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 (G?/?/?) 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 G?12/13 and G?i 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 G?12 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-G? signaling.
In Aim 1 we will determine how CLICs couple GPCR-G? (G?12/13 and G?i) signaling to Rho and Rac. We will survey the requirement for CLICs in different cell and signaling contexts to define key GPCR-G? 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-G???-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-G???-Rho/Rac signaling we will significantly increase our knowledge of how GPCRs influence human biology and uncover new ways of targeting these pathways.
Signal transduction via G-protein-coupled receptors (GPCRs) regulates many aspects of development, physiology and disease. Over 30% of FDA-approved drugs, and many undergoing clinical trials, target GPCRs and the pathways they regulate. Our proposed work defines chloride intracellular channel (CLIC) proteins as novel effectors of GPCR signaling that regulate Rho/Rac, and these studies will provide new avenues for targeting and manipulating these important developmental and pathophysiological pathways.