FR2823762A1 - New nucleic acid encoding G protein-coupled receptor, useful for identifying specific modulators, potentially useful for treating e.g. diabetes - Google Patents

Abstract

Nucleic acid (I), having a 1044 bp sequence (S1), reproduced, that encodes a G protein coupled receptor (R), is new. Independent claims are also included for the following: (1) Nucleic acid (Ia) that hybridizes to (I) under stringent conditions and encodes a protein with essentially the same properties as (R); (2) Nucleic acid (Ib) deduced from a 347 amino acid sequence (S2), given in the specification; (3) Antisense nucleic acid (Ic) that inhibits expression of (I)-(Ib); (4) Recombinant vector (III) containing (I)-(Ib); (5) Host cell (IV) transformed with (III); (6) Polypeptide (II), specifically (S2), encoded by (I)-(Ib); (7) Fragments of (II) that have receptor activity, can stimulate G proteins directly, or can inhibit the action of an agonist; (8) Primers (V) for amplifying a sequence encoding (II); (9) Hybridization probes (VI) for specific recognition of (I)-(Ib); (10) Antibody (Ab) directed against (II) or its fragments; (11) Identifying (M1) agonists or antagonists of (R); (12) Identifying (M2) pathological allelic variants of the gene encoding (R); (13) Identifying (M3) sequences in other species that are homologous with (S1); (14) Kit for diagnosis, prognosis and monitoring treatment of patients with, or susceptible to, diseases associated with over or underexpression of (I)-(Ib); (15) DNA chip containing (I)-(Ib); (16) Kit for detecting an abnormal structure for (R); and (17) Protein chip containing Ab or (II), or its fragments.

Description

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 The invention relates to the identification of a new receptor coupled to GTP-binding proteins or G proteins. It relates in particular to the identification of a nucleic acid sequence encoding this receptor as well as to the recombinant vectors comprising these sequences and host cells containing the vectors. The uses of host cells for the screening of agonist or antagonist molecules of the receptor coupled to G proteins are also part of the invention.

 By receptors coupled to G proteins, is meant monomeric, dimeric or multimeric membrane proteins expressed on the surface of cells, capable of receiving an extracellular signal and of transmitting it to the cell via their interaction with specific heterotrimeric proteins with which they are associated and called G proteins. Heterotrimeric G proteins are composed of an a subunit and an inseparable ss [gamma] dimer. The a subunit carries a GDP or GTP nucleotide. The G proteins are submernan proteins associated with the membrane by lipids. Schematically, the activation mechanism of a receptor coupled to G proteins by an agonist molecule is summarized as follows: when an agonist binds to a receptor coupled to G proteins, said receptor associates with the GDP form of the G protein heterotrimeric.

The latter then loses its GDP which is replaced by a GTP, following a transient state.

The fixation of the GTP causes the dissociation of the three partners, the receptor, the subunit a. and the ss [gamma] dimer of protein G. The [alpha] subunit and the ss [gamma] dimer are then capable of stimulating one or more effectors. The hydrolysis of GTP, sometimes stimulated by the effector, marks the end of the activation of the effector by the a subunit.

 Among the different families of membrane receptors involved in cellular communications, the family of receptors coupled to G proteins is the most numerous.

 It is divided into five distinct subgroups according to molecular structure and function: (i) receptors for peptide hormones (somatostatin, TRH, etc.); (ii) receptors for glycoprotein hormones (TSH, LH, FSH, etc.); (iii) receptors for secretin-type peptide hormones; (iv) olfactory receptors and (v) bioamine receptors (serotonin, adrenergic). The receptors coupled to G proteins are thus involved in the reception of external stimuli (vision, taste, odor), functions

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 endocrines (pituitary and adrenal), exocrine functions (pancreas), certain neurological functions, cardiac function, lipolysis and carbohydrate metabolism (for reviews, Wilson S. et al., British Journal of Pharmacology 1998 125: 1387-1392 ; Stadel JM et al., TIPS 1997 18: 430-437).

 Considering the importance and the diversity of the communication systems controlled by the receptors coupled to the G proteins, and in particular their implication in numerous pathologies, we understand the advantage of looking for possible sequences capable of encoding these receptors in order to isolate them, to verify that they correspond to new genes, to express their product and to build the tools to identify their ligand (s).

 The research and identification of new receptors coupled to G proteins is based in particular on the analysis of genomic DNA sequences from the various mammalian genome sequencing projects currently underway, and stored in databases generally accessible by the internet network. These include data from high throughput genome sequencing that are available through the GenBank database.

 In general, the sequences initially deposited in the databases are so-called raw or non-annotated sequences. The annotation phase which consists of identifying, using bioinformatics tools, the significant sequences, and in particular the potential coding sequences, is carried out subsequently. Knowing that in the human genome, only 1 to 2% of the sequences are coding, one of the difficulties consists in locating the coding phases among the raw genomic sequences available in the databases.

 In addition, it should be noted that the available genomic DNA sequences are likely to be the juxtaposition of unordered partial sequences from different sequence clones. This can therefore constitute an additional obstacle in the identification of coding sequences.

 We then use computer analysis of sequences using different software available and appropriate for the search for open reading phases (ORFs)

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 potential. The choice and configuration of the software used is decisive for the identification of potentially coding sequences. It is however important to emphasize that the identification of a potential ORF using bioinformatics programs (in silico) is only a first step in the identification of a new functional gene. Those skilled in the art must then verify that the potential ORF identified in silico corresponds to a gene actually expressed and functional in the cell. We know for example that there are pseudo-genes, the remainder of evolution, whose portions of sequences are homologous to certain genes, however these pseudo-genes are not expressed in the cell.

 The inventors have now identified, by computer analysis of the database of sequences from high-throughput genome sequencing (HTGS), an open potential reading phase whose deduced protein sequence has homology with receptors coupled to G proteins The deduced sequence indeed has the structural criteria typical of receptors coupled to G proteins, including 7 domains which each contain about 20-30 hydrophobic amino acids and the LRY motif present in the third trans-membrane domain.

 By RT-PCR analysis, the inventors revealed a specific tissue expression of the identified sequence. By RT-PCR is meant a PCR amplification on the cDNA fragments obtained by retrotranscription of the transcriptome. They thus showed that the sequence identified by bioinformatics analysis effectively corresponded to a sequence of an expressed gene. They also showed that the gene encoding the new identified receptor is mainly expressed in the brain, and in particular in the hippocampus, the amygdala, the substantia nigra, the caudate nucleus and the cercbellum. It is also expressed at the periphery in the intestines, the pancreas, the lungs and the liver.

 The cDNA encoding the complete receptor was cloned after amplification by RT-PCR from RNA of total human brain, then sequenced.

