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

描述（由申请方提供）：心血管功能中的β-抑制素和GRKs。G蛋白偶联受体（GPCR），如用于儿茶酚胺和血管紧张素II的那些，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）有丝分裂和迁移;以及心脏变力性和变时性。这些功能在高血压、旁路手术或血管成形术后病理性内膜增生或心力衰竭时可能受到干扰。在GPCR激活后，受体被几种G蛋白偶联受体激酶（GRK）之一磷酸化，然后结合β-抑制蛋白的两种同种型之一。β-arrestin结合空间阻断进一步的信号传导至G蛋白，导致受体脱敏和第二信使产生的减弱，β-arrestins在信号传导中也发挥积极作用，作为衔接子和支架组织受体介导的MAP激酶级联和其他途径的活化。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-抑制蛋白介导的信号传导的能力可能有很大差异。然而，在心血管（或其他）系统中β-抑制蛋白/GRK介导的信号传导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-抑制蛋白/GRK介导的信号传导对心血管功能的短期和长期调节有显着贡献。我们的方法将包括使用突变受体和特异性激动剂，其可以选择性地激活β-抑制蛋白而不是G蛋白信号传导;针对β-抑制蛋白和GRKs的siRNA;我们先前开发的缺乏β-抑制蛋白和GRKs中的每一种的敲除小鼠;以及从其衍生的细胞。利用β-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）在脱敏G蛋白介导的第二信使产生中个别β-抑制蛋白和GRKs的特异性，并将其与它们在介导信号传导中的作用（例如通过ERK活化）进行比较。2)β-抑制蛋白介导的信号传导的生理作用，a）在体外对心肌细胞和血管平滑肌细胞的作用，和B）在体内对心脏变力性和变时性以及对全身血压的作用，c）通过突变的β-肾上腺素能受体和血管紧张素II 1A受体的心脏信号传导的体内结果，所述受体与G蛋白解偶联，但通过β-抑制蛋白进行信号传导。3)仅通过β-arrestins传递信号的Ang类似物（SII Ang）对心脏中过表达钙螯合蛋白的转基因小鼠CHF的发展和病程的慢性影响。这些实验有可能描绘一个全新的和一般的受体介导的信号传导模式，并指出了新的治疗方法，目标是最近发现的信号传导机制的发展。

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.