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

Abstract

Thrombopoietin (TPO) is the major regulator of growth and differentiation of megakaryocytes. To identify functionally important regions in the cytoplasmic domain of the TPO receptor, mpl, we introduced wild-type mpl and deletion mutants of murine mpl into the granulocyte-macrophage 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, CD61, and CD42b. When UT7-mpl cells were cultured long-term in EPO instead of GM-CSF, the TPO effect was dominant over that of EPO. Moreover, the differentiation induced by TPO was more pronounced for cells shifted 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 cytoplasmic domain of mpl showed that proliferation and maturation functions of mpl can be uncoupled. Two functional regions were identified: (i) the first 69 amino acids comprising the cytokine receptor motifs, box 1 and box 2, which are necessary for both TPO-induced mitogenesis and 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 differentiation, suggesting a close relationship between EPO and TPO signaling.

Biogenesis of endoplasmic reticulum proteins involved in Ca2+ signalling during megakaryocytic differentiation: an in vitro study

“…Because of the restricted expression of the TPO receptor c-Mpl in megakaryoblastic cells [45], experiments were carried out using the UT7-Mpl cell line expressing the murine c-Mpl. TPO treatment of these cells has been shown to induce increased levels of specific megakaryocytic differentiation antigens and to reduce those of erythroid markers [46,47]. Control and TPO-treated UT7-Mpl cell lysates were tested for their expression of GPIIIa, SERCAs and calreticulin as in Figure 1 expression level of SERCA3 was also observed under physiologically induced megakaryocytic differentiation.…”

The Prolactin Receptor and Severely Truncated Erythropoietin Receptors Support Differentiation of Erythroid Progenitors

“…Our finding that the minimal domain of EpoR able to support differentiation is the same as that required to support cell proliferation raises the possibility that the role of the EpoR-activated signals is only to support the survival and proliferation of the differentiating progenitors, as proposed by the stochastic hypothesis. It is possible, however, by analogy with studies on the granulocyte-colony-stimulating factor or thrombopoietin receptors (44,45), that, to support erythroid gene expression, additional signals are needed beyond those required for proliferation. If this is the case, these signals must arise from the membrane-proximal domain of the EpoR and cannot be unique to the EpoR.…”

Terminal megakaryocytic differentiation of TF-1 cells is induced by phorbol esters and thrombopoietin and is blocked by expression of PML/RARα fusion protein

“…33,34 Previous studies have shown that UT-7, a growth factordependent cell line established from a patient with acute megakaryoblastic leukemia, 35 represents a useful in vitro model to study megakaryocytopoiesis. [36][37][38][39][40] Particularly, UT-7 TPO and UT-7 GM 38 sublines undergo megakaryocytic differentiation following incubation with Tpo. UT-7 cells have many similarities with TF-1 cells.…”