Tyrosine Kinases in G Protein Mediated Signaling

描述（由申请人提供）：在过去的十年中，对G蛋白偶联受体（GPCR）如何控制细胞生长、增殖和分化的研究从根本上改变了我们对GPCR信号转导的看法。与GPCR仅作为异源三聚体G蛋白激活剂的典型模型不同，我们现在认识到它们是传递G蛋白依赖性和非依赖性信号的通用信号平台。我们的研究最初集中在ERK 1/2 MAP激酶级联的GPCR调控。我们确定GPCR使用几种不同的机制来控制ERK 1/2活性，包括由第二信使依赖性蛋白激酶和“反式激活”EGF受体传递的G蛋白依赖性信号，以及由多蛋白“信号体”的抑制蛋白依赖性组装产生的新的G蛋白独立性信号。这些结果已经定义了两种不同的GPCR信号传导“模式”，并且在某些情况下，我们已经确定了使它们解离的路径选择性“偏向激动剂”。此外，我们发现这些途径在功能上并不冗余。相反，激活机制决定了GPCR调节激酶的时间进程、空间分布和最终功能。这个建议的中心假设是，异源三聚体G蛋白和-arrestins作为独立的GPCR信号转导介导的不同方面的细胞响应GPCR刺激。该提案分为三个具体目标，前两个重点是GPCR-抑制蛋白“信号体”的结构和功能，第三个重点是G蛋白依赖性和抑制蛋白依赖性信号如何整合以确定细胞反应。在每一个目标，我们将集中在血管紧张素AT 1A受体，它利用两种信号机制。目的I和II采用转染的细胞系统，使我们能够使用受体和抑制蛋白突变体和快速siRNA沉默的蛋白质表达，以最大限度地发挥优势。实验将确定组成的AT 1AR-arrestin '信号体'和结构特征的受体和arrestin，决定信号体的组成和稳定性。我们将采用先进的蛋白质组学方法，以确定如何G蛋白独立的信号影响蛋白磷酸化，并确定如何抑制信号影响基因转录。目的III将集中于内源性AT 1A受体在原代主动脉血管平滑肌细胞中的信号传导。我们将采用途径选择性激动剂，药理学抑制剂和shRNA表达沉默来研究在生理相关背景下由每种类型的信号调节的细胞过程。实验将确定不同类型信号的时间，空间和功能特征，以及它们如何整合以产生与动脉粥样硬化血管疾病发展相关的细胞变化。这些研究的完成将解决我们对GPCR如何工作的理解中的一个根本性差距，并可能为具有路径选择性激动剂或拮抗剂性质的GPCR配体的新治疗应用提供见解。 公共卫生相关性：与G蛋白偶联受体（GPCR）仅通过激活异源三聚体G蛋白来传递信号的传统观点相反，最近的研究表明，它们也传递通过与衔接子或支架蛋白结合而启动的G蛋白非依赖性信号。本项目的重点是arrestins在血管紧张素AT 1A受体信号传导中的作用。抑制蛋白与激活的GPCR结合，使它们与G蛋白“解偶联”，同时促进影响蛋白磷酸化和基因转录的“信号体”的组装。我们的研究将确定控制AT 1A受体- arrestin信号体的组装和功能的因素，并确定G蛋白依赖性和arrestin依赖性信号是如何整合的。这些研究解决了我们对GPCR如何工作的理解中的根本差距，并可能为具有路径选择性激动剂或拮抗剂性质的GPCR配体的新治疗应用提供见解。

DESCRIPTION (provided by applicant): Over the past decade, the study of how G protein-coupled receptors (GPCRs) control cell growth, proliferation and differentiation has fundamentally changed our view of GPCR signal transduction. Far from the canonical model in which GPCRs function solely as activators of heterotrimeric G proteins, we now recognize that they are versatile signaling platforms that transmit both G protein-dependent and -independent signals. Our research originally focused on GPCR regulation of the ERK1/2 MAP kinase cascade. We established that GPCRs use several mechanistically distinct pathways to control ERK1/2 activity, including G protein- dependent signals transmitted by second messenger-dependent protein kinases and 'transactivated' EGF receptors, and novel G protein-independent signals that result from the -arrestin-dependent assembly of multiprotein 'signalsomes'. These results have defined two distinct GPCR signaling 'modes', and in some cases we have identified pathway-selective 'biased agonists' that dissociate them. Moreover, we have found that these pathways are not functionally redundant. Rather, the mechanism of activation determines the time course, spatial distribution, and ultimately the function of GPCR-regulated kinases. The central hypothesis of this proposal is that heterotrimeric G proteins and -arrestins serve as independent GPCR signal transducers that mediate distinct facets of the cellular response to GPCR stimulation. The proposal is organized into three Specific Aims, the first two focused on the structure and function of the GPCR-arrestin 'signalsome' and the third on how G protein-dependent and -arrestin-dependent signals are integrated to determine the cellular response. In each aim, we will focus on the angiotensin AT1A receptor, which utilizes both signaling mechanisms. Aims I and II employ transfected cell systems that allow us to use receptor and -arrestin mutants and rapid siRNA silencing of protein expression to maximum advantage. Experiments will determine the composition of the AT1AR--arrestin 'signalsome' and the structural features of the receptor and -arrestin that dictate signalsome composition and stability. We will employ advanced proteomic methodology to determine how G protein-independent signaling affects protein phosphorylation and determine how -arrestin signaling affects gene transcription. Aim III will concentrate on signaling by endogenous AT1A receptors in primary aortic vascular smooth muscle cells. We will employ pathway-selective agonists, pharmacologic inhibitors and shRNA expression silencing to study the cellular processes regulated by each type of signal in a physiologically relevant context. Experiments will determine the temporal, spatial and functional characteristics of the different types of signal, and how they are integrated to produce cellular changes associated with the development of atherosclerotic vascular disease. Completion of these studies will address a fundamental gap in our understanding of how GPCRs work and may provide insights into novel therapeutic applications of GPCR ligands with pathway-selective agonist or antagonist properties. PUBLIC HEALTH RELEVANCE: Contrary to the traditional view that G protein-coupled receptors (GPCRs) only signal by activating heterotrimeric G proteins, recent research has shown that they also transmit G protein-independent signals that are initiated by binding to adapter or scaffold proteins. This project focuses on the role of arrestins in angiotensin AT1A receptor signaling. Arrestins bind to activated GPCRs, 'uncoupling' them from G proteins while at the same time promoting the assembly of 'signalsomes' that affect protein phosphorylation and gene transcription. Our research will define the factors that control the assembly and function of the AT1A receptor- arrestin signalsome and determine how G protein-dependent and arrestin-dependent signals are integrated. These studies address fundamental gaps in our understanding of how GPCRs work and may provide insights into novel therapeutic applications of GPCR ligands with pathway-selective agonist or antagonist properties.