%0 Journal Article %1 Hoffmann2005 %A Hoffmann, C. %A Gaietta, G. %A Bunemann, M. %A Adams, S. R. %A Oberdorff-Maass, S. %A Behr, B. %A Vilardaga, J. P. %A Tsien, R. Y. %A Ellisman, M. H. %A Lohse, M. J. %D 2005 %J Nat Methods %K A2A Adenosine Animals COS Cercopithecus Cultured Dyes Energy Fluorescence Fluorescent G-Protein-Coupled/*metabolism GTP-Binding Green Hela Humans Interaction Kidney/*metabolism Mapping/*methods Protein Proteins Resonance Transfer/*methods aethiops Receptor Cell Proteins/metabolism %N 3 %P 171-6 %T A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15782185 %V 2 %X Fluorescence resonance energy transfer (FRET) from cyan to yellow fluorescent proteins (CFP/YFP) is a well-established method to monitor protein-protein interactions or conformational changes of individual proteins. But protein functions can be perturbed by fusion of large tags such as CFP and YFP. Here we use G protein-coupled receptor (GPCR) activation in living cells as a model system to compare YFP with the small, membrane-permeant fluorescein derivative with two arsen-(III) substituents (fluorescein arsenical hairpin binder; FlAsH) targeted to a short tetracysteine sequence. Insertion of CFP and YFP into human adenosine A(2A) receptors allowed us to use FRET to monitor receptor activation but eliminated coupling to adenylyl cyclase. The CFP/FlAsH-tetracysteine system gave fivefold greater agonist-induced FRET signals, similar kinetics (time constant of 66-88 ms) and perfectly normal downstream signaling. Similar results were obtained for the mouse alpha(2A)-adrenergic receptor. Thus, FRET from CFP to FlAsH reports GPCR activation in living cells without disturbing receptor function and shows that the small size of the tetracysteine-biarsenical tag can be decisively advantageous.

@article{Hoffmann2005, abstract = {Fluorescence resonance energy transfer (FRET) from cyan to yellow fluorescent proteins (CFP/YFP) is a well-established method to monitor protein-protein interactions or conformational changes of individual proteins. But protein functions can be perturbed by fusion of large tags such as CFP and YFP. Here we use G protein-coupled receptor (GPCR) activation in living cells as a model system to compare YFP with the small, membrane-permeant fluorescein derivative with two arsen-(III) substituents (fluorescein arsenical hairpin binder; FlAsH) targeted to a short tetracysteine sequence. Insertion of CFP and YFP into human adenosine A(2A) receptors allowed us to use FRET to monitor receptor activation but eliminated coupling to adenylyl cyclase. The CFP/FlAsH-tetracysteine system gave fivefold greater agonist-induced FRET signals, similar kinetics (time constant of 66-88 ms) and perfectly normal downstream signaling. Similar results were obtained for the mouse alpha(2A)-adrenergic receptor. Thus, FRET from CFP to FlAsH reports GPCR activation in living cells without disturbing receptor function and shows that the small size of the tetracysteine-biarsenical tag can be decisively advantageous.}, added-at = {2010-12-14T18:12:02.000+0100}, author = {Hoffmann, C. and Gaietta, G. and Bunemann, M. and Adams, S. R. and Oberdorff-Maass, S. and Behr, B. and Vilardaga, J. P. and Tsien, R. Y. and Ellisman, M. H. and Lohse, M. J.}, biburl = {https://www.bibsonomy.org/bibtex/2be3237c9c61bb9eec69c6ce39fc456d3/pharmawuerz}, endnotereftype = {Journal Article}, interhash = {c1261d5f22021607bb7fe3ceb3d5c239}, intrahash = {be3237c9c61bb9eec69c6ce39fc456d3}, issn = {1548-7091 (Print) 1548-7091 (Linking)}, journal = {Nat Methods}, keywords = {A2A Adenosine Animals COS Cercopithecus Cultured Dyes Energy Fluorescence Fluorescent G-Protein-Coupled/*metabolism GTP-Binding Green Hela Humans Interaction Kidney/*metabolism Mapping/*methods Protein Proteins Resonance Transfer/*methods aethiops Receptor Cell Proteins/metabolism}, month = Mar, note = {Hoffmann, Carsten Gaietta, Guido Bunemann, Moritz Adams, Stephen R Oberdorff-Maass, Silke Behr, Bjorn Vilardaga, Jean-Pierre Tsien, Roger Y Ellisman, Mark H Lohse, Martin J P41RR004050/RR/NCRR NIH HHS/United States Comparative Study Evaluation Studies Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. United States Nature methods Nat Methods. 2005 Mar;2(3):171-6. Epub 2005 Feb 17.}, number = 3, pages = {171-6}, shorttitle = {A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells}, timestamp = {2010-12-14T18:21:42.000+0100}, title = {A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells}, url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15782185}, volume = 2, year = 2005 }