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 journalArticlepeer-review

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

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

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