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.
Understanding the development of human cancers and birth defect disorders remains dependent on identifying key signaling molecules and their signal transduction pathways that contribute to this pathology. One key-signaling molecule that has been demonstrated to play causative roles in human cancer is the Wnt protein. Our studies are focused on understanding the role of the Wnt receptor molecule Frizzled in transducing Wnt signaling. We propose that these studies can provide important new insights into the mechanisms regulating embryogenesis as well as gaining insights into how deregulated Wnt signaling contributes to cancer formation and birth defect disorders.
