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  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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  • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
• • A—HUMAN NECESSITIES
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  • A01K2217/00—Genetically modified animals
  • A01K2217/07—Animals genetically altered by homologous recombination
  • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out

Definitions

• 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.
• the invention is also related to the polynucleic acid sequence (polynucleotide) encoding said receptor.
• the invention is further related to methods using receptor polypeptide and polynucleotide applicable to diagnostic and treatment in receptor-mediated disorders.
• 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.
• the invention further encompasses unknown agonists and antagonists detected and recovered based on the receptor polypeptide and polynucleotide.
• 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
• G-protein coupled receptors 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.
• 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 .
• 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.
• G proteins represent a family of heterotrimeric proteins composed of ⁇ , ⁇ and ⁇ subunits, that bind guanine nucleotides. These proteins are usually linked to cell surface receptors (receptors containing seven transmembrane domains) .
• the GTP-bound form of the ⁇ , ⁇ and ⁇ -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.
• second messengers such as cAMP (e.g. by activation of adenyl cyclase) , diacylglycerol or inositol phosphates.
• 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 .
• G protein coupled receptors which could be used for the screening of new agonists and antagonists having advantageous potential prophylactic and therapeutical properties .
• GPCRs 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
• 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:
• 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) .
• the present invention is also related to nucleotidic and/or amino acid sequence homologous to the sequences corresponding to the receptor described hereafter.
• 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.
• 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.
• 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.
• 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.
• 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.
• the invention is preferably related to said
• agonist, reverse agonist and antagonist compounds or inhibitors of said receptor 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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,
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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
• 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.
• 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,
• 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
• GPCRxll G-protein coupled receptor
• 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) .
• NBI public high-throughput genomic sequence databases
• 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)
• RT-PCR Reverse transcription-polymerase chain reaction
• 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 .
• 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 ⁇ l) 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.
• GPCRxll expressing clones have been obtained by transfection of CH0-K1 cells coexpressing mitochondrial apoaequorin and Galphal ⁇ , 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.
• somatostatin analog D2-NaI-Cys-Tyr-D-trp-Lys-Val-Cys-D2- NaI-NH2
• Somatostatin 14 has no activity on GPCRxll.
• Aequorin assays CHO-K1 cell lines expressing GPCRxll receptors, Galpha 16 and mitochondrial apoaequorin were established. A functional assay based on the luminescence of mitochondrial aequorin following intracellular Ca 2+ 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 ⁇ M Coelenterazine H (Molecular Probes) for 4 hours at room temperature.
• 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
• GPCR8 GPR8 , ChemR23, HM74 and GPR14 were used as queries to search for homologies in public high-throughput genomic sequence databases (NCBI) .
• GPCRxl ⁇ SEQ ID NO 17 GPCRxl9, SEQ ID NO 19 GPCRX20, SEQ ID NO 21
• PCR polymerase chain reaction
• 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.
• 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;
• the tissues are the following: Li, liver; Lu, lung; Sp, Spleen; Te, testis;
• Pi.G Pituitary gland
• Sp.C spinal cord
• Th Thymus
• Pa Pancreas
• S.In Small intestine
• Ut Uterus
• Pi Plancenta
• 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.
• 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 QRAIiQDAS ⁇ VDEGGGQ PEEILE SGSRL EQ
• the human GPCRxS is 31% identical to the human mas receptor.
• the human GPCRx7 is 29% identical to the rat RTA receptor.
• 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.
• 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.
• the human GPCRxl ⁇ 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.
• the human GPCRxl7 is 28% identical to the human EDG6 receptor
• Nucleotide and deduced amino acid sequence of human GPCRxl ⁇ (SEQ ID NO: 17 and 18 respectively) . This nucleotide sequence is located on the chromosome 2.
• the human GPCRxl ⁇ is 25% identical to the rabbit 5HTlD- ⁇ 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.
• the human GPCRxl9 is 25% identical to the C. Elegans F21C10.9 G-protein coupled receptor.
• 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.
• the human GPCRx20 is 20% identical to the mouse galanin 2 receptor.

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