WO2001098330A2

WO2001098330A2 - A RECOMBINANT CELL LINE EXPRESSING GPCRx11 AS A FUNCTIONAL RECEPTOR VALIDATED BY ANGIOPEPTIN AND USEFUL FOR SCREENING OF AGONISTS AND ANTAGONISTS

Info

Publication number: WO2001098330A2
Application number: PCT/BE2001/000104
Authority: WO
Inventors: Vincent Lannoy; Stéphane BREZILLON; Michel Detheux; Marc Parmentier; Cédric GOVARTS
Original assignee: Euroscreen S.A.
Priority date: 2000-06-20
Filing date: 2001-06-20
Publication date: 2001-12-27
Also published as: EP1297003A2; JP2004500125A; AU2001265717A1; CA2413435A1; WO2001098330A3

Abstract

The present invention is related to a G-protein coupled receptor or GPCRx11 similar to rat RTA receptor (37 %) and expressed in testis, thymus and uterus. Aequorin cell line expressing GPCRx11 has been used for screening of tissue extracts and reference ligands. GPCRx11 cells gave a specific signal with synthetic angiopeptin and a somatostatin analog allowing to validate this cell line for screening of natural or synthetic agonists and antagonists. In parallel, extended tissue distribution and polyclonal antibodies have been produced to facilitate GPCRx11 characterisation.

Description

A RECOMBINANT CELL LINE EXPRESSING GPCRxll AS A FUNCTIONAL RECEPTOR VALIDATED BY ANGIOPEPTIN AND USEFUL FOR SCREENING

OF AGONISTS AND ANTAGONISTS

Field of the invention

[0001] The present invention is related to a newly identified member of the superfamily of G-protein-coupled receptors as well as to the various uses that can be made of said receptor.

[0002] The invention is also related to the polynucleic acid sequence (polynucleotide) encoding said receptor.

[0003] The invention is further related to methods using receptor polypeptide and polynucleotide applicable to diagnostic and treatment in receptor-mediated disorders.

[0004] The invention is further related to drug- screening methods using the receptor polypeptide and polynucleotide, to identify agonists and antagonists applicable to diagnostic, prevention and/or treatment of said various disorders.

[0005] The invention further encompasses unknown agonists and antagonists detected and recovered based on the receptor polypeptide and polynucleotide. [0006] The invention is further related to procedures for producing the receptor polypeptide and polynucleotide according to the invention, preferably by genetic recombinant methods. Background of the invention

[0007] G-protein coupled receptors (GPCRs) are proteins responsible for transducing a signal within a cell. GPCRs have usually seven transme brane domains. Upon binding of a ligand to an extra-cellular portion or fragment of a GPCR, a signal is transduced within the cell that results in a change in a biological or physiological property or behaviour of the cell. GPCRs, along with G- proteins and effectors (intracellular enzymes and channels modulated by G-proteins) , are the components of a modular signalling system that connects the state of intra-cellular second messengers to extra-cellular inputs.

[0008] GPCR genes and gene products are potential causative agents of disease and these receptors seem to be of critical importance to both the central nervous system and peripheral physiological processes .

[0009] The GPCR protein superfamily is represented in five families : Family I, receptors typified by rhodopsin and the beta2-adrenergic receptor and currently represented by over 200 unique members; Family II, the parathyroid hormone/calcitonin/secretin receptor family; Family III, the metabotropic glutamate receptor family, Family IV, the CAMP receptor family, important in the chemotaxis and development of D. discoideum; and Family V, the fungal mating pheromone receptor such as STE2.

[0010] G proteins represent a family of heterotrimeric proteins composed of α, β and γ subunits, that bind guanine nucleotides. These proteins are usually linked to cell surface receptors (receptors containing seven transmembrane domains) .

[0011] Following ligand binding to ^• the GPCR, a conformational change is transmitted to the G protein, which caused the α-subunit to exchange a bound GDP molecule for a GTP molecule and to dissociate from the βγ-subunits.

[0012] The GTP-bound form of the α, β and γ-subunits typically functions as an effector-modulating moiety, leading to the production of second messengers, such as cAMP (e.g. by activation of adenyl cyclase) , diacylglycerol or inositol phosphates.

[0013] Greater than 20 different types of α-subunits are known in humans. These subunits associate with a small pool of β and γ subunits. Examples of mammalian G proteins include Gi, Go, Gq, Gs and Gt . G proteins are described extensively in Lodish et al . , Molecular Cell Biology, (Scientific American Books Inc., New York, N.Y., 1995) , the contents of which are incorporated herein by reference .

[0014] Known and unknown GPCRs constitute now major targets for drug action and development .

[0015] Therefore, it exists a need for providing new

G protein coupled receptors which could be used for the screening of new agonists and antagonists having advantageous potential prophylactic and therapeutical properties .

[0016] More than 300 GPCRs have been cloned thus far and it is generally assumed that it exists well over 1000 such receptors. Mechanistically, approximately 50-60% of all clinically relevant drugs act by modulating the functions of various GPCRs (Cudermann et al . , J. Mol . Med . , Vol. 73, pages 51-63, 1995). Summary of the invention

[0017] The present invention is related to newly identified member of G-protein-coupled receptor, preferably a human receptor, as well as to the polynucleotide sequence encoding said human receptor described hereafter (SEQ ID

NO. 1 and 2) .

[0018] The present invention is also related to other newly identified members of G-protein-coupled receptors, preferably human receptors, as well as to the polynucleotide sequence encoding said other human receptor described hereafter (SEQ ID NO. 3 to SEQ ID NO . 22) .

[0019] The present invention is also related to nucleotidic and/or amino acid sequence homologous to the sequences corresponding to the receptor described hereafter.

[0020] An homologous sequence (which may exist in other mammal species) means a sequence which presents a high sequence identity or homology (which presents an identity higher than 70%, 75%, 80%, 85%, 90% or 95%) with the complete human sequence described hereafter, and preferably characterised by a similar pharmacology, especially a preference for binding angiopeptin and/or somatostatin analogs.

[0021] Another aspect of the present invention is related to a specific active portion of said sequence. Said active portion could be a receptor which comprises a partial deletion upon the complete nucleotide or amino acid sequence and which still maintains the active site(s) necessary for the binding of specific ligands able to interact with said receptor.

[0022] Homologous sequences of the sequence according to the invention may comprise similar receptors which exist in other animal (rat, mouse, dog, etc.) or specific human populations, but which are involved in the same biochemical pathway.

[0023] Such homologous sequences may comprise addition, deletion or substitution of one or more amino acids or nucleotides, which does not substantially alter the functional characteristics of the receptor according to the invention.

[0024] Thus, the invention encompasses also a receptor and corresponding nucleotide sequence having exactly the same amino acid or nucleotide sequences as shown in the enclosed sequence listing, as well as molecules which differ, but which are retaining the basic qualitative binding properties of the complete receptor according to the invention. [0025] The invention is preferably related to said

(human) receptor characterised by the complete nucleotide and amino acid sequences described hereafter, to unknown

(and not previously described in the state of the art) agonist, reverse agonist and antagonist compounds or inhibitors of said receptor. Preferably, said inhibitors are antisens RNAs, rybozymes or antibodies (or specific hypervariable (FAB, FAB ' 2 , ...) portions thereof) that bind specifically to said receptor or its encoding nucleotide sequence (i.e. that have at least a 10 fold greater affinity for said receptors than any other naturally occurring antibody) . Said specific antibodies are preferably obtained by a process involving the injection of a pharmaceutically acceptable preparation of such amino acid sequence into a animal capable of producing antibodies directed against said receptor.

[0026] For instance, a monoclonal antibody directed to the receptor according to the invention is obtained by injecting of an expression plasmid comprising the DNA encoding said receptor into a mouse and than fusing mouse spleen cells with myeloma cells.

[0027] The present invention is also related to the polynucleotide according to the invention, possibly linked to other expression sequences and incorporated into a vector (plasmids, viruses, liposomes, cationic vesicles,...) and host cells transformed by such vector. [0028] The present invention is also related to the recombinant, preferably human receptor according to the invention, produced by such host cells according to the method well known by the person skilled in the art, as well as a functional assay (diagnostic kit) comprising all the means and media for the identification of the receptor, its nucleotide sequence, as well as agonist, reverse agonist, antagonist and inhibitor of said receptor or its nucleotide sequence. Said diagnostic kit comprises preferably the following elements : the receptor, its encoding nucleotide sequence, antibodies directed against said receptor or its nucleotide sequence, as well as possible agonist, reverse agonist, antagonist or inhibitor compounds of said receptor. Said diagnostic kit comprises means and media for performing said diagnostic preferably through a measure of dosage/activity of said receptor, by genetic analysis of the receptor nucleotide sequence, preferably by RT/PCR or by immuno-analysis, preferably by the use of antibodies directed against said receptor.

[0029] The present invention is also related to a transgenic non-human mammal comprising a partial or total deletion of the genetic sequence encoding the receptor according to the invention, preferably a non human mammal comprising an homologous recombination "knock-out" of the nucleotide sequence (polynucleotide) according to the invention or a transgenic non human mammal overexpressing above natural level said polynucleotide sequence. [0030] Said transgenic non-human mammal can be obtained by methods well known by the person skilled in the art, for instance by the one described in the document WO98/20112 using classical techniques based upon the transfection of embryonic stem cells, preferably according to the method described by Carmeliet et al . , Nature, Vol. 380, p. 435-439, 1996.

[0031] Preferably, in said transgenic non human mammal overexpressing, the polynucleotide according to the invention or active portions thereof has been previously incorporated in a DNA construct with an inducible promoter allowing its overexpression and possibly with tissues and other specific regulatory elements.

[0032] Another aspect of the present invention is related to a method and kit for performing said method for the screening (detection and possibly recovering) of compounds or a natural extract which are unknown (not yet described in the state of the art) or not known to be agonists, reverse agonists, antagonists or inhibitors of natural compounds to the receptor according to the invention, said method comprising : contacting a cell or cell extract from the cell transfected with a vector expressing the polynucleotide encoding the receptor according to the invention or active portion (s) thereof, possibly isolating a membrane fraction from the cell extract or the complete cell with a compound or molecules present in said natural extract under conditions permitting binding of said compound or said mixture of molecules to said receptor, possibly by the activation of a functional response and detecting the presence (and possibly the binding) of said compound or said mixture of molecules to said receptor by means of a bioassay, (preferably a modification in the production of a second messenger or an increase in the receptor activity) in the presence of another compound working as an agonist, reverse agonist, antagonist or inhibitor to the receptor according to the invention and thereby possibly recovering and determining whether said compound or mixture of molecules is (are) able to work as agonist, reverse agonist, antagonist, or inhibitor of the compound to its receptor. [0033] Preferably, the second messenger assay comprises the measurement of intra-cellular cAMP, intracellular inositol phosphates, intra-cellular diacylglycerol concentrations, arachinoid acid concentration, MAP kinase(s) or tyrosine kinase (s) pathways activation or intra-cellular calcium mobilisation.

