Regulation of G Protein Signaling by RGS Proteins

描述（申请人提供）：G蛋白介导的信号系统是最普遍的细胞通讯机制，几乎参与所有细胞过程。G蛋白信号调节蛋白（regulatory of G protein signaling， RGS）在G蛋白介导的信号通路中发挥着多种重要的调控作用。galphaq介导的plcβ（磷脂酶c - β）激活调节各种组织的关键过程，包括心血管系统或神经系统。然而，由于缺乏制备足够量的重组Galphaq用于生化研究的方法，人们对其信号转导及其受RGS蛋白调控的分子机制知之甚少。最近，我们开发了一种改进的纯化方法来大规模制备功能性重组Galphaq。利用这种方法，我们成功地分离了Galphaq-GRK2-Gbetagamma配合物，并测定了其晶体结构。这一结构揭示了GRK2的RGS结构域像效应器一样与Galphaq相互作用。它还提供了异三聚体G蛋白在膜上受体激活后如何移动并形成信号复合物的第一个观点。此外，我们最近发现p63RhoGEF是Galphaq的效应物。基于这些结果，我们计划进一步表征Galphaq与RGS蛋白、GRK2、p63RhoGEF或PLCbeta相互作用的分子机制。在Aim 1中，我们将分离各种Galphaq突变体，这些突变体具有与具有GAP活性的RGS蛋白、GRK2、PLCbeta或p63RhoGEF相互作用的特定缺陷。在Aim 2中，使用这些突变体，我们将表征Galphaq-RGS/GRK2相互作用在受体介导的细胞反应中的生理功能。我们还计划应用SPR（表面等离子体共振）方法分析Gaphaq-RGS/GRK2与固定化galphaq生物传感器芯片相互作用的动力学。我们还将通过x射线晶体学来阐明Galphaq-RGS相互作用的分子机制。在Aim 3中，我们将尝试进一步提高重组Galphaq的PLCbeta刺激活性。我们将应用SPR方法进一步了解Galphaq-PLCbeta相互作用的动力学和热力学。我们还试图用x射线晶体学来确定Galphaq-PLCbeta3配合物的结构。

DESCRIPTION (provided by applicant): G protein-mediated signaling system is the most universal mechanism of cell communication and is involved in almost all cellular processes. RGS (regulator of G protein signaling) proteins play diverse and crucial functions to regulate G protein mediated signaling pathways. Galphaq-mediated activation of PLCbeta (phospholipase C-beta) regulates critical processes in various tissues, including cardiovascular system or nervous system. However, the molecular mechanism of its signal transduction and its regulation by RGS proteins have been poorly understood, mainly due to the lack of method to prepare enough amount of recombinant Galphaq for biochemical studies. Recently we developed an improved purification method to prepare functional recombinant Galphaq in large scale. Using this method, we have successfully isolated the complex of Galphaq-GRK2-Gbetagamma and determined its crystal structure. This structure revealed that RGS domain of GRK2 interacts with Galphaq like an effector. It also provided the first view of how heterotrimeric G protein moves and forms a signaling complex after receptor activation at the membrane. In addition, we recently identified p63RhoGEF as an effector for Galphaq. In this proposal, based on these results, we plan to further characterize the molecular mechanism of the interactions of Galphaq with RGS proteins, GRK2, p63RhoGEF, or PLCbeta. In Aim 1, we will isolate various Galphaq mutants that have specific defect to interact with RGS protein with GAP activity, GRK2, PLCbeta or p63RhoGEF. In Aim 2, using these mutants, we will characterize the physiological function of Galphaq-RGS/GRK2 interaction in receptor-mediated responses in cell. We also plan to apply SPR (surface plasmon resonance) method to analyze the kinetics of Gaphaq-RGS/GRK2 interaction with Galphaq-immobilized biosensor chip. We will also elucidate the molecular mechanism of Galphaq-RGS interaction by X-ray crystallography. In Aim 3, we will attempt to further improve PLCbeta stimulating activity of our recombinant Galphaq. We will apply SPR method to further understand the kinetics and thermodynamics of Galphaq-PLCbeta interaction. We also aim to determine the structure of Galphaq-PLCbeta3 complex by X-ray crystallography. PUBLIC HEALTH RELEVANCE: This proposal aims to understand the molecular mechanism of cells to respond to extracellular signals. The study will help to advance our understanding of various physiological functions. It will also contribute to develop novel drugs in future.