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Effects of palmitoylation of Cys(415) in helix 8 of the CB(1) cannabinoid receptor on membrane localization and signalling.
Br J Pharmacol. 2012 Apr; 165(8):2635-51.BJ

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 [(35) S]GTPγS, 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.

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Authors+Show Affiliations

Oddi S
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.
Dainese E
No affiliation info available
Sandiford S
No affiliation info available
Fezza F
No affiliation info available
Lanuti M
No affiliation info available
Chiurchiù V
No affiliation info available
Totaro A
No affiliation info available
Catanzaro G
No affiliation info available
Barcaroli D
No affiliation info available
De Laurenzi V
No affiliation info available
Centonze D
No affiliation info available
Mukhopadhyay S
No affiliation info available
Selent J
No affiliation info available
Howlett AC
No affiliation info available
Maccarrone M
No affiliation info available

MeSH

AnimalsCell LineCell MembraneCysteineGreen Fluorescent ProteinsHEK293 CellsHumansLipoylationMutationProsencephalonRatsReceptor, Cannabinoid, CB1Signal Transduction

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

21895628

Citation

Oddi, Sergio, et al. "Effects of Palmitoylation of Cys(415) in Helix 8 of the CB(1) Cannabinoid Receptor On Membrane Localization and Signalling." British Journal of Pharmacology, vol. 165, no. 8, 2012, pp. 2635-51.
Oddi S, Dainese E, Sandiford S, et al. Effects of palmitoylation of Cys(415) in helix 8 of the CB(1) cannabinoid receptor on membrane localization and signalling. Br J Pharmacol. 2012;165(8):2635-51.
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), 2635-51. https://doi.org/10.1111/j.1476-5381.2011.01658.x
Oddi S, et al. Effects of Palmitoylation of Cys(415) in Helix 8 of the CB(1) Cannabinoid Receptor On Membrane Localization and Signalling. Br J Pharmacol. 2012;165(8):2635-51. PubMed PMID: 21895628.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Effects of palmitoylation of Cys(415) in helix 8 of the CB(1) cannabinoid receptor on membrane localization and signalling. AU - Oddi,Sergio, AU - Dainese,Enrico, AU - Sandiford,Simone, AU - Fezza,Filomena, AU - Lanuti,Mirko, AU - Chiurchiù,Valerio, AU - Totaro,Antonio, AU - Catanzaro,Giuseppina, AU - Barcaroli,Daniela, AU - De Laurenzi,Vincenzo, AU - Centonze,Diego, AU - Mukhopadhyay,Somnath, AU - Selent,Jana, AU - Howlett,Allyn C, AU - Maccarrone,Mauro, PY - 2011/9/8/entrez PY - 2011/9/8/pubmed PY - 2012/7/28/medline SP - 2635 EP - 51 JF - British journal of pharmacology JO - Br J Pharmacol VL - 165 IS - 8 N2 - 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 [(35) S]GTPγS, 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. SN - 1476-5381 UR - https://www.unboundmedicine.com/medline/citation/21895628/Effects_of_palmitoylation_of_Cys_415__in_helix_8_of_the_CB_1__cannabinoid_receptor_on_membrane_localization_and_signalling_ L2 - https://doi.org/10.1111/j.1476-5381.2011.01658.x DB - PRIME DP - Unbound Medicine ER -