[0034] Preferably, said bioassay is validated by the addition of angiopeptin and any other suitable related peptides to the receptor according to the invention by a method well-known by the person skilled in the art and described hereafter.

[0035] The screening method according to the invention could be performed by well known methods to the person skilled in the art, preferably by high-throughput screening, diagnostic and dosage devices based upon the method described in the International patent application

WO00/02045 performed upon various solid supports such as micro-titer plates or biochips (microarrays) according to known techniques by the person skilled in the art.

[0036] The present invention is also related .to the known or unknown compound or molecules characterised and possibly recovered by said method for its (their) use as a medicament in therapy and is related to the pharmaceutical composition comprising a sufficient amount of said compound or molecule (s) and a pharmaceutically acceptable carrier or diluent for the preparation of a medicament in the prevention and/or the treatment of various diseases. [0037] In the pharmaceutical composition, the carrier or the adequate pharmaceutical carrier or diluant can be any solid, liquid or gaseous support which is non- toxic and adapted for the administration (in vivo or ex vivo) to the patient, including the human, through various administration roots such as oral administration, intravenous administration, intradermal administration, etc.

[0038] Said pharmaceutical composition may comprise also various vesicles or adjuvants well known by the person skilled in the art, able to modulate the immune response of the patient. The percentage of active compound-molecules/ pharmaceutical carriers can vary, the range being only limited by the tolerance and the efficiency of the active compounds to the patient . Said ranges of administration are also limited by the frequency of administration and the possible side effects of the compound or molecules. [0039] A further aspect of the present invention is related to said unknown compound or molecule (s) identified by said screening method, to the pharmaceutical composition comprising it and to their use in the treatment of viral infections or diseases induced by various viruses or bacteria, the treatment or prevention of disturbances of cell migration, diseases or perturbations of the immune system, including cancer, development of tumours and tumour metastasis, inflammatory and neo-plastic processes, bacterial and fungal infections, for wound and bone healing and dysfunction of regulatory growth functions, pains, diabetes, obesity, anorexia, bulimia, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, angina pectoris, myocardial infarction, restenosis, atherosclerosis, diseases characterised by excessive smooth muscle cell proliferation, aneurysms, wound healing, diseases characterised by loss of smooth muscle cells or reduced smooth muscle cell proliferation, stroke, ischemia, ulcers, allergies, benign prostatic hypertrophy, migraine, vomiting, psychotic and neurological disorders, including anxiety, schizophrenia, maniac depression, depression, delirium, dementia and severe mental retardation, degenerative diseases, neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease, and dyskinasias, such as Huntington's disease or Gilles de la Tourett's syndrome and other related diseases. [0040] Among the mentioned diseases the preferred applications are related to therapeutic agents targeting 7TM receptor that can play a function in preventing, improving or correcting dysfunctions or diseases, including, but not limited to fertility, foetal development, infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV1 and HIV2 , pain, cancer, anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypertension, urinary retention, osteoporosis, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, psychotic and neurological disorders including anxiety, depression, migraine, vomiting, stroke, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette's syndrome.

[0041] This invention relates to the use of a human

G protein-coupled receptor as a screening tool to identify agonists or antagonists of the aequorin luminescence resulting from expression of this receptor. Example 1; Cloning of human GPCRxll receptor [0042] In order to identify and clone novel human GPCR (G-protein coupled receptor) the following approach was used. Sequences of the following GPCR: GPR8 , ChemR23, HM74 and GPR14 were used as queries to search for homologies in public high-throughput genomic sequence databases (NCBI) . [0043] Using the above strategies, a novel human sequence of GPCR was identified. We called this new GPCR: GPCRxll (SEQ ID number 1 and 2) . [0044] In order to clone the GPCRxll sequence we performed a polymerase chain reaction (PCR) on total human genomic DNA. Primers were synthetized based upon the GPCRxll human sequence and were as follows:

SEQ ID 23 GPCRxll fw: 5 ' -ccggaattcaccatggatccaaccaccccg-3 ' SEQ ID 24 GPCRxll rv: 5 ' -ctagtctagactctacaccagactgcttctc-3 '

[0045] Amplification resulted in a fragments of 0.99 kilobase containing the entire coding sequence of the GPCRxll gene. This fragment was subcloned into the pCDNA3 (Invitrogen) vector for DNA sequencing analysis. [0046] Nucleotide and deduced amino acid sequence of human GPCRxll (SEQ ID NO 1)

1 M D P T T P A W G T E S T T V

15

1 ATG GAT CCA ACC ACC CCG GCC TGG GGA ACA GAA AGT ACA ACA GTG 45 16 N G N D Q A L L L L C G K E T

30

46 AAT GGA AAT GAG CAA GCC CTT CTT CTG CTT TGT GGC AAG GAG ACC 90 31 I P V F L I F I A L V G L

45

91 CTG ATC CCG GTC TTC CTG ATC CTT TTC ATT GCC CTG GTC GGG CTG 135 46 V G N G F V L W L G F R M R

60 136 GTA GGA AAC GGG TTT GTG CTC TGG CTC CTG GGC TTC CGC ATG CGC

180

61 R N A F S V Y V L S L A G A D

75 181 AGG AAC GCC TTC TCT GTC TAC GTC CTC AGC CTG GCC GGG GCC GAC

225

76 F L F L C F Q I I N C L V Y L

90 226 TTC CTC TTC CTC TGC TTC CAG ATT ATA AAT TGC CTG GTG TAC CTC

270

91 S N F F C S I S I N F P S F F

105 271 AGT AAC TTC TTC TGT TCC ATC TCC ATC AAT TTC CCT AGC TTC TTC

315

106 T T V M T C A Y L A G L S M L

120 316 ACC ACT GTG ATG ACC TGT GCC TAC CTT GCA GGC CTG AGC ATG CTG

360

121 S T V S T Ξ R C S V L W P I

135 361 AGC ACC GTC AGC ACC GAG CGC TGC CTG TCC GTC CTG TGG CCC ATC

405

136 W Y R C R P R H L S A V V C

150 406 TGG TAT CGC TGC CGC CGC CCC AGA CAC CTG TCA GCG GTC GTG TGT

450

151 V L L A L S L L S I L E G

165 451 GTC CTG CTC TGG GCC CTG TCC CTA CTG CTG AGC ATC TTG GAA GGG

495

166 K F C G F L F S D G D S G W C

180 496 AAG TTC TGT GGC TTC TTA TTT AGT GAT GGT GAC TCT GGT TGG TGT

540

181 Q T F D F I T A A W L I F L F

195

541 CAG ACA TTT GAT TTC ATC ACT GCA GCG TGG CTG ATT TTT TTA TTC

585

196 M V L C G S S L A L L V R I L

210 586 ATG GTT CTC TGT GGG TCC AGT CTG GCC CTG CTG GTC AGG ATC CTC

630

211 C G S R G L P L T R L Y L T I 25 631 TGT GGC TCC AGG GGT CTG CCA CTG ACC AGG CTG TAC CTG ACC ATC

675

226 L L T V L V F L L C G L P F G 40 676 CTG CTC ACA GTG CTG GTG TTC CTC CTC TGC GGC CTG CCC TTT GGC

720

241 I Q F I L I K D S D V 255 721 ATT CAG TGG TTC CTA ATA TTA TGG ATC TGG AAG GAT TCT GAT GTC 765

256 L F C H I H P V S V V L S S L 270 766 TTA TTT TGT CAT ATT CAT CCA GTT TCA GTT GTC CTG TCA TCT CTT

810

271 N S S A N P I I Y F F V G S F 285 811 AAC AGC AGT GCC AAC CCC ATC ATT TAC TTC TTC GTG GGC TCT TTT 855

286 R K Q R L Q Q P I L K L A 300 856 AGG AAG CAG TGG CGG CTG CAG CAG CCG ATC CTC AAG CTG GCT CTC

900

301 Q R A L Q D I A E V D H S E G 315 901 CAG AGG GCT CTG CAG GAC ATT GCT GAG GTG GAT CAC AGT GAA GGA

945

316 C F R Q G T P E M S R S S L V 330 946 TGC TTC CGT CAG GGC ACC CCG GAG ATG TCG AGA AGC AGT CTG GTG

990

331 * 331 991 TAG 993

[0047] Amino acid sequence of human GPCRxll (330 amino acids) (SEQ ID NO: 2) . The seven predicted transmembrane domaines are underlined.

MDPTTPAWGTESTTVNGNDQALLLLCGKETLIPVFLI FIALVGLVGNGFVLW LGFRM RRNAFSVYVLSLAGADFLFLCFQIINCLVYLSNFFCSISINFPSFFTTVMTCAYLAGLS MLSTVSTERCLSV WPIWYRCRRPRHLSAWCVLLWA SLLLSILEGKFCGFLFSDGDS GWCQTFDFITAAW IFLFMVLCGSSLALLVRILCGSRGLPLTRLYLTILLTVLVFLLCG LPFGIQWFLILWIWKDSDVLFCHIHPVSWLSSLNSSANPIIYFFVGSFRKQWRLQQPI LKLALQRALQDIAEVDHSEGCFRQGTPEMSRSSLV [0048] At the amino acid sequence level, the human GPCRxll is 37% identical to the rat RTA receptor. The gene coding GPCRxll is located on chromosome 11.

Alignment of GPCRxll (fig.l) [0049] Alignment of the amino acid sequence of GPCRxll with RTA and other RTA related sequences were performed using ClustalX algorithm. Then, the dendrogram was constucted using TreeView algorithm.

GPR1

RTA

GPCRxll

JVIAS

ΓVIRG

Tissular distribution of GPCRxll [0050] Reverse transcription-polymerase chain reaction (RT-PCR) experiments were carried out using a panel of polyA⁺ RNA (Clontech) . The primers were as follows: GPCRxll sense primer (SEQ ID NO 25: 5'- TTCTCTGTCTACGTCCTCAG-3') and GPCRxll antisense primer (SEQ ID NO 26: 5 ' -GTCCTGTCATCTCTTAACAG-3 ') . The expected size of the amplified DNA band was 586 bp . Approximately 75 ng of poly A⁺ RNA was reverse transcribed with superscript II

(Life Technologies) and used for PCR. PCR was performed under the following conditions: denaturation at 94°C for 3 min, 38 cycles at 94°C for 1 min, 58°C for 2 min and 72°C for 2 min. Aliquots (10 μl) of the PCR reaction were analysed by 1% agarose gel electrophoresis . [0051] GPCRxll mRNA was assayed by RT-PCR in 16 human tissues. A strong band of expected size (586 bp) was detected in testis, at lower levels in uterus and thymus, while not in pituitary gland, spinal cord, pancreas, small intestine, placenta, stomach, liver, lung, spleen, brain, heart, kidney and skeletal muscle.

