Modulation of endocannabinoid-binding receptors in human neuroblastoma cells by tunicamycin

Cinzia Rapino, Annalisa Castellucci, Anna Rita Lizzi, Annalaura Sabatucci, Clotilde B. Angelucci, Daniel Tortolani, Gianna Rossi, Gabriele D’Andrea, Mauro Maccarrone

Research output: Contribution to journalArticle

Abstract

Endocannabinoid (eCB)-binding receptors can be modulated by several ligands and membrane environment, yet the effect of glycosylation remains to be assessed. In this study, we used human neuroblastoma SH-SY5Y cells to interrogate whether expression, cellular localization, and activity of eCB-binding receptors may depend on N-linked glycosylation. Following treatment with tunicamycin (a specific inhibitor of N-linked glycosylation) at the non-cytotoxic dose of 1 µg/mL, mRNA, protein levels and localization of eCB-binding receptors, as well as N-acetylglucosamine (GlcNAc) residues, were evaluated in SH-SY5Y cells by means of quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR), fluorescence-activated cell sorting (FACS), and confocal microscopy, respectively. In addition, the activity of type-1 and type-2 cannabinoid receptors (CB 1 and CB 2 ) was assessed by means of rapid binding assays. Significant changes in gene and protein expression were found upon tunicamycin treatment for CB 1 and CB 2 , as well as for GPR55 receptors, but not for transient receptor potential vanilloid 1 (TRPV1). Deglycosylation experiments with N-glycosidase-F and immunoblot of cell membranes derived from SH-SY5Y cells confirmed the presence of one glycosylated form in CB 1 (70 kDa), that was reduced by tunicamycin. Morphological studies demonstrated the co-localization of CB 1 with GlcNAc residues, and showed that tunicamycin reduced CB 1 membrane expression with a marked nuclear localization, as confirmed by immunoblotting. Cleavage of the carbohydrate side chain did not modify CB receptor binding affinity. Overall, these results support N-linked glycosylation as an unprecedented post-translational modification that may modulate eCB-binding receptors’ expression and localization, in particular for CB 1 .

Original languageEnglish
Article number1432
JournalMolecules
Volume24
Issue number7
DOIs
Publication statusPublished - Apr 11 2019

Fingerprint

Glycosylation
Tunicamycin
Endocannabinoids
Neuroblastoma
Modulation
modulation
cells
N-Acetylglucosamine Receptors
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Membranes
Cannabinoid Receptors
Acetylglucosamine
Confocal microscopy
Polymerase chain reaction
RNA-Directed DNA Polymerase
Cell membranes
Post Translational Protein Processing
Reverse Transcriptase Polymerase Chain Reaction
Sorting
Immunoblotting

Keywords

  • Endocannabinoid-binding receptors
  • N-acetylglucosamine
  • SH-SY5Y cells
  • Tunicamycin

ASJC Scopus subject areas

  • Analytical Chemistry
  • Chemistry (miscellaneous)
  • Molecular Medicine
  • Pharmaceutical Science
  • Drug Discovery
  • Physical and Theoretical Chemistry
  • Organic Chemistry

Cite this

Rapino, C., Castellucci, A., Lizzi, A. R., Sabatucci, A., Angelucci, C. B., Tortolani, D., ... Maccarrone, M. (2019). Modulation of endocannabinoid-binding receptors in human neuroblastoma cells by tunicamycin. Molecules, 24(7), [1432]. https://doi.org/10.3390/molecules24071432

Modulation of endocannabinoid-binding receptors in human neuroblastoma cells by tunicamycin. / Rapino, Cinzia; Castellucci, Annalisa; Lizzi, Anna Rita; Sabatucci, Annalaura; Angelucci, Clotilde B.; Tortolani, Daniel; Rossi, Gianna; D’Andrea, Gabriele; Maccarrone, Mauro.

In: Molecules, Vol. 24, No. 7, 1432, 11.04.2019.

Research output: Contribution to journalArticle

Rapino, C, Castellucci, A, Lizzi, AR, Sabatucci, A, Angelucci, CB, Tortolani, D, Rossi, G, D’Andrea, G & Maccarrone, M 2019, 'Modulation of endocannabinoid-binding receptors in human neuroblastoma cells by tunicamycin', Molecules, vol. 24, no. 7, 1432. https://doi.org/10.3390/molecules24071432
Rapino C, Castellucci A, Lizzi AR, Sabatucci A, Angelucci CB, Tortolani D et al. Modulation of endocannabinoid-binding receptors in human neuroblastoma cells by tunicamycin. Molecules. 2019 Apr 11;24(7). 1432. https://doi.org/10.3390/molecules24071432
Rapino, Cinzia ; Castellucci, Annalisa ; Lizzi, Anna Rita ; Sabatucci, Annalaura ; Angelucci, Clotilde B. ; Tortolani, Daniel ; Rossi, Gianna ; D’Andrea, Gabriele ; Maccarrone, Mauro. / Modulation of endocannabinoid-binding receptors in human neuroblastoma cells by tunicamycin. In: Molecules. 2019 ; Vol. 24, No. 7.
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