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: 7681732
• 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/7681732
• 关键词: ADRBK1 gene; Biochemical; Biosensor; Cardiovascular system; Cell Communication; Cell physiology; Cells; Central Nervous System Diseases; Collaborations; Complex; 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; RGS Domain; RGS Proteins; RGS2 gene; Receptor Activation; Recombinants; Regulation; Rho Factor; Role; Signal Pathway; Signal Transduction; Smooth Muscle; Structure; Surface Plasmon Resonance; System; Thermodynamics; Tissues; X-Ray Crystallography; base; drug development; extracellular; improved; mutant; neurotransmitter release; novel; phospholipase C beta; public health relevance; 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(申请人提供)：G蛋白介导的信号系统是细胞通讯中最普遍的机制，几乎参与所有的细胞过程。G蛋白信号转导调节蛋白(RGS)在调节G蛋白介导的信号转导通路中发挥着多种重要作用。Galphaq介导的磷脂酶C-β(PLCbeta)激活调节包括心血管系统和神经系统在内的各种组织中的关键过程。然而，由于缺乏制备足够数量的重组Galphaq用于生化研究的方法，其信号转导的分子机制以及RGS蛋白对其的调控一直知之甚少。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。用这种方法，我们成功地分离了Galphaq-GRK2-Gbetagamma的配合物，并确定了它的晶体结构。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。这也提供了异源三聚体G蛋白在膜上受体激活后如何运动并形成信号复合体的第一个视角。此外，我们最近发现p63Rhogef是Galphaq的效应器。在这项建议中，基于这些结果，我们计划进一步表征Galphaq与RGS蛋白、GRK2、p63Rhogef或PLCβ相互作用的分子机制。在目标1中，我们将分离出各种Galphaq突变体，它们可以与具有GAP活性的RGS蛋白、GRK2、PLCbeta或p63Rhogef相互作用。在目标2中，我们将利用这些突变体，研究Galphaq-RGS/GRK2相互作用在细胞内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/GRK2与Galphaq固定化生物传感器芯片的相互作用动力学。我们还将通过X射线结晶学来阐明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PLCβ刺激活性。我们将应用SPR方法进一步了解Galphaq-PLCβ相互作用的动力学和热力学。我们还旨在通过X射线结晶学确定Galphaq-PLCbeta3络合物的结构。 与公共健康相关：这项提议旨在了解细胞对细胞外信号做出反应的分子机制。这项研究将有助于增进我们对各种生理功能的理解。这也将为未来新药的开发做出贡献。