1. Gs Activation Is Time-limiting in Initiating Receptor-mediated Signaling*.
- Author
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Hein, Peter, Rochais, Francesca, Hoffmann, Carsten, Dorsch, Sandra, Nikolaev, Viacheslav O., Engeihardt, Stefan, Beriot, Catherine H., Lohse, Martin J., and Bünemann, Moritz
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RECEPTOR antibodies , *G proteins , *ACTIVATION (Chemistry) , *ENERGY transfer , *RESONANCE , *FLUORESCENCE - Abstract
To analyze individual steps of GS-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 GS activation in living cells. This was done by coexpressing a Gαs mutant, in which a yellow fluorescent protein was inserted, together with cyan fluorescent protein-tagged Gβγ 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 GS signaling cascade. When A2A-adenosine or β1-adrenergic receptors are coexpressed with GS in HEK293T cells, the receptor-GS interaction was on the same time scale as A2A receptor activation with a time constant of <50 ms. GS activation was markedly slower and around 450 ms with similar kinetics following activation of A2A- or β1-receptors. Taken together, our kinetic measurements demonstrate that the rate of GS activation limits initiation of GS-coupled receptor signaling. [ABSTRACT FROM AUTHOR]
- Published
- 2006
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