FRET-based monitoring of conformational change of the β₂ adrenergic receptor in living cells

The β₂ adrenergic receptor (β₂AR) is a G protein-coupled receptor that is selective to epinephrine. We demonstrate herein monitoring of an agonist-induced conformational change of β₂AR in living cells. The monitoring method is based on fluorescence resonance energy transfer from a cyan fluorescent protein (CFP) to a biarsenical fluorophore, FlAsH, attached to the C-terminus, and the third intracellular loop (ICL3), respectively. Recombinant β₂ARs exhibited agonist-induced increases in the FlAsH/CFP emission ratio, indicating that the ICL3 approached the C-terminus upon activation. Since the emission ratio changes were on a time scale of seconds, the conformational change of β₂AR in living cells was more rapid than that of purified β₂AR measured in vitro. Interestingly, the direction of the emission ratio change of β₂AR was opposite to that of the norepinephrine-responsive α_2A adrenergic receptor reported recently. It was suggested that this discrepancy corresponds directly to the diametric biological functions, i.e., the activation or inactivation of adenylyl cyclase.