We designed a fluorescence resonance energy transfer (FRET)-based approach to study the ligand binding constants of the adenosine A_2A receptor (A_2AR). Our assay is based in the interaction of a fluorescent A_2AR agonist ligand (MRS5424) with an A_2AR tagged with the cyan fluorescent protein (CFP) at the N-terminus (i.e. A_2AR^CFP) and expressed in living cells. Thus, upon fast superfusion of the A_2AR^CFP expressing cells with MRS5424, the ligand-receptor interaction is determined by single-cell FRET in a real-time mode. Accordingly, our approach allowed immediate ‘real-time’ readout of the ligand-receptor interaction, thus allowing kinetic binding experiments, a feature impossible to achieve using conventional radioisotope-labelled ligands. In addition, since our assay permitted the visual confirmation of receptor localization it also allowed localized saturation binding experiments.

FRET (烦恼)

Fluorescent ligands (荧光配体)

GPCR (G蛋白偶联受体)

Real-time binding (实时结合)

Kinetics (动力学)