Activation of GPR18 by cannabinoid compounds: A tale of biased agonism

Background and Purpose GPR18 is a candidate cannabinoid receptor, but its classification as such is controversial. The rationale of the study presented herein was to consider the effects of N-arachidonoyl glycine (NAGly) and cannabinoids via differential G-protein coupled pathways, in addition to β-arrestin signalling. Cellular localization of GPR18 receptors was also examined. Experimental Approach Calcium mobilization and ERK1/2 phosphorylation were quantified in a cell line stably expressing GPR18 (HEK293/GPR18 cells). In addition, using the DiscoveRx PathHunter® CHO-K1 GPR18 β-arrestin cell line, recruitment of β-arrestin was quantified. Key Results Concentration-dependent increases in intracellular calcium and ERK1/2 phosphorylation were observed in the presence of NAGly, abnormal cannabidiol (AbnCBD), O-1602, O-1918 and Δ⁹-tetrahydrocannabinol (Δ⁹-THC) in HEK293/GPR18 cells. The initial rise in intracellular calcium in the presence of NAGly, O1918 and THC was blocked by either Gα_q or Gα_i/o inhibition. The ERK1/2 phosphorylation was inhibited by Pertussis toxin and N-arachidonoyl-L-serine (NARAS). Recruitment of β-arrestin in the PathHunter CHO-K1 GPR18 cell line revealed a differential pattern of GPR18 activation; of all the ligands tested, only Δ⁹-THC produced a concentration-dependent response. The localization of GPR18 receptors within the HEK293/GPR18 cells is both intracellular, and on the plasma membrane. Conclusions and Implications These findings suggest that GPR18 activation involves several signal transduction pathways indicative of biased agonism, thereby providing a plausible explanation for the apparent discrepancies in GPR18 activation found in the literature. Additionally, the results presented herein provide further evidence for GPR18 as a candidate cannabinoid receptor.