Partial purification and characterization of erythropoietin receptors from erythroid progenitor cells.
We have partially purified and characterized erythropoietin (Epo) receptors of erythroid progenitor cells which were obtained from the spleens of anemia-inducing Friend virus infected mice. Membrane proteins of splenic erythroid progenitor cells were solubilized with 1% Triton X-102. Upon chromatography on DEAE-Sephacel anion-exchange columns, two distinct Epo receptor peak fractions referred to as Peak I and Peak II were identified by 125I-Epo binding assays using the polyethylene glycol precipitation method. The Peak I and Peak II samples were then individually chromatographed on an S-Sepharose column. The S-Sepharose-purified Peak I and Peak II samples were crosslinked with 125I-Epo in the presence and absence of excess unlabeled Epo by disuccinimidyl suberate treatment, and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. Both Peak I and Peak II samples showed a radiolabeled peptide with a Mr 135K and the labeling was blocked by excess unlabeled Epo. Since the Mr of Epo is about 35K, Epo receptor peptide has a Mr approximately 100K. To determine whether Epo stimulates autophosphorylation of the receptors, the S-Sepharose-purified Peak I and Peak II samples were incubated with or without Epo, and then briefly incubated in the presence of [gamma-32P]ATP and Mn2+. The tyrosine residue phosphorylated protein was isolated by an immunochemical technique, and then analyzed by SDS-PAGE and autoradiography. The result showed that Epo stimulates phosphorylation of a 100-kDa peptide.
Im
J H
JH
Department of Pharmacology, School of Medicine, University of Missouri, Columbia 65212.
Lee
S J
SJ
Kim
H D
HD
D000818:Animals; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D015672:Erythroid Precursor Cells / Q000032:analysis* / Q000187:drug effects; D004921:Erythropoietin / Q000494:pharmacology; D051379:Mice; D010766:Phosphorylation; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000302:isolation & purification*; D017467:Receptors, Erythropoietin; D013154:Spleen / Q000032:analysis / Q000187:drug effects
D003432:Cross-Linking Reagents; D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D004921:Erythropoietin