title: Effects of palmitoylation of Cys(415)  in helix 8 of the CB(1)  cannabinoid receptor on membrane localization and signalling.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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 [(35) S]GTPγS, cAMP and co-immunoprecipitation assays.
  
  
  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
  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.
  
  
  BACKGROUND
  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.
authors: Department of Biomedical Sciences, University of Teramo, Teramo, Italy European Center for Brain Research (CERC)/Santa Lucia Foundation I.R.C.C.S., Rome, Italy.
authors: Sergio
authors: S
authors: Oddi
authors: Enrico
authors: E
authors: Dainese
authors: Simone
authors: S
authors: Sandiford
authors: Filomena
authors: F
authors: Fezza
authors: Mirko
authors: M
authors: Lanuti
authors: Valerio
authors: V
authors: Chiurchiù
authors: Antonio
authors: A
authors: Totaro
authors: Giuseppina
authors: G
authors: Catanzaro
authors: Daniela
authors: D
authors: Barcaroli
authors: Vincenzo
authors: V
authors: De Laurenzi
authors: Diego
authors: D
authors: Centonze
authors: Somnath
authors: S
authors: Mukhopadhyay
authors: Jana
authors: J
authors: Selent
authors: Allyn C
authors: AC
authors: Howlett
authors: Mauro
authors: M
authors: Maccarrone
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D054878:Lipoylation; D009154:Mutation; D016548:Prosencephalon / Q000378:metabolism; D051381:Rats; D043884:Receptor, Cannabinoid, CB1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D043884:Receptor, Cannabinoid, CB1; D049452:Green Fluorescent Proteins; D003545:Cysteine