N-glycan-mediated quality control in the endoplasmic reticulum is required for the expression of correctly folded alfa-opioid receptors at the cell surface

Abstract

<div lang="en" class="abs"><p class="abs_header">Abstract</p><p>A great majority of G protein-coupled receptors are modified by <em>N</em>-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 <em>N</em>-glycosylation sites (Asn¹⁸ and Asn³³) of the human δ-opioid receptor, and expressing the mutants from the same chromosomal integration site in stably transfected inducible HEK293 cells. Both <em>N</em>-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-<em>N</em>-glycosylated receptors were exported from the endoplasmic reticulum with enhanced kinetics. The results also revealed differences in the significance of the individual <em>N</em>-glycans, as the one attached to Asn³³ was found to be more important for endoplasmic reticulum retention of the receptor. The non-<em>N</em>-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 <em>N</em>-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 <em>N</em>-glycosylation is disrupted. We conclude that <em>N</em>-glycosylation of the δ-opioid receptor is needed to maintain the expression of fully functional and stable receptor molecules at the cell surface.</p></div>