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）：我的实验室的长期目标是了解细胞信号传导的机理，始于激素介导的G蛋白的异构三聚体家族的激活。我的实验室努力确定分子细节与鸟嘌呤核苷酸交换有关的激素受体结合。当前的工作范例是β2-肾上腺素能受体（BETA2R）和刺激性异三聚体G蛋白GSABG。在理解视紫红蛋白活性的机理，原型G蛋白偶联受体（GPCR）方面已取得了长足的进步。相比之下，其他GPCR知之甚少。此外，虽然很明显，视紫红质和几种G蛋白的结构揭示了其独立功能的分子细节，但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蛋白的激活的理解是不完整的。我建议采用生化和结构方法来描述激素结合位点结构，beta2r：gsabg复合物的第四纪结构，并开发出一种模型来阐明激素依赖性核苷酸的交换是如何发生的。我将开发一个表达式和纯化系统，用于与GSABG复合体中活性beta2r的生产。这些试剂将用于评估BetA2R：GSABG活性的实时分光光度测量。我将采用各种方法来确定和表征beta2r和gsabg之间相互作用的位点，并使用交联，定位的诱变和荧光共振能量传递光谱法。我进一步扩展了β2R作为低聚物存在，二聚化的功能固有地与受体激活的机理有关，从而使g蛋白激活。我将努力描述各种激活状态下Beta2R：GSABG复合物的低分辨率结构。最后，我将整合这些数据，以制定一个模型，该模型是如何导致核苷酸交换激活的激素激活，并提出该模型将扩展到整个GPCR家族。