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First published online 6 May 2003
doi: 10.1242/jcs.00451

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Frizzled receptor dimerization is sufficient to activate the Wnt/ß-catenin pathway

Clémence Carron, Aude Pascal, Alexandre Djiane, Jean-Claude Boucaut, De-Li Shi and Muriel Umbhauer*

Laboratoire de Biologie du Développement, Groupe de Biologie Expérimentale, UMR CNRS 7622, Université Paris VI, 9 quai Saint-Bernard, 75005, Paris, France



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  		Fig. 1. Structure of Xfz3 and Xfz7 derivatives used in this study. The myc-tagged proteins contain six myc epitopes inserted at the N-terminus immediately after the peptide signal. The flag and HA tags are located at the C-terminus end of the proteins. Xfz3-{Delta}C-myc corresponds to Xfz3 but retains only five amino acids after the seventh transmembrane domain (Umbhauer et al., 2000aGo). Extra3-myc and extra3-flag correspond to the 196 first amino acids of Xfz3 bearing myc and flag tags, respectively. Extra3-R1 is a chimeric receptor in which the 196 N-terminal amino acids of Xfz3 are fused with the transmembrane and cytoplasmic domains of FGFR-1 (Musci et al., 1990Go). Xfz3{Delta}C-R1 corresponds to the first 505 N-terminal amino acids of Xfz3 fused with the cytoplasmic domain of FGFR-1. Xf7{Delta}C-flag corresponds to Xfz7 truncated immediately after the seventh transmembrane domain at residue 525. In Xfz7-BD, the CRD of Xfz7 is swapped with the oligomerization domain (B domain) of the ETS protein, TEL (Lopez et al., 1999Go).

 


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  		Fig. 2. Xfz3 receptor forms homodimers in Xenopus embryo. (A) Embryos were injected at the two-cell stage with mRNA encoding the Xfz3-myc receptor or the C-terminal truncated form Xfz3{Delta}C-myc. Protein extracts derived from gastrula were immunoblotted following SDS-PAGE under unreduced conditions, using the 9E10 anti-myc antibody. For both constructs, the blot reveals immunoreactive bands corresponding to the expected monomeric form (arrows) as well as a species of higher molecular mass corresponding to twice that of the monomer (arrowhead) or even higher (star). (B) Embryos were injected at the two-cell stage with mRNA encoding Xfz3-HA. Embryonic extracts were immunoblotted using 12CA5 monoclonal antibody as describe in (A). A band corresponding to the expected monomeric form (arrow) plus a higher form corresponding to the expected size for a dimer (arrowhead) are shown. (C) Xfz3{Delta}C-myc mRNA (200 pg) was injected alone or co-injected with Xfz3-HA mRNA (800 pg). Protein extracts were analyzed with the anti-myc antibody. Co-expression of a high amount of Xfz3-HA leads to the disappearance of the band corresponding to the dimeric form of Xfz3{Delta}C-myc. A higher band appears (arrowhead), revealing the presence of Xfz3{Delta}C-myc/Xfz3-HA heterodimer.

 


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  		Fig. 3. Co-immunoprecipitation of different epitope-tagged wild-type and truncated Xfz3 receptors. Embryos were injected with the mRNA encoding the indicated receptor (Xfz3-myc or Xfz3{Delta}C-myc) alone or in combination with Xfz3-HA. Protein extracts from injected embryos were subjected to immunoprecipitation by using the anti-HA antibody (lanes 1, 3, 5, 7) or the anti-myc antibody (lanes 2, 4, 6, 8). Immunoprecipitates were then immunoblotted with the anti-myc antibody. Anti-HA antibody does not immunoprecipitate any myc-positive bands from embryos expressing only Xfz3-HA (lane 7) or Xfz3{Delta}C-myc (lane 1) but does precipitate bands corresponding to Xfz3{Delta}C-myc in co-injected embryos (lane 3). Similar results are obtained when Xfz3{Delta}C-myc is replaced by the full-length Xfz3-myc (lane 5).

 


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  		Fig. 4. Xfz3 dimerization is sensitive to ß-mercaptoethanol. Protein extracts from embryos overexpressing Xfz3{Delta}C-myc were analyzed by western blot with the anti-myc antibody. Before SDS-PAGE, protein samples were incubated with 2% or 20% ß-mercaptoethanol. This treatment leads to a diminution of the dimer/monomer ratio. The dimeric form of the receptor is still strongly detected in extracts treated with 10 mM iodoacetamide (IAA), which prevents the artificial formation of disulfide bonds during sample preparation.

