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WO2000077047A1 - Recepteur de l'hormone de liberation de la gonadotropine du chien - Google Patents

Recepteur de l'hormone de liberation de la gonadotropine du chien Download PDF

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Publication number
WO2000077047A1
WO2000077047A1 PCT/US2000/015496 US0015496W WO0077047A1 WO 2000077047 A1 WO2000077047 A1 WO 2000077047A1 US 0015496 W US0015496 W US 0015496W WO 0077047 A1 WO0077047 A1 WO 0077047A1
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Prior art keywords
dog
receptor
gnrh receptor
cells
dog gnrh
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PCT/US2000/015496
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English (en)
Inventor
Jisong Cui
Jane-Ling Lo
George R. Mount
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Merck and Co Inc
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Merck and Co Inc
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Publication of WO2000077047A1 publication Critical patent/WO2000077047A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/721—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • nucleic acids which encode the receptor or a functional equivalent are nucleic acids which encode the 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 the receptor or a functional equivalent.
  • a further aspect of this invention relates to vectors which comprise nucleic acids encoding the dog GnRH receptor or a functional equivalent. These vectors may be comprised of DNA or RNA; however, for most cloning purposes, DNA vectors are preferred.
  • mammalian cell lines such as COS, HEK-293, CHO, HeLa, NS/O, 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.
  • FIGURE 2 is the DNA of the dog GnRH receptor that encodes the entire open reading frame, from Met Initiation codon to the Stop codon as set forth in SEQ ID no. 9.
  • FIGURE 3 is the amino acid sequence of the dog GnRH receptor encoded by the DNA in FIGURE 2, as set forth in SEQ ID no. 10.
  • FIGURE 4 is a graph demonstrating the expression of the dog GnRH receptor in transfected CHO-Kl cells using the membrane binding assay.
  • This invention relates to the cloning of the dog gonadotropin- releasing hormone (GnRH) receptor, and also to mutant or polymorphic forms of the receptor and recombinant nucleic acids encoding the same.
  • the invention also relates to genetically engineered host cells which express the receptor as well as antibodies against the receptor and polypeptides thereof.
  • the invention also relates to uses of the receptor, recombinant nucleic acids and recombinant host cells in drug screening and development, diagnosis and therapeutic applications.
  • an "agonist” is a compound or molecule that interacts with and activates a polypeptide of the receptor.
  • polynucleotide is a nucleic acid of more than one nucleotide.
  • a polynucleotide can be made up of multiple poly- nucleotide units that are referred to by description of the unit.
  • a polynucleotide can comprise within its bounds a polynucleo- tide(s) having a coding sequence(s), a polynucleotide (s) that is a regulatory region(s) and/or other polynucleotide units commonly used in the art.
  • an "expression vector” is a polynucleotide having regulatory regions operably linked to a coding region such that, when in a host cell, the vector can direct the expression of the coding sequence.
  • the use of expression vectors is well known in the art. Expression vectors can be used in a variety of host cells and, therefore, the regulatory regions are preferably chosen as appropriate for the particular host cell.
  • a “regulatory region” is a polynucleotide that can promote or enhance the initiation or termination of transcription or translation of a coding sequence.
  • a regulatory region includes a sequence that is recognized by the RNA polymerase, ribosome, or associated transcription or translation initiation or termination factors of a host cell. Regulatory regions that direct the initiation of transcription or translation can direct constitutive or inducible expression of a coding sequence.
  • the isolated nucleic acid molecule of the present invention can include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which can be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide.
  • the isolated nucleic acid molecule of the present invention can also include a ribonucleic acid molecule (RNA).
  • RNA ribonucleic acid molecule
  • the present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification.
  • Polynucleotides of this invention contain full length or partial length sequences of the receptor gene.
  • Polynucleotides of this invention can be single or double stranded. If single stranded, the polynucleotides can be a coding, "sense,” strand or a complementary, "antisense,” strand.
  • Antisense strands can be useful as modulators of the receptor by interacting with RNA encoding the receptor. Antisense strands are preferably less than full length strands having sequences unique or highly specific for RNA encoding the receptor.
