title: Cys-27 variant of human δ-opioid receptor modulates maturation and cell surface delivery of Phe-27 variant via heteromerization.
abstract: The important role of G protein-coupled receptor homo/heteromerization in receptor folding, maturation, trafficking, and cell surface expression has become increasingly evident. Here we investigated whether the human δ-opioid receptor (hδOR) Cys-27 variant that shows inherent compromised maturation has an effect on the behavior of the more common Phe-27 variant in the early secretory pathway. We demonstrate that hδOR-Cys-27 acts in a dominant negative manner and impairs cell surface delivery of the co-expressed hδOR-Phe-27 and impairs conversion of precursors to the mature form. This was demonstrated by metabolic labeling, Western blotting, flow cytometry, and confocal microscopy in HEK293 and human SH-SY5Y neuroblastoma cells using differentially epitope-tagged variants. The hδOR-Phe-27 precursors that were redirected to the endoplasmic reticulum-associated degradation were, however, rescued by a pharmacological chaperone, the opioid antagonist naltrexone. Co-immunoprecipitation of metabolically labeled variants revealed that both endoplasmic reticulum-localized precursors and mature receptors exist as homo/heteromers. The existence of homo/heteromers was confirmed in living cells by bioluminescence resonance energy transfer measurements, showing that the variants have a similar propensity to form homo/heteromers. By forming both homomers and heteromers, the hδOR-Cys-27 variant may thus regulate the levels of receptors at the cell surface, possibly leading to altered responsiveness to opioid ligands in individuals carrying the Cys-27 variant.
authors.affiliation: Department of Anatomy and Cell Biology, Institute of Biomedicine, University of Oulu, FI-90014 Oulu, Finland.
authors.forename: Tarja T
authors.initials: TT
authors.lastname: Leskelä
authors.forename: Jarkko J
authors.initials: JJ
authors.lastname: Lackman
authors.forename: Miia M
authors.initials: MM
authors.lastname: Vierimaa
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kobayashi
authors.forename: Michel
authors.initials: M
authors.lastname: Bouvier
authors.forename: Ulla E
authors.initials: UE
authors.lastname: Petäjä-Repo
mesh_terms: D045744:Cell Line, Tumor; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D009271:Naltrexone / Q000494:pharmacology; D009292:Narcotic Antagonists / Q000494:pharmacology; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization / Q000187:drug effects / Q000502:physiology*; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D059748:Proteolysis / Q000187:drug effects; D017465:Receptors, Opioid, delta / Q000235:genetics / Q000378:metabolism*
chemical_list: D009292:Narcotic Antagonists; C529706:OPRD1 protein, human; D011498:Protein Precursors; D017465:Receptors, Opioid, delta; D010649:Phenylalanine; D009271:Naltrexone