EP1097169A1 - Recepteur secretagogue d'hormones de croissance chez la souris - Google Patents

Recepteur secretagogue d'hormones de croissance chez la souris

Info

Publication number
EP1097169A1
EP1097169A1 EP99933758A EP99933758A EP1097169A1 EP 1097169 A1 EP1097169 A1 EP 1097169A1 EP 99933758 A EP99933758 A EP 99933758A EP 99933758 A EP99933758 A EP 99933758A EP 1097169 A1 EP1097169 A1 EP 1097169A1
Authority
EP
European Patent Office
Prior art keywords
growth hormone
receptor
mouse
hormone secretagogue
nucleic acid
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Withdrawn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
EP99933758A
Other languages
German (de)
English (en)
Inventor
Roy G. Smith
Leonardus H. T. Van Der Ploeg
Andrew D. Howard
Hui Zheng
Karen Kulju Mckee
Michael Minghao Jiang
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.)
Merck and Co Inc
Original Assignee
Merck and Co Inc
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 Merck and Co Inc filed Critical Merck and Co Inc
Publication of EP1097169A1 publication Critical patent/EP1097169A1/fr
Withdrawn legal-status Critical Current

Links

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

Definitions

  • This invention relates to a newly identified receptor, the mouse growth hormone secretagogue receptor (mGHS-R), nucleic acids encoding this receptor; and to the use of a mGHS-R to identify growth hormone secretagogues and compounds that modulate mGHS-R function.
  • mGHS-R mouse growth hormone secretagogue receptor
  • GHSs Growth hormone secretagogues
  • GPC-R G protein-coupled receptor
  • GHRH growth hormone releasing hormone
  • SST somatostatin
  • GHS-R growth hormone secretagogue receptor
  • GHS-R growth hormone secretagogues
  • This invention relates to a novel receptor, mouse growth hormone secretagogue receptor (mGHS-R), which is free from receptor associated proteins.
  • mGHS-R mouse growth hormone secretagogue receptor
  • a further aspect of this invention is mGHS-R which is isolated or purified.
  • mGHS-Rs which are encoded by substantially the same nucleic acid sequence, but which have undergone changes in splicing or other RNA processing-derived modifications or mutagenesis induced changes, so that the expressed protein has a homologous, but different amino acid sequence from the native form.
  • These variant forms may have different and/or additional functions in animal physiology or in vitro in cell based assays.
  • Growth hormone secretagogue receptors are proteins containing various functional domains, including one or more domains which anchor the receptor in the cell membrane, and at least one ligand binding domain. As with many receptor proteins, it is possible to modify many of the amino acids, particularly those which are not found in the ligand binding domain, and still retain at least a percentage of the biological activity of the original receptor. Thus, this invention specifically includes modified functionally equivalent mGHS-Rs which have deleted, truncated, or mutated N-terminal portions. This invention also specifically includes modified functionally equivalent mGHS-Rs which contain modified and/or deletions in other domains, which are not accompanied by a loss of functional activity.
  • a further aspect of this invention are nucleic acids which encode a mouse growth hormone secretagogue receptor or a functional equivalent. These nucleic acids may be free from associated nucleic acids, or they may be isolated or purified. For most cloning purposes, cDNA is a preferred nucleic acid, but this invention specifically includes other forms of DNA as well as RNAs which encode a mGHS-R or a functional equivalent.
  • vectors which comprise nucleic acids encoding mGHS-R or a functional equivalent.
  • These vectors may be comprised of DNA or RNA; for most cloning purposes DNA vectors are preferred.
  • Typical vectors include plasmids, modified viruses, bacteriophage and cosmids, yeast artificial chromosomes, transposable elements and other forms of episomal or integrated DNA that can encode a mGHS-R. It is well within the skill of the ordinary artisan to determine an appropriate vector for a particular gene transfer or other use.
  • a further aspect of this invention are host cells which are transformed with a vector comprising a gene which encodes a mouse growth hormone secretagogue receptor or a functional equivalent.
  • the host cell may or may not naturally express a GHS-R on the cell membrane.
  • the host cells are able to express the mouse growth hormone secretagogue receptor or a functional equivalent on the cell membrane.
  • mammalian cell lines such as COS, HEK-293, CHO, HeLa, NS/0, CV-1, GC, GH3 or VERO cells are preferred host cells, but other cells and cell lines such as Xenopus oocytes or insect cells, may also be used.