Functional assay for GPCRxll

[0052] GPCRxll expressing clones have been obtained by transfection of CH0-K1 cells coexpressing mitochondrial apoaequorin and Galphalδ, limit dilution and selection by northern blotting. Positive clones were used for screening with a reference peptidic library containing 250 peptides and neuropeptides at a concentration of 100 nM. A specific activity was obtained with angiopeptin (D-Nal-Cys-Tyr-D- trp-Lys-Val-Cys-Thr-NH2 with a disulfide bridge between the two cysteines) and confirmed by a dose respone curve (see figure 1) . Additional related peptides were tested using the same cells. Amongst the different peptides tested, somatostatin analog (D2-NaI-Cys-Tyr-D-trp-Lys-Val-Cys-D2- NaI-NH2) exhibited similar affinity. Somatostatin 14 has no activity on GPCRxll.

Material . All chemicals were obtained from Sigma, unless stated. The cell culture media were from Gibco BRL and the peptides from bachem

Aequorin assays . CHO-K1 cell lines expressing GPCRxll receptors, Galpha₁₆ and mitochondrial apoaequorin were established. A functional assay based on the luminescence of mitochondrial aequorin following intracellular Ca²⁺ release (1) was performed as described (2) . Briefly, cells were collected from plates with PBS containing 5 mM EDTA, pelleted and resuspended at 5 X 10^s cells/ml in DMEM-F12 medium, incubated with 5 μM Coelenterazine H (Molecular Probes) for 4 hours at room temperature. Cells were then washed in DMEM-F12 medium and resuspended at a concentration of 0.5 X 10^s cells/ml. Cells were then mixed with the peptides and the light emission recorded during 30 sec. using a Microlumat luminometer (Perkin Elmer). Results are expressed as Relative Light Units (RLU) .

Antibodies

[0053] Antibodies directed against GPCRxll have been produced by repeated injections of plasmid encoding GPCRxll to mice. Serum has been collected following 5 injections and used for flow cytometry analysis with cells transfected with GPCRxll. Several sera were positive and can be used for immunohistochemistry and other related applications

Example 2 : Cloning of the other sequences related to

G-protein-coupled receptors

[0054] In order to identify and clone novel human DNA sequences related to GPCR, the following approche was used.

Sequences of the following GPCR: GPR8 , ChemR23, HM74 and GPR14 were used as queries to search for homologies in public high-throughput genomic sequence databases (NCBI) .

[0055] Using the above strategies, ten novel human sequences of GPCR were identified. None of these clones contain introns .-

GPCRX2, SEQ ID NO 3

GPCRX5, SEQ ID NO 5

GPCRX7, SEQ ID NO 7

GPCRX9, SEQ ID NO 9 GPCRX14, SEQ ID NO 11

GPCRxl6, SEQ ID NO 13

GPCRX17, SEQ ID NO 15

GPCRxlδ, SEQ ID NO 17 GPCRxl9, SEQ ID NO 19 GPCRX20, SEQ ID NO 21

[0056] In order to clone these GPCRx sequences, a polymerase chain reaction (PCR) was performed on total human genomic DNA. Primers were synthetized based upon the human sequences described above and were as follows:

SEQ ID NO 27 GPCRx2 fw: 5 ' -ccggaattcaccatggagtcctcacccatc-3 ' SEQ ID NO 28 GPCRx2 rv: 5' -ctagtctagacatcatgactccagccggg-3 '

SEQ ID NO 29 GPCRx5 fw: 5' -ccggaattcaccatggatccaaccatctcaacc-3 ' SEQ ID NO 30 GPCRxΞ rv: 5' -ctagtctagatcactgctccaatctgcttc-3 '

SEQ ID NO 31 GPCRx7 fw:

5 ' -ccggaattcaccatgaaccagactttgaatagcagtgg-3 '

SEQ ID NO 32 GPCRx7 rv:

5 ' -ctagtctagatctcaagcccccatctcattggtgccc-3 '

SEQ ID NO 33 GPCRx9 fw: 5 ' -ccggaattcaccatggaagctgacctgg-3 ' SEQ ID NO 34 GPCRx9 rv: 5 ' -ctagtctagactcacgtggggcctgcgcc-3 '

SEQ ID NO 35 GPCRxl4 fw: 5 ' -ccggaattcgccatgtacaacgggtcg-3 ' SEQ ID NO 36 GPCRxl4 rv: 5 ' -ctagtctagattcagtgccactcaacaatg-3 ' [0057] Amplification resulted in a fragments of approximately 1 - 1.5 kilobase containing the entire coding sequence of the human genes . These fragments obtained were subcloned into the pCDNA3 (Invitrogen) vector for DNA sequencing analysis.

Tissue distribution of identified (GPCRx) receptors [0058] To determine the tissue distribution of different

GPCRx mRNA, reverse transcriptase -polymerase chaine reaction (RT-PCR) were performed with 200 ng of mRNA isolated from human tissues (Clontech) . The oligo (dT) primer was used in the reverse transcription step. Then, different GPCRx cDNA were amplified with specifics primers.

Table 1: Tissue distribution of GPCRxs: The presence or absence of differents GPCRx was determined by RT-PCR analysis. ++, strong signal; +, signal clearly detected;

+/-, weak signal; -, signal not detected. The tissues are the following: Li, liver; Lu, lung; Sp, Spleen; Te, testis;

Br, Brain; He, Heart; Ki, Kidney; Sk.M, Skeletal muscle;

Pi.G, Pituitary gland; Sp.C, spinal cord; Th, Thymus; Pa, Pancreas; S.In, Small intestine; Ut, Uterus; Pi, Plancenta;

St, Stomach. Reference

1. Stables, J., A. Green, F. Marshall, N. Fraser, E. Knight, M. Sautel, G. Milligan, M. Lee, and S. Rees. 1997. A bioluminescent assay for agonist activity at potentially any G-protein-coupled receptor. Anal . Biochem . 252:115-126.

2. Blanpain, C, I. Migeotte, B. Lee, J. Vakili, B.J. Doranz, C. Govaerts, G. Vassart, R.W. Doms, and M. Parmentier. 1999 CCR5 binds multiple CC-chemokines : MCP- 3 acts as a natural antagonist. Blood 94:1899-1905.

Nucleotide and deduced amino acid sequence of human GPCRx2 (SEQ ID NO : 3 and 4 respectively)

1 M E S S P I P Q S S G N S S T 15

1 ATG GAG TCC TCA CCC ATC CCC CAG TCA TCA GGG AAC TCT TCC ACT 45

16 L G R V P Q T P G P S T A S G 30

46 TTG GGG AGG GTC CCT CAA ACC CCA GGT CCC TCT ACT GCC AGT GGG 90

31 V P E V G L R D V A S E S V A 45

91 GTC CCG GAG GTG GGG CTA CGG GAT GTT GCT TCG GAA TCT GTG GCC 135

46 L F F M L L D L T A V A G N 60

136 CTC TTC TTC ATG CTC CTG CTG GAC TTG ACT GCT GTG GCT GGC AAT 180

61 A A V M A V I A K T P A L R K 75

181 GCC GCT GTG ATG GCC GTG ATC GCC AAG ACG CCT GCC CTC CGA AAA 225

76 F V F V F H L C V D L L A A 90

226 TTT GTC TTC GTC TTC CAC CTC TGC CTG GTG GAC CTG CTG GCT GCC 270

91 T M P L A M L S S S A L F 105

271 CTG ACC CTC ATG CCC CTG GCC ATG CTC TCC AGC TCT GCC CTC TTT 315

106 D H A L F G E V A C R L Y L F 120

316 GAC CAC GCC CTC TTT GGG GAG GTG GCC TGC CGC CTC TAC TTG TTT 360

121 L S V C F V S L A I L S V S A 135

361 CTG AGC GTG TGC TTT GTC AGC CTG GCC ATC CTC TCG GTG TCA GCC 405

136 I N V E R Y Y Y V V H P M R Y 150

406 ATC AAT GTG GAG CGC TAC TAT TAC GTA GTC CAC CCC ATG CGC TAC 450

151 Ξ V R M T L G L V A S V L V G 165

451 GAG GTG CGC ATG ACG CTG GGG CTG GTG GCC TCT GTG CTG GTG GGT 495

166 V W V K A L A M A S V P V G 180

496 GTG TGG GTG AAG GCC TTG GCC ATG GCT TCT GTG CCA GTG TTG GGA 540

181 R V S E E G A P S V P P G C 195

541 AGG GTC TCC TGG GAG GAA GGA GCT CCC AGT GTC CCC CCA GGC TGT 585

196 S L Q S H S A Y C Q L F V V 210

586 TCA CTC CAG TGG AGC CAC AGT GCC TAC TGC CAG CTT TTT GTG GTG 630

211 V F A V Y F L L P L L L I 225

631 GTC TTT GCT GTC CTT TAC TTT CTG TTG CCC CTG CTC CTC ATA CTT 675

226 V V Y C S F R V A R V A A M 240

676 GTG GTC TAC TGC AGC ATG TTC CGA GTG GCC CGC GTG GCT GCC ATG 720

241 Q H G P L P T W M E T P R Q R 255

721 CAG CAC GGG CCG CTG CCC ACG TGG ATG GAG ACA CCC CGG CAA CGC 765

256 S E S L S S R S T M V T S S G 270

766 TCC GAA TCT CTC AGC AGC CGC TCC ACG ATG GTC ACC AGC TCG GGG 810 271 A P Q T T P H R T F G G G K A 285

811 GCC CCC CAG ACC ACC CCA CAC CGG ACG TTT GGG GGA GGG AAA GCA 855

286 A V V L L A V G G Q F L C 300

856 GCA GTG GTT CTC CTG GCT GTG GGG GGA CAG TTC CTG CTC TGT TGG 900

301 L P Y F S F H L Y V A L S A Q 315

901 TTG CCC TAC TTC TCT TTC CAC CTC TAT GTT GCC CTG AGT GCT CAG 945

316 P I S T G Q V E S V V T I G 330

946 CCC ATT TCA ACT GGG CAG GTG GAG AGT GTG GTC ACC TGG ATT GGC 990

331 Y F C F T S N P F F Y G C L N 345

991 TAC TTT TGC TTC ACT TCC AAC CCT TTC TTC TAT GGA TGT CTC AAC 1035

346 R Q I R G E L S K Q F V C F F 360

1036 CGG CAG ATC CGG GGG GAG CTC AGC AAG CAG TTT GTC TGC TTC TTC 1080

361 K P A P E E E R L P S R E G 375

1081 AAG CCA GCT CCA GAG GAG GAG CTG AGG CTG CCT AGC CGG GAG GGC 1125

376 S I E E N F Q F L Q G T G C 390

1126 TCC ATT GAG GAG AAC TTC CTG CAG TTC CTT CAG GGG ACT GGC TGT 1170

391 P S E S W V S R P L P S P Q 405

1171 CCT TCT GAG TCC TGG GTT TCC CGA CCC CTA CCC AGC CCC AAG CAG 1215

406_, E P P A V D F R I P G Q I A E 420

1216 GAG CCA CCT GCT GTT GAC TTT CGA ATC CCA GGC CAG ATA GCT GAG 1260

421 E T S E F L E Q Q L T S D I I 435

1261 GAG ACC TCT GAG TTC CTG GAG CAG CAA CTC ACC AGC GAC ATC ATC 1305

436 M S D S Y R P A A S P R E 450

1306 ATG TCA GAC AGC TAC CTC CGT CCT GCC GCC TCA CCC CGG CTG GAG 1350

451 S * 452

1351 TCA TGA 1356

Amino acid sequence of human GPCRx2 (451 amino acids) (SEQ ID NO: 4) . The seven predicted transmembrane domaines are underlined.

