CA2413435A1

CA2413435A1 - A recombinant cell line expressing gpcrx11 as a functional receptor validated by angiopeptin and useful for screening of agonists and antagonists

Info

Publication number: CA2413435A1
Application number: CA002413435A
Authority: CA
Inventors: Vincent Lannoy; Stephane Brezillon; Michel Detheux; Marc Parmentier; Cedric Govaerts
Original assignee: Individual
Current assignee: Ogeda SA
Priority date: 2000-06-20
Filing date: 2001-06-20
Publication date: 2001-12-27
Also published as: WO2001098330A3; AU2001265717A1; WO2001098330A2; JP2004500125A; EP1297003A2

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.
[00027 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 transmembrane 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/calcitoninjsecretin 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 a, (3 and y 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 a-subunit to exchange a bound GDP molecule for a GTP molecule and to dissociate from the (3y-subunits.
[0012] The GTP-bound form of the a, (3 and y-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 a-subunits are known in humans. These subunits associate with a small pool of (3 and y 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 50-600 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).
[00l8] 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).
[00l9] 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 0, 75°s, 80 0, 85%, 90 o 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 sites) 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 5 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, rybo~ymes 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 suoh 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 W098/20112 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 portions) 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 molecules) 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 molecules) 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, 5 vomiting, psychotic arid 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 10 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, fetal 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 l: Cloning of human GPCRxII 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] Tn order to clone the GPCRxII 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 GPCRxII fw: 5'-ccggaattcaccatggatccaaccaccccg-3' SEQ ID 24 GPCRxII rv: 5'-ctagtctagactctacaccagactgcttctc-3' [0045] Amplification resulted in a fragments of 0.99 kilobase containing the entire coding sequence of the GPCRxIl gene. This fragment was subcloned into the pCDNA3 (Invitrogen) vector for DNA sequencing analysis.
[0046] Nucleotide and deduced amino acid sequence of human GPCRxII (SEQ ID NO 1) l36 GTA GGAAACGGG TTTGTGCTC TGGCTCCTG GGCTTCCGC ATGCGC

lso [0047] Amino acid sequence of human GPCRxII (330 amino acids) (SEQ ID N0:2). The seven predicted transmembrane domaines are underlined.
MDPTTPAWGTESTTVNGNDQALLLLCGKETLIPVFLILFIALVGLVGNGFVLWLLGFRM
RRNAFSVYVLSLAGADFLFLCFQIINCLVYLSNFFCSISINFPSFFTTVMTCAYLAGLS
MLSTVSTERCLSVLWPIWYRCRRPRHLSAVVCVLLWALSLLLSILEGKFCGFLFSDGDS
GWCQTFDFITAAWLIFLFMVLCGSSLALLVRILCGSRGLPLTRLYLTILLTVLVFLLCG
LPFGIQWFLILWIWKDSDVLFCHIHPVSWLSSLNSSANPIIYFFVGSFRKQWRLQQPI
LKLALQRALQDTAEVDHSEGCFRQGTPEMSRSSLV

[0048] At the amino acid sequence level, the human GPCRxII is 37% identical to the rat RTA receptor. The gene coding GPCRxl1 is located on chromosome 11.
Alignment of GPCRxII (fig. l) [0049] Alignment of the amino acid sequence of GPCRxl1 with RTA and other RTA related sequences were performed using ClustalX algorithm. Then, the dendrogram was constucted using TreeView algorithm.
GPRI
l vt~ ~
Tissular distribution of GPCRxl2 [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 GPCRxII 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 mina 38 cycles at 94°C for 1 min, 58°C for 2 min and 72°C
for 2 min. Aliquots (10 ~.l) of the PCR reaction were analysed by 1o agarose gel electrophoresis.