  • the polynucleotides can include deoxyribonucleotides, ribonucleotides or mixtures of both.
  • the polynucleotides can be produced by cells, in cell-free biochemical reactions or through chemical synthesis.
  • Non-natural or modified nucleotides including inosine, methyl-cytosine, deaza-guanosine, etc., can be present.
  • Natural phosphodiester internucleotide linkages can be appropriate.
  • polynucleotides can have non-natural linkages between the nucleotides.
  • Non-natural linkages are well known in the art and include, without limitation, methylphosphonates, phosphorothioates, phosphorodithionates, phosphoroamidites and phosphate ester linkages.
  • Dephospho-linkages are also known, as bridges between nucleotides. Examples of these include siloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, and thioether bridges.
  • Plastic DNA having, for example, N-vinyl, methacryloxytethyl, methacrylamide or ethyleneimine internucleotide linkages, can be used.
  • PNA Peptide Nucleic Acid
  • PNA is also useful and resists degradation by nucleases. These linkages can be mixed in a polynucleotide.
  • nucleic acids claimed herein can be present in whole cells or in cell lysates or in a partially purified or substantially purified form.
  • a polynucleotide is considered purified when it is purified away from environmental contaminants.
  • a polynucleotide isolated from cells is considered to be substantially purified when purified from cellular components by standard methods while a chemically synthesized nucleic acid sequence is considered to be substantially purified when purified from its chemical precursors.
  • this invention is also directed to those DNA sequences encode RNA comprising alternative codons which code for the eventual translation of the identical amino acid, as shown below:
  • the present invention discloses codon redundancy which can result in differing DNA molecules expressing an identical protein.
  • a sequence bearing one or more replaced codons will be defined as a degenerate variation.
  • mutations either in the DNA sequence or the translated protein which do not substantially alter the ultimate physical properties of the expressed protein. For example, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine may not cause a change in functionality of the polypeptide.
  • DNA sequences coding for a peptide can be altered so as to code for a peptide having properties that are different than those of the naturally occurring peptide.
  • Methods of altering the DNA sequences include but are not limited to site directed mutagenesis. Examples of altered properties include but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand.
  • a "biologically active equivalent” or “functional derivative” of a wild-type dog GnRH receptor possesses a biological activity that is substantially similar to the biological activity of the wild type dog receptor.
  • the term “functional derivative” is intended to include the “fragments,” “mutants,” “variants,” “degenerate variants,” “analogs” and “homologues” or to “chemical derivatives” of the wild type dog receptor.
  • fragment is meant to refer to any polypeptide subset of wild-type dog receptor.
  • mutant is meant to refer to a molecule that may be substantially similar to the wild-type form but possesses distinguishing biological characteristics.
  • Such altered characteristics include but are in no way limited to altered substrate binding, altered substrate affinity and altered sensitivity to chemical compounds affecting biological activity of the dog receptor or dog receptor functional derivative.
  • variant is meant to refer to a molecule substantially similar in structure and function to either the entire wild- type protein or to a fragment thereof.
  • a molecule is "substantially similar” to a wild-type dog receptor if both molecules have substantially similar structures or if both molecules possess similar biological activity. Therefore, if the two molecules possess substantially similar activity, they are considered to be variants even if the structure of one of the molecules is not found in the other or even if the two amino acid sequences are not identical.
  • analog refers to a molecule substantially similar in function to either the full-length dog receptor or to a biologically active fragment thereof.