MESSPIPQSSGNSST GRVPQTPGPSTASGVPEVGLRDVASESVA FFM LDLTAVAGNAAVMAVIAKTPALRKFVFVF HLCLVDLLAALT MPLAMLSSSALFDHA FGEVACRLY FLSVCFVSLAILSVSAINVERYYYWHPMRYEVR T GLVA SV VGV VKALA ASVPVLGRVSWEEGAPSVPPGCSLQ SHSAYCQIFVvVFAV YF PL LI VVYCSiyiFRVARVAAM QHGP PT METPRQRSESLSSRSTMVTSSGAPQTTPHRTFGGGKAAWLLAVGGQFL C PYFSFHLYVALSAQPISTG QVESWTWIGYFCFTSNPFFYGCLNRQIRGE SKQFVCFFKPAPEEELRLPSREGSIEΞNF QFLQGTGCPSES VSRPL PSPKQEPPAVDFRIPGQIAEETSEFLEQQLTSDIIMSDSYLRPAASPR ΞS

At the amino acid sequence level, the human GPCRx2 is 23% identical to the human histamine H2 receptor. Nucleotide and deduced amino acid sequence of human GPCRxS (SEQ ID NO : 5 and 6 respectively)

1 M D P T I S T L D T E L T P I 15

1 ATG GAT CCA ACC ATC TCA ACC TTG GAC ACA GAA CTG ACA CCA ATC 45

16 G T E Ξ T C Y K Q T S L 30

46 AAC GGA ACT GAG GAG ACT CTT TGC TAC AAG CAG ACC TTG AGC CTC 90

31 T V L T C I V S L V G L T G N 45

91 ACG GTG CTG ACG TGC ATC GTT TCC CTT GTC GGG CTG ACA GGA AAC 135

46 A V V L W L L G C R M R R N A 60

136 GCA GTT GTG CTC TGG CTC CTG GGC TGC CGC ATG CGC AGG AAC GCC 180

61 F S I Y I L N L A A A D F L F 75

181 TTC TCC ATC TAC ATC CTC AAC TTG GCC GCA GCA GAC TTC CTC TTC 225

76 L S G R I Y S L L S F I S I 90

226 CTC AGC GGC CGC CTT ATA TAT TCC CTG TTA AGC TTC ATC AGT ATC 270

91 P H T I S K I L Y P V M M F S 105

271 CCC CAT ACC ATC TCT AAA ATC CTC TAT CCT GTG ATG ATG TTT TCC 315

106 Y F A G S F L S A V S T E R 120

316 TAC TTT GCA GGC CTG AGC TTT CTG AGT GCC GTG AGC ACC GAG CGC 360

121 C L S V W P I W Y R C H R P 135

361 TGC CTG TCC GTC CTG TGG CCC ATC TGG TAC CGC TGC CAC CGC CCC 405

136 T H L S A V V C V L A L S 150

406 ACA CAC CTG TCA GCG GTG GTG TGT GTC CTG CTC TGG GCC CTG TCC 450

151 L R S I L E M L C G F L F 165

451 CTG CTG CGG AGC ATC CTG GAG TGG ATG TTA TGT GGC TTC CTG TTC 495

166 S G A D S A C Q T S D F I T 180

496 AGT GGT GCT GAT TCT GCT TGG TGT CAA ACA TCA GAT TTC ATC ACA 540

181 V A L I F C V V L C G S S 195

541 GTC GCG TGG CTG ATT TTT TTA TGT GTG GTT CTC TGT GGG TCC AGC 585

196 L V I R I L C G S R K I P 210

586 CTG GTC CTG CTG ATC AGG ATT CTC TGT GGA TCC CGG AAG ATA CCG 630

211 L T R L Y V T I L L T V V F 225

631 CTG ACC AGG CTG TAC GTG ACC ATC CTG CTC ACA GTA CTG GTC TTC 675

226 L L C G P F G I Q F F L F 240

676 CTC CTC TGT GGC CTG CCC TTT GGC ATT CAG TTT TTC CTA TTT TTA 720

241 I H V D R E V L F C H V H L 255

721 TGG ATC CAC GTG GAC AGG GAA GTC TTA TTT TGT CAT GTT CAT CTA 765

256 V S I F L S A L N S S A N P I 270

766 GTT TCT ATT TTC CTG TCC GCT CTT AAC AGC AGT GCC AAC CCC ATC 810 271 I Y F F V G S F R Q R Q N R Q 285

811 ATT TAC TTC TTC GTG GGC TCC TTT AGG CAG CGT CAA AAT AGG CAG 855

286 N L K L V L Q R A L Q D A S E 300

856 AAC CTG AAG CTG GTT CTC CAG AGG GCT CTG CAG GAC GCG TCT GAG 900

301 V D E G G G Q L P E E I L E L 315

901 GTG GAT GAA GGT GGA GGG CAG CTT CCT GAG GAA ATC CTG GAG CTG 945

316 S G S R E Q * 323

946 TCG GGA AGC AGA TTG GAG CAG TGA 969

Amino acid sequence of human GPCRx5 (322 amino acids) (SEQ ID NO:6) . The seven predicted transmembrane domaines are underlined.

MDPTISTLDTELTPINGTEETLCYKQT S TV TCIVSLVGLTGNAWL LLGCRMRRNAFSIYILNLAAADF F SGRL IYSLLSFISIPHTISKILYPV MFSYFAG SFLSAVSTERCLSVL PI YRCHRPTH SAVVCVLIi ALS RSI E L CGFLFSGADSA CQTSDFITVA IF CW CGSSLVL IRILCGSRKIPLTR YVTI LTVLVF LCGLPFGIQFFLFL IHVDREVLFCHVHLVSIFLSA NSSANPIIYFFVGSFRQRQNRQN KLV QRAIiQDASΞVDEGGGQ PEEILE SGSRL EQ

At the amino acid sequence level, the human GPCRxS is 31% identical to the human mas receptor.

Nucleotide and deduced amino acid sequence of human GPCRx7 (SEQ ID NO: 7 and ϊ respectively)

1 M N Q T L N S S G T V E S A 15

1 ATG AAC CAG ACT TTG AAT AGC AGT GGG ACC GTG GAG TCA GCC CTA 45

16 Y S R G S T V H T A Y L V L 30

46 AAC TAT TCC AGA GGG AGC ACA GTG CAC ACG GCC TAC CTG GTG CTG 90

31 S S L A M F T C L C G M A G N 45

91 AGC TCC CTG GCC ATG TTC ACC TGC CTG TGC GGG ATG GCA GGC AAC 135

46 S M V I L G F R M H R N P 60

136 AGC ATG GTG ATC TGG CTG CTG GGC TTT CGA ATG CAC AGG AAC CCC 180

61 F C I Y I L N A A A D L L F 75

181 TTC TGC ATC TAT ATC CTC AAC CTG GCG GCA GCC GAC CTC CTC TTC 225

76 F S M A S T L S L E T Q P L 90

226 CTC TTC AGC ATG GCT TCC ACG CTC AGC CTG GAA ACC CAG CCC CTG 270

91 V N T T D K V H E L M K R M 105

271 GTC AAT ACC ACT GAC AAG GTC CAC GAG CTG ATG AAG AGA CTG ATG 315

106 Y F A Y T V G S L T A I S 120

316 TAC TTT GCC TAC ACA GTG GGC CTG AGC CTG CTG ACG GCC ATC AGC 360

121 T Q R C L S V F P I W F K C 135

361 ACC CAG CGC TGT CTC TCT GTC CTC TTC CCT ATC TGG TTC AAG TGT 405

136 H R P R H L S A V C G 150

406 CAC CGG CCC AGG CAC CTG TCA GCC TGG GTG TGT GGC CTG CTG TGG 450

151 T L C L L M N G T S S F C S 165

451 ACA CTC TGT CTC CTG ATG AAC GGG TTG ACC TCT TCC TTC TGC AGC 495

166 K F L K F N E D R C F R V D M 180

496 AAG TTC TTG AAA TTC AAT GAA GAT CGG TGC TTC AGG GTG GAC ATG 540

181 V Q A A I M G V T P V M T 195

541 GTC CAG GCC GCC CTC ATC ATG GGG GTC TTA ACC CCA GTG ATG ACT 585

196 L S S L T L F V V R R S S Q 210

586 CTG TCC AGC CTG ACC CTC TTT GTC TGG GTG CGG AGG AGC TCC CAG 630

211 Q R R Q P T R L F V V V L A 225

631 CAG TGG CGG CGG CAG CCC ACA CGG CTG TTC GTG GTG GTC CTG GCC 675

226 S V L V F L I C S L P L S I Y 240

676 TCT GTC CTG GTG TTC CTC ATC TGT TCC CTG CCT CTG AGC ATC TAC 720

241 F V L Y W L S L P P E M Q V 255

721 TGG TTT GTG CTC TAC TGG TTG AGC CTG CCG CCC GAG ATG CAG GTC 765

256 L C F S L S R L S S S V S S S 270

766 CTG TGC TTC AGC TTG TCA CGC CTC TCC TCG TCC GTA AGC AGC AGC 810 271 A N P V I Y F L V G S R R S H 285

811 GCC AAC CCC GTC ATC TAC TTC CTG GTG GGC AGC CGG AGG AGC CAC 855

286 R P T R S L G T V L Q Q A L 300

856 AGG CTG CCC ACC AGG TCC CTG GGG ACT GTG CTC CAA CAG GCG CTT 900

301 R E E P E L E G G E T P T V G 315

901 CGC GAG GAG CCC GAG CTG GAA GGT GGG GAG ACG CCC ACC GTG GGC 945

316 T N E M G A * 322

946 ACC AAT GAG ATG GGG GCT TGA 966

Amino acid sequence of human GPCRx7 (321 amino acids) (SEQ ID N0.-8) . The seven predicted transmembrane domaines are underlined.

