The hypothesis to be tested is that the effects of interleukin-3 (IL-3) on proliferation of murine IL-3-dependent myeloid cell lines (and, by inference, normal murine myeloid stem cells) may be explained in terms of signal transduction mediated by guanine nucleotide binding regulatory proteins (G proteins). G proteins mediate the transduction of a remarkably diverse group of extracellular signals, to a relatively limited number of intracellular effector enzymes. The signal of interest in the proposed study is the growth factor peptide, IL-3, that exerts its action via binding to specific plasma membrane receptors on murine myeloid stem cells. As a result of our preliminary studies of IL-3 signal transduction at the cellular level, phosphatidylinositol 4,5, bisphosphate-specific phospholipase C will be a major focus of the proposed studies, in addition to the G proteins themselves. Using IL-3 dependent murine myeloid cell lines as the principal cellular system, we propose to: 1) characterize the early metabolic and late cellular responses to IL- 3 recetpor-ligand interaction, 2) purify and characterize the major G proteins from these cells, 3) purify and characterize the (IL-3)-stimulated phosphatidylinositol 4,5, bisphosphate-specific phsopholipase C from these cells. The early metabolic responses to IL-3 to be studied are those resulting from changes in phosphatidylinositol 4,5, bisphosphate-specific phospholipase C activity (as reflected by phosphoinositide 4,5, bisphosphate hyrolysis, sn 1, 2, discylglycerol generation, and inositol 1, 4, 5 trisphosphate generation). The molecular basis for IL-3 induced PIP2 hydroylsis will be studied by development of a reconstitution assay for G protein-coupled PIP2-specific phospholipase C enzymatic activity. This assay will be used in the subsequent purification of reconstitutively active G protein-coupled PIP2- specific phospholipase C from the plasma membranes of IL-3 dependent cell lines.
