Gs activation is time-limiting in initiating receptor-mediated signaling
P. Hein, F. Rochais, C. Hoffmann, S. Dorsch, V. Nikolaev, S. Engelhardt, C. Berlot, M. Lohse, and M. Bunemann. J Biol Chem, 281 (44):
33345-51(November 2006)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..
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.
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.
%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
}