Signaling through Rho GTP/GDP Exchange Factors

描述(由申请人提供)：细胞内信号通路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响应细胞外刺激而从一个亚细胞位置移动到另一个位置(例如，从细胞质到质膜或从细胞质到细胞核)。错误定位的蛋白质可能会阻止特定信号通路的完成，或者可能导致信号失控，如细胞生长失控。了解细胞蛋白是如何在特定的时间和亚细胞位置聚集在一起的，将有助于深入了解如何在疾病状态下阻止不适当的信号传递。 异三聚体G蛋白充当分子开关，将激活的细胞表面受体的信息传递给适当的细胞内效应器。一个由α12和α13组成的G蛋白a亚单位家族可以激活Rho家族的小GTP酶。Rho反过来激活刺激细胞生长和形态变化的信号通路。一个Rho鸟嘌呤核苷酸交换因子家族激活Rho，一个称为RGS-RhoGEF的亚家族由p115-Rhogef、PDZ-Rhogef和LARG组成，在α12/13和Rho之间起着直接的连接作用。然而，α12和α13激活RGS-RhoGEF的机制尚不清楚。本应用的主要目的是阐明控制RGS-RhoGEF亚细胞定位的机制，确定RGS-RhoGEF的差异和受调控的亚细胞定位如何有助于信号功能，并定义α12/13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，并确定定位随激活的GA和GPCRs的变化；(2)定义PDZ-Rhogef的肌动蛋白结合域的功能意义；(3)定义Rho在依赖于字母13的p115-Rhogef的PM招募中的参与；(4)确定RGS-RhoGEF与α13-α12/13或αq之间的相互作用，并确定α12/13的Rhogef结合缺陷突变体的信号功能。本研究将以培养的哺乳动物细胞为模型系统，利用细胞生物学和生化技术来检验中心假设，即亚细胞定位在理解RGS-Rhogef功能以及alpha12/13如何通过p115-Rhogef、PDZ-Rhogef和LARG激活Rho信号通路方面起着关键作用。

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.