(0051] GPCRxIl mRNA was assayed by RT-PCR in 16 human tissues. A strong band of expected sire (586 bp) was detected in testis, at lower levels in uterus and thymus, while not in pituitary gland, spinal cord, pancreas, small 5 intestine, placenta, stomach, liver, lung, spleen, brain, heart, kidney and skeletal muscle.
Functional assay for GPCRxII
(0052] GPCRxII expressing clones have been obtained by transfection of CHO-K1 cells coexpressing mitochondrial 10 apoaequorin and Galphal6, 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-NaI-Cys-Tyr-D-15 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, Galphal6 and mitochondrial apoaequorin were established. A functional assay based on the luminescence of mitochondrial aequorin following intracellular Caz+
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 106 cells/ml in DMEM-F12 medium, incubated with 5 ~.M 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 106 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 GPCRxl1 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 GPCRxS, SEQ ID NO 5 GPCRx7, SEQ ID NO 7 GPCRx9, SEQ ID NO 9 GPCRxI4, SEQ ID NO 11 GPCRxI6, SEQ ID NO 13 GPCRxl7, SEQ ID NO 15 GPCRxI8, SEQ ID NO 17 GPCRxI9, 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 GPCRx5 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 GPCRxI4 fw: 5'-ccggaattcgccatgtacaacgggtcg-3' SEQ ID NO 36 GPCRxI4 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.
GPCRx GPCRx GPCRx GPCRx GPCRx GPCRx GPCRxGPCRx GPCRx Li _ _ _ _ _ _ _ _ +

Lu .+. _ + + - ++ - - ++
/
_ Sp _ - ++ + - _ _ _ +

Te - + - ++ - ++ - -I-/- +

Br ++ - - - - - ++ - ++

He _ _ _ _ _ _ _ - ++

Ki +/ - - + - ++ - - +
_ Sk.m _ _ _ _ _ + _ - ++

Pi.G _ - - - - - ++ +/- +

Sp.C ++ _ - - - ++ +/- +/- +~-Th +/ - + - - +-I- - - ++
_ Pa _ _ _ _ - ++ .+/ _ -_ S +/ - + - - ++ - - +
. _ In Ut _ _ _ _ _ .+.+ _ +/ +, _ P1 - - - ++ ++ - - - +

St - - + +/- - ..~+ - - +

Table l: 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; Pl, 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.
Doran~, C. Govaerts, G. Vassart, R.W. Doms, and M.
Parmentier. 1999 CCRS binds multiple CC-chemokines: MCP
3 acts as a natural antagonist. Blood 94:1899-1905.

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

respectively) 9l GTCCCG GAGGTGGGG CTACGG GATGTTGCT TCGGAATCT GTGGCC 135 406,E P P A V D F R I P G Q I A E 420 451 S * 452 Amino acid sequence of human GPCRx2 (451 amino acids) (SEQ ID N~: 4). The seven predicted transmembrane domaines are underlined.
MESSPIPQSSGNSSTLGRVPQTPGPSTASGVPEVGLRDVASESVALFFMLLLDLTAVAGNAAVMAVIAKTPALRKFV_F
'VF
HLCLVDLLAALTLMPLAMLSSSALFDHALFGEVACRLYLFLSVCFVSLAILSVSAINVERYYYVVHPMRYEVRMTLGLV
A
SVLVGVWKALAMASVPVLGRVSWEEGAPSVPPGCSLQWSHSAYCQLFWVFAVLYFLLPLLLILVWCSMFRVARVAAM
QHGPLPTWMETPRQRSESLSSRSTMVTSSGAPQTTPHRTFGGGKAAWLLAVGGQFLLCWLPYFSFHLWALSAQPISTG
QVESWTWIGYFCFTSNPFFYGCLNRQIRGELSKQFVCFFKPAPEEELRLPSREGSIEENFLQFLQGTGCPSESWVSRPL

PSPKQEPPAVDFRIPGQIAEETSEFLEQQLTSDIIMSDSYLRPAASPRLES
At the amino acid sequence level, the human GPCRx2 is 23o identical to the human histamine H2 receptor.

