title: Opioid receptor pharmacological chaperones act by binding and stabilizing newly synthesized receptors in the endoplasmic reticulum.
abstract: Accumulating evidence has indicated that membrane-permeable G protein-coupled receptor ligands can enhance cell surface targeting of their cognate wild-type and mutant receptors. This pharmacological chaperoning was thought to result from ligand-mediated stabilization of immature receptors in the endoplasmic reticulum (ER). In the present study, we directly tested this hypothesis using wild-type and mutant forms of the human delta-opioid receptor as models. ER-localized receptors were isolated by expressing the receptors in HEK293 cells under tightly controlled tetracycline induction and blocking their ER export with brefeldin A. The ER-retained delta-opioid receptor precursors were able to bind [(3)H]diprenorphine with high affinity, and treatment of cells with an opioid antagonist naltrexone led to a 2-fold increase in the number of binding sites. After removing the transport block, the antagonist-mediated increase in the number of receptors was detectable at the cell surface by flow cytometry and cell surface biotinylation assay. Importantly, opioid ligands, both antagonists and agonists, were found to stabilize the ER-retained receptor precursors in an in vitro heat inactivation assay and the treatment enhanced dissociation of receptor precursors from the molecular chaperone calnexin. Thus, we conclude that pharmacological chaperones facilitate plasma membrane targeting of delta-opioid receptors by binding and stabilizing receptor precursors, thereby promoting their release from the stringent ER quality control.
authors.affiliation: Biocenter Oulu and Department of Anatomy and Cell Biology, University of Oulu, FI-90014 Oulu, Finland.
authors.forename: Tarja T
authors.initials: TT
authors.lastname: Leskelä
authors.forename: Piia M H
authors.initials: PM
authors.lastname: Markkanen
authors.forename: E Maritta
authors.initials: EM
authors.lastname: Pietilä
authors.forename: Jussi T
authors.initials: JT
authors.lastname: Tuusa
authors.forename: Ulla E
authors.initials: UE
authors.lastname: Petäjä-Repo
mesh_terms: D001665:Binding Sites; D019921:Biotinylation; D020126:Brefeldin A / Q000737:chemistry; D037281:Calnexin / Q000737:chemistry; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D018832:Molecular Chaperones / Q000378:metabolism; D011485:Protein Binding; D011957:Receptors, Opioid / Q000737:chemistry / Q000378:metabolism*; D017465:Receptors, Opioid, delta / Q000737:chemistry
chemical_list: D008024:Ligands; D018832:Molecular Chaperones; D011957:Receptors, Opioid; D017465:Receptors, Opioid, delta; D037281:Calnexin; D020126:Brefeldin A; D004247:DNA