 The nucleotide sequence obtained by the sequencing of the eDNA is presented in FIG. 1 (SEQ ID NO 1).

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 The amino acid sequence deduced from the sequence of Figure 1 is presented in Figure 2 (SEQ ID NO 2).

 The subject of the invention is therefore a nucleic acid sequence encoding a receptor coupled to the G proteins, characterized by the sequence of FIG. 1.

 A nucleic acid sequence according to the invention notably comprises: - a genomic DNA sequence, characterized by the sequence of FIG. 1, - a messenger RNA sequence, product of transcription of the genomic DNA sequence, - or a cDNA sequence, a product of the reverse transcription of the messenger RNA sequence.

 By nucleic acid sequence, there must be understood a nucleotide sequence isolated from its natural context. These are in particular isolated, amplified and / or purified sequences and possibly modified by genetic engineering.

 By cDNA or complementary DNA is meant a nucleic acid resulting from the reverse transcription of a messenger RNA.

 The inventors observed that the genomic DNA coding sequence of the G protein-coupled receptor according to the invention was identical to the isolated cDNA sequence and therefore did not contain introns.

A nucleic acid sequence according to the invention can be easily isolated by specific amplification by RT-PCR from extracts of messenger RNA from human brain cells, or by amplification of human genomic DNA, for example using the pair of primers below:
5'-GGGGATGAGCTGGCACCTTGC-3 '5'-ATGGTAAAGATGGCCTGGTGC-3'
The invention also relates to variants of the nucleic acid sequence of FIG. 1.

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 The variants of the nucleic acid sequence are in particular: - sequences capable of hybridizing under stringent conditions with a nucleic acid sequence of FIG. 1 or a sequence complementary to it, and encoding a polypeptide having essentially the same properties as the receiver coded by the sequence of Figure 1; - the nucleic acid sequences deduced from the amino acid sequence of FIG. 2, according to the genetic code, - pathological allelic forms of the sequence of FIG. 1; again, - sequences of a mammalian species homologous to the sequence of FIG. 1 isolated in humans.

 By stringent conditions is meant the conditions which allow the specific hybridization of two single-stranded DNA sequences at approximately 65 ° C. for example in a solution of 6 x SSC, 0.5% SDS, 5X Denhardt's solution and 100 g of Non-specific carrier DNA or any other solution of equivalent ionic strength and after washing at 65 ° C., for example in a solution of at most 0.2 × SSC and 0.1% SDS or any other solution of equivalent ionic strength. The parameters defining the stringency conditions depend on the temperature at which 50% of the paired strands separate (Tm). For sequences comprising more than 30 bases, Tm is defined by the relation: Tm = 81.5 + 0.41 (% G + C) + 16.6 Log (cation concentration) - 0.63 (% formamide) - ( 600 / number of bases). For sequences of length less than 30 bases, Tm is defined by the relation: Tm = 4 (G + C) + 2 (A + T). The stringency conditions can therefore be adapted by those skilled in the art according to the size of the sequence, the GC content and any other parameter, in particular according to the protocols described in Sambrook et al., 2001 (Molecular Cloning: A laboratory Manual, 3rd Ed., Cold Spring Harbor, laboratory press, Cold Spring Harbor, New York).

 The expression pathological allelic forms is understood to mean any sequence isolated in humans corresponding to a mutated form of the sequence of FIG. 1, and the mutations of which are involved in a specific pathology. These mutations can be one or more nucleotide change (s) or one or more deletion (s) or insertion (s) of fragments of nucleotide sequences.

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 The expression “sequences homologous to the nucleic acid sequence of FIG. 1” is intended to mean a sequence of structure similar to the sequence of FIG. , rat or mouse. The percentage of identity between two homologous sequences in the functional regions is generally greater than 80%, preferably greater than 90%.

 The homologous sequences are in particular useful for the production of animal models for the functional study of the receptor coupled to the G proteins and its pathological variants.

The invention thus relates to a method for identifying pathological allelic forms of a human gene encoding a receptor coupled to G proteins, said method comprising the following steps: a) isolation of a nucleic acid sequence encoding a receptor coupled to G proteins according to the invention from total nucleic acids (RNA or
DNA) from patients with pathology and healthy subjects; b) the sequencing of the nucleic acids isolated in a), and, c) the detection of one or more mutations common to the nucleic acids originating from patients suffering from a pathology, the said mutation (s) n not being present on nucleic acids from healthy subjects.

 The invention also relates to a method for identifying sequences homologous to the nucleic acid sequence of FIG. 1 in a non-human species, comprising the following steps: a) the incubation under stringent conditions of a probe hybridization consisting of at least 50 nucleotides of the sequence of FIG. 1, preferably between 150 and 250 nucleotides, with genomic DNA or extracts of total RNA from non-human mammal species; b) detecting a hybridization reaction; and,

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 c) isolating the nucleic acid sequence homologous and encoding a receptor coupled to the G proteins from the genomic DNA of the species for which a hybridization reaction has been detected.

 The hybridization probes for the specific recognition of the nucleic acid sequences encoding a receptor coupled to the G proteins according to the invention also form part of the invention. Such probes are characterized in that they correspond to a fragment with a length of at least 50 nucleotides, preferably between 150 and 250 nucleotides of a nucleic acid sequence according to the invention. These probes are in particular useful in implementing the methods of the invention mentioned above.

The probes can be coupled directly or indirectly with a radioactive or non-radioactive marker, mention may in particular be made of 32P or 35S among radioactive compounds or else biotin, avidin and streptavidin or any type of fluorescent compounds or bioluminescent or chemiluminescent among the non-radioactive compounds.

The invention also provides primers useful for the amplification and isolation of the nucleic acid sequences encoding a receptor coupled to the G proteins according to the invention. Primers for the amplification of a sequence encoding a receptor coupled to the G proteins according to the invention comprise in particular the following sequences: 5'-GGGGATGAGCTGGCACCTTGC-3 '5'-ATGGTAAAGATGGCCTGGTGC-3'
The primers can also comprise, in their 5 ′ part, sequences which are not specific for the gene, in particular restriction sites to facilitate the cloning of the sequences amplified in a vector, or alternatively sequences coding for tags, epitopes or signal sequences to facilitate the expression and / or purification of the polypeptides. Primers comprising a tag sequence and suitable restriction sites for cloning the amplification product into a plasmid are also described in Example 1.

 The invention also relates to the nucleic acid sequences capable of being obtained by amplification using the primers defined above.

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 The methods of amplification using the primers of the invention are known to those skilled in the art and are in particular the PCR method which comprises the use of a thermostable polymerase or the RT-PCR method which allows the amplification of DNA from a messenger RNA sample by performing a reverse transcription step before amplification. There are also many alternative techniques to PCR. Examples include isothermal amplification methods such as TMA (transcription mediated amplification), NASBA (nucleic acid sequence based amplification), 3SR (self sustained sequence replication) or strand displacement amplification ( strand displacement amplification).

