FUNCTIONAL REGIONS OF THE MOUSE THROMBOPOIETIN RECEPTOR CYTOPLASMIC DOMAIN - EVIDENCE FOR A CRITICAL REGION WHICH IS INVOLVED IN DIFFERENTIATION AND CAN BE COMPLEMENTED BY ERYTHROPOIETIN

Thrombopoietin (TPO) is the major regulator of growth and differentiat ion of megakaryocytes. To identify functionally important regions in t he cytoplasmic domain of the TPO receptor, mpl, we introduced wild-typ e mpl and deletion mutants of murine mpl into the granulocyte-macropha ge colony-stimulating factor (GM-CSF)or erythropoietin (EPO)-dependent human cell line UT7, TPO induced differentiation of UT7-Wtmpl cells, not parental UT7 cells, along the megakaryocytic lineage, as evidenced by decreased proliferation, changes in cell morphology, and increased surface expression and mRNA levels of megakaryocytic markers CD41, CD 61, and CD42b, When UT7-mpl cells were cultured long-term in EPO inste ad of GM-CSF, the TPO effect was dominant over that of EPO, Moreover, the differentiation induced by TPO was more pronounced for cells shift ed from EPO to TPO than for cells shifted from GM-CSF to TPO, as shown by the appearance of polyploid cells, Mutational analysis of the cyto plasmic domain of mpl showed that proliferation and maturation functio ns of mpl can be uncoupled, Two functional regions were identified: (i ) the first 69 amino acids comprising the cytokine receptor motifs, bo x 1 and box 2, which are necessary for both TPO-induced mitogenesis an d maturation; and (ii) amino acids 71 to 94, which are dispensable for proliferation but required for differentiation, Surprisingly, however , EPO could complement this latter domain for TPO-induced differentiat ion, suggesting a close relationship between EPO and TPO signaling.