Abstract
To analyze individual steps of G(S)-linked signaling in intact cells,
we used fluorescence resonance energy transfer (FRET)-based assays
for receptor-G protein interaction, G protein activation, and cAMP
effector activation. To do so, we developed a FRET-based sensor to
directly monitor G(S) activation in living cells. This was done by
coexpressing a Galpha(s) mutant, in which a yellow fluorescent protein
was inserted, together with cyan fluorescent protein-tagged Gbetagamma
subunits and appropriate receptors in HEK293 cells. Together with
assays for receptor activation and receptor-G protein interaction,
it is possible to characterize large parts of the G(S) signaling
cascade. When A(2A)-adenosine or beta(1)-adrenergic receptors are
coexpressed with G(S) in HEK293T cells, the receptor-G(S) interaction
was on the same time scale as A(2A) receptor activation with a time
constant of <50 ms. G(S) activation was markedly slower and around
450 ms with similar kinetics following activation of A(2A)- or beta(1)-receptors.
Taken together, our kinetic measurements demonstrate that the rate
of G(S) activation limits initiation of G(S)-coupled receptor signaling.
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