The cannabinoid CB₂receptor (CB₂R) represents a promising therapeutic target for various forms of tissue injury and inflammatory diseases. Although numerous compounds have been developed and widely used to target CB₂R, their selectivity, molecular mode of action and pharmacokinetic properties have been poorly characterized. Here we report the most extensive characterization of the molecular pharmacology of the most widely used CB₂R ligands to date. In a collaborative effort between multiple academic and industry laboratories, we identify marked differences in the ability of certain agonists to activate distinct signalling pathways and to cause off-target effects. We reach a consensus that HU910, HU308 and JWH133 are the recommended selective CB₂R agonists to study the role of CB₂R in biological and disease processes. We believe that our unique approach would be highly suitable for the characterization of other therapeutic targets in drug discovery research.

Background and Purpose: CB₁receptor signalling is canonically mediated through inhibitory Gα_iproteins, but occurs through other G proteins under some circumstances, Gα_sbeing the most characterized secondary pathway. Determinants of this signalling switch identified to date include Gα_iblockade, CB₁/D₂receptor co‐stimulation, CB₁agonist class and cell background. Hence, we examined the effects of receptor number and different ligands on CB₁receptor signalling.Experimental Approach: CB₁receptors were expressed in HEK cells at different levels, and signalling characterized for cAMP by real‐time BRET biosensor –CAMYEL – and for phospho‐ERK by AlphaScreen. Homogenate and whole cell radioligand binding assays were performed to characterize AM6544, a novel irreversible CB₁receptor antagonist.Key Results: In HEK cells expressing high levels of CB₁receptors, agonist treatment stimulated cAMP, a response not known to be mediated by receptor number. Δ⁹‐THC and BAY59‐3074 increased cAMP only in high‐expressing cells pretreated with pertussis toxin, and agonists demonstrated more diverse signalling profiles in the stimulatory pathway than the canonical inhibitory pathway. Pharmacological CB₁receptor knockdown and Gα_i1 supplementation restored canonical Gα_isignalling to high‐expressing cells. Constitutive signalling in both low‐ and high‐expressing cells was Gα_i‐mediated.Conclusion and Implications: CB₁receptor coupling to opposing G proteins is determined by both receptor and G protein expression levels, which underpins a mechanism for non‐canonical signalling in a fashion consistent with Gα_ssignalling. CB₁receptors mediate opposite consequences in endpoints such as tumour viability depending on expression levels; our results may help to explain such effects at the level of G protein coupling.

Arrestin translocation and signaling have come to the fore of the G protein-coupled receptor molecular pharmacology field. Some receptor–arrestin interactions are relatively well understood and considered responsible for specific therapeutic or adverse outcomes. Coupling of arrestins with cannabinoid receptors 1 (CB₁) and 2 (CB₂) has been reported, though the majority of studies have not systematically characterized the differential ligand dependence of this activity. In addition, many prior studies have utilized bovine (rather than human) arrestins, and the most widely applied assays require reporter-tagged receptors, which prevent meaningful comparison between receptor types. We have employed a bioluminescence resonance energy transfer (BRET) method that does not require the use of tagged receptors and thereby allows comparisons of arrestin translocation between receptor types, as well as with cells lacking the receptor of interest – an important control. The ability of a selection of CB₁ and CB₂ agonists to stimulate cell surface translocation of human and bovine β-arrestin-1 and -2 was assessed. We find that some CB₁ ligands induce moderate β-arrestin-2 translocation in comparison with vasopressin V₂ receptor (a robust arrestin recruiter); however, CB₁ coupling with β-arrestin-1 and CB₂ with either arrestin elicited low relative efficacies. A range of efficacies between ligands was evident for both receptors and arrestins. Endocannabinoid 2-arachidonoylglycerol stood out as a high efficacy ligand for translocation of β-arrestin-2 via CB₁. Δ⁹-tetrahydrocannabinol was generally unable to elicit translocation of either arrestin subtype via CB₁ or CB₂; however, control experiments revealed translocation in cells not expressing CB₁/CB₂, which may assist in explaining some discrepancy with the literature. Overexpression of GRK2 had modest influence on CB₁/CB₂-induced arrestin translocation. Results with bovine and human arrestins were largely analogous, but a few instances of inconsistent rank order potencies/efficacies between bovine and human arrestins raise the possibility that subtle differences in receptor conformation stabilized by these ligands manifest in disparate affinities for the two arrestin species, with important potential consequences for interpretation in ligand bias studies. As well as contributing important information regarding CB₁/CB₂ ligand-dependent arrestin coupling, our study raises a number of points for consideration in the design and interpretation of arrestin recruitment assays.

Experimental pharmacologists rely on the application of models to describe biological observations in order to learn about a drug's effective concentration, the strength with which it binds its target and drives a response (at either molecular or system level), and the nature of more complex drug actions (allosterism/functional selectivity). Models in current use build upon decades of basic principles, going back to the beginning of the last century. Yet often, researchers are only partially familiar with these underlying principles, creating the potential for confusion due to failure to recognise the underpinning assumptions of the models that are used. Here, we describe the history of receptor theory as it underpins receptor stimulus–response models in use today, emphasising particularly attributes and models relevant to GPCRs—and point to some current aims of model development.

The orphan receptor GPR18 has become a research target following the discovery of a putative endogenous agonist, N-arachidonoyl glycine (NAGly). Chemical similarity between NAGly and the endocannabinoid anandamide suggested the hypothesis that GPR18 is a third cannabinoid receptor. GPR18-mediated cellular signalling through inhibition of cyclic adenosine monophosphate (cAMP) and phosphorylation of extracellular signal-regulated kinase (ERK), in addition to physiological consequences such as regulation of cellular migration and proliferation/apoptosis have been described in response to both NAGly and anandamide. However, discordant findings have also been reported. Here we sought to describe the functional consequences of GPR18 activation in heterologously-expressing HEK cells. GPR18 expression was predominantly intracellular in stably transfected cell lines, but moderate cell surface expression could be achieved in transiently transfected cells which also had higher overall expression. Assays were employed to characterise the ability of NAGly or anandamide to inhibit cAMP or induce ERK phosphorylation through GPR18, or induce receptor trafficking. Positive control experiments, which utilised cells expressing hCB1 receptors (hCB1R), were performed to validate assay design and performance. While these functional pathways in GPR18-expressing cells were not modified on treatment with a panel of putative GPR18 ligands, a constitutive phenotype was discovered for this receptor. Our data reveal that GPR18 undergoes rapid constitutive receptor membrane trafficking—several-fold faster than hCB1R, a highly constitutively active receptor. To enhance the likelihood of detecting agonist-mediated receptor signalling responses, we increased GPR18 protein expression (by tagging with a preprolactin signal sequence) and generated a putative constitutively inactive receptor by mutating the hGPR18 gene at amino acid site 108 (alanine to asparagine). This A108N mutant did cause an increase in surface receptor expression (which may argue for reduced constitutive activity), but no ligand-mediated effects were detected. Two glioblastoma multiforme cell lines (which endogenously express GPR18) were assayed for NAGly-induced pERK phosphorylation, with negative results. Despite a lack of ligand-mediated responses in all assays, the constitutive trafficking of GPR18 remains an interesting facet of receptor function and will have consequences for understanding the role of GPR18 in physiology.