Structure and mechanism of beta2-adrenergic receptor

β2-肾上腺素受体的结构和机制

基本信息

批准号：
7487178
负责人：
Roger K Sunahara
金额：
$ 4.56万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-11-01 至 2008-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7487178
关键词：
Adrenergic Receptor Binding Binding Sites Biochemical Biological Assay Chemicals Chimeric Proteins Complex Condition Coupled Crystallization Crystallography Data Dimerization Endopeptidases Family Fluorescence Resonance Energy Transfer Fluorescence Spectroscopy G-Protein-Coupled Receptors GTP-Binding Proteins Gel Chromatography Goals Guanine Nucleotides Heterotrimeric GTP-Binding Proteins Hormone Receptor Hormones Laboratories Libraries Location Luciferases Mass Spectrum Analysis Measurement Mediating Microscopy Modeling Molecular Nucleotides Peptide Hydrolases Production Property Proteins Proteolysis Reagent Receptor Activation Resolution Rhodopsin Role Screening procedure Signal Transduction Site Site-Directed Mutagenesis Spectrometry Spectrum Analysis Structure System Technology Time Work crosslink fluorophore gel electrophoresis protein activation receptor receptor binding reconstruction spatial relationship stoichiometry structural biology

项目摘要

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 (b2R) 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 nucieotide exchange is incomplete. I propose to take a biochemical and structural approach to delineate the hormone-binding site structure, the quaternary structure of the b2R: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 b2R in a complex with Gsabg. These reagents will be used to assess real time spectrophotometic measurements of b2R:Gsabg activity. ! will take various approaches to determine and characterize the site of interaction between b2R and Gsabg using cross-linking, site-directed mutagenesis and fluorescence resonance energy transfer spectrometry. I further extend that the b2R 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 b2R:Gsabg complex in various activation states. Finally, I will integrate these data to formulate a model of how hormone activation of b2R leads to activation of nucleotide exchange and propose that this model will extend to the entire family of GPCRs.

我实验室的长期目标是了解细胞信号传导的机制，从激素介导的 G 蛋白异源三聚体家族的激活。我的实验室致力于确定与激素受体结合与鸟嘌呤核苷酸交换相关的分子细节。这目前的工作范例是 β2-肾上腺素受体 (b2R) 和刺激性异三聚体 G 蛋白，格萨布格。在了解视紫红质活性机制方面已经取得了相当大的进展，原型 G 蛋白偶联受体 (GPCR)。相比之下，人们对其他方面知之甚少。 GPCR。此外，虽然很明显视紫红质和几种 G 蛋白的结构揭示了尽管它们的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 的激活如何导致 G 蛋白激活的理解是：核苷酸交换的刺激是不完全的。我建议采取生化和结构方法描绘激素结合位点结构、b2R:Gsabg 复合物的四级结构，以及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。我将开发一个表达和纯化系统，用于在与 Gsabg 的复合物中生产活性 b2R。这些试剂将用于评估 b2R:Gsabg 活性的实时分光光度测量。！将采取各种确定和表征 b2R 和 Gsabg 之间相互作用位点的方法交联、定点诱变和荧光共振能量转移光谱测定。我进一步延伸出 b2R 作为寡聚物存在，并且二聚化的功能本质上与受体激活的机制，从而G蛋白激活。我将努力描绘低点以及各种激活状态下 b2R:Gsabg 复合物的高分辨率结构。最后我会整合这些数据建立了一个模型，说明 b2R 的激素激活如何导致核苷酸交换的激活并建议将该模型扩展到整个 GPCR 系列。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Cloning and characterization of the G protein betagamma subunits from Trichoplusia ni (High Five cells).

Trichoplusia ni（高五细胞）G 蛋白 betaamma 亚基的克隆和表征。

DOI：
10.1016/j.ibmb.2004.12.011
发表时间：
2005
期刊：
Insect biochemistry and molecular biology.
影响因子：
0
作者：
Vadakkadathmeethal,Kannan;Felczak,Aimee;Davignon,Isabelle;Collins,Julie;Sunahara,RogerK
通讯作者：
Sunahara,RogerK

A CE assay for the detection of agonist-stimulated adenylyl cyclase activity.

用于检测激动剂刺激的腺苷酸环化酶活性的 CE 测定。

DOI：
10.1002/elps.200600571
发表时间：
2007
期刊：
Electrophoresis
影响因子：
2.9
作者：
Cunliffe,JenniferM;Whorton,MatthewR;Sunahara,RogerK;Kennedy,RobertT
通讯作者：
Kennedy,RobertT

Ligand-regulated oligomerization of beta(2)-adrenoceptors in a model lipid bilayer.

DOI：
10.1038/emboj.2009.267
发表时间：
2009-11-04
期刊：
EMBO JOURNAL
影响因子：
11.4
作者：
Fung, Juan Jose;Deupi, Xavier;Pardo, Leonardo;Yao, Xiao Jie;Velez-Ruiz, Gisselle A.;DeVree, Brian T.;Sunahara, Roger K.;Kobilka, Brian K.
通讯作者：
Kobilka, Brian K.

Detection of g protein coupled receptor mediated adenylyl cyclase activity by capillary electrophoresis using fluorescently labeled ATP.

使用荧光标记的 ATP 通过毛细管电泳检测 g 蛋白偶联受体介导的腺苷酸环化酶活性。