Molecular Genetics of Drosophila MAGUK's

果蝇 MAGUK 的分子遗传学

• 批准号: 6320466
• 负责人: PETER J BRYANT
• 金额: $ 23.92万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-14 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6320466
• 关键词: Drosophilidae; cell growth regulation; cell proliferation; developmental genetics; developmental neurobiology; enzyme activity; enzyme induction /repression; epidermal growth factor; fluorescence resonance energy transfer; gene mutation; green fluorescent proteins; growth factor receptors; imaginal disc; immunoprecipitation; larva; molecular genetics; nerve stem cell; neurogenesis; neurogenetics; phosphotransferases; receptor expression; tumor suppressor proteins; yeast two hybrid system

This project concerns the Drosophila Membrane-Associated Guanylate Kinase homolog (MAGUK) Dlg and its function in controlling cell proliferation in imaginal discs and the larval brain. Assays of binding to peptides in vitro indicate that Dlg binds through its PDZ domains to the C- terminal regions of the Epidermal Growth Factor Receptor (EGFR) and to two regulators of the EGFR pathway, Kek-1 and D-Cbl. Immunoprecipiation and yeast two-hybrid assays will be used to confirm and further characterize these interactions. The hypothesis that Dlg regulates the EGFR pathway by controlling the subcellular localization of these proteins will be tested by investigating the effects of dlg mutations on their localization and on activation of the EGFR pathway in vivo. The prediction that EGFR, Kek-1 and D-Cbl are localized and their activity regulated via their C-terminal regions will be tested by expressing C- terminal truncated versions in vivo, and examining the effect on localization of each protein and on its functions in pathway activation and proliferation control. Two proteins (Lin-7 and Lin-10), identified in Caenorhabditis as controlling EGFR localization, have close homologs in Drosophila and they form a complex with Dlg. Their subcellular localization, interactions with EGFR, and their other binding partners will be investigated, and their functions will be identified by isolation and characterization of loss-of-function mutations. The role of Dlg and its binding partners, as well as some other tumor suppressor genes, in controlling growth of the larval brain will be analyzed. This work will include genetic and molecular studies of a known binding partner (Pins) for the Dlg-SH3 domain which, like Dlg, is required for cell proliferation control in the larval brain. An effort will be mad to develop the use of Fluorescent Resonance Energy Transfer Microscopy to examine dynamics of partner binding in live cells, and mislocalization of binding partners in dlg and other mutants. The work will provide a genetic approach to understanding the functions of Dlg and its mammalian homologs, some of which are strongly implicated in a variety of human cancers.

该项目涉及果蝇膜相关的鸟苷酸激酶同源物（MAGUK）DLG及其在控制想象盘和幼虫大脑中细胞增殖方面的功能。 在体外与肽结合的测定表明，DLG通过其PDZ结构域与表皮生长因子受体（EGFR）的C-末端区域结合，并与EGFR途径Kek-1和D-CBL的两个调节剂结合。免疫沉淀和酵母双杂交测定法将用于确认并进一步表征这些相互作用。 DLG通过控制这些蛋白质的亚细胞定位来调节EGFR途径的假设将通过研究DLG突变对其定位的影响以及在体内激活EGFR途径的影响。 EGFR，KEK-1和D-CBL的局部化预测，通过其C末端区域调节其活性，将通过表达c-末端截断版本的体内进行测试，并检查对每种蛋白质及其功能在途径中的位置的影响。 在Caenorhabditis中鉴定为控制EGFR定位的两种蛋白质（LIN-7和LIN-10）在果蝇中具有密切的同源物，它们与DLG形成了复合物。 将研究它们的亚细胞定位，与EGFR的相互作用及其其他结合伙伴的相互作用，并通过隔离和表征功能丧失突变来识别它们的功能。 将分析DLG及其结合伴侣以及其他一些肿瘤抑制基因在控制幼虫大脑生长中的作用。 这项工作将包括针对DLG-SH3结构域的已知结合伴侣（PIN）的遗传和分子研究，该结构域像DLG一样，是幼虫大脑中细胞增殖所必需的。 努力将对使用荧光共振能量转移显微镜来检查活细胞中伴侣结合的动力学以及DLG和其他突变体中结合伴侣的错误定位。 这项工作将为理解DLG及其哺乳动物同源物的功能提供一种遗传方法，其中有些与各种人类癌症有关。