Structure and mechanism of beta2-adrenergic receptor

β2-肾上腺素受体的结构和机制

• 批准号: 6784230
• 负责人: Roger K Sunahara
• 金额: $ 32.52万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6784230
• 关键词: G protein; G protein coupled receptor kinase; SDS polyacrylamide gel electrophoresis; X ray crystallography; adrenergic receptor; binding sites; biological signal transduction; crosslink; cryoelectron microscopy; enzyme activity; fluorescence resonance energy transfer; fluorescence spectrometry; gel filtration chromatography; guanine nucleotide exchange factors; hormone regulation /control mechanism; mass spectrometry; matrix assisted laser desorption ionization; site directed mutagenesis; two dimensional gel electrophoresis

DESCRIPTION (provided by applicant): The long-term goal of my laboratory is to understand the mechanism of cellular signaling, beginning at hormone-mediated activation of the heterotrimeric family of G proteins. My laboratory endeavors to determine the molecular details relating hormone-receptor binding with guanine-nucleotide exchange. The current working paradigm is the beta2-adrenergic receptor (beta2R) and the stimulatory heterotrimeric G protein, Gsabg. Considerable progress has been made toward understanding the mechanism of rhodopsin activity, the prototypical G protein-coupled receptor (GPCR). In contrast, comparatively little is known about other GPCRs. Furthermore, while it is clear that the structures of rhodopsin and several G proteins reveal the molecular details of their independent function, little is known about their structure and function as a complex. Our understanding of how activation of rhodopsin, and all GPCRs, leads to activation of G proteins, through stimulation of nucleotide exchange is incomplete. I propose to take a biochemical and structural approach to delineate the hormone-binding site structure, the quaternary structure of the beta2R:Gsabg complex, and develop a model to elucidate how hormone-dependent nucleotide exchange occurs. I will develop an expression and purification system for the production of active beta2R in a complex with Gsabg. These reagents will be used to assess real time spectrophotometric measurements of beta2R:Gsabg activity. I will take various approaches to determine and characterize the site of interaction between beta2R and Gsabg using cross-linking, site-directed mutagenesis and fluorescence resonance energy transfer spectrometry. I further extend that the beta2R exists as an oligomer and that the function of dimerization relates inherently to the mechanism of receptor activation, and thus G protein activation. I will engage in an effort to delineate the low and high resolution structures of the beta2R:Gsabg complex in various activation states. Finally, I will integrate these data to formulate a model of how hormone activation of beta2R leads to activation of nucleotide exchange and propose that this model will extend to the entire family of GPCRs.

描述（由申请人提供）：我实验室的长期目标是了解细胞信号传导机制，从激素介导的 G 蛋白异三聚体家族的激活开始。我的实验室致力于确定激素受体结合与鸟嘌呤核苷酸交换相关的分子细节。目前的工作范例是 β2 肾上腺素受体 (beta2R) 和刺激性异三聚体 G 蛋白 Gsabg。在了解视紫红质活性（原型 G 蛋白偶联受体 (GPCR)）的机制方面已经取得了相当大的进展。相比之下，人们对其他 GPCR 知之甚少。此外，虽然视紫红质和几种 G 蛋白的结构清楚地揭示了它们独立功能的分子细节，但人们对它们作为复合物的结构和功能知之甚少。我们对视紫红质和所有 GPCR 的激活如何通过刺激核苷酸交换导致 G 蛋白激活的理解并不完整。我建议采用生化和结构方法来描述激素结合位点结构、β2R:Gsabg 复合物的四级结构，并开发一个模型来阐明激素依赖性核苷酸交换是如何发生的。我将开发一种表达和纯化系统，用于在与 Gsabg 的复合物中生产活性 beta2R。这些试剂将用于评估 beta2R:Gsabg 活性的实时分光光度测量。我将采用多种方法，使用交联、定点诱变和荧光共振能量转移光谱法来确定和表征 beta2R 和 Gsabg 之间的相互作用位点。我进一步认为，β2R 作为寡聚物存在，二聚化功能本质上与受体激活机制相关，从而与 G 蛋白激活机制相关。我将致力于描绘各种激活状态下 beta2R:Gsabg 复合物的低分辨率和高分辨率结构。最后，我将整合这些数据来制定一个模型，说明 beta2R 的激素激活如何导致核苷酸交换的激活，并建议该模型将扩展到整个 GPCR 家族。