~G
Nucleotide acidsequence human 5 and and of GPCRx5 6 deduced (SEQ
amino ID
NO:

respectively) l81 V A W L I F L C V V L C G S S 195 316S G S R L E Q * 323 Amino acid sequence of human GPCRx5 (322 amino acids) (SEQ ID N0:6) . The seven predicted transmembrane domaines are underlined.
MDPTISTLDTELTPINGTEETLCYKQTLSLTVLTCIVSLVGLTGNAVVLWLLGCRMRRNAFSIYILNLAAADFLFLSGR
L
IYSLLSFISIPHTISKILYPVMMFSYFAGLSFLSAVSTERCLSVLWPIWYRCHRPTHLSAWCVLLWALSLLRSILEWML

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

Nucleotide acidsequence human 7 and and of GPCRx7 8 deduced (SEQ
amino ID
NO:

respectively) 316T N E M G A * 322 Amino acid sequence of human GPCRx7 (321 amino acids) (SEQ ID N0:8). The seven predicted transmembrane domaines are underlined.
MNQTLNSSGTVESALNYSRGSTVHTAYLVLSSLAMFTCLCGMAGNSMVIWLLGFRMHRNPFCIYILNLAAADLLFLFSM
A
STLSLETQPLVNTTDKVHELMKRLMYFAYTVGLSLLTAISTQRCLSVLFPIWfKCHRPRHLSAWVCGLLWTLCLLMNGL
T
SSFCSKFLKFNEDRCFRVDMVQAALIMGVLTPVMTLSSLTLFVWVRRSSQQWRRQPTRLFVVVLASVLVFLICSLPLSI
Y
WFVLYWLSLPPEMQVLCFSLSRLSSSVSSSANPVIYFLVGSRRSHRLPTRSLGTVLQQALREEPELEGGETPTVGTNEM
G
A
At the amino acid sequence level, the human GPCRx7 is 29o identical to the rat RTA receptor.

Nucleotide acidsequence human 9 and and of GPCRx9 10 deduced (SEQ
amino ID
NO:

respectively) 136 ATG GCGTGGCTG GCCGGCTCC CAGGCCCGGCAT GGAGCTGGC ACG l80 GCC TAC AGC

AGC CTC CGG

CTG GTG TGC

CGG AGC CAG

TCT CCA GCC

CAG CCT ACA

CCA GAT CCT

CAG TCG AAC

GCC CAG GCA

AAC ACC CCC

TGT GCT ACC

GCC GAC GGA

CCC ACC GGC

466 T * 467 TGA

Amino f humanGPCRx9 (466amino cids) . The acid a (SEQ six sequence ID
o N0:10) predictedtransmembrane domaines are underlined.

MEADLGATGHRPRTELDDEDSYPQGGWDTVFLVALLLLGLPANGLMAWLAGSQARHGAGTRLALLLLSLALSDFLFLAA
A

A_FQILEIRHGGHWPLGTAACRFYYFLWGVSYSSGLFLLAALSLDRCLLALCPHWYPGHRPVRLPLWVCAGVWVLATLF
SV

PWLVFPEAAVWWYDLVICLDFWDSEELSLRMLEVLGGFLPFLLLLVCHVLTQATACRTCHRQQQPAACRGFARVARTIL
S

AYVVLRLPYQLAQLLYLAFLWDVYSGYLLWEALVYSDYLILLNSCLSPFLCLMASADLRTLLRSVL
SSFAAALCEERPGS

FTPTEPQTQLDSEGPTLPEPMAEAQSQMDPVAQPQVNPTLQPRSDPTAQP
QLNPTAQPQSDPTAQP QLNLMAQPQSDSVA

QPQADTNVQTPAPAASSVPSPCDEASPTPS SHPTPGALEDPATPPASEGE
SPSSTPPEAAPGAGPT

At the no acidsequence el,the human is 33aidentical the human ami lev GPCRx9 to ChemR23 receptor.

Nucleotide acid GPCRxI4 (SEQID 11 and sequence NO: and deduced of amino human 12 respectively) TCA TTT

AGT AAG

GAA ATT

GTG TTC

331 ~Q S Q S D G Q W D P H I V E W 345 CAC TGG

346 H * 347 Amino acid sequence of human GPCRxI4 (346 (SEQ ID N0:12).The amino acids) seven predicted transmembrane domaines are underlined.

