A FlAsH-based FRET approach to determine G protein–coupled receptor activation in living cells (original) (raw)
- Article
- Published: 17 February 2005
- Guido Gaietta2,
- Moritz Bünemann1,
- Stephen R Adams3,
- Silke Oberdorff-Maass1,
- Björn Behr1,
- Jean-Pierre Vilardaga1,
- Roger Y Tsien3,
- Mark H Ellisman2 &
- …
- Martin J Lohse1
Nature Methods volume 2, pages 171–176 (2005)Cite this article
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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 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 α2A-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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Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft (Leibniz award to M.J.L. and grant HO 2357/1-1 to C.H.), a Bavaria California Technology Center (BaCaTeC) grant to C.H., a Fonds der Chemischen Industrie grant to M.J.L. and US National Institutes of Health grant P41RR004050 to M.H.E.
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Authors and Affiliations
- Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Str. 9, Würzburg, D-97078, Germany
Carsten Hoffmann, Moritz Bünemann, Silke Oberdorff-Maass, Björn Behr, Jean-Pierre Vilardaga & Martin J Lohse - Department of Neuroscience, National Center of Microscopy and Imaging Research, University of California, San Diego, 9500 Gilman Drive, La Jolla, 92093, California, USA
Guido Gaietta & Mark H Ellisman - Department of Pharmacology, University of California, San Diego, 9500 Gilman Drive, La Jolla, 92093, California, USA
Stephen R Adams & Roger Y Tsien
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Correspondence toMartin J Lohse.
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The University of Würzburg has applied for a patent covering the technology described in this manuscript.
Supplementary information
Supplementary Video 1
FRET imaging of receptor activation in HEK-293 cell transiently transfected with α2A-AR-Flash/CFP. (MOV 1801 kb)
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Hoffmann, C., Gaietta, G., Bünemann, M. et al. A FlAsH-based FRET approach to determine G protein–coupled receptor activation in living cells.Nat Methods 2, 171–176 (2005). https://doi.org/10.1038/nmeth742
- Received: 14 October 2004
- Accepted: 26 January 2005
- Published: 17 February 2005
- Issue Date: March 2005
- DOI: https://doi.org/10.1038/nmeth742