Characterization of the structure of the erythropoietin receptor by ligand
blotting
HL Atkins, VC Broudy and T Papayannopoulou
University of Washington, Seattle.
Erythropoietin (Epo) regulates the growth and differentiation of erythroid
cells by binding to a specific receptor. We characterized the native Epo
receptor on erythroleukemia cell lines by ligand blotting. Solubilized cell
membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis, transferred onto nitrocellulose, and probed with
125I-Epo. Specificity was demonstrated by inhibition of 125I-Epo binding by
unlabeled excess Epo but not other peptide growth factors and by the
cellular distribution of the Epo binding protein. A single membrane protein
of 61 Kd +/- 4 Kd was sufficient to bind 125I Epo in both human (OCIM2,
K562) and murine (GM979, Rauscher, DA-1) cell lines. This finding is
consistent with the predicted size of the Epo receptor from the murine cDNA
clone. However, chemical crosslinking of 125I-Epo to its receptor has
identified two Epo binding proteins of 105 Kd and 85 Kd. This difference
may occur because the receptor is size fractionated before Epo binding in
the ligand blot, but after Epo binding in crosslinking studies. Ligand
blotting demonstrates that the native Epo receptor is composed of a single
61-Kd Epo binding protein, and suggests the presence of additional proteins
of 20 to 25 Kd that associate with the receptor after Epo binding.
Volume 77,
Issue 12,
pp. 2577-2582,
06/15/1991
Copyright © 1991 by The American Society of Hematology
