CB1 allosteric modulator Org27569 is an antagonist/inverse agonist of ERK1/2 signaling.

Allosteric modulation of cannabinoid type-1 receptors (CB1) is a novel means through which signaling bias may be exerted. Org27569 remains the most-characterized CB1 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 CB1 (hCB1) 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 hCB1 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 CB1 receptors. Org27569 reduced basal ERK phosphorylation in hCB1 HEK293 cells but not in untransfected cells, demonstrating that Org27569 acts via the CB1 receptor to produce this effect. Furthermore, inverse agonism was through inhibition of Gi/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 CB1-mediated ERK1/2 activation is Gi/o-dependent and that Org27569 is an inverse agonist of CB1 receptors. To our knowledge this is the first reported demonstration of inverse agonism of ERK signaling by Org27569.