FR2821619A1 - New isolated human gonadotropin-releasing hormone receptors and nucleic acids, useful for treating disorders associated with gonadal steroids, e.g. endometriosis - Google Patents

Abstract

Isolated gonadotropin-releasing hormone receptors (GnRH-R2): (i) comprising a 354 amino acid (aa) sequence (A1) or 379 aa sequence (A2); or (ii) encoded by a sequence that hybridizes with a 1174 bp sequence (N1) or 1249 bp sequence (N2). All sequences are reproduced in the specification. Independent claims are also included for: (1) isolated polynucleotide (I) that: (i) is (N1) or (N2); (ii) encodes (A1) or (A2); (iii) encodes a natural GnRH receptor and hybridizes with (i) or (ii); or (iv) encodes a natural GnRH receptor and is obtained by a polymerase chain reaction (PCR) method; (2) natural GnRH receptor encoded by the sequence of (a)(iv); and (3) recombinant vector containing (I); and (d) genetically modified cell containing (I).

Description

<Desc / Clms Page number 1>

The present invention relates to a gene coding for a new human receptor GnRH (Gonadotropin Releasing Hormone Receptor or receptor for the hormone releasing gonadotropin). It also relates to genetically modified host cells which express said receptor, as well as their use for evaluating and screening GnRH products and analogs involved in the activation, regulation and decoupling of said receptor.

A large number of GnRH-R family receptors (GnRH receptors) have been identified recently in many species. Often, a number of GnRH receptor subtypes have been isolated from the same species. In humans, there is currently only one known GnRH receptor, namely that isolated by S. Kakar and his collaborators (Kakar et al., Biochem. Biophys. Res. Commun. (1992), 189,289-295 ).

The Applicant has now just found a new human GnRH receptor, hereinafter designated by GnRH-R2.

The invention relates firstly to said isolated GnRH-R2 receptor, which comprises the amino acid sequence SEQ. ID. NO. 4. Said sequence SEQ. ID. NO. 4 is as follows:
1 MSAGNGTPWG VEVEGSELPT FSAAAKVRVG VTIVLFVSSA GGNLAVLWSV
51 TRREPSQLRP SPVRRLFIHL AAADLLVTFV VMPLDATWNI TVQWLAVDIA
101 CRTLMFLKLM ATYSAAFLPV VIGLDRQAAV LNPLGSRSGV RKLLGAAWGL
151 SFLLAFPDPF TQCVTKGSFK AQWQETTYNL FTFCCLFLLP LTAMAICYSR
201 IVLSVSRPQT RKGSHAPAGE FALPRSFDNC PRVRLRALRL ALLILLTFIL
251 CWTPYYLLGM WYWFSPTMLT EVPPSLSHIL FLLGLLNAPL DPLLYGAFTL
301 GCRRGHQELS IDSSKEGSGR MLQEEIHAFR QLEVQKTVTS RRAGETKGIS
351 ITSI Preferably, said receptor consists of the amino acid sequence SEQ. ID. NO. 4. The invention also relates to an isolated GnRH receptor encoded by a nucleotide sequence such that it hybridizes with the polynucleotide of sequence SEQ. ID. NO. 3

<Desc / Clms Page number 2>

 (described later). Preferably, said isolated GnRH receptor is coded by a nucleotide sequence such that it hybridizes under conditions of high stringency with said polynucleotide of sequence SEQ. ID. NO. 3.

