Regulation of G Protein Signaling by RGS Proteins

RGS 蛋白对 G 蛋白信号传导的调节

• 批准号: 7522360
• 负责人: TOHRU KOZASA
• 金额: $ 31.4万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7522360
• 关键词: ADRBK1 gene; Biochemical; Biosensor; Cardiovascular system; Cell Communication; Cell physiology; Cells; Central Nervous System Diseases; Class; Collaborations; Complex; Condition; Defect; Disease; Functional disorder; Future; G alpha q Protein; GTP-Binding Protein Regulators; GTP-Binding Proteins; Generations; Guanine Nucleotides; Heart Hypertrophy; Heart failure; Heterotrimeric GTP-Binding Proteins; Hypertension; Kinetics; Mediating; Membrane; Methods; Molecular; Muscle Contraction; Nervous system structure; Pathway interactions; Pharmaceutical Preparations; Physiological; Physiological Processes; Platelet Activation; Play; Process; Public Health; RGS Domain; RGS Proteins; RGS2 gene; Receptor Activation; Recombinants; Regulation; Rho Factor; Role; Signal Pathway; Signal Transduction; Structure; Surface Plasmon Resonance; System; Thermodynamics; Tissues; X-Ray Crystallography; base; drug development; extracellular; human RGS2 protein; improved; mutant; neurotransmitter release; novel; phospholipase C beta; receptor; response; rho; sensor

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.

描述（由申请人提供）：G蛋白介导的信号传导系统是细胞通讯的最普遍机制，几乎参与所有细胞过程。G蛋白信号调节蛋白（regulator of G protein signaling，RGS）在调节G蛋白介导的信号通路中发挥着重要的作用。Galphaq介导的PLC β（磷脂酶C-β）激活调节各种组织中的关键过程，包括心血管系统或神经系统。然而，其信号转导的分子机制及其通过RGS蛋白的调节一直知之甚少，主要是由于缺乏方法来制备足够量的重组Galphaq的生化研究。最近，我们开发了一种改进的纯化方法，以大规模制备功能性重组Galphaq。利用此方法，我们成功地分离出了Galphaq-GRK 2-Gbetagamma配合物，并测定了其晶体结构。该结构揭示GRK 2的RGS结构域与Galphaq像效应物一样相互作用。它还提供了异源三聚体G蛋白如何移动并在膜上受体激活后形成信号复合物的第一个观点。此外，我们最近确定p63 RhoGEF作为Galphaq的效应子。在本提案中，基于这些结果，我们计划进一步表征Galphaq与RGS蛋白、GRK 2、p63 RhoGEF或PLC β相互作用的分子机制。在目的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、GRK 2、PLC β或p63 RhoGEF相互作用的特异性缺陷。在目的2中，使用这些突变体，我们将表征Galphaq-RGS/GRK 2相互作用在细胞中受体介导的反应中的生理功能。我们还计划应用表面等离子体共振（SPR）方法来分析Gaphaq-RGS/GRK 2与固定化Galphaq的生物传感器芯片的相互作用动力学。我们还将通过X射线晶体学阐明Galphaq-RGS相互作用的分子机制。在目标3中，我们将尝试进一步提高我们的重组Galphaq的PLC β刺激活性。我们将应用SPR方法进一步了解Galphaq-PLC β相互作用的动力学和热力学。我们还旨在通过X射线晶体学确定Galphaq-PLC β 3复合物的结构。 公共卫生相关性：该提案旨在了解细胞响应细胞外信号的分子机制。这项研究将有助于促进我们对各种生理功能的理解。它也将有助于开发新的药物在未来。