Figure 1.

Application of Förster resonance energy transfer (FRET) for recording initial steps of G protein-coupled receptors (GPCR) signaling in live cells. (A) Kinetics of ligand/receptor interaction are measured under a fluorescence microscope where a single cell was selectively excited at 480 nm [excitation of green fluorescent protein (GFP)], and the changes of the GFP emission fluorescence (515 nm) caused by FRET between a receptor N-terminally labeled by GFP (•) and a tetramethylrhodamine (TMR•)-tagged ligand were recorded over time. (B) Example of ligand/receptor interaction measured by FRET between GFP-tagged PTH-receptor (^GFPN-PTHR) and TMR-labeled PTH analog, M-PTH(1–14)^TMR. Shown are the changes of GFP emission by FRET (green signal) in response to rapid superfusion of a PTH analog, M-PTH(1–14)^TMR (red signal). (C) Relationship between the rate constants k (s⁻¹) and ligand concentration. k values for the first component (k fast) are directly proportional to the ligand concentration, whereas k values for the second step (k slow) follow an hyperbolic dependence on ligand concentration and reach a maximal value. (D) For receptor activation/deactivation, experiments are performed under a fluorescence microscope, where light at 436 nm selectively excited a single cell expressing GPCR^FlAsH/CFP or GPCR^CFP/YFP (for recordings of receptor activation) to induce donor (CFP•) and acceptor (FlAsH or yellow•) emission fluorescences simultaneously recorded over time. FRET is calculated as the ratio of emission intensities F_YFP/F_CFP after correction for the donor bleed-through into the acceptor emission, the direct acceptor excitation by light at 436 nm, and photobleaching effect. (E) Example of FRET experiments showing direct recordings of norepinephrine (NE)-mediated activation of α_2AAR^FlAsH/CFP in a single HEK-293 cell. Activation of α_2AAR is monitored upon NE application (horizontal bar) by a decrease in the FRET signal (red). (F) Relationship between the time constant of α_2A-AR^FlAsH/CFP activation after stimulation by NE at a saturating concentration, and receptor concentrations. (G) Receptor/G protein interactions are measured by recording the time course of FRET between a GPCR C-terminally labeled by yellow fluorescent protein and a CFP-labeled Gγ in combination with Gα and Gβ subunits. The principal of these experiments are similar than those described in Figure 1D. (H) Example of recordings showing the interaction between α_2AAR and G_i proteins in response to NE measured as an increase in FRET between YFP-labeled α_2AAR and CFP-labeled Gγ₂ in combination with Gα_i1 and Gβ₁ proteins. (I) Relationship between the time constant of α_2A-AR^CFP/Gα_i1b₁g₂^YFP interaction after stimulation by NE (100 mM) and G protein concentrations. In this case the kinetics of receptor/G protein interaction depend on expression levels of G_i. (Adapted from ref (20,23,24).) FlAsH, Fluorescein Arsenical Hairpin binder; PTHR, parathyroid hormone type 1 receptor.