Journal
EMBO JOURNAL
Volume 24, Issue 23, Pages 4106-4114Publisher
WILEY
DOI: 10.1038/sj.emboj.7600870
Keywords
alpha(2A) receptors; FRET; GIRK; G protein; kinetics
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The interaction of activated G protein-coupled receptors with G proteins is a key event in signal transduction. Here, using a fluorescence resonance energy transfer (FRET)based assay, we measure directly and in living cells the interaction of YFP-labeled alpha(2A)-adrenergic receptors with CFP-labeled G proteins. Upon agonist stimulation, a small, concentration-dependent increase in FRET was observed. No specific basal FRET was detected in the absence of agonist. Kinetics of the onset of receptor/G protein interaction were < 100 ms and depended on expression levels of Got. Simultaneously recorded G protein-regulated inwardly rectifying K+ channel currents revealed a maximal current response already at agonist concentrations producing submaximal FRET amplitudes. By analyzing FRET signals in the presence of a G alpha mutant, which dissociates more slowly from activated receptors, it was demonstrated that only a fraction of wild-type G proteins interacts with the activated receptor at any time. Our data suggest that alpha(2A)-adrenergic receptors and G proteins interact by rapid collision coupling and indicate that there is no significant precoupling between these receptors and G proteins.
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