Identifying and characterizing new components for Wnt signaling

 描述（由申请人提供）：受精卵发育为多细胞生物体是几种信号通路相互作用的结果。胚胎发生所需的这些信号传导途径之一是Wnt途径，该途径调节细胞命运决定、增殖、迁移和极性。Wnt配体及其信号传导组分除了调节胚胎发生之外，还与肿瘤发生有关，并在人类结肠癌和乳腺癌以及出生缺陷疾病中起致病作用。因此，阐明Wnt信号转导机制对于我们理解调控胚胎发生和肿瘤发生的分子机制仍然至关重要。 研究表明，Wnt信号传导通过卷曲（Fz）受体沿着LRP 5/6共受体在质膜上启动。在Wnt结合后，通过细胞质蛋白Disheveled（Dvl）的信号传导诱导β-连环蛋白（β-cat）的稳定化和核转位，其然后调节Wnt靶基因的转录诱导。该途径被称为经典Wnt途径。此外，存在称为非经典途径的β-cat独立途径，其功能是调节细胞极性和运动性。非经典途径也由Fz蛋白和Dvl介导。然而，是什么决定了Wnt信号如何分支到这些不同的通路仍然是一个知之甚少的现象。Fz的作用在其抑制Wnt信号传导的能力方面仍然是一个谜，因为迄今为止只有少数Fz相互作用蛋白被鉴定。是否存在与Fz相互作用的其他蛋白及其在调节Wnt信号传导中的作用仍不清楚。我们假设，额外的身份不明的Fz相互作用蛋白存在，发挥功能性调节作用的Wnt信号转导。 为此，我们采用了一种新的基于膜的筛选方法来分离Fz相互作用蛋白，我们的初步研究已经显示出这种方法的良好可行性，因为我们已经分离出了新的Fz相互作用蛋白。我们已经进一步开始使用快速和简便的检测方法在非洲爪蟾和我们的初步表征这些分子的功能特性揭示了一些这些确定的蛋白质的推定的调节作用。在这个应用中，我们提出了两个具体的目标，集中在扩大这个屏幕和执行生化，细胞生物学和体内表征所确定的Fz相互作用蛋白 来描述它们在Wnt信号通路中的作用。我们的研究将主要利用哺乳动物培养细胞和非洲爪蟾胚胎作为模型系统。 我们提出的研究共同致力于揭示和理解新的Fz相互作用蛋白作为Wnt信号传导的关键转导子的作用。我们的初步数据强烈支持，这种筛选方法将提供重要的新的组件，最终将有助于确定从质膜到细胞内环境的Wnt信号的分子性质。最终，这些研究可以导致对Wnt信号失调如何导致癌症形成和出生缺陷疾病的清晰理解。

 DESCRIPTION (provided by applicant): The development of a fertilized egg into a multicellular organism results from the interplay between several signaling pathways. One of these signaling pathways required for embryogenesis is the Wnt pathway and this pathway regulates cell fate determination, proliferation, migration and polarity. The Wnt ligand and its signaling components, in addition to regulating embryogenesis, are implicated in tumorigenesis and play causative roles in human colon and breast cancers as well as birth defect disorders. The elucidation of the mechanisms of Wnt signaling therefore remains vital for our understanding of the molecular mechanisms regulating embryogenesis and tumorigenesis. Studies have shown that Wnt signaling is initiated at the plasma membrane via the Frizzled (Fz) receptor along with the LRP5/6 co-receptors. Upon Wnt binding, signaling through the cytoplasmic protein Disheveled (Dvl) induces the stabilization and nuclear translocation of beta-catenin (beta-cat), which then regulates transcriptional induction of Wnt-target genes. This pathway is termed the canonical Wnt pathway. Additionally, there exists a beta-cat independent pathway termed the non-canonical pathway that functions to regulate cell polarity and motility. The non-canonical pathway is also mediated by the Fz protein and Dvl. However what determines how the Wnt signaling branches into these distinct pathways remains a poorly understood phenomenon. The role of Fz remains somewhat of a puzzle in terms of its ability to transduce Wnt signaling as only a handful of Fz-interacting proteins have been identified to date. Whether additional proteins that interact with Fz exist and their roles in modulating Wnt signaling remains unclear. We hypothesize that additional unidentified Fz-interacting proteins exist that play functional modulatory roles for transducing Wnt signaling. Towards this end, we have employed a novel membrane-based screening methodology to isolate Fz-interacting proteins and our preliminary studies have already shown excellent feasibility of this approach as we have isolated novel Fz-interacting proteins. We have further begun to functionally characterize these molecules using rapid and facile assays in Xenopus and our preliminary characterization reveals putative modulatory roles for some of these identified proteins. In this application, we propose two specific aims centered on expanding this screen and performing biochemical, cell biological and in-vivo characterization of the identified Fz-interacting proteins to delineate their roles in the Wnt signaling pathway. Our studies will utilize mammalian culture cells and the Xenopus embryo primarily as model systems. Our proposed studies together are directed towards uncovering and understanding the role of the novel Fz-interacting proteins as key transducers of Wnt signaling. Our preliminary data strongly supports that this screening methodology will provide important new components that will ultimately help to define the molecular nature of Wnt signaling from the plasma membrane to the intracellular milieu. Ultimately these studies can lead to a lucid understanding of how deregulated Wnt signaling results in cancer formation and birth defect disorders.