Cannabidiol, a novel inverse agonist for GPR12

Abstract

GPR12 is a constitutively active, G_s protein-coupled receptor that currently has no confirmed endogenous ligands. GPR12 may be involved in physiological processes such as maintenance of oocyte meiotic arrest and brain development, as well as pathological conditions such as metastatic cancer. In this study, the potential effects of various classes of cannabinoids on GPR12 were tested using a cAMP accumulation assay. Our data demonstrate that cannabidiol (CBD), a major non-psychoactive phytocannabinoid, acted as an inverse agonist to inhibit cAMP accumulation stimulated by the constitutively active GPR12. Thus, GPR12 is a novel molecular target for CBD. The structure-activity relationship studies of CBD indicate that both the free hydroxyl and the pentyl side chain are crucial for the effects of CBD on GPR12. Furthermore, studies using cholera toxin, which blocks G_s protein and pertussis toxin, which blocks G_i protein, revealed that G_s, but not G_i is involved in the inverse agonism of CBD on GPR12. CBD is a promising novel therapeutic agent for cancer, and GPR12 has been shown to alter viscoelasticity of metastatic cancer cells. Since we have demonstrated that CBD is an inverse agonist for GPR12, this provides novel mechanism of action for CBD, and an initial chemical scaffold upon which highly potent and efficacious agents acting on GPR12 may be developed with the ultimate goal of blocking cancer metastasis.

Keywords: Cannabidiol; GPR12; Inverse agonist; Orphan receptor.

Figure 1. Constitutive activity of GPR12

HEK293 parental cells and HEK293 cells stably expressing GPR12 were incubated for one hour, and cAMP accumulation assays were performed. The open bar represents parental HEK293 cells, while the striped bar represents HEK293 cells stably expressing GPR12. Data shown represent the mean ± SEM of three independent experiments performed in quadruplicate.

Figure 2. Effects of various classes of cannabinoids on cAMP accumulation mediated by GPR12

(A) Effects of endocannabinoids. (B) Effects of phytocannabinoids (C) Effects of synthetic cannabinoids. HEK293 cells stably expressing GPR12 were treated with different concentrations of the various classes of cannabinoids for one hour. Data shown represent the mean ± SEM of three independent experiments performed in quadruplicate.

Figure 3. Cannabidiol structure-activity relationship at GPR12

HEK293 cells stably expressing GPR12 were treated with different concentrations of CBD derivatives for one hour. Data shown represent the mean ± SEM of three independent experiments performed in quadruplicate.

Figure 4. Involvement of G protein in cAMP accumulation mediated by GPR12

HEK293 cells stably expressing GPR12 were treated with vehicle, CTX or PTX overnight prior to stimulation with different concentrations of CBD for one hour. Data shown represent the mean ± SEM of three independent experiments performed in quadruplicate.