Cys-27 Variant of Human δ-Opioid Receptor Modulates Maturation and Cell Surface Delivery of Phe-27 Variant via Heteromerization
Cys-27 Variant of Human δ-Opioid Receptor Modulates Maturation and Cell Surface Delivery of Phe-27 Variant via Heteromerization
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.
- University of Montreal Canada
- University of Oulu Finland
- Institute for Research in Immunology and Cancer Canada
- Oulu University Hospital Finland
Narcotic Antagonists, Phenylalanine, Endoplasmic Reticulum, Naltrexone, Protein Transport, HEK293 Cells, Cell Line, Tumor, Receptors, Opioid, delta, Proteolysis, Humans, Protein Multimerization, Protein Precursors
Narcotic Antagonists, Phenylalanine, Endoplasmic Reticulum, Naltrexone, Protein Transport, HEK293 Cells, Cell Line, Tumor, Receptors, Opioid, delta, Proteolysis, Humans, Protein Multimerization, Protein Precursors
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