Signal transduction through growth factor receptor complexes regulates cell proliferation and when signaling elements are genetically altered, cell transformation is a frequent consequence. Among the large number of signal transducing proteins downstream from activated growth factor receptors is the tyrosine kinase substrate phospholipase C-gamma 1 (PLC-gamma 1), an enzyme which generates second messenger molecules that regulate intracellular calcium levels and protein kinase C activity. The research proposed in this application addresses the regulation of PLC-gamma 1 and its role in growth factor signalling. The first objective concerns structure/function analysis of the PLC-gamma 1 molecule. Mutagenesis of various structural domains within the molecule will be used to explore the role of individual domains in the control of PLC enzyme activity. Src homology domains, located in the center of the PLC-gamma 1 sequence, will be explored as a possible source of intramolecular regulation of basal and growth factor-stimulated enzyme activity. The experiments will involve analysis of PLC-gamma 1 mutants in cultured cells and of selected PLC-gamma 1 mutant molecules expressed in the baculovirus system. Additionally, we will collaborate with crystallography group to produce x-ray quality crystals of this protein for structural determination. The second and third areas of investigation concern the biological function of PLC-gamma 1 in cells and in the intact animal. The latter is addressed by disrupting the gene for PLC-gamma 1 in mice, which produces an embryonic lethal phenotype. We propose experiments to rescue this phenotype and to induce PLC-gamma 1 gene disruption in the adult animal. PLC-gamma 1 null cell lines have been generated from transgenic animals. These cells lines will be used to address the role of PLC-gamma 1 in growth factor-induced proliferation in cell culture systems and to analyze the behavior of PLC-gamma 1 mutants within the context of the intact cell.
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