Molecular genetics of Drosophila Wnt signaling

果蝇 Wnt 信号传导的分子遗传学

• 批准号: 6654398
• 负责人: MARCEL WEHRLI
• 金额: $ 26.43万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-02 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6654398
• 关键词: DNA footprinting; Drosophilidae; SDS polyacrylamide gel electrophoresis; biological signal transduction; carcinogenesis; cell cell interaction; cell growth regulation; developmental genetics; gene interaction; genetically modified animals; immunoprecipitation; mass spectrometry; membrane proteins; polymerase chain reaction; protein binding; protein protein interaction; protein structure function; receptor expression; western blottings; yeast two hybrid system

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to understand how Wnt signals regulate normal development and cancer. The immediate goal is to refine our understanding of how Wnt signals are transduced by studying Arrow, a single pass transmembrane protein recently identified by the PI. Arrow, together with members of the Frizzled (Fz) family, is a co-receptor for the fly Wnt, Wingless (Wg). A fusion of the Arrow cytoplasmic tail to the Fz2 carboxy-terminus (Fz2-Arr[i]) recapitulates the proximity normally generated during receptor activation by the Wnt ligand, demonstrating that Arrow and Fz2 function as co-receptors. Fz2-Arr[i] functions as an activated receptor that is necessary and sufficient to generate ligand- independent signaling. In the first aim, structure/function analyses of this chimeric receptor, which is locked in the signaling conformation, will be performed to identify domains that may be required for assembly of intracellular complexes that transduce Wnt signals from the membrane to the nucleus. The second aim will identify Arrow-interacting proteins by (a) Immunoprecipitating and micro sequencing Arrow-associated proteins from fly embryos, and (b) a novel and highly sensitive yeast two-hybrid system that allows the isolation of components interacting with a membrane protein, the activated receptor. Fly genetics will then be used to identify the in vivo function of Arrow- interacting proteins. The third aim is based on our finding that Axin, a far downstream component of the Wnt cascade, directly interacts with the Arrow cytoplasmic domain (and therefore the activated receptor). This interaction was unexpected since Axin was previously shown to bind and negatively regulate Armadillo (fly B-catenin), the final cytoplasmic Wg/Wnt pathway component. This raises the possibility that Arrow not only functions as a receptor subunit but also regulates the final step in signaling by binding Axin, thereby freeing Armadillo to enter the nucleus and signal. This hypothesis will be tested by analyzing Wnt signaling in transgenic flies made to express mutant Axin proteins that discriminate between binding to Arrow or Armadillo. Collectively, these studies will provide insights into a signaling pathway that is required for normal development of all species and insights into cancers due to deregulation of these processes.

描述（由申请人提供）：拟议研究的长期目标是了解Wnt信号如何调节正常发育和癌症。 直接的目标是完善我们对Wnt信号如何通过研究箭头转导的理解，这是PI最近通过PI鉴定的单个通过跨膜蛋白。 箭与卷曲（FZ）家族的成员一起是苍蝇，无翅（WG）的共同受体。 箭头细胞质尾巴融合到FZ2羧基末端（FZ2-arr [i]）概述了Wnt配体在受体激活期间正常产生的接近度，这表明箭头和FZ2功能充当共受体。 FZ2-arr [i]充当活化受体，足以产生配体独立信号传导。 在第一个目的中，将进行该嵌合受体的结构/功能分析，该嵌合受体将锁定在信号构象中，以识别将Wnt信号从膜转移到核的细胞内复合物可能需要的域。 第二个目标将通过（a）从蝇胚胎中鉴定出（a）免疫沉淀和微测序箭头相关蛋白，（b）一种新颖且高度敏感的酵母两杂交系统，允许分离与膜蛋白（激活受体）与膜蛋白相互作用的组件分离。 然后，将使用蝇遗传学来识别箭头相互作用蛋白的体内功能。 第三个目的是基于我们的发现，即Wnt Cascade的Axin（远下游）与箭头细胞质结构域（因此是活化受体）直接相互作用。 这种相互作用是出乎意料的，因为先前显示AXIN结合并负调节Armadillo（Fly B-catenin），这是最终的细胞质WG/WNT途径成分。 这增加了箭头不仅充当受体亚基，而且还通过结合轴结合轴来调节信号传导的最后一步，从而使armadillo释放了armadillo进入核和信号。 该假设将通过分析用于表达突变轴蛋白的转基因果蝇中的Wnt信号传导来检验，该蛋白会区分与箭头或arrawillo的结合。 总的来说，这些研究将提供有关信号通路的见解 由于这些过程的放松管制，所有物种的正常发展和对癌症的见解。