Abstract
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 Galpha. 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 Galpha
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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