A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells

Nat Methods. 2005 Mar;2(3):171-6. doi: 10.1038/nmeth742. Epub 2005 Feb 17.


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.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • COS Cells
  • Cells, Cultured
  • Chlorocebus aethiops
  • Fluorescence Resonance Energy Transfer / methods*
  • Fluorescent Dyes
  • GTP-Binding Proteins / metabolism*
  • Green Fluorescent Proteins
  • HeLa Cells
  • Humans
  • Kidney / metabolism*
  • Protein Interaction Mapping / methods*
  • Receptor, Adenosine A2A
  • Receptors, G-Protein-Coupled / metabolism*


  • Cyan Fluorescent Protein
  • Fluorescent Dyes
  • Receptor, Adenosine A2A
  • Receptors, G-Protein-Coupled
  • Green Fluorescent Proteins
  • GTP-Binding Proteins