 The invention also relates to a recombinant vector comprising a nucleic acid sequence according to the invention. The term “vector” should be understood to mean any type of vector allowing the introduction of the nucleic acid sequence into a host cell and possibly the expression of the polypeptide encoded by the nucleic acid sequence in the host cell.

 Such a vector is for example the plasmid pS3040 deposited at the CNCM on March 12, 2001 under the number 1-2648.

 In a preferred embodiment, the recombinant vector according to the invention is characterized in that it comprises DNA sequences necessary for the expression of the receptor coupled to the G proteins in a host cell. These sequences are in particular promoter sequences suitable for the expression of genes in host cells and terminator sequences downstream of the coding sequence which include a polyadenylation site. Examples of promoter sequences are the sequence of the cytomegalovirus promoter (CMV) or the promoter sequence SV40 which allow constitutive and strong expression of the coding sequences placed downstream in mammalian cells and insect cells respectively.

 There are a large number of usable expression vectors. Such expression vectors include, inter alia, chromosomal, episomal or virus-derived vectors, in particular, vectors derived from bacterial plasmids, bacteriophages, transposons, plasmids and chromosomes of yeast, viruses such as baculoviruses , papoviruses and in particular SV40, adenoviruses, and retroviruses and

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 vectors derived from their combinations, in particular cosmids and phagemids. Among the expression vectors particularly preferred for the heterologous expression of membrane receptors in mammalian cells, there may be mentioned the vectors pCI-neo (promega; N accession U47120) and pCDNA3 comprising the CMV promoter, the bicistronic vector pIRESneo / pIREShyg (Clontech N cat 6061-1) or also the vector pSG5 (Stratagene) comprising the promoter SV40.

 To allow secretion of the proteins translated into the lumen of the endoplasmic reticulum of eukaryotic cells or in the periplasmic space of bacteria or in any other extracellular environment, the vectors can comprise sequences coding the appropriate secretion signals on the expressed polypeptide.

 The invention also relates to host cells transformed by the recombinant vectors. In a particular embodiment, the host cells are bacteria, such as, for example, E. coli, streptococci, staphylococci, Streptomyces and B. subtilis, preferably Escherichia coli. The transformation of bacterial host cells is in particular useful for the amplification and storage of the recombinant vectors of the invention.

 In another embodiment of the invention, the host cells are eukaryotic cells. Eukaryotic host cells which can be used are in particular yeast cells such as S. cerevisiae or filamentous fungi such as Aspergillus, insect cells such as Drosphila S2 cells or Spodoptera Sf9 cells, or even plant cells.

 Preferably, the eukaryotic host cells are mammalian cells and in particular mammalian cell lines, in particular CHO, COS, HeLa, C127,3T3, HepG2, L (TK-) cells and more preferably, the lines. CHO-K1 or HEK 293.

 It is thus possible to express the receptor coupled to the G proteins in an appropriate host cell, preferably a eukaryotic host cell, by the introduction into the host cell of the recombinant vector of the invention.

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 Those skilled in the art also know how to purify said receptor from eukaryotic cells expressing it. Different methods of purifying G protein-coupled receptors are described in the prior art (Eckard CP and Beck-sickinger AG, Curr Med Chem 2000 7: 897-910; Ohtaki T et al., J Biol Chem 1998 273: 15464-15473; Hayashi MK, Haga TJ, Biochem (tokyo) 1996 120: 1232-1238). By way of example, let us cite the purifications by precipitation with ammonium sulphate or with ethanol, on an ion exchange chromatography column, by affinity chromatography, by size exclusion chromatography, by reverse phase or by high performance liquid chromatography. It is also possible to purify the receptor of the invention using electrophoretic techniques for separating proteins according to their size and / or their charge.

 The affinity chromatography method consists in particular in expressing in a host cell, a recombinant receptor having an epitope or a specific tag sequence (tag) upstream or downstream of the coding sequence, in recovering the membrane proteins or the total proteins of the host cell and then to purify the receptors on an affinity column fixing the proteins expressing the specific epitope or tag. According to this particular purification mode, the expression vector additionally comprises the tag sequences for the purification of the polypeptide expressed in the host cells.

 The invention also relates to a polypeptide characterized in that it is encoded by a nucleic acid sequence according to the sequence of FIG. 1 (SEQ ID NO 1) or by any variant according to the invention of the sequence of FIG. 1 .

 In particular, the invention relates to a polypeptide comprising the amino acid sequence of FIG. 2 (SEQ ID NO 2).

 The invention also relates to the fragments of this polypeptide exhibiting receptor activity coupled to G proteins, in particular, the fragments of this polypeptide comprising the seven transmembrane domains.

 It is further known that certain fragments of the G protein coupled receptors are capable of directly stimulating the G protein independently of the presence of an agonist. For example, Hebert et al. showed that the second and third loop

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 intracellular receptor coupled to G proteins HT1AR allow the coupling of said receptor to G proteins and their stimulation (Hebert TE et al., J Biol Chim 1996 271: 16384-16392). Thus, the invention relates to the fragments of the polypeptide described above, said fragments being capable of directly stimulating the G proteins, and thus mimicking the activity of the receptor coupled to the G proteins in the presence of an agonist.

 Conversely, the fragments of the receptor coupled to the G proteins deleted from all or part of the regions involved in the dimerization of the receptor or in the interaction with the G proteins can be advantageously used in order to inhibit the action of an agonist (Varrault A . et al., J Biol Chem 1994 269: 16720-16725). Consequently, the invention also relates to the fragments of the polypeptide described above, capable of inhibiting the action of an agonist. Thus, according to the embodiments of the invention, the fragments of polypeptide sequences of the invention are included in one of the 7 transmembrane domains or in the intracytoplasmic part (first, second and third intracellular loop) or even in the part external to the cell (N-terminal part or first, second or third extracellular loop).

 The polypeptides or fragments of polypeptides of the invention are in the form of native proteins or are part of a larger fusion protein. In particular, the polypeptides of the invention are fused to additional amino acid sequences which contain the signals necessary for the translation, stability, secretion and / or purification of the corresponding recombinant protein.

 The antibodies directed against the polypeptides and fragments of polypeptides described above also form part of the invention. These antibodies find their interest in detecting and / or quantifying the expression of receptors on the surface of cells. When the antibodies are specifically directed against the recognition site of the G protein-coupled receptor ligand, these antibodies can also be used as receptor antagonists.

 The invention relates to both polyclonal and monoclonal antibodies.

