N-glycan-mediated quality control in the endoplasmic reticulum is required for the expression of correctly folded delta-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 delta-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 delta-opioid receptor is needed to maintain the expression of fully functional and stable receptor molecules at the cell surface.
Markkanen
Piia M H
PM
Department of Anatomy and Cell Biology, Institute of Biomedicine, University of Oulu, FI-90014, Oulu, Finland.
Petäjä-Repo
Ulla E
UE
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005786:Gene Expression Regulation; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008954:Models, Biological; D011134:Polysaccharides / Q000737:chemistry*; D017465:Receptors, Opioid, delta / Q000378:metabolism*
D008024:Ligands; D011134:Polysaccharides; D017465:Receptors, Opioid, delta