Nature Methods2, 171 - 176 (2005)
Published online: 17 February 2005; | doi:10.1038/nmeth742
A FlAsH-based FRET approach to determine G protein−coupled receptor activation in living cells
Carsten Hoffmann1, Guido Gaietta2, Moritz Bünemann1, Stephen R Adams3, Silke Oberdorff-Maass1, Björn Behr1, Jean-Pierre Vilardaga1, Roger Y Tsien3, Mark H Ellisman2
& Martin J Lohse1
1
Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Str. 9, D-97078 Würzburg, Germany.
2
National Center of Microscopy and Imaging Research, Department of Neuroscience, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA.
3
Department of Pharmacology, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA.
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 A2A 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 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.
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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.
