The development of blood cells requires the interplay of hematopoietic stem and progenitor cells, marrow stroma and polypeptide growth factors. Although many proteins support the expansion of megakaryocytic precursor cells, identification of the late acting, lineage specific growth factor for platelet production, termed Thrombopoietin (Tpo), has remained elusive. Recently, characterization of the proto-oncogene c-mpl revealed structural homology with the hematopoietic cytokine receptor family. Based on the cell of origin of its cDNA, we hypothesized that the ligand for c- mpl might be identical with Tpo, and in collaboration with scientists at ZymoGenetics recently cloned its cDNA. Using recombinant protein we have shown that the mpl-ligand displays all of the expected biological properties of the major regulator of megakaryocyte development, and proposed that it be termed Thrombopoietin (Tpo). In the present proposal we hope to expand upon these results and study its cellular and molecular biology. Specifically, we propose a research plan of three specific aims. In the first, we will determine whether Tpo gene expression is regulated, localize the cellular site(s) of its production, identify the proximate signal for Tpo release and establish an in vitro model to study the molecular mechanisms underlying its expression. Although Tpo was initially cloned based on its ability to stimulate cells expressing c-mpl, multiple lines of evidence suggest that alone, this cell surface protein may not constitute the high affinity signalling Tpo receptor. To precisely define the molecular composition of the complete Tpo receptor, our second specific aim is designed to determine the binding affinity of ligand for the various component parts, reconstitute a cell surface complex which transmits a Tpo signal in naive cells, and identify the critical secondary messengers responsible for Tpo-induced proliferation and differentiation of target cells. We strongly suspect that this involves potentially novel transcription factors and propose to clone cDNA for the proteins involved in Tpo-induced signalling. And in the third specific aim, we will study the effects of Tpo on megakaryocytes and their purified progenitors, on both the cellular and molecular levels. We anticipate that successful completion of the proposed research will provide insights into the biology of platelet production and in a more general way, contribute to our understanding of the regulation of cell proliferation and differentiation.
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