MOLECULAR INTERACTIONS IN G PROTEIN COUPLED SIGNALING

描述（申请人的描述）：细胞外信号的整合是 这是质膜的基本功能。对于七个跨膜片段 受体这一功能是由异源三聚体G蛋白完成的， 将受体激活与特定效应物偶联。虽然许多受体和 已经鉴定出效应子，并且几种G蛋白的晶体结构 这些分子与膜相互作用的方式， 参与包括信号转导级联的化学反应 仍然知之甚少。该项目的主要目标是提供 对这些过程的理解。全内反射荧光 支持膜的显微镜（TIRFM）将用于研究 荧光团标记的G蛋白α和β γ亚基与脂质的相互作用 膜，无论是纯的或掺入分子与G蛋白 相互作用，以了解G蛋白的物理化学 在化学定义的环境中相互作用。亚基脂质修饰， 包括Gi/α的N-末端豆蔻酰化和硫代酰化， 检查，因为这些可能在亚基的动力学中起主要作用 与膜的相互作用。膜的成分接近于 将检查质膜的抗洗涤剂亚结构域， 确定可能特异性靶向G蛋白的分子特征， 地区与G蛋白相关的蛋白质，如RGS、小窝蛋白和a 通道效应子，也将检查它们对亚基横向的影响。 流动性和互动。在一种补充方法中，切除的细胞片 表达G蛋白的膜调节毒蕈碱钾通道（KACh） 将不仅用于测定荧光标记的 和/或化学修饰的G蛋白亚基， 在我们的TIRFM结果的基础上在体内调节。的组合 定量生物物理、生物化学和生理学方法被认为是 作为拟议研究的一个独特方面，应提供现实的， G蛋白偶联信号转导的互补观点。此外，委员会认为， 这些研究应该有助于建立结构特征之间的联系， 参与G蛋白偶联信号传导的分子和 监管的回应。将结构特征与功能联系起来是 为了解和改善因气候变化而造成的状况 破坏了细胞膜的信号转导过程。

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.