title: A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells.
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 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.
authors.affiliation: Institute of Pharmacology and Toxicology, University of Würzburg, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Hoffmann
authors.forename: Guido
authors.initials: G
authors.lastname: Gaietta
authors.forename: Moritz
authors.initials: M
authors.lastname: Bünemann
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Adams
authors.forename: Silke
authors.initials: S
authors.lastname: Oberdorff-Maass
authors.forename: Björn
authors.initials: B
authors.lastname: Behr
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Vilardaga
authors.forename: Roger Y
authors.initials: RY
authors.lastname: Tsien
authors.forename: Mark H
authors.initials: MH
authors.lastname: Ellisman
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Lohse
mesh_terms: 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*
chemical_list: C485182:Cyan Fluorescent Protein; D005456:Fluorescent Dyes; D043705:Receptor, Adenosine A2A; D043562:Receptors, G-Protein-Coupled; D049452:Green Fluorescent Proteins; D019204:GTP-Binding Proteins