Tyrosine Kinases in G Protein Mediated Signaling

在过去的十年中，关于G蛋白偶联受体（GPCRs）如何控制细胞生长、增殖 分化从根本上改变了我们对GPCR信号转导的看法。远离规范 GPCR仅作为异源三聚体G蛋白的激活剂起作用的模型，我们现在认识到它们 是通用的信号平台，既传输G蛋白依赖性和非依赖性信号。我们 研究集中在ERK 1/2 MAP激酶级联的GPCR调节上。我们确定 GPCR使用许多机制上不同的途径来控制ERK 1/2活性，包括G蛋白非依赖性途径。 第二信使依赖性蛋白激酶和<$transactivated <$EGF传递的信号 受体，和新的G蛋白独立的信号，导致？？-抑制蛋白依赖性支架 ERK 1/2激活复合物。这些结果定义了两种不同的GPCR信号传导模式，在某些情况下， 我们已经确定了一些病例，这些病例是通路选择性的偏向性激动剂使它们解离的。此外，我们还发现， ERK 1/2激活的途径在功能上不是冗余的。相反，激活机制 决定了激酶的时间进程、空间分布以及最终的功能。中央 这一建议的假设是，异源三聚体G蛋白和？？-抑制蛋白是独立GPCR信号 这些传感器介导对GPCR刺激的细胞应答的不同方面。该提案 分为三个具体目标，前两个侧重于GPCR抑制的结构和功能 <$signalsome <$and the third on how G protein-dependent and？？-抑制蛋白依赖性信号被整合， 确定细胞反应。在每个目标中，我们将重点关注血管紧张素AT 1A受体，它利用 这两种信号传导机制控制ERK 1/2活性。目的I和II使用转染的细胞系统，其允许 我们使用受体和？？抑制蛋白突变体和快速siRNA沉默蛋白表达，以达到最大 优势实验将确定AT 1AR-？？-逮捕信号和 受体的结构特征和？？-控制信号成分和稳定性的抑制蛋白。我们将 识别信号体特异性ERK 1/2底物，并确定如何？抑制蛋白信号传导影响基因 转录。目的III将集中于内源性AT 1A受体在原发性主动脉血管中的信号传导 平滑肌细胞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学上由每种类型的信号调节的细胞过程。 相关背景。实验将确定时间，空间和功能特性的不同 信号的类型，以及它们如何整合以产生与发展相关的细胞变化。 动脉粥样硬化性血管疾病这些研究的完成将解决我们的一个根本差距， 了解GPCR如何工作，并可能为GPCR的新治疗应用提供见解 具有通路选择性激动剂或拮抗剂性质的配体。

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 has focused on GPCR regulation of the ERK1/2 MAP kinase cascade. We have established that GPCRs use a number of mechanistically distinct pathways to control ERK1/2 activity, including G proteindependent signals transmitted by second messenger-dependent protein kinases and ¿transactivated¿ EGF receptors, and novel G protein-independent signals that result from ??-arrestin-dependent scaffolding of an ERK1/2 activation complex. 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 the pathways of ERK1/2 activation are not functionally redundant. Rather, the mechanism of activation determines the time course, spatial distribution, and ultimately the function of the kinase. The central hypothesis of this proposal is that heterotrimeric G proteins and ??-arrestins are 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 to control ERK1/2 activity. 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 identify signalsome-specific ERK1/2 substrates 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.