Structure and mechanism of beta2-adrenergic receptor

β2-肾上腺素受体的结构和机制

• 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
• 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

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的机制，从 激素介导的G蛋白异三个家族的激活。我的实验室努力 确定与鸟嘌呤核苷酸交换有关激素受体结合的分子细节。这 当前的工作范例是β2-肾上腺素能受体（B2R）和刺激异三聚体G蛋白， GSABG。在理解视紫红质活性机制方面已取得了很大进展， 原型G蛋白偶联受体（GPCR）。相比之下，其他关于其他的知之甚少 GPCR。此外，虽然很明显，视紫红质和几种G蛋白的结构揭示了 它们独立功能的分子细节，对它们作为复合物的结构和功能知之甚少。 我们对Rhodopsin和所有GPCR的激活如何导致G蛋白的激活的理解 刺激核苷酸交换是不完整的。我建议采用生化和结构性方法 描述激素结合位点结构，B2R：GSABG复合物的第四纪结构， 开发一个模型，以阐明如何发生激素依赖性核苷酸交换。我会发展一个 在与GSABG复合物中生产活性B2R的表达和纯化系统。这些试剂 将用于评估B2R：GSABG活性的实时分光体测量。呢会带来各种各样的 确定和表征B2R和GSABG之间相互作用位点的方法 交联，位置定向的诱变和荧光共振能量传递光谱法。我进一步 扩展了B2R作为低聚物的存在，并且二聚化的功能固有地与 受体激活的机制，从而G蛋白激活。我将努力描绘低 B2R：GSABG复合物在各种激活状态下的高分辨率结构。最后，我将整合 这些数据以制定了一种模型，该模型是B2R激活如何导致核苷酸交换激活的模型 并建议该模型将扩展到整个GPCR家族。

项目成果

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.
• 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。

• DOI: 10.1021/ac071203
• 发表时间: 2007
• 期刊: Analytical chemistry
• 影响因子: 7.4
• 作者: Cunliffe,JenniferM;Sunahara,RogerK;Kennedy,RobertT
• 通讯作者: Kennedy,RobertT