G-coupled receptor showing selective affinity for ATP
The present invention relates to an isolated G-protein coupled receptor, nucleic acid sequence encoding the receptor, and host cells capable of expressing the receptor. The invention further comprises methods for detecting the expression of a G-protein coupled receptor, and methods for identifying agonists or antagonists of the receptor. The invention still further encompasses methods for preparing an isolated G-protein coupled receptor.
1. A method for determining whether a ligand can specifically bind to a receptor comprising an amino acid sequence that is at least 95% identical to amino acid residues 424-795 of SEQ ID NO: 1, comprising the steps of:
(a) contacting a cell transfected with a nucleic acid molecule encoding said receptor or cell extract obtained from cells transfected with the nucleic acid molecule encoding said receptor with said ligand under conditions which permit the binding of said ligand to said receptor; and
(b) detecting the presence of any of said ligand bound to said receptor, thereby determining whether said ligand binds to said receptor.
2. The method of claim 1 , wherein said ligand is an agonist or antagonist of said receptor.
3. The method of claim 1 , wherein said binding is detected by measuring a modification of cell metabolism resulting from the stimulation of an intracellular cascade.
4. The method according to claim 3 , wherein said modification of cell metabolism is detected by monitoring the acidification rate of the medium in which said cells, are cultured.
5. The method of claim 1 , wherein said binding is detected by measuring a change in second messenger concentration.
6. The method of claim 5 , wherein said measuring a change in second messenger concentration is performed by a measuring step selected from the group consisting of measuring a change in intracellular cAMP concentration, measuring a change in intracellular inositol phosphate concentration, measuring a change in intracellular diacylglycerol concentration, and measuring a change in intracellular calcium concentration.
7. A method for determining whether a ligand can specifically bind and modulate the activity of a receptor comprising an amino acid sequence that is at least 95% identical to amino acid residues 424-795 of SEQ ID NO: 1, comprising the steps of:
(a) contacting a cell transfected with a nucleic acid molecule encoding said receptor or cell extract obtained from cells transfected with the nucleic acid molecule encoding said receptor with said ligand under conditions which permit the binding of said ligand to said receptor; and
(b) detecting a change in a second messenger concentration, wherein said change in a second messenger concentration is indicative of said ligand binding and modulation of the activity of said receptor.
8. The method of claim 7 , wherein said measuring a change in second messenger concentration is performed by a measuring step selected from the group consisting of measuring a change in intracellular cAMP concentration, measuring a change in intracellular inositol phosphate concentration, measuring a change in intracellular diacyiglycerol concentration, and measuring a change in intracellular calcium concentration.
9. The method of claim 7 , wherein said ligand is an agonist or antagonist of said receptor.
10. A method for determining whether a ligand can specifically bind and modulate the activity of a receptor comprising an amino acid sequence that is at least 95% identical to amino acid residues 424-795 of SEQ ID NO: 1, comprising the steps of:
(a) contacting a cell transfected with a nucleic acid molecule encoding said receptor or cell extract obtained from cells transfected with the nucleic acid molecule encoding said receptor with said ligand under conditions which permit the binding of said ligand to said receptor; and
(b) detecting a modification in cellular metabolism, wherein modification of cellular metabolism is indicative of said ligand binding and modulation of the activity of said receptor.
11. The method of claim 10 , wherein said modification in cellular metabolism is determined by measuring the acidification rate of the medium in which said cell is cultured.
12. The method of claim 10 , wherein said ligand is an agonist or antagonist of said receptor.