Membrane Targeting of G proteins

G 蛋白的膜靶向

• 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
• 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

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（由申请人提供）：细胞内信号通路依赖于其组成蛋白的适当和独特的亚细胞位置。外周膜蛋白可逆靶向不同细胞膜的机制尚不清楚。此外，目前尚不清楚不同的亚细胞定位如何影响信号蛋白的功能。目前的资助重点是了解控制异三聚体G蛋白的组装、膜定位和细胞内运输的机制，此外，还确定了G蛋白在细胞中非规范位置的作用，例如G¿？调节高尔基膜的囊泡运输。修订申请将通过研究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。