The long term objectives of this project are to determine the mechanisms by which erythropoietin (EPO) contributes to the control of the proliferation and differentiation of erythrocytes. Erythroid lineage differentiation initiates with the commitments of a pluripotential progenitor. One marker of lineage commitment is the expression of a high-affinity receptor for EPO and the acquisition of an ability to proliferate in response to EPO and to begin to express additional erythroid lineage specific markers, including the globin genes. The studies proposed in this project focus on the signalling events involved in the response of cells to EPO. In the first specific aim, studies are proposed to identify the functional domains of the EPO receptor that are required for differentiation. Conversely, experiments will be done to define the domains of the IL-3 receptor that can dominantly suppress EPO-induced expression of erythroid markers. In particular, both receptors contain membrane distal, carboxyl-domains that are required for the activation of the ras signalling pathway and a membrane proximal domain that is required for activation of the JAK/STAT pathway. Various mutated receptors will be used to determine which pathways are involved in promoting or suppressing expression of erythroid lineage specific markers. In the second specific aim, the studies focus on the role of novel STAT proteins in signalling in erythroid lineage cells. Recent studies have demonstrated that the substrates of JAK kinases include transcriptional factors of the STAT family. Recently a family member (STAT4) has been cloned that is expressed in myeloid cells and whose expression is extinguished during the continued commitment to the erythroid lineage. Studies are proposed to determine the role of STAT4 in erythroid lineage commitment. Since additional serologically and functionally related proteins exist, experiments are proposed to clone additional STAT proteins and to assess their role in signal transduction through the EPO receptor. Collectively, these studies will contribute to an understanding of the initial signal transduction events that occur through the EPO receptor and contribute to proliferation and differentiation of erythrocytes.
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