GPR3, GPR6, and GPR12 as novel molecular targets: their biological functions and interaction with cannabidiol.

The G protein-coupled receptors 3, 6, and 12 (GPR3, GPR6, and GPR12) comprise a family of closely related orphan receptors with no confirmed endogenous ligands. These receptors are constitutively active and capable of signaling through G protein-mediated and non-G protein-mediated mechanisms. These orphan receptors have previously been reported to play important roles in many normal physiological functions and to be involved in a variety of pathological conditions. Although they are orphans, GPR3, GPR6, and GPR12 are phylogenetically most closely related to the cannabinoid receptors. Using ß-arrestin2 recruitment and cAMP accumulation assays, we recently found that the nonpsychoactive phytocannabinoid cannabidiol (CBD) is an inverse agonist for GPR3, GPR6, and GPR12. This discovery highlights these orphan receptors as potential new molecular targets for CBD, provides novel mechanisms of action, and suggests new therapeutic uses of CBD for illnesses such as Alzheimer's disease, Parkinson's disease, cancer, and infertility. Furthermore, identification of CBD as a new inverse agonist for GPR3, GPR6, and GPR12 provides the initial chemical scaffolds upon which potent and efficacious agents acting on these receptors can be developed, with the goal of developing chemical tools for studying these orphan receptors and ultimately new therapeutic agents.

Novel inverse agonists for the orphan G protein-coupled receptor 6.

The orphan G protein-coupled receptor 6 (GPR6) displays unique promise as a therapeutic target for the treatment of neuropsychiatric disorders due to its high expression in the striatopallidal neurons of the basal ganglia. GPR6, along with closely related orphan receptors GPR3 and GPR12, are phylogenetically related to CB1 and CB2 cannabinoid receptors. In the current study, we performed concentration-response studies on the effects of three different classes of cannabinoids: endogenous, phyto-, and synthetic, on both GPR6-mediated cAMP accumulation and ß-arrestin2 recruitment. In addition, structure-activity relationship studies were conducted on cannabidiol (CBD), a recently discovered inverse agonist for GPR6. We have identified four additional cannabinoids, cannabidavarin (CBDV), WIN55212-2, SR141716A and SR144528, that exert inverse agonism on GPR6. Furthermore, we have discovered that these cannabinoids exhibit functional selectivity toward the ß-arrestin2 recruitment pathway. These novel, functionally selective inverse agonists for GPR6 can be used as research tools and potentially developed into therapeutic agents.

Cannabidiol, a novel inverse agonist for GPR12.

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.

Mass Spectrometry Analysis of Human CB2 Cannabinoid Receptor and Its Associated Proteins.

Studying the assemblies and dynamics of the complex formed by cannabinoid receptors and their associated proteins is important for understanding the molecular basis for the functions of these receptors. In the absence of the crystal structures of these macromolecular complexes, mass spectrometry is a sensitive technique that can be used to study cannabinoid receptors and their associated proteins. In this chapter, three aspects of the work are presented: (1) mass spectrometry analysis of the primary sequence of the human CB2 cannabinoid receptor after affinity chromatography purification of the receptor proteins, (2) functional proteomic analysis of CB2-associated proteins coimmunoprecipitated with CB2, and (3) chemical cross-linking and mass spectrometry analysis of CB2-G protein complex. The mass spectrometry approaches, in combination with mutagenesis and molecular modeling techniques, have been successfully used for studying the CB2-G protein complex. Similar approaches can also be applied for studying other G protein-coupled receptors in general.

GPR3 and GPR6, novel molecular targets for cannabidiol.

GPR3 and GPR6 are members of a family of constitutively active, Gs protein-coupled receptors. Previously, it has been reported that GPR3 is involved in Alzheimer's disease whereas GPR6 plays potential roles in Parkinson's disease. GPR3 and GPR6 are considered orphan receptors because there are no confirmed endogenous agonists for them. However, GPR3 and GPR6 are phylogenetically related to the cannabinoid receptors. In this study, the activities of endocannabinoids and phytocannabinoids were tested on GPR3 and GPR6 using a ß-arrestin2 recruitment assay. Among the variety of cannabinoids tested, cannabidiol (CBD), the major non-psychoactive component of marijuana, significantly reduced ß-arrestin2 recruitment to both GPR3 and GPR6. In addition, the inhibitory effects of CBD on ß-arrestin2 recruitment were concentration-dependent for both GPR3 and GPR6, with a higher potency for GPR6. These data show that CBD acts as an inverse agonist at both GPR3 and GPR6 receptors. These results demonstrate for the first time that both GPR3 and GPR6 are novel molecular targets for CBD. Our discovery that CBD acts as a novel inverse agonist on both GPR3 and GPR6 indicates that some of the potential therapeutic effects of CBD (e.g. treatment of Alzheimer's disease and Parkinson's disease) may be mediated through these important receptors.