Detection and isolation of the erythropoietin receptor using biotinylated erythropoietin.
Procedures have been developed to label human erythropoietin (Ep) with biotin to detect and isolate the Ep-receptor. The labeling method used the abundant carbohydrate groups on Ep and resulted in biologically active biotin-Ep (b-Ep) containing 8 to 10 biotins per Ep molecule. Specific binding of b-Ep to cells from spleens of mice made anemic by phenylhydrazine injections was demonstrated using 125I-labeled streptavidin. B-Ep, together with fluorescently tagged streptavidin, was found to specifically detect Ep-receptor-bearing cells by flow cytometry. This was demonstrated in several ways. First, approximately 90% of nucleated spleen cells from phenylhydrazine-treated mice were clearly fluorescent after staining with b-Ep and streptavidin-phycoerythrin, whereas only background fluorescence was detected using spleen cells from untreated mice. In addition, Ep-receptors were detected on 5% to 10% of normal mouse bone marrow cells, and these cells could be identified as erythroid in nature by separating the cells into subpopulations based on light-scatter properties. Third, Ep-receptor expression was found to correlate positively with expression of transferrin receptors, confirming the erythroid nature of these cells. B-Ep was also used to isolate Ep-receptors from monkey COS cells transfected with the murine Ep-receptor cDNA. In these experiments a cell-surface-bound protein of approximately 65 Kd and an intracellular protein of approximately 60 Kd were isolated from these cells. The procedures described in this report for detecting Ep-receptor expressing cells and for isolating the Ep-receptor should be valuable for purifying erythroid cells from heterogeneous cell populations, for elucidating the structure of the Ep-receptor, and for studying the biological activities of Ep at the cellular and molecular level.
Wognum
A W
AW
Terry Fox Laboratory, B.C. Cancer Research Centre, Vancouver, Canada.
Lansdorp
P M
PM
Humphries
R K
RK
Krystal
G
G
D000818:Animals; D001426:Bacterial Proteins; D001710:Biotin / Q000378:metabolism; D002460:Cell Line; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D004921:Erythropoietin / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D000882:Haplorhini; D051379:Mice; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism; D017467:Receptors, Erythropoietin; D013154:Spleen / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D019809:Streptavidin; D014162:Transfection
D001426:Bacterial Proteins; D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D004921:Erythropoietin; D001710:Biotin; D019809:Streptavidin