G-coupled receptor showing selective affinity for atp

Abstract

The present invention concerns a G-coupled receptor which has an amino acid sequence having more than 50% homology with the amino acid sequence shown in FIG. 1.

1 . Receptor which has an amino acid sequence having more than 50% homology with the amino acid sequence shown in FIG. 1.  
     
     
         2 . Receptor according to  claim 1 , which has at least the amino acid sequence shown in FIG. 1 or a portion thereof.  
     
     
         3 . Receptor according to  claim 1  or  2  having a selective affinity ADP.  
     
     
         4 . Receptor according to any of the preceding claims, belonging to the P2Y receptor family.  
     
     
         5 . Receptor according to any of the preceding claims, being a G protein-coupled receptor.  
     
     
         6 . Receptor according to any of the preceding claims, being a human receptor.  
     
     
         7 . Nucleic acid molecule encoding the receptor according to any of the preceding claims.  
     
     
         8 . Nucleic acid molecule according to  claim 7 , wherein the nucleic acid molecule is DNA or RNA molecule.  
     
     
         9 . DNA molecule according to  claim 8 , which is a cDNA molecule or a genomic DNA molecule.  
     
     
         10 . Nucleic acid molecule according to any of the  claims 7  to  9 , having more than 50% homology to the DNA sequence shown in FIG. 1.  
     
     
         11 . DNA molecule according to  claim 10 , which has at least the DNA sequence as shown in FIG. 1 or a portion thereof.  
     
     
         12 . Vector comprising the nucleic acid molecule according to any of the  claims 7  to  11 .  
     
     
         13 . Vector according to  claim 12 , adapted for expression in a cell, which comprises the regulatory elements necessary for expression of the nucleic acid molecule in said cell operatively linked to the nucleic acid molecule according to any of the  claims 7  to  11  as to permit expression thereof.  
     
     
         14 . Vector of  claim 13 , wherein the cell is selected from the group consisting of bacterial cells, yeast cells, insect cells or mammalian cells.  
     
     
         15 . Vector according to any of the  claims 12  to  14 , wherein the vector is a plasmid or a virus, preferably a baculovirus, an adenovirus or a Semliki Forest virus.  
     
     
         16 . Cell comprising the vector according to any of the  claims 12  to  15 .  
     
     
         17 . Cell of  claim 16 , wherein the cell is a mammalian cell, preferably non neuronal in origin.  
     
     
         18 . Cell of  claim 16 , wherein the cell is selected from the group consisting of COS-7 cells, CHO cells, LM(tk-) cells, NIH-3T3 cells or 1321N1 astrocytoma cells.  
     
     
         19 . Nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridising with a unique sequence included within the nucleic acid molecule according to any of the  claims 11  to  15 .  
     
     
         20 . Nucleic acid probe of  claim 19 , wherein the nucleic acid is DNA or RNA.  
     
     
         21 . Antisense oligonucleotide having a sequence capable of specifically hybridising to a mRNA molecule of  claim 8 , so as to prevent translation of the mRNA molecule.  
     
     
         22 . Antisense oligonucleotide having a sequence capable of specifically hybridising to the DNA molecule of  claim 9 .  
     
     
         23 . Antisense oligonucleotide according to  claim 21  or  22 , comprising chemical analogs of nucleotides.  
     
     
         24 . Ligand other than purine and pyridine nucleotides capable of binding to a receptor according to any of the  claims 1  to  6 .  
     
     
         25 . Anti-ligand capable of competitively inhibiting the binding of the ligand according to  claim 24  to the receptor according to any of the  claims 1  to  6 .  
     
     
         26 . Ligand according to  claim 24 , which is an antibody.  
     
     
         27 . Anti-ligand according to  claim 25 , which is an antibody.  
     
     
         28 . Antibody according to  claim 26  or  27 , which is a monoclonal antibody.  
     