I^QTLNSSGTVESALNYSRGSTVHTAYLVLSSLAMFTCLCGMAGNSMVIWLLGFRMHRNPFCIYILNLAAADLLFLFSMA STLSLETQPLVNTTDKVHELMKRLMYFAYTVGLSLLTAISTQRCLSVLFPI fKCHRPRHLSA VCGLLWTLCLLMNGLT SSFCS KFLKFNEDRCFRVDMVQAALIMGVLTPVMTLSSLTLFVWVRRS S QQ RRQPTRLFVWLASVLVFLI CSLPLS I Y FVLY LSLPPEMQVLCFSLSRLSSSVSSSANPVIYFLVGSRRSHRLPTRSLGTVLQQALiREEPELEGGETPTVGTNEMG A

At the amino acid sequence level, the human GPCRx7 is 29% identical to the rat RTA receptor.

Nucleotide and deduced amino acid sequence of human GPCRx9 (SEQ ID NO: 9 and 10 respectively)

1 M E A D L G A T G H R P R T E 15

1 ATG GAA GCT GAC CTG GGT GCC ACT GGC CAC AGG CCC CGC ACA GAG 45

16 L D D E D S Y P Q G G W D T V 30

46 CTT GAT GAT GAG GAC TCC TAC CCC CAA GGT GGC TGG GAC ACG GTC 90

31 F L V A L L L G L P A N G L 45

91 TTC CTG GTG GCC CTG CTG CTC CTT GGG CTG CCA GCC AAT GGG TTG 135

46 M A W L A G S Q A R H G A G T 60

136 ATG GCG TGG CTG GCC GGC TCC CAG GCC CGG CAT GGA GCT GGC ACG 180

61 R L A L L L L S L A L S D F L 75

181 CGT CTG GCG CTG CTC CTG CTC AGC CTG GCC CTC TCT GAC TTC TTG 225

76 F L A A A A F Q I L E I R H G 90

226 TTC CTG GCA GCA GCG GCC TTC CAG ATC CTA GAG ATC CGG CAT GGG 270

91 G H W P L G T A A C R F Y Y F 105

271 GGA CAC TGG CCG CTG GGG ACA GCT GCC TGC CGC TTC TAC TAC TTC 315

106 L W G V S Y S S G L F L L A A 120

316 CTA TGG GGC GTG TCC TAC TCC TCC GGC CTC TTC CTG CTG GCC GCC 360

121 L S L D R C L L A L C P H Y 135

361 CTC AGC CTC GAC CGC TGC CTG CTG GCG CTG TGC CCA CAC TGG TAC 405

136 P G H R P V R L P L W V C A G 150

406 CCT GGG CAC CGC CCA GTC CGC CTG CCC CTC TGG GTC TGC GCC GGT 450

151 V W V L A T L F S V P W L V F 165

451 GTC TGG GTG CTG GCC ACA CTC TTC AGC GTG CCC TGG CTG GTC TTC 495

166 P E A A V W Y D L V I C L D 180

496 CCC GAG GCT GCC GTC TGG TGG TAC GAC CTG GTC ATC TGC CTG GAC 540

181 F W D S E E L S L R M L E V L) 195

541 TTC TGG GAC AGC GAG GAG CTG TCG CTG AGG ATG CTG GAG GTC CTG 585

196 G G F L P F L L L L V C H V L 210

586 GGG GGC TTC CTG CCT TTC CTC CTG CTG CTC GTC TGC CAC GTG CTC 630

211 T Q A T A C R T C H R Q Q Q P 225

631 ACC CAG GCC ACA GCC TGT CGC ACC TGC CAC CGC CAA CAG CAG CCC 675

226 A A C R G F A R V A R T I L S 240

676 GCA GCC TGC CGG GGC TTC GCC CGT GTG GCC AGG ACC ATT CTG TCA 720

241 A Y V V L R L P Y Q L A Q L L 255

721 GCC TAT GTG GTC CTG AGG CTG CCC TAC CAG CTG GCC CAG CTG ^' CTC 765

256 Y L A F L D V Y S G Y L L .270

766 TAC CTG GCC TTC CTG TGG GAC GTC TAC TCT GGC TAC CTG CTC TGG 810 271 E A L V Y S D Y L I L L N S C 285 811 GAG GCC CTG GTC TAC TCC GAC TAC CTG ATC CTA CTC AAC AGC TGC 855

286 L S P F L C L M A S A D L R T 300 856 CTC AGC CCC TTC CTC TGC CTC ATG GCC AGT GCC GAC CTC CGG ACC 900

301 L L R S V L S S F A A A L C E 315 901 CTG CTG CGC TCC GTG CTC TCG TCC TTC GCG GCA GCT CTC TGC GAG 945

316 E R P G S F T P T E P Q T Q L 330 946 GAG CGG CCG GGC AGC TTC ACG CCC ACT GAG CCA CAG ACC CAG CTA 990

331 D S E G P T L P E P A E A Q 345 991 GAT TCT GAG GGT CCA ACT CTG CCA GAG CCG ATG GCA GAG GCC CAG 1035

346 S Q M D P V A Q P Q V N P T L 360 1036 TCA CAG ATG GAT CCT GTG GCC CAG CCT CAG GTG AAC CCC ACA CTC 1080

361 Q P R S D P T A Q P Q L N P T 375 1081 CAG CCA CGA TCG GAT CCC ACA GCT CAG CCA CAG CTG AAC CCT ACG 1125

376 A Q P Q S D P T A Q P Q L N L 390 1126 GCC CAG CCA CAG TCG GAT CCC ACA GCC CAG CCA CAG CTG AAC CTC 1170

391 M A Q P Q S D S V A Q P Q A D 405 1171 ATG GCC CAG CCA CAG TCA GAT TCT GTG GCC CAG CCA CAG GCA GAC 1215

406 T N V Q T P A P A A S S V P S 420 1216 ACT AAC GTC CAG ACC CCT GCA CCT GCT GCC AGT TCT GTG CCC AGT 1260

421 P C D E A S P T P S S H P T P 435 1261 CCC TGT GAT GAA GCT TCC CCA ACC CCA TCC TCG CAT CCT ACC CCA 1305

436 G A L E D P A T P P A S E G Ξ 450 1306 GGG GCC CTT GAG GAC CCA GCC ACA CCT CCT GCC TCT GAA GGA GAA 1350

451 S P S S T P P E A A P G A G P 465 1351 AGC CCC AGC AGC ACC CCG CCA GAG GCG GCC CCG GGC GCA GGC CCC 1395

466 T * 467 1396 ACG TGA 1401

Amino acid sequence of human GPCRx9 (466 amino acids) (SEQ ID NO: 10). The six predicted transmembrane domaines are underlined.

MEADLGATGHRPRTELDDEDSYPQGGWDTVFLVALLLLGLPANGL A LAGSQARHGAGTRLALLLLSLALSDFLFLAAA AFQILEIRHGGHWPLGTAACRFYYFLWGVSYSSGLFLLAALSLDRCLLALCPHWYPGHRPVRLPLWVCAGVWVLATLFSV P LVFPEAAVW YDLVICLDFWDSEELSLR LEVLGGFLPFLLLLVCHVLTQATACRTCHRQQQPAACRGPARVARTILS AYWLRLPYQLAQLLYLAFLWDVYSGYLL EALVYSDYLILLNSCLSPFLCLMASADLRTLLRSVLSSFAAALCEERPGS FTPTEPQTQLDSEGPTLPEPMAEAQSQ DPVAQPQVNPTLQPRSDPTAQPQLNPTAQPQSDPTAQPQLNLMAQPQSDSVA QPQADTNVQTPAPAASSVPSPCDEASPTPSSHPTPGALEDPATPPASEGESPSSTPPEAAPGAGPT

At the amino acid sequence level, the human GPCRx9 is 33% identical to the human ChemR23 receptor. Nucleotide and deduced amino acid sequence of human GPCRxl4 (SEQ ID NO: 11 and 12 respectively)

1 M Y N G S C C R I E G D T I S 15

1 ATG TAC AAC GGG TCG TGC TGC CGC ATC GAG GGG GAC ACC ATC TCC 45

16 Q V M P P L L I V A F V L G A 30

46 CAG GTG ATG CCG CCG CTG CTC ATT GTG GCC TTT GTG CTG GGC GCA 90

31 L G N G V A L C G F C F H M K 45

91 CTA GGC AAT GGG GTC GCC CTG TGT GGT TTC TGC TTC CAC ATG AAG 135

46 T W K P S T V Y L F N L A V A 60

136 ACC TGG AAG CCC AGC ACT GTT TAC CTT TTC AAT TTG GCC GTG GCT 180

61 D F L L M I C L P F R T D Y Y 75

181 GAT TTC CTC CTT ATG ATC TGC CTG CCT TTT CGG ACA GAC TAT TAC 225

76 L R R R H W A F G D I P C R V 90

226 CTC AGA CGT AGA CAC TGG GCT TTT GGG GAC ATT CCC TGC CGA GTG 270

91 G L F T L A N R A G S I V F 105

271 GGG CTC TTC ACG TTG GCC ATG AAC AGG GCC GGG AGC ATC GTG TTC 315

106 L T V V A A D R Y F K V V H P 120

316 CTT ACG GTG GTG GCT GCG GAC AGG TAT TTC AAA GTG GTC CAC CCC 360

121 H H A V N T I S T R V A A G I 135

361 CAC CAC GCG GTG AAC ACT ATC TCC ACC CGG GTG GCG GCT GGC ATC 405

136 V C T L A L V I L G T V Y L 150

406 GTC TGC ACC CTG TGG GCC CTG GTC ATC CTG GGA ACA GTG TAT CTT 450

151 L L E N H L C V Q E T A V S C 165

451 TTG CTG GAG AAC CAT CTC TGC GTG CAA GAG ACG GCC GTC TCC TGT 495

166 E S F I M E S A N G W H D I M 180

496 GAG AGC TTC ATC ATG GAG TCG GCC AAT GGC TGG CAT GAC ATC ATG 540

181 F Q L E F F M P L G I I L F C 195

541 TTC CAG CTG GAG TTC TTT ATG CCC CTC GGC ATC ATC TTA TTT TGC 585

196 S F I V S L R R R Q Q L A 210

586 TCC TTC AAG ATT GTT TGG AGC CTG AGG CGG AGG CAG CAG CTG GCC 630

211 R Q A R K K A T R F I M V V 225

631 AGA CAG GCT CGG ATG AAG AAG GCG ACC CGG TTC ATC ATG GTG GTG 675

226 A I V F I T C Y L P S V S A R 240

676 GCA ATT GTG TTC ATC ACA TGC TAC CTG CCC AGC GTG TCT GCT AGA 720

241 L Y F L W T V P S S A C D P S 255

721 CTC TAT TTC CTC TGG ACG GTG CCC TCG AGT GCC TGC GAT CCC TCT 765

256 V H G A L H I T L S F T Y M N 270

766 GTC CAT GGG GCC CTG CAC ATA ACC CTC AGC TTC ACC TAC ATG AAC 810 271 S M L D P L V Y Y F S S P S F 285 811 AGC ATG CTG GAT CCC CTG GTG TAT TAT TTT TCA AGC CCC TCC TTT 855

286 P K F Y N K L K I C S L K P K 300 856 CCC AAA TTC TAC AAC AAG CTC AAA ATC TGC AGT CTG AAA CCC AAG 900

301 Q P G H S K T Q R P E E M P I 315 901 CAG CCA GGA CAC TCA AAA ACA CAA AGG CCG GAA GAG ATG CCA ATT 945

316 S N L G R R S C I S V A N S F 330 946 TCG AAC CTC GGT CGC AGG AGT TGC ATC AGT GTG GCA AAT AGT TTC 990

331 'Q S Q S D G Q W D P H I V E W 345 991 CAA AGC CAG TCT GAT GGG CAA TGG GAT CCC CAC ATT GTT GAG TGG 1035

346 H * 347

1036 CAC TGA 1041

Amino acid sequence of human GPCRX14 (346 amino acids) (SEQ ID NO:12). The seven predicted transmembrane domaines are underlined.

