Tyrosine Kinases in G Protein Mediated Signaling

描述（由申请人提供）：在过去的十年中，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蛋白的激活剂的标准模型不同，我们现在认识到它们是多功能的信号平台，可以传递G蛋白依赖性和非依赖性信号。我们的研究最初集中在GPCR对ERK1/2 MAP激酶级联的调控。我们发现，gpcr使用几种机制上不同的途径来控制ERK1/2活性，包括由第二信使依赖蛋白激酶和“反激活”的EGF受体传递的G蛋白依赖信号，以及由多蛋白“信号体”的-抑制蛋白依赖组装产生的新型G蛋白独立信号。这些结果定义了两种不同的GPCR信号“模式”，在某些情况下，我们已经确定了分离它们的途径选择性“偏倚激动剂”。此外，我们发现这些通路在功能上并不是冗余的。相反，激活机制决定了gpcr调节激酶的时间过程、空间分布，并最终决定了其功能。该建议的中心假设是异三聚体G蛋白和-阻滞蛋白作为独立的GPCR信号转导器，介导细胞对GPCR刺激反应的不同方面。该提案被组织成三个特定目标，前两个专注于gpcr -抑制蛋白“信号体”的结构和功能，第三个专注于如何整合G蛋白依赖和-抑制蛋白依赖的信号来确定细胞反应。在每个目标中，我们将重点关注血管紧张素AT1A受体，它利用这两种信号机制。目标I和II采用转染的细胞系统，使我们能够利用受体和-阻滞蛋白突变体和快速siRNA沉默蛋白表达，以最大限度地发挥优势。实验将确定AT1AR-抑制素“信号体”的组成，以及受体和抑制素的结构特征，这些特征决定了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来确定G蛋白独立信号如何影响蛋白质磷酸化，并确定-阻滞蛋白信号如何影响基因转录。Aim III将集中于内源性AT1A受体在原发性主动脉血管平滑肌细胞中的信号传导。我们将使用途径选择性激动剂，药物抑制剂和shRNA表达沉默来研究在生理相关背景下每种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性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如何工作的理解的根本空白，并可能为具有途径选择性激动剂或拮抗剂特性的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.