     
         29 . Monoclonal antibody according to  claim 28 , directed to an epitope of the receptor according to any of the  claims 1  to  6 , present on the surface of a cell expressing said receptor.  
     
     
         30 . Pharmaceutical composition comprising an amount of the oligonucleotide according to  claim 21 , effective to decrease activity of the receptor according to any of the  claims 1  to  6  by passing through a cell membrane and binding specifically with mRNA encoding said receptor in the cell so as to prevent its translation, and a pharmaceutically acceptable carrier capable of passing through a cell membrane.  
     
     
         31 . Pharmaceutical composition of  claim 30 , wherein the oligonucleotide is coupled to a substance which inactivates mRNA.  
     
     
         32 . Pharmaceutical composition of  claim 31 , wherein the substance which inactivates mRNA is a ribozyme.  
     
     
         33 . Pharmaceutical composition according to any of the  claims 30  to  32 , wherein the pharmaceutically acceptable carrier comprises a structure which binds to a receptor on a cell capable of being taken up by cell after binding to the structure.  
     
     
         34 . Pharmaceutical composition of  claim 33 , wherein the structure of the pharmaceutically acceptable carrier is capable of binding to a receptor which is specific for a selected cell type.  
     
     
         35 . Pharmaceutical composition which comprises an effective amount of the anti-ligand of  claim 30 , effective to block binding of a ligand to the receptor according to any of the  claims 1  to  6  and a pharmaceutically acceptable carrier.  
     
     
         36 . Transgenic non human mammal expressing the nucleic acid molecule according to any of the  claims 7  to  11 .  
     
     
         37 . Transgenic non human mammal comprising a homologous recombination knockout of the native receptor according to any of the  claims 1  to  6 .  
     
     
         38 . Transgenic non human mammal whose genome comprises antisense nucleic acid complementary to the nucleic acid molecule according to any of the  claims 7  to  11  so placed as to be transcripted into antisense mRNA which is complementary to the mRNA of  claim 8  and which hybridises to said mRNA thereby reducing its translation.  
     
     
         39 . Transgenic non human mammal according to any of the  claims 36  to  38 , wherein the nucleic acid according to any of the  claims 7  to  11  additionally comprises an inducible promoter.  
     
     
         40 . Transgenic non human mammal according to any of the  claims 36  to  39 , wherein the nucleic acid according to  claim 7  to  11  additionally comprises tissue specific regulatory elements.  
     
     
         41 . Transgenic non human mammal according to any of the  claims 36  to  40 , which is a mouse.  
     
     
         42 . Method for determining whether a ligand can specifically bind to a receptor according to any of the  claims 1  to  6 , possibly as an agonist or an antagonist of said receptor; said method comprising the steps of contacting a cell or cell extract from cells transfected with a vector expressing the nucleic acid molecule encoding said receptor, possibly isolating a membrane fraction from the cell extract, with the ligand under conditions permitting binding of said ligand to said receptor, possibly by the activation of a functional response, and detecting the presence of any such ligand bound specifically to said receptor, possibly by means of a bioassay such as a modification of the production of a second messenger or an increasing in the receptor activity, thereby determining whether the ligand binds specifically to said receptor, possibly as an agonist or an antagonist of said receptor.  
     
     
         43 . A method according to  claim 42 , wherein the second messenger assay comprises measurement of intracellular cAMP, intracellular Inositol phosphate, intracellular diacylglycerol concentration or intracellular calcium mobilisation.  
     
     
         44 . Method according to  claim 42  or  43 , wherein the cell is a mammalian cell, preferably non neuronal in origin, and selected from the group consisting of COS-7 cells, CHO cells, LM(tk-) cells, NIH-3T3 cells or 1321N1 cells.  
     
     
         45 . Method according to any of the preceding  claims 42  to  44 , wherein the ligand is not previously known.  
     
     
         46 . Ligand detected by the method according to any of the preceding  claims 42  to  45 .  
     
