PMID: 17711303

 

    Legend: Gene, Sites

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

Abstract :
  1. We examined glycosylation of FLAG- hKOR expressed in CHO cells and determined its functional significance
  2. 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
  3. 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
  4. 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
  5. FLAG- hKOR was reduced to lower Mr bands by neuraminidase and O-glycosidase, indicating that the hKOR contains O-linked glycan
  6. 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
  7. The double mutant hKOR-N25/39Q was resolved as a 43-kDa (mature form) and a 38-kDa (intermediate form) band
  8. When transiently expressed, hKOR-N25/39Q had a lower expression level than the wild type
  9. 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
  10. 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
  11. 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
  12. 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
Output (sent_index, trigger, protein, sugar, site):
  • 0. N-Glycosylation, , kappa opioid receptor, -, -
  • 1. glycosylation, , hKOR, -, -
  • 12. N-glycosylation, , hKOR, -, -
  • 2. glycosylated, , receptor, -, -
  • 5. contains, , hKOR, O-linked glycan, -
  • 6. glycosylated, , -, -, residues
Output(Part-Of) (sent_index, protein, site):
*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

 

Protein NCBI ID SENTENCE INDEX
form of hKOR 4986 10
hKOR 4986 1,2,4,5,7,8,9,11,12
neuraminidase 4758 5
kappa opioid receptor 4986 0