Binding and regulation of thrombopoietin to human megakaryocytes.
Thrombopoietin (TPO, c-Mpl ligand) is considered to play an important role in the regulation of megakaryocytopoiesis and platelet production by activating the cytokine receptor c-Mpl. We have examined the binding of 125I-TPO to the human megakaryocytic cell line, CMK, and to primary human megakaryocytes. Scatchard analysis of TPO binding to its cognate receptor in megakaryocytic cells suggested the existence of a single class of c-Mpl receptors. CMK cells exhibited 1223 receptors per cell with a dissociation constant (Kd) of Kd = 223 pM, whereas primary human megakaryocytes exhibited 12140 receptors per cell and a dissociation constant of Kd = 749 pM. The pretreatment of CMK cells and primary bone marrow megakaryocytes with TPO resulted in a decreased binding of TPO to the c-Mpl receptors. This down-regulation was observed within 3 h and was not inhibited by cycloheximide. Phorbol ester, an activator of protein kinase C, also inhibited TPO binding to the c-Mpl receptors by reducing the number of these receptors. The pretreatment of CMK cells with IL-3, IL-6 and DMSO, all of which induced the differentiation of CMK cells, did not affect the binding of TPO to the c-Mpl receptors. These results suggest an additional mechanism, where protein kinase C may help to regulate the binding of TPO to these cells.
Sato
T
T
Department of Paediatrics, School of Medicine, Chiba, Japan.
Fuse
A
A
Niimi
H
H
Fielder
P J
PJ
Avraham
H
H
D004121:Dimethyl Sulfoxide / Q000378:metabolism; D006801:Humans; D007377:Interleukin-3 / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D007947:Leukemia, Megakaryoblastic, Acute / Q000378:metabolism*; D008533:Megakaryocytes / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D013926:Thrombopoietin / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D007377:Interleukin-3; D015850:Interleukin-6; D013926:Thrombopoietin; D013755:Tetradecanoylphorbol Acetate; D004121:Dimethyl Sulfoxide