     
         47 . Pharmaceutical composition which comprises the ligand according to  claim 46  and a pharmaceutically acceptable carrier.  
     
     
         48 . Method of screening drugs to identify drugs which specifically bind to the receptor according to any of the  claims 1  to  6  on the surface of the cell, which comprises contacting a cell transfected with a vector expressing the nucleic acid molecule encoding said receptor with a plurality of drugs under conditions permitting binding of said drugs to the receptor, and determining those drugs which specifically bind to the transfected cell, thereby identifying drugs which specifically bind to the receptor.  
     
     
         49 . Method of screening drugs to identify drugs which specifically bind to the receptor according to any of the  claims 1  to  6  on the surface of the cell, which comprises preparing a cell extract from cells transfected with a vector expressing the nucleic acid molecule encoding said receptor, isolating a membrane fraction from the cells extract, contacting the membrane fraction with a plurality of drugs and determining those drugs which bind to the transfected cell, thereby identifying drugs which specifically bind to said receptor.  
     
     
         50 . Method of screening drugs to identify drugs which act as agonists of the receptor according to any of the  claims 1  to  6 , which comprises contacting a cell transfected with a vector expressing the nucleic acid molecule encoding said receptor with a plurality of drugs under conditions permitting the activation of a functional receptor response, and determining those drugs which activate such receptor using a bio-assay, such as a modification in a second messenger concentration or modification in the cellular metabolism, thereby identifying drugs which act as receptor agonists.  
     
     
         51 . Method of screening drugs to identify drugs which act as agonists of the receptor according to any of the  claims 1  to  6 , which comprises preparing a cell extract from cells transfected with a vector expressing the nucleic acid molecule encoding said receptor, isolating a membrane fraction from the cell extract, contacting the membrane fraction with a plurality of drugs under conditions permitting the activation of a functional receptor response, and determining those drugs which activate such receptor using a bio-assay, such as a modification in a second messenger concentration, thereby identifying drugs which act as receptor agonists.  
     
     
         52 . Method of screening drugs to identify drugs which act as antagonists of the receptor according to any of the  claims 1  to  6 , which comprises contacting a cell transfected with a vector expressing the nucleic acid molecule encoding said receptor with a plurality of drugs in the presence of a known receptor agonist, under conditions permitting the activation of a functional receptor response, and determining those drugs which inhibit the activation of the receptor using a bio-assay, such as a modification in a second messenger concentration or modification in the cellular metabolism, thereby identifying drugs which act as receptor antagonists.  
     
     
         53 . Method of screening drugs to identify drugs which act as antagonists of the receptor according to any of the  claims 1  to  6 , which comprises preparing a cell extract from cells transfected with a vector expressing the nucleic acid molecule encoding said receptor, isolating a membrane fraction from the cell extract, contacting the membrane fraction with a plurality of drugs in presence of a known receptor agonist, under conditions permitting the activation of a functional receptor response, and determining those drugs which inhibit the activation of the receptor using a bio-assay, such as a modification in a second messenger concentration, thereby identifying drugs which act as receptor antagonists.  
     
     
         54 . Drug detected by any of the methods according to  claims 48  to  53 .  
     
     
         55 . Pharmaceutical composition comprising a drug according to  claim 54 .  
     
     
         56 . Method of detecting the expression of the receptor according to any of the  claims 1  to  6 , by detecting the presence of -mRNA coding said receptor, which comprises obtaining total RNA or total mRNA from the cell and contacting the RNA or mRNA so obtained with the nucleic acid probe according to  claim 19  under hybridising conditions, and detecting the presence of mRNA hybridised to the probe, thereby detecting the expression of the receptor by the cell.  
     
     
         57 . Method of detecting the presence of the receptor according to any of the  claims 1  to  6  on the surface of a cell, which comprises contacting the cell with the antibody of  claim 26  under conditions permitting binding of the antibody to the receptor, and detecting the presence of the antibody bound to the cell, thereby detecting the presence of the receptor on the surface of the cell.  
     
