CB1 Allosteric Modulator Org27569 Is an Antagonist/Inverse Agonist of ERK1/2 Signaling

Allosteric modulation of cannabinoid type-1 receptors (CB 1 ) is a novel means through which signaling bias may be exerted. Org27569 remains the most-characterized CB 1 allosteric modulator yet there are conflicting reports regarding its effects on extracellular signal-regulated kinase (ERK) signaling. We conducted a systematic evaluation of Org27569 signaling through ERK. We have found that Org27569 is an antagonist of human CB 1 (hCB 1 ) mediated ERK signaling in HEK293 cells where it fully blocks CP55,940- but does not completely inhibit THC- and 2-AG-stimulated ERK1/2 activation. In hCB 1 HEK293 cells, CP55,940 (1 μM) treatment produced a significant increase in puncta at 20, 40, 60, and 120 min, consistent with receptor internalization. Org27569 (10 μM) co-treatment prevented internalization at each time point and alone had no effect. These data demonstrate that Org27569 can block the CP55,940- induced internalization of CB 1 receptors. Org27569 reduced basal ERK phosphorylation in hCB 1 HEK293 cells but not in untransfected cells, demonstrating that Org27569 acts via the CB 1 receptor to produce this effect. Furthermore, inverse agonism was through inhibition of G i/o as overnight treatment with pertussis toxin abated this response. Finally, to delineate Org27569's effects on ERK1/2 in subcellular compartments, subcellular fractionation was performed; Org27569 produced a significant decrease in ERK phosphorylation in the nuclear-enriched and cytosolic fractions. Altogether, these data are consistent with previous studies demonstrating that CB 1 -mediated ERK1/2 activation is G i/o -dependent and that Org27569 is an inverse agonist of CB 1 receptors. To our knowledge this is the first reported demonstration of inverse agonism of ERK signaling by Org27569.