Signaling mechanism and regulation of Frizzled

Wnts are secreted proteins important to many biological processes. frizzled genes encode a family of Wnt receptors that signal to the intracellular compartment through the cytosolic protein Dishevelled. Frizzled proteins are predicted to form seven transmembrane segments, similar to receptors that couple to heterotrimeric G-proteins, leading to the hypothesis that Frizzleds also couple with G-proteins to conduct Wnt signaling. If this is the case, like other G-protein coupled receptors (GPCRs), Frizzleds could be regulated by phosphorylation. In my thesis work, I address two questions. First, is phosphorylation involved in the regulation of Frizzled signaling in a manner similar to GPCRs? Second, do G-proteins conduct Wnt/Frizzled signaling? We found that Xenopus Frizzled-3 is phosphorylated in a Dishevelled-dependent manner that appears to require the DEP domain of Dishevelled. Serine-576 in the C-terminal tail is required for maximal phosphorylation of Frizzled-3. Mutation of serine-576 enhances Frizzled-3 mediated neural crest induction, suggesting that C-terminal phosphorylation plays a role in down regulating Frizzled signaling. Additional sites are also phosphorylated in Frizzled-3; mutation of multiple, predicted phosphorylation sites in the C-terminal tail further reduces phosphorylation and significantly increases the potency of Frizzled-3 to disrupt convergence/extension movements during gastrulation, an assay of the planar cell polarity function of Frizzleds. To explore whether G-proteins are involved in Wnt signaling, we used loss-of-function approaches in tissue culture cells and in Xenopus embryos. Our preliminary data support a role for Gαq/11 in canonical Wnt signaling, but further work is required to substantiate these findings