  • a protein or fragment thereof is considered purified or isolated when it is obtained at a concentration at least about five-fold to ten-fold higher than that found in nature
  • a protein or fragment thereof is considered substantially pure if it is obtained at a concentration of at least about 100-fold higher than that found in nature
  • a protein or fragment thereof is considered essentially pure if it is obtained at a concentration of at least about 1000-fold higher than that found m nature
  • Probes can be labeled by any number of ways known in the art including isotopes, enzymes, substrates, chemilummescent, electrochemilummescent, biotm and fret pairs among many others
  • a probe so labeled can generate a detectable signal directly (e.g., isotopes), or upon hybridization (fret pairs), or indirectly after a chemical (e g , luminescence) or biochemical reaction (e g., enzyme-substrate) or after binding a strepavidin linked moiety that can generate a detectable signal direcl
  • probes and the generation of detectable signals are well known techniques in the art and include Polymerase Chain Reaction and Reverse Transc ⁇ ptase Polymerase Cham Reaction (See e.g., PCR Primer, edited by C W Dieffenbach and G S Dveksler, (1995) Cold Spring Harbor Laboratory Press.), Strand Displacement Amplification, Self-Sustained Sequence Reaction, and any other amplification known to one of skill in the art that uses primers
  • the present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification. Therefore, the present invention also relates to methods of expressing the dog GnRH receptor and biological equivalents disclosed herein, assays employing these recombinantly expressed gene products, cells expressing these gene products, and modulators, agonistic and/or antagonistic compounds identified through the use of assays utilizing these recombinant forms, including, but not limited to, one or more compounds or molecules that act through direct contact with the receptor, particularly with the ligand binding domain, or through direct or indirect contact with a ligand which either interacts with the receptor or with the transcription or translation of GnRH, thereby modulating GnRH expression.
  • a variety of expression vectors can be used to express the dog GnRH receptor in host cells.
  • Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned DNA and the translation of their mRNAs in an appropriate host.
  • Such vectors can be used to express eukaryotic DNA in a variety of hosts such as bacteria, bluegreen algae, plant cells, insect cells and animal cells. Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells.
  • An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters.
  • a promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis.
  • a strong promoter is one which causes mRNAs to be initiated at high frequency.
  • Expression vectors can include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
  • mammalian expression vectors which can be suitable for the dog GnRH receptor expression, include but are not limited to, pcDNA3.1 (Invitrogen), pIRESl/ieo (Clontech), pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Biolabs), pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO- pSV2-neo (ATCC 37593) pBPV- 1(8-2) (ATCC 37110), pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC
  • a variety of bacterial expression vectors can be used to express the dog GnRH receptor in bacterial cells.
  • Commercially available bacterial expression vectors which are suitable for recombinant dog GnRH receptor expression include, but are not limited to pQE (Qiagen), pETlla (Novagen), lambda gtll (Invitrogen), and pKK223-3 (Pharmacia).
  • a variety of fungal cell expression vectors can be used to express the dog GnRH receptor in fungal cells.
  • Commercially available fungal cell expression vectors which are suitable for the dog GnRH receptor expression include but are not limited to pYES2 (Invitrogen) and Pichia expression vector (Invitrogen).
  • insect cell expression vectors can be used to express recombinant receptor in insect cells.
  • Commercially available insect cell expression vectors which are suitable for the dog GnRH receptor include but are not limited to pBlueBacIII and pBlueBacHis2 (Invitrogen), and pAcG2T (Pharmingen).
  • An expression vector containing DNA encoding the dog GnRH receptor can be used for expression of the dog GnRH receptor in a recombinant host cell.
  • Recombinant host cells can be prokaryotic or eukaryotic, including but not limited to bacteria such as E. coli, fungal cells such as yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila- and silkworm-derived cell lines.
  • L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), Saos-2 (ATCC HTB-85), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-Kl (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171) and CPAE (ATCC CCL 209).
  • the expression vector can be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, protoplast fusion, and electroporation.
  • the expression vector-containing cells are individually analyzed to determine whether they produce the dog GnRH receptor. Identification of the dog GnRH receptor expressing cells can be done by several means, including but not limited to immunological reactivity with anti-dog GnRH antibodies, labeled ligand binding and the presence of host cell-associated GnRH activity
  • the cloned dog GnRH receptor cDNA obtained through the methods described herein can be recombinantly expressed by molecular cloning into an expression vector (such as pcDNA3 1, pIRESlzieo, pQE, pBlueBacH ⁇ s2 and pLITMUS28) containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant human HG38 Techniques for such manipulations can be found described in Sambrook, et al , supra , and are well known and easily available to the one of ordinary skill in the art
  • RNA from dog pituitaries was prepared using the TRIzol reagents (Promega, Madison, WI) following the manufacturer's instruction Typically, 0 5 mg of total RNA was isolated from 350 mg wet weight of pituitary tissue Poly (A) RNA was isolated from total RNA by column chromatography on oligo (dT) cellulose (Promega, Madison, WI) The yield of poly (A) mRNA from total RNA was usually 0.5%.
