N-Glycan Dependent and Independent Quality Control of Human Delta Opioid Receptor N-Terminal Variants [Signal Transduction]

May 5th, 2014 by Lackman, J. J., Markkanen, P. M. H., Hogue, M., Bouvier, M., Petaȷa–Repo, U. E.

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 delta-opioid receptor (hδOR) is subjected to ERQC in both N-glycan dependent and independent manner. This was shown by investigating the biosynthesis and trafficking of wild-type and non-N-glycosylated Phe27Cys 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.