Background and purpose: Although GPR55 is potently activated by the endogenous lysophospholipid, L-alysophosphatidylinositol (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 Ca 2+ 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-kB (NF-kB) 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. (2010) AM281, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide; CREB, cAMP response element binding protein; ERK1/2, extracellular signal regulated kinase 1/2; G13, Ga13 protein; GFP, green fluorescent protein; GPR55, G protein-coupled receptor 55; GPR55-HEK293, stable HEK293 cells expressing 3xHA-GPR55; HA, haemaglutinin; HBS, HEPES-buffered saline; LPI, L-alysophosphatidylinositol; MAP, mitogen activated kinase; NFAT, nuclear factor of activated T British Journal of Pharmacology

The directional migration of neutrophils towards inflammatory mediators, such as chemokines and cannabinoids, occurs via the activation of seven transmembrane G protein coupled receptors (7TM/GPCRs) and is a highly organized process. A crucial role for controlling neutrophil migration has been ascribed to the cannabinoid CB 2 receptor (CB 2 R), but additional modulatory sites distinct from CB 2 R have recently been suggested to impact CB 2 R-mediated effector functions in neutrophils. Here, we provide evidence that the recently de-orphanized 7TM/GPCR GPR55 potently modulates CB 2 R-mediated responses. We show that GPR55 is expressed in human blood neutrophils and its activation augments the migratory response towards the CB 2 R agonist 2-arachidonoylglycerol (2-AG), while inhibiting neutrophil degranulation and reactive oxygen species (ROS) production. Using HEK293 and HL60 cell lines, along with primary neutrophils, we show that GPR55 and CB 2 R interfere with each other's signaling pathways at the level of small GTPases, such as Rac2 and Cdc42. This ultimately leads to cellular polarization and efficient migration as well as abrogation of degranulation and ROS formation in neutrophils. Therefore, GPR55 limits the tissueinjuring inflammatory responses mediated by CB 2 R, while it synergizes with CB 2 R in recruiting neutrophils to sites of inflammation.

Background: Lung eosinophilia is a hallmark of asthma, and eosinophils are believed to play a crucial role in the pathogenesis of allergic inflammatory diseases. Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are produced in high amounts in the gastrointestinal tract by commensal bacteria and can be absorbed into the bloodstream. Although there is recent evidence that SCFAs are beneficial in allergic asthma models, the effect on eosinophils has remained elusive. Objective: The role of SCFAs was investigated in human eosinophil function and a mouse model of allergic asthma. Methods: Eosinophils were purified from self-reported allergic or healthy donors. Migration, adhesion to the endothelium, and

BACKGROUND AND PURPOSEHeteromerization of GPCRs 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 lysophosphatidylinositol GPCR 55 (GPR55) has been shown to affect the function of the cannabinoid receptor subtype 2 (CB2 receptor) in human neutrophils, we investigated the possible heteromerization of CB2 receptors with GPR55. EXPERIMENTAL APPROACHThe direct interaction of human GPR55 and CB2 receptors heterologously expressed in HEK293 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 and CellKey impedance assays), ERK1/2-MAPK activation and gene reporter assays. KEY RESULTSGPR55 and CB2 receptors co-localized on the surface of HEK293 cells, co-precipitated in membrane extracts and formed heteromers in living HEK293 cells. Whereas heteromerization led to a reduction in GPR55-mediated activation of transcription factors (nuclear factor of activated T-cells, NF-κB and cAMP response element), ERK1/2-MAPK activation was potentiated in the presence of CB2 receptors. CB2 receptor-mediated signalling was also affected by co-expression with GPR55. Label-free assays confirmed cross-talk between the two receptors.

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