Hormones and neurotransmitters can activate intracellular signal transduction by binding receptors linked to heterotrimeric guanine nucleotide-binding or """"""""G"""""""" proteins. G protein a subunits cycle between active, GTP-bound and inactive, GDP-bound states, and thus signal duration is controlled by their intrinsic GTPase activity. """"""""Regulator of G-protein signaling"""""""" (RGS) proteins are considered key desensitizers of G protein-coupled signaling given the ability of their hallmark """"""""RGS-box"""""""" domains to accelerate Ga GTPase activity. However, as RGS proteins have only recently been identified, their physiological functions in the overall dynamics of signal onset, integration, and termination are poorly defined. Recent identification of Ras-family GTPase- and Ga-interaction domains (""""""""RFL"""""""" and """"""""GoLoco"""""""" domains) within RGS 12 and RGS 14 presents the opportunity to define the molecular mechanisms these two RGS proteins use to transact higher-order functions in G protein signaling modulation. This proposal is focused on determining the binding specificities and structural determinants of RGS 12/14 GoLoco and RFL domains, as well as the effects of domain interactions both on the nucleotide cycle of the bound G protein and on RGS-box GTPase-accelerating activity. Studies in Aim 1 test the hypothesis that the GoLoco region binds to GDP-bound Gi-class Ga subunits and acts as a receptor-independent guanine-nucleotide exchange factor. Studies in Aim 2 test the hypothesis that the RFL domains bind GTP-bound Ras-family G proteins and inhibit nucleotide dissociation. Studies in Aim 3 will define the Ga selectivites of RGS12/14 ROS-boxes as well as test the hypothesis that GoLoco/Gax and/or RFL/Ras-family protein interactions modify RGS box function. In all three Aims, binding specificity and affinity will be determined by a combination of yeast two-hybrid analyses, coprecipitation studies, biosensor measurements, and cell co-immunoprecipitation assays; effects of these interactions on nucleotide binding/hydrolysis will be analysed by in vitro nucleotide binding assays, single-turnover and steady state GTP hydrolysis measurements, and cellular readouts of receptor signaling outcomes. As perturbation of G protein-coupled signal transduction can cause human disease, yet forms the basis of many drug actions, defining the mechanisms by which RGS12 and RGS14 assemble and regulate specific heterotrimeric and Ras-family G proteins should ultimately lead to novel drug discovery targets with exquisite specificity.
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