Modulation of Endocannabinoid-Binding Receptors in Human Neuroblastoma Cells by Tunicamycin.
Cinzia RapinoAnnalisa CastellucciAnna Rita LizziAnnalaura SabatucciClotilde B AngelucciDaniel TortolaniGianna RossiGabriele D'AndreaMauro MaccarronePublished in: Molecules (Basel, Switzerland) (2019)
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.
Keyphrases
- binding protein
- induced apoptosis
- cell cycle arrest
- dna binding
- poor prognosis
- endothelial cells
- oxidative stress
- endoplasmic reticulum stress
- cell death
- single cell
- spinal cord
- spinal cord injury
- cell proliferation
- high throughput
- signaling pathway
- genome wide
- heat shock protein
- pi k akt
- induced pluripotent stem cells
- subarachnoid hemorrhage
- replacement therapy
- pluripotent stem cells