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

Show simple item record Finlay, David en Joseph, Wayne en Grimsey, Natasha en Glass, Michelle en 2016-11-02T02:56:22Z en 2016 en
dc.identifier.citation PeerJ, 2016, 4: e1835, pp. 1-29 en
dc.identifier.uri en
dc.description.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. en
dc.description.uri en
dc.publisher PeerJ en
dc.relation.ispartofseries PeerJ en
dc.rights Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher. Details obtained from en
dc.rights.uri en
dc.rights.uri en
dc.title GPR18 undergoes a high degree of constitutive trafficking but is unresponsive to N-Arachidonoyl Glycine en
dc.type Journal Article en
dc.identifier.doi 10.7717/peerj.1835 en
pubs.begin-page 1 en
pubs.volume 4 en
dc.description.version VoR – Version of Record en
dc.rights.holder Copyright: The Author(s) en
dc.identifier.pmid 27018161 en en
pubs.end-page 29 en
pubs.publication-status Published en
dc.rights.accessrights en
pubs.subtype Article en
pubs.elements-id 525560 en Medical and Health Sciences en Medical Sciences en Auckland Cancer Research en Pharmacology en
dc.identifier.eissn 2167-8359 en
pubs.number e1835 en
pubs.record-created-at-source-date 2016-11-02 en
pubs.dimensions-id 27018161 en

Files in this item

Find Full text

This item appears in the following Collection(s)

Show simple item record


Search ResearchSpace