B-Arrestins and G Protein-Coupled Receptor Kinases in Cardiovascular Function

描述（由申请人提供）：P-ARRESTINS和GRKs在心血管功能中的作用。G蛋白偶联受体（gpcr）如儿茶酚胺和血管紧张素II调节心血管功能，包括血管收缩和血管舒张；血管平滑肌细胞（VSM）的有丝分裂和迁移；以及心肌收缩性和慢性。在高血压、病理性内膜增生（如搭桥手术或血管成形术后）或心力衰竭时，这些功能可能受到干扰。GPCR激活后，受体被几种G蛋白偶联受体激酶（GRKs）中的一种磷酸化，然后结合p-阻滞蛋白的两种亚型之一。p-arrestins结合立体阻断G蛋白的进一步信号传导，导致受体脱敏和第二代信使的衰减，p-arrestins也在信号传导中发挥积极作用，作为接头和支架组织受体介导的MAP激酶级联反应等途径的激活。这些MAP激酶调节血管平滑肌细胞的有丝分裂和迁移。最近，我们发现受体配体在刺激G蛋白和p-阻滞蛋白介导的信号传导方面的能力可能有很大差异。然而，p-骤停素/GRK介导的信号在心血管（或其他）系统中的细胞和生理后果在很大程度上是未知的。因此，我们将使用几种方法来验证我们的中心假设，即p-骤停蛋白/GRK介导的信号传导对心血管功能的短期和长期调节有重要作用。我们的方法将包括使用突变受体和特异性激动剂，它们可以选择性地激活p-阻滞蛋白，但不能激活G蛋白信号；siRNA对p-arrest和GRKs的影响；我们之前开发的缺失p-阻滞蛋白和GRKs的敲除小鼠；细胞来源于。利用p-肾上腺素能受体和血管紧张素IMA受体作为我们的模型，我们的具体目标是确定：1)个体p-arrestins和GRKs在脱敏G蛋白介导的第二信使生成中的特异性，并将其与它们在介导信号传导（例如通过ERK激活）中的作用进行比较。2) p-骤停素介导的信号传导的生理效应，a)体外对心肌细胞和血管平滑肌细胞的影响，b)体内对心脏肌力、慢变性和全身血压的影响，c)突变的p-肾上腺素能受体和血管紧张素II1A受体的心脏信号传导在体内的影响，这些受体与G蛋白解偶联，但通过p-骤停素信号传导。3)一种仅通过p-骤停因子发出信号的ang类似物（SII ang）在心脏过表达calsequestrin的转基因小鼠中对CHF的发展和过程的慢性影响。这些实验有可能描绘出一种全新的、普遍的受体介导的信号传导模式，并为开发针对这种最近发现的信号传导机制的新疗法指明道路。

DESCRIPTION (provided by applicant): P-ARRESTINS AND GRKs IN CARDIOVASCULAR FUNCTION. G protein coupled receptors (GPCRs) such as those for catecholamines and angiotensin II regulate cardiovascular functions including vasoconstriction and vasodilation; vascular smooth muscle cell (VSM) mitogenesis and migration; and cardiac inotropy and chronotropy. These functions may be perturbed in hypertension, in pathological intimal hyperplasia as after bypass surgery or angioplasty, or in heart failure. Following GPCR activation, the receptors are phosphorylated by one of several G protein coupled receptor kinases (GRKs) and then bind one of the two isoforms of p-arrestin. p-arrestin binding sterically interdicts further signaling to G proteins leading to receptor desensitization and attenuation of second messenger generation, p-arrestins also play positive roles in signaling, serving as adaptors and scaffolds to organize receptor-mediated activation of MAP kinase cascades and other pathways. These MAP kinases regulate mitogenesis and migration of vascular smooth muscle cells. Recently we have found that receptor ligands may vary dramatically in their ability to stimulate G protein versus p-arrestin-mediated signaling. However, the cellular and physiological consequences of p-arrestin/GRK mediated signaling in cardiovascular (or other) systems are largely unknown. Accordingly, we will use several approaches to test our central hypothesis that p-arrestin/GRK- mediated signaling contributes significantly to the short and long-term regulation of cardiovascular function. Our approaches will include the use of mutant receptors and specific agonists which can selectively activate p-arrestin but not G protein signaling; siRNA to p-arrestins and GRKs; knock-out mice which we have previously developed lacking each of the p-arrestins and GRKs; and cells derived there from. Utilizing the p-adrenergic receptors and angiotensin IMA receptor as our models our specific aims are to determine: 1) the specificity of individual p-arrestins and GRKs in desensitizing G protein mediated second messenger generation and to compare this with their roles in mediating signaling, as for example through ERK activation. 2) The physiological effects of P-arrestin-mediated signaling, a) In vitro on cardiac myocytes and vascular smooth muscle cells, and b) In vivo on cardiac inotropy and chronotropy and on systemic blood pressure, c) The in vivo consequences of cardiac signaling by mutant p-adrenergic receptors and angiotensin II1A receptors which are uncoupled from G proteins but signal through p-arrestins. 3) The chronic effects of an ang analog (SII ang) which signals only through p-arrestins, on the development and course of CHF in a transgenic mouse overexpressing calsequestrin in the heart. These experiments have the potential to delineate an entirely novel and general mode of receptor-mediated signaling and to point the way to the development of novel therapeutics which target this recently discovered signaling mechanism.