PURIFICATION AND PHOSPHORYLATION OF A P2Y-PURINOCEPTOR

P2Y-嘌呤受体的纯化和磷酸化

• 批准号: 2749751
• 负责人: ROY K PALMER
• 金额: $ 1.57万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-25 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2749751
• 关键词: G protein; phosphorylation; protein engineering; protein kinase; protein purification; purinergic receptor; receptor sensitivity; recombinant proteins

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.

P2 Y-嘌呤受体（p2 Y-R），像许多其他受体，是脱敏， 长时间接触兴奋剂 脱敏现象经常被发现 这是由受体水平发生的变化引起的， 是由蛋白质磷酸化引发的。G蛋白受体激酶（GRK） 识别并磷酸化G蛋白连接的受体，包括那些 与磷酸肌醇特异性磷脂酶C-β结合。GRK- 人P2 Y-R（hP 2 Y-R）介导的调节将在 提议的研究。该项目的具体目标是：a）建造一个 含有氨基和羧基修饰的重组hP 2 Y-R 增强受体表达和纯化的末端; B） 表达重组hP 2 Y-R，并开发用于表达hP 2 Y-R的体外系统。 GRK催化的hP 2 Y-R磷酸化的研究; c）纯化 重组hP 2 Y-R，并鉴定其氨基酸残基， 磷酸化，和d）确认激动剂促进的位点 在体外系统中鉴定的GRK-磷酸化是生理性的， 与激动剂诱导的hP 2 Y-R脱敏过程有关， 完整的细胞

项目成果

Agonist action of adenosine triphosphates at the human P2Y1 receptor.

三磷酸腺苷对人类 P2Y1 受体的激动作用。

• 发表时间: 1998
• 期刊: Molecular pharmacology
• 影响因子: 3.6
• 作者: Palmer,RK;Boyer,JL;Schachter,JB;Nicholas,RA;Harden,TK
• 通讯作者: Harden,TK