GPCR signaling complexes in living cells

描述(申请人提供)：G蛋白偶联受体(GPCRs)是临床上最常见的药物靶点。GPCR功能的标准模型认为，受体、G蛋白和效应器分子相互作用(通过碰撞耦合)，允许每个受体激活几个G蛋白分子，每个G蛋白亚基激活几个效应器分子。然而，最近的工作让人们对碰撞耦合的普遍性产生了怀疑。相反，有人认为GPCR信号分子可以预先偶联在“信号体”复合体中，这些复合体在信号传递过程中保持完整。复合体的预组装可以促进快速信号传递，并提供正常细胞功能所需的受体-效应器特异性。这项研究的长期目标是了解活细胞中信号分子的空间排列和时间动力学。因此，该项目的目标是确定GPCR信号是通过碰撞耦合、信号分子的稳定复合体还是通过这些机制的组合来调节的。我们已经开发了一种简单的技术来检测膜蛋白之间的相互作用，并在活细胞中量化这种相互作用的稳定性。这项技术测量了当潜在的相互作用伙伴被实验固定时，膜蛋白横向迁移率的变化。跨膜蛋白(如GPCRs和离子通道)被固定在完整的细胞中，潜在相互作用的蛋白质的横向迁移率通过监测光漂白后的荧光恢复来测量。我们将使用这种方法和标准的电生理学技术来测试关于GPCR信号复合体的特定假说。其具体目的是：(1)检验假设，即不活跃的GPCRs和G-蛋白异源三聚体形成促进信号转导的特定复合体；(2)检验G-蛋白异源三聚体与内向整流钾(GIRK)通道形成复合体的假说；(3)检验RGS蛋白通过与GPCRs和/或G-蛋白异源三聚体形成稳定的复合体来加速信号启动的假说；以及(4)确定G-蛋白在信号传递过程中是否解离成GET和GBG亚基。目前可用的药物作用于GPCR信号的第一步，即药物(或阻滞剂)与受体的结合。预计未来的治疗药物将针对信号转导的后续步骤。开发这类药物将需要详细了解这些步骤，例如，受体、G蛋白和效应器分子何时何地相互作用以传递信号。该项目的目标是提供这方面的信息。

DESCRIPTION (provided by applicant): G-protein coupled receptors (GPCRs) are, as a class, the most common target of clinically used drugs. The standard model of GPCR function holds that receptors, G-proteins and effector molecules interact with each other sequentially and transiently (by collision coupling), allowing each receptor to activate several G-protein molecules, and each G-protein subunit to activate several effector molecules. Recent work has, however, cast doubt on the generality of collision coupling. Instead, it has been suggested that GPCR signaling molecules can be precoupled in "signalosome" complexes that remain intact during signaling. Preassembly of complexes could facilitate rapid signaling and provide the receptor-effector specificity necessary for normal cell function. The long term objective of this research is to understand the spatial arrangement and temporal dynamics of signaling molecules in living cells. Accordingly, the goal of this project is to determine if GPCR signaling is mediated by collision coupling, by stable complexes of signaling molecules, or by a combination of these mechanisms. We have developed a simple technique to detect interactions between membrane proteins and to quantify the stability of such interactions in live cells. This technique measures changes in the lateral mobility of a membrane protein when a potential interacting partner is experimentally immobilized. Transmembrane proteins (e.g. GPCRs and ion channels) are immobilized in intact cells, and the lateral mobility of potentially interacting proteins is measured by monitoring fluorescence recovery after photobleaching. We will use this method together with standard electrophysiological techniques to test specific hypotheses about GPCR signaling complexes. The specific aims are (i) to test the hypothesis that inactive GPCRs and G-protein heterotrimers form specific complexes that facilitate signaling; (2) to test the hypothesis that G-protein heterotrimers form complexes with inwardly-rectifying potassium (GIRK) channels; (3) to test the hypothesis that RGS proteins accelerate signal onset by forming stable complexes with GPCRs and/or G-protein heterotrimers; and (4) to determine if G-proteins dissociate into component Get and Gbg subunits during signaling. Currently available drugs act at the first step of GPCR signaling, namely binding of the drug (or a blocker) to the receptor. It is anticipated that future therapeutic drugs will target the subsequent steps of signaling. Development of such drugs will require a detailed understanding of these steps, e.g. when and where receptors, G-proteins and effector molecules interact with each other to transmit signals. The goal of this project is to provide this information.