Journal
NATURE METHODS
Volume 2, Issue 3, Pages 171-176Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/NMETH742
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- NCRR NIH HHS [P41RR004050] Funding Source: Medline
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Fluorescence resonance energy transfer (FRET) from cyan to yellow fluorescent proteins (CFP/YFP) is a welt-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-coupied 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.
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