A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells.
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.
Hoffmann
Carsten
C
Institute of Pharmacology and Toxicology, University of Würzburg, Germany.
Gaietta
Guido
G
Bünemann
Moritz
M
Adams
Stephen R
SR
Oberdorff-Maass
Silke
S
Behr
Björn
B
Vilardaga
Jean-Pierre
JP
Tsien
Roger Y
RY
Ellisman
Mark H
MH
Lohse
Martin J
MJ
D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D019204:GTP-Binding Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods*; D043705:Receptor, Adenosine A2A; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*
C485182:Cyan Fluorescent Protein; D005456:Fluorescent Dyes; D043705:Receptor, Adenosine A2A; D043562:Receptors, G-Protein-Coupled; D049452:Green Fluorescent Proteins; D019204:GTP-Binding Proteins