MYNGSCCRIEGDTISQVMPPLLIVAFVLGALGNGVALCGFCFHMKT KPSTVYLFNLAVADFLLMICLPFRTDYYLRRRH AFGDIPCRVGLFTLAMNRAGSIVFLTvVAADRYFKVVHPHHAVNTISTRVAAGIVCTLWALVILGTVYLLLENHLCVQE TAVSCESFIMESANG HDIMFQLEFFMPLGIILFCSFKIVWSLRRRQQLARQARMKKATRFIMWAIVFITCYLPSVSAR LYFLWTVPSSACDPS\ GALHITLSFTYiyaTSMLDPLVYYFSSPSFPKFYNKLKICSLKPKQPGHSKTQRPEEMPISNLGR RSCISVANSFQSQSDGQ DPHIVE H

At the amino acid sequence level, the human GPCRxl4 is 50% identical to the human HM74 receptor.

Nucleotide and deduced amino acid sequence of human GPCRxl6 (SEQ ID NO: 13 and 14 respectively) . This nucleotide sequence is located on the chromosome 4.

1 M G P G E A L L A G L L V M V 15 1 ATG GGC CCC GGC GAG GCG CTG CTG GCG GGT CTC CTG GTG ATG GTA 45

16 L A V A L L S N A L V L L C C 30 46 CTG GCC GTG GCG CTG CTA TCC AAC GCA CTG GTG CTG CTT TGT TGC 90

31 A Y S A E L R T R A S G V L L 45 91 GCC TAC AGC GCT GAG CTC CGC ACT CGA GCC TCA GGC GTC CTC CTG 135

46 V N L S L G H L L L A A L D M 60 136 GTG AAT CTG TCT CTG GGC CAC CTG CTG CTG GCG GCG CTG GAC ATG 180

61 P F T L L G V M R G R T P S A 75 181 CCC TTC ACG CTG CTC GGT GTG ATG CGC GGG CGG ACA CCG TCG GCG 225

76 P G A C Q V I G F L D T F L A 90

226 CCC GGC GCA TGC CAA GTC ATT GGC TTC CTG GAC ACC TTC CTG GCG 270

91 S N A A L S V A A L S A D Q W 105

271 TCC AAC GCG GCG CTG AGC GTG GCG GCG CTG AGC GCA GAC CAG TGG 315

106 L A V G F P L R Y A G R L R P 120 316 CTG GCA GTG GGC TTC CCA CTG CGC TAC GCC GGA CGC CTG CGA CCG 360

121 R Y A G L L L G C A W G Q S L 135

361 CGC TAT GCC GGC CTG CTG CTG GGC TGT GCC TGG GGA CAG TCG CTG 405

136 A F S G A A L G C S L G Y S 150

406 GCC TTC TCA GGC GCT GCA CTT GGC TGC TCG TGG CTT GGC TAC AGC 450

151 S A F A S C S L R L P P E P E 165

451 AGC GCC TTC GCG TCC TGT TCG CTG CGC CTG CCG CCC GAG CCT GAG 495

166 R P R F A A F T A T L H A V G 180 496 CGT CCG CGC TTC GCA GCC TTC ACC GCC ACG CTC CAT GCC GTG GGC 540

181 F V L P L A V L C L T S L Q V 195

541 TTC GTG CTG CCG CTG GCG GTG CTC TGC CTC ACC TCG CTC CAG GTG 585

196 H R V A R R H C Q R M D T V T 210

586 CAC CGG GTG GCA CGC AGA CAC TGC CAG CGC ATG GAC ACC GTC ACC 630

211 M K A L A L L A D L H P R Y 225 631 ATG AAG GCG CTC GCG CTG CTC GCC GAC CTG CAC CCC AGG TAT TGG 675

226 P S A C R Q A Q A R D L G A P 240

676 CCC AGT GCA TGC CGA CAG GCC CAG GCC AGG GAC TTG GGC GCT CCC 720

241 A V G L R S L W A S P P L L 255

721 TGG GCA GTT GGC TTG AGG AGC CTG TGG GCA TCA CCA CCG TTA CTC 765

256 C P E F T S H S T A P A R C S 270 766 TGC CCA GAG TTC ACC AGC CAC AGC ACT GCC CCT GCA CGC TGC TCA 810 271 Q G F P V G S L V Q T L R G P 285

811 CAG GGG TTT CCT GTT GGT TCA TTG GTG CAG ACA CTG CGG GGG CCT 855

286 L P P G I C A H S A Q G A L R 300

856 CTG CCT CCT GGG ATA TGT GCT CAC AGT GCA CAG GGA GCT TTG CGC 900

301 R A V G C A S P G G V P R A L 315

901 AGA GCT GTG GGG TGT GCT TCT CCG GGA GGG GTT CCG CGG GCT CTG 945

316 L A A R H T P P V H G C G S 330

946 CTG TGG GCG GCC AGA CAC ACC CCT CCT GTG CAT GGC TGT GGG TCT 990

331 E A S A C F C P L L T Q C P C 345

991 GAG GCA TCT GCT TGT TTC TGC CCA CTG CTG ACC CAG TGC CCT TGC 1035

346 M D L G F K S * 352

1036 ATG GAC TTG GGC TTC AAG TCT TGA 1059

Amino acid sequence of human GPCRxlβ (352 amino acids) (SEQ ID NO: 14) . The six predicted transmembrane domaines are underlined.

MGPGEALLAGLLV VLAVALLSNALVLLCCAYSAELRTRASGVLLVNLSLGHLLLAALDiyiPFTLLGvMRGRTPSAPGACQ VIGFLDTFLASNAALSVAALSADQWLAVGFPLRYAGRLRPRYAGLLLGCAWGQSLAFSGAALGCS LGYSSAFASCSLRL PPEPERPRFAAFTATLHAVGFVLPLAVLCLTSLQVHRVARRHCQRMDTVTMKALALLADLHPRY PSACRQAQARDLGAP AVGLRSL ASPPLLCPEFTSHSTAPARCSQGFPVGSLVQTLRGPLPPGICAHSAQGALRRAVGCASPGGVPRALLWAAR HTPPVHGCGSEASACFCPLLTQCPCMDLGFKS

At the amino acid sequence level, the human GPCRxlδ is 50% identical to the rat GPR 26 receptor.

Nucleotide and deduced amino acid sequence of human GPCRxl7 (SEQ ID NO: 15 and 16 respectively) . This nucleotide sequence is located on the chromosome 2.

1 M T P N S T G E V P S P I P K 15 1 ATG ACG CCC AAC AGC ACT GGC GAG GTG CCC AGC CCC ATT CCC AAG 45

16 G A L G L S L A L A S L I I T 30

46 GGG GCT TTG GGG CTC TCC CTG GCC CTG GCA AGC CTC ATC ATC ACC 90

31 A N L L L A L G I A G T A A C 45

91 GCG AAC CTG CTC CTA GCC CTG GGC ATC GCT GGG ACC GCC GCC TGC 135

46 A A T C L L L P E P T A G 60

136 GCA GCC ACC TGC TGG CTG CTT CTT CCT GAG CCT ACT GCT GGC TGG 180

61 A A H G S G I A T L P G L W N 75

181 GCT GCT CAC GGG TCT GGC ATT GCC ACA TTG CCA GGG CTG TGG AAC 225

76 Q S R R G Y S C L L V Y L A 90

226 CAG AGT CGC CGG GGT TAC TGG TCC TGC CTC CTC GTC TAC TTG GCT 270

91 N F S F L S L L A N L L L V 105

271 CCC AAC TTC TCC TTC CTC TCC CTG CTT GCC AAC CTC TTG CTG GTG 315

106 H G Ξ R Y M A V L R P L Q P P 120

316 CAC GGG GAG CGC TAC ATG GCA GTC CTG AGG CCA CTC CAG CCC CCT 360

121 G S I R L A L L L T A G P L 135

361 GGG AGC ATT CGG CTG GCC CTG CTC CTC ACC TGG GCT GGT CCC CTG 405

136 L F A S L P A L G N H W T P 150

406 CTC TTT GCC AGT CTG CCC GCT CTG GGG TGG AAC CAC TGG ACC CCT 450

151 G A N C S S Q A I F P A P Y L 165

451 GGT GCC AAC TGC AGC TCC CAG GCT ATC TTC CCA GCC CCC TAC CTG 495

166 Y L E V Y G L L L P A V G A A 180

496 TAC CTC GAA GTC TAT GGG CTC CTG CTG CCC GCC GTG GGT GCT GCT 540

181 A F L S V R V L A T A H R Q L 195

541 GCC TTC CTC TCT GTC CGC GTG CTG GCC ACT GCC CAC CGC CAG CTG 585

196 Q D I C R L E R A V C R D E P 210

586 CAG GAC ATC TGC CGG CTG GAG CGG GCA GTG TGC CGC GAT GAG CCC 630

211 S A L A R A L T R Q A R A Q 225

631 TCC GCC CTG GCC CGG GCC CTT ACC TGG AGG CAG GCA AGG GCA CAG 675

226 A G A M L L F G L C G P Y V 240

676 GCT GGA GCC ATG CTG CTC TTC GGG CTG TGC TGG GGG CCC TAC GTG 720

241 A T L L L S V L A Y E Q R P P 255

721 GCC ACA CTG CTC CTC TCA GTC CTG GCC TAT GAG CAG CGC CCG CCA 765

256 L G P G T L L S L L S L G S A 270

766 CTG GGG CCT GGG ACA CTG TTG TCC CTC CTC TCC CTA GGA AGT GCC 810 271 S A A A V P V A M G L G D Q R 285 811 AGT GCA GCG GCA GTG CCC GTA GCC ATG GGG CTG GGC GAT CAG CGC 855

286 Y T A P W R Q P P K G A C R G 300 856 TAC ACA GCC CCC TGG AGG CAG CCG CCC AAA GGT GCC TGC AGG GGC 900

301 C G E E P P G T V P A P A L P 315 901 TGT GGG GAA GAG CCT CCC GGG ACA GTC CCG GCC CCA GCA TTG CCT 945

316 T T Q A A K A V S T T * 327

946 ACC ACC CAA GCA GCC AAA GCA GTG TCG ACC TGG ACT TGA 984

Amino acid sequence of human GPCRxl7 (327 amino acids) (SEQ ID NO: 16) . The seven predicted transmembrane domaines are underlined.