  • Another aspect of this invention is a process for identifying nucleic acids encoding mouse growth hormone secretagogue related receptors comprising hybridizing a first nucleic acid encoding a mouse growth hormone secretagogue receptor with a second nucleic acid suspected of comprising nucleic acids encoding a growth hormone secretagogue receptor, wherein the hybridizing takes place under relaxed or moderate post hybridizational washing conditions; and identify areas of the second nucleic acid where hybridization occurred.
  • FIGURE 1 is the DNA sequence encoding the mouse GHS-R, 5' and 3' flanking regions and the intron; SEQ ID NO:l.
  • FIGURE 2 is the DNA sequence encoding the open reading frame (ORF) of the mouse GHS-R; SEQ ID NO:2.
  • FIGURE 3 is the deduced amino acid sequence of the mouse GHS- R; SEQ K> NO:3.
  • FIGURE 4 is an amino acid alignment of the mouse GHS-R with other GHS-R's from several species (human - SEQ ID NO:4, rat - SEQ ID NO: 5, and swine - SEQ ID NO:6).
  • Growth Hormone Secretagogue any compound or agent that directly or indirectly stimulates or increases the release of growth hormone in an animal.
  • Ligands any molecule which binds to the mGHS-R of this invention. These ligands can have either agonist, partial agonist, partial antagonist or antagonist activity. Free from receptor-associated proteins—the receptor protein is not in a mixture or solution with other membrane receptor proteins.
  • nucleic acid Free from associated nucleic acids—the nucleic acid is not covalently linked to DNA which it is naturally covalently linked in the organism's chromosome.
  • Isolated receptor the protein is not in a mixture or solution with any other proteins.
  • Isolated nucleic acid the nucleic acid is not in a mixture or solution with any other nucleic acid.
  • Functional equivalent a receptor which does not have the exact same amino acid sequence of a naturally occurring mouse growth hormone secretagogue receptor due to alternative splicing, deletions, mutations, or additions, but retains at least 1%, preferably 10%, and more preferably 25% of the biological activity of the naturally occurring receptor.
  • Such derivatives will have a significant homology with a natural mGHS-R and can be detected by reduced stringency hybridization with a DNA sequence obtained from a mGHS-R.
  • the nucleic acid encoding a functional equivalent has at least about 50% homology at the nucleotide level to a naturally occurring receptor nucleic acid.
  • the receptor is at least about 95% pure.
  • nucleic acid is at least about 95% pure. Standard or high stringency post hybridizational washing conditions - 6 X SSC at 55°C.
  • Moderate post hybridizational washing conditions —6 X SSC at 45°C.
  • Relaxed post hybridizational washing conditions — 6 X SSC at
  • the mouse isoform of the previously identified GHS-R was cloned from two genomic DNA libraries for the generation of a GHS-R knock-out mouse.
  • This isoform has been shown to be functionally activated by secretagogues such as growth hormone releasing peptide GHRP-6 and MK-0677 through expression studies of the complete and contiguous open reading frame of mGHS-R using the aequorin biolumenescence assay.
  • the proteins of this invention were found to have structural features which are typical of the 7-transmembrane domain (TM)- containing G-protein linked receptor superfamily (GPC-R' s or 7-TM receptors), including seven transmembrane regions, three intra- and extracellular loops, and the GPC-R protein signature sequence.
  • TM 7-transmembrane domain
  • GPC-R' s or 7-TM receptors G-protein linked receptor superfamily
  • mGHS-R as an additional member of the growth hormone secretagogue family of receptors, constitutes a new member of the GPC-R family of receptors. Note not all regions are required for functioning, and therefore this invention also comprises functional receptors which lack one or more non-essential domains.
  • the mGHS-Rs of this invention also share some sequence homology with previously cloned GPC-receptors including the rat and human neurotensin receptor (approximately 32 % identity) and the rat and human thyrotropin releasing hormone (TRH) receptor (approximately 29 % identity).
  • the mGHS-R and fragments are immunogenic.
  • another aspect of this invention is antibodies and antibody fragments which can bind to mGHS-R or a mGHS-R fragment. These antibodies may be monoclonal antibodies and produced using either hybridoma technology or recombinant methods. They may be used as part of assay systems or to deduce the function of a mGHS-R present on a cell membrane.
  • a further aspect of this invention are antisense oligonucleotides - nucleotides which can bind to mGHS-R nucleotides and modulate receptor function or expression.
  • a further aspect of this invention is a method of increasing the amount of mGHS-Rs on a cell membrane comprising, introducing into the cell a nucleic acid encoding a mGHS-R, and allowing expression of the mGHS-R.
