CB(1) Allosteric Modulator Org27569 Is an Antagonist/Inverse Agonist of ERK1/2 Signaling

Introduction: 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'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(i/o) in Org27569's inverse agonist effects. HEK293 cells transfected with green fluorescent protein tagged rat CB(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(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 rat CB(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(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(1)-mediated ERK1/2 activation is G(i/o)-dependent and that Org27569 is an inverse agonist of CB(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(1)-G(i/o) to produce this effect. To our knowledge, this is the first reported demonstration of inverse agonism of ERK signaling by Org27569.