It further relates to an isolated polynucleotide comprising either the nucleotide sequence SEQ. ID. NO. 3, or the sequence complementary to said nucleotide sequence SEQ. ID. NO. 3. This sequence SEQ. ID. NO. 3 is as follows:
1 tttctctcca ggccaccatg tctgcaggca acggcacccc ttggggagtg
51 gaggtggagg gctcagagct gcccaccttc tcggcagcag ccaaggtccg
101 agtgggagtg accattgtgc tgtttgtttc ttcggctgga gggaacctgg
151 cagtcctgtg gtcagtgaca cggcgggaac ccagccagct ccgcccctct
201 ccggtcagga gactcttcat ccatttagca gccgccgact tactagtcac
251 ttttgtggtt atgcccctag atgccacctg gaatatcact gttcaatggc
301 tggctgtgga catcgcatgt cggacactga tgttcctgaa actaatggcc
351 acgtattctg cagctttcct gcctgtggtc attggattgg accgccaggc
401 agcagtactc aacccgcttg gatcccgttc aggtgtaagg aaacttctgg
451 gggcagcctg gggacttagt ttcctgcttg ccttccccga ccctttcact
501 cagtgtgtca ccaaaggcag cttcaaggct caatggcaag agaccaccta
551 taacctcttc accttctgct gcctctttct gctgccactg actgccatgg
601 ccatctgcta tagccgcatt gtcctcagtg tgtccaggcc ccagacaagg
651 aaggggagcc atgcccctgc tggtgaattt gccctccccc gctcctttga
701 caattgtccc cgtgttcgtc tccgggccct gagactggcc ctgcttatct
751 tgctgacctt catcctctgc tggacacctt attacctact gggtatgtgg
801 tactggttct cccccaccat gctaactgaa gtccctccca gcctgagcca
851 catccttttc ctcttgggcc tcctcaatgc tcctttggat cctctcctct
901 atggggcctt cacccttggc tgccgaagag ggcaccaaga acttagtata
951 gactcttcta aagaagggtc tgggagaatg ctccaagagg agattcatgc
1001 ctttagacag ctggaagtac aaaaaactgt gacatcaaga agggcaggag
1051 aaacaaaagg catttctata acatctatct gatcctaaca gagtatgtag
1101 gaacagaata gtaagtcttt agtgccataa gatcttaaca tctcacttct
1151 actcctgctc tcctagttcc cccc

<Desc / Clms Page number 3>

 Preferably, said isolated polynucleotide consists of a nucleotide sequence comprising the nucleotide sequence SEQ. ID. NO. 3. Even more preferably, said isolated polynucleotide consists of the nucleotide sequence SEQ. ID. NO. 3.

The invention also includes polynucleotides whose sequence is at least 75% homologous, preferably at least 85% and even more preferably at least 90% or even 95%, to the sequence SEQ. ID. NO. 3.

The invention likewise relates to an isolated polynucleotide which codes for a natural GnRH receptor, said polynucleotide comprising a nucleotide sequence such that it hybridizes with an isolated polynucleotide comprising the nucleotide sequence SEQ. ID. NO. 3 or the sequence complementary to said sequence of nucleotides SEQ. ID. NO. 3.

The invention also relates to an isolated polynucleotide comprising a nucleotide sequence coding either for a polypeptide of amino acid sequence SEQ. ID. NO. 4, or for the polypeptide of amino acid sequence complementary to the amino acid sequence SEQ. ID. NO. 4.

The subject of the invention is also an isolated polynucleotide which codes for a natural GnRH receptor, said polynucleotide being capable of hybridizing with an isolated polynucleotide comprising a nucleotide sequence coding either for a polypeptide of amino acid sequence SEQ. ID. NO. 4, or for the polypeptide of amino acid sequence complementary to the amino acid sequence SEQ. ID. NO. 4.

A further object of the invention is a recombinant vector containing the nucleotide sequence of one of the polynucleotides described above. The invention further relates to a recombinant vector containing the nucleotide sequence of one of said polynucleotides associated with a regulatory nucleotide sequence containing transcriptional and translational regulatory information which controls expression of the nucleotide sequence in a host cell.

The invention also provides a genetically modified cell containing the nucleotide sequence of one of the polynucleotides described above. It also provides a genetically modified cell containing the nucleotide sequence of one of said polynucleotides associated with a regulatory nucleotide sequence containing transcriptional and translational regulatory information which controls the expression of the nucleotide sequence in a host cell.

<Desc / Clms Page number 4>

The invention further relates to the use of one of the receptors defined above for the discovery of therapeutic agents.

The invention further relates, as a medicament, to said receptor or a biologically active fragment thereof.

Other objects and interests of the present invention will become apparent from the detailed description which follows.

DETAILED DESCRIPTION OF THE INVENTION According to the present invention, the human GnRH-R2 receptor can be cloned and expressed.

