Molecular Dissection of Mouse Wnt3a-Frizzled8 Interaction Reveals Essential and Modulatory Determinants of Wnt Signaling Activity

Wnt/β-catenin signaling plays a critical role in animal development and adult tissue homeostasis, including regulation of cell fate decisions, axial pattering, organogenesis and stem cell maintenance. Deregulation of Wnt signaling causes many human diseases, including cancer and osteoporosis. Wnt proteins constitute a large family of growth factors characterized by conserved cysteine and a lipid modification at their N-terminal half. Upon transport through a specialized secretary route they act as morphogens in variety of tissues by forming concentration gradient in the extracellular space. The post-translational addition of palmitate or palmitoic acid to Wnts renders them highly hydrophobic and is believed to control their membrane localization, it is unknown, however, how Wnts are recruited from the membrane to signaling complexes and how their signaling range is regulated. Wnts bind to Frizzled (Fz) seven transmembrane receptors and LRP5/6 co-receptors, which leads to stabilization of the transcription coactivation of β-catenin and activation of target gene. The structural basis of Wnt signaling by receptor binding has long been elusive. Only recently, the crystal structure of the Xenopus Wnt8- Frizzled8-CRD complex was solved, but the significance of the interaction sites for signaling has not been assessed. In the present thesis, by using a structure-based mutagenesis approach, I present an extensive structure-function analysis of mouse Wnt3a by in vitro and in vivo evaluation. Evidence is provided for an essential role of Serine 209, Glycine 210 (site 1) and Tryptophan 333 (site 2) in Fz binding. Importantly, I discovered that Valine 337 in the site 2 binding loop is critical for signaling without contributing to binding. Mutations in the presumptive second CRDbinding site (site 3) partly abolished Wnt binding, suggesting Fz dimerization as a necessary step in signaling. Intriguingly, most site 3 mutations increased Wnt signaling, probably by inhibiting Wnt-CRD oligomerization. In accordance, it was observed that increasing amounts of soluble Fz8-CRD protein modulated Wnt3a signaling in a biphasic manner. Based on these finding, a model was developed, in which a concentration-dependent switch in Wnt-CRD complex formation from an inactive aggregation state to an activated, high mobility state, represents a modulatory mechanism in Wnt signaling gradients