Background: The endocannabinoid system functions through two well characterized receptor systems, the CB 1 and CB 2 receptors. Work by a number of groups in recent years has provided evidence that the system is more complicated and additional receptor types should exist to explain ligand activity in a number of physiological processes. Experimental approach: Cells transfected with the human cDNA for GPR55 were tested for their ability to bind and to mediate GTPgS binding by cannabinoid ligands. Using an antibody and peptide blocking approach, the nature of the Gprotein coupling was determined and further demonstrated by measuring activity of downstream signalling pathways.Key results: We demonstrate that GPR55 binds to and is activated by the cannabinoid ligand CP55940. In addition endocannabinoids including anandamide and virodhamine activate GTPgS binding via GPR55 with nM potencies. Ligands such as cannabidiol and abnormal cannabidiol which exhibit no CB 1 or CB 2 activity and are believed to function at a novel cannabinoid receptor, also showed activity at GPR55. GPR55 couples to Ga13 and can mediate activation of rhoA, cdc42 and rac1. Conclusions: These data suggest that GPR55 is a novel cannabinoid receptor, and its ligand profile with respect to CB 1 and CB 2 described here will permit delineation of its physiological function(s).

GPR55 is a G protein-coupled receptor recently shown to be activated by certain cannabinoids and by lysophosphatidylinositol (LPI). However, the physiological role of GPR55 remains unknown. Given the recent finding that the cannabinoid receptors CB 1 and CB2 affect bone metabolism, we examined the role of GPR55 in bone biology. GPR55 was expressed in human and mouse osteoclasts and osteoblasts; expression was higher in human osteoclasts than in macrophage progenitors. Although the GPR55 agonists O-1602 and LPI inhibited mouse osteoclast formation in vitro, these ligands stimulated mouse and human osteoclast polarization and resorption in vitro and caused activation of Rho and ERK1/2. These stimulatory effects on osteoclast function were attenuated in osteoclasts generated from GPR55 ؊/؊ macrophages and by the GPR55 antagonist cannabidiol (CBD). Furthermore, treatment of mice with this non-psychoactive constituent of cannabis significantly reduced bone resorption in vivo. Consistent with the ability of GPR55 to suppress osteoclast formation but stimulate osteoclast function, histomorphometric and microcomputed tomographic analysis of the long bones from male GPR55 ؊/؊ mice revealed increased numbers of morphologically inactive osteoclasts but a significant increase in the volume and thickness of trabecular bone and the presence of unresorbed cartilage. These data reveal a role of GPR55 in bone physiology by regulating osteoclast number and function. In addition, this study also brings to light an effect of both the endogenous ligand, LPI, on osteoclasts and of the cannabis constituent, CBD, on osteoclasts and bone turnover in vivo.A role for the endocannabinoid system (1) in the regulation of bone mass has been demonstrated recently, because mice lacking either of the cannabinoid receptors CB 1 or CB 2 have abnormal bone phenotypes. Furthermore, cannabinoid receptor agonists and inverse agonists reduce bone loss in mice following ovariectomy and have direct effects on both bone-resorbing cells (osteoclasts) and bone-forming cells (osteoblasts) in vitro (2, 3).In some systems such as the vasculature, there is considerable evidence for a role of non-CB 1 /non-CB 2 receptors in mediating some of the effects of certain cannabinoid ligands (for review, see ref. 4). Such non-CB 1 /non-CB 2 effects have been observed with a range of cannabinoid ligands including certain endocannabinoids and the phytocannabinoid-like compound O-1602; these effects are antagonized by the cannabis constituent cannabidiol (CBD) (4). Recently, the G protein-coupled receptor GPR55 has been shown to be activated by O-1602 (EC 50 ϭ 13 nM) and antagonized by CBD (IC 50 ϭ 445 nM) (5-8). In contrast, these compounds have low affinity (5-30 M) for CB 1 and CB 2 receptors (9, 10). GPR55 also is activated by the bioactive lipid, L-␣-lysophosphatidylinositol (LPI) (11,12).The physiological role(s) of GPR55 remains unknown. Given the apparent role of CB 1 and CB 2 in regulating bone mass, we examined whether GPR55 is expressed by osteoblasts and osteoc...

Cannabinoid ligands are implicated in many physiological processes and to date two receptors have been identified. However, a growing body of evidence exists that suggests the presence of additional receptors. Whilst cloning the previously described hCB1a, we have identified a novel variant that we call hCB1b. Characterising these two splice variants demonstrates that they have a unique pharmacological profile and that their RNA's are expressed at low levels in a variety of tissues.

N-arachidonoyl-L-serine (AraS) is a brain component structurally related to the endocannabinoid family. We investigated the neuroprotective effects of AraS following closed head injury induced by weight drop onto the exposed fronto-parietal skull and the mechanisms involved. A single injection of AraS following injury led to a significant improvement in functional outcome, and to reduced edema and lesion volume compared with vehicle. Specific antagonists to CB2 receptors, transient receptor potential vanilloid 1 (TRPV1) or large conductance calcium-activated potassium (BK) channels reversed these effects. Specific binding assays did not indicate binding of AraS to the GPR55 cannabinoid receptor. N-arachidonoyl-L-serine blocked the attenuation in phosphorylated extracellular-signal-regulated kinase 1/2 (ERK) levels and led to an increase in pAkt in both the ipsilateral and contralateral cortices. Increased levels of the prosurvival factor Bcl-xL were evident 24 hours after injury in AraS-treated mice, followed by a 30% reduction in caspase-3 activity, measured 3 days after injury. Treatment with a CB2 antagonist, but not with a CB1 antagonist, reversed this effect. Our results suggest that administration of AraS leads to neuroprotection via ERK and Akt phosphorylation and induction of their downstream antiapoptotic pathways. These protective effects are related mostly to indirect signaling via the CB2R and TRPV1 channels but not through CB1 or GPR55 receptors.

Edited by Lukas HuberKeywords: GPCR GPR91 Succinate PLCb PTX a b s t r a c t Succinate has been reported as the endogenous ligand for GPR91. In this study, succinate was confirmed to activate GPR91 resulting in both 3 0 -5 0 -cyclic adenosine monophosphate (cAMP) inhibition and inositol phosphate formation in a pertussis toxin (PTX)-sensitive manner. GPR91 agonist-mediated effects detected using dynamic mass redistribution (DMR) were inhibited with PTX, edelfosine and U73122 demonstrating the importance of not only the Ga i pathway but also PLCb. These results show that GPR91 when expressed in HEK293s cells couples exclusively through the Ga i pathway and acts through Ga i not only to inhibit cAMP production but also to increase intracellular Ca 2+ in an inositol phosphate dependent mechanism via PLCb activation.