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 understanding of how Mpl activation stimulates proliferation of megakaryocyte-lineage cells is based largely on the engineered expression of Mpl in nonmegakaryocyte-lineage cell lines. However, the relevance of these findings to Mpl signaling in primary megakaryocyte-lineage cells remains largely unknown. Therefore, a system was developed to study Mpl function in primary mpl(-/-) megakaryocyte-lineage cells. Expressing avian retroviral receptors on the surfaces of mammalian cells overcomes their natural block to avian retroviral infection; 815 bp of human GPIIb regulatory sequence was used to generate transgenic mice with megakaryocyte-lineage expression of the subgroup A avian leukosis virus receptor, TVA. Avian retroviral infection of unfractionated bone marrow from these mice is restricted to megakaryocyte-lineage cells. The transgenic mice were crossed to an mpl(-/-) background generating GPIIb-tva+mpl(-/-) mice. By using avian retroviruses to express wild-type or mutant Mpl on the surfaces of primary megakaryocyte-lineage cells, it was demonstrated that (1) the 10 membrane-proximal, cytoplasmic amino acids of Mpl 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 activation, yet it does not alter the synergistic effect of stem cell factor on TPO-induced proliferation; and (4) TPO-induced proliferation of early, primary megakaryocyte-lineage cells does not require Stat-5 phosphorylation. The system reported provides an improved approach for Mpl structure-function studies, and the method can be applied to any hematopoietic lineage.
Department of Laboratory Medicine, University of California, San Francisco 94143-0100, USA.
M
M
Gaur
G J
GJ
Murphy
F J
FJ
deSauvage
A D
AD
Leavitt
D000595:Amino Acid Sequence; D000818:Animals; D001354:Avian Leukosis Virus / Q000235:genetics; D030161:Avian Proteins; D002455:Cell Division / Q000187:drug effects; D019070:Cell Lineage; D004357:Drug Synergism; D005822:Genetic Vectors; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008817:Mice, Mutant Strains / Q000235:genetics*; D008822:Mice, Transgenic / Q000235:genetics*; D023421:Models, Animal; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D011991:Receptors, Virus / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D019089:Stem Cell Factor / Q000494:pharmacology; D013926:Thrombopoietin / Q000494:pharmacology; D014162:Transfection / Q000379:methods
D030161:Avian Proteins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D011991:Receptors, Virus; D019089:Stem Cell Factor; C088745:Tva receptor; C082218:MPL protein, human; D013926:Thrombopoietin