  • a mGHS receptor preferably immobilized on a solid support, may be used diagnostically for the determination of the concentration of growth hormone secretagogues, or metabolites thereof, in physiological fluids, e.g. body fluids, including serum, and tissue extracts, as for example in patients who are undergoing therapy with a growth hormone secretagogue.
  • physiological fluids e.g. body fluids, including serum, and tissue extracts, as for example in patients who are undergoing therapy with a growth hormone secretagogue.
  • the administration of a mGHS receptor to a patient may also be employed for purposes of amplifying the net effect of a growth hormone secretagogue by providing increased downstream signaling following administration of the growth hormone secretagogue thereby diminishing the required dosage of growth hormone secretagogue; or diminishing the effect of an overdosage of a growth hormone secretagogue during therapy.
  • Yet a further aspect of the present invention is a method of identifying ligands comprising contacting the mGHS-R with a compound suspected of being a ligand specific for said receptor and determining whether binding occurs, binding constituting a positive indication of the presence of a ligand.
  • Ligands detected using assays described herein may be used in the treatment of conditions which occur when there is a shortage of growth hormone, such as observed in growth hormone deficient children, elderly patients with musculo skeletal impairment and those recovering from hip fracture, and osteoporosis.
  • Targeted disruption of the mouse GHS-R gene may also prove useful in elucidation of the mechanism of action and role of the growth hormone secretagogues in human and animal physiology.
  • the BAC clone was sequenced with ABI Prism BigDye terminator cycle sequencing ready reaction mix (P/N 4303149; PE Applied Biosystems, Foster City, CA) using l ⁇ g DNA/reaction, 5% DMSO, 100 ng primer - standard cycle sequencing. Reactions were run on an ABI Prism 377 DNA Sequencer with XL Upgrade (ABI Prism 377XL). DNA from the positive lambda clones was prepared from a liquid lysate of the E. coli strain XLBlue MRA minus. For DNA sequencing, 500 ng of DNA was used under the same conditions as given above.
  • GHS-R into an amino-terminal segment (encompassing the extracellular domain, TM-1 through TM-5, and the first two intra-and extra-cellular loops) and a carboxyl-terminal segment containing TM-6, TM-7, the third intra- and extra-cellular loops, and the intracellular domain.
  • the point of insertion and flanking DNA sequence are highly conserved between human, swine, rat and mouse.
  • Comparison of the complete ORF encoding the murine GHS-R type la protein sequence (Fig. 4) to rat, human and swine GHS-R homologs reveals a high degree of sequence identity (mouse vs. rat, 99.5%; mouse vs. human 95%; mouse vs. swine 94%).
  • Figs. 2 and 3 was assembled in the vector pcDNA-3 (Invitrogen) by overlapping PCR to remove the single intron present following nucleotide 790 of the ORF.
  • the Advantage HF PCR kit K 1909-1; Clonetech Laboratories, Inc, Palo Alto, CA
  • 94°C for lmin was used under the following conditions: 94°C for lmin;, then 25 cycles of the following: 94°C for 15 sec, 55°C for 15 sec, and 68°C for 3 min.
  • primers used were: primer 1- 5'GGG CCC GAA TTC GCC GCC ATG TGG AAC GCG ACG CCC AGC 3' (SEQ ID NO: 7, including EcoR I site, Kozak initation sequence, and translational start Met); primer 2- 5'G4C CAC CAC AG C AAG CAT CTT CAC TGT CTG3' (SEQ ID NO:8; nucleotides shown in italic type overlap exon 2); primer 3- 5'AAG ATG CTTG CT GTG GTG GTG TTT GCT TTC ATC3' (SEQ ID NO:9; nucleotides shown in italic type overlap exon 1); and primer 4- 5'AGT TTA GCG GCC GCT CAT GTA TTG ATG CTC GAC TTT GT3' (SEQ ID NO: 10, including Not I site and stop codon).
  • “Overlapping" PCR was performed.
  • the first PCR reactions were performed with primers 1 and 2 (exon 1) or 3 and 4 (exon 2).
  • the second PCR reactions were performed with primers 1 and 4 (ORF).
  • the second product was digested with EcoRI and Notl, agarose gel purified, ethanol precipitated, phenol extracted, and ligated into pcDNA3 with Ready-to-Go T4 Ligase (27-0361-01; Pharmacia, Piscataway, NJ), and transformed into SCSI cells (200231; Stratagene, La Jolla, CA).
  • DNA was isolated with Wizard Plus miniprep (A1460; Promega, Madison, WI) and 500ng was sequenced as above, but without DMSO.
  • mice GHS-R expression in the aequorin- expressing stable reporter cell line 293-AEQ17 was performed using a Luminoskan RT luminometer (Labsystems Inc., Gaithersburg, MD. 293-AEQ17 cells (8 x 105 cells plated 18 hr. before transfection in a T75 flask) were transfected with 22 ⁇ g of pcDNA-3 /mouse GHS-R plasmid DNA and 264 ⁇ g lipofectamine (Life Technologies).
  • the cells were harvested, washed once in ECB medium and resuspended to 500,000 cells/ml.

