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

Introduction: Allosteric modulation of cannabinoid type-1 receptors (CB[sub]1) is a novel means through which signaling bias may be exerted. Org27569 remains the most-characterized CB[sub]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's effects on cannabinoid agonists and ERK signaling. Materials and Methods: HEK293 cells transfected with the human cannabinoid type-1 receptor (hCB1) were treated with Org27569 alone or in combination with the endocannabinoid 2-arachidonoylglycerol (2-AG), the synthetic cannabinoid CP55,940, or the phytocannabinoid delta-9-tetrahydrocannabinol (THC) and ERK activation was measured by western blot. Overnight treatment with pertussis toxin (PTX) was used to determine the role of G[sub]i/o in Org27569's inverse agonist effects. HEK293 cells transfected with green fluorescent protein tagged hCB[sub]1 receptor were used to assess effects of Org27569 on CP55,940-induced receptor internalization. Subcellular fractionation was used to determine effects of Org27569 on ERK phosphorylation in both nuclear and cytosolic compartments. Results: We found that Org27569 is an antagonist of hCB[sub]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 following 5 min treatment. In hCB[sub]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. Org27569 reduced basal ERK phosphorylation in hCB[sub]1 HEK293 cells but not in untransfected cells following 20 min treatment. Overnight treatment with PTX abated this response. Following subcellular fractionation, Org27569 produced a significant decrease in ERK phosphorylation in the nuclear-enriched and cytosolic fractions. Conclusions: These data are consistent with previous studies demonstrating that CB[sub]1-mediated ERK1/2 activation is G[sub]i/o-dependent and that Org27569 is an inverse agonist of CB[sub]1 receptors. Abrogation of Org27569's ability to reduce basal ERK phosphorylation following treatment with PTX and lack of inverse agonist effects in untransfected HEK293 cells demonstrates that Org27569 acts via CB[sub]1-G[sub]i/o to produce this effect. To our knowledge,