PURIFICATION AND PHOSPHORYLATION OF A P2Y-PURINOCEPTOR

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P2Y-purinoceptors (p2Y-R), like many other receptors, are desensitized by prolonged exposure to agonists. Desensitization has frequently been found to result from changes occurring at the level of the receptor, and likely is initiated by protein phosphorylation. G-protein receptor kinases (GRK) recognize and phosphorylate G-protein-linked receptors, including those that couple to phosphoinositide-specific phospholipase C-beta. GRK- mediated regulation of the human P2Y-R (hP2Y-R) will be examined in the proposed research. The specific aims of the project are a) to construct a recombinant hP2Y-R containing modifications to the amino and carboxy termini that will enhance receptor expression and purification; b) to express the recombinant hP2Y-R and to develop an in vitro system for the study of GRK-catalyzed hP2Y-R phosphorylation; c) to purify the recombinant hP2Y-R and identify the amino acid residue(s) that is phosphorylated, and d) to confirm that the site(s) of agonist-promoted GRK-phosphorylation identified in the in vitro system is physiologically relevant to the process of agonist-induced desensitization of hP2Y-R in intact cells.