 


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  		Fig. 5. The N-terminal extracellular region of Xfz3 is sufficient to induce dimerization. (A) Co-immunoprecipition of tagged N-terminal extracellular region of Xfz3. mRNA encoding extra3-myc and extra3-flag were injected alone or in combination in two-cell-stage embryos. Protein extracts from injected embryos were subjected to immunoprecipitation by using the anti-myc antibody (lanes 1 and 4) or the anti-flag antibody (lanes 2, 3 and 5). Immunoprecipitates were then immunoblotted with the anti-myc antibody. Extra3-myc is indicated with an arrow. (B) Analysis of Xbra expression in animal caps in response to the wild-type FGFR-1, a constitutive form of FGF-R1 receptor (torso-R1), Xfz3{Delta}C-R1 or extra3-R1. Total RNA extracted from injected animal caps was assayed for Xbra expression at the early gastrula stage by RT-PCR. ODC is a loading control. RT–, control without reverse transcriptase. In each case, 50 pg of synthetic mRNA were injected. (C) Analysis of extra-R1 and Xfz3{Delta}C-R1 phosphorylation. Embryos were injected with mRNA encoding the myc-tagged extra3-R1 or Xfz3{Delta}C-R1 proteins. Protein extracts from the early gastrula stage were immunoprecipitated by the anti-myc antibody, separated by SDS-PAGE and blotted on nitrocellulose. Blots were analyzed with either anti-myc or anti-phosphotyrosine (4G10, UBI) antibodies.

 


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  		Fig. 6. Dimeric forms of Xfz7 are not detected after overexpression in Xenopus embryo. (A) Direct immunoblot analysis using anti-myc antibody of protein extracts from gastrula embryos overexpressing Xfz7-myc, Xfz3{Delta}C-myc or Xfz3-myc. In protein extracts derived from Xfz7-myc-injected embryos, the anti-myc antibody revealed one group of three major bands corresponding to the expected molecular weight for Xfz7-myc; no band at the size of the putative dimeric form is clearly detected. By contrast, monomeric and dimeric forms of Xfz3-myc and Xfz3{Delta}C-myc are observed. (B) Embryos were injected with mRNA encoding Xfz7-myc or Xfz7{Delta}C-flag, or both. Protein extracts from injected embryos were subjected to immunoprecipitation by using the anti-myc antibody (lanes 1 and 5) or the anti-flag antibody (lanes 2, 3 and 4). Immunoprecipitates were then immunoblotted with the anti-myc antibody (left panel) or anti-flag antibody (right panel). Anti-flag antibody does not immunoprecipitate any myc-positive bands from embryos expressing both Xfz7-myc and Xfz7{Delta}C-flag.

 


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  		Fig. 7. The N-terminal extracellular domain of Xfz3 is unable to inhibit Xfz3 dimerization and activation of the Wnt/ß-catenin pathway in response to overexpression of Xfz3 in Xenopus embryo. Embryos were injected with mRNA encoding Xfz7-myc, Xfz3-HA or a combination of Xfz3-HA and extra3-myc mRNA. (A) RT-PCR analysis of the Wnt/ß-catenin target gene siamois in animal caps cultured to the early gastrula stage. Siamois expression in whole embryo is used as a positive control and in noninjected caps as a negative control. ODC is a loading control. (B) Immunoprecipitation and western blot analysis of protein extracts from injected embryos. Expression of Xfz3-HA is revealed by immunoprecipitation using the anti-HA antibody followed by western blot analysis with the same antibody. The dimeric form of Xfz3-HA (arrow) is detected after injection of Xfz3-HA mRNA alone (lane 2) or in combination with extra3-myc mRNA (lane3). Expression of the extra3-myc protein in co-injected embryos is checked by immunoprecipitation and western blot with the anti-myc antibody (lane 5, arrowhead).

 


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  		Fig. 8. Oligomerization of Xfz7 is sufficient to activate the Wnt/ß-catenin pathway in Xenopus embryos. (A) Embryos were injected with mRNA encoding Xfz7-BD-myc, Xfz7-myc and Xfz3{Delta}C-myc. Protein extracts from injected embryos were separated by SDS-PAGE and immunoblotted with the anti-myc antibody. Xfz7-BD-myc dimer and oligomer are detected (arrowhead). (B) RT-PCR analysis of the Wnt/ß-catenin target gene siamois in animal caps after overexpression of different frizzled constructs as indicated. RT–: control without reverse transcriptase. ODC is a loading control. Analysis was performed at the early gastrula stage. (C) Localization of Xfz7-BD at the plasma membrane. Immunocytochemistry with the anti-myc antibody, performed on blastula animal caps isolated from Xfz7-myc or Xfz7-BD-myc injected embryos. The subcellular localization was analysed by confocal microscopy.

 

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© The Company of Biologists Ltd 2003