Characterization of the structure of the erythropoietin receptor by ligand blotting.
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.
Atkins
H L
HL
University of Washington, Seattle.
Broudy
V C
VC
Papayannopoulou
T
T
D000818:Animals; D004910:Erythrocyte Membrane / Q000737:chemistry; D004921:Erythropoietin / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D004915:Leukemia, Erythroblastic, Acute; D051379:Mice; D008970:Molecular Weight; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D004921:Erythropoietin