Palmitoylation of cysteine 415 of CB1 receptor affects ligand-stimulated internalization and selective interaction with membrane cholesterol and caveolin 1

We previously demonstrated that CB1 receptor is palmitoylated at cysteine 415, and that such a post-translational modification affects its biological activity. To assess the molecular mechanisms responsible for modulation of CB1 receptor function by S-palmitoylation, in this study biochemical and morphological approaches were paralleled with computational analyses. Molecular dynamics simulations suggested that this acyl chain stabilizes helix 8 as well as the interaction of CB1 receptor with membrane cholesterol. In keeping with these in silico data, experimental results showed that the non-palmitoylated CB1 receptor was unable to interact efficaciously with caveolin 1, independently of its activation state. Moreover, in contrast with the wild-type receptor, the lack of S-palmitoylation in the helix 8 made the mutant CB1 receptor completely irresponsive to agonist-induced effects in terms of both lipid raft partitioning and receptor internalization. Overall, our results support the notion that palmitoylation of cysteine 415 modulates the conformational state of helix 8 and influences the interactions of CB1 receptor with cholesterol and caveolin 1, suggesting that the palmitoyl chain may serve as a functional interface for CB1 receptor localization and function. •Molecular dinamics simulations suggest a pivotal role of Cys415 palmitoylation in stabilizing H8.•Computational analyses show that specific cholesterol molecules are stabilized by the presence of the palmitoyl acyl chain.•C415A mutation impairs the receptor's ability to functionally interact with lipid rafts.•C415A mutation fully eliminates the agonist-promoted internalization of CB1 receptor.•The lack of palmitoyl chain alters the capability of CB1 to dynamically interact with caveolin 1.