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Detection and isolation of the erythropoietin receptor using biotinylated
erythropoietin
AW Wognum, PM Lansdorp, RK Humphries and G Krystal
Terry Fox Laboratory, B.C. Cancer Research Centre, Vancouver, Canada.
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.
Volume 76,
Issue 4,
pp. 697-705,
08/15/1990
Copyright © 1990 by The American Society of Hematology
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