GnRH-R2 is characterized by seven trans-membrane segments characteristic of receptors coupled to G proteins; but, unlike the GnRH-R receptor, GnRH-R2 has a C-terminal intracellular end with in final sequence the presence of a PDZ domain for anchoring to the cytoskeleton (PSD-95 / large discs / ZO-1, described in Kornau et al., Science (1995), 269, 1737-1740).

GnRH-R2 produced here can be used to assess and screen GnRH products and analogs involved in the activation, regulation or decoupling of GnRH-R2. Alternatively, the DNA of GnRH-R2, sense or antisense oligonucleotides, GnRH-R2, peptide fragments thereof, or antibodies to it can be used in diagnostic and / or therapeutic methods .

The coding nucleic acid sequence (SEQ. ID. NO. 3) and the deduced amino acid sequence (SEQ. ID. NO. 4) for the human receptor GnRH-R2 are described in the present application. The complete open reading phase codes for a protein of 354 amino acids of approximately 39,000 daltons. The hydrophobicity analysis of the deduced protein reveals 7 portions composed of highly hydrophobic amino acids. Unlike the first human receptor GnRH-R discovered, GnRH-R2 has a long C-terminal end with the presence of a PDZ recognition motif (ITSI-Stop) at the end of the C-terminal chain.

The present invention relates to isolated polynucleotides or polypeptides. A polynucleotide or a polypeptide is said to be isolated if it is taken out of its original environment. In particular, a polynucleotide or a polypeptide is isolated if it is separated from the biological material with which it coexists in the natural system.

<Desc / Clms Page number 5>

 According to the invention, the sequences which code for GnRH-R2 and fragments or fusion proteins of GnRH-R2 can be used to generate recombinant DNA molecules which direct the expression of GnRH-R2, or of a active portion of it, in appropriate host cells. Alternatively, sequences which hybridize with portions of the GnRH-R2 sequences can also be used in nucleic acid hybridization tests, Southem blot, Northem blot, etc.

Due to the degeneration of the genetic code, other DNA sequences encoding substantially for the amino acid sequence of GnRH-R2 can be used for cloning and expression of GnRH-R2. Such DNA sequences include those capable of hybridizing the GnRH-R2 sequences under certain stringency conditions which can be adjusted in several ways. For example, during a polymerase chain reaction (PCR), the temperature at which the primers hybridize to the matrix or the concentrations of MgCL, in the reaction buffer, can be adjusted. When using radio-labeled DNA fragments, or oligonucleotides to probe membranes, the stringency can be adjusted by changing the ionic strengths of the washing solutions or by carefully controlling the washing temperature.

The invention particularly includes polynucleotides having at least 75% homology with the polynucleotide of sequence SEQ. ID. NO. 3. The degree of homology expressed in% is calculated as follows: 100-100 x (N ′ with N ′ representing the number of nucleotides modified relative to the sequence SEQ. ID. NO. 3 and N the number of nucleotides of SEQ. ID. NO. 3.

Preferably, such a homologous nucleotide sequence hybridizes specifically with the sequence complementary to the sequence SEQ. ID. NO. 3 under stringent conditions. The parameters defining the stringency conditions depend on the temperature at which 50% of the paired strands separate (T.

For sequences comprising more than 30 bases, and according to Sambrook et al.

(Molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989), Tm is defined by the relation: Tm = 81.5 + 0.41 x (% G + C) + 16.6 x log [cations] -0.63 x (% formamide) - (600 / number of bases)

<Desc / Clms Page number 6>

 For the present invention, the stringency conditions will be said to be strong when a hybridization temperature of 10 ° C. is used below Tm and hybridization buffers containing a solution 6 × SSC (0.9 M sodium chloride and 0.09 M sodium citrate). Under such conditions, the polynucleotides of aspecific sequences will not hybridize with the polynucleotide of the sequence complementary to the sequence SEQ. ID. NO. 3.

Altered DNA sequences which can be used in accordance with the present invention include deletions, additions or substitutions of different nucleotide residues resulting in a sequence which codes for the same gene product or equivalent function. The gene product may also contain deletions, additions or substitutions of amino acid residues in the GnRH-R2 sequence, which result in so-called silent changes, thereby producing a GnRH-R2 of equivalent function.

