RGS Protein Regulation of G Protein Coupled Receptors

描述(申请人提供)：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 M M胆碱能受体(M1 AChR)在调节血管高血压、心肌肥厚、交感神经控制心脏功能和血管张力方面发挥着突出的作用。然而，这些蛋白质如何相互作用的分子模型还没有被很好地理解。RGS蛋白直接与激活的GA亚基结合，调节和整合它们的功能。对于细胞中目标Ga的RGS选择性是如何确定的，我们知之甚少。最近的研究表明，RGS和GPCR在细胞中功能上是相连的，但还没有显示出直接的相互作用。在这些观察的基础上，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)直接和选择性地与GQ/11连锁的M1AChR和A1A-AR的第三胞内环(I3)结合，而不与A1B-AR或a1D-AR的I3或Gi/o连锁的M2-或M4AChR结合。我们的研究表明，RGS2、M1-13和GqA形成了一个稳定的杂三聚体复合体，RGS2的N端负责RGS与两个受体的I3的结合。我的工作假设是，RGS蛋白与首选的GPCR形成稳定的功能复合体，选择性地调节这些受体和连接的G蛋白的信号功能。利用分子、细胞和生化方法，具体目标将是：目标1：识别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GQ/11a功能偶联中的作用。目的：研究RGS2对GRK2结合/磷酸化和arrestin与受体结合脱敏、内化和细胞内转运的影响。目的：研究抑制天然RGS2 mRNA/蛋白对天然表达A1AChR或M1AChR和RGS2的细胞/VSM组织中天然M1AChR和A1AAR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，并为心血管疾病的治疗干预确定潜在的新分子靶点。

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.