Cannabigerol Action at Cannabinoid CB1 and CB2 Receptors and at CB1-CB2 Heteroreceptor Complexes

Abstract

Cannabigerol (CBG) is one of the major phytocannabinoids present in Cannabis sativa L. that is attracting pharmacological interest because it is non-psychotropic and is abundant in some industrial hemp varieties. The aim of this work was to investigate in parallel the binding properties of CBG to cannabinoid CB₁ (CB₁R) and CB₂ (CB₂R) receptors and the effects of the compound on agonist activation of those receptors and of CB₁-CB₂ heteroreceptor complexes. Using [³H]-CP-55940, CBG competed with low micromolar K_i values the binding to CB₁R and CB₂R. Homogeneous binding in living cells, which is only technically possible for the CB₂R, provided a 152 nM K_i value. Also interesting, CBG competed the binding of [³H]-WIN-55,212-2 to CB₂R but not to CB₁R (K_i: 2.7 versus >30 μM). The phytocannabinoid modulated signaling mediated by receptors and receptor heteromers even at low concentrations of 0.1-1 μM. cAMP, pERK, β-arrestin recruitment and label-free assays in HEK-293T cells expressing the receptors and treated with endocannabinoids or selective agonists proved that CBG is a partial agonist of CB₂R. The action on cells expressing heteromers was similar to that obtained in cells expressing the CB₂R. The effect of CBG on CB₁R was measurable but the underlying molecular mechanisms remain uncertain. The results indicate that CBG is indeed effective as regulator of endocannabinoid signaling.

Keywords: G-protein-coupled receptor; TR-FRET; cannabigerol; cannabinoid receptor; partial agonist; phytocannabinoid.

FIGURE 1

Radioligand binding assays to CB₁R and CB₂R. (A–D) Saturation curves of either [³H]-CP-55940 or [³H]-WIN-55,212-2 binding on membranes from CHO cells stably expressing human CB₁R (A,C) or CB₂R (B,D). (E,F) Competition curves for WIN-55,212-2 in radioligand-based assays using either [³H]-CP-55940 (E) or [³H]-WIN-55,212-2 (F) binding on membranes from CHO cells stably expressing human CB₁R or CB₂R. Data are expressed as the mean ± SEM of five independent experiments performed in duplicate. K_D (obtained from saturation isotherms) are shown in Table 1.

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.

FIGURE 3

Cannabigerol action in cells expressing CB₁R or CB₂R. HEK-293T cells were transfected with 0.75 μg cDNA for CB₁R (red line) or 1 μg cDNA for CB₂R (blue line). Dose–effect curves for cAMP production are expressed as % of levels obtained by 0.5 μM forskolin treatment (A). Dose-effect curves for ERK1/2 phosphorylation are expressed as % respect to basal levels (B). Dose-effect curves for β-arrestin recruitment (C) and label-free (D) assays are expressed, respectively, in mBRET units and pm. In β-arrestin-2 recruitment assays cells were transfected with 1 μg cDNA for β-arrestin-Rluc and either 0.75 μg cDNA for CB₁R-YFP or 1 μg cDNA for CB₂R-YFP. Data are the mean ± SEM of a representative experiment in triplicates (n = 6).

FIGURE 4

Effect of CBG on the action of CB₁R and CB₂R agonists. (A–D) HEK-293T cells were transfected with 0.75 μg cDNA for CB₁R and treated with 100 nM AEA or a selective CB₁R ligand (100 nM ACEA) in the absence (black bars) or presence of 100 nM (white bars) or 1 μM (gray bars) CBG. (E–H) HEK-293T cells were transfected with 1 μg cDNA for CB₂R and treated with 100 nM AEA or a selective CB₂R ligand (100 nM JWH133) in the absence (black bars) or presence of 100 nM (white bars) or 1 μM (gray bars) CBG. cAMP production (A,E) is expressed as % of levels obtained by 0.5 μM forskolin. ERK1/2 phosphorylation data are expressed as % respect to basal levels (B,F). In β-arrestin-2 recruitment assays cells were transfected with 1 μg cDNA for β-arrestin-Rluc and either 0.75 μg cDNA for CB₁R-YFP or 1 μg cDNA for CB₂R-YFP. Data for β-arrestin recruitment (C,G) and label-free (D,H) assays are expressed, respectively, in mBRET units and pm. Data represent the mean ± SEM of six different experiments performed with six replicates. One-way ANOVA and Bonferroni’s multiple comparison post hoc test were used for statistical analysis (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001; versus treatment with AEA, ACEA, or JWH133 alone).

FIGURE 5

Effect of CBG in cells expressing CB₁ and CB₂ receptors. (A–D) Effect of CBG in HEK-293T cells transfected with 0.75 μg cDNA for CB₁R and 1 μg cDNA for CB₂R (A,B,D) or 1 μg cDNA for β-arrestin-Rluc, 0.75 μg cDNA for CB₁R and 1 μg cDNA for CB₂R-YFP (C). Dose–effect curves for cAMP production are expressed as % of levels obtained by 0.5 μM forskolin treatment (A). Dose-effect curves for ERK1/2 phosphorylation are expressed as % respect of basal levels (B). Dose-effect curves for β-arrestin recruitment (C) and label-free (D) assays are expressed, respectively, in mBRET units and pm. Dotted lines (red and blue) are the same than those shown in Figure 3 and serve as a reference for differential effects in cells coexpressing both receptors. Data are the mean ± SEM of a representative experiment in triplicates (n = 6). (E–H) HEK-293T cells transfected with 0.75 μg cDNA for CB₁R and 1 μg cDNA for CB₂R (E,F,H) or 1 μg cDNA for β-arrestin-Rluc, 0.75 μg cDNA for CB₁R and 1 μg cDNA for CB₂R-YFP (G) were treated with 100 nM AEA or a selective CB₂R ligand (100 nM JWH133) in the absence (black bars) or presence (white bars) of 100 nM CBG. cAMP production (E) is expressed as % of levels obtained by 0.5 μM forskolin. ERK1/2 phosphorylation data are expressed as % respect of basal levels (F). Data for β-arrestin recruitment (G) and label-free (H) assays are expressed, respectively, in mBRET units and pm. Data represent the mean ± SEM of six different experiments performed with three replicates. One-way ANOVA and Bonferroni’s multiple comparison post hoc test were used for statistical analysis (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001; versus treatment with AEA, ACEA, or JWH133 alone).