REGULATION OF ADENOSINE RECEPTOR SIGNALING

腺苷受体信号传导的调节

• 批准号: 2635027
• 负责人: AMY L TUCKER
• 金额: $ 8.34万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-15 至 1999-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2635027
• 关键词: CHO cells; G protein; SDS polyacrylamide gel electrophoresis; adenosine; allosteric site; biological signal transduction; cell type; chimeric proteins; dogs; ligands; messenger RNA; phosphorylation; receptor coupling; tissue /cell culture

The objective of the research proposal is to understand the regulation of adenosine receptor signal transduction. These mechanisms could be fundamental in the roles of adenosine as a cardioprotector and a smooth muscle and endothelial cell mitogen. A1 and A2a adenosine receptor subtypes have opposite effects on adenylyl cyclase. Subtypes also appear to have distinct desensitization profiles. These features will be exploited in designing canine A1/A2a receptor chimeric receptors in whiCh the intracellular domains thought to regulate G protein coupling and desensitization have been switched from one subtype to the other. The chimeric receptors will be compared to wildtype recombinant receptors using a number of different experimental approaches to determine G protein coupling selectivity and desensitization patterns. Classic indirect markers for coupling to particular G proteins, such as alterations in high affinity ligand binding in the presence of Gi-selective uncoupling agents, and changes in adenylyl cyclase responses, will be supplemented with newer, more direct methodologies based on purification of receptor-G protein complexes. The G proteins associated with the purified complexes will be identified and quantitated using subtype specific antibodies. Protein phosphorylation is thought to play an important role in agonist- induced receptor desensitization. The kinetics of desensitization for chimeric and wildtype adenosine receptors will be compared in CHO cells and related to the extent and location of phosphorylation of the receptors and their associated G proteins. Changes in native receptor mRNA levels and G protein mRNA levels during desensitization will be assayed using solution hybridization. Immunocytochemistry will be used to identify changes in the localization of G protein subunits following adenosine receptor stimulation. Finally, using ligand binding and cAMP assays, the chimeras will be used to explore the mechanism of action of compounds which are allosteric enhancers of agonist ligand binding to A1 adenosine receptors.

研究建议的目的是了解 腺苷受体信号转导。这些机制可能是 腺苷作为心脏保护剂的角色和平滑的基础 肌肉和内皮细胞有丝分裂原。 A1和A2A腺苷受体 亚型对腺苷酸环化酶具有相反的影响。还出现了亚型 具有独特的脱敏轮廓。这些功能将是 在设计犬A1/A2A受体嵌合受体中被利用 被认为调节G蛋白偶联的细胞内结构域和 脱敏已从一个亚型切换到另一个亚型。这 将将嵌合受体与野生型重组受体进行比较 使用多种不同的实验方法来确定G蛋白 耦合选择性和脱敏模式。 经典间接 与特定G蛋白耦合的标记物，例如高改变 在存在GI选择性解偶联剂的情况下，亲和力配体结合， 并将补充腺苷酸环化酶反应的变化 基于受体-G的纯化，更新的，更直接的方法论 蛋白质复合物。与纯化复合物相关的G蛋白 将使用亚型特异性抗体识别和定量。 蛋白质磷酸化被认为在激动剂中起重要作用 诱导受体脱敏。脱敏的动力学 将比较CHO细胞中的嵌合和野生型腺苷受体 并与受体磷酸化的程度和位置有关 及其相关的G蛋白。天然受体mRNA水平的变化 将使用脱敏期间的G蛋白mRNA水平进行测定 溶液杂交。免疫细胞化学将用于识别 腺苷后G蛋白亚基的定位变化 受体刺激。最后，使用配体装订和营地测定法 嵌合体将用于探索化合物的作用机理 是激动剂配体与A1腺苷结合的变构增强剂 受体。