Functional selectivity in CB ₂ cannabinoid receptor signaling and regulation: Implications for the therapeutic potential of CB ₂ ligands

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)-(+)-[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 β-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.