The endogenous phospholipid l ‐α‐lyso‐phosphatidylinositol (LPI) was recently identified as a novel ligand for the orphan G protein‐coupled receptor 55 (GPR55). In this study we define the downstream signaling pathways activated by LPI in a human embryonic kidney (HEK) 293 cell line engineered to stably express recombinant human GPR55. We find that treatment with LPI induces marked GPR55 internalization and stimulates a sustained, oscillatory Ca ²⁺ release pathway, which is dependent on Gα13 and requires RhoA activation. We then establish that this signaling cascade leads to the efficient activation of NFAT (nu‐clear factor of activated T cells) family transcription factors and their nuclear translocation. Analysis of cannabinoid ligand activity at GPR55 revealed no clear effect of the endocannabinoids anandamide and 2‐arachidonoylglycerol;however, the classical CB ₁ antagonist AM251 evoked GPR55‐mediated Ca ²⁺ signaling. Thus, LPI is a potent and efficacious ligand at GPR55, which is likely to be a key plasma membrane mediator of LPI‐mediated signaling events and changes in gene expression.—Henstridge, C. M., Balenga, N. A. B., Ford, L. A., Ross, R. A., Waldhoer, M., Irving, A. J. The GPR55 ligand l ‐α‐lysophosphatidylinositol promotes RhoA‐dependent Ca ²⁺ signaling and NFAT activation. FASEB J. 23, 183‐193 (2009)

Background and purpose:  Although GPR55 is potently activated by the endogenous lysophospholipid, L‐α‐lysophosphatidylinositol (LPI), it is also thought to be sensitive to a number of cannabinoid ligands, including the prototypic CB1 receptor antagonists AM251 and SR141716A (Rimonabant^®). In this study we have used a range of functional assays to compare the pharmacological activity of selected cannabinoid ligands, AM251, AM281 and SR141716A with LPI in a HEK293 cell line engineered to stably express recombinant, human GPR55.Experimental approach:  We evaluated Ca²⁺ signalling, stimulation of extracellular signal regulated kinase (ERK1/2) mitogen activated kinase MAP‐kinases, induction of transcriptional regulators that are downstream of GPR55, including nuclear factor of activated T cells (NFAT), nuclear factor‐κB (NF‐κB) and cAMP response element binding protein (CREB), as well as receptor endocytosis. In addition, we assessed the suitability of a novel, label‐free assay for GPR55 ligands that involves optical measurement of dynamic mass redistribution following receptor activation.Key results:  GPR55 linked to a range of downstream signalling events and that the activity of GPR55 ligands was influenced by the functional assay employed, with differences in potency and efficacy observed.Conclusions and implications:  Our data help to resolve some of the issues surrounding the pharmacology of cannabinoid ligands at GPR55 and highlight some differences in effector coupling associated with distinct GPR55 ligands.

Although the endocannabinoid anandamide is frequently described to act predominantly in the cardiovascular system, the molecular mechanisms of its signaling remained unclear. In human endothelial cells, two receptors for anandamide were found, which were characterized as cannabinoid 1 receptor (CB₁R; CNR1) and G-protein-coupled receptor 55 (GPR55). Both receptors trigger distinct signaling pathways. It crucially depends on the activation status of integrins which signaling cascade becomes promoted upon anandamide stimulation. Under conditions of inactive integrins, anandamide initiates CB₁R-derived signaling, including G_i-protein-mediated activation of spleen tyrosine kinase (Syk), resulting in NFκB translocation. Furthermore, Syk inhibits phosphoinositide 3-kinase (PI3K) that represents a key protein in the transduction of GPR55-originated signaling. However, once integrins are clustered, CB₁R splits from integrins and, thus, Syk cannot further inhibit GPR55-triggered signaling resulting in intracellular Ca²⁺ mobilization from the endoplasmic reticulum (ER) via a PI3K-Bmx-phospholipase C (PLC) pathway and activation of nuclear factor of activated T-cells. Altogether, these data demonstrate that the physiological effects of anandamide on endothelial cells depend on the status of integrin clustering.

Emerging data suggest that off-target cannabinoid effects may be mediated via novel seven-transmembrane spanning/G protein-coupled receptors. Due to its cannabinoid sensitivity, the G protein-coupled receptor 55 (GPR55) was recently proposed as a candidate; however, GPR55 is phylogenetically distinct from the traditional cannabinoid receptors, and the conflicting pharmacology, signaling, and functional data have prevented its classification as a novel cannabinoid receptor. Indeed, the most consistent and potent agonist to date is the noncannabinoid lysophospholipid, lysophosphatidylinositol. Here we present new human GPR55 mRNA expression data, providing supportive evidence of GPR55 expression in a vast array of tissues and cell types. Moreover, we summarize major recent developments in GPR55 research and aim to update the reader in the rapidly expanding fields of GPR55 pharmacology, physiology, and pathology.

Background and Purpose: Heteromerization ofGPCRs is key to the integration of extracellular signals and the subsequent cell response via several mechanisms including heteromer‐selective ligand binding, trafficking and/or downstream signalling. As the lysophosphatidylinositolGPCR55 (GPR55) has been shown to affect the function of the cannabinoid receptor subtype 2 (CB₂receptor) in human neutrophils, we investigated the possible heteromerization ofCB₂receptorswith GPR55.Experimental Approach: The direct interaction of humanGPR55 andCB₂receptors heterologously expressed inHEK293 cells was assessed by co‐immunoprecipitation and bioluminescence resonance energy transfer assays. The effect of cross‐talk on signalling was investigated at downstream levels by label‐free real‐time methods (Epic dynamic mass redistribution andCellKey impedance assays),ERK1/2‐MAPKactivation and gene reporter assays.Key Results: GPR55 andCB₂receptors co‐localized on the surface ofHEK293 cells, co‐precipitated in membrane extracts and formed heteromers in livingHEK293 cells. Whereas heteromerization led to a reduction inGPR55‐mediated activation of transcription factors (nuclear factor of activatedT‐cells,NF‐κBandcAMPresponse element),ERK1/2‐MAPKactivation was potentiated in the presence ofCB₂receptors.CB₂receptor‐mediated signalling was also affected by co‐expression withGPR55. Label‐free assays confirmed cross‐talk between the two receptors.Conclusions and Implications: Heteromers, unique signalling units, form inHEK293 cells expressingGPR55 andCB₂receptors. The signalling by agonists of either receptor was governed (i) by the presence or absence of the partner receptors (with the consequent formation of heteromers) and (ii) by the activation state of the partner receptor.