EFFECT OF PALMITOYLATION ON MEMBRANE LOCALISATION AND SIGNALLING OF CB1 CANNABINOID RECEPTOR

The CB1 cannabinoid receptor is regulated by its association with membrane microdomains like the lipidrafts. The purpose of this study was to investigate the role of CB1 palmitoylation by analyzing the functionalconsequences of site-specific mutation of cysteine 415, the likely palmitoylated residue at the end of helix 8,in terms of membrane association, raft targeting and signalling of the receptor.The palmitoylation state of the CB1 receptor was assessed in rat forebrain bydepalmitoylation/repalmitoylation experiments. Cysteine 415 was replaced with alanine by site directedmutagenesis. Green fluorescence protein chimeras of both wild-type and mutant receptors were transientlyexpressed and functionally characterised in human neuronal cells by means of confocal microscopy,cytofluorimetry and competitive binding assays. Confocal fluorescence recovery after photobleaching wasused to assess receptor membrane dynamics, whereas cAMP and co-immunoprecipitation assays wereemployed to assess signalling activity.Endogenous CB1 receptors were found to be palmitoylated in rat brain. Mutation of cysteine 415significantly reduced receptor recruitment at both the plasma membrane and the lipid rafts; it also increasedprotein diffusional mobility, whereas the immobile fraction remained unaltered. The same mutationmarkedly reduced the functional coupling of CB1 receptor with adenylyl cyclase, whereas depalmitoylationabrogated receptor association with G proteins.Taken together, we found that CB1 receptor is post-translationally modified by palmitoylation and thatmutation of cysteine 415 gives rise to a form of receptor that is functionally impaired in terms of membranetargeting and signalling.[...