CLIC function in GPCR-mediated Rho/Rac signaling

G蛋白偶联受体（GPCR）介导的GTP酶Rho和Rac的调节是一种保守的信号传导途径， 重要细胞行为所需的模块。然而，连接GPCR及其异源三聚体的机制， G-蛋白（Gα/β/γ）伴侣与Rho/Rac的关系尚未完全阐明。我们在C.线虫和人内皮细胞 细胞揭示了Rho/Rac信号传导所需的一个新的保守参与者：细胞内氯离子通道 （CLIC）蛋白质家族。我们的数据显示CLIC在两个重要的内皮GPCR途径中是必需的 (S1P/S1 P受体和凝血酶/PAR），其通过Gα12/13和Gαi起作用以激活Rho和Rac。这 CLIC功能在进化上是保守的，因为我们发现在C. elegans的CLIC直系同源物exc-4 与Gα12直系同源物gpa-12以及与Rac直系同源物ced-10和mig-2遗传相互作用。分子 CLIC的功能长期以来一直是一个谜。基于序列和结构的相似性， 提出作为氯离子通道和/或谷胱甘肽S-转移酶（GST）起作用。以前的工作表明 EXC-4膜定位是由N末端结构域介导的，这种定位对于功能至关重要。 我们现在发现CLIC膜定位对于其在GPCR介导的Rho/Rac中的作用也是关键的 在内皮细胞中的活化，以及用豆蔻酰化取代膜靶向N-末端 信号足以恢复该功能。由于CLIC的通道和GST活动需要完整的N- 我们发现了CLIC的一种新活性。我们假设CLIC是膜定位的， 响应GPCR-Gα信号传导的Rho和Rac调节子。在目标1中，我们将确定CLIC如何耦合 GPCR-Gα（Gα12/13和Gαi）向Rho和Rac的信号传导。我们将调查不同单元对CLIC的需求 和信号传导上下文来定义利用CLIC来调节Rho和Rac的关键GPCR-Gα组合。我们 将使用尖端的生物传感器和遗传工具来测量和调节信号，以确定哪一步 在GPCR-Gαβγ-Rho/Rac级联中，需要CLIC。最后，我们将测试CLIC是否与 Rho/Rac调节信号传导。在目标2中，我们将定义CLIC调节Rho/Rac的决定因素， 人细胞和C. elegans的结构-功能分析，重点是EXC-4/CLIC C- 终点站将测试本目标中定义的关键域与Rho/Rac（如定义）相互作用的能力 目标1）。在目标3中，我们将在C. elegans发现保守的 基因和物理上与EXC-4/CLIC相互作用的参与者，以进一步阐明CLIC如何调节Rho/Rac 发信号。然后，我们将测试在这些筛选中鉴定的基因的人类直系同源物是否影响Rho/Rac 哺乳动物细胞中的信号传导。通过定义CLIC在GPCR-Gαβγ-Rho/Rac信号传导中的新作用机制， 我们将大大增加我们对GPCR如何影响人类生物学的了解，并发现新的方法， 针对这些路径。

G-protein-coupled receptor (GPCR)-mediated regulation of the GTPases Rho and Rac is a conserved signaling module required for vital cell behaviors. However, the mechanisms that connect GPCRs, and their heterotrimeric G-protein (Gα/β/γ) partners, to Rho/Rac are not fully delineated. Our studies in C. elegans and human endothelial cells have revealed a new conserved player required for Rho/Rac signaling: the Chloride Intracellular Channel (CLIC) family of proteins. Our data shows that CLICs are required in two important endothelial GPCR pathways (S1P/S1P Receptor and thrombin/PAR) that function through Gα12/13 and Gαi to activate Rho and Rac. This CLIC function is evolutionarily conserved, because we found that in C. elegans the CLIC ortholog exc-4 genetically interacts with the Gα12 ortholog gpa-12 and with the Rac orthologs ced-10 and mig-2. The molecular function of CLICs has long remained a mystery. Based on sequence and structural similarities, they have been proposed to function as chloride channels and/or as glutathione S-transferases (GST). Previous work has shown that EXC-4 membrane localization is mediated by an N-terminal domain and this localization is critical for function. We have now found that CLIC membrane localization is also critical for its role in GPCR-mediated Rho/Rac activation in endothelial cells, and that replacement of the membrane-targeting N-terminus with a myristoylation signal is sufficient to restore this function. Since the channel and GST activities of CLICs require an intact N- terminus we have discovered a novel activity for CLICs. We hypothesize that CLICs are membrane-localized regulators of Rho and Rac that respond to GPCR-Gα signaling. In Aim 1 we will determine how CLICs couple GPCR-Gα (Gα12/13 and Gαi) signaling to Rho and Rac. We will survey the requirement for CLICs in different cell and signaling contexts to define key GPCR-Gα combinations that utilize CLICs to regulate of Rho and Rac. We will use cutting-edge bio-sensors and genetic tools to measure and modulate signaling to determine which step in the GPCR-Gαβγ-Rho/Rac cascade requires CLICs. Finally, we will test whether CLICs physically interact with Rho/Rac to modulate signaling. In Aim 2 we will define the determinants by which CLICs regulate Rho/Rac in human cells and in C. elegans by performing structure-function analyses, focused on the EXC-4/CLIC C- terminus. Critical domains defined in this Aim will be tested for their ability to interact with Rho/Rac (as defined in Aim 1). In Aim 3 we will carry out unbiased genetic and proteomic screens in C. elegans to find conserved players that genetically and physically interact with EXC-4/CLIC to further elucidate how CLICs regulate Rho/Rac signaling. We will then test whether human orthologs of genes identified in these screens influence Rho/Rac signaling in mammalian cells. By defining new mechanisms of action for CLICs in GPCR-Gαβγ-Rho/Rac signaling we will significantly increase our knowledge of how GPCRs influence human biology and uncover new ways of targeting these pathways.