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J. Biol. Chem., Vol. 281, Issue 44, 33345-33351, November 3, 2006

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G_S Activation Is Time-limiting in Initiating Receptor-mediated Signaling^*

Peter Hein, Francesca Rochais, Carsten Hoffmann, Sandra Dorsch, Viacheslav O. Nikolaev, Stefan Engelhardt, Catherine H. Berlot^¶, Martin J. Lohse, and Moritz Bünemann¹

From the University Würzburg, Institute of Pharmacology and Toxicology, Versbacher Strasse 9, 97078 Würzburg, Germany, the University of Würzburg, Rudolf-Virchow-Center, Deutsche Forschungsgemeinschaft (DFG) Research Center for Experimental Biomedicine, Germany, and the ^¶Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822-2623

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_s mutant, in which a yellow fluorescent protein was inserted, together with cyan fluorescent protein-tagged G 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 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 ₁-receptors. Taken together, our kinetic measurements demonstrate that the rate of G_S activation limits initiation of G_S-coupled receptor signaling.

Received for publication, July 14, 2006 , and in revised form, August 25, 2006.

^* This work was supported by the DFG Grant SFB487 (to M. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 49-931-201-48854; Fax: 49-931-201-48539; E-mail: m-buenemann{at}toxi.uni-wuerzburg.de.

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