Activation of GPR18 by cannabinoid compounds: a tale of biased agonism.
OBJECTIVE
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.


METHODS
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.


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 Δ(9)-tetrahydrocannabinol (Δ(9)-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 Δ(9)-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
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.
Center for Substance Abuse Research, Temple University School of Medicine, Philadelphia, PA, USA.
Linda
L
Console-Bram
Eugen
E
Brailoiu
Gabriela Cristina
GC
Brailoiu
Haleli
H
Sharir
Mary E
ME
Abood
D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology; D019390:Arrestins / Q000378:metabolism; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002185:Cannabidiol / Q000031:analogs & derivatives; D063386:Cannabinoid Receptor Agonists / Q000494:pharmacology*; D003412:Cricetulus; D003510:Cyclohexanes / Q000494:pharmacology; D013759:Dronabinol / Q000494:pharmacology; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000378:metabolism; D012118:Resorcinols / Q000494:pharmacology; D015398:Signal Transduction; D000071557:beta-Arrestins
C479832:4-(3-3,4-p-menthadien-(1,8)-yl)olivetol; D001095:Arachidonic Acids; D019390:Arrestins; D063386:Cannabinoid Receptor Agonists; D003510:Cyclohexanes; C107515:GPR18 protein, human; C465623:N-arachidonylglycine; D043562:Receptors, G-Protein-Coupled; D012118:Resorcinols; D000071557:beta-Arrestins; D002185:Cannabidiol; C568537:O-1602 compound; D013759:Dronabinol; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D002118:Calcium; D005998:Glycine