Regulation of the AGS3·Gαi Signaling Complex by a Seven-transmembrane Span Receptor,

G-protein signaling modulators (GPSM) play diverse functional roles through their interaction with G-protein subunits. AGS3 (GPSM1) contains four G-protein regulatory motifs (GPR) that directly bind G alpha(i) free of G alpha gamma providing an unusual scaffold for the "G-switch" and signaling complexes, but the mechanism by which signals track into this scaffold are not well understood. We report the regulation of the AGS3 center dot G alpha(i) signaling module by a cell surface, seven-transmembrane receptor. AGS3 and G alpha(i1) tagged with Renilla luciferase or yellow fluorescent protein expressed in mammalian cells exhibited saturable, specific bioluminescence resonance energy transfer indicating complex formation in the cell. Activation of alpha(2)-adrenergic receptors or mu-opioid receptors reduced AGS3-RLuc center dot G alpha(i1)-YFP energy transfer by over 30%. The agonist- mediated effects were inhibited by pertussis toxin and co-expression of RGS4, but were not altered by G beta gamma sequestration with the carboxyl terminus of GRK2 center dot G alpha(i)-dependent and agonist-sensitive bioluminescence resonance energy transfer was also observed between AGS3 and cell-surface receptors typically coupled to G alpha(i) and/or G alpha(o) indicating that AGS3 is part of a larger signaling complex. Upon receptor activation, AGS3 reversibly dissociates from this complex at the cell cortex. Receptor coupling to both G alpha beta gamma and GPR center dot G alpha(i) offer additional flexibility for systems to respond and adapt to challenges and orchestrate complex behaviors.