Cannabidiol, a novel inverse agonist for GPR12

https://doi.org/10.1016/j.bbrc.2017.09.001 Get rights and content

Highlights

  • GPR12 is a novel molecular target for CBD.

  • CBD acts as a new inverse agonist on GPR12.

  • Both free hydroxyl and the pentyl side chain are crucial for the effects of CBD.

  • Gs, but not Gi is involved in inverse agonistic activity of CBD on GPR12.

  • Potent and efficacious ligands for GPR12 may be developed based on CBD scaffold.

Abstract

GPR12 is a constitutively active, Gs 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 Gs protein and pertussis toxin, which blocks Gi protein, revealed that Gs, but not Gi 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.

Introduction

G protein-coupled receptor 12 (GPR12) belongs to the rhodopsin families of G protein-coupled receptors (GPCRs), which are important therapeutic targets [1], [2]. GPR12 was first cloned from a mouse cDNA library in 1993 and was originally named GPCR01 [3]. This was followed by cloning of human GPR12, along with the two related orphan receptors GPR3 and GPR6, from a human genomic DNA library [4]. In the brain, GPR12 is located in the medial habenular nucleus, and to a lesser extent in hippocampus, cerebral cortex, olfactory bulb, and striatum [3]. Peripherally GPR12 is found in the testis [3] and oocytes [5]. GPR12 is involved in several physiological processes. For example, GPR12 plays an important role in meiotic arrest in rat oocytes [5]. In addition, GPR12 participates in the process of neurite outgrowth and may contribute to brain development [6], [7].

GPR12 is constitutively active and couples to both Gs and Gi proteins [7], [8], [9]. However, GPR12 is an orphan receptor with no confirmed endogenous ligands. Initially, lysophospholipids sphingosine-1-phosphate (S1P) and sphingosylphosphorylcholine (SPC) were identified as agonists for GPR12 [7], [8]. However, a later study was unable to confirm either S1P or SPC as ligand for GPR12 [10].

In spite of being orphans, GPR12 share about 35% amino acid sequence identity in the transmembrane regions with the CB1 and CB2 cannabinoid receptors [11], [12]. Therefore, it is considered a “cannabinoid receptor-like orphan GPCR” [11]. In the present study, we first tested various classes of cannabinoids for their potential effects on GPR12 using a cAMP accumulation assay. After revealing GPR12 as a novel target for cannabidiol (CBD), we then studied the structure-activity relationship and the involvement of G proteins in the inverse agonistic effects of CBD on GPR12.

Section snippets

Materials

Dulbecco's Modified Eagles's Medium (DMEM), penicillin/streptomycin, l-glutamine, trypsin, and geneticin were purchased from Mediatech (Manassas, VA). Fetal bovine serum was obtained from Atlanta Biologicals (Lawrenceville, GA). Dichlorodimethylsilane for silanizing glass tubes was purchased from Sigma-Aldrich (St. Louis, Mo). 384-well, round bottom, low volume white plates were purchased from Grenier Bio One (Monroe, NC). The HTRF cAMP Hirange kits were purchased from CisBio International

Constitutive activity of GPR12

As shown in Fig. 1, HEK293 cells stably expressing GPR12 exhibited 8.5-fold cAMP levels compared to that of parental HEK293 cells.

Effects of various classes of cannabinoids on GPR12 mediated cAMP accumulation

To determine whether endocannabinoids are capable of altering cAMP accumulation mediated by GPR12, various concentrations of endocannabinoids 2-arachidonoylglycerol (2-AG), anandamide (AEA), virodhamine, and noladin ether (NE) were tested. As shown in Fig. 2A, neither AEA nor NE had any significant effects on cAMP accumulation at any of the concentrations tested.

Discussion

GPR12 is an orphan GPCR with no confirmed endogenous ligands. Since GPR12 is phylogenetically related to cannabinoid receptors, in this study we tested various classes of cannabinoids on GPR12.

First we sought to confirm the constitutive activity of GPR12. Our data showed that HEK293 cells stably expressing GPR12 exhibit 8.5-fold cAMP levels compared to parental HEK293 cells. This confirms the previous reports of GPR12 constitutively activating adenylate cyclase [8], [9].

We then tested several

Acknowledgments

This study was supported in part by the National Institutes of Health Grants DA11551 and EY13632 (to Z.H.S.), CA134283 (to David W. Hein) and University of Louisville Research Infrastructure Fund R5385 (to Z.H.S.).

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