Identification and analysis of human erythropoietin receptors on a factor-dependent cell line, TF-1.
We have recently established a novel cell line, TF-1, from bone marrow cells of a patient with erythroleukemia, that showed an absolute growth dependency on each of three hematopoietic growth factors: erythropoietin (EPO) granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin 3 (IL-3). EPO stimulated the proliferation of TF-1 cells even at the physiologic concentration (0.03 U/mL). We performed binding experiments on TF-1 cells using radioiodinated EPO. The binding of radioiodinated EPO to TF-1 was specific, time- and temperature-dependent, and saturable. Scatchard analysis of the saturation binding data suggested the existence of a single class of binding sites (kd = 0.40 nmol/L; number of binding sites = 1,630 per cell). TF-1 cells were usually maintained in RPMI 1640 containing 10% fetal bovine serum and 5 ng/mL GM-CSF. The kd and the number of the EPO receptors were not changed by incubating the cells with IL-3, although culturing the cells in the presence of EPO resulted in down-modulation of EPO receptors. The chemical cross-linking study demonstrated that two molecules with apparent molecular weights of 105 kilodalton (Kd) and 90 Kd were the binding components of EPO. Present data suggest that human EPO receptors are very similar to the previously reported murine EPO receptors.
Kitamura
T
T
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Tojo
A
A
Kuwaki
T
T
Chiba
S
S
Miyazono
K
K
Urabe
A
A
Takaku
F
F
D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003115:Colony-Stimulating Factors / Q000494:pharmacology; D003432:Cross-Linking Reagents; D004921:Erythropoietin / Q000378:metabolism* / Q000502:physiology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D006133:Growth Substances / Q000494:pharmacology; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D007457:Iodine Radioisotopes; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism / Q000473:pathology; D008970:Molecular Weight; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000187:drug effects; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins / Q000494:pharmacology
D003115:Colony-Stimulating Factors; D003432:Cross-Linking Reagents; D006133:Growth Substances; D007377:Interleukin-3; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D004921:Erythropoietin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor