Subunit structure of the erythropoietin receptor.
Chemical cross-linking of the red blood cell hormone, erythropoietin (Epo), to its receptor on erythroid cells has revealed the presence of two proteins closely associated with Epo, but the relationship between these two proteins is controversial. Using the cross-linking reagents disuccinimidyl suberate and dithiobissuccinimidyl propionate, we show that 125I-Epo can be specifically conjugated in a complex of 224kDa using mouse fetal liver cells, bone marrow cells, and Friend virus-induced splenic erythroblasts as demonstrated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. Under reducing conditions, the 224-kDa complex appeared as two Epo conjugates of 136 kDa and 119 kDa, and these bands were also observed to a variable extent in some nonreducing gels. Disulfide linking of the 136-kDa and 119-kDa bands was confirmed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis run under nonreducing followed by reducing conditions. With increasing time of 125I-Epo binding to Friend virus erythroblasts in the presence of sodium azide to inhibit receptor internalization, the 136-kDa and 119-kDa bands seen under reducing conditions increased markedly in intensity, whereas the 224-kDa band seen under nonreducing conditions declined. These results suggest that the 224-kDa Epo conjugate is inefficiently solubilized under nonreducing conditions following prolonged periods of Epo binding. A single class of saturable, high affinity receptors for Epo on each of the cell types tested is demonstrated. It is concluded that the two disulfide-linked Epo-binding proteins which can be independently cross-linked to Epo form a single ligand binding site.
McCaffery
P J
PJ
Malaghan Institute of Medical Research, Wellington School of Medicine, Wellington Hospital, New Zealand.
Fraser
J K
JK
Lin
F K
FK
Berridge
M V
MV
D000818:Animals; D002472:Cell Transformation, Viral; D003432:Cross-Linking Reagents / Q000378:metabolism; D004921:Erythropoietin / Q000378:metabolism*; D005333:Fetus; D005622:Friend murine leukemia virus / Q000235:genetics; D005865:Gestational Age; D007700:Kinetics; D008099:Liver / Q000196:embryology / Q000378:metabolism*; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C; D008815:Mice, Inbred Strains; D008970:Molecular Weight; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins / Q000378:metabolism; D013154:Spleen / Q000378:metabolism
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D004921:Erythropoietin