Such amino acid substitutions can be made on the basis of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipatic nature of the residues involved.

d'hydrophobicité voisines incluent la leucin, l'isoleucine, la valine ; la glycine, l'alanine ; l'asparagine, la glutamin ; la serine, la thréonine ; la phénylalanine, la tyrosine. Un récepteur ayant une fonction équivalente à GnRH-R2 réfère à un récepteur qui fixe GnRH mais pas nécessairement avec la même affinité de son équivalent natif GnRH-R2. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, amino acids with polar groups having values

neighboring hydrophobicity include leucin, isoleucine, valine; glycine, alanine; asparagine, glutamin; serine, threonine; phenylalanine, tyrosine. A receptor having a function equivalent to GnRH-R2 refers to a receptor which fixes GnRH but not necessarily with the same affinity of its native equivalent GnRH-R2.

The DNA sequences of the present invention can be modified to alter the sequence encoding GnRH-R2 for a number of reasons including, but not limited to, alterations which modify the process and expression of the gene product.

For example, mutations can be introduced using techniques well known to those skilled in the art, for example site-directed mutagenesis, insertion of new restriction sites, alteration of glycosylations, phosphorylation, etc.

In particular, in certain expression systems such as yeast, the host cell can over-glycosylate the gene product. In such a system, it is preferable to alter the coding sequence of GnRH-R2 to eliminate the glycosylation sites. Also within the scope of the disclosure of the present invention is a modified GnRH-R2 or GnRH-R2 sequence linked to a heterologous sequence to code for a protein of

<Desc / Clms Page number 7>

 fusion. The fusion protein can be modified to contain a cleavage site located between the sequence of GnRH-R2 and the sequence of the heterologous protein, such that GnRH-R2 can be cleaved from the heterologous portion.

The sequence coding for GnRH-R2, which is part of the present invention, can be synthesized entirely or in part using chemical methods well known to those skilled in the art who may in particular refer to the following publications: Caruthers et al., nuc. Acids Res. Symp. Ser. (1980), 7,215-233; Crea and Hom, Nuc. Acids Res., 9 (10), 2331; Matteucci and Caruthers, Tetrahedron Litt. (1980), 21,719; and Chow and Kempe, Nuc. Acids Res. (1981), 9 (12), 2807-2817.

Alternatively, the protein itself can be produced using chemical methods to synthesize the amino acid sequence of GnRH-R2 in whole or in part.

In order to express the biologically active GnRH-R2 receptor, the nucleotide sequence coding for GnRH-R2 or a functional equivalent as described above, is inserted into an appropriate expression vector, i. e., a vector containing the elements necessary for transcription and translation of the inserted coding sequence. The GnRH-R2 gene products as well as host cells or cell lines transfected or transformed with the recombinant expression vector GnRH-R2 can be used in many cases which include but are not limited to the generation of antibodies ( ie monoclonal or polyclonal) which bind the receptor, including those which competitively inhibit the binding of GnRH and neutralize its activity, the screening and selection of GnRH analogs or products which act via the GnRH-R2 receptor, etc. .

coll., Current Protocols in Molecular Biology, Greene Publishing Associates et Wiley Interscience (1989), New York). Methods well known to those skilled in the art can be used to construct expression vectors containing the sequence coding for GnRH-R2 and the transcription / translation control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and genetic recombinations (see for example the techniques described in Maniatis et al., Molecular Cloning Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989) and Ausubel and

coll., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience (1989), New York).

A variety of host / expression vector systems can be used to express the sequence encoding GnRH-R2. Said systems include but are not limited to microorganisms such as bacteria transformed by recombinant bacteriophage DNA, plasmid or cosmid DNA vectors containing the sequence

<Desc / Clms Page number 8>

 encoding GnRH-R2, yeasts transformed with expression vectors containing the sequence encoding GnRH-R2, insect cell systems infected with a viral expression vector (for example a baculovirus) containing the sequence encoding GnRH-R2, plant cells infected with a viral expression vector (for example the cauliflower mosaic virus (CaMV) or the tobacco mosaic virus (TMV)) or transformed with a plasmid expression vector (for example Ti plasmid) containing the sequence encoding GnRH-R2, or an animal cell system infected with a recombinant viral expression vector (eg adenovirus or vaccinia virus).