 Polyclonal antibodies can be obtained by immunization of a suitable living host with said purified polypeptides or their fragments containing the

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 epitopes that one wishes to identify and / or quantify. The antibodies formed from a serum of the immunized host are then recovered in particular by bringing the sera into contact with the purified polypeptides and by recovering these antibodies from the antigen-antibody complexes formed.

 The monoclonal antibodies are in particular obtained according to the conventional method of culture of hybridomas of Kohler and Milstein.

 The antibodies according to the invention can also be chimeric antibodies, humanized antibodies or Fab or F (ab ') 2 fragments. It is in particular possible to clone the genes coding for the two chains of monoclonal antibodies produced by hybridomas, which can be manipulated in vitro and then reintroduced into secretory cells, such as lymphoid lines or others, after their reinsertion into the expression vectors suitable for the selected cells. New monoclonal antibodies are thus constructed with variable regions of mice or rats and constant human regions. Different types of immunoglobulins can be obtained.

Their specificity in all cases will be the same as that of the variable regions used but they will have the effector properties of human immunoglobulins according to the isotype chosen in the construction.

 The antibodies of the invention can also be reconstituted (Reshaped antibodies) (Riechmann et al. (1988), Nature 332: 323-327). Schematically, the starting point of this approach is the observation that the contact sites of the antibodies with the epitopes of the antigens which they recognize are localized on certain sequences of the so-called hypervariable variable regions. The technique consists in grafting the hypervariable regions of a murine immunoglobulin called "region determining the complementarity with the antigen" or "CDR" on the variable regions of a human class G immunoglobulin.

 The antibodies according to the invention are also capable of being coupled to another compound, in particular a marker, in order to obtain a detectable or quantifiable signal.

 The host cells expressing the receptor coupled to the G proteins according to the invention can be advantageously used for the screening of compounds which bind to

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 receptor and activates it (agonists of this receptor) or which inhibit activation of said receptor (antagonist).

The invention thus relates to a screening method for the identification of agonist molecules of the receptor coupled to the G proteins, characterized in that it comprises the following steps: a) the culture of eukaryotic host cells transformed by a recombinant vector according to l invention allowing expression of the G protein coupled receptor; b) bringing the host cells in culture into contact with a candidate compound; c) the determination of a possible signal generated by the receptor coupled to the proteins
G in the presence of the candidate compound.

 According to the embodiments of the method, the signal to be determined, generated by the activation of the receptor coupled to the G proteins by the candidate compound can be a stimulation or an inhibition of adenylate cyclase, a binding of 35SGTPy, a MAP kinase activity. or an increase in intracellular inositol triphosphate, and more generally any type of signal capable of being modulated positively or negatively by the G proteins.

 In a preferred embodiment, the signal generated by the receptor coupled to the G proteins in the presence of one of its agonists is determined using a reporter system comprising the use of a recombinant cell expressing the subunit has protein G16 in a stable or transient manner and the quantification of the intracellular calcium concentration. The constitutive expression of the a subunit of the G16 protein allows any antagonistic response to be reflected essentially by an increase in the concentration of intracellular calcium.

 Thus, the invention relates to a screening method for the identification of agonist molecules comprising the following steps: a) the culture of eukaryotic host cells transformed by a recombinant vector according to the invention allowing the expression of the receptor coupled to G proteins ,

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 said host cells additionally expressing an α subunit of protein G constitutively; b) the quantification of the intracellular calcium concentration in the cells in culture in the presence and in the absence of the molecules to be screened; c) the selection of molecules in the presence of which the concentration of intracellular calcium is increased.

 In the above method, the host cells are modified in such a way that any activation of the receptor leads to an increase in the concentration of intracellular calcium. Use, for example, COS7, CHO or HEK293 cells expressing the G [alpha] 16 subunit in a stable or transient manner. The concentration of intracellular calcium is determined using a marker, in particular a fluorescent marker.

 The invention also relates to a method for identifying the presence of antagonistic molecules comprising the following steps: a) the culture of eukaryotic host cells transformed by a recombinant plasmid according to the invention allowing the expression of the receptor coupled to G proteins presence of an agonist; b) bringing the host cells in culture into contact with a candidate compound; c) determining a decrease in the possible signal generated by the G protein-coupled receptor in the presence of the candidate compound and the agonist compared to the signal generated with the agonist alone.

 The agonists and antagonists identified by such a method are also targeted by the invention.

 The invention also relates to the use of a nucleic acid sequence encoding a receptor coupled to G proteins according to the invention for the screening of agonist and antagonist molecules of a receptor coupled to G proteins.

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 It also relates to the use of the host cells mentioned above for the screening of agonist and antagonist molecules of a receptor coupled to G proteins.

 The polypeptides encoded by the nucleic acid sequences of the invention are involved in different biological functions, and possibly certain pathologies.

In particular, the demonstration by the inventors of the expression of the receptor coupled to the G proteins specifically in the hippocampus, the substantia nigra, the amygdala, the caudate nucleus, the cerebellum, the pancreas or the liver supposes a role of receptor in functions linked to these structures and possibly in certain pathologies linked to an abnormality in the expression of said receptor, in particular in these tissues.

 Consequently, the invention also relates to methods of diagnosis, prognosis or monitoring the progress of a pathology linked to an abnormal expression of the receptor in a tissue.

 By abnormal expression is meant any qualitatively or quantitatively different expression between a sick individual and a healthy individual.

 In a first particular embodiment, the diagnostic method consists in comparing the sequence encoding the receptor in the cells of a particular tissue in a sick individual and a healthy individual and in detecting a particular allelic form of the sequence in the individual sick in relation to the healthy subject.

 In a second particular embodiment, the method consists in comparing the expression of the messenger RNA encoding the receptor in the cells of a particular tissue in a sick individual and a healthy individual and detecting an abnormal expression of said messenger RNA in the sick individual in relation to the healthy subject.

 Techniques for quantifying the expression of a messenger RNA are well known to those skilled in the art. Mention will in particular be made of the Northern Blot or quantitative RT-PCR technique.

 More particularly, the invention relates to a method of diagnosis, prognosis or monitoring the development of a pathology linked to an abnormal expression of the receptor and

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 comprising detecting abnormal expression of the messenger RNA of the receptor using DNA chips.

 The invention relates to kits for quantitative and / or qualitative measurement of the expression of the gene encoding the receptor coupled to G proteins in tissues or cells of a particular tissue. Such kits can be used for the diagnosis, prognosis or therapeutic monitoring of patients presenting, or likely to present a pathology linked to an overexpression or under-expression of the nucleic acid sequence according to the invention.

A kit according to the invention comprises: - either primers according to the invention, in particular for the RT-PCR amplification of the gene encoding the receptor and the quantification of the amplified cDNA; - or a probe according to the invention, in particular for the quantification in Northem Blot of the messenger RNA encoding the receptor coupled to the G proteins.

