GPCR signaling complexes in living cells

描述（由申请人提供）：g蛋白偶联受体（gpcr）作为一类药物，是临床上最常见的靶点。GPCR功能的标准模型认为，受体、g蛋白和效应分子之间的相互作用是顺序的、短暂的（通过碰撞耦合），每个受体可以激活几个g蛋白分子，每个g蛋白亚基可以激活几个效应分子。然而，最近的研究对碰撞耦合的普遍性提出了质疑。相反，有人认为GPCR信号分子可以在“信号体”复合物中预偶联，在信号传导过程中保持完整。复合物的预组装可以促进快速信号传导，并提供正常细胞功能所必需的受体-效应特异性。本研究的长期目标是了解活细胞中信号分子的空间分布和时间动态。因此，本项目的目标是确定GPCR信号是通过碰撞偶联介导的，还是通过信号分子的稳定复合物介导的，还是通过这些机制的组合介导的。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，并量化这种相互作用在活细胞中的稳定性。当潜在的相互作用伙伴被实验固定时，该技术测量膜蛋白横向迁移率的变化。跨膜蛋白（如gpcr和离子通道）固定在完整的细胞中，通过监测光漂白后的荧光恢复来测量潜在相互作用蛋白的横向迁移率。我们将使用这种方法与标准的电生理技术一起测试关于GPCR信号复合物的特定假设。具体目的是：(i)验证无活性gpcr和g蛋白异源三聚体形成促进信号传导的特定复合物的假设；(2)验证g蛋白异源三聚体与内向纠偏钾（GIRK）通道形成复合物的假设；(3)验证RGS蛋白通过与gpcr和/或g蛋白异源三聚体形成稳定复合物加速信号启动的假设；(4)确定g蛋白在信号传导过程中是否解离成组分Get和Gbg亚基。目前可用的药物作用于GPCR信号传导的第一步，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传导的后续步骤。开发此类药物需要详细了解这些步骤，例如，受体、g蛋白和效应分子何时何地相互作用以传递信号。这个项目的目标就是提供这些信息。

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.