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In this study, we investigated the role of CB 1 palmitoylation in modulating the functional interaction with G proteins both in the absence and presence of agonist binding. Our data show that the nonpalmitoylated CB 1 receptor significantly reduced its association with Gα i2 . The agonist stimulation induced a partial dissociation of Gα i2 proteins from the wild-type receptor, while on the C415A mutant the agonist binding was not able to induce a significant dissociation of Gα i2 from the receptor. The lack of palmitoyl chain seems to hamper the ability of the receptor to functionally interact with the Gα i2 and indicate that the palmitoyl chain is responsible for the functional transmission of the agonist-induced conformational change in the receptor of the G protein. These data were further corroborated by molecular dynamics simulations. Overall these results suggest that palmitoylation of the CB 1 receptor finely tunes its interaction with G proteins and serves as a targeting signal for its functional regulation. Of note, the possibility to reversibly modulate the palmitoylation of CB 1 receptor may offer a coordinated process of regulation and could open new therapeutic approaches., (© 2017 International Union of Biochemistry and Molecular Biology, Inc.)

We previously demonstrated that CB 1 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 CB 1 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 CB 1 receptor with membrane cholesterol. In keeping with these in silico data, experimental results showed that the non-palmitoylated CB 1 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 CB 1 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 CB 1 receptor with cholesterol and caveolin 1, suggesting that the palmitoyl chain may serve as a functional interface for CB 1 receptor localization and function., (Copyright © 2017 Elsevier B.V. All rights reserved.)