Effect of tunicamycin treatment on ligand binding to the erythropoietin receptor: conversion from two classes of binding sites to a single class.
Scatchard analyses of erythropoietin (EPO) binding to its receptor (EPO-R) have shown that some erythroid cells display a biphasic nature of the ligand-saturation curve, indicating the presence of two classes of binding sites with different affinities. The biochemical basis accounting for this observation is unknown. We found that the culture of a human erythroleukemia cell line with tunicamycin, an inhibitor of N-glycosylation, converted the biphasic Scatchard plot to a single phase with high-affinity sites. Scatchard plots of baby hamster kidney (BHK) cells that had been engineered to express cloned mouse EPO-R were also biphasic and the plots of cells cultured with tunicamycin became a single phase with high-affinity sites. Mouse EPO-R is glycosylated at one asparagine residue in the extracellular region. The mutant EPO-R, in which asparagine residue responsible for N-glycosylation was replaced with glutamine residue, was expressed on BHK cells. Unexpectedly, mutant EPO-R was similar in ligand binding to wild-type EPO-R. BHK cells that expressed mutant EPO-R showed biphasic Scatchard plots that were converted to single-phase plots with only high-affinity sites by tunicamycin treatment. These results indicate that the N-linked sugar of EPO-R is not involved in the manifestation of two classes of binding sites, and that there is a yet unidentified glycoprotein crucial for the ligand-saturation characteristics of EPO-R.
Nagao
M
M
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Japan.
Matsumoto
S
S
Masuda
S
S
Sasaki
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D006224:Cricetinae; D004921:Erythropoietin / Q000378:metabolism*; D005973:Glutamine; D006031:Glycosylation; D007668:Kidney; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D017467:Receptors, Erythropoietin / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology*
D009838:Oligodeoxyribonucleotides; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D005973:Glutamine; D014415:Tunicamycin; D004921:Erythropoietin; D001216:Asparagine