Molecular Genetics of Drosophila MAGUK's

该项目涉及果蝇膜相关鸟苷酸激酶同源物 (MAGUK) Dlg 及其在控制成虫盘和幼虫大脑中细胞增殖的功能。 体外与肽的结合分析表明，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 途径的两个调节因子 Kek-1 和 D-Cbl 结合。免疫沉淀和酵母双杂交测定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 途径的假设将通过研究 dlg 突变对其定位和体内 EGFR 途径激活的影响进行检验。 EGFR、Kek-1 和 D-Cbl 是定位的，并且它们的活性通过其 C 末端区域调节的预测将通过在体内表达 C 末端截短版本来测试，并检查对每种蛋白质的定位及其在通路激活和增殖控制中的功能的影响。 在秀丽隐杆线虫中被鉴定为控制 EGFR 定位的两种蛋白（Lin-7 和 Lin-10）在果蝇中具有密切的同源性，并且它们与 Dlg 形成复合物。 我们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伴侣，并通过分离和表征功能丧失突变来鉴定它们的功能。 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。 这项工作将包括对 Dlg-SH3 结构域的已知结合伴侣 (Pins) 进行遗传和分子研究，与 Dlg 一样，Dlg-SH3 结构域是幼虫大脑中细胞增殖控制所必需的。 人们将疯狂地努力开发使用荧光共振能量转移显微镜来检查活细胞中配偶体结合的动态，以及 dlg 和其他突变体中结合配偶体的错误定位。 这项工作将提供一种遗传学方法来理解 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.