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.

We describe a new method for analyzing embryonic events dependent on a specific peptide recognition signal. A short, specific amino acid sequence in fibronectin has been implicated as a recognition site in fibronectin-mediated interactions. Fibroblast adhesion to fibronectin is competitively inhibited by certain synthetic peptides, including the decapeptide Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro, which appears to contain the cell recognition sequence. We found that this peptide inhibited both amphibian gastrulation and avian neural crest cell migration in vivo, as well as the attachment and migration of neural crest cells in vitro. These processes are major cell migratory events previously suggested to involve fibronectin. Negative controls included another conserved fibronectin peptide from the collagenbinding region containing the sequence Cys-GIn-Asp-Ser-Glu-Thr-Arg-Thr-Phe-Tyr and another peptide. Our results demonstrate the feasibility of using synthetic peptides directed at recognition sites in extracellular proteins as probes of morphogenetic processes, and they provide further support for the hypothesis that fibronectin is involved in gastrulation and neural crest cell migration.

Wnt signaling has an important role in cell-fate determination, tissue patterning and tumorigenesis. Wnt proteins signal through seven-pass transmembrane receptors of the frizzled family to activate β-catenindependent transcription of target genes. Using early Xenopus embryos, we show that frizzled receptors can dimerize and that dimerization is correlated with activation of the Wnt/β-catenin pathway. Co-immunoprecipitation studies revealed that the receptor Xfz3 exists as a dimer when expressed in Xenopus embryos, and it has been shown to activate the Wnt/β-catenin pathway as revealed by expression of the target gene siamois. Xfz3 dimerization requires intramolecular and/or intermolecular disulfide linkages, and the N-terminal extracellular region of the receptor, including the cysteine-rich domain (CRD), is sufficient for dimerization. The receptor Xfz7 behaves differently from Xfz3 when overexpressed in the embryo as Xfz7 is monomeric and is unable to directly activate the Wnt/β-catenin pathway. However, activation of this pathway can be achieved by artificially forcing Xfz7 dimerization. These results provide the first direct evidence for the dimerization of frizzled receptors and suggest that dimerization contributes to transducing the Wnt/β-catenin signal. Research Article 2542 the cytoplasm and nuclei of dorsal blastomeres during early cleavage stages (Larabell et al., 1997). Moreover, overepression of GSK3 and Axin or depletion of maternal β-catenin RNA causes deficiencies in dorsal structures (He et al., 1995; Heasman et al., 1994;Yost et al., 1998;Zeng et al., 1997).The biochemical mechanisms by which the binding of the Wnt ligand to its frizzled receptor elicits signal transduction within the cell are poorly characterized. Numerous studies have suggested that several G-protein-coupled receptor (GPCR) families exist as dimers or even higher structures (Devi, 2001; Gouldson et al., 2000; Hebert and Bouvier, 1998;. Recently, biophysical methods based on luminescence and fluorescence energy transfer have confirmed the existence of such oligomeric complexes in living cells (Angers et al., 2000;Angers et al., 2001; Kroeger et al., 2001;Overton and Blumer, 2000). However, whether dimerization is a general property of this class of receptors and whether this is functionally relevant for signal transduction remains controversial (Cvejic and Devi, 1997; George et al., 1998; Gouldson et al., 1998; Hebert et al., 1998;Marshall et al., 1999). In several cases, receptors appear to fold as constitutive dimers shortly after biosynthesis, whereas ligand-promoted dimerization at the cell surface has been proposed for others (Jones et al., 1998; Kaupmann et al., 1998; Kuner et al., 1999;White et al., 1998). Dimerization is required for normal functioning of β-adrenergic receptors and has been shown to rescue the function of mutant forms of β-adrenergic and angiotensin type I receptors (Hebert et al., 1998).In this report, we address the question of the potential dimerization property of two Xenopus frizzled receptors...