%0 Journal Article %1 Hein2006 %A Hein, P. %A Rochais, F. %A Hoffmann, C. %A Dorsch, S. %A Nikolaev, V. O. %A Engelhardt, S. %A Berlot, C. H. %A Lohse, M. J. %A Bunemann, M. %D 2006 %J J Biol Chem %K *Signal A2A/genetics/*metabolism AMP/metabolism Activation Adenosine Binding Cell Cyclic Enzyme Factors GTP-Binding Gs/genetics/*metabolism Humans Kinetics Line Protein Subunits, Time Transduction alpha beta-1/genetics/*metabolism Receptor Adrenergic %N 44 %P 33345-51 %T Gs activation is time-limiting in initiating receptor-mediated signaling %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16963443 %V 281 %X 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.

@article{Hein2006, 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.}, added-at = {2010-12-14T18:12:02.000+0100}, author = {Hein, P. and Rochais, F. and Hoffmann, C. and Dorsch, S. and Nikolaev, V. O. and Engelhardt, S. and Berlot, C. H. and Lohse, M. J. and Bunemann, M.}, biburl = {https://www.bibsonomy.org/bibtex/2ef85cb3c2dce5ece5000e56fd543ec8a/pharmawuerz}, endnotereftype = {Journal Article}, interhash = {8922452862095194268b43ba5b265c91}, intrahash = {ef85cb3c2dce5ece5000e56fd543ec8a}, issn = {0021-9258 (Print) 0021-9258 (Linking)}, journal = {J Biol Chem}, keywords = {*Signal A2A/genetics/*metabolism AMP/metabolism Activation Adenosine Binding Cell Cyclic Enzyme Factors GTP-Binding Gs/genetics/*metabolism Humans Kinetics Line Protein Subunits, Time Transduction alpha beta-1/genetics/*metabolism Receptor Adrenergic}, month = {Nov 3}, note = {Hein, Peter Rochais, Francesca Hoffmann, Carsten Dorsch, Sandra Nikolaev, Viacheslav O Engelhardt, Stefan Berlot, Catherine H Lohse, Martin J Bunemann, Moritz Research Support, Non-U.S. Gov't United States The Journal of biological chemistry J Biol Chem. 2006 Nov 3;281(44):33345-51. Epub 2006 Sep 8.}, number = 44, pages = {33345-51}, shorttitle = {Gs activation is time-limiting in initiating receptor-mediated signaling}, timestamp = {2010-12-14T18:22:40.000+0100}, title = {Gs activation is time-limiting in initiating receptor-mediated signaling}, url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16963443}, volume = 281, year = 2006 }