MECHANISM OF ACTION OF A HUMAN GRANULOPOIETIN--GM-CSF

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Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the proliferation and maturation of normal bone marrow hematopoietic progenitor cells. GM-CSF is currently employed to ameliorate myelosuppression caused by chemotherapy and radiation and to facilitate recovery following bone marrow transplantation. More recently, GM-CSF has been employed as a "harvest hormone" to mobilize progenitor cells to the peripheral blood. These strategies provide powerful new tools in cancer therapy, and for future gene therapy strategies. The goal of the studies described in this competitive renewal application is to continue our studies on the mechanism of action of human GM-CSF. Based on the progress achieved over the past funding period, three Specific Aims are proposed: l. To define the critical regions required for function of the alpha and beta subunits of the GM-CSF receptor. These studies employ site-directed and truncation mutants of both the alpha and beta subunits of the GM-CSF receptor to identify regions of the molecules required for ligand binding, internalization and signal transduction. In addition, cell lines of neural crest origin which express non-functional but intermediate-affinity receptors will be utilized to characterize the structure of alpha and beta subunits found in these cells. 2. To identify and characterize additional molecular components necessary for the function of the GM-CSF receptor. Strategies are outlined to identify putative tyrosine kinases and other intracellular components which can interact with the alpha and beta subunits to generate functional GM-CSF receptor. Cell lines derived from neural crest tissues will be used as a biological background to introduce additional putative receptor components required to generate high-affinity functional GM-CSF receptors. 3. Employ early response gene induction as an endpoint to define biochemical pathways mediating GM-CSF action. We will continue our studies to identify cis-acting DNA sequences in the promoter of the early response gene, EGR-l, which are responsive to GM-CSF. Proteins interacting with these sequences will be identified and examined for post- translational modification in response to GM-CSF.