 Advantageously, the expression of the transcript encoding the receptor coupled to the G proteins in cells or tissues is studied by hybridization of the cDNAs or mRNAs in kits made up of DNA chips, or micro-networks or macro-networks on which are fixed probes specific for the gene of the invention whose expression is sought.

 By DNA chip, it is necessary to understand any type of support allowing the stable fixation of a quantity of nucleic acids, the hybridization of the nucleic acids fixed on the support with a sequence contained in a DNA or RNA extract beforehand. labeled, then quantifying the hybridization. DNA chip technologies are in particular described by Sambrook et al. (see above).

 Likewise, kits allowing the detection of abnormal forms of the receptor coupled to the G proteins are also part of the invention. These tests can be of an immunological nature including the use of antibodies specific for regions of the receptor which may contain abnormal structures.

 These kits can also be a protein chip consisting of a support on which is fixed an amino acid sequence according to the invention or an antibody or

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 antibody fragment directed against a polypeptide or a polypeptide fragment according to the invention. Such protein chips allow the large-scale detection of abnormal structures of several proteins, or the detection of specific interactions of the receptor with other molecules.

 The invention also relates to methods of therapeutic treatment of pathologies linked to an abnormal, excessive or insufficient expression of the receptor coupled to the G proteins encoded by the sequences of the invention, in particular in the hippocampus, the cerebellum, the substantia nigra, the caudate nucleus, the amygdala, the liver, the intestines and the pancreas, characterized in that they comprise the administration of an effective therapeutic dose of an agonist or an antagonist of the G protein-coupled receptor encoded by the sequences of the invention.

 In particular, the invention relates to an antisense nucleotide sequence, capable of inhibiting the expression of the nucleic acid sequences according to the invention, and characterized in that it corresponds to a single-stranded nucleic acid sequence capable of hybridizing under stringent conditions to the strand coding for a nucleic acid sequence according to the invention. It also relates to the use of these sequences for the therapeutic treatment of pathologies linked to excessive expression of the receptor coupled to the G proteins.

 Abnormal functioning of the hippocampus, the amygdala, the substantia nigra, the caudate nucleus, the cerebellum or the pancreas can lead to a certain number of disorders or pathologies, these include obesity, bulimia, anorexia , Parkinson's disease, hypotension, hypertension and psychotic and neurological disorders such as anxiety, schizophrenia, manic depression, delirium, dementia, epilepsy, migraine, insomnia, circadian rhythm disorders and attenuation of cognitive performance.

 Consequently, the invention also relates to the use of the nucleic acid sequences and polypeptides according to the invention for the screening of active molecules in the treatment of diabetes, obesity, bulimia, anorexia , Parkinson's disease, hypotension, hypertension and psychotic and neurological disorders such as anxiety, schizophrenia, manic depression, delirium,

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 dementia, epilepsy, migraine, insomnia, circadian rhythm disorders and attenuation of cognitive performance.

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 DESCRIPTION OF FIGURES Figure1: Sequence of nucleic acids encoding a receptor coupled to G proteins (S3040).

 (SEQ ID NO 1) Figure 2: Amino acid sequence of the receptor coupled to G proteins.

 (SEQ ID NO 2) Figure 3: Hydrophilic profile of the protein encoding the receptor coupled to G proteins.

Figure 4: Tissue expression.

Example 1 Identification and isolation of a nucleic acid sequence encoding a new receptor coupled to the G proteins
Sequences exhibiting homologies with the sequences encoding receptors coupled to the G proteins were sought from the sequences of the HTGS (high throuput genome sequencing) library. For this research, the Lassap V2 sequence comparison software (from the company Gene-IT) was used. This software is based on a Blast 2 type algorithm.

 The screening made it possible to identify a fragment of genomic DNA sequence potentially encoding a receptor coupled to the G proteins. The analysis of the sequence made it possible to reconstruct an open theoretical reading phase containing an initiation codon and a stop codon. .

 The translation of the theoretical nucleotide sequence into amino acids produces a protein sequence of 347 amino acids. This sequence has a certain number of structural criteria typical of receptors coupled to G proteins: including 7 transmembrane domains each containing approximately 20-30 hydrophobic amino acids and the LRY motif present in the third transmembrane domain.

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 Furthermore, the comparison of this sequence with the sequences accessible in the databases using the Clustal W algorithm reveals an identity of 26% with the serotoninergic receptor 5HT-1D.

A cDNA was isolated by RT-PCR from brain mRNA using the sense primers 5'-CGCGGATCCACCATGGGGGATGAGCTGGCACCTTGC-3 'and antisense
5'-GCTCTAGAGCTTACTACTTGTCATCGTCGTCCTTGTAATCGGAAATGG TAAAGATGGCCTGGTGC-3 '.

These primers were defined from the theoretical sequence obtained, and additionally contain the sequences of the restriction sites Bam HI and Xba I (underlined in the sequence of oligonucleotides) allowing cloning oriented in the expression vector named pcDNA3. 1 (+) (Invitrogen). The antisense primer also contains a sequence in phase with that coding for the receptor and coding for the peptide DYKDDDDK.

 The sequence amplified by RT-PCR was inserted into the expression vector pcDNA3.1 (+) then the nucleotide sequence was determined from a chain termination reaction and using an automatic DNA sequencer ABI 377 (Applied Biosystems).

 The nucleotide sequence (S3040) thus obtained is presented in Figure 1 (SEQ ID NO 1). The amino acid sequence, deduced from the nucleotide sequence is presented in Figure 2 (SEQ ID NO 2). The analysis of the hydrophilic profile of the sequence using the Kyte and Doolittle method is presented in Figure 3. It confirms the existence of seven hydrophobic transmembrane domains as well as the presence of the LRY motif in the third transmembrane domain. These results attest that the isolated nucleic acid sequence codes for a new receptor coupled to G proteins.

Example 2 Study of the tissue expression of the mRNA encoding the new receptor
In order on the one hand to verify that the identified sequence actually corresponds to a sequence transcribed under normal physiological conditions and on the other hand to

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 to know the tissues in which the sequence is more particularly transcribed, an analysis of tissue expression was carried out by RT-PCR and hybridization of the specific amplified products.

 The human RNAs used are extracted from the total brain, the hippocampus, the cerebellum, the substantia nigra, the caudate nucleus, the amygdala, the thalamus, the liver, the hypothalamus, the heart, the muscle. skeletal, placenta, uterus, lung, intestines, kidney, pancreas, lymph nodes, bone marrow and adipose tissue. Four micrograms of total RNA are denatured for 10 minutes at 65 ° C. and then immediately placed on ice. The reversion reaction of the first strand cDNA mRNA is carried out in a final volume of 17 μl for 1 h 15 min at 37 ° C. in the presence of First Strand cDNA kit IX buffer (Amersham Pharmacia) supplemented with 1 mM final DTT, 50 ng of dT oligonucleotides and 25 ng of random primers according to the protocol described by the supplier. Five minutes at 65 C are required to inactivate the enzyme.

