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Effect of tunicamycin treatment on ligand binding to the erythropoietin
receptor: conversion from two classes of binding sites to a single class
M Nagao, S Matsumoto, S Masuda and R Sasaki
Department of Food Science and Technology, Faculty of Agriculture, Kyoto
University, Japan.
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.
Volume 81,
Issue 10,
pp. 2503-2510,
05/15/1993
Copyright © 1993 by The American Society of Hematology
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