MTPNSTGEVPSPIPKGALGLSLALASLIITANLLLALGIAGTAACAATC LLLPEPTAG AAHGSGIATLPGL NQSRRG Y SCLLVYLAPNFSFLSLLANLLLVHGERYMAVLRPLQPPGSIRLALLLTWAGPLLFASLPALGWNH TPGANCSSQAIF PAPYLYLEVYGLLLPAVGAAAFLSVRVLATAHRQLQDICRLERAVCRDEPSALARALT RQARAQAGAMLLFGLC GPYV ATLLLSVLAYEQRPPLGPGTLLSLLSLGSASAAAVPVA GLGDQRYTAP RQPPKGACRGCGEEPPGTVPAPALPTTQAA KAVST T

At the amino acid sequence level, the human GPCRxl7 is 28% identical to the human EDG6 receptor

Nucleotide and deduced amino acid sequence of human GPCRxlδ (SEQ ID NO: 17 and 18 respectively) . This nucleotide sequence is located on the chromosome 2.

1 M G D E L A P C P V G T T A 15 1 ATG GGG GAT GAG CTG GCA CCT TGC CCT GTG GGC ACT ACA GCT TGG 45

16 P A L I Q L I S K T P C M P Q 30

46 CCG GCC CTG ATC CAG CTC ATC AGC AAG ACA CCC TGC ATG CCC CAA 90

31 A A S N T S L G L G D L R V P 45

91 GCA GCC AGC AAC ACT TCC TTG GGC CTG GGG GAC CTC AGG GTG CCC 135

46 S S M L Y L F L P S S L L A 60

136 AGC TCC ATG CTG TAC TGG CTT TTC CTT CCC TCA AGC CTG CTG GCT 180

61 A A T L A V S P L L L V T I L 75

181 GCA GCC ACA CTG GCT GTC AGC CCC CTG CTG CTG GTG ACC ATC CTG 225

76 R N Q R L R Q Ξ P H Y L L P A 90

226 CGG AAC CAA CGG CTG CGA CAG GAG CCC CAC TAC CTG CTC CCG GCT 270

91 N I L L S D L A Y I L L H L 105

271 AAC ATC CTG CTC TCA GAC CTG GCC TAC ATT CTC CTC CAC ATG CTC 315

106 I S S S S L G G E L G R M A 120

316 ATC TCC TCC AGC AGC CTG GGT GGC TGG GAG CTG GGC CGC ATG GCC 360

121 C G I L T D A V F A A C T S T 135

361 TGT GGC ATT CTC ACT GAT GCT GTC TTC GCC GCC TGC ACC AGC ACC 405

136 I L S F T A I V L H T Y L A V 150

406 ATC CTG TCC TTC ACC GCC ATT GTG CTG CAC ACC TAC CTG GCA GTC 450

151 I H P L R Y L S F M S H G A A 165

451 ATC CAT CCA CTG CGC TAC CTC TCC TTC ATG TCC CAT GGG GCT GCC 495

166 K A V A L I L V A C C F P 180

496 TGG AAG GCA GTG GCC CTC ATC TGG CTG GTG GCC TGC TGC TTC CCC 540

181 T F L I L S K W Q D A Q L E 195

541 ACA TTC CTT ATT TGG CTC AGC AAG TGG CAG GAT GCC CAG CTG GAG 585

196 E Q G A S Y I L P P S M G T Q 210

586 GAG CAA GGA GCT TCA TAC ATC CTA CCA CCA AGC ATG GGC ACC CAG 630

211 P G C G L L V I V T Y T S I L 225

631 CCG GGA TGT GGC CTC CTG GTC ATT GTT ACC TAC ACC TCC ATT CTG 675

226 C V L F L C T A L I A N C F 240

676 TGC GTT CTG TTC CTC TGC ACA GCT CTC ATT GCC AAC TGT TTC TGG 720

241 R I Y A E A K T S G I G Q G 255

721 AGG ATC TAT GCA GAG GCC AAG ACT TCA GGC ATC TGG GGG CAG GGC 765

256 Y S R A R G T L L I H S V L I 270

766 TAT TCC CGG GCC AGG GGC ACC CTG CTG ATC CAC TCA GTG CTG ATC 810 271 T L Y V S T G V V F S L D M V 285 811 ACA TTG TAC GTG AGC ACA GGG GTG GTG TTC TCC CTG GAC ATG GTG 855

286 L T R Y H H I D S G T H T L 300 856 CTG ACC AGG TAC CAC CAC ATT GAC TCT GGG ACT CAC ACA TGG CTC 900

301 L A A N S E V L M M L P R A M 315 901 CTG GCA GCT AAC AGT GAG GTA CTC ATG ATG CTT CCC CGT GCC ATG 945

316 L T Y L Y L L R Y R Q L L G M 330 946 CTC ACA TAC CTG TAC CTG CTC CGC TAC CGG CAG CTG TTG GGC ATG 990

331 V R G H L P S R R H Q A I F T 345 991 GTC CGG GGC CAC CTC CCA TCC AGG AGG CAC CAG GCC ATC TTT ACC 1035

346 I S * 347

1036 ATT TCC TAG 1044

Amino acid sequence of human GPCRxlS (347 amino acids) (SEQ ID NO: 18) . The seven predicted transmembrane domaines are underlined.

MGDELAPCPVGTTA PALIQLISKTPCMPQAASNTSLGLGDLRVPSSMLY LFLPSSLLAAATLAVSPLLLVTILRNQRL RQEPHYLLPANILLSDLAYILLHMLISSSSLGG ELGRMACGILTDAVFAACTSTILSFTAIVLHTYLAVIHPLRYLSFM SHGAA KAVALI LVACCFPTFLI LSK QDAQLEEQGASYILPPSMGTQPGCGLLVIVTYTSILCVLFLCTALIANCF RIYAEAKTSGIWGQGYSRARGTLLIHSVLITLYVSTGWFSLDMVLTRYHHIDSGTHT LLAANSEVLMMLPRAMLTYLY LLRYRQLLGMVRGHLPSRRHQAIFTIS

At the amino acid sequence level, the human GPCRxlδ is 25% identical to the rabbit 5HTlD-β receptor.

Nucleotide and deduced amino acid sequence (partial sequence) of human GPCRxl9 (SEQ ID NO: 19 and 20 respectively). This nucleotide sequence is located on the chromosome 16.

1 G P H R S Q R S H L C F R A K 15 1 GGC CCC CAT AGG AGC CAA CGA AGT CAT CTT TGC TTC AGA GCT AAA 45

16 P V F L L S T A N I L T V I I 30

46 CCA GTT TTT CTT CTC TCC ACA GCA AAT ATC TTG ACA GTG ATC ATC 90

31 S Q L V A R R Q K S S Y N Y 45

91 CTC TCC CAG CTG GTG GCA AGA AGA CAG AAG TCC TCC TAC AAC TAT 135

46 L A L A A A D I L V L F F I 60

136 CTC TTG GCA CTC GCT GCT GCC GAC ATC TTG GTC CTC TTT TTC ATA 180

61 V F V D F L L E D F I L N M Q 75

181 GTG TTT GTG GAC TTC CTG TTG GAA GAT TTC ATC TTG AAC ATG CAG 225

76 P Q V P D K I I E V L E F S 90

226 ATG CCT CAG GTC CCC GAC AAG ATC ATA GAA GTG CTG GAA TTC TCA 270

91 S I H T S I I T V P L T I D 105

271 TCC ATC CAC ACC TCC ATA TGG ATT ACT GTA CCG TTA ACC ATT GAC 315

106 R Y I A V C H P L K Y H T V S 120

316 AGG TAT ATC GCT GTC TGC CAC CCG CTC AAG TAC CAC ACG GTC TCA 360

121 Y P A R T R K V I V S V Y I T 135

361 TAC CCA GCC CGC ACC CGG AAA GTC ATT GTA AGT GTT TAC ATC ACC 405

136 C F L T S I P Y Y W P N I 150

406 TGC TTC CTG ACC AGC ATC CCC TAT TAC TGG TGG CCC AAC ATC TGG 450

151 T E D Y I S T S V H H V L I 165

451 ACT GAA GAC TAC ATC AGC ACC TCT GTG CAT CAC GTC CTC ATC TGG 95

166 I H C F T V Y L V P C S I F F 180

496 ATC CAC TGC TTC ACC GTC TAC CTG GTG CCC TGC TCC ATC TTC TTC 540

181 I L N S I I V Y K L R R S N 195

541 ATC TTG AAC TCA ATC ATT GTG TAC AAG CTC AGG AGG AAG AGC AAT 585

196 F R L R G Y S T G K T T A I L 210

586 TTT CGT CTC CGT GGC TAC TCC ACG GGG AAG ACC ACC GCC ATC TTG 630

211 F T I T S I F A T L A P R I 225

631 TTC ACC ATT ACC TCC ATC TTT GCC ACA CTT TGG GCC CCC CGC ATC 675

226 I M I L Y H L Y G A P I Q N R 240

676 ATC ATG ATT CTT TAC CAC CTC TAT GGG GCG CCC ATC CAG AAC CGC 720

241 L V H I M S D I A N M L A L 255

721 TGG CTG GTA CAC ATC ATG TCC GAC ATT GCC AAC ATG CTA GCC CTT 765

256 L N T A I N F F L Y C F I S K 270

766 CTG AAC ACA GCC ATC AAC TTC TTC CTC TAC TGC TTC ATC AGC AAG 810 271 R F R T A A A T L K A F F K 285

811 CGG TTC CGC ACC ATG GCA GCC GCC ACG CTC AAG GCT TTC TTC AAG 855

286 C Q K Q P V Q F Y T N H N F S 300

856 TGC CAG AAG CAA CCT GTA CAG TTC TAC ACC AAT CAT AAC TTT TCC 900

301 I T S S P W I S P A N S H C I 315

901 ATA ACA AGT AGC CCC TGG ATC TCG CCG GCA AAC TCA CAC TGC ATC 945

316 K M L V Y Q Y D K N G K P I K 330

946 AAG ATG CTG GTG TAC CAG TAT GAC AAA AAT GGA AAA CCT ATA AAA 990

331 V S P * 333

991 GTA TCC CCG TGA 1002

Partial amino acid sequence of human GPCRxl9 (333 amino acids) (SEQ ID NO:20) . The seven predicted transmembrane domaines are underlined.

