title: Partial purification and characterization of erythropoietin receptors from erythroid progenitor cells.
abstract: 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.
authors.affiliation: Department of Pharmacology, School of Medicine, University of Missouri, Columbia 65212.
authors.forename: J H
authors.initials: JH
authors.lastname: Im
authors.forename: S J
authors.initials: SJ
authors.lastname: Lee
authors.forename: H D
authors.initials: HD
authors.lastname: Kim
mesh_terms: 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
chemical_list: D003432:Cross-Linking Reagents; D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D004921:Erythropoietin