-03-17 2006-04-18 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7279553B2 (en) * 1998-05-13 2007-10-09 Genentech, Inc. PRO1083 polypeptides

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
LOK S., ET AL.: "THE HUMAN GLUCAGON RECEPTOR ENCODING GENE: STRUCTURE, CDNA SEQUENCE AND CHROMOSOMAL LOCALIZATION.", GENE., ELSEVIER, AMSTERDAM., NL, vol. 140., no. 02., 1 January 1994 (1994-01-01), NL, pages 203 - 209., XP002921264, ISSN: 0378-1119, DOI: 10.1016/0378-1119(94)90545-2 *
MACNEIL D J, ET AL.: "CLONING AND EXPRESSION OF A HUMAN GLUCAGON RECEPTOR", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 198, no. 01, 14 January 1994 (1994-01-14), US, pages 328 - 334, XP002947733, ISSN: 0006-291X, DOI: 10.1006/bbrc.1994.1046 *
MAYER H, ET AL.: "ISOLATION MOLECULAR CHARACTERIZATION AND TISSUE-SPECIFIC EXPRESSIONOF A NOVEL PUTATIVE G PROTEIN-COUPLED RECEPTOR", BIOCHIMICA ET BIOPHYSICA ACTA., ELSEVIER, NL, vol. 1395, 1 January 1998 (1998-01-01), NL, pages 301 - 308, XP002922512, ISSN: 0006-3002 *
OSTERHOFF C, IVELL R, KIRCHHOFF C: "CLONING OF A HUMAN EPIDIDYMIS-SPECIFIC MRNA, HE6, ENCODING A NOVEL MEMBER OF THE SEVEN TRASMEMBRANE- DOMAIN RECEPTOR SUPERFAMILY", DNA AND CELL BIOLOGY, MARY ANN LIEBERT, NEW YORK, NY, US, vol. 16, no. 04, 1 January 1997 (1997-01-01), US, pages 379 - 389, XP002925525, ISSN: 1044-5498 *
See also references of EP0973800A4 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7029874B2 (en) * 1998-03-17 2006-04-18 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7279553B2 (en) * 1998-05-13 2007-10-09 Genentech, Inc. PRO1083 polypeptides
WO2000020590A3 (fr) * 1998-10-06 2000-07-20 Incyte Pharma Inc Proteines de recepteurs couples a des proteines g
WO2000034473A3 (fr) * 1998-12-10 2000-08-17 Zymogenetics Inc Domaine transmembranaire 7 zsig56
WO2001025430A3 (fr) * 1999-10-01 2001-10-18 Lexicon Genetics Inc Nouvelles proteines membranaires humaines
WO2001044281A3 (fr) * 1999-12-15 2001-10-11 Zymogenetics Inc Recepteur humain de type secretine
WO2001083558A1 (fr) * 2000-05-03 2001-11-08 Astrazeneca Ab Agonistes/antagonistes du recepteur gpr56 utilises comme agents de regulation de l'appetit
WO2002034781A1 (fr) * 2000-10-20 2002-05-02 Solvay Pharmaceuticals Gmbh Recepteur de diadenosine tetraphosphate (ap4ar) couple a la proteine g humaine

Also Published As

Publication number Publication date
EP0973800A4 (fr) 2001-10-04
JP2001513653A (ja) 2001-09-04
EP0973800A1 (fr) 2000-01-26
CA2271700A1 (fr) 1999-04-01
AUPO938697A0 (en) 1997-10-16

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