Signaling through Rho GTP/GDP Exchange Factors

描述（由申请方提供）：细胞内信号传导途径取决于其组成蛋白的适当和独特的亚细胞位置。通常，关键的细胞内信号蛋白从一个亚细胞位置移动到另一个亚细胞位置（例如，从细胞质到质膜或从细胞质到细胞核）。不正确定位的蛋白质可以阻止特定信号通路的完成，或者可以导致不受调节的信号传导，例如不受控制的细胞生长。了解细胞蛋白质如何在特定时间和亚细胞位点聚集在一起，将有助于深入了解阻止疾病状态中不适当信号传导的方法。 异源三聚体G蛋白作为分子开关将信息从激活的细胞表面受体传递到适当的细胞内效应物。一个由α 12和α 13组成的G蛋白α亚基家族可以激活Rho家族的小GTP酶。反过来，Rho激活刺激细胞生长和形态变化的信号通路。Rho鸟嘌呤核苷酸交换因子（GEF）的一个大家族激活Rho，并且称为RGS-RhoGEF的亚家族由p115-RhoGEF、PDZ-RhoGEF和LARG组成，作为α 12/13和Rho之间的直接连接起作用。然而，负责通过alpha12和alpha13激活RGS-RhoGEFs的机制知之甚少。本申请的主要目的是阐明控制RGS-RhoGEF亚细胞定位的机制，确定RGS-RhoGEF的差异和调节的亚细胞定位如何有助于信号传导功能，并定义alpha12/13和RGS-RhoGEF之间的相互作用。这些目标将通过以下具体目标来实现：（1）定义LARG的亚细胞定位，并确定响应于激活的Ga和GPCR的定位变化;（2）定义PDZ-RhoGEF的肌动蛋白结合结构域的功能意义;（3）定义Rho在p115-RhoGEF的α 13依赖性PM募集中的参与;（4）确定RGS-RhoGEFs与alpha13-alpha12/13或alphaq之间的相互作用，并确定alpha12/13的RhoGEF结合缺陷突变体的信号功能。本研究将利用培养的哺乳动物细胞作为模型系统，并将采用细胞生物学和生物化学技术来研究中心假设，即亚细胞定位在理解RGS-RhoGEF功能中起着关键作用，以及alpha12/13如何通过p115-RhoGEF，PDZ-RhoGEF和LARG激活Rho信号通路。

DESCRIPTION (provided by applicant): Intracellular signaling pathways depend upon appropriate and unique subcellular locations of their constituent proteins. Frequently, key intracellular signaling proteins move from one subcellular location to another (e.g., from cytoplasm to plasma membranes or from cytoplasm to nucleus) in response to extracellular stimuli. Incorrectly localized proteins can prevent completion of a particular signaling pathway or can cause unregulated signaling, such as uncontrolled cell growth. Understanding how cellular proteins come together at specific times and subcellular sites will lead to insight into ways to block inappropriate signaling in disease states. The heterotrimeric (alphabetagamma) G proteins act as molecular switches to relay information from activated cell-surface receptors to appropriate intracellular effectors. One family of G protein a subunits, consisting of alpha12 and alpha13, can activate the Rho family of small GTPases. Rho, in turn, activates signaling pathways that stimulate cell growth and morphological changes. A large family of Rho guanine nucleotide exchange factors (GEF) activates Rho, and a sub-family, termed RGS-RhoGEFs, and consisting of p115-RhoGEF, PDZ-RhoGEF, and LARG, function as direct links between alpha12/13 and Rho. However, the mechanisms responsible for activation of the RGS-RhoGEFs by alpha12 and alpha13 are poorly understood. The main objectives of this application are to elucidate the mechanisms that control subcellular localizations of the RGS-RhoGEFs, determine how differential and regulated subcellular localization of the RGS-RhoGEFs contribute to signaling function, and define the interactions between alpha12/13 and the RGS-RhoGEFs. These objectives will be addressed through the following specific aims: (1) Define subcellular localization of LARG, and determine changes in localization in response to activated Ga and GPCRs; (2) Define the functional significance of the actin-binding domain of PDZ-RhoGEF; (3) Define the involvement of Rho in alpha13- dependent PM recruitment of p115-RhoGEF; (4) Define interactions between RGS-RhoGEFs and alpha13-alpha12/13 or alphaq, and determine signaling functions of RhoGEF binding-defective mutants of alpha12/13. This research will utilize cultured mammalian cells as the model systems and will employ cell biology and biochemical techniques to examine the central hypothesis that subcellular localization plays a critical role in understanding RGS-RhoGEF function and how alpha12/13 activate Rho signaling pathways through p115-RhoGEF, PDZ-RhoGEF and LARG.