PHOSPHOLIPASE C GAMMA 1--BIOCHEMISTRY AND BIOLOGY

磷脂酶 C GAMMA 1--生物化学和生物学

• 批准号: 6362629
• 负责人: GRAHAM F CARPENTER
• 金额: $ 41.83万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-19 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6362629
• 关键词: X ray crystallography; biological signal transduction; cell differentiation; crystallization; enzyme activity; enzyme structure; gene deletion mutation; genetically modified animals; growth factor; laboratory mouse; mutant; phenotype; phospholipase C; protein structure function; tissue /cell culture

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.

通过生长因子受体复合物转导信号转导 调节细胞增殖以及信号传导元素 基因改变，细胞转化是经常的结果。 在大量信号转导蛋白中 活化生长因子受体的下游是酪氨酸 激酶底物磷脂酶C-Gamma 1（PLC-GAMMA 1），A 生成调节的第二信使分子的酶 细胞内钙水平和蛋白激酶C活性。 这 本申请中提出的研究解决了 PLC-GAMMA 1及其在生长因子信号传导中的作用。 第一个 PLC-GAMMA 1的客观问题的结构/功能分析1 分子。 诱变的各种结构结构域 分子将用于探索单个域中的作用 PLC酶活性的控制。 SRC同源域， 位于PLC-GAMMA 1序列的中心，将是 作为基础分子内调节的可能来源 和生长因子刺激的酶活性。 实验 将涉及对培养细胞中PLC-GAMMA 1突变体的分析 和选定的PLC-GAMMA 1突变体分子 杆状病毒系统。 此外，我们将与 结晶组生产X射线质量晶体 蛋白质用于结构测定。 第二和第三区 调查涉及PLC-GAMMA的生物学功能1 在细胞和完整动物中。 后者是由 破坏小鼠中PLC-GAMMA 1的基因，这会产生一个 胚胎致死表型。 我们建议实验以营救 这种表型并诱导PLC-GAMMA 1基因破坏 成人动物。 PLC-GAMMA 1无效细胞系已产生 来自转基因动物。 这些单元线将用于解决 PLC-GAMMA 1在生长因子诱导的增殖中的作用 细胞培养系统并分析PLC-GAMMA 1的行为 在完整细胞的背景下突变体。