Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 Sep 15;19(18):4944-54.
doi: 10.1093/emboj/19.18.4944.

The C-terminal cytoplasmic Lys-thr-X-X-X-Trp motif in frizzled receptors mediates Wnt/beta-catenin signalling

M Umbhauer  1 , A Djiane, C Goisset, A Penzo-Méndez, J F Riou, J C Boucaut, D L Shi
Affiliations

The C-terminal cytoplasmic Lys-thr-X-X-X-Trp motif in frizzled receptors mediates Wnt/beta-catenin signalling

M Umbhauer et al. EMBO J. .

Abstract

Frizzled receptors are components of the Wnt signalling pathway, but how they activate the canonical Wnt/beta-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/beta-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/beta-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/beta-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/beta-catenin signalling. These findings 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.

PubMed Disclaimer

Figures

None
Fig. 1. Differential activation of Wnt/β-catenin target genes Siamois and Xnr3 by Xfz3, Xfz4 and Xfz7. Embryos at the 2-cell stage were injected at the animal pole region with the mRNAs indicated, whole embryos and animal caps were analysed by RT–PCR at stage 10.5 for the expression of Siamois and Xnr3. (A) Injection of Xfz3 and Xwnt8 mRNAs (500 pg and 5 pg, respectively), but not Xfz4 and Xfz7 mRNAs (500 pg), induces the expression of Siamois and Xnr3 in the animal caps. Coinjection of GSK3β mRNA (500 pg) blocks the effect of Xfz3. (B) Xwnt5A mRNA (5 pg) was either injected alone or coinjected with Xfz4 or Xfz7 mRNA (500 pg). Notice that Xwnt5A synergizes with Xfz4 and Xfz7 to induce Siamois and Xnr3 expression in the animal caps. ODC was used as a control for the level of input RNA. RT–, whole embryo control sample without reverse transcriptase.
None
Fig. 2. Ectopic chordin expression induced by Xfz3 and Xwnt5A plus Xfz7 in stage 10.5 gastrulae. Four-cell stage embryos were injected at the ventral vegetal region with the mRNAs indicated and allowed to develop to stage 10.5 for in situ hybridization. (A) Expression of chordin in the Spemann organizer in an uninjected embryo. (B and C) Ventral injection of Xwnt8 or Xfz3 (5 pg and 500 pg mRNAs, respectively) induces ectopic chordin expression. (D and E) Ventral injection of Xwnt5A or Xfz7 alone (5 pg and 500 pg mRNAs, respectively) has no effect. (F) Coinjection of Xwnt5A and Xfz7 mRNAs induces ectopic chordin expression.
None
Fig. 3. Induction of the Wnt/β-catenin target gene Siamois by chimeric frizzled receptors. (A) Schematic representation of wild-type and chimeric frizzled constructs. Wild-type Xfz3, Xfz4 and Xfz7 contain an N-terminal signal peptide (SP) and an extracellular cysteine-rich domain (CRD), the seven transmembrane domains (TM 1–7) are followed by a C-terminal cytoplasmic region. Xfz3/4 and Xfz3/7 are chimeric receptors in which the C-terminal cytoplasmic region of Xfz3 is replaced by the corresponding region of Xfz4 or Xfz7; Xfz7/3 is the converse. (B) Xfz3, Xfz3/4 and Xfz3/7 induce the expression of Siamois in animal cap cells, while Xfz4 and Xfz7/3 have no effect. Note that Xfz7/3 synergizes with Xwnt5A to induce Siamois expression.
None
Fig. 4. Functional mapping of the C-terminal cytoplasmic portion of frizzled receptors. (A) Schematic representation of the Xfz3 deletion constructs. The C-terminal cytoplasmic region of Xfz3 protein has 165 amino acids. Xfz3-ΔC153 and Xfz3-ΔC160 proteins retain 12 and five amino acids after the seventh TM, respectively. (B) Embryos were injected with the mRNAs indicated and animal caps were analysed at stage 10.5 by RT–PCR for Siamois expression. Injection of 500 pg Xfz3-ΔC153 mRNA, but not the same amount of Xfz3-ΔC160 mRNA, induces Siamois expression.
