Effects of palmitoylation of Cys 415 in helix 8 of the CB 1 cannabinoid receptor on membrane localization and signalling

Sergio Oddi, Enrico Dainese, Simone Sandiford, Filomena Fezza, Mirko Lanuti, Valerio Chiurchiù, Antonio Totaro, Giuseppina Catanzaro, Daniela Barcaroli, Vincenzo De Laurenzi, Diego Centonze, Somnath Mukhopadhyay, Jana Selent, Allyn C. Howlett, Mauro Maccarrone

Research output: Contribution to journalArticle

Abstract

BACKGROUND AND PURPOSE: The CB 1 cannabinoid receptor is regulated by its association with membrane microdomains such as lipid rafts. Here, we investigated the role of palmitoylation of the CB 1 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 APPROACH: The palmitoylation state of CB 1 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 [ 35S]GTPgS, cAMP and co-immunoprecipitation assays. KEY RESULTS: Endogenous CB 1 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 CB 1 receptors with G-proteins and adenylyl cyclase, whereas depalmitoylation abolished receptor association with a specific subset of G-proteins. CONCLUSIONS AND IMPLICATIONS: CB 1 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. 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.

Original languageEnglish
Pages (from-to)2635-2651
Number of pages17
JournalBritish Journal of Pharmacology
Volume165
Issue number8
DOIs
Publication statusPublished - Apr 2012

Keywords

  • CB cannabinoid receptor
  • Drug receptor mechanisms
  • GFP-tagged receptors
  • Lipid rafts
  • Palmitoylation
  • Receptor trafficking
  • Signalling

ASJC Scopus subject areas

  • Pharmacology

Fingerprint Dive into the research topics of 'Effects of palmitoylation of Cys 415 in helix 8 of the CB 1 cannabinoid receptor on membrane localization and signalling'. Together they form a unique fingerprint.

  • Cite this

    Oddi, S., Dainese, E., Sandiford, S., Fezza, F., Lanuti, M., Chiurchiù, V., Totaro, A., Catanzaro, G., Barcaroli, D., De Laurenzi, V., Centonze, D., Mukhopadhyay, S., Selent, J., Howlett, A. C., & Maccarrone, M. (2012). Effects of palmitoylation of Cys 415 in helix 8 of the CB 1 cannabinoid receptor on membrane localization and signalling. British Journal of Pharmacology, 165(8), 2635-2651. https://doi.org/10.1111/j.1476-5381.2011.01658.x