The cytoplasmic protein Dishevelled (Dvl) and the associated membrane-bound receptor Frizzled (Fz) are essential in canonical and noncanonical Wnt signaling pathways. However, the molecular mechanisms underlying this signaling are not well understood. By using NMR spectroscopy, we determined that an internal sequence of Fz binds to the conventional peptide binding site in the PDZ domain of Dvl; this type of site typically binds to C-terminal binding motifs. The C-terminal region of the Dvl inhibitor Dapper (Dpr) and Frodo bound to the same site. In Xenopus, Dvl binding peptides of Fz and Dpr/Frodo inhibited canonical Wnt signaling and blocked Wnt-induced secondary axis formation in a dose-dependent manner, but did not block noncanonical Wnt signaling mediated by the DEP domain. Together, our results identify a missing molecular connection within the Wnt pathway. Differences in the binding affinity of the Dvl PDZ domain and its binding partners may be important in regulating signal transduction by Dvl.

M.Umbhauer and A.Djiane contributed equally to this workFrizzled receptors are components of the Wnt signalling pathway, but how they activate the canonical Wnt/b-catenin pathway is not clear. Here we use three distinct vertebrate frizzled receptors (Xfz3, Xfz4 and Xfz7) and describe whether and how their C-terminal cytoplasmic regions transduce the Wnt/b-catenin signal. We show that Xfz3 activates this pathway in the absence of exogenous ligands, while Xfz4 and Xfz7 interact with Xwnt5A to activate this pathway. Analysis using chimeric receptors reveals that their C-terminal cytoplasmic regions are functionally equivalent in Wnt/b-catenin signalling. Furthermore, a conserved motif (Lys-Thr-X-X-X-Trp) located two amino acids after the seventh transmembrane domain is required for activation of the Wnt/b-catenin pathway and for membrane relocalization and phosphorylation of Dishevelled. Frizzled receptors with point mutations affecting either of the three conserved residues are defective in Wnt/b-catenin signalling. These ®ndings provide functional evidence supporting a role of this conserved motif in the modulation of Wnt signalling. They are consistent with the genetic features exhibited by Drosophila Dfz3 and Caenorhabditis elegans mom-5 in which the tryptophan is substituted by a tyrosine.