The expression elements of these systems vary according to the host / vector system used.

For example, when cloning takes place in a mammalian cell system, the promoters which are derived from the genome of mammalian cells (for example a metallothionein promoter) or from mammalian viruses (for example the late adenovirus promoter ( adenovirus late promoter) or the vaccinia virus promoter 7.5 K) can be used.

construction vecteur/GnRH-R2 de manière transitoire ou de façon stable en utilisant un réactif Effectene selon les recommandations du fabricant (Qiagen). De nombreuses procédures connues de l'homme du métier peuvent être utilisées pour la production d'anticorps contre des épitopes du récepteur GnRH-R2 produit de manière recombinante. Des anticorps neutralisants, ceux qui réalisent une compétition avec les sites de fixation à GnRH sur le récepteur GnRH-R2 sont préférés pour une utilisation dans des méthodes de diagnostic ou thérapeutiques. The invention further relates to the cloning of the cDNA of GnRH-R2 into an expression vector pCDNA 3.1. HEK cells can be transfected with

vector / GnRH-R2 construct transiently or stably using an Effectene reagent according to the manufacturer's recommendations (Qiagen). Many procedures known to those skilled in the art can be used for the production of antibodies against epitopes of the recombinantly produced GnRH-R2 receptor. Neutralizing antibodies, those which compete with GnRH binding sites on the GnRH-R2 receptor are preferred for use in diagnostic or therapeutic methods.

For the production of antibodies, different species of animals can be immunized by injection of GnRH-R2 (including, in particular, rabbits, mice, rats, etc.).

Adjuvants can be used to increase the immunological response, depending on the host species, in particular the Freund's reagent, mineral gels like aluminum hydroxide, substances with activated surfaces like lysoelicithin, pluronic polyols, polyanions, peptides, mineral oils, dinitrophenol, etc.

Monoclonal antibodies against GnRH-R2 can be prepared using any technique which allows the production of antibody molecules in cell line cultures such as, for example, the hybridoma techniques originally described by Kohler and Milstein (Nature (1975), 256, 495-497), the

<Desc / Clms Page number 9>

technique d'hybridome de cellules B (Kosbor et coll., Immunology Today (1983), 4, 72 ; Cote et coll., Proc. NatM. Acad. Sec. (1983), 80, 2026-2030). Des fragments d'anticorps reconnaissant les sites spécifiques de liaison de GnRH-R2 peuvent être générés par des techniques classiques bien connues de l'homme du métier. De tels fragments comprennent notamment les fragments F (ab') 2 qui peuvent être obtenus par digestion trypsique de la molécule d'anticorps.

B cell hybridoma technique (Kosbor et al., Immunology Today (1983), 4, 72; Cote et al., Proc. NatM. Acad. Sec. (1983), 80, 2026-2030). Antibody fragments recognizing the specific binding sites of GnRH-R2 can be generated by standard techniques well known to those skilled in the art. Such fragments include in particular the F (ab ') 2 fragments which can be obtained by tryptic digestion of the antibody molecule.

GnRH-R2 DNA, antisense oligonucleotides, GnRH-R2 expression products, antibodies and host cell lines expressing GnRH-R2 described above are useful in the diagnostic field and in therapeutics, but also for pharmacological research and drug discovery.

For example, the DNA sequence of GnRH-R2 can be used in biopsy hybridization assays to diagnose an abnormal expression of GnRH-R2, for example analysis by Southem and Northem techniques, including assays of in situ hybridization. In the therapeutic field, antisense molecules defined on the basis of the DNA sequence of GnRH-R2 can be used to block the transport and expression of GnRH-R2. Alternatively, GnRH-R2 DNA can be used in gene therapy to introduce the recombinant normal gene into defective cells or to correct mutations in order to reconstruct GnRH-R2 and its function.

Also according to the present invention, specific antibodies against GnRH-R2 can be used to determine the expression profile of the receptor in tissue biopsies, or to allow diagnostics from medical imaging carried out in vivo. For example, an antibody coupled to a detection entity in imaging can be administered to a patient to localize the distribution of GnRH-R2 in vivo.

