N-Glycan-dependent and -independent quality control of human δ opioid receptor N-terminal variants

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-Cys(27) 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-Cys(27) 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-Cys(27), 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-Cys(27) 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 Cys(27) 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.

Keywords: Calnexin; ER Quality Control; ER-associated Degradation; G Protein-coupled Receptor (GPCR); Genetic Polymorphism; Glycosylation; Opioid Receptor; Pharmacological Chaperone; Ubiquitination.