 The PCR reactions are carried out in the presence of a pair of oligonucleotides located 5 '(5'-GCCTACATTCTCCTCCACATGC-3') and 3 '(5'-AAGCTCCTTGCTCCTC CAGC-3') of the theoretical sequence obtained (S3040). Two microliters of the reverse transcription reaction are amplified with the enzyme Ampli Taq Gold kit (Perkin Elmer) in a volume of 100 l in the presence of 1x buffer, 0.2 mM dNTP, 0.2 M of each oligonucleotide , 10% v / v of DMSO and 2.5 Units of Taq polymerase. The amplification program is as follows: a denaturation step at 94 C for 9 min precedes 30 cycles of denaturation (1 min. At 94 C), hybridization (2 min. At 58 C) and elongation (2 min at 72 C). The PCR ends with a final elongation of 8 min. at 72 C.

 The PCR products are then separated by agarose electrophoresis gel (1%, weight / volume) and transferred to nylon membranes Hybon N + (Amersham). The membranes are hybridized with an oligonucleotide (5'-TCTCACTGATGCTGTCTTCG-3 ') labeled with adCTP 32P (3000 Ci / mmol, NEN). Hybridization continues at 42 ° C overnight with agitation. The membrane is then subjected to 2 washes in SSC 2X / SDS 0.1% buffer at room temperature, then to 2 washes at 42 C for 30 min and finally to a wash at 50 C for 30 min. Surrounded by Saran Wrap plastic film, the membrane is

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 put in the presence of an X-Omat autoradiographic film (Kodak) and the film is revealed in a Hyperprocessor processor (Amersham).

 The results presented in FIG. 4 indicate the expression of the messenger RNA in the various tissues. The receptor coded by the sequence S3040 of FIG. 1 is mainly expressed in the brain including for example the hippocampus, the cerebellum, the substantia nigra, the caudate nucleus, the amygdala, the liver, the intestines, the lungs and the pancreas . However, it is absent in adipose tissue, the heart and the pancreas.

Example 3 Recombinant Vector and Cell Lines Stably Expressing the Sequence Coding for a Receptor Coupled with G Proteins According to the Invention
The cDNA as isolated in Example 1 was cloned into the expression vector pcDNA3.1 (+). This vector, named pS3040, comprises an origin of bacterial replication making it possible to multiply the recombinant plasmid pcDNA3-S3040 flag obtained in large number of copies. The vector further comprises an ampicillin resistance gene for the selection of bacterial clones, a geneticin resistance gene (G418) allowing the selection of clones of eukaryotic cells having incorporated the S3040 cDNA coding for the receptor coupled to proteins G. Finally, the sequences cloned in the vector are under the control of the CMV promoter which allows a strong expression of the cloned gene in the eukaryotic host cell.

 The recombinant plasmid described above is introduced into CHO-K1 cells (American culture collection type, CCL61) maintained in Ham-F12 medium with 10% fetal calf serum, 2 mM glutamine, 500 IU / ml of penicillin and 500 g / ml streptomycin using the lipofectamine transfection method as described by the supplier (Life Technologies, France). The clones expressing the receptor are selected with 500 g / ml of G418 and tested for the expression of the receptor, by an immunofluorescence technique, using an IgG antibody recognizing the peptide DYKDDDDK. These experiments make it possible to confirm the expression of the receptor in the CHO-K1 cell.

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EXAMPLE 4 Screening Method by Identification of Agonist Molecule of a Receptor Coupled with G Proteins Measurement of Intracellular Calcium at FLIPR The CHO-K1 Cells Expressing the Product of the Gene Coded by the Sequence of FIG. a of the G16 protein are dissociated the day before the experiment and seeded (45,000 cells / well) in a volume of 100 l / well in black 96-well culture plates (costar) with light background.

On the day of the experiment, 1001 of calcium kit (Molecular Devices), 2.5 mM final of probenecid and 10 mM final of Hepes buffer are added to each well and the cells are incubated for 1 hour at 37 C. The cell plates are then placed in a fluorometer of the FLIPR type (Fluorometric Imaging Plate Reader). The experiment is carried out at room temperature. The exposure time (0.4 sec) and the intensity of the laser (15 to 18 amps) are adjusted in order to obtain basic fluorescence values between 5000 and 10000 units. The fluorescence measurement is carried out on the entire plate every second for the first 60 seconds and then every 6 seconds for 1 minute.

Ligands of different categories, previously dissolved and distributed at a defined concentration on 96-well plates, are brought into contact with the cells. The addition of the products to be tested is carried out during the experiment at 10 seconds using a distribution device comprising 96 black cones (50 l of a solution [x5] are added to each well at the speed of 70 μl / dry and a height of 200 l). The calcium response is characterized by a transient increase in fluorescence immediately after the addition of the active ligands. Solutions of 10 µM ATP and HBSS buffer are included in each ligand plate and serve as positive and negative control of the calcium response, respectively.

Claims (33)

