Receptor-regulated interaction of activator of G-protein signaling-4 and Galphai

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 re...

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Bibliographic Details
Published in:The Journal of biological chemistry 2010-07, Vol.285 (27), p.20588
Main Authors: Oner, Sukru Sadik, Maher, Ellen M, Breton, Billy, Bouvier, Michel, Blumer, Joe B
Format: Article
Language:English
Subjects:
Animals
Cell Line
Genes, Reporter
GTP-Binding Protein Regulators - physiology
Homeostasis
Humans
Kidney
Luciferases - genetics
Luminescent Proteins - physiology
Mutagenesis, Site-Directed
Receptors, Adrenergic, alpha-2 - physiology
Receptors, G-Protein-Coupled - genetics
Receptors, G-Protein-Coupled - metabolism
Renilla - enzymology
RGS Proteins - genetics
RGS Proteins - metabolism
Transfection
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Summary: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.
ISSN:1083-351X
DOI:10.1074/jbc.C109.088070