Functional analysis of the cytoplasmic domain of the human Mpl receptor for tyrosine-phosphorylation of the signaling molecules, proliferation and differentiation.
To investigate the functional domains for signal transduction of human Mpl, we constructed a series of human c-mpl cDNAs with various deletions in the cytoplasmic domain, and then introduced each cDNA into murine IL-3-dependent myeloid leukemia FDC/P2 cells to establish stable transformants. We examined the growth and differentiation responses and tyrosine phosphorylation of the intracellular signaling proteins including Jak2, Tyk2, Stat3, Stat5, Vav, SHPTP2, Cbl, Shc and Shc-associated p145 when receptor stimulation occurred after thrombopoietin (TPO) binding. TPO stimulated cell proliferation and induced the expression of megakaryocyte lineage-specific AP-51 and CD61 cell surface antigens and tyrosine phosphorylation of the signaling proteins in transformants expressing full length human Mpl. These results suggested that Mpl not only induced proliferation but also transduced megakaryocyte-specific differentiation signals into FDC/P2 cells. Mutational analysis of human Mpl indicated that the N-terminal region of its cytoplasmic domain is necessary and sufficient to transduce proliferation and differentiation signals into cells, while the C-terminal region may also play important roles in transducing the differentiation signals.
Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., Gunma, Japan.
H
H
Morita
T
T
Tahara
A
A
Matsumoto
T
T
Kato
H
H
Miyazaki
H
H
Ohashi
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D006224:Cricetinae; D003600:Cytosol; D017931:DNA Primers; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D051379:Mice; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D018121:Receptors, Cytokine / Q000737:chemistry / Q000502:physiology*; D053628:Receptors, Thrombopoietin; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D017384:Sequence Deletion; D015398:Signal Transduction; D013926:Thrombopoietin / Q000378:metabolism / Q000494:pharmacology*; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D007377:Interleukin-3; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D011994:Recombinant Proteins; C082218:MPL protein, human; D019000:Phosphotyrosine; D013926:Thrombopoietin