  • First-strand cDNA was synthesized from poly (A) + mRNA using Superscript II reverse transc ⁇ ptase (GIBCO-BRL, Grand Island, NY) as per the manufacture's instruction One-tenth of the volume was used for each RT-PCR reaction.
  • Cloning of the Dog GnRH Receptor cDNA The steps for cloning the dog GnRH receptor cDNA included: (1) isolation of the genomic clones of the dog GnRH receptor; (2) PCR amplifying partial coding sequences from the genomic clones by degenerate primers; and (3) cloning of the entire open reading frame (ORF) of the dog receptor by RT-PCR from the dog pituitary mRNA.
  • ORF open reading frame
  • the genomic clones that contained the dog GnRH receptor were pulled out from a dog genomic library (Stratagene, La Jolla, CA) under low stringency hybridization conditions. Approximately 10 6 phage plaques of a dog genomic library in Lambda DASH were plated on the E. coli strain XLl-Blue MRA (Stratagene, La Jolla, CA). The plaques were transferred to Nitrocellulose membranes (Schleicher & Schuell, Keene, NH), denatured, neutralized, and screened with a 1 kb cDNA of the human GnRH receptor (Kakar et al., 1992, Biochem. Biophys. Res. Commun. 189, 289-295).
  • the membranes were incubated at 42°C in prehybridization solution (40% formamide, 1 x Denhardts, 6 x SSC, 0.5% SDS, 100 ⁇ g/ml salmon sperm DNA) for 3 hours followed by overnight incubation in hybridization solution (40% formamide, 1 x Denhardts, 6 x SSC, 100 ⁇ g/ml salmon sperm DNA) with 1 x 10 6 cpm/ml of [ 32 P]-labeled probe.
  • the probe was labeled with [ 32 P]-dCTP using a random priming kit (Amersham, Arlington Heights, IL).
  • the membranes were first washed three times with 2 x SSC, 0.1% SDS at room temperature, and finally washed once with 0.5 x SSC, 0.1% SDS at 50°C for 30 minutes.
  • a single positive clone was isolated following three rounds of plaque purification.
  • Phage containing the dog GnRH receptor was eluted from plate plaques with 1 x lambda buffer (0.1 M NaCl, 10 mM MgSO 4 7H 2 O, 35 mM Tris-HCl, PH7.5) following overnight growth of approximately 10 10 pfu/150 mm dish. After a 10 minute centrifugation at 10,000 x g to remove debris, the phage solution was treated with 1 mg/ml RNAse A and DNAse I for 30 minutes at 24°C, followed by precipitation with 20% PEG (8000)/2 M NaCl for two hours on ice, and collection by centrifugation at 10,000 x g for 20 minutes.
  • 1 x lambda buffer 0.1 M NaCl, 10 mM MgSO 4 7H 2 O, 35 mM Tris-HCl, PH7.5
  • Phage DNA was isolated by incubation in 0.1% SDS, 30 mM EDTA, 50 mg/ml proteinase K for one hour at 68°C, with subsequent phenol (three times) and chloroform (twice) extraction before isopropanol precipitation overnight.
  • the inserts in lambda DASH were individually sub-cloned into the plasmid vector pBlueSc ⁇ pt (GIBCO- BRL. Grand Island, NY). 2 ⁇ g of phage DNA was heated to 65°C for 10 minutes, then digested with 10 units of Sail at 37°C for 4 hours. A fragment of 10 kb (containing exon 1) or 12 kb (containing exon 3) was subcloned into vector pBlueScript.