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  • Gastroenterology & Hepatology (AREA)
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  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Toxicology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Endocrinology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un récepteur sécrétagogue d'hormones de croissance chez la souris qui a été isolé, cloné et séquencé. Ce récepteur est caractéristique de la famille des récepteurs de la protéine G. Les récepteurs sécrétagogues d'hormones de croissance chez la souris peuvent être utilisés pour cribler et identifier des composés se liant au récepteur sécrétagogue d'hormones de croissance chez la souris. De tels composés peuvent être utilisés pour traiter des affections survenant dans des cas de déficit d'hormones de croissance, telles qu'elles peuvent être observées chez des enfants présentant un tel déficit, chez des patients âgés souffrant de déficience musculo-squelettique, ou chez des personnes convalescentes d'une fracture de la hanche ou de l'ostéoporose. Le dérèglement ciblé du gène du GHS-R peut contribuer efficacement à l'élucidation du mécanisme d'action et du rôle des sécrétagogues d'hormones de croissance dans la physiologie humaine ou animale.
EP99933758A 1998-07-10 1999-07-08 Recepteur secretagogue d'hormones de croissance chez la souris Withdrawn EP1097169A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US9236198P 1998-07-10 1998-07-10
US92361P 1998-07-10
PCT/US1999/015375 WO2000002918A1 (fr) 1998-07-10 1999-07-08 Recepteur secretagogue d'hormones de croissance chez la souris

Publications (1)

Publication Number Publication Date
EP1097169A1 true EP1097169A1 (fr) 2001-05-09

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EP99933758A Withdrawn EP1097169A1 (fr) 1998-07-10 1999-07-08 Recepteur secretagogue d'hormones de croissance chez la souris

Country Status (4)

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EP (1) EP1097169A1 (fr)
JP (1) JP2002520337A (fr)
CA (1) CA2335272A1 (fr)
WO (1) WO2000002918A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2399831T3 (es) 2004-12-24 2013-04-03 National Cerebral And Cardiovascular Center Nuevo polipéptido y su uso

Family Cites Families (1)

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Publication number Priority date Publication date Assignee Title
US6242199B1 (en) * 1995-12-13 2001-06-05 Merck & Co., Inc. Assays for growth hormone secretagogue receptors

Non-Patent Citations (1)

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Title
See references of WO0002918A1 *

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CA2335272A1 (fr) 2000-01-20
WO2000002918A1 (fr) 2000-01-20
JP2002520337A (ja) 2002-07-09

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