A FlAsH-based FRET approach to determine G protein–coupled receptor activation in living cells (original) (raw)

Nature Methods volume 2, pages 171–176 (2005)Cite this article

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

  1. 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
  2. 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
  3. Department of Pharmacology, University of California, San Diego, 9500 Gilman Drive, La Jolla, 92093, California, USA
    Stephen R Adams & Roger Y Tsien

Authors

  1. Carsten Hoffmann
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  2. Guido Gaietta
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  3. Moritz Bünemann
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  4. Stephen R Adams
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  5. Silke Oberdorff-Maass
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  6. Björn Behr
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  7. Jean-Pierre Vilardaga
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  8. Roger Y Tsien
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  9. Mark H Ellisman
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  10. Martin J Lohse
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Corresponding author

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

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