The goals of this research program are to better understand the mechanisms which regulate the expression of early acting hematopoietic growth factors and to characterize the interaction of these proteins with their cell surface receptors. Three of these proteins will be studied in detail, stem cell factor (SCF, or KIT ligand or mast cell growth factor), interleukin (IL-) 3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). Having previously described the cis-acting genetic elements involved in IL- 3 transcriptional enhancement, we propose to clone and characterize the trans-acting proteins responsible for the enhancer function detected 160 BP upstream of the transcriptional start site, a site necessary but not sufficient for IL-3 gene expression. We will utilize two expression cloning strategies, and will characterize the functional capacity of the resultant clones using in vitro transcription assays, transient transfection studies and antisense inhibitor technology. Second, to better understand the control of early events in hematopoiesis, we will characterize the cis- and trans-acting elements involved in the tissue- specific regulation of human SCF. Using DNase I hypersensitivity site mapping, mobility shift and DNase footprinting assays, and reporter gene analysis we will correlate the sites of DNA-nuclear protein interaction with their capacity to activate the SCF and heterologous promoters. Third, we will express normal, truncated and mutated GM-CSF receptors in non receptor bearing cells to better understand the structural features of the two receptor polypeptides responsible for subunit association and for high affinity GM-CSF binding. the mechanism of high affinity GM-CSF-GM-CSF receptor interaction will also be explored using conformation dependent anti-receptor monoclonal antibodies. And fourth, we will develop a series of interspecies chimera, point mutations, and quaternary structure mutants of human and murine SCF to define the site(s) of SCF interaction with the SCF receptor, the role of the homodimeric structure on SCF signal transduction, and the structural signals responsible for release of soluble SCF from its membrane bound intermediate.
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