Functional Selectivity in CB₂ Cannabinoid Receptor Signaling and Regulation: Implications for the Therapeutic Potential of CB₂ Ligands

Abstract

Receptor internalization increases the flexibility and scope of G protein-coupled receptor (GPCR) signaling. CB 1 and CB 2 cannabinoid receptors undergo internalization after sustained exposure to agonists. However, it is not known whether different agonists internalize CB 2 to different extents. Because CB 2 is a promising therapeutic target, understanding its trafficking in response to different agonists is necessary for a complete understanding of its biology. Here we profile a number of cannabinoid receptor ligands and provide evidence for marked functional selectivity of cannabinoid receptor internalization. Classic, aminoalkylindole, bicyclic, cannabilactone, iminothiazole cannabinoid, and endocannabinoid ligands varied greatly in their effects on CB 1 and CB 2 trafficking. Our most striking finding was that (R)-(ϩ)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo-[1,2,3-d,e]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone (WIN55,212-2) (and other aminoalkylindoles) failed to promote CB 2 receptor internalization, whereas 5-(1,1-dimethylheptyl)-2-(5-hydroxy-2-(3-hydroxypropyl)cyclohexyl)phenol (CP55,940) robustly internalized CB 2 receptors. Furthermore, WIN55,212-2 competitively antagonized CP55,940-induced CB 2 internalization. Despite these differences in internalization, both compounds activated CB 2 receptors as measured by extracellular signal-regulated kinase 1/2 phosphorylation and recruitment of ␤-arrestin 2 to the membrane. In contrast, whereas CP55,940 inhibited voltage-gated calcium channels via CB 2 receptor activation, WIN55,212-2 was ineffective on its own and antagonized the effects of CP55,940. On the basis of the differences we found between these two ligands, we also tested the effects of other cannabinoids on these signaling pathways and found additional evidence for functional selectivity of CB 2 ligands. These novel data highlight that WIN55,212-2 and other cannabinoids show strong functional selectivity at CB 2 receptors and suggest that different classes of CB 2 ligands may produce diverse physiological effects, emphasizing that each class needs to be separately evaluated for therapeutic efficacy.

CB2Cannabinoid Receptors as a Therapeutic Target—What Does the Future Hold?

“…Although initial experiments failed to detect functional coupling of CB 2 receptors to G protein-gated inwardly rectifying potassium channels and calcium channels (Felder et al, 1995;Pertwee, 1997), other reports suggest that CB 2 receptors can modulate the activity of these channels (Ho et al, 1999;McAllister et al, 1999;Atwood et al, 2012b). Atwood et al (2012b) showed CB 2 receptor-mediated inhibition of voltagegated calcium channels in AtT20 cells.…”

“…Thus, the reason some of the earlier studies failed to find ion channel modulation can likely be attributed to the functional selectivity of the ligands used in the earliest studies (see below) (Atwood et al, 2012b).…”

“…Atwood et al (2012b) showed CB 2 receptor-mediated inhibition of voltagegated calcium channels in AtT20 cells.…”

“…Thus, internalization of a GPCR in response to a ligand can be considered a form of signaling. CB 2 receptors exhibit variable internalization in response to an agonist, with some agonists promoting marked internalization and others being inactive (Grimsey et al, 2011;Atwood et al, 2012b;Petrov et al, 2013).…”

Synthesis and spectroscopic characterization of emerging synthetic cannabinoids and cathinones

“…The main aim of these studies was to synthesize compounds with high specificity for the second cannabinoid receptor (CB2), transmitting antiinflammatory effects and pain release [35][36][37] . However, these efforts resulted in a large number of structurally different drug candidates that exhibited undesirable psychoactive effects [38][39][40] due to their binding affinity to the first cannabinoid receptor (CB1).…”