Still according to the invention, the GnRH-R2 receptor, or a fragment thereof containing its binding site, can be administered in vivo. The free receptor or the peptide fragment thereof can compete for the binding of GnRH and inhibit its interaction with the native receptor in vivo.

The following paragraphs describe a method for cloning a cDNA encoding the GnRH-R2 receptor.

<Desc / Clms Page number 10>

Clo. of the GnRH-R2 receptor Initially, a polymerase chain reaction (PCR) is carried out on a series of commercially available cDNA libraries using the primers FI of sequence SEQ. ID. NO. 1 and RI of sequence SEQ. ID. NO. 2. These sequences, reproduced below, contain HindIII and EcoRI restriction site sequences respectively.

RI (de séquence SEQ. ID. NO. 2), 200 u. M de déoxynucléotides triphosphate (ou dNTPs : dATP, dCTP, dGTP et dTTP), 10 mM Tris-HCl (pH 8, 3), 50 mM KCI, 1, 5 mM MgClz et 0, 5 U Taq ADN polymérase dans un volume final de 50 gel. Les paramètres de cycles thermiques comprennent une dénaturation à 94 C pendant 30 s, une hybridation des amorces à 60 C pendant 1 minute et une extension de polymérisation à 72 C pendant 30 s (avec une extension finale de 5 minutes). SEQ sequences. ID. NO. 1 and SEQ. ID. NO. 2 of the primers FI and RI are the following: - SEQ. ID. NO. 1: 5'-GCA AGC TTC CAC CAT GTC TGC AGG C-3 '- SEQ. ID. NO. 2: 5'-CGG AAT TCG GGG GGA ACT AGG AG-3 'The reaction conditions include 0.5 μM of FI (of sequence SEQ. ID. NO. 1) and of

RI (of sequence SEQ. ID. NO. 2), 200 u. M deoxynucleotide triphosphate (or dNTPs: dATP, dCTP, dGTP and dTTP), 10 mM Tris-HCl (pH 8, 3), 50 mM KCI, 1.5 mM MgClz and 0.5 U Taq DNA polymerase in a final volume of 50 gel. The thermal cycle parameters include denaturation at 94 C for 30 s, hybridization of the primers at 60 C for 1 minute and polymerization extension at 72 C for 30 s (with a final extension of 5 minutes).

The PCR products are then separated on 1% agarose gel and visualized by staining with ethidium bromide.

The nucleic acid sequence thus obtained contains the HindIII restriction sites in the 5 ′ part and EcoRI in the 3 ′ part, allowing insertion directly into an expression vector, for example an expression vector of pCDNA3 type. 1 (InVitrogen).

After the PCR amplification step presented above, the products are subjected to HindIII and EcoRI hydrolysis in order to release ends containing these sequences and then purified from the agarose gel by electroelution. The sequence obtained is then inserted into an expression vector such as for example pCDNA3. 1 (InVitrogen). Analysis by digestion with suitable restriction enzymes of different bacterial clones having received said expression vector makes it possible to isolate the bacterial clones containing the new expression vector / polynucleotide construction coding for GnRH-R2.

<Desc / Clms Page number 11>

tests pharmacologiques décrits ci-après. The preparation in large quantities of these plasmid constructs is carried out using a plasmid DNA purification kit (Qiagen). The advantage of the invention in the therapeutic field can be appreciated using the

pharmacological tests described below.

avec 0, 2 nM de ['I] D-Trp6-GnRH (NEN) dans un volume final de 200 ul pendant 1 heure à 4 C. La réaction est arrêtée par une filtration rapide sur des filtres 96 puits GF/1 pré-chargés à 0,5 % en polyéthylènimine. Les filtres sont ensuite lavés trois fois à 4 C avec du tampon de lavage contenant 50 mM Tris (pH 7,4) en utilisant une station de filtration Packard 96 puits. Les filtres ainsi séchés sont submergés de 40 ul de cocktail scintillant (Microscint 0, Packard) et sont soumis à un comptage sur le Topcount (Packard). L'activité non-spécifique est déterminée en présence de 100 nM de D-Trp6-GnRH. Une courbe dose-réponse est générée pour D-Trp6-GnRH (0,001 nM à 100 nM) et permet de montrer l'affinité de GnRH pour le récepteur GnRH-R2. . 7'coMay A) Affinity of GnRH for the GnRH-R2 receptor: The cells are recovered 48 h after transfection by treatment with trypsin. For the membrane preparation, the membranes of the cells transfected with the human GnRH-R2 receptor are diluted to the concentration of 100 Jlglml in the reaction buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM of MgCl2, 100 Jlglml of bacitracin and 0.1% bovine serum albumin (BSA). The membranes are incubated