 CLAIMS 1. A nucleic acid sequence encoding a G protein-coupled receptor, characterized by the following sequence (SEQ ID NO 1): 5'-ATGGGGGATGAGCTGGCACCTTGCCCTGTGGGCACTACAGCTTGGCCGGCCCTGATCC AGCTCATCAGCAAGACACCCTGCATGCCCCAAGCAGCCAGCAACACTTCCTTGGGCCTGGG GGACCTCAGGGTGCCCAGCTCCATGCTGTACTGGCTTTTCCTTCCCTCAAGCCTGCTGGCT GCAGCCACACTGGCTGTCAGCCCCCTGCTGCTGGTGACCATCCTGCGGAACCAACGGCTGC GACAGGAGCCCCACTACCTGCTCCCGGCTAACATCCTGCTCTCAGACCTGGCCTACATTCT CCTCCACATGCTCATCTCCTCCAGCAGCCTGGGTGGCTGGGAGCTGGGCCGCATGGCCTGT GGCATTCTCACTGATGCTGTCTTCGCCGCCTGCACCAGCACCATCCTGTCCTTCACCGCCA TTGTGCTGCACACCTACCTGGCAGTCATCCATCCACTGCGCTACCTCTCCTTCATGTCCCA TGGGGCTGCCTGGAAGGCAGTGGCCCTCATCTGGCTGGTGGCCTGCTGCTTCCCCACATTC CTTATTTGGCTCAGCAAGTGGCAGGATGCCCAGCTGGAGGAGCAAGGAGCTTCATACATCC TACCACCAAGCATGGGCACCCAGCCGGGATGTGGCCTCCTGGTCATTGTTACCTACACCTC CATTCTGTGCGTTCTGTTCCTCTGCACAGCTCTCATTGCCAACTGTTTCTGGAGGATCTAT GCAGAGGCCAAGACTTCAGGCATCTGGGGGCAGGGCTATTCCCGGGCCAGGGGCACCCTGC TGATCCACTCAGTGCTGATCACATTGTACGTGAGCACAGGG GTGGTGTTCTCCCTGGACAT GGTGCTGACCAGGTACCACCACATTGACTCTGGGACTCACACATGGCTCCTGGCAGCTAAC AGTGAGGTACTCATGATGCTTCCCCGTGCCATGCTCCCATACCTGTACCTGCTCCGCCCCGGAGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGCCAGGAGACAGGAGAGAGAGAGAGAGAGAGAGAGAGAGCCAGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGCCAGGAGCCAGGAGCCAGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGCCAGGAGCCAGGAGAGCCAGGAGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGCCAGGAGCCAGGAGCCAGGAGCCAGGCCCC 2. A nucleic acid sequence capable of hybridizing under stringent conditions with the sequence of claim 1 and encoding a polypeptide having essentially the same properties as the receptor encoded by the sequence of claim 1. <Desc / Clms Page number 25> 3. Nucleic acid sequence deduced according to the genetic code for the following amino acid sequence (SEQ ID NO 2): 5 10 15 Met Gly Asp Glu Leu Ala Pro Cys Pro Val Gly Thr Thr Ala Trp 20 25 30 Pro Ala Leu Ile Gln Leu Ile Ser Lys Thr Pro Cys Met Pro Gln 35 40 45 Ala Ala Ser Asn Thr Ser Leu Gly Leu Gly Asp Leu Arg Val Pro 50 55 60 Ser Ser Met Leu Tyr Trp Leu Phe Leu Pro Ser Ser Leu Leu Ala 65 70 75 Ala Ala Thr Leu Ala Val Ser Pro Leu Leu Leu Val Thr Ile Leu 80 85 90 Arg Asn Gln Arg Leu Arg Gln Glu Pro His Tyr Leu Leu Pro Ala 95 100 105 Asn Ile Leu Leu Ser Asp Leu Ala Tyr Ile Leu Leu His Met Leu 110 115 120 Ile Ser Ser Ser Ser Leu Gly Gly Trp Glu Leu Gly Arg Met Ala 125 130 135 Cys Gly Ile Leu Thr Asp Ala Val Phe Ala Ala Cys Thr Ser Thr 140 145 150 Ile Leu Ser Phe Thr Ala Ile Val Leu His Thr Tyr Leu Ala Val 155 160 165 Ile His Pro Leu Arg Tyr Leu Ser Phe Met Ser His Gly Ala Ala 170 175 180 Trp Lys Ala Val Ala Leu Ile Trp Leu Val Ala Cys Cys Phe Pro 185 190 195 Thr Phe Leu Ile Trp Leu Ser Lys Trp Gln Asp Ala Gln Leu Glu 200 205 210 Glu Gln Gly Ala Ser Tyr Ile Leu Pro Pro Ser Met Gly Thr Gln <Desc / Clms Page number 26> 335 340 345 Val Arg Gly His Leu Pro Ser Arg Arg His Gln Ala Ile Phe Thr Ile Ser ***> 4. Antisense nucleic acid sequence capable of inhibiting the expression of a nucleic acid sequence according to one of claims 1 to 3, characterized in that it corresponds to a simple nucleic acid sequence strand, capable of hybridizing under stringent conditions to the strand coding for the nucleic acid sequence according to one of claims 1 to 3. 320 325 330 Leu Pro Tyr Leu Tyr Leu Leu Arg Arg Arg Arg Gln Leu Leu Gly Met 305 310 315 Leu Ala Ala Asn Ser Glu Val Leu Met Met Leu Pro Arg Ala Met 290 295 300 Leu Thr Arg Tyr His Hia Ile Asp Ser Gly Thr His Thr Trp Leu 275 280 285 Thr Leu Tyr Val Ser Thr Gly Val Val Phe Ser Leu Asp Met Val 260 265 270 Tyr Ser Arg Ala Arg Gly Thr Leu Leu Ile His Ser Val Leu Ile 245 250 255 Arg Ile Tyr Ala Glu Ala Lys Thr Ser Gly Ile Trp Gly Gln Gly 230 235 240 Cys Val Leu Phe Leu Cys Thr Ala Leu Ile Ala Asn Cys Phe Trp 215 220 225 Pro Gly Cys Gly Leu Leu Val Ile Val Thr Tyr Thr Ser Ile Leu 5. Recombinant vector characterized in that it comprises a nucleic acid sequence according to one of claims 1 to 3. 6. Recombinant vector according to claim 5, characterized in that the vector is a plasmid comprising the sequences necessary for the expression of the receptor coupled to the G proteins in a host cell. <Desc / Clms Page number 27> 7. Recombinant vector according to claim 6, characterized in that the sequences necessary for the expression of the receptor coupled to the G proteins comprise a sequence of the cytomegalovirus promoter (CMV) or a sequence of the promoter SV40. 8. Plasmid pS3040 deposited at the CNCM on March 12, 2001 under the number I-2648. 9. Host cell characterized in that it is transformed by the recombinant vector according to one of claims 5 to 8. 10. Host cell according to claim 9, characterized in that said host cell is a bacterium. 11. Host cell according to claim 9, characterized in that said host cell is a eukaryotic cell. 12. Host cell according to claim 11, characterized in that said host cell comes from a CHO-K1 line (American type culture collection, CCL61). 13. Host cell according to claim 12, characterized in that said host cell additionally expresses the α subunit of protein G in a stable or transient manner. 14. Polypeptide characterized in that it is coded by a nucleic acid sequence according to one of claims 1 to 3. 15. A polypeptide according to claim 14 comprising the following amino acid sequence (SEQ ID NO 2): 5 10 15 Met Gly Asp Glu Leu Ala Pro Cys Pro Val Gly Thr Thr Ala Trp 20 25 30 Pro Ala Leu Ile Gln Leu Ile Ser Lys Thr Pro Cys Met Pro Gln 35 40 45 Ala Ala Ser Asn Thr Ser Leu Gly Leu Gly Asp Leu Arg Val Pro 50 55 60 Ser Ser Met Leu Tyr Trp Leu Phe Leu Pro Ser Ser Leu Leu Ala <Desc / Clms Page number 28> 290 295 300 Leu Thr Arg Tyr His His Ile Asp Ser Gly Thr His Thr Trp Leu 275 280 285 Thr Leu Tyr Val Ser Thr Gly Val Val Phe Ser Leu Asp Met Val 260 265 270 Tyr Ser Arg Ala Arg Gly Thr Leu Leu Ile His Ser Val Leu Ile 245 250 255 Arg Ile Tyr Ala Glu Ala Lys Thr Ser Gly Ile Trp Gly Gln Gly 230 235 240 Cys Val Leu Phe Leu Cys Thr Ala Leu Ile Ala Asn Cys Phe Trp 215 220 225 Pro Gly Cys Gly Leu Leu Val Ile Val Thr Tyr Thr Ser Ile Leu 200 205 210 Glu Gln Gly Ala Ser Tyr Ile Leu Pro Pro Ser Met Gly Thr Gln 185 190 195 Thr Phe Leu Ile Trp Leu Ser Lys Trp Gln Asp Ala Gin Leu Glu 170 175 180 Trp Lys Ala Val Ala Leu Ile Trp Leu Val Ala Cys Cys Phe Pro 155 160 165 Ile His Pro Leu Arg Tyr Leu Ser Phe Met Ser His Gly Ala Ala 140 145 150 Ile Leu Ser Phe Thr Ala Ile Val Leu His Thr Tyr Leu Ala Val 125 130 135 Cys Gly Ile Leu Thr Asp Ala Val Phe Ala Ala Cys Thr Ser Thr 110 115 120 Ile Ser Ser Ser Ser Leu Gly Gly Trp Glu Leu Gly Arg Met Ala 95 100 105 Asn Ile Leu Leu Ser Asp Leu Ala Tyr Ile Leu Leu His Met Leu 80 85 90 Arg Asn Gln Arg Leu Arg Gln Glu Pro His Tyr Leu Leu Pro Ala 65 70 75 Ala Ala Thr Leu Ala Val Ser Pro Leu Leu Leu Val Thr Ile Leu <Desc / Clms Page number 29> 335 340 345 Val Arg Gly His Leu Pro Ser Arg Arg His Gln Ala Ile Phe Thr Ile Ser ***> 16. Polypeptide characterized in that it corresponds to a fragment of a polypeptide according to one of claims 14 or 15 and in that it exhibits receptor activity coupled to G proteins. 