FIGURE 2.

Competition by CBG of agonist binding to CB₁R and/or CB₂R. (A,B) Competition curves for CBG in radioligand-based assays using either [³H]-CP-55940 (A) or [³H]-WIN-55,212-2 (B) binding on membranes from CHO cells stably expressing human CB₁R or CB₂R. (C) Scheme of the HTRF-based competitive binding assay. The GPCR of interest with the SNAP-tagged enzyme fused to its N-terminal domain is expressed at the cell surface. SNAP is a commercially available tag consisting of circa 180 amino acids, that can be labeled with fluorophores or other probes in a covalent fashion. The GPCR–SNAP-tagged cells are subsequently labeled with a Tb-containing probe (SNAP-Lumi4-Tb) through a covalent bond between the Tb and the reactive side of the SNAP enzyme. The Tb acts as FRET donor of an acceptor covalently linked to a selective CB2 receptor ligand. Thus, upon binding of a fluorophore-conjugated ligand (FRET acceptor) on the donor-labeled SNAP-tagged/GPCR fusion protein, an HTRF signal from the sensitized acceptor can be detected since the energy transfer can occur only when the donor and the acceptor are in close proximity. In competition binding assays using CM-157, the unlabelled specific ligand competes for receptor binding site with the fluorophore-conjugated ligand, leading to a decrease in the HTRF signal detected. (D–G) HEK-293T were transiently transfected with 1 μg cDNA for SNAP-CB₂R in the absence (D,E) or presence of 0.5 μg cDNA for CB₁R (F,G). Competition curves of specific binding of 20 nM fluorophore-conjugated CM-157 using CM-157 (0–10 μM) (D,F) or of CBG (0–10 μM) (E,G) as competitors are shown. Data represent the mean ± SEM of five experiments in triplicates.