RGS Protein Regulation of G Protein Coupled Receptors

G 蛋白偶联受体的 RGS 蛋白调节

• 批准号: 7860719
• 负责人: JOHN R HEPLER
• 金额: $ 33.09万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-15 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7860719
• 关键词: ADRBK1 gene; Adrenergic Agents; Amino Acids; Animals; Area; Arrestins; Binding; Biochemical; Biological Assay; Blood Vessels; Cardiac; Cardiovascular Diseases; Cardiovascular Physiology; Cell Nucleus; Cell membrane; Cells; Cholinergic Receptors; Complex; Coupling; Cytosol; Doctor of Philosophy; Employee Strikes; Endothelium; Event; Exhibits; Family; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; G-substrate; GRK; GTP-Binding Proteins; Genes; Goals; Heart Hypertrophy; Hormones; Human Cell Line; Hypertension; Knock-out; Knockout Mice; Knowledge; Ligand Binding; Link; Messenger RNA; Modeling; Molecular; Molecular Models; Molecular Target; Mus; Muscle Contraction; Muscle function; Neurotransmitters; Phosphorylation; Property; Protein Binding; Proteins; RGS Proteins; RGS1 gene; RGS2 gene; RNA Interference; Recruitment Activity; Regulation; Research Personnel; Role; Signal Transduction; Signaling Protein; Smooth Muscle; Smooth Muscle Myocytes; Solutions; Testing; Therapeutic Intervention; Tissues; Work; adrenergic; base; desensitization; genetic regulatory protein; hypertension control; molecular modeling; novel; receptor; receptor binding; trafficking

DESCRIPTION (provided by applicant): Recent studies with gene knock-out mice indicate a prominent role for the functionally linked signaling proteins RGS2, Gq-alpha (Gqa), alpha-1A-adrenergic (a1A-AR) and m1 muscarininc cholinergic receptors (M1 AChR) in regulation of vascular hypertension, cardiac hypertrophy, sympathetic control of cardiac function, and vascular tone. However, molecular models for how these proteins interact are not well understood. RGS proteins bind directly to activated Ga subunits to modulate and integrate their functions. Very little is known about how RGS selectivity for target Ga is determined in cells. Recent studies suggest that RGS and GPCR are functionally linked in cells but direct interactions have not been shown. Based on these observations, we tested whether RGS proteins and GPCR interact directly. We found that RGS2 (but not RGS1 or RGS16) binds directly and selectively to the third intracellular loop (i3) of the Gq/11-linked M1AChR and a1A-AR, but not i3 of a1B-AR or a1D-AR or Gi/o-linked M2- or M4AChR. Our studies show that RGS2, M1-13, and Gqa form a stable heterotrimer complex, and that the N-terminus of RGS2 is responsible for RGS binding to the i3 of both receptors. My working hypothesis is that RGS proteins form stable, functional complexes with preferred GPCR to selectively modulate the signaling functions of those receptors and linked G proteins. Using molecular, cellular and biochemical approaches, the Specific Aims will be to: Aim 1: Identify amino acids on the M1AChR, a1A-AR and RGS2 responsible for direct binding. Aim 2: Determine roles for RGS2 on receptor/ligand binding and functional Gq/11 a coupling. Aim 3: Determine the effects of RGS2 on GRK2 binding/phosphorylation and arrestin binding to receptors on their desensitization, internalization, and intracellular trafficking. Aim 4: Determine the effects of suppressing native RGS2 mRNA/protein on native M1AChR and a1A-AR signaling functions in cells/VSM tissues that natively expresses a1A-AR or M1AChR and RGS2. These studies will define novel cellular mechanisms for regulating neurotransmitter and hormone signaling, and identify potential new molecular targets for therapeutic intervention in cardiovascular diseases.

描述（由申请者提供）：最近对基因敲除小鼠的研究表明，功能相关的信号蛋白RGS2、gq - α (Gqa)、α - 1a -肾上腺素能（a1A-AR）和m1 muscarinin胆碱能受体（m1 AChR）在调节血管高血压、心脏肥厚、交感神经控制心功能和血管张力方面发挥着重要作用。然而，这些蛋白质如何相互作用的分子模型还没有得到很好的理解。RGS蛋白直接与活化的Ga亚基结合，调节和整合其功能。细胞中RGS对靶Ga的选择性是如何确定的，目前所知甚少。最近的研究表明，RGS和GPCR在细胞中具有功能联系，但尚未显示直接相互作用。基于这些观察结果，我们测试了RGS蛋白和GPCR是否直接相互作用。我们发现RGS2（而不是RGS1或RGS16）直接和选择性地结合到Gq/11连接的M1AChR和a1A-AR的第三胞内环（i3），而不是a1B-AR或a1D-AR的i3或Gi/o连接的M2-或M4AChR。我们的研究表明，RGS2、M1-13和Gqa形成了稳定的异源三聚体复合物，RGS2的n端负责RGS与这两种受体的i3结合。我的工作假设是，RGS蛋白与首选GPCR形成稳定的功能性复合物，选择性地调节这些受体和相关G蛋白的信号功能。使用分子，细胞和生化方法，具体目标将是：目标1：鉴定M1AChR， a1A-AR和RGS2上负责直接结合的氨基酸。目的2：确定RGS2在受体/配体结合和功能性Gq/ 11a偶联中的作用。目的3：确定RGS2对GRK2结合/磷酸化和受体阻滞蛋白结合对其脱敏、内化和细胞内运输的影响。目的4：确定抑制天然RGS2 mRNA/蛋白对天然表达a1A-AR或M1AChR和RGS2的细胞/VSM组织中天然M1AChR和a1A-AR信号功能的影响。这些研究将确定调节神经递质和激素信号的新细胞机制，并确定心血管疾病治疗干预的潜在新分子靶点。