Altered trafficking and unfolded protein response induction as a result of M3 muscarinic receptor impaired N-glycosylation

Wilber Romero-Fernandez*, Dasiel O. Borroto-Escuela, Mileidys Pérez-Alea, Yoelvis Garcia-Mesa, Pere Garriga

*Corresponding author for this work

Research output: Contribution to journalArticle

Abstract

The human M3 muscarinic acetylcholine receptor is present in both the central and peripheral nervous system, and it is involved in the pathophysiology of several neurodegenerative and autoimmune diseases. We suggested a possible N-glycosylation map for the M3 muscarinic receptor expressed in COS-7 cells. Here, we examined the role that N-linked glycans play in the folding and in the cell surface trafficking of this receptor. The five potential asparagine-linked glycosylation sites in the muscarinic receptor were mutated and transiently expressed in COS-7 cells. The elimination of N-glycan attachment sites did not affect the cellular expression levels of the receptor. However, proper receptor localization to the plasma membrane was affected as suggested by reduced [3H]-N-methylscopolamine binding. Confocal microscopy confirmed this observation and showed that the nonglycosylated receptor was primarily localized in the intracellular compartments. The mutant variant showed an increase in phosphorylation of the α-subunit of eukaryote initiation factor 2, and other well-known endoplasmic reticulum stress markers of the unfolded protein response pathway, which further supports the proposal of the improper intracellular accumulation of the nonglycosylated receptor. The receptor devoid of glycans showed more susceptibility to events that culminate in apoptosis reducing cell viability. Our findings suggest up-regulation of pro-apoptotic Bax protein, down-regulation of anti-apoptotic Bcl-2, and cleavage of caspase-3 effectors. Collectively, our data provide experimental evidence of the critical role that N-glycan chains play in determining muscarinic receptor distribution, localization, as well as cell integrity.

Original languageEnglish
Pages (from-to)1663-1672
Number of pages10
JournalGlycobiology
Volume21
Issue number12
Early online date28 Jul 2011
DOIs
Publication statusPublished - Dec 2011

Keywords

  • apoptosis
  • endoplasmic reticulum stress
  • muscarinic receptor
  • N-glycosylation
  • unfolded protein response

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    Romero-Fernandez, W., Borroto-Escuela, D. O., Pérez-Alea, M., Garcia-Mesa, Y., & Garriga, P. (2011). Altered trafficking and unfolded protein response induction as a result of M3 muscarinic receptor impaired N-glycosylation. Glycobiology, 21(12), 1663-1672. https://doi.org/10.1093/glycob/cwr105