Membrane Targeting of G proteins

G 蛋白的膜靶向

基本信息

批准号：
7809731
负责人：
PHILIP B WEDEGAERTNER
金额：
$ 38.63万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2012-09-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7809731
关键词：
ADRBK1 gene ADRBK2 gene Address Agonist Amino Acids Animal Model Arrestins Binding Biochemical Biological Assay Bioluminescence Blindness Brain Diseases C-terminal Cardiovascular Physiology Cardiovascular system Cell Differentiation process Cell membrane Cell physiology Cells Cellular Membrane Cellular biology Co-Immunoprecipitations Cultured Cells Data Dimerization Disease Endocrine System Diseases Energy Transfer Equilibrium Fluorescence Fractionation G Protein-Coupled Receptor Signaling G protein coupled receptor kinase G-Protein-Coupled Receptors GRK GRK6 gene GTP-Binding Proteins Goals Golgi Apparatus Grant Heart Diseases Heart failure Heterotrimeric GTP-Binding Proteins Homodimerization Hypertension Immune Immunoblotting Immunofluorescence Microscopy Immunoprecipitation Individual Laboratories Lead Location Malignant Neoplasms Mediating Membrane Membrane Proteins Mental disorders Mutation Nature Neoplasm Metastasis Neurons Neurotransmitters Opiate Addiction Pathway interactions Peripheral Phosphorylation Physiological Physiological Processes Play Point Mutation Proteins RGS Domain Recombinant Proteins Regulation Reporting Research Project Grants Role Signal Pathway Signal Transduction Signaling Protein Site-Directed Mutagenesis Smell Perception Structure System Taste Perception Techniques Testing Transport Vesicles Vision Work cell motility chemical addition crosslink desensitization dimer effective therapy extracellular human GRK5 protein hypertensive heart disease membrane assembly membrane model mutant novel prevent protein function research study response trafficking

ADRBK1 gene ADRBK2 gene Address Agonist Amino Acids Animal Model Arrestins Binding Biochemical Biological Assay Bioluminescence Blindness Brain Diseases C-terminal Cardiovascular Physiology Cardiovascular system Cell Differentiation process Cell membrane Cell physiology Cells Cellular Membrane Cellular biology Co-Immunoprecipitations Cultured Cells Data Dimerization Disease Endocrine System Diseases Energy Transfer Equilibrium Fluorescence Fractionation G Protein-Coupled Receptor Signaling G protein coupled receptor kinase G-Protein-Coupled Receptors GRK GRK6 gene GTP-Binding Proteins Goals Golgi Apparatus Grant Heart Diseases Heart failure Heterotrimeric GTP-Binding Proteins Homodimerization Hypertension Immune Immunoblotting Immunofluorescence Microscopy Immunoprecipitation Individual Laboratories Lead Location Malignant Neoplasms Mediating Membrane Membrane Proteins Mental disorders Mutation Nature Neoplasm Metastasis Neurons Neurotransmitters Opiate Addiction Pathway interactions Peripheral Phosphorylation Physiological Physiological Processes Play Point Mutation Proteins RGS Domain Recombinant Proteins Regulation Reporting Research Project Grants Role Signal Pathway Signal Transduction Signaling Protein Site-Directed Mutagenesis Smell Perception Structure System Taste Perception Techniques Testing Transport Vesicles Vision Work cell motility chemical addition crosslink desensitization dimer effective therapy extracellular human GRK5 protein hypertensive heart disease membrane assembly membrane model mutant novel prevent protein function research study response trafficking

