ABSTRACT
Low density lipoprotein receptor-related protein 5 (LRP5) has been identified as a Wnt co-receptor involved in the activation of the beta-catenin signaling pathway. To improve our understanding of the molecular mechanisms by which LRP5 triggers the canonical Wnt signaling cascade, we have screened for potential partners of LRP5 using the yeast two-hybrid system and identified Frat1 as a protein interacting with the cytoplasmic domain of LRP5. We demonstrate here that LRP5/Frat1 interaction is involved in beta-catenin nuclear translocation and TCF-1 transcriptional activation. The addition of Wnt3a or overexpression of constitutively active truncated LRP5 (LRP5C) induces Frat1 recruitment to the cell membrane. Overexpression of a dominant negative form of disheveled (Dvl) shows that this protein positively affects LRP5/Frat1 interaction. Furthermore, the fact that dominant negative Dvl does not interfere with LRP5C/Frat1 interaction can explain how LRP5C is capable of acting independently of this major Wnt signaling player. Axin, which has been shown to interact with LRP5 and to be recruited to the membrane through this interaction, was found to co-immunoprecipitate with Frat1 and LRP5. We propose that recruitment of Axin and Frat1 to the membrane by LRP5 leads to both Axin degradation and Frat1-mediated inhibition of glycogen synthase kinase-3. As a consequence, beta-catenin is no longer bound to Axin or phosphorylated by glycogen synthase kinase-3, resulting in TCF-1 activation.
Subject(s)
Carrier Proteins/metabolism , Neoplasm Proteins/metabolism , Proteins/metabolism , Receptors, LDL/metabolism , Signal Transduction/physiology , Adaptor Proteins, Signal Transducing , Animals , Axin Protein , Carrier Proteins/genetics , Cell Line , Cell Membrane/metabolism , Dishevelled Proteins , LDL-Receptor Related Proteins , Low Density Lipoprotein Receptor-Related Protein-5 , Mice , Mice, Knockout , Neoplasm Proteins/genetics , Phosphoproteins , Protein Binding , Proteins/genetics , Proto-Oncogene Proteins , Receptors, LDL/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Repressor Proteins/metabolism , Two-Hybrid System Techniques , Wnt Proteins , Wnt3 Protein , Wnt3A ProteinABSTRACT
Activation of the Wnt signaling cascade provides key signals during development and in disease. Wnt signals are transduced by seven-transmembrane Frizzleds (Fzs) and the single transmembrane low density lipoprotein receptor-related proteins 5 or 6. In the course of the analysis of genes regulated by bone morphogenetic protein 2 in mesenchymal cells we found a significant induction of murine Frizzled-1 (mFz1) gene expression. Unexpectedly overexpression of mFz1 dramatically repressed the induction of alkaline phosphatase mediated by either bone morphogenetic protein 2 or Wnt3a in these cells. Moreover mFz1 overexpression significantly repressed both beta-catenin translocation into the nucleus and T cell factor signaling mediated by Wnt3a. Importantly microinjection of mFz1 transcript in Xenopus embryo inhibited the ability of Wnt1 to induce the expression of the Wnt/beta-catenin target gene Siamois in animal cap assay and secondary axis formation in whole embryo. By using chimeric constructs in which N- and C-terminal segments of mFz1 were replaced by the corresponding parts of Xfz3 we demonstrated that the antagonistic activity resides in the cysteine-rich domain of the N-terminal part. The antagonist activity of mFz1 could be prevented by overexpression of Galphaq-(305-359), which specifically uncouples Gq-coupled receptors, suggesting that Galphaq signaling contributes to the inhibition of Wnt/beta-catenin pathway by mFz1. This is the first time that a Frizzled receptor has been reported to antagonize Wnt/beta-catenin.