  • the ORF of human GnRH receptor spins three exons (1-3) on the genome.
  • the exon 1 contains the translational initiator and the exon 3 contains the stop codon (Fan et al., 1994, Molecular and Cellular Endocrinology. 103, R1-R6). Since the gene organization across species is well conserved, it was assumed that the dog GnRH receptor is also contained in three exons.
  • Degenerate primers were designed based on the sequences of cloned GnRH receptors from various mammalian species.
  • the PCR was performed on the above phage clones using PCR kit (GIBCO-BRL, Grand Island, NY) under the condition of 94°C for 80 seconds, 50°C for 1 minute, and 72°C for 2 minutes (30 cycles) with the final extension at 72°C for 10 minutes.
  • PCR products of partial exon 1 by primers of JCl-1 + JC3, Figure 1 and exon 3 (by primers JC5 + JC8, Figure 1) were obtained.
  • the PCR products were extracted by equal volume of phenol/chloroform, followed by ethanol precipitation.
  • the purified DNA fragments were individually ligated into the plasmid vector pCR2.1 (Invitrogen Corporation, San Diego, CA) by T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN), and transformed into the DH5 ⁇ cells (GIBCO-BRL, Grand Island, NY). Plasmid DNA was prepared by Qiagen kit (Qiagen Inc., Valencia, CA). The cloned cDNAs were sequenced (ACGT, Inc., Northbrook, Illinois) at both strands. Based on these sequences, primers Dog 1 and Dog 3 ( Figure 1) were designed and used to determine the 5' and 3' sequences of the dog GnRH receptor from these genomic clones.
  • the purified DNA fragment was directly ligated into the plasmid vector pCR2.1 (Invitrogen Corporation, San Diego, CA) by T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN), and transformed into the DH5 ⁇ cells (GIBCO-BRL, Grand Island, NY).
  • Plasmid DNA was prepared by Qiagen kit (Qiagen Inc., Valencia, CA). The DNA of the dog GnRH receptor was sequenced (ACGT, Inc., Northbrook, Illinois) at both strands. Four clones that were derived from independent PCRs had identical sequences ( Figure 2).
  • the dog receptor contains 327 amino acids (Figure 3), one residue shorter than that of the human receptor. Surprisingly, the dog receptor lacks an asparagine equivalent to the third residue of the human receptor. The asparagine residue at this position is well conserved in all other known GnRH receptors of mammalian species.
  • the dog receptor DNA has 91% homology with the human GnRH receptor, and its protein has 92.6% identity and 96.6% similarity with the human receptor.
  • the dog GnRH receptor has the characteristic of the G-protein coupled receptors with seven putative tramsmembrance domains. The sites for glycosylation and for disulfide bond formation are well conserved compared with other mammalian GnRH receptors. Similar to the human receptor, the dog GnRH receptor also lacks a C-terminal cytoplamic tail.
  • the coding cDNA of the dog GnRH receptor was subcloned into a polycistronic expression vector pIRESl. ⁇ ,eo (Clontech Laboratories, Palo Alto, CA) to generate cells stably expressing the receptor.
  • the plasmid DNA of pIRESl./ieo-dog-GnRHR was transfected into CHO-Kl cells by lipofectin (GIBCO-BRL, Grand Island, NY) following the manufacturer's instruction. Transfection was carried out overnight and the cells were split 48 hours later. G418 at 1 mg/ml was added 24 hours after splitting, and its concentration was increased to 2 mg/ml in 2-3 days. After 10 days selection by G418, colonies were picked and transferred to 24-well plates.
  • the clones that expressed the dog GnRH receptor were screened by a whole cell binding assay using a radiolabeled peptide agonist of GnRH, 5-( 125 Iodo-Tyr)-Buserelin (Woods Assay, Portland, Oregon). Seven of the 71 neo ⁇ clones screened had a specific binding activity of 5-fold above the non-specific binding. The best one, 1B2-1F9 had a specific binding activity of more than 10-fold above the background. After single cell isolation, the membrane from this clone was prepared, and the membrane binding assay was performed using 125 I-buserelin. Figure 4 shows that the ligand binding activity was dose-dependent.
