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. 1988 Dec;9:31–35.

Purification of erythroid progenitor cells and characterization of erythropoietin receptors.

S B Krantz 1, S T Sawyer 1, K I Sawada 1
PMCID: PMC2149105  PMID: 2855465

Abstract

Highly purified murine and human erythroid progenitor cells at the colony-forming unit-erythroid (CFU-E) stage of development were prepared and recombinant human erythropoietin (rEp) was radioiodinated with retention of full biological activity. Specific binding of 125I-rEp to the murine cells revealed 950 receptors on the cell surface. Three hundred had a Kd of 0.09 nM while the remaining receptors had a Kd of 0.57 nM. The human erythroid progenitor cells also had two classes of receptors with a similar number per cell and similar distribution. The high affinity receptors had a Kd of 0.15 nM while the remaining receptors had a Kd of 0.37 nM. 125I-rEp was rapidly internalized into the cells at 37 C and metabolized to iodotyrosine. When cross-linking of 125I-rEp to the murine erythroid progenitor cells was performed with disuccinimidyl suberate, two labelled bands of 100 and 85 kDa were demonstrated. The radioactivity of both bands was reduced when binding was performed in the presence of excessive unlabelled rEp indicating a specific interaction of 125I-rEp with the receptor.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bondurant M. C., Lind R. N., Koury M. J., Ferguson M. E. Control of globin gene transcription by erythropoietin in erythroblasts from friend virus-infected mice. Mol Cell Biol. 1985 Apr;5(4):675–683. doi: 10.1128/mcb.5.4.675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Clarke B. J., Housman D. Characterization of an erythroid precursor cell of high proliferative capacity in normal human peripheral blood. Proc Natl Acad Sci U S A. 1977 Mar;74(3):1105–1109. doi: 10.1073/pnas.74.3.1105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dessypris E. N., Krantz S. B. Effect of pure erythropoietin on DNA-synthesis by human marrow day 15 erythroid burst forming units in short-term liquid culture. Br J Haematol. 1984 Feb;56(2):295–306. doi: 10.1111/j.1365-2141.1984.tb03957.x. [DOI] [PubMed] [Google Scholar]
  4. Hassan M. W., Lutton J. D., Levere R. D., Rieder R. F., Cederqvist L. L. In vitro culture of erythroid colonies from human fetal liver and umbilical cord blood. Br J Haematol. 1979 Apr;41(4):477–484. doi: 10.1111/j.1365-2141.1979.tb05885.x. [DOI] [PubMed] [Google Scholar]
  5. Iscove N. N., Sieber F., Winterhalter K. H. Erythroid colony formation in cultures of mouse and human bone marrow: analysis of the requirement for erythropoietin by gel filtration and affinity chromatography on agarose-concanavalin A. J Cell Physiol. 1974 Apr;83(2):309–320. doi: 10.1002/jcp.1040830218. [DOI] [PubMed] [Google Scholar]
  6. Koury M. J., Sawyer S. T., Bondurant M. C. Splenic erythroblasts in anemia-inducing Friend disease: a source of cells for studies of erythropoietin-mediated differentiation. J Cell Physiol. 1984 Dec;121(3):526–532. doi: 10.1002/jcp.1041210311. [DOI] [PubMed] [Google Scholar]
  7. Krantz S. B., Goldwasser E. Specific binding of erythropoietin to spleen cells infected with the anemia strain of Friend virus. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7574–7578. doi: 10.1073/pnas.81.23.7574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Krantz S. B., Sawada K., Sawyer S. T., Civin C. I. Specific binding of erythropoietin to human erythroid colony-forming cells. Trans Assoc Am Physicians. 1987;100:164–172. [PubMed] [Google Scholar]
  9. Metcalf D. The molecular biology and functions of the granulocyte-macrophage colony-stimulating factors. Blood. 1986 Feb;67(2):257–267. [PubMed] [Google Scholar]
  10. Ogawa M., MacEachern M. D., Avila L. Human marrow erythropoiesis in culture: II. Heterogeneity in the morphology, time course of colony formation, and sedimentation velocities of the colony-forming cells. Am J Hematol. 1977;3:29–36. doi: 10.1002/ajh.2830030104. [DOI] [PubMed] [Google Scholar]
  11. Sawada K., Krantz S. B., Kans J. S., Dessypris E. N., Sawyer S., Glick A. D., Civin C. I. Purification of human erythroid colony-forming units and demonstration of specific binding of erythropoietin. J Clin Invest. 1987 Aug;80(2):357–366. doi: 10.1172/JCI113080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Sawyer S. T., Koury M. J., Bondurant M. C. Large-scale procurement of erythropoietin-responsive erythroid cells: assay for biological activity of erythropoietin. Methods Enzymol. 1987;147:340–352. doi: 10.1016/0076-6879(87)47123-1. [DOI] [PubMed] [Google Scholar]
  13. Sawyer S. T., Krantz S. B., Goldwasser E. Binding and receptor-mediated endocytosis of erythropoietin in Friend virus-infected erythroid cells. J Biol Chem. 1987 Apr 25;262(12):5554–5562. [PubMed] [Google Scholar]
  14. Sawyer S. T., Krantz S. B., Luna J. Identification of the receptor for erythropoietin by cross-linking to Friend virus-infected erythroid cells. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3690–3694. doi: 10.1073/pnas.84.11.3690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Sieff C. A., Emerson S. G., Mufson A., Gesner T. G., Nathan D. G. Dependence of highly enriched human bone marrow progenitors on hemopoietic growth factors and their response to recombinant erythropoietin. J Clin Invest. 1986 Jan;77(1):74–81. doi: 10.1172/JCI112305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Spivak J. L. The mechanism of action of erythropoietin. Int J Cell Cloning. 1986 May;4(3):139–166. doi: 10.1002/stem.5530040302. [DOI] [PubMed] [Google Scholar]
  17. Stephenson J. R., Axelrad A. A., McLeod D. L., Shreeve M. M. Induction of colonies of hemoglobin-synthesizing cells by erythropoietin in vitro. Proc Natl Acad Sci U S A. 1971 Jul;68(7):1542–1546. doi: 10.1073/pnas.68.7.1542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Tepperman A. D., Curtis J. E., McCulloch E. A. Erythropietic colonies in cultures of human marrow. Blood. 1974 Nov;44(5):659–669. [PubMed] [Google Scholar]

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