Cytokines act through specific membrane receptors to regulate hematopoietic cell development. A number of intracellular signaling pathways are stimulated by activated cytokine receptors; however, the molecular events involved in initiation of signal transduction, and how the different pathways control hematopoietic cell survival, proliferation, and differentiation, are largely unknown. Abnormal hematopoietic cell growth can lead to the development of disease, including myeloproliferative disorders, leukemia, or lymphoma. In addition, underproduction of crucial hematopoietic cell types can compromise health and survival. The overall goals of this project are to define the role specific protein:protein oligomerization events play in activation of cytokine receptor signal transduction, and to determine the specificity of Jak and Stat signals in erythroid development.
In Aim 1 the role dimerization of the receptor cytoplasmic region plays in activation of downstream signal transduction pathways will be examined, using the erythropoietin receptor (EpoR) as a model of hemodimeric cytokine receptors. Specific dimerization motifs will be fused to the EpoR cytoplasmic tail. The effects of cytoplasmic tail dimerization, in the absence or presence of plasma membrane association, on the activation of signal transduction pathways leading to cell proliferation will be determined.
In Aim 2 the role of homo-or heterodimerization of Jak protein tyrosine kinases in the role of regulation of intrinsic kinase activity will be studied. Chimeric EpoRs containing Jak1 or Jak3 binding domains will be generated and it will be determined if ligand-induced receptor dimerization activates the respective Jak kinase. Dimerization motifs will also be fused directly to Jak1, Jak2, or Jak3 cDNAs to determine if homo- or heterodimerization between specific Jaks regulates their activity. In the third aim, the investigator will determine if signals from Jak2 and Stat5 are specific for erythroid development by generating chimeric EpoRs with altered Jak or Stat signal transduction potential. These will be introduced into growth-factor dependent hematopoietic cells to analyze effects on cell proliferation in vitro. Constitutively active forms of the chimeric receptors will be introduced into hematopoietic progenitor cells and in vitro erythroid differentiation analyzed by colony-forming assays. Effects on in vivo erythropoiesis and contribution to development of leukemia will be analyzed in mice infected with recombinant retroviruses.
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