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项目摘要

DESCRIPTION (provided by applicant): Intracellular signaling pathways depend upon appropriate and unique subcellular locations of their constituent proteins. Mechanisms responsible for reversibly targeting peripheral membrane proteins to different cellular membranes are poorly understood. Moreover, it is not clear how different subcellular localizations influence a signaling protein's function. The current grant is focused on understanding the mechanism that govern assembly, membrane localization, and intracellular trafficking of heterotrimeric G proteins, and, in addition, is determining the role of G proteins at non-canonical locations in the cell, such as a role for G¿? in regulating vesicle transport at Golgi membranes. The Revision application will expand the scope of these studies by investigating mechanisms of membrane localization for G protein-coupled receptor kinase 5 (GRK5). G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate agonist-activated GPCRs. Phosphorylation of activated GPCRs by GRKs plays a critical role in GPCR desensitization; phosphorylation promotes recruitment of arrestins to the GPCR which prevents further activation of heterotrimeric G proteins and promotes the internalization of the GPCR. Thus, GRKs are universally important for regulating signaling mediated by GPCRs and G protein pathways. Because of the ubiquitous importance of GPCR signaling in regulating a vast number of physiological functions, GRKs have essential roles in the proper regulation of a multitude of physiological processes that are initiated by extracellular signals. Work in this laboratory has recently provided data to support a novel mechanism that regulates membrane targeting of GRK5: dimerization of GRK5 is critical for its plasma membrane localization. This application will focus on defining this new model for membrane localization of GRK5. To address this goal, the major objectives of the proposal are 1) test the hypothesis that GRK5 forms dimers in cells; and 2) test the hypothesis that dimerization of GRK5 is critical for plasma membrane localization and function. These objectives will be pursued by a variety of experimental approaches, including expression of recombinant proteins in cultured cells, fluorescence and immunofluorescence microscopy localization of proteins, immunoblotting, immunoprecipitations, bimolecular fluorescence complementation (BiFC), bioluminescence resonance energy transfer (BRET), inducible homodimerization systems, G protein-mediated signaling assays in intact cells, and phosphorylation assays. PUBLIC HEATH RELEVANCE: G protein-mediated signaling pathways regulate numerous physiological responses, including cardiovascular function, neurotransmitter responses, cell differentiation, cell migration, immune cell function, and smell, taste, and vision; and dysregulation of G protein-mediated signaling pathways contribute to numerous disease states, including heart disease, hypertension, cancer, metastasis, endocrine disorders, brain disorders and blindness. G protein-coupled receptor (GPCR) kinases (GRKs) are key proteins involved in turning-off a signaling response to insure that the response is of the proper duration. To function properly, the GRKs must interact tightly with cellular membranes. Blocking membrane recruitment of one GRK, called GRK2, has been used as an effective treatment for heart disease in animal models. GRK5, the focus of the research project, is important in neuronal, as well as cardiovascular and other responses. Thus, it is clear that understanding the mechanisms that regulate GRK localization could lead to novel ways to therapeutically target individual GRKs.

描述（由适用提供）：细胞内信号通路取决于其组成蛋白的适当和独特的亚细胞位置。对不同细胞膜的可逆性靶向外周膜蛋白的机制知之甚少。此外，尚不清楚不同的亚细胞局部化如何影响信号蛋白的功能。当前的赠款集中在理解杂点G蛋白的组装，膜定位和细胞内运输的机制上，此外，还在确定G蛋白在细胞中非传统位置的作用，例如G⿿的作用？在高尔基机制的调节场地运输中。修订应用将通过研究G蛋白偶联受体激酶5（GRK5）的膜定位机制来扩大这些研究的范围。 G蛋白偶联受体（GPCR）激酶（GRKS）磷酸化激动剂激活的GPCR。 GRKS激活的GPCR磷酸化在GPCR脱敏中起关键作用。磷酸化促进抑制蛋白募集到GPCR上，从而防止了异三聚体G蛋白的进一步激活，并促进了GPCR的内在化。这对于控制由GPCR和G蛋白途径介导的信号传导普遍重要。由于GPCR信号在确定大量生理功能中的无处不在，因此GRK在适当调控由细胞外信号引发的多种物理过程中具有重要作用。该实验室的工作最近提供了数据，以支持一种新的机制，该机制调节了GRK5的膜靶向：GRK5的二聚化对其质膜定位至关重要。该应用程序将着重定义用于GRK5膜定位的新模型。为了解决这一目标，该提案的主要目标是1）检验以下假设：GRK5在细胞中形成二聚体； 2）检验以下假设：GRK5的二聚化对于质膜定位和功能至关重要。 These objectives will be pursued by a variety of experimental approaches, including expression of recombinant proteins in cultured cells, fluorescence and immunofluorescence microscopy localization of proteins, immunoblotting, immunoprecipitations, biolecular fluorescence completion (BiFC), bioluminescence resonance energy transfer (BRET), inducible homodimerization systems, G protein-mediated signaling assays in完整的细胞和磷酸化测定。公共荒地相关性：G蛋白介导的信号通路调节了许多身体反应，包括心血管功能，神经递质反应，细胞分化，细胞迁移，免疫电池功能以及气味，口味和视力； G蛋白介导的信号通路的失调和失调导致许多疾病状态，包括心脏病，高血压，癌症，转移，内分泌疾病，脑部疾病和失明。 G蛋白偶联受体（GPCR）激酶（GRKS）是与关闭信号反应相关的关键蛋白，以确保响应的适当持续时间。为了正常运行，GRK必须与细胞膜紧密相互作用。在动物模型中，阻断一种称为GRK2的膜募集（称为GRK2）已被用作心脏病的有效治疗方法。 GRK5是研究项目的重点，在神经元，心血管和其他反应中很重要。这很明显，了解调节GRK定位的机制可能会导致新颖的方法来靶向单个GRK。