title: Altered trafficking and unfolded protein response induction as a result of M3 muscarinic receptor impaired N-glycosylation.
abstract: The human M(3) 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 M(3) 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 [(3)H]-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.
authors.affiliation: Department d'Enginyeria Química, Centre de Biotecnologia Molecular, Universitat Politècnica de Catalunya, Edifici Gaia, Rbla San Nebridi s/n, Terrassa, 08222 Barcelona, Spain. wilber.romero@upc.edu
authors.forename: Wilber
authors.initials: W
authors.lastname: Romero-Fernandez
authors.forename: Dasiel O
authors.initials: DO
authors.lastname: Borroto-Escuela
authors.forename: Mileidys Perez
authors.initials: MP
authors.lastname: Alea
authors.forename: Yoelvis
authors.initials: Y
authors.lastname: Garcia-Mesa
authors.forename: Pere
authors.initials: P
authors.lastname: Garriga
mesh_terms: D000818:Animals; D017209:Apoptosis; D019556:COS Cells; D002470:Cell Survival; D002522:Chlorocebus aethiops; D006031:Glycosylation; D006801:Humans; D021381:Protein Transport; D058767:Protein Unfolding; D043587:Receptor, Muscarinic M3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D056811:Unfolded Protein Response / Q000502:physiology*
chemical_list: D043587:Receptor, Muscarinic M3