Characterization of MPl mutants using primary megakaryocyte-lineage cells from mpl(-/-) mice: a new system for Mpl structure-function studies

Mpl is the thrombopoietin (TPO) receptor. The current molecular understandi ng of how Mpl activation stimulates proliferation of megakaryocyte-lineage cells is based largely on the engineered expression of Mpl in non megakaryo cyte-lineage cell lines. However, the relevance of these findings to Mpl si gnaling in primary megakaryocyte-lineage cells remains largely unknown. The refore, a system was developed to study Mpl function in primary mpl(-/-) me gakaryocyte-lineage cells. Expressing avian retroviral receptors on the sur faces of mammalian cells overcomes their natural block to avian retroviral infection; 815 bp of human GPIIb regulatory sequence was used to generate t ransgenic mice with megakaryocyte-lineage expression of the subgroup A avia n leukosis virus receptor, TVA. Avian retroviral infection of unfractionate d bone marrow from these mice is restricted to megakaryocyte-lineage cells. The transgenic mice were crossed to an mpl(-/-) background generating GPII b-tva(+)mpl(-/-) mice. By using avian retroviruses to express wild-type or mutant Mpl on the surfaces of primary megakaryocyte-lineage cells, it was d emonstrated that (1) the 10 membrane-proximal, cytoplasmic amino acids of M pl are required for TPO-induced proliferation; (2) Y582F mutation confers a proliferative advantage over wild-type Mpl and imparts a constitutive anti -apoptotic signal; (3) truncating the 50 C-terminal Mpl amino acids reduces but does not eliminate TPO-induced mitogen-activated protein kinase activa tion, yet it does not alter the synergistic effect of stem cell factor on T PO-induced proliferation; and (4) TPO-induced proliferation of early, prima ry megakaryocyte-lineage cells does not require Stat-5 phosphorylation, The system reported provides an improved approach for Mpl structure-function s tudies, and the method can be applied to any hematopoietic lineage, (Blood, 2001;97:1653-1661) (C) 2001 by The American Society of Hematology.