None
Fig. 5. Induction of the Wnt/β-catenin target gene Siamois by Xfz3 point mutants in the C-terminal cytoplasmic region. (A) Alignment of 12 amino acids after the seventh TM among 16 frizzled homologues. Note that three amino acids (bold) are well conserved, except in Dfz3 and mom-5. (B) Xfz3 point mutants (see text for detail) and their ability to induce Siamois expression in animal caps. Mutations or deletions of either of the three conserved amino acids (Lys, Thr and Trp) abolish the function of Xfz3 to activate Siamois expression in animal caps, while mutations in other non-conserved amino acids have no effect. Note that deletion (Xfz3-ΔC503) or addition (Xfz3 + F504) of an amino acid between the threonine and tryptophan also affects Xfz3 activity.
None
Fig. 6. Expression of myc-tagged wild-type and mutant Xfz3 proteins at the cell surface. Synthetic mRNAs encoding myc-tagged Xfz3 (A), Xfz3-ΔC160 (B) and Xfz3-W506G (C) were injected at 2-cell stage near the animal pole region and animal caps were analysed at stage 9 by whole-mount immunostaining using 9E10 monoclonal antibody and confocal microscopy. All three proteins are correctly expressed at the cell surface.
None
Fig. 7. Recruitment of Xdshmyc to the plasma membrane by wild-type and mutant frizzled receptors. Embryos at 4-cell stage were either injected with 250 pg Xdshmyc mRNA alone or coinjected with mRNAs (500 pg) encoding the indicated wild-type and mutant frizzled receptors into the animal pole region of all four blastomeres. Animal caps were analysed at stage 9 by whole-mount immunostaining using 9E10 monoclonal antibody and confocal microscopy for the subcellular localization of Xdshmyc. (A) Expression of Xdshmyc alone results in diffuse fluorescent staining in the cytoplasm, no membrane localization is apparent. (B and C) Coexpression of Xdshmyc with Xfz3 (B) or Xfz3-ΔC153 (C) results in qualitative membrane localization of Xdshmyc. (D) Xfz3-ΔC160 does not recruit Xdshmyc to the plasma membrane. (E and F) Xfz4 and Xfz7 recruit Xdshmyc to the plasma membrane similarly to Xfz3. (G, H and I) Xdshmyc does not relocalize to the plasma membrane in response to expression of Xfz3-K501M (G), Xfz3-T502V (H) or Xfz3-W506G (I). (J) Xfz3-C503S significantly recruits Xdshmyc to the plasma membrane. (K and L) Xfz3-A507V (K) and Xfz3-F509L (L) recruit Xdshmyc at the plasma membrane similarly to the wild-type Xfz3.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Adler P.N. (1992) The genetic control of tissue polarity in Drosophila. BioEssays, 14, 735–741. - PubMed
    1. Axelrod J.D., Miller,J.R., Shulman,J.M., Moon,R.T. and Perrimon,N. (1998) Differential recruitment of Dishevelled provides signaling specificity in the planar cell polarity and Wingless signaling pathways. Genes Dev., 12, 2610–2622. - PMC - PubMed
    1. Bassez T., Paris,J., Omilli,F., Dorel,C. and Osborne,H.B. (1990) Post-transcriptional regulation of ornithine decarboxylase in Xenopus laevis. Development, 110, 955–962. - PubMed
    1. Bhanot P., Brink,M., Samos,C.H., Hsieh,J.C., Wang,Y.S., Macke,J.P., Andrew,D., Nathans,J. and Nusse,R. (1996) A new member of the frizzled family from Drosophila functions as a Wingless receptor. Nature, 382, 225–230. - PubMed
    1. Bhanot P., Fish,M., Jemison,J.A., Nusse,R., Nathans,J. and Cadigan,K.M. (1999) Frizzled and Dfrizzled-2 function as redundant receptors for Wingless during Drosophila embryonic development. Development, 126, 4175–4186. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources