J. Neurochem. (2011) 116, 858–865. Abstract Endocannabinoid signaling modulates a variety of neuroinflammatory and neurodegenerative diseases, mainly through the activation of type‐1 and type‐2 (CB1R and CB2R) cannabinoid receptors. CB1R is negatively regulated by membrane cholesterol, while CB2R is unaffected. Here, we identified in the transmembrane helix 7 of human CBRs a consensus sequence already known in other proteins as cholesterol recognition/interaction amino acid sequence and consensus pattern. As this motif is different in the two CBR subtypes, we mutated lysine 402 of CB1R into glycine, to obtain a cholesterol recognition/interaction amino acid sequence and consensus similar to that of CB2R. Both mutated and wild‐type receptors were transiently expressed in human neuronal SH‐SY5Y cells, and their localization and functioning were investigated using biochemical assays and immunofluorescence labelling. We found a reduced propensity of the mutant CB1R to reside in cholesterol‐rich microdomains and, by means of fluorescence recovery after photobleaching analysis, we documented its loss of sensitivity to increased membrane cholesterol content. These results seem to uncover the existence of a new structural determinant in cannabinoid receptors, that is likely implicated in directing their interaction with cholesterol‐rich microdomains of cell membranes.

G protein-coupled receptor 55 (GPR55) is activated by endogenous, plant-derived and synthetic cannabinoids. Recent studies reported a broad tissue distribution for GPR55 and found prominent roles for this receptor in inflammatory pain, gut and bone physiology, as well as cancer. However, little is known about the expression and function of GPR55 in immune cells. To address this question, we performed a detailed characterization of GPR55 in different human innate and adaptive immune populations using polychromatic flow cytometry and we found that monocytes and NK cells expressed remarkable levels of this receptor compared to several cells of adaptive immunity. GPR55 activation by the specific agonist O-1602 boosted IL-12 and TNF-α production, and decreased endocytic activity, in LPS-activated monocytes. In addition, it increased CD69 activation marker expression, granzyme B and CD107a-dependent cytotoxicity and IFN-γ and TNF-α production in NK cells activated by both IL-2 and IL-12. These over-stimulatory effects of GPR55 were antagonized by its selective antagonist cannabidiol. Altogether, our data thus unveil a proinflammatory role for GPR55 in innate immunity that may be important for the design of new immune therapeutic strategies.

BACKGROUND AND PURPOSEThe CB1 cannabinoid receptor is regulated by its association with membrane microdomains such as lipid rafts. Here, we investigated the role of palmitoylation of the CB1 receptor by analysing the functional consequences of site-specific mutation of Cys 415 , the likely site of palmitoylation at the end of helix 8, in terms of membrane association, raft targeting and signalling. EXPERIMENTAL APPROACHThe palmitoylation state of CB1 receptors in rat forebrain was assessed by depalmitoylation/repalmitoylation experiments. Cys 415 was replaced with alanine by site-directed mutagenesis. Green fluorescence protein chimeras of both wild-type and mutant receptors were transiently expressed and functionally characterized in SH-SY5Y cells and HEK-293 cells by means of confocal microscopy, cytofluorimetry and competitive binding assays. Confocal fluorescence recovery after photobleaching was used to assess receptor membrane dynamics, whereas signalling activity was assessed by [ 35 S]GTPgS, cAMP and co-immunoprecipitation assays. KEY RESULTSEndogenous CB1 receptors in rat brain were palmitoylated. Mutation of Cys 415 prevented the palmitoylation of the receptor in transfected cells and reduced its recruitment to plasma membrane and lipid rafts; it also increased protein diffusional mobility. The same mutation markedly reduced the functional coupling of CB1 receptors with G-proteins and adenylyl cyclase, whereas depalmitoylation abolished receptor association with a specific subset of G-proteins. CONCLUSIONS AND IMPLICATIONSCB1 receptors were post-translationally modified by palmitoylation. Mutation of Cys 415 provides a receptor that is functionally impaired in terms of membrane targeting and signalling.