Subunit structure of the erythropoietin receptor analyzed by 125I-Epo cross-linking in cells expressing wild-type or mutant receptors.
To analyze the structure of the murine erythropoietin receptor (EpoR), wild-type or mutant EpoR cDNAs were expressed in cell lines, and the proteins that cross-linked with 125I-labeled erythropoietin (Epo) were analyzed by immunoprecipitation using an antibody against the intracellular region of the cloned EpoR. COS-7 cell transfectants expressing the wild-type EpoR showed two major cross-linked species of 145 and 110 Kd, both of which were recognized by the antibody against the cloned EpoR after denaturation under reducing conditions. Furthermore, a reduction in sizes of both cross-linked bands was observed in COS-7 transfectants expressing a mutant receptor with an internal deletion, thus indicating that both species contain the cloned EpoR. COS-7 cells expressing mutant receptors with carboxy-terminal deletions showed cross-linked bands corresponding to the smaller species of the two observed in cells expressing the wild-type receptor. In contrast to COS-7 cell transfectants, DA3 cells expressing wild-type or mutant EpoR cDNAs showed an additional cross-like species of 130 Kd. The size of this species was not altered by deletions in EpoR, showing that it did not contain EpoR. The 130-Kd cross-linked band, which would contain a 95-Kd protein, was also observed in a murine erythroleukemia cell line, D1B. These results suggest that Epo associates with a second component of 95 Kd, which is specifically expressed in hematopoietic cells.
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Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN 38105.
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D000818:Animals; D002460:Cell Line; D004921:Erythropoietin / Q000737:chemistry* / Q000235:genetics; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism; D051379:Mice; D009154:Mutation; D010766:Phosphorylation; D017467:Receptors, Erythropoietin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D017467:Receptors, Erythropoietin; D004921:Erythropoietin