Modulation of Endocannabinoid-Binding Receptors in Human Neuroblastoma Cells by Tunicamycin

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 (CB1 and CB2) was assessed by means of rapid binding assays. Significant changes in gene and protein expression were found upon tunicamycin treatment for CB1 and CB2, 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 CB1 (70 kDa), that was reduced by tunicamycin. Morphological studies demonstrated the co-localization of CB1 with GlcNAc residues, and showed that tunicamycin reduced CB1 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 CB1.