MYNGSCCRIEGDTISQVMPPLLIVAFVLGALGNGVALCGFCFHMKTWKPSTVYLFNLAVADFLLMICLPFRTDYYLRRR
H

WAFGDIPCRVGLFTLAMNRAGSIVFLTWAADRYFKVVHPHHAVNTISTRVAAGIVCTLWALVILGTVYLL
LENHLCVQE

TAVSCESFIMESANGWHDIMFQLEFFMPLGIILFCSFKIVWSLRRRQQLARQARMKKATRFIMVVAIVFITCYLPSVSA
R

LYFLWTVPSSACDPSVHGALHITLSFTYMNSMLDPLVYYFSSPSFPKFYNKLKICSLKPKQPGHSKTQRPEEMPISNLG
R

RSCISVANSFQSQSDGQWDPHIVEWH

At the amino acid sequence level, the human 50o identical the GPCRxI4 is to human HM74 receptor.

Nucleotide acidsequence human ID 13 and of GPCRxI6 N0: and deduced (SEQ
amino 14 respectively). This tidesequence located on the chromosome4.
nucleo is 136 GTGAAT CTGTCT CTGGGCCAC CTGCTG'CTGGCGGCGCTG GACATG 180 91 S N A A L S V A A h 5 A D Q W 105 27l TCCAAC GCGGCG CTGAGCGTG GCGGCGCTG AGCGCAGAC CAGTGG 315 7.06 L A V G F P L R Y A G R L R P 120 301R A V G C A S P G G V P R A L 3l5 346M D L G F K S * 352 1036ATG GACTTGGGC TTCAAG'PCTTGA 1059 Amino acid sequence of human GPCRxl6 (352 amino acids) (SEQ ID N0: 14). The six predicted transmembrane domaines are underlined.
MGPGEALLAGLLVMVLAVALLSNALVLLCCAYSAELRTRASGVLLVNLSLGHLLLAALDMPFTLLGVMRGRTPSAPGA_ CQ
VIGFLDTFLASNAALSVAALSADQWLAVGFPLRYAGRLRPRYAGLLLGCAWGQSLAFSGAALGCSWLGYSSAFASCSLR
L
PPEPERPRFAAFTATLHAVGFVLPLAVLCLTSLQVHRVARRHCQRMDTVTMKALALLADLHPRYWPSACRQAQARDLGA
P
WAVGLRSLWASPPLLCPEFTSHSTAPARCSQGFPVGSLVQTLRGPLPPGICAHSAQGALRRAVGCASPGGVPRALLWAA
R
HTPPVHGCGSEASACFCPLLTQCPCMDLGFKS
At the amino acid sequence level, the human GPCRxI6 is 50o identical to the rat GPR 26 receptor.

Nucleotide and deduced amino acid sequence of human GPCRxI7 (SEQ TD N0: 1S and 16 respectively). This nucleotide sequence is located on the chromosome 2.

241 A T' L L L S V L A Y E Q R P P 255 GCA

CCC

GAG

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

Amino acid sequence of human GPCRxI7 (327 amino acids) (SEQ TD N0:16). The seven predicted transmembrane domaines are underlined.
MTPNSTGEVPSPIPKGALGLSLALASLIITANLLLALGIAGTAACAATCWLLLPEPTAGWAAHGSGIATLPGLWNQSRR
G
YWSCLLVYLAPNFSFLSLLANLLLVHGERYMAVLRPLQPPGSIRLALLLTWAGPLLFASLPALGWNHWTPGANCSSQAI
F
PAPYLYLEVYGLLLPAVGAAAFLSVRVLATAHRQLQDICRLERAVCRDEPSALARALTWRQARAQAGAMLLFGLCWGPY
V
ATLLLSVLAYEQRPPLGPGTLLSLLSLGSASAAAVPVAMGLGDQRYTAPWRQPPKGACRGCGEEPPGTVPAPALPTTQA
A
At the amino acid sequence level, the human GPCRxI7 is 28's identical to the human EDGE receptor 34 ' Nucleotide acidsequence human ID 17 and of GPCRxl8 NO: and deduced (SEQ
amino 18 respectively). This tidesequence located on the chromosome 2.
nucleo is TCC GTG

ACT CTC

CTT ATG

CAG ATG

CAG ACC

346 I S * 347 Amino acid sequence of human GPCRxI8 (347 (SEQ ID N0:18).The amino acids) seven predicted transmembrane domaines are underlined.

