The role of heterotrimeric G proteins in the signaling of Wnts has been established, from flies to mammals. Wnts signal, in part, via heptahelical G protein coupled receptors (GPCR) to effectors systems that control fundamental processes, such as adipogenesis, bone formation, cell progression, cell fate, and other aspects of early development. Dysregulation of Wot signaling provides a basis for human disease, including cancer. The phosphoprotein Dishevelled (Dvl) is essential to Wnt/p-catenin (canonical), Wnt/Ca2+/cGMP, and Wnt/planar cell polarity (PCP) pathways. Wnts promote mouse F9 embryonic teratocarcinoma cells (F9) to primitive endoderm (PE) and activate each of these effector pathways, via 1/more of 3 mammalian Dvls. We propose 3 specific aims: (i) to define functional roles of mammalian Dvls on each major Wnt-stimulated pathway using siRNA and read outs of p-catenin stability, Lef/Tcf-sensitive transcription, Ca2+ imaging and cGMP analysis, INK activation via Rho, and PE formation;(ii) to establish multivalency of Dvl interactions with GPCR, G proteins, kinases/phosphatases, and adaptor molecules critical to Wnt signaling making use of tandem affinity-based purification (TAP) and advanced proteomics (M ALDI, QToF, and nanospray mass spectrometry), siRNA-based knock downs, enzyme inhibitors/dominant negative constructs, and eventual mutagenesis of potential docking sites;and (iii) to establish the functional consequences of spatial localization and trafficking of Dvl in na'ive and Wnt-activated F9 cells, focusing on defining the dynamic aspects of activation /deactivation using fluorescence-based strategies (including 2-photon,/cs, and TIRF microscopy), energy transfer measurements In live cells (via BRET), and cell fractionation coupled with TAP-tagged Dvl followed by proteomics. The overarching hypothesis is that Dvls function as dynamic scaffolds for integration of cell signaling to a spectrum of diverse pathways, much like the AKAP protein family members function. Dvls both dock and are candidate substrates for many kinases/phosphatases, although the details of these interactive regulations remain to be established by proteomics. Real-time measurements of docking in live cells are made possible by BRET and parallel biochemical /proteomics assays that can define the status of Dvl with respect to protein phosphorylation. The functional basis for 3 mammalian Dvls, for their spatial and temporal trafficking, and for cell membrane association are addressed using in vivo and in vitro strategies. Understanding the multivalency and docking of Dvl open opportunity to intervene in the primary signaling pathways controlled by Wnts, including those whose dysregulation leads to cancer, birth defects, and altered bone formation. Dvls, as multivalent dynamic scaffolds, are high-value likely targets for new therapies.
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