PURIFICATION AND PHOSPHORYLATION OF A P2Y-PURINOCEPTOR

像许多其他受体一样，p2Y-嘌呤受体(p2Y-R)通过 长期接触激动剂。脱敏作用经常被发现 由受体水平上发生的变化引起，并且很可能 是由蛋白质磷酸化启动的。G蛋白受体激酶(GRK) 识别和磷酸化G蛋白相关受体，包括那些 与磷脂酰肌醇特异性磷脂酶C-β偶联。GRK- 人类P2Y-R(hP2Y-R)的中介调节将在 拟开展的研究。该项目的具体目标是a)建立一个 含有氨基和羧基修饰的重组hP2Y-R 将增强受体表达和纯化的Termini；b) 重组hP2Y-R的表达及其体外系统的建立 GRK催化hP2Y-R磷酸化的研究；c)纯化 重组hP2Y-R并鉴定其氨基酸残基(S)，即 磷酸化，以及d)确认激动剂促进的位点(S) 在体外系统中确定的GRK-磷酸化是生理学的 与激动剂诱导的hP2Y-R脱敏过程有关 完好无损的细胞。

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.