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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.

Pubmed ID: 11238104

Authors

  • Gaur M
  • Murphy GJ
  • deSauvage FJ
  • Leavitt AD

Journal

Blood

Publication Data

March 15, 2001

Associated Grants

  • Agency: NHLBI NIH HHS, Id: P50 HL54476

Mesh Terms

  • Amino Acid Sequence
  • Animals
  • Avian Proteins
  • Avian leukosis virus
  • Cell Division
  • Cell Lineage
  • Drug Synergism
  • Genetic Vectors
  • Humans
  • Megakaryocytes
  • Mice
  • Mice, Mutant Strains
  • Mice, Transgenic
  • Models, Animal
  • Neoplasm Proteins
  • Proto-Oncogene Proteins
  • Receptors, Cytokine
  • Receptors, Thrombopoietin
  • Receptors, Virus
  • Signal Transduction
  • Stem Cell Factor
  • Thrombopoietin
  • Transfection