G protein-coupled receptors (GPCRs) serve as catalytic activators of heterotrimeric G-proteins by exchanging GTP for the bound GDP on the G? subunit. This guanine nucleotide exchange factor (GEF) activity of GPCRs is the initial step in the G-protein cycle and determines the onset of various intracellular signaling pathways that govern critical physiological responses to extracellular cues. The structural basis for several steps in the G-protein nucleotide cycle have been made clear over the past decade, including intrinsic GTP hydrolysis by G? and acceleration of this hydrolysis (`GAP activity') by RGS domains;however, the precise structural determinants underlying receptor-mediated G-protein activation, and facilitation of signal onset by RGS proteins, remain incompletely defined. As GPCRs represent a rich set of drug targets, more thorough understanding of their mechanism of activating intracellular signaling should provide valuable further avenues for drug discovery. Currently, several distinct (and somewhat conflicting) models have been proposed to explain the communication between activated GPCRs and G-protein heterotrimers that leads to the structural changes required for guanine nucleotide exchange. This research effort is focused on a high-resolution elucidation of the structural details underlying heterotrimeric G-protein activation via nucleotide exchange. Aim 1 is to resolve the structural determinants of nucleotide exchange within G? via protein crystallography of fast-exchanging G? subunits we recently identified from the genomes of A. thaliana and C. elegans, as well as additional G? mutants with enhanced GDP release or propensity to exist in a stable, nucleotide-free state. In Aim 2, three complementary cellular systems (yeast pheromone signaling, mammalian cell GIRK currents, Dictyostelium cAMP responses) will be used to ascertain the structural determinants underlying non-GAP actions of RGS proteins that facilitate GPCR/heterotrimer signal onset kinetics. This second aim relies on our recent crystallographic evidence that the fast-hydrolyzing phenotype of the G?i1 mutant G202A arises from mimicry of the transition state for GTP hydrolysis normally stabilized by RGS domains. Aim 3 is to resolve the structural determinants of receptor-catalyzed nucleotide exchange via protein crystallography of functional receptor loop peptides bound to heterotrimeric G-protein subunits. This latter aim will be facilitated by our discovery of a G?i subfamily GEF peptide, KB-752, which acts as a surrogate for G??-mediated switch region changes;we have recently used KB-752 to establish the first crystal structure of a receptor loop bound to its G- protein target - the dopamine D2-receptor ic3 loop peptide D2N bound to G?i1. High-resolution structural models derived from these pursuits will be validated in biochemical and cellular studies of point mutants predicted from structural details to abrogate or enhance nucleotide exchange or switch receptor/G-protein coupling specificity. Success of this research program will lead to a new understanding of the precise structural determinants of GPCR/G-protein coupling, agonist-induced activation, and RGS protein facilitation. The Public Health Relevance: The family of proteins known as G protein-coupled receptors represent the largest single fraction of targets for current drug therapies, including key medicines that control schizophrenia, bipolar disorder, and depression. While critically important for these drugs'actions, the precise molecular details by which these receptor proteins activate biochemical processes inside cells is poorly understood. This research is thus directed towards building and validating structural models that describe the details of how receptors activate their coupled G-proteins, with such new knowledge providing valuable further avenues for drug discovery and design.