title: Gs activation is time-limiting in initiating receptor-mediated signaling.
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.
authors.affiliation: University Würzburg, Institute of Pharmacology and Toxicology, Versbacher Strasse 9, 97078 Würzburg, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Hein
authors.forename: Francesca
authors.initials: F
authors.lastname: Rochais
authors.forename: Carsten
authors.initials: C
authors.lastname: Hoffmann
authors.forename: Sandra
authors.initials: S
authors.lastname: Dorsch
authors.forename: Viacheslav O
authors.initials: VO
authors.lastname: Nikolaev
authors.forename: Stefan
authors.initials: S
authors.lastname: Engelhardt
authors.forename: Catherine H
authors.initials: CH
authors.lastname: Berlot
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Lohse
authors.forename: Moritz
authors.initials: M
authors.lastname: Bünemann
mesh_terms: 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
chemical_list: D043705:Receptor, Adenosine A2A; D018342:Receptors, Adrenergic, beta-1; D000242:Cyclic AMP; D019205:GTP-Binding Protein alpha Subunits, Gs