MECHANISMS OF G PROTEIN COUPLED RECEPTOR REGULATION

G蛋白偶联蛋白调控着许多重要的生理过程。该家族的两个典型成员，β 2-肾上腺素能受体（beta2AR）和P物质受体分别是心血管系统和外周神经系统和中枢神经系统疼痛传递的关键介质。虽然这些受体对不同的配体有反应，但它们具有共同的结构特征。此外，G蛋白偶联受体激酶（GRKS）和β -抑制素调节了它们信号能力的脱敏和再敏以及内化能力。几乎所有关于GRKs和β -抑制素调控gpcr的信息都来自于使用细胞膜提取物或重组蛋白系统的研究。本研究的总体目标是通过对活细胞中GPCR信号传导过程的研究，明确调控GPCR信号传导的分子机制，最终了解GPCR异常调控引起的细胞生理和病理生理。β -肾上腺素能受体和P物质受体将作为代表性受体，因为它们的整体行为应该反映许多其他GPCR家族成员的行为。基于可视化β 2ar或P物质受体GRK2和β -抑制素1和2的行为的检测，利用它们的荧光嵌合体，将用于实时跟踪受体脱敏和细胞再分配。目的1：利用GRKs和β -抑制因子的新型绿色荧光蛋白变体，实时研究活细胞中β 2-肾上腺素能和P物质受体中激动剂介导的信号脱敏。这些结果应该为复杂细胞系统中gpcr的分子调控提供重要的见解。目的II-是研究β -肾上腺素能受体和P物质受体下调的细胞内吞通路。该结果将为了解gpcr是如何作为长期激动剂暴露的结果而降解或再循环的目标提供基础。提出的研究应该加强我们对基本分子和细胞特性的理解，这些特性对于维持正常的GPCR信号转导很重要。从中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。

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.