MECHANISMS OF G PROTEIN COUPLED RECEPTOR REGULATION

G蛋白偶联调节许多重要的生理过程。该家族的两个典型成员，β2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和中枢神经系统疼痛传递的关键介质。尽管这些受体中的每一个都对不同的配体做出反应，但它们有共同的结构特征。此外，它们的信号能力的脱敏和再增敏以及它们的内化能力受到G蛋白偶联受体激酶(GRKS)和β-拦阻蛋白的调节。几乎所有关于GRKs和β-拦阻蛋白调控GPCRs的信息都来自于使用细胞膜提取液或重组蛋白质系统的研究。这项研究的总体目标是通过研究活细胞中的这些过程来确定调控GPCR信号转导的分子机制，最终目的是了解异常GPCRs调控引起的细胞生理学和病理生理学。β2-肾上腺素能受体和P物质受体将被用作代表性受体，因为它们的整体行为应该反映许多其他GPCR家族成员的行为。基于利用其荧光嵌合体可视化β2AR或P物质受体GRK2和β-arrestin 1和2行为的能力的分析将被用于实时跟踪受体脱敏和细胞重新分布。目的利用新型绿色荧光蛋白突变体GRKs和β-arrestins在活细胞中实时研究激动剂介导的β2-肾上腺素能受体和P物质受体信号的脱敏作用。这一结果将为复杂细胞系统中GPCRs的分子调控提供重要的见解。目的研究β2肾上腺素能受体和P物质受体下调的细胞内吞途径。这一结果将为理解由于长期接触激动剂而如何针对GPCRs进行降解或回收提供基础。建议的研究将加强我们对分子和细胞的基本性质的理解，这些性质对于维持正常的GPCR信号转导是重要的。从他们那里获得的洞察力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.