br Introduction Activation of the

Introduction Activation of the lipid sensing receptor, GPR55, by lysophosphatidyl inositol (LPI) has been well documented, and implicated in endocannabinoid signaling [1]. Intracellular events resulting from GPR55 activation include; enhanced β-arrestin activity, calcium mobilization and ERK1/2 phosphorylation. Despite the efforts of many investigations, a variety of potent, selective, pharmacologically useful GPR55 ligands are not yet available. LPI remains the principle ligand that synthetic agonists are compared to, and synthetic antagonists are measured against. The role of N-arachidonoyl Tylosin phosphate (NAAA) as modulators of GPCR activity has been suggested as a consequence of NAAA involvement in a variety of physiologic processes [2], [3], [4], [5]. 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. Employing a triple hemagglutinin-tagged GPR55 stable Chinese Hamster cell line (HAGPR55/CHO), we investigated alterations in levels of intracellular calcium and ERK 1/2 (extracellular receptor kinase 1/2) phosphorylation induced by NAGly in the presence and absence of a previously identified and selective GPR55 antagonist, ML193 [10].
Materials and methods
Results
Discussion Findings from the current study are the first, to our knowledge, to report activation of GPR55 by the N-arachidonoyl amino acid, NAGly. Activation of GPR55 by NAGly was determined using two different signal transduction assays, calcium mobilization and MAPK activity. In the presence of the previously reported GPR55 antagonist, ML193 [10], increases in intracellular calcium and MAPK activity were abrogated. This indicates a specific GPR55 mediated event. Further support for a receptor mediated response is the finding that wild-type CHO cells do not demonstrate an increase in calcium mobilization nor MAPK activity in the presence of NAGly. Since NAGly/GPR55-mediated increases in calcium were observed in the presence and absence of extracellular calcium, we chose to ascertain the contribution by intracellular calcium stores as well as by TRP channels. The increase in intracellular calcium is not a consequence of calcium release from lysosomal stores as the vacuolar H+ ATPase inhibitor bafilomycin A1 [16] was without effect on the amplitude and duration of the NAGly-GPR55 mediated calcium mobilization. To parse out the influence of IP3 receptors and TRP channels, the inhibitors 2-APB and XeC were employed. Whereas 2-APB inhibits both IP3 receptors and TRP channels [17], [18], XeC is a specific inhibitor of IP3 receptors [19]. Our findings implicate IP3 receptors rather than TRP channels as NAGly does not activate these plasma membrane channels [20]. Additionally, abolishment of the NAGly/GPR55-mediated response was observed in calcium free buffer. Since CHO cells have been shown to be devoid of ryanodine receptors [21], and ryanodine was without effect on calcium mobilization, IP3 receptors are the likely candidate involved in NAGly/GPR55-mediated calcium mobilization. The results presented herein indicate that the NAAA, NAGly, is another endogenous ligand of GPR55. NAGly/GPR55-mediated cell signaling events include augmentation of ERK 1/2 phosphorylation and intracellular calcium. Both of these signal transduction pathways have been identified in lysophosphatidyl inositol-induced GPR55 activation [22], [23].
Introduction
GPR55 expression and signalling Since the cloning of GPR55 in 1999 and its identification as a novel G-protein coupled receptor (GPCR) highly expressed in the human brain (Sawzdargo et al., 1999), mRNA encoding GPR55 has been detected in various regions of the brain and spinal cord including the hippocampus, caudate and putamen (Sawzdargo et al., 1999), brain stem, cerebellum, frontal cortex, hypothalamus, striatum (Ryberg et al., 2007) and dorsal root ganglion (DRG) neurons (Lauckner et al., 2008). Confirmation that the mRNA is translated into protein has been demonstrated by immunohistochemistry in DRG neurons using an antibody whose specificity was determined by immunostaining of GPR55-expressing HEK293 cells but not native HEK293 cells (Lauckner et al., 2008). Intriguingly, in mouse striatum, hypothalamus and brain stem, expression levels of GPR55 mRNA are comparable to those of CB1, suggestive of an important role for GPR55 in these regions (Ryberg et al., 2007, Henstridge et al., 2011). Consistent with this, it has recently been reported that GPR55 plays a role in motor co-ordination (Wu et al., 2013).