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J. Biol. Chem., Vol. 283, Issue 43, 29086-29098, October 24, 2008

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

From the Department of Anatomy and Cell Biology, Institute of Biomedicine, University of Oulu, FI-90014, Oulu, Finland

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 (Asn¹⁸ and Asn³³) of the human -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 Asn³³ 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 -opioid receptor is needed to maintain the expression of fully functional and stable receptor molecules at the cell surface.

Received for publication, March 7, 2008 , and in revised form, July 16, 2008.

^* This work was supported by the Biocenter Oulu and Grant 206230 from the Academy of Finland. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–3.

¹ To whom correspondence should be addressed: Institute of Biomedicine, Dept. of Anatomy and Cell Biology, University of Oulu, P. O. Box 5000, FI-90014, Oulu, Finland. Tel.: 358-8-537-5193; Fax: 358-8-537-5172; E-mail: Ulla.Petaja-Repo{at}oulu.fi.

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