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

说明(申请人提供)：P-阻滞剂和GRKs在心血管功能。G蛋白偶联受体(GPCRs)，如儿茶酚胺和血管紧张素II受体，调节心血管功能，包括血管收缩和血管扩张；血管平滑肌细胞(VSM)有丝分裂和迁移；心脏变力和变时性。这些功能可能在高血压、搭桥手术或血管成形术后的病理性内膜增生或心力衰竭中受到干扰。在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，然后与p-arrestin的两种异构体之一结合。P-arrestin与G蛋白的空间结合可以进一步阻断G蛋白的信号传递，导致受体脱敏和第二信使生成的减弱，p-arrestins还在信号传递中发挥积极作用，作为适配器和支架，组织受体介导的MAP激活级联通路和其他途径。这些MAP激酶调节血管平滑肌细胞的有丝分裂和迁移。最近，我们发现受体配体刺激G蛋白的能力与p-arrestin介导的信号转导能力可能有很大的不同。然而，p-arrestin/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检验我们的中心假设，即p-arrestin/GRK介导的信号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的受体和特定的激动剂，它们可以选择性地激活p-arrestin，但不能激活G蛋白信号；siRNA到p-arrestins和GRKs；我们以前培育的缺乏p-arrestins和GRKs的基因敲除小鼠；以及来自那里的细胞。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，我们的具体目的是确定：1)单个p-arrestins和GRKs在脱敏G蛋白介导的第二信使产生中的特异性，并将其与它们在介导信号转导中的作用进行比较，例如通过ERK激活。2)P-arrestin介导的信号转导的生理效应，a)体外对心肌细胞和血管平滑肌细胞的作用，b)体内对心脏变力和变时性以及对全身血压的影响，c)通过突变的p-肾上腺素能受体和血管紧张素II1a受体传递心脏信号的体内后果。3)血管紧张素转换酶类似物(SII Ang)对心脏过度表达钙调素的转基因小鼠心力衰竭的发生和发展的慢性影响。这些实验有可能描绘出一种全新的、通用的受体介导的信号传递模式，并为开发针对这一新近发现的信号机制的新疗法指明方向。

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.