with 0.2 nM of ['I] D-Trp6-GnRH (NEN) in a final volume of 200 μl for 1 hour at 4 C. The reaction is stopped by rapid filtration on 96-well filters GF / 1 pre- 0.5% loaded with polyethyleneimine. The filters are then washed three times at 4 ° C. with washing buffer containing 50 mM Tris (pH 7.4) using a Packard 96-well filtration station. The filters thus dried are submerged with 40 μl of scintillating cocktail (Microscint 0, Packard) and are subjected to counting on the Topcount (Packard). The non-specific activity is determined in the presence of 100 nM of D-Trp6-GnRH. A dose-response curve is generated for D-Trp6-GnRH (0.001 nM to 100 nM) and makes it possible to show the affinity of GnRH for the GnRH-R2 receptor.

B) Modulation of the production of Inositol Phosphate: The production of Inositol Phosphate (IP) stimulated by GnRH is determined according to the method of Davidson et al., Endocrinology (1990), 126,80-87. In summary, the cells transfected with GnRH-R2 are incubated for 18 h in the presence of [H3] Inositol and stimulated with 100 nM of GnRH in the presence of lithium chloride. The reaction is stopped by the addition of 0.1 M formic acid at 4 C. After neutralization with 1 M Tris / Base, the Inositol Phosphates are separated on an ion exchange column (Biorad) and counted. The results obtained make it possible to appreciate the modulation of the production of Inositol Phosphate by the GnRH-R2 receptor.

Claims (10)

CLAIMS 1. Isolated GnRH-R2 receptor, which comprises the amino acid sequence SEQ. ID. NO. 4. 2. Isolated GnRH receptor encoded by a nucleotide sequence such that it hybridizes with the polynucleotide of sequence SEQ. ID. NO. 3. 3. Isolated polynucleotide comprising either the nucleotide sequence SEQ. ID. NO. 3, or the sequence complementary to said nucleotide sequence SEQ. ID. NO. 3. 4. An isolated polynucleotide which codes for a natural GnRH receptor, said polynucleotide comprising a nucleotide sequence such that it hybridizes with a polynucleotide according to claim 3. 5. Isolated polynucleotide which codes for a natural GnRH receptor, said polynucleotide being capable of being obtained by a process comprising the following successive steps: a) polymerization chain reaction (PCR) on a cDNA library with 0.5 u . M of primer FI (of sequence SEQ. ID. NO. 1) and of primer RI (of sequence SEQ. ID. NO. 2), 200 μM deoxynucleotide triphosphate (or dNTPs: dATP, dCTP, dGTP and dTTP), 10 mM Tris-HCl (pH 8.3), 50 mM KCI, 1.5 mM MgClz and 0.5 U of Taq DNA polymerase in a final volume of 50 u. l, the thermal cycle parameters comprising denaturation at 940 C for 30 s, hybridization of the primers at 60 C for 1 minute and polymerization extension at 72 C for 30 s (with a final extension of 5 minutes); b) hydrolysis of the HindIII and EcoRI sequences; and c) separation on 1% agarose gel. 6. GnRH receptor encoded by an isolated polynucleotide according to claim 5. 7. Recombinant vector containing the nucleotide sequence of a polynucleotide according to one of claims 3 to 5. <Desc / Clms Page number 13>  8. Genetically modified cell containing the nucleotide sequence of a polynucleotide according to one of claims 3 to 5. 9. Use of a receptor according to claim 1,2 or 6 for the discovery of therapeutic agents. 10. As a medicament, a receptor according to claim 1,2 or 6 or a biologically active fragment thereof.