title: Receptor-regulated interaction of activator of G-protein signaling-4 and Galphai.
abstract: Activator of G-protein signaling-4 (AGS4), via its three G-protein regulatory motifs, is well positioned to modulate G-protein signal processing by virtue of its ability to bind Galpha(i)-GDP subunits free of Gbetagamma. Apart from initial observations on the biochemical activity of the G-protein regulatory motifs of AGS4, very little is known about the nature of the AGS4-G-protein interaction, how this interaction is regulated, or where the interaction takes place. As an initial approach to these questions, we evaluated the interaction of AGS4 with Galpha(i1) in living cells using bioluminescence resonance energy transfer (BRET). AGS4 and Galpha(i1) reciprocally tagged with either Renilla luciferase (RLuc) or yellow fluorescent protein (YFP) demonstrated saturable, specific BRET signals. BRET signals observed between AGS4-RLuc and Galpha(i1)-YFP were reduced by G-protein-coupled receptor activation, and this agonist-induced reduction in BRET was blocked by pertussis toxin. In addition, specific BRET signals were observed for AGS4-RLuc and alpha(2)-adrenergic receptor-Venus, which were Galpha(i)-dependent and reduced by agonist, indicating that AGS4-Galpha(i) complexes are receptor-proximal. These data suggest that AGS4-Galpha(i) complexes directly couple to a G-protein-coupled receptor and may serve as substrates for agonist-induced G-protein activation.
authors.affiliation: From the Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, SC 29425, USA.
authors.forename: Sukru Sadik
authors.initials: SS
authors.lastname: Oner
authors.forename: Ellen M
authors.initials: EM
authors.lastname: Maher
authors.forename: Billy
authors.initials: B
authors.lastname: Breton
authors.forename: Michel
authors.initials: M
authors.lastname: Bouvier
authors.forename: Joe B
authors.initials: JB
authors.lastname: Blumer
mesh_terms: D000818:Animals; D002460:Cell Line; D020555:GTP-Binding Protein Regulators / Q000502:physiology*; D017930:Genes, Reporter; D006706:Homeostasis; D006801:Humans; D007668:Kidney; D008156:Luciferases / Q000235:genetics; D008164:Luminescent Proteins / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D020710:RGS Proteins / Q000235:genetics / Q000378:metabolism*; D018341:Receptors, Adrenergic, alpha-2 / Q000502:physiology; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D048790:Renilla / Q000201:enzymology; D014162:Transfection
chemical_list: D020555:GTP-Binding Protein Regulators; D008164:Luminescent Proteins; D020710:RGS Proteins; D018341:Receptors, Adrenergic, alpha-2; D043562:Receptors, G-Protein-Coupled; C098799:RGS4 protein; D008156:Luciferases
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