MGDELAPCPVGTTAWPALIQLISKTPCMPQAASNTSLGLGDLRVPSSMLYWLFLPSSLLAAATLAVSPLLLVTILRNQR
L

RQEPHYLLPANILLSDLAYILLHMLISSSSLGGWELGRMACGILTDAVFAACTSTILSFTAIVLHTYLAVT
HPLRYLSFM

SHGAAWKAVALIWLVACCFPTFLIWLSKWQDAQLEEQGASYILPPSMGTQPGCGLLVIVTYTSILCVLFLC
TALIANCFW

RIYAEAKTSGIWGQGYSRARGTLLIHSVLITLYVSTGWFSLDMVLTRYHHIDSGTHTWLLAANSEVLMMLPRAMLTYLY

LLRYRQLLGMVRGHLPSRRHQAIFTIS

At the amino acid sequence level, the human 25% identical the GPCRxIB is to rabbit 5HT1D-(3 receptor.

Nucleotide acid sequence) GPCR.xl9 and sequence of deduced (partial human amino (SEQ 9 respective ly).This tidesequenc e locatedon ID NO: and nucleo is the chromosome .

271 TCCATCCAC ACCTCC ATATGGATTACT GTACCG TTAACCATTGAC 3l5 331 V S P * 333 Partial amino acid sequence of human GPCRxI9 (333 amino acids) (SEQ ID N0:20).
The seven predicted transmembrane domaines are underlined.
GPHRSQRSHLCFRAKPVFLLSTANILTVIILSQLVARRQKSSYNYLLALAAADILVLFFIVFVDFLLEDFILNMQMPQV
P
DKIIEVLEFSSIHTSIWITVPLTIDRYIAVCHPLKYHTVSYPARTRKVIVSVYITCFLTSIPYYWWPNIWTEDYISTSV
H
HVLIWIHCFTVYLVPCSIFFILNSIIVYKLRRKSNFRLRGYSTGKTTAILFTITSIFATLWAPRIIMILYHLYGAPIQN
_R
WLVHIMSDIANMLALLNTAINFFLYCFISKRFRTMAAATLKAFFKCQKQPVQFYTNHNFSITSSPWISPANSHCIKMLV
Y
QYDKNGKPIKVSP
At the amino acid sequence level, the human GPCRxI9 is 25o identical to the C.
EleganS F21C10.9 G-protein coupled receptor.

3a Nucleotide acid GPCRx20 (SEQ 21 and sequence ID and deduced of N0:
amino human 22 respectively) . s cated thechromosome 5.
This lo on nucleotide sequence i l M L A A A F A D S N S S S M N 15 GCT CCG CAA

GTC TTT TCA

GTG GAA GAA

TGG ACC CCA

ACA GGC GGT

CCA CCA CCA CCA

AGC CCC GGC ACT

CCC CTT GTA TGG

GTC TCT GAC ATC

406 P W E H E D Q E T G E G V K * 419 GAT GAG GAA TAG

Amino acid sequence of human amino acids)(SEQ ID N0:22).The seven GPCRx20 (419 predicted transmembrane domaines are underlined.

MLAAAFADSNSSSMNVSFAHLHFAGGYLPSDSQDWRTITPALLVAVCLVGFVGNLCVIGILLHNAWKGKPS
MIHSlILNL

SLADLSLLLFSAPIRATAYSKSVWDLGWFVCKSSDWFIHTCMAAKSLTIVWAKVCFMYASDPAKQVSIHN
YTIWSVLVA

IWTVASLLPLPEWFFSTIRHHEGVEMCLVDVPAVAEEFMSMFGKLYPLLAFGLPLFFASFYFWRAYDQCKKRGTKTQNL
R

NQTRSKQVTVMLLSIAIISALLWLPEWVAWLWVWHLKAAGPAPPQGFIALSQVLMFSISSANPLIFLVMSE
EFREGLKGV

WKWMITKKPPTVSESQETPAGNSEGLPDKVPSPESPASTPEKEKPSSPSSGKGKTEKAEIPILPDVEQFWHERDTVPSV
Q

DNDPIPWEHEDQETGEGVK

At the amino acid sequence the 20s identical the level, human to GPCRx20 is 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 SEA 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.