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

Abstract

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␣ i free of G␤␥ 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 AGS3⅐ G␣ i signaling module by a cell surface, seven-transmembrane receptor. AGS3 and G␣ 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 ␣ 2 -adrenergic receptors or -opioid receptors reduced AGS3-RLuc⅐G␣ 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␤␥ sequestration with the carboxyl terminus of GRK2. G␣ i -dependent and agonist-sensitive bioluminescence resonance energy transfer was also observed between AGS3 and cell-surface receptors typically coupled to G␣ i and/or G␣ 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␣␤␥ and GPR-G␣ i offer additional flexibility for systems to respond and adapt to challenges and orchestrate complex behaviors.

Direct Coupling of a Seven-Transmembrane-Span Receptor to a Gαi G-Protein Regulatory Motif Complex

“…*P , 0.05 compared with vehicle. Direct Coupling of a 7TM Receptor to GaiGPR previous observations using untethered GaiYFP (Oner et al, 2010a). Furthermore, the Gbg inhibitor gallein also did not alter the basal or agonist-regulated BRET between AGS3Rluc or AGS4Rluc and either untethered Gai 2 YFP or Gai 2 YFP tethered to the a 2A AR (W. G. Robichaux, III and J.…”

“…4). AGS3-Q/A-Rluc, which cannot bind Gai (Oner et al, 2010a), did not coimmunoprecipitate with a 2A AR-Gai 2 YFP, thus serving as an important internal negative control. Treatment with the a 2A AR agonist UK14304 resulted in an ∼30% decrease in coimmunoprecipitation of AGS3Rluc with a 2A AR-Gai 2 YFP compared with vehicle treatment.…”

“…Protease inhibitor mixture tablets (Complete Mini) were obtained from Roche Applied Science (Indianapolis, IN). AGS3 and AGS4 fused at the carboxyl terminus to Renilla luciferase (Rluc) and a 2A adrenergic receptor (a 2A AR) constructs were generated as previously described (Oner et al, 2010a(Oner et al, ,b, 2013a. Rat Gai 2 -yellow fluorescent protein (YFP) was generated by Dr. Scott Gibson (Gibson and Gilman, 2006) and provided by Dr. Nathan Dascal (Tel Aviv University, Tel Aviv, Israel).…”

“…pcDNA3::GRK2-carboxyl terminus (CT) (GRK2-CT), which encodes amino acids Tyr 466 -Leu 689 in the carboxyl terminus of GRK2, was provided by Dr. Jeffrey Benovic (Thomas Jefferson University, Philadelphia, PA). All other reagents and materials were obtained as described elsewhere (Oner et al, 2010a(Oner et al, ,b, 2013a.…”

“…BRET measurements and immunoblotting were performed as previously described (Oner et al, 2010a(Oner et al, ,b, 2013a . Forty-eight hours after cell transfection, cells were dispensed in triplicate at 1 Â 10 5 cells/well in gray 96-well Optiplates (Perkin Elmer).…”