GS activation is time-limiting in initiating receptor-mediated signaling

被引:112
作者
Hein, Peter
Rochais, Francesca
Hoffmann, Carsten
Dorsch, Sandra
Nikolaev, Viacheslav O.
Engelhardt, Stefan
Berlot, Catherine H.
Lohse, Martin J.
Buenemann, Moritz
机构
[1] Univ Wurzburg, Inst Pharmacol & Toxicol, D-97078 Wurzburg, Germany
[2] Univ Wurzburg, Rudolf Virchow Ctr, DFG, Res Ctr Expt Biomed, Wurzburg, Germany
[3] Weis Ctr Res, Geisinger Clin, Danville, PA 17822 USA
关键词
D O I
10.1074/jbc.M606713200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 G alpha s mutant, in which a yellow fluorescent protein was inserted, together with cyan fluorescent protein-tagged G beta gamma 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.
引用
收藏
页码:33345 / 33351
页数:7
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