Molecular Genetics of Drosophila MAGUK's

本项目涉及果蝇膜相关鸟苷酸激酶同源物(MAGUK)DLG及其在控制成像盘和幼虫脑细胞增殖中的功能。体外与多肽的结合分析表明，DLG通过其PDZ结构域与表皮生长因子受体(EGFR)的C末端区域以及EGFR途径的两个调节子Kek-1和D-Cbl结合。免疫沉淀和酵母双杂交分析将用于确认和进一步表征这些相互作用。DLG通过控制这些蛋白的亚细胞定位来调控EGFR途径的假设将通过研究DLG突变对它们的定位和体内EGFR途径的激活的影响来检验。EGFR、KEK-1和D-Cbl被定位并通过其C-末端调控活性的预测将通过体内表达C-末端截短的版本来检验，并检测对每个蛋白质定位及其在通路激活和增殖控制中功能的影响。在线虫中发现的控制EGFR定位的两个蛋白(LIN-7和LIN-10)在果蝇中具有密切的同源性，它们与DLG形成复合体。将研究它们的亚细胞定位、与EGFR的相互作用以及它们的其他结合伙伴，并通过分离和鉴定功能丧失突变来确定它们的功能。将分析DLG及其结合伙伴以及其他一些肿瘤抑制基因在控制幼虫大脑生长方面的作用。这项工作将包括对DLG-SH3结构域的已知结合伙伴(Pins)的遗传和分子研究，DLG-SH3结构域与DLG一样，是控制幼虫大脑细胞增殖所必需的。将努力开发使用荧光共振能量转移显微镜来研究活细胞中伙伴结合的动力学，以及DLG和其他突变体中结合伙伴的错误定位。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学方法，其中一些与多种人类癌症密切相关。

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.