  • the ligand binding assay was conducted with the dog GnRH receptor obtained from 1B2-1F9. Crude membrane suspensions were prepared from 1B2-1F9 cells and stored as aliquots at -80°C.
  • the radioactive peptide ligand [5-( 12 ⁇ Iodo-Tyr)-Buserelin] was obtained from Woods Assay (Portland, Oregon) and had a radioactive specific activity of 1000 Ci/mmol.
  • the membranes and radioligand were diluted in assay buffer which consists of 50 mM Tris-HCl (PH 7.5), 2 mM MgCl 2 and 0.1% bovine serum albumin.
  • the ligand binding assay was performed at 22°C for 1 hour in borosilicate glass tubes in a final volume of 500 ml. Each tube contained an appropriate concentration of the radioligand (from 0.025 to 2.5 nM) and 15-20 mg of 1B2-1F9 membrane protein. The incubation was terminated by vacuum filtration onto GF/C Whatman filter paper (pretreated with 0.1% BSA in PBS) by a Brandel 48-well Harvester and then washed with 2 ml of cold phosphate saline (PH 7.5). The filter strips were counted in a Gamma Counter.
  • 1B2-1F9 cells are functionally coupled to phospholipase C.
  • 1B2-1F9 cells were seeded into 24-well plates at a concentration of 80,000 cells/ml/well in inositol-free F12 medium (Gibco Life Technology, Grand Island, NY) containing 10% dialyzed fetal bovine serum (Gibco Life Technology, Grand Island, NY), 1% Pen/Strep, 2 mM glutamine, 2 mg/ml G418 and 1 ⁇ Ci ( 3 H) inositol (NEN Life Sciences, Boston, MA).
  • the column was washed with 2 x 1 ml H 2 O to remove free ( 3 H)inositol, and ( 3 H)inositol phosphates were eluted with 3 x 1 ml 2 M ammonium formate in 1 M formic acid. The eluate was then counted in a scintillation counter.

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Abstract

Le récepteur de la GnRH du chien a été isolé, cloné et séquencé. Le récepteur de la GnRH du chien convient à la recherche systématique et l'identification de composés qui se lient au récepteur de la GnRH. De tels composés identifiés conviennent au traitement d'états liés aux hormones sexuelles tels que l'endométriose, les fibroïdes de l'utérus, le syndrome des ovaires polykystiques, l'hirsutisme, la puberté précoce, l'adénome hypophysaire gonadotrophe, l'apnée du sommeil, le syndrome du côlon irritable, le syndrome prémenstruel et l'hypertrophie bénigne de la prostate. Les protéines et polypeptides récepteurs, les acides nucléiques, les cellules et essais de cette invention conviennent à la recherche systématique et à la mise au point de médicaments, ainsi qu'à des applications de diagnostic et thérapeutiques.
PCT/US2000/015496 1999-06-10 2000-06-06 Recepteur de l'hormone de liberation de la gonadotropine du chien Ceased WO2000077047A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994000590A1 (fr) * 1992-06-23 1994-01-06 The Mt. Sinai School Of Medicine Of The City University Of New York CLONAGE ET EXPRESSION D'UN RECEPTEUR D'HORMONE LIBERANT DE LA GONADOTROPINE (R-HLGn)
US5750366A (en) * 1992-06-23 1998-05-12 Mount Sinai School Of Medicine Of The City University Of New York Cloning and expression of gonadotropin-releasing hormone receptor

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994000590A1 (fr) * 1992-06-23 1994-01-06 The Mt. Sinai School Of Medicine Of The City University Of New York CLONAGE ET EXPRESSION D'UN RECEPTEUR D'HORMONE LIBERANT DE LA GONADOTROPINE (R-HLGn)
US5750366A (en) * 1992-06-23 1998-05-12 Mount Sinai School Of Medicine Of The City University Of New York Cloning and expression of gonadotropin-releasing hormone receptor

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