title: 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.
authors.affiliation: From the Department of Anatomy and Cell Biology and the Medical Research Center Oulu, Institute of Biomedicine, University of Oulu, FI-90014 Oulu, Finland and.
authors.forename: Jarkko J
authors.initials: JJ
authors.lastname: Lackman
authors.affiliation: From the Department of Anatomy and Cell Biology and the Medical Research Center Oulu, Institute of Biomedicine, University of Oulu, FI-90014 Oulu, Finland and.
authors.forename: Piia M H
authors.initials: PM
authors.lastname: Markkanen
authors.affiliation: the Department of Biochemistry, Institute for Research in Immunology and Cancer and Groupe de Recherche Universitaire sur le Médicament, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
authors.forename: Mireille
authors.initials: M
authors.lastname: Hogue
authors.affiliation: the Department of Biochemistry, Institute for Research in Immunology and Cancer and Groupe de Recherche Universitaire sur le Médicament, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
authors.forename: Michel
authors.initials: M
authors.lastname: Bouvier
authors.affiliation: From the Department of Anatomy and Cell Biology and the Medical Research Center Oulu, Institute of Biomedicine, University of Oulu, FI-90014 Oulu, Finland and ulla.petaja-repo@oulu.fi.
authors.forename: Ulla E
authors.initials: UE
authors.lastname: Petäjä-Repo
mesh_terms: D037281:Calnexin / Q000378:metabolism*; D060746:Endoplasmic Reticulum-Associated Degradation / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D011134:Polysaccharides / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D017465:Receptors, Opioid, delta / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology
chemical_list: D011134:Polysaccharides; D017465:Receptors, Opioid, delta; D037281:Calnexin
keywords: Calnexin; ER Quality Control; ER-associated Degradation; G Protein-coupled Receptor (GPCR); Genetic Polymorphism; Glycosylation; Opioid Receptor; Pharmacological Chaperone; Ubiquitination