PHOSPHOLIPASE C GAMMA 1--BIOCHEMISTRY AND BIOLOGY

通过生长因子受体复合物的信号转导 调节细胞增殖，当信号传导元件 基因改变，细胞转化是一个常见的后果。 在众多的信号转导蛋白中， 活化的生长因子受体的下游是酪氨酸 激酶底物磷脂酶C-γ 1（PLC-γ 1）， 产生第二信使分子的酶， 细胞内钙水平和蛋白激酶C活性。 的 本申请中提出的研究解决了以下问题的调节： PLC-γ 1及其在生长因子信号传导中的作用。 第一 目的涉及PLC-γ 1的结构/功能分析 分子。 基因组中各种结构域的突变 分子将被用来探索单个结构域在 PLC酶活性的控制。 Src同源结构域， 位于PLC-γ 1序列的中心， 探索作为一个可能的来源，分子内调节的基础 和生长因子刺激的酶活性。 实验 将涉及分析培养细胞中的PLC-γ 1突变体 和选择的PLC-γ 1突变体分子表达在 杆状病毒系统 此外，我们将与 晶体学小组，以生产x射线质量的晶体， 蛋白质结构测定。 第二和第三区域 关于PLC-γ 1的生物学功能的研究 在细胞和完整的动物中。 后一个问题由 破坏小鼠中PLC-γ 1的基因， 胚胎致死表型 我们提出实验来拯救 这种表型，并诱导PLC-γ 1基因破坏， 成年动物已生成PLC-γ 1无效细胞系 从转基因动物。 这些细胞系将用于解决 PLC-γ 1在生长因子诱导增殖中的作用 细胞培养系统，并分析PLC-γ 1的行为 在完整细胞的背景下突变。

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.