MOLECULAR INTERACTIONS IN G PROTEIN COUPLED SIGNALING

G 蛋白偶联信号传导中的分子相互作用

• 批准号: 6286733
• 负责人: GABOR SZABO
• 金额: $ 24.5万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6286733
• 关键词: Escherichia coli; G protein; Rana; Sf9 cell line; caveolins; fluorescence microscopy; heart cell; lipid bilayer membrane; muscarinic receptor; potassium channel; receptor coupling; recombinant proteins; transfection; voltage /patch clamp

DESCRIPTION (applicant's description): Integration of extracellular signals is an essential function of the plasma membrane. For seven transmembrane segment receptors this function is accomplished by heterotrimeric G proteins that couple receptor activation to specific effectors. While numerous receptors and effectors have been identified and the crystal structure of several G proteins has been solved, the way in which these molecules interact with membranes or participate in chemical reactions comprising the signal transduction cascade remains poorly understood. The main objective of this project is to provide insights into these processes. Total internal reflection fluorescence microscopy (TIRFM) of supported membranes will be used to investigate interactions of fluorophore-labeled G protein alpha and (3y subunits with lipid membranes, either pure or incorporating molecules with which G proteins interact, in order to understand the physical chemistry of G protein interactions in a chemically defined environment. Subunit lipid modifications, including N-terminal myristoylation and thioacylation of Gi/oalpha will be examined since these may play a primary role in the dynamics of subunit interactions with membranes. Membranes approximating the composition of detergent resistant subdomains of the plasma membrane will be examined to identify molecular features that may target G proteins specifically to these areas. Proteins that associate with G proteins, such as RGS, caveolin and a channel effector, will also be examined for their effects on subunit lateral mobility and interactions. In a complementary approach, excised patches of cell membranes expressing G protein regulated muscarinic potassium channels (KACh) will be used not only to assay the functionality of fluorescently labeled and/or chemically modified G protein subunits but also to understand channel regulation in vivo on the basis of our TIRFM results. A combination of quantitative biophysical, biochemical and physiological approaches is believed to be a unique aspect of the proposed studies that should provide realistic and complementary perspectives of G protein coupled signal transduction. Moreover, these studies should help to establish a link between structural features of molecules participating in G protein coupled signaling and the physiology of the regulatory responses. Linking structural features to function is an essential step towards understanding and ameliorating conditions stemming from compromised signal transduction processes in cell membranes.

描述（申请人的描述）：细胞外信号的集成是 质膜的重要功能。对于七个跨膜段 受体这种功能是通过异三聚体G蛋白来完成的 对特定效应子的夫妇受体激活。而许多受体和 已经鉴定出效应子，并且几种G蛋白的晶体结构 已经解决了这些分子与膜或 参与包括信号转导级联的化学反应 仍然很了解。该项目的主要目的是提供 对这些过程的见解。总内反射荧光 支撑膜的显微镜（TIRFM）将用于研究 荧光团标记的G蛋白α和3岁亚基与脂质的相互作用 膜，纯或掺入与g蛋白的分子 相互作用，以了解G蛋白的物理化学 化学定义环境中的相互作用。亚基脂质修饰， 包括gi/oalpha的N末端肉豆蔻酰化和硫代酰化 检查由于这些可能在亚基的动力学中起主要作用 与膜的相互作用。近似于成分的膜 将检查质膜的清洁剂耐洗涤剂的子域 识别可能针对G蛋白的分子特征 区域。与G蛋白相关的蛋白质，例如RGS，小窝蛋白和A 通道效应子也将检查其对亚基侧面的影响 流动性和互动。在互补的方法中，切除细胞的斑块 表达G蛋白调节的毒蕈碱钾通道（KACH）的膜 将不仅用于测定荧光标记的功能 和/或化学修饰的G蛋白亚基，但也了解通道 根据我们的TIRFM结果，体内调节。组合 据信定量生物物理，生化和生理方法 成为拟议研究的独特方面，应该提供现实和 G蛋白耦合信号转导的互补观点。而且， 这些研究应有助于在结构特征之间建立联系 参与G蛋白耦合信号传导的分子和 监管反应。将结构特征链接到功能是 迈向理解和改善条件的基本步骤 细胞膜中信号转导过程受损。