Molecular Genetics of Drosophila MAGUK's

本项目主要研究果蝇膜相关鸟苷酸激酶同源物（MAGUK）Dlg及其在控制成虫盘和幼虫脑细胞增殖中的作用。 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末端区域和EGFR途径的两种调节剂Kek-1和D-Cbl结合。将使用免疫印迹和酵母双杂交试验来确认和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突变对其定位和体内EGFR途径活化的影响来检验。 EGFR、Kek-1和D-Cbl被定位并且它们的活性通过它们的C-末端区域调节的预测将通过在体内表达C-末端截短形式来测试，并检查对每种蛋白质的定位及其在途径活化和增殖控制中的功能的影响。 在小杆线虫中鉴定为控制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，并且它们与Dlg形成复合物。 将研究其亚细胞定位、与EGFR及其其他结合伴侣的相互作用，并通过分离和表征功能丧失突变来鉴定其功能。 Dlg及其结合伙伴，以及其他一些肿瘤抑制基因，在控制幼虫脑的生长的作用进行了分析。 这项工作将包括一个已知的结合伙伴（针）的Dlg-SH 3域，像Dlg，是需要在幼虫脑细胞增殖控制的遗传和分子研究。 将努力开发荧光共振能量转移显微镜的使用，以检查在活细胞中的伴侣结合的动力学，以及在dlg和其他突变体中的结合伴侣的错误定位。 这项工作将提供一种遗传学方法来了解Dlg及其哺乳动物同源物的功能，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.