MECHANISMS OF G PROTEIN COUPLED RECEPTOR REGULATION

G 蛋白偶联受体调节机制

• 批准号: 6390099
• 负责人: LAWRENCE S. BARAK
• 金额: $ 24.57万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6390099
• 关键词: G protein; arrestins; beta adrenergic receptor; biological signal transduction; chimeric proteins; confocal scanning microscopy; flow cytometry; green fluorescent proteins; neuropeptide receptor; nucleic acid sequence; phosphorylation; protein localization; protein structure function; receptor coupling; receptor expression; receptor mediated endocytosis; receptor sensitivity; substance P; tissue /cell culture

G protein-coupled regulate many critical physiological processes. Two prototypical members of this family, the beta2-adrenergic receptor (beta2AR) and substance P receptor are the key mediators of the cardiovascular system and of pain transmission in the peripheral and central nervous systems, respectively. Although each of these receptors responds to a different ligand, they share common structural features. Moreover, the desensitization and resensitization of their signaling abilities and their ability to internalize are regulated by G protein- coupled receptor kinases (GRKS) and beta arrestins. Almost all of the information concerning the regulation of GPCRs by the GRKs and beta- arrestins derives from studies using either cellular membrane extracts or reconstituted system of proteins. The overall objective of the proposed research is to define the molecular mechanisms governing GPCR signaling by studying these processes in live cells, with the ultimate goal of understanding the cell physiology and pathophysiology arising from abnormal GPCR regulation. The beta2-adrenergic receptor and substance P receptor will be used as representative receptors, since their overall behavior should reflect that of many other GPCR family members. Assays based upon the ability to visualize the behavior of the beta2AR or the substance P receptor, GRK2, and beta-arrestin 1 and 2, utilizing their fluorescent chimeras, will be employed to follow receptor desensitization and cell redistribution in real-time. Aim I-is to study the agonist-mediated desensitization of signaling in the beta2- adrenergic and substance P receptors in real-time in living cells using novel green fluorescent protein variants of GRKs and beta arrestins. The results should provide significant insight into the molecular regulation of GPCRs in complex cellular systems. Aim II- is to study the cellular endocytic pathway responsible for beta2-adrenergic receptor and substance P receptor down regulation. The results will provide a basis to understand how GPCRs are targeted for degradation or recycling as a result of prolonged agonist exposure. The proposed studies should enhance our understanding of the fundamental molecular and cellula properties that are important for maintenance of normal GPCR signal transduction. Insight gained from them should provide a foundation for the development of new treatment regiments that target diseases such as hypertension, heart failure, and chronic pain syndromes.

G蛋白偶联调节许多重要的生理过程。该家族的两个原型成员，β 2-肾上腺素能受体（β 2AR）和P物质受体分别是心血管系统和外周及中枢神经系统疼痛传递的关键介质。虽然这些受体中的每一个对不同的配体作出反应，但它们具有共同的结构特征。此外，它们的信号传导能力和它们的内化能力的脱敏和再敏化由G蛋白偶联受体激酶（GRKS）和β抑制蛋白调节。几乎所有关于GRK和β-抑制蛋白调节GPCR的信息都来自于使用细胞膜提取物或蛋白质重构系统的研究。拟议研究的总体目标是通过研究活细胞中的这些过程来确定GPCR信号传导的分子机制，最终目标是了解异常GPCR调控引起的细胞生理学和病理生理学。β 2-肾上腺素能受体和P物质受体将被用作代表性受体，因为它们的总体行为应反映许多其他GPCR家族成员的行为。将采用基于可视化β 2 AR或P物质受体、GRK 2和β-抑制蛋白1和2的行为的能力的测定，利用其荧光嵌合体，实时跟踪受体脱敏和细胞再分布。目的利用GRKs和β-arrestins的新型绿色荧光蛋白变体，在活细胞中实时研究激动剂介导的β 2-肾上腺素能受体和P物质受体信号转导的脱敏作用。这些结果将为复杂细胞系统中GPCR的分子调控提供重要的见解。目的II-研究β 2-肾上腺素能受体和P物质受体下调的细胞内吞途径。这些结果将为理解GPCR如何作为长期激动剂暴露的结果而被靶向降解或回收提供基础。拟议的研究应提高我们的基本分子和cellula属性，是重要的维持正常的GPCR信号转导的理解。从他们那里获得的见解应该为开发针对高血压、心力衰竭和慢性疼痛综合征等疾病的新治疗方案提供基础。