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（由申请方提供）：细胞内信号传导途径取决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知之甚少。此外，目前还不清楚不同的亚细胞定位如何影响信号蛋白的功能。目前的赠款是集中在了解的机制，管理装配，膜定位，和细胞内贩运的异源三聚体G蛋白，此外，是确定G蛋白的作用在非典型的位置在细胞中，如G的作用？在调节囊泡运输在高尔基体膜。Revision应用程序将通过研究G蛋白偶联受体激酶5（GRK 5）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）使激动剂激活的GPCR磷酸化。 GRK对激活的GPCR的磷酸化在GPCR脱敏中起关键作用;磷酸化促进抑制蛋白向GPCR的募集，这防止异源三聚体G蛋白的进一步激活并促进GPCR的内化。因此，GRK对于调节由GPCR和G蛋白途径介导的信号传导是普遍重要的。由于GPCR信号在调节大量生理功能中的普遍重要性，GRKs在由细胞外信号引发的多种生理过程的适当调节中具有重要作用。 该实验室的工作最近提供了数据支持一种调节GRK 5膜靶向的新机制：GRK 5的二聚化对其质膜定位至关重要。该应用程序将专注于定义GRK 5膜定位的新模型。为了实现这一目标，该提案的主要目标是1）测试GRK 5在细胞中形成二聚体的假设;和2）测试GRK 5的二聚化对质膜定位和功能至关重要的假设。这些目标将通过各种实验方法来实现，包括在培养细胞中表达重组蛋白、蛋白质的荧光和免疫荧光显微镜定位、免疫印迹、免疫沉淀、双分子荧光互补（BiFC）、生物发光共振能量转移（BRET）、诱导性同源二聚化系统、完整细胞中G蛋白介导的信号传导测定和磷酸化测定。 公共卫生相关性：G蛋白介导的信号传导途径调节许多生理反应，包括心血管功能、神经递质反应、细胞分化、细胞迁移、免疫细胞功能以及嗅觉、味觉和视觉;并且G蛋白介导的信号传导途径的失调导致许多疾病状态，包括心脏病、高血压、癌症、转移、内分泌紊乱、脑紊乱和失明。G蛋白偶联受体（GPCR）激酶（GRKs）是参与关闭信号应答以确保应答具有适当持续时间的关键蛋白。为了正常工作，GRKs必须与细胞膜紧密相互作用。在动物模型中，阻断一种称为GRK 2的GRK的膜募集已被用作心脏病的有效治疗方法。GRK 5是该研究项目的重点，在神经元以及心血管和其他反应中很重要。因此，很明显，了解调节GRK定位的机制可能会导致治疗靶向个体GRK的新方法。