GPHRS QRSHLCFRAKPVFLLSTANILTVIILS QLVARRQKSSYNYLLALAAADILVLFF I VF VDFLLEDF I LNMQMPQVP DKIIEVLΞFSSIHTSI ITVPLTIDRYIAVCHPLKYHTVSYPARTRKVIVSArYITCFLTSIPYY WPNI TΞDYISTSVH HVLI IHCFTVYLVPCSIFFILNSIIVYKLRRKSNFRLRGYSTGKTTAILFTITSIFATLWAPRIIMILYHLYGAPIQNR LVHIMSDIANMLALLNTAINFFLYCFISKRFRTMAAATLKAFFKCQKQPVQFYTNHNFSITSSP ISPANSHCIKMLVY QYDKNGKPIKVSP

At the amino acid sequence level, the human GPCRxl9 is 25% identical to the C. Elegans F21C10.9 G-protein coupled receptor.

oo

Nucleotide and deduced amino acid sequence of human GPCRx20 (SEQ ID NO: 21 and 22 respectively) . This nucleotide sequence is located on the chromosome 5.

1 M L A A A F A D S N S S S M N 15

1 ATG CTG GCA GCT GCC TTT GCA GAC TCT AAC TCC AGC AGC ATG AAT 45

16 V S F A H L H F A G G Y L P S 30

46 GTG TCC TTT GCT CAC CTC CAC TTT GCC GGA GGG TAC CTG CCC TCT 90

31 D S Q D W R T I I P A L L V A 45

91 GAT TCC CAG GAC TGG AGA ACC ATC ATC CCG GCT CTC TTG GTG GCT 135

46 V C L V G F V G N L C V I G I 60

136 GTC TGC CTG GTG GGC TTC GTG GGA AAC CTG TGT GTG ATT GGC ATC 180

61 L L H N A W K G K P S M I H S 75

181 CTC CTT CAC AAT GCT TGG AAA GGA AAG CCA TCC ATG ATC CAC TCC 225

76 L I L N L S L A D L S L L L F 90

226 CTG ATT CTG AAT CTC AGC CTG GCT GAT CTC TCC CTC CTG CTG TTT 270

91 S A P I R A T A Y S S V D 105

271 TCT GCA CCT ATC CGA GCT ACG GCG TAC TCC AAA AGT GTT TGG GAT 315

106 L G F V C K S S D W F I H T 120

316 CTA GGC TGG TTT GTC TGC AAG TCC TCT GAC TGG TTT ATC CAC ACA 360

121 C M A A K S L T I V V V A K V 135

361 TGC ATG GCA GCC AAG AGC CTG ACA ATC GTT GTG GTG GCC AAA GTA 405

136 C F M Y A S D P A K Q V S I H 150

406 TGC TTC ATG TAT GCA AGT GAC CCA GCC AAG CAA GTG AGT ATC CAC 450

151 N Y T I S V L V A I W T V A 165

451 AAC TAC ACC ATC TGG TCA GTG CTG GTG GCC ATC TGG ACT GTG GCT 495

166 S L L P L P E F F S T I R H 180

496 AGC CTG TTA CCC CTG CCG GAA TGG TTC TTT AGC ACC ATC AGG CAT 540

181 H E G V E M C L V D V P A V A 195

5 1 CAT GAA GGT GTG GAA ATG TGC CTC GTG GAT GTA CCA GCT GTG GCT 585

196 E E F M S M F G K L Y P L L A 210

586 GAA GAG TTT ATG TCG ATG TTT GGT AAG CTC TAC CCA CTC CTG GCA 630

211 F G L P L F F A S F Y F W R A 225

631 TTT GGC CTT CCA TTA TTT TTT GCC AGC TTT TAT TTC TGG AGA GCT 675

226 Y D Q C K K R G T K T Q N L R 240

676 TAT GAC CAA TGT AAA AAA CGA GGA ACT AAG ACT CAA AAT CTT AGA 720

241 N Q I R S K Q V T V M L L S I 255

721 AAC CAG ATA CGC TCA AAG CAA GTC ACA GTG ATG CTG CTG AGC ATT 765

256 A I I S A L L L P E W V A W 270

766 GCC ATC ATC TCT GCT CTC TTG TGG CTC CCC GAA TGG GTA GCT TGG 810 271 L W V W H L K A A G P A P P Q 285 811 CTG TGG GTA TGG CAT CTG AAG GCT GCA GGC CCG GCC CCA CCA CAA 855

286 G F I A L S Q V L M F S I S S 300 856 GGT TTC ATA GCC CTG TCT CAA GTC TTG ATG TTT TCC ATC TCT TCA 900

301 A N P L I F L V M S E E F R E 315 901 GCA AAT CCT CTC ATT TTT CTT GTG ATG TCG GAA GAG TTC AGG GAA 945

316 G L K G V W K W M I T K K P P 330 946 GGC TTG AAA GGT GTA TGG AAA TGG ATG ATA ACC AAA AAA CCT CCA 990

331 T V S E S Q E T P A G N S E G 345 991 ACT GTC TCA GAG TCT CAG GAA ACA CCA GCT GGC AAC TCA GAG GGT 1035

346 L P D K V P S P Ξ S P A S I P 360 1036 CTT CCT GAC AAG GTT CCA TCT CCA GAA TCC CCA GCA TCC ATA CCA 1080

361 E K E K P S S P S S G K G K T 375 1081 GAA AAA GAG AAA CCC AGC TCT CCC TCC TCT GGC AAA GGG AAA ACT 1125

376 E K A E I P I L P D V E Q F W 390 1126 GAG AAG GCA GAG ATT CCC ATC CTT CCT GAC GTA GAG CAG TTT TGG 1170

391 H E R D T V P S V Q D N D P I 405 1171 CAT GAG AGG GAC ACA GTC CCT TCT GTA CAG GAC AAT GAC CCT ATC 1215

406 P E H E D Q E T G E G V K * 419 1216 CCC TGG GAA CAT GAA GAT CAA GAG ACA GGG GAA GGT GTT AAA TAG 1260

Amino acid sequence of human GPCRx20 (419 amino acids) (SEQ ID NO: 22) . The seven predicted transmembrane domaines are underlined.

MLAAAFADSNSSSI^NVSFAHLHFAGGYLPSDSQDWRTIIPALLVAVCLVGFVGNLCVIGILLHNA KGKPSMIHSIILNL SLADLSLLLFSAPIRATAYSKSVWDLGWFVCKSSD FIHTCMAAKSLTI WAKVCFMYASDPAKQVSIHNYTIWSVLVA IWTVASLLPLPE FFSTIRHHΞGVE CLVDVPAVAEEFMSMFGKLYPLLAFGLPLFFASFYF RAYDQCKKRGTKTQNLR NQIRSKQVTVMLLSIAIISALLWLPE VA L VWHLKAAGPAPPQGFIALSQVLMFSISSANPLIFLVMSEΞFREGLKGV KWMITKKPPTVSESQETPAGNSEGLPDKVPSPESPASIPEKEKPSSPSSGKGKTEKAEIPILPDVEQF HERDTVPSVQ DNDPI WEHEDQETGEGVK

At the amino acid sequence level, the human GPCRx20 is 20% identical to the mouse galanin 2 receptor.

Claims

1. A G-protein coupled receptor having an amino acid sequence which presents more than 75% sequence identity with the sequence SEQ ID NO. 1.

2. The G-protein coupled receptor according to claim 1, having an amino acid sequence which presents more than 80% sequence identity with the sequence SEQ ID NO. 1.

3. The G-protein coupled receptor according to claim 1, having an amino acid sequence which presents more than 85% sequence identity with the sequence SEQ ID NO. 1.

4. The G-protein coupled receptor according to claim 1, having an amino acid sequence which presents more than 90% sequence identity with the sequence SEQ ID NO. 1.

5. The G-protein coupled receptor according to claim 1, having an amino acid sequence which presents more than 95% sequence identity with the sequence SEQ ID NO. 1.

6. The G-protein coupled receptor having the amino acid sequence SEQ ID NO. 1 or a specific active portion thereof.

7. A polynucleotide encoding any of the amino acid sequences of the G-protein coupled receptor according to any of the preceding claims 1 to 6.

8. An agonist, reverse agonist, antagonist or inhibitor of the receptor or the polynucleotide according to any of the preceding claims 1 to 7.

9. A vector comprising the polynucleotide according to the claim 7.

10. A cell transformed by the vector according to the claim 9.

11. A non-human mammal comprising a partial or total deletion of the polynucleotide according to the claim 8 encoding the receptor according to any of the preceding claims 1 to 6, preferably an non-human mammal comprising an homologous recombination "knock-out" of said polynucleotide or a transgenic non-human mammal overexpressing above natural level said polynucleotide.

12. A method for the screening (detection and possibly recovering) of compounds or natural extract which are known or not known to be agonists, antagonists or inhibitors to the receptor according to any of the preceding claims 1 to 6, said method comprising : contacting a cell or cell extract from the cell transfected with a vector according to the claim 9, - possibly isolating a membrane fraction from the cell extract or the complete cell with a compound binding to said receptor under conditions permitting binding of said compound or molecules present in said natural extract to said receptor, possibly by the activation of a functional response, and detecting the presence of any such compound or molecules by means of a bioassay (preferably a modification in the production of a second messenger or an increase in the receptor activity) in the presence of the other known compound working as an agonist, reverse agonist, antagonist or inhibitor to the receptor and thereby recovering and determining whether said unknown compound or molecule (s) is (are) able to work as an agonist, antagonist or inhibitor of the compound to its receptor.

13. An unknown compound or molecule (s), identified by the screening method according to the claim 12.

14. A pharmaceutical composition comprising an adequate pharmaceutical carrier and a sufficient amount of the compound or molecules according to the claim 8 or 13.

15. Use of the pharmaceutical composition according to the claim 14, for the manufacture of a medicament in the prevention and/or the treatment of a disease selected from the group consisting of viral infections or diseases induced by various viruses or bacteria, the treatment of disturbances of cell migration, diseases or perturbations of the immune system, including cancer, development of tumours and tumour metastasis, inflammatory and neo-plastic processes, bacterial and fungal infections, for wound and bone healing and dysfunction of regulatory growth functions, pains, diabetes, obesity, anorexia, bulimia, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, angina pectoris, myocardial infarction, restenosis, atherosclerosis, diseases characterised by excessive smooth muscle cell proliferation, aneurysms, wound healing, diseases characterised by loss of smooth muscle cells or reduced smooth muscle cell proliferation, stroke, ischemia, ulcers, allergies, benign prostatic hypertrophy, migraine, vomiting, psychotic and neurological disorders, including anxiety, schizophrenia, maniac depression, depression, delirium, dementia and severe mental retardation, degenerative diseases, neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease, and dyskinasias, such as Huntington's disease or Gilles de la Tourett's syndrome and other related diseases.

16. Use of the pharmaceutical composition according to the claim 14, for the manufacture of a medicament in the prevention and/or the treatment of blood circulating affections, including acute heart failure, hypotension, hypertension or myocardial infarction.

17. Diagnostic kit comprising all the media and means for detecting the receptor and nucleotide sequence encoding it or an activity of said receptor and nucleotide sequence encoding it according to any of the preceding claims 1 to 8.