320 325 330 Leu Pro Tyr Leu Tyr Leu Leu Arg Arg Arg Arg Gln Leu Leu Gly Met 305 310 315 Leu Ala Ala Asn Ser Glu Val Leu Met Met Leu Pro Arg Ala Met 17. Polypeptide characterized in that it corresponds to a fragment of a polypeptide according to one of claims 14 or 15, and in that it is capable of directly stimulating the G proteins. 18. A polypeptide characterized in that it corresponds to a fragment of a polypeptide according to one of claims 14 or 15, and in that it is capable of inhibiting the action of an agonist. 19. Primers for the amplification of a sequence encoding a polypeptide according to one of claims 14 to 16, characterized in that they comprise the following sequences: 5'-GGGGATGAGCTGGCACCTTGC-3 '5'-ATGGTAAAGATGGCCTGGTGC-3' 20. Hybridization probe for the specific recognition of the nucleic acid sequences encoding a receptor coupled to the G proteins according to one of claims 1 or 3, characterized in that it corresponds to a fragment with a length of at least at least 50 nucleotides, preferably between 150 and 250 nucleotides, of a nucleic acid sequence according to one of claims 1 or 3. 21. Antibody characterized in that it is directed: <Desc / Clms Page number 30>  - either against a polypeptide encoded by a nucleic acid sequence according to any one of claims 1 to 3; - or against a polypeptide according to any one of claims 14 to 18. 22. Antibody according to claim 21, characterized in that it is a monoclonal antibody. 23. Method for identifying the presence of agonist molecules of the receptor coupled to G proteins, characterized in that it comprises the following steps: a. the culture of eukaryotic host cells transformed with a recombinant vector according to one of claims 6,7 or 8; b. contacting the host cells in culture with a candidate compound; vs. determining a possible signal generated by the receptor coupled to the G proteins in the presence of the candidate compound. 24. A method of screening an agonist of a receptor coupled to G proteins, characterized in that it comprises the following steps: a. the culture of host cells according to claim 13 in the presence or absence of one or more molecules to be screened; b. quantifying the concentration of intracellular calcium in cells in culture in the presence and absence of the molecules to be wired; vs. the selection of molecules in the presence of which the concentration of intracellular calcium is increased. 25. A method of identifying the presence of antagonistic molecules comprising the following steps: a. the culture of eukaryotic host cells transformed by a recombinant vector according to one of claims 5 to 8 under conditions allowing expression of the receptor coupled to G proteins in the presence of an agonist; b. bringing the host cells in culture into contact with a candidate compound; <Desc / Clms Page number 31>  vs. determining a decrease in the possible signal generated by the G protein-coupled receptor in the presence of the candidate compound and the agonist compared to the signal generated with the agonist alone. 26. A method of identifying pathological allelic forms of a gene encoding a receptor coupled to G proteins, said method comprising the following steps: a. the isolation of a nucleic acid sequence encoding a receptor coupled to the G proteins according to one of claims 1 or 3 from total nucleic acids (RNA or DNA) originating from patients suffering from a pathology and from subjects healthy; b. the sequencing of the nucleic acids isolated in a); vs. the detection of one or more mutation (s) common to the nucleic acids originating from patients suffering from a pathology, the said mutation (s) not being present on the nucleic acids originating from the healthy subjects. 27. A method of identifying sequences homologous to the nucleic acid sequence according to claim 1 in another species comprising the following steps: a. incubation under stringent conditions of a hybridization probe according to claim 20 with genomic DNA or extracts of total RNA from non-human mammalian species; b. detecting a hybridization reaction; vs. isolating the homologous nucleic acid sequence from the genomic DNA of the species for which a hybridization reaction has been detected. 28. Use of a nucleic acid sequence according to one of claims 1 to 3 for the screening of an agonist of a receptor coupled to the protein G. 29. Use of the host cells according to claim 13 for the screening of a G protein-coupled receptor agonist. <Desc / Clms Page number 32>  30. Kit for the diagnosis, prognosis or therapeutic follow-up of patients presenting, or likely to present, a pathology linked to an overexpression or under-expression of the nucleic acid sequence according to one of claims 1 to 3, characterized in that it comprises: - either primers according to claim 19; - or a probe according to claim 20; 31. DNA chip containing sequences according to one of claims 1 to 3. 32. Kit for the detection of an abnormal structure of the receptor coupled to G proteins, characterized in that it comprises: - either antibodies according to one of claims 21 or 22; - or polypeptides according to one of claims 14 to 18 33. Protein chip containing an antibody according to one of claims 21 or 22 or a polypeptide according to one of claims 14 to 18.