The erythropoietin receptor of rat erythroid progenitor lens. Characterization and affinity cross-linkage.
Commercially available 125I-labeled erythropoietin, obtained by genetic engineering from a human gene, was used to characterize receptors for this hormone on the cell surface of rat erythroid progenitor cells. A low number of high affinity binding sites (487 +/- 32 sites/cell, Kd = 167 +/- 14 pm) were found. Nonerythroid cells and erythrocytes did not exhibit specific binding. The high affinity binding was reversible and displaced by unlabeled erythropoietin, but not by other hormones and growth factors. After incubation at 37 degrees C, nearly 35% of the specifically bound erythropoietin seemed to be internalized, as judged by resistance to acidic buffer treatment. Thus, binding showed characteristics of a hormone-receptor association. 125I-Erythropoietin-labeled cells were treated with the bifunctional reagent dissucinimidyl suberate. Analysis of the cellular extracts by polyacrylamide gel electrophoresis under denaturing and reducing conditions revealed that erythropoietin can be cross-linked to two molecules of 94 and 78 kDa, respectively. Both labeled bands disappeared when the cells were labeled in the presence of an excess of unlabeled erythropoietin. Under nonreducing conditions, a cross-linked band of 230-255 kDa was observed. The relationships between these bands are discussed.
Mayeux
P
P
Laboratoire de Physiologie Animale, Faculte des Sciences, Reims, France.
Billat
C
C
Jacquot
R
R
D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004921:Erythropoietin / Q000378:metabolism*; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D007700:Kinetics; D004915:Leukemia, Erythroblastic, Acute; D007942:Leukemia, Experimental; D008099:Liver / Q000196:embryology / Q000378:metabolism; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins / Q000378:metabolism*; D013388:Succinimides / Q000494:pharmacology*
D003432:Cross-Linking Reagents; D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D013388:Succinimides; D004921:Erythropoietin; C019358:disuccinimidyl suberate