N-glycan-dependent and -independent quality control of human δ opioid receptor N-terminal variants
Lackman, Jarkko J.; Markkanen, Piia M. H.; Hogue, Mireille; Bouvier, Michel; Petäjä-Repo, Ulla E. (2014-06-20)
Lackman, J. J., Markkanen, P. M. H., Hogue, M., Bouvier, M., & Petäjä-Repo, U. E. (2014). N-Glycan-dependent and -independent Quality Control of Human δ Opioid Receptor N-terminal Variants. Journal of Biological Chemistry, 289(25), 17830–17842. https://doi.org/10.1074/jbc.m114.566273
This research was originally published in the Journal of Biological Chemistry. Lackman, J. J., Markkanen, P. M. H., Hogue, M., Bouvier, M., & Petäjä-Repo, U. E.. N-Glycan-dependent and -independent Quality Control of Human δ Opioid Receptor N-terminal Variants. J. Biol. Chem. 2012; 289:17830-17842. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
https://rightsstatements.org/vocab/InC/1.0/
https://urn.fi/URN:NBN:fi-fe2019120545771
Tiivistelmä
Abstract
Quality control (QC) in the endoplasmic reticulum (ER) scrutinizes newly synthesized proteins and directs them either to ER export or ER-associated degradation (ERAD). Here, we demonstrate that the human δ-opioid receptor (hδOR) is subjected to ERQC in both N-glycan-dependent and -independent manners. This was shown by investigating the biosynthesis and trafficking of wild-type and non-N-glycosylated F27C variants in metabolic pulse-chase assays coupled with flow cytometry and cell surface biotinylation. Both QC mechanisms distinguished the minute one-amino acid difference between the variants, targeting a large fraction of hδOR-Cys²⁷ to ERAD. However, the N-glycan-independent QC was unable to compensate the N-glycan-dependent pathway, and some incompletely folded non-N-glycosylated hδOR-Cys²⁷ reached the cell surface in conformation incompatible with ligand binding. The turnover of receptors associating with the molecular chaperone calnexin (CNX) was significantly slower for the hδOR-Cys²⁷, pointing to an important role of CNX in the hδOR N-glycan-dependent QC. This was further supported by the fact that inhibiting the co-translational interaction of hδOR-Cys²⁷ precursors with CNX led to their ERAD. Opioid receptor pharmacological chaperones released the CNX-bound receptors to ER export and, furthermore, were able to rescue the Cys²⁷ variant from polyubiquitination and retrotranslocation to the cytosol whether carrying N-glycans or not. Taken together, the hδOR appears to rely primarily on the CNX-mediated N-glycan-dependent QC that has the capacity to assist in folding, whereas the N-glycan-independent mechanism constitutes an alternative, although less accurate, system for directing misfolded/incompletely folded receptors to ERAD, possibly in altered cellular conditions.
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