Gs activation is time-limiting in initiating receptor-mediated signaling.
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.
Hein
Peter
P
University Würzburg, Institute of Pharmacology and Toxicology, Versbacher Strasse 9, 97078 Würzburg, Germany.
Rochais
Francesca
F
Hoffmann
Carsten
C
Dorsch
Sandra
S
Nikolaev
Viacheslav O
VO
Engelhardt
Stefan
S
Berlot
Catherine H
CH
Lohse
Martin J
MJ
Bünemann
Moritz
M
D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D043705:Receptor, Adenosine A2A / Q000235:genetics / Q000378:metabolism*; D018342:Receptors, Adrenergic, beta-1 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors
D043705:Receptor, Adenosine A2A; D018342:Receptors, Adrenergic, beta-1; D000242:Cyclic AMP; D019205:GTP-Binding Protein alpha Subunits, Gs