MOLECULAR BASIS OF HEMATOPOIETIC GROWTH FACTOR SIGNALING
Hematopoiesis is a complex, dynamic process in which blood cells of all types are produced from a primitive progenitor cell. This process is regulated at multiple levels. The mechanism for one of the earliest echelons of control is mediated by a glycoprotein called interleukin-3 (IL-3), which stimulates the proliferation and differentiation of these primitive progenitors. The cascade of cellular events elicited by the binding of IL-3 to the cell surface is incompletely understood. Although a potential cell surface receptor molecule has been biochemically characterized, and molecularly cloned, the inability of cDNA encoding the receptor to confer high affinity ligand binding, receptor phosphorylation, and ligand-induced down-modulation suggest that additional polypeptide subunits are required to form a biologically active receptor complex. A novel monoclonal antibody to myeloid cells, designated HIM-1, was found to inhibit the binding of IL-3 to various target cells, suggesting that it is physically linked to the structure that binds the ligand. The demonstration that this antibody inhibited the proliferative effect of IL-3 on hematopoietic progenitors and bound to factor-dependent myeloid cells in dramatically reduced levels following IL-3 stimulation further implicates the involvement of the molecule recognized by HIM-1 in the association of IL-3 with the surface of myeloid cells. This proposal outlines studies to biochemically and genetically characterize the role of the 220 kiloDalton glycoprotein (gp220) recognized by HIM-1 in the specific binding of IL-3 to the surface of normal and leukemic hematopoietic cells and in the transduction of the signal to proliferate. Immunoprecipitation experiments will be performed using strategies that preserve inter- molecular associations to demonstrate that gp220 binds directly to IL-3 or is physically associated with polypeptides that bind IL-3. The participation of gp220 in a biologically active IL-3 receptor complex will determined by genetically manipulating its expression. We will test the ability of cDNA encoding gp220 and the low affinity receptor to reconstitute a structure that binds IL-3 at high affinity and transduces its biological activity. This effect of molecular abrogation of gp220 expression on high affinity ligand binding and transduction of biologic activity, since IL-3 stimulation elicits tyrosine-specific protein kinase activity, the participation of this mechanism in ligand-induced down- modulation of gp220 will be determined. Together, these experiments should elucidate the proximal events that control the proliferation of normal hematopoietic progenitors and provide insight into the molecular basis for dysregulated proliferation as occurs in myeloproliferative disorders.