SEARCH

SEARCH BY CITATION

Keywords:

  • drug receptor mechanisms;
  • CB1 cannabinoid receptor;
  • palmitoylation;
  • lipid rafts;
  • receptor trafficking;
  • signalling;
  • GFP-tagged receptors

BACKGROUND AND PURPOSE The 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 Cys415, the likely site of palmitoylation at the end of helix 8, in terms of membrane association, raft targeting and signalling.

EXPERIMENTAL APPROACH The palmitoylation state of CB1 receptors in rat forebrain was assessed by depalmitoylation/repalmitoylation experiments. Cys415 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 [35S]GTPγS, cAMP and co-immunoprecipitation assays.

KEY RESULTS Endogenous CB1 receptors in rat brain were palmitoylated. Mutation of Cys415 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 IMPLICATIONS CB1 receptors were post-translationally modified by palmitoylation. Mutation of Cys415 provides a receptor that is functionally impaired in terms of membrane targeting and signalling.

LINKED ARTICLES This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7