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及其在控制图像盘和幼虫大脑细胞增殖中的功能。体外肽结合实验表明，Dlg通过其PDZ结构域与表皮生长因子受体（EGFR）的C端区域以及EGFR通路的两种调节因子Kek-1和D-Cbl结合。免疫沉淀和酵母双杂交试验将用于确认和进一步表征这些相互作用。Dlg通过控制这些蛋白质的亚细胞定位来调节EGFR通路的假设将通过研究Dlg突变对其定位和体内EGFR通路激活的影响来验证。EGFR， Kek-1和D-Cbl的定位及其活性通过其C端区域调节的预测将通过在体内表达C端截断版本来验证，并检查对每种蛋白质定位的影响及其在途径激活和增殖控制中的功能。两种蛋白（Lin-7和Lin-10）在隐杆线虫中被鉴定为控制EGFR定位，它们在果蝇中有密切的同源物，并与Dlg形成复合物。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将被研究，它们的功能将通过分离和功能丧失突变的特征来确定。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脑生长中的作用。这项工作将包括Dlg- sh3结构域的已知结合伙伴（Pins）的遗传和分子研究，该结构域与Dlg一样，是幼虫大脑中细胞增殖控制所必需的。将努力发展荧光共振能量转移显微镜的使用，以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。这项工作将为了解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.