N-Glycan-mediated Quality Control in the Endoplasmic Reticulum Is Required for the Expression of Correctly Folded δ-Opioid Receptors at the Cell Surface

A great majority of G protein-coupled receptors are modified by N -glycosylation, but the functional significance of this modification for receptor folding and intracellular transport has remained elusive. Here we studied these phenomena by mutating the two N-terminal N -glycosylation sites (Asn 18 and Asn 33 ) of the human δ-opioid receptor, and expressing the mutants from the same chromosomal integration site in stably transfected inducible HEK293 cells. Both N -glycosylation sites were used, and their abolishment decreased the steady-state level of receptors at the cell surface. However, pulse-chase labeling, cell surface biotinylation, and immunofluorescence microscopy revealed that this was not because of intracellular accumulation. Instead, the non- N -glycosylated receptors were exported from the endoplasmic reticulum with enhanced kinetics. The results also revealed differences in the significance of the individual N -glycans, as the one attached to Asn 33 was found to be more important for endoplasmic reticulum retention of the receptor. The non- N -glycosylated receptors did not show gross functional impairment, but flow cytometry revealed that a fraction of them was incapable of ligand binding at the cell surface. In addition, the receptors that were devoid of N -glycans showed accelerated turnover and internalization and were targeted for lysosomal degradation. The results accentuate the importance of protein conformation-based screening before export from the endoplasmic reticulum, and demonstrate how the system is compromised when N -glycosylation is disrupted. We conclude that N -glycosylation of the δ-opioid receptor is needed to maintain the expression of fully functional and stable receptor molecules at the cell surface.