Cys-27 variant of human δ-opioid receptor modulates maturation and cell surface delivery of Phe-27 variant via heteromerization

J Biol Chem. 2012 Feb 10;287(7):5008-20. doi: 10.1074/jbc.M111.305656. Epub 2011 Dec 19.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Tumor
  • Endoplasmic Reticulum / genetics
  • Endoplasmic Reticulum / metabolism
  • HEK293 Cells
  • Humans
  • Naltrexone / pharmacology
  • Narcotic Antagonists / pharmacology
  • Phenylalanine / genetics
  • Phenylalanine / metabolism
  • Protein Multimerization / drug effects
  • Protein Multimerization / physiology*
  • Protein Precursors / genetics
  • Protein Precursors / metabolism*
  • Protein Transport / drug effects
  • Protein Transport / physiology
  • Proteolysis / drug effects
  • Receptors, Opioid, delta / genetics
  • Receptors, Opioid, delta / metabolism*

Substances

  • Narcotic Antagonists
  • OPRD1 protein, human
  • Protein Precursors
  • Receptors, Opioid, delta
  • Phenylalanine
  • Naltrexone