GPR18 undergoes a high degree of constitutive trafficking but is unresponsive to N-Arachidonoyl Glycine

Abstract

The orphan receptor GPR18 has become a research target following the discovery of a putative endogenous agonist, N-arachidonoyl glycine (NAGly). Chemical similarity between NAGly and the endocannabinoid anandamide suggested the hypothesis that GPR18 is a third cannabinoid receptor. GPR18-mediated cellular signalling through inhibition of cyclic adenosine monophosphate (cAMP) and phosphorylation of extracellular signal-regulated kinase (ERK), in addition to physiological consequences such as regulation of cellular migration and proliferation/apoptosis have been described in response to both NAGly and anandamide. However, discordant findings have also been reported. Here we sought to describe the functional consequences of GPR18 activation in heterologously-expressing HEK cells. GPR18 expression was predominantly intracellular in stably transfected cell lines, but moderate cell surface expression could be achieved in transiently transfected cells which also had higher overall expression. Assays were employed to characterise the ability of NAGly or anandamide to inhibit cAMP or induce ERK phosphorylation through GPR18, or induce receptor trafficking. Positive control experiments, which utilised cells expressing hCB1 receptors (hCB1R), were performed to validate assay design and performance. While these functional pathways in GPR18-expressing cells were not modified on treatment with a panel of putative GPR18 ligands, a constitutive phenotype was discovered for this receptor. Our data reveal that GPR18 undergoes rapid constitutive receptor membrane trafficking—several-fold faster than hCB1R, a highly constitutively active receptor. To enhance the likelihood of detecting agonist-mediated receptor signalling responses, we increased GPR18 protein expression (by tagging with a preprolactin signal sequence) and generated a putative constitutively inactive receptor by mutating the hGPR18 gene at amino acid site 108 (alanine to asparagine). This A108N mutant did cause an increase in surface receptor expression (which may argue for reduced constitutive activity), but no ligand-mediated effects were detected. Two glioblastoma multiforme cell lines (which endogenously express GPR18) were assayed for NAGly-induced pERK phosphorylation, with negative results. Despite a lack of ligand-mediated responses in all assays, the constitutive trafficking of GPR18 remains an interesting facet of receptor function and will have consequences for understanding the role of GPR18 in physiology.

N-arachidonoyl glycine, another endogenous agonist of GPR55

“…Our group has previously reported activation of GPR18 by NAGly (N-arachidonoyl glycine), supporting studies by McHugh et al [6, 7], yet in conflict with the reports of others [8, 9]. Since both GPR18 and GPR55 have been implicated in endocannabinoid signaling, and are regarded as candidate cannabinoid receptors, we examined the effect of NAGly on GPR55-mediated intracellular events.…”

Model‐free and kinetic modelling approaches for characterising non‐equilibrium pharmacological pathway activity: Internalisation of cannabinoid CB1 receptors

“…Image analysis in MetaXpress® was performed by the application of an analysis method first reported by Grimsey et al (), with modifications reported in Finlay, Joseph, Grimsey, and Glass (). This analysis entailed calculating fluorescence intensity per cell above background (based on a user‐defined intensity threshold) and is reported in arbitrary units for total grey value per cell.…”

The Effect of Chronic Activation of the Novel Endocannabinoid Receptor GPR18 on Myocardial Function and Blood Pressure in Conscious Rats

“…While GPR18 is considered the abn-cbd receptor 5, 7 , failure of abn-cbd or NAGly to produce GPR18-dependent biochemical responses in some studies 8, 9 fueled a debate on whether GPR18 is indeed the abn-cbd receptor. It is possible that the biochemical responses are tissue/cell type specific due to differences in the constitutive activity of GPR18 9 .…”

“…It is possible that the biochemical responses are tissue/cell type specific due to differences in the constitutive activity of GPR18 9 . Importantly, many studies supported the role of GPR18 in regulating the cardiovascular function because NAGly caused relaxation of isolated rat mesenteric arteries 10 and abn-cbd induced vasorelaxant and hypotensive responses that were abrogated by the GPR18 antagonist O-1918 2–4, 11 , and are not dependent on CB 1 /CB 2 receptors 2, 3, 12 .…”