Biased agonism at G protein–coupled receptors describes the phenomenon whereby some drugs can activate some downstream signaling activities to the relative exclusion of others. Descriptions of biased agonism focusing on the differential engagement of G proteins versus β-arrestins are commonly limited by the small response windows obtained in pathways that are not amplified or are less effectively coupled to receptor engagement, such as β-arrestin recruitment. At the μ-opioid receptor (MOR), G protein–biased ligands have been proposed to induce less constipation and respiratory depressant side effects than opioids commonly used to treat pain. However, it is unclear whether these improved safety profiles are due to a reduction in β-arrestin–mediated signaling or, alternatively, to their low intrinsic efficacy in all signaling pathways. Here, we systematically evaluated the most recent and promising MOR-biased ligands and assessed their pharmacological profile against existing opioid analgesics in assays not confounded by limited signal windows. We found that oliceridine, PZM21, and SR-17018 had low intrinsic efficacy. We also demonstrated a strong correlation between measures of efficacy for receptor activation, G protein coupling, and β-arrestin recruitment for all tested ligands. By measuring the antinociceptive and respiratory depressant effects of these ligands, we showed that the low intrinsic efficacy of opioid ligands can explain an improved side effect profile. Our results suggest a possible alternative mechanism underlying the improved therapeutic windows described for new opioid ligands, which should be taken into account for future descriptions of ligand action at this important therapeutic target.

BACKGROUND AND PURPOSEThe 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 APPROACHStudies 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 RESULTSORG27569 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 IMPLICATIONSContrary to previous studies characterizing allosteric modulators at CB1, 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. Abbreviations

CB receptor 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 signalling. CB receptors 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.

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Cannabinoid receptor 2 (CB2) is a GPCR highly expressed on the surface of cells of the immune system, supporting its role in immunomodulation. This study has investigated the trafficking properties of this receptor when stably expressed by HEK-293 cells. As previously reported, cell surface CB2 rapidly internalized upon exposure to agonist. Direct evidence of CB2 recycling was observed upon competitive removal of the stimulating agonist by inverse agonist. CB2 also underwent slow constitutive internalization when agonist was absent and was up-regulated in the presence of inverse agonist. Co-expression of CB2 and dominant negative Rab5 resulted in a significantly reduced capacity for receptors to internalize with no effect on recycling of the internalized receptors. Conversely, co-expression with dominant negative Rab11 did not alter the ability of CB2 to internalize but did impair their ability to return to the cell surface. Co-expression of wild-type, dominant negative or constitutively active Rab4 with CB2 did not alter basal surface expression, extent of internalization, or extent of recycling. These results suggest that Rab5 is involved in CB2 endocytosis and that internalized receptors are recycled via a Rab11 associated pathway rather than the rapid Rab4 associated pathway. This report provides the first comprehensive description of CB2 internalization and recycling to date.

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 (CB 1 ) and 2 (CB 2 ) 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 CB 1 and CB 2 agonists to stimulate cell surface translocation of human and bovine β-arrestin-1 and -2 was assessed. We find that some CB 1 ligands induce moderate β-arrestin-2 translocation in comparison with vasopressin V 2 receptor (a robust arrestin recruiter); however, CB 1 coupling with β-arrestin-1 and CB 2 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 CB 1 . Δ 9 -tetrahydrocannabinol was generally unable to elicit translocation of either arrestin subtype via CB 1 or CB 2 ; however, control experiments revealed translocation in cells not expressing CB 1 /CB 2 , which may assist in explaining some discrepancy with the literature. Overexpression of GRK2 had modest influence on CB 1 /CB 2 -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 CB 1 /CB 2 ligand-dependent arrestin coupling, our study raises a number of points for consideration in the design and interpretation of arrestin recruitment assays.

Pharmacological modulation of cannabinoid type 2 receptor (CB2R) holds promise for the treatment of numerous conditions, including inflammatory diseases, autoimmune disorders, pain, and cancer. Despite the significance of this receptor, researchers lack reliable tools to address questions concerning the expression and complex mechanism of CB2R signaling, especially in cell-type and tissue-dependent context. Herein, we report for the first time a versatile ligand platform for the modular design of a collection of highly specific CB2R fluorescent probes, used successfully across applications, species and cell types. These include flow cytometry of endogenously expressing cells, real-time confocal microscopy of mouse splenocytes and human macrophages, as well as FRET-based kinetic and equilibrium binding assays. High CB2R specificity was demonstrated by competition experiments in living cells expressing CB2R at native levels. The probes were effectively applied to FACS analysis of microglial cells derived from a mouse model relevant to Alzheimer's disease and to the detection of CB2R in human breast cancer cells.

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