SOURCES AND ACTIONS OF HUMAN GM-CSF AND MULTI-CSF

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DESCRIPTION: (Adapted from investigator's abstract). Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3) are two members of a family of hematopoietic growth factors (HGFs). They stimulate the proliferation and differentiation of primitive hematopoietic stem cells, and the function of mature cells; furthermore, they act synergistically both in vitro and in vivo. Our long-term goal is to understand the molecular events that mediate GM-CSF and IL-3 show reciprocal cross competition for binding to the surface of cells that bear both receptors; this occurs at 4 degrees-C, indicating that it is probably due to interaction at the cell surface. Crosslinking studies suggest that both the GM-CSF and the IL-3 receptors are complex heterodimeric or heterotrimeric proteins. We wish to test the hypothesis that high affinity binding of GM-CSF and IL-3 requires expression, for each receptor, of a unique and a common subunit; and that on cells that express both receptors they interact to form a GM-CSF/IL-3 complex linked by the common subunit. We plan specifically to test this hypothesis by isolating cDNAs that encode the GM-CSF an IL-3 receptor subunits. We will investigate structure function relationships of the receptor subunits by transfection of mutants into factor-dependent murine cell lines and by antipeptide monoclonal antibodies. We aim to determine the structural requirements for high/low affinity binding and cross competition by cotransfection of cDNA's into receptor negative cells. We plan to identify proteins that associate with the receptor after ligand binding by phosphoprotein and receptor immunoprecipitation analyses. Last, we aim to investigate receptor function in patients with inherited bone marrow failure syndromes such as Kostmann's and Diamond Blackfan anemia. We will use PCR methods to carry out linkage analysis in affected families and sequence putative mutant receptors. We believe that such studies of HGF receptor function in both normal and abnormal individuals will provide insight into the molecular regulation of hematopoiesis and lead to new therapeutic possibilities.