N-Glycosylation of the human kappa opioid receptor enhances its stability but slows its trafficking along the biosynthesis pathway

Biochemistry. 2007 Sep 25;46(38):10960-70. doi: 10.1021/bi700443j. Epub 2007 Aug 21.

Abstract

We examined glycosylation of FLAG-hKOR expressed in CHO cells and determined its functional significance. FLAG-hKOR was resolved as a broad and diffuse 55-kDa band and a less diffuse 45-kDa band by immunoblotting, indicating that the receptor is glycosylated. Endoglycosidase H cleaved the 45-kDa band to approximately 38 kDa but did not change the 55-kDa band, demonstrating that the 45-kDa band is N-glycosylated with high-mannose or hybrid-type glycan. Peptide-N-glycosidase F digestion of solubilized hKOR or incubation of cells with tunicamycin resulted in two species of 43 and 38 kDa, suggesting that the 43-kDa band is O-glycosylated. FLAG-hKOR was reduced to lower Mr bands by neuraminidase and O-glycosidase, indicating that the hKOR contains O-linked glycan. Mutation of Asn25 or Asn39 to Gln in the N-terminal domain reduced the Mr by approximately 5 kDa, indicating that both residues were glycosylated. The double mutant hKOR-N25/39Q was resolved as a 43-kDa (mature form) and a 38-kDa (intermediate form) band. When transiently expressed, hKOR-N25/39Q had a lower expression level than the wild type. In CHO cells stably expressing the hKOR-N25/39Q, pulse-chase experiments revealed that the turnover rate constants (ke) of the intermediate and mature forms were approximately 3 times those of the wild type. In addition, the maturation rate constant (ka) of the 43-kDa form of hKOR-N25/39Q was 6 times that of the mature form of the wild type. The hKOR-N25/39Q mutant showed increased agonist-induced receptor phosphorylation, desensitization, internalization, and downregulation, without changing ligand binding affinity or receptor-G protein coupling. Thus, N-glycosylation of the hKOR plays important roles in stability and trafficking along the biosynthesis pathway of the receptor protein as well as agonist-induced receptor regulation.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer / pharmacology
  • Amino Acid Substitution
  • Analgesics, Non-Narcotic / pharmacology
  • Animals
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • Diprenorphine / pharmacology
  • Down-Regulation
  • Electrophoresis, Polyacrylamide Gel
  • Gene Expression Regulation* / physiology
  • Glycoside Hydrolases / antagonists & inhibitors
  • Glycoside Hydrolases / metabolism
  • Glycosylation
  • Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
  • Humans
  • Immunoprecipitation
  • Kinetics
  • Mutagenesis, Site-Directed
  • Protein Processing, Post-Translational* / physiology
  • Protein Structure, Tertiary
  • Protein Transport / physiology
  • Receptors, Cell Surface / biosynthesis
  • Receptors, Cell Surface / metabolism*
  • Receptors, Opioid, kappa / biosynthesis
  • Receptors, Opioid, kappa / metabolism*
  • Recombinant Fusion Proteins / drug effects
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction
  • Sulfur Radioisotopes
  • Tritium
  • Tunicamycin / pharmacology

Substances

  • Analgesics, Non-Narcotic
  • Receptors, Cell Surface
  • Receptors, Opioid, kappa
  • Recombinant Fusion Proteins
  • Sulfur Radioisotopes
  • Tritium
  • Tunicamycin
  • Diprenorphine
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer
  • Glycoside Hydrolases