Low intrinsic efficacy can explain the reduced side effects of apparently biased μ-opioid receptor agonists.

Background and Purpose: The cannabinoid receptor type 1 (CB₁) has an allosteric binding site. The drugs ORG27569 {5‐chloro‐3‐ethyl‐N‐[2‐[4‐(1‐piperidinyl)phenyl]ethyl]‐1H‐indole‐2‐carboxamide} and PSNCBAM‐1 {1‐(4‐chlorophenyl)‐3‐[3‐(6‐pyrrolidin‐1‐ylpyridin‐2‐yl)phenyl]urea} have been extensively characterized with regard to their effects on signalling of the orthosteric ligand CP55,940 {(−)‐cis‐3‐[2‐hydroxy‐4‐(1,1‐dimethylheptyl)phenyl]‐trans‐4‐(3‐hydroxypropyl)cyclohexanol}, and studies have suggested that these allosteric modulators increase binding affinity but act as non‐competitive antagonists in functional assays. To gain a deeper understanding of allosteric modulation of CB₁, we examined real‐time signalling and trafficking responses of the receptor in the presence of allosteric modulators.Experimental Approach: Studies of CB₁ signalling were carried out in HEK 293 and AtT20 cells expressing haemagglutinin‐tagged human and rat CB₁. We measured real‐time accumulation of cAMP, activation and desensitization of potassium channel‐mediated cellular hyperpolarization and CB₁ internalization.Key Results: ORG27569 and PSNCBAM‐1 produce a complex, concentration and time‐dependent modulation of agonist‐mediated regulation of cAMP levels, as well as an increased rate of desensitization of CB₁‐mediated cellular hyperpolarization and a decrease in agonist‐induced receptor internalization.Conclusions and Implications: Contrary to previous studies characterizing allosteric modulators at CB_1, this study suggests that the mechanism of action is not non‐competitive antagonism of signalling, but rather that enhanced binding results in an increased rate of receptor desensitization and reduced internalization, which results in time‐dependent modulation of cAMP signalling. The observed effect of the allosteric modulators is therefore dependent on the time frame over which the signalling response occurs. This finding may have important consequences for the potential therapeutic application of these compounds.

Cannabinoid receptor 2 (CB₂) is a promising therapeutic target for immunological modulation. There is, however, a deficit of knowledge regarding CB₂ signaling and function in human primary immunocompetent cells. We applied an experimental paradigm which closely models the in situ state of human primary leukocytes (PBMC; peripheral blood mononuclear cells) to characterize activation of a number of signaling pathways in response to a CB₂-selective ligand (HU308). We observed a “lag” phase of unchanged cAMP concentration prior to development of classically expected Gα_i-mediated inhibition of cAMP synthesis. Application of G protein inhibitors revealed that this apparent lag was a result of counteraction of Gα_i effects by concurrent Gα_s activation. Monitoring downstream signaling events showed that activation of p38 was mediated by Gα_i, whereas ERK1/2 and Akt phosphorylation were mediated by Gα_i-coupled βγ. Activation of CREB integrated multiple components; Gα_s and βγ mediated ∼85% of the response, while ∼15% was attributed to Gα_i. Responses to HU308 had an important functional outcome—secretion of interleukins 6 (IL-6) and 10 (IL-10). IL-2, IL-4, IL-12, IL-13, IL-17A, MIP-1α, and TNF-α were unaffected. IL-6/IL-10 induction had a similar G protein coupling profile to CREB activation. All response potencies were consistent with that expected for HU308 acting via CB₂. Additionally, signaling and functional effects were completely blocked by a CB₂-selective inverse agonist, giving additional evidence for CB₂ involvement. This work expands the current paradigm regarding cannabinoid immunomodulation and reinforces the potential utility of CB₂ ligands as immunomodulatory therapeutics.

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.