     
         58 . Method of determining the physiological effects of expressing varying levels of the receptor according to any of the  claims 1  to  6 , which comprises producing a transgenic non human mammal according to any of the  claims 36  to  41  whose levels of receptor expression are varied by use of an inducible promoter which regulates the receptor expression.  
     
     
         59 . Method of determining the physiological effects of expressing varying levels of the receptor according to any of the  claims 1  to  6 , which comprises producing a panel of transgenic non human mammals according to any of the  claims 36  to  41 , each expressing a different amount of said receptor.  
     
     
         60 . Method for identifying an antagonist of the receptor according to any of the  claims 1  to  6  capable of alleviating an abnormality in a subject wherein the abnormality is alleviated by decreasing the activity of the receptor, which comprises administering the antagonist to a transgenic non human mammal according to any of the  claims 36  to  41  and determining whether the antagonist alleviates the physical and behavioural abnormalities displayed by the transgenic non human mammal as a result of receptor activity, thereby identifying the antagonist.  
     
     
         61 . Antagonist identified by the method of  claim 60 .  
     
     
         62 . Pharmaceutical composition comprising an antagonist according to  claim 61  and a pharmaceutically acceptable carrier.  
     
     
         63 . Method for identifying an agonist of the receptor according to any of the  claims 1  to  6  capable of alleviating an abnormality in a subject wherein the abnormality is alleviated by activation of said receptor, which comprises administering the agonist to a transgenic non human mammal according to any of the  claims 36  to  41  and determining whether the antagonist alleviates the physical and behavioural abnormalities displayed by the transgenic non human mammal, the alleviation of the abnormalities indicating the identification of the agonist.  
     
     
         64 . Agonist identified by the method of  claim 63 .  
     
     
         65 . Pharmaceutical composition comprising an agonist according to  claim 64  and a pharmaceutically acceptable carrier.  
     
     
         66 . Method for diagnosing a predisposition to a disorder associated with the activity of a specific allele of the receptor according to any of the  claims 1  to  6 , which comprises: 
 a) obtaining nucleic acid molecules of subjects suffering from said disorder;  
 b) performing a restriction digest of said nucleic acid molecules with a panel of restriction enzymes;  
 c) electrophoretically separating the resulting nucleic acid fragments on a sized gel;  
 d) contacting the resulting gel with a nucleic acid probe capable of specifically hybridising to said nucleic acid molecule and labelled with a detectable marker;  
 e) detecting labelled bands which have hybridised to the said nucleic acid molecule labelled with a detectable marker to create a unique band pattern specific to subjects suffering from said disorder;  
 f) preparing nucleic acid molecules obtained for diagnosis by step a-e; and  
 g) comparing the unique band pattern specific to the nucleic acid molecule of subjects suffering from the disorder from step e and the nucleic acid molecule obtained for diagnosis from step f to determine whether the patterns are the same or different and to diagnose thereby predisposition to the disorder if the patterns are the same.  
 
     
     
         67 . Method of preparing the purified receptor according to any of the  claims 1  to  6 , which comprises: 
 a) constructing a vector adapted for expression in a cell which comprises the regulatory elements necessary for the expression of nucleic acid molecules in the cell operatively linked to nucleic acid molecule encoding said receptor so as to permit expression thereof, wherein the cell is selected from the group consisting of bacterial cells, yeast cells, insect cells and mammalian cells;  
 b) inserting the vector of step a in a suitable host cell;  
 c) incubating the cell of step b under conditions allowing the expression of the receptor according to the invention;  
 d) recovering the receptor so obtained; and  
 e) purifying the receptor so recovered, thereby preparing an isolated receptor according to the invention.  
 
     
     
         68 . Use of the pharmaceutical composition according to  claim 47 ,  55 ,  62  or  65 , in the preparation of a medicament for the treatment and/or the prevention of the neutropenie, agranulocytose infections or cancer.