title: N-Arachidonyl glycine does not activate G protein-coupled receptor 18 signaling via canonical pathways.
abstract: Recent studies propose that N-arachidonyl glycine (NAGly), a carboxylic analogue of anandamide, is an endogenous ligand of the Gα(i/o) protein-coupled receptor 18 (GPR18). However, a high-throughput β-arrestin-based screen failed to detect activation of GPR18 by NAGly (Yin et al., 2009; JBC, 18:12328). To address this inconsistency, this study investigated GPR18 coupling in a native neuronal system with endogenous signaling pathways and effectors. GPR18 was heterologously expressed in rat sympathetic neurons, and the modulation of N-type (Ca(v)2.2) calcium channels was examined. Proper expression and trafficking of receptor were confirmed by the "rim-like" fluorescence of fluorescently tagged receptor and the positive staining of external hemagglutinin-tagged GPR18-expressing cells. Application of NAGly on GPR18-expressing neurons did not inhibit calcium currents but instead potentiated currents in a voltage-dependent manner, similar to what has previously been reported (Guo et al., 2008; J Neurophysiol, 100:1147). Other proposed agonists of GPR18, including anandamide and abnormal cannabidiol, also failed to induce inhibition of calcium currents. Mutants of GPR18, designed to constitutively activate receptors, did not tonically inhibit calcium currents, indicating a lack of GPR18 activation or coupling to endogenous G proteins. Other downstream effectors of Gα(i/o)-coupled receptors, G protein-coupled inwardly rectifying potassium channels and adenylate cyclase, were not modulated by GPR18 signaling. Furthermore, GPR18 did not couple to other G proteins tested: Gα(s), Gα(z), and Gα(15). These results suggest NAGly is not an agonist for GPR18 or that GPR18 signaling involves noncanonical pathways not examined in these studies.
authors.affiliation: Laboratory of Molecular Physiology, National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism, 5625 Fishers Lane, Room TS-11, MSC 9411, Bethesda, MD 20892, USA. sikeda@mail.nih.gov
authors.forename: Van B
authors.initials: VB
authors.lastname: Lu
authors.forename: Henry L
authors.initials: HL
authors.lastname: Puhl
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Ikeda
mesh_terms: D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology*; D020864:Calcium Channels, N-Type / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism; D005998:Glycine / Q000031:analogs & derivatives* / Q000494:pharmacology; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D009154:Mutation; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D051381:Rats; D017208:Rats, Wistar; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D017783:Superior Cervical Ganglion / Q000166:cytology
chemical_list: D001095:Arachidonic Acids; C503279:Cacna1b protein, rat; D020864:Calcium Channels, N-Type; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; C107515:GPR18 protein, human; D044385:GTP-Binding Protein alpha Subunits; C465623:N-arachidonylglycine; D043562:Receptors, G-Protein-Coupled; D000242:Cyclic AMP; D005998:Glycine