Premium
Regulation of AGS3 and Gialpha1 interaction in living cells
Author(s) -
Oner Sukru Sadik,
Ningfei An,
Breton Billy,
Bouvier Michel,
Blumer Joe,
Stephen Lanier
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.584.4
Subject(s) - chemistry , hek 293 cells , microbiology and biotechnology , yellow fluorescent protein , docking (animal) , förster resonance energy transfer , protein–protein interaction , cell , cell signaling , signal transduction , biophysics , receptor , biochemistry , biology , gene , physics , fluorescence , medicine , nursing , quantum mechanics
Activator of G Protein Signaling 3 (AGS3) and related GPR proteins provide unexpected regulatory mechanisms for G‐protein signaling systems. AGS3 contains 4 GPR motifs, each of which can serve as a docking site for Giα‐GDP free of Gβγ. Two of the key questions for AGS3 is what controls its interaction with G‐protein and where does this interaction occur within the cell. As an initial approach to this question, we evaluated the interaction of AGS3 with Giα1 in living cells using bioluminescence resonance energy transfer (BRET) to measure interaction between proteins tagged with Renilla luciferase (RLuc) and the YFP‐variant Venus in HEK cells. AGS3 and Giα1 reciprocally tagged with either RLuc or YFP exhibited a BRET signal that was saturable and specific. The net BRET signal was blocked by Gβγ expression and it was not observed with the AGS3‐Q/A mutant in which each of the GPR motifs are rendered incapable of binding Giα. BRET was observed with AGS3‐YFP and Giα1‐RLuc or with AGS3‐RLuc and Giα1‐YFP pairs. The latter donor and acceptor pair exhibited a significantly stronger net BRET signal potentially reflecting the assembly of a Giα1‐YFP acceptor on each of the four GPR docking sites in an individual AGS3‐RLuc molecule. These data indicate a robust interaction of GPR proteins and Giα1 in the living cell and provide a platform to determine how this interaction is regulated and where it occurs within the cell.