Wnt/β-catenin signaling requires interaction of the Dishevelled DEP domain and C terminus with a discontinuous motif in Frizzled

Abstract

Wnt binding to members of the seven-span transmembrane Frizzled (Fz) receptor family controls essential cell fate decisions and tissue polarity during development and in adulthood. The Fz-mediated membrane recruitment of the cytoplasmic effector Dishevelled (Dvl) is a critical step in Wnt/β-catenin signaling initiation, but how Fz and Dvl act together to drive downstream signaling events remains largely undefined. Here, we use an Fz peptide-based microarray to uncover a mechanistically important role of the bipartite Dvl DEP domain and C terminal region (DEP-C) in binding a three-segmented discontinuous motif in Fz. We show that cooperative use of two conserved motifs in the third intracellular loop and the classic C-terminal motif of Fz is required for DEP-C binding and Wnt-induced β-catenin activation in cultured cells and Xenopus embryos. Within the complex, the Dvl DEP domain mainly binds the Fz C-terminal tail, whereas a short region at the Dvl C-terminal end is required to bind the Fz third loop and stabilize the Fz-Dvl interaction. We conclude that Dvl DEP-C binding to Fz is a key event in Wnt-mediated signaling relay to β-catenin. The discontinuous nature of the Fz-Dvl interface may allow for precise regulation of the interaction in the control of Wnt-dependent cellular responses.

Fig. 1.

Identification of a discontinuous Dvl1 binding site in Fz5. (A) (Left) Schematic depiction of the topology of Fz proteins. iLoop3 and the C-terminal tail are boxed. (Right) Design of the Fz5 iLoop3-tail peptide library, based on 30 amino acids from mouse Fz5 iLoop3 (white) and 30 membrane-proximal amino acids from the Fz5 tail (black). Linear 9-mer peptides (22 each) were combined in cis, flanked and separated by Gly or Cys residues. CLIPS technology was used on Cys-based combinations, yielding bicyclical peptides; Gly-linked peptides were left linear. The combinatory peptides were immobilized in 455-well plates. Binding of recombinant His-tagged Dvl1-ΔDIX (G86–M695) was determined by an ELISA-based assay. (B) Binding signals were ranked by binding intensity (BI), and the 10 best binding peptides are depicted with their absolute score and normalized BI (relative to the screen average). AU, Arbitrary Units. (C) Frequency analysis of the top 5% (48 best binding peptides) reveals a strong preference for binding two motifs in iLoop3 (motifs I and II; pink and green) and one in the C terminus (motif III; blue). Motif III overlaps with the classic PDZ binding motif. Depicted are the frequencies of iLoop3 peptides in the top 5%, whether combined to motif III (blue) or not (white; other). (D) Diagram of the three Dvl1 binding motifs in the mFz5 iLoop3 and C-terminal tail. (E) Alignment of regions encompassing motifs I and II of mouse Fz family members and Drosophila Fz (DFz). Motifs I and II are highly conserved among most Fzs.

Fig. 2.

iLoop3 motifs I and II in Fz are essential for Wnt/β-catenin signaling and PM recruitment of Dvl. (A) Alanine scan of iLoop3 residues within full-length Fz5. Fz5 mutants were tested in the TOPFlash luciferase reporter assay and compared with WT Fz5. HEK293T cells were transfected with either TOPFlash or FOPFlash firefly luciferase reporter, thymidine kinase-Renilla control plasmid, and the indicated mFz5 constructs or empty vector (Mock). Cells were treated overnight with Wnt3a-conditioned medium. Firefly/Renilla values of TOPFlash/FOPFlash are given as normalized averages from at least three individual experiments; error bars depict SDs. Critical single residues within motifs I and II for Fz function are indicated with arrows. (B) Immunoblot expression analysis of V5-Fz5 variants, transfected in HEK293T cells; actin loading controls are shown. Immature (ER) and fully glycosylated mature Fz as well as SDS-resistant Fz dimers are indicated. (C) Color-coded luciferase activity map of iLoop3 alanine-scanning data. Fz5 mutations that caused ER retention are indicated in gray. (D) Categories of Flag-Dvl1 recruitment to Fz5 mutants as analyzed by confocal immunofluorescence microscopy are shown. (Scale bars: 10 μm.) (E) Inactive Fz5 iLoop3 point mutants display impaired Flag-Dvl1 recruitment. Motif I mutants show an intermediate recruitment phenotype, and motif II mutations L443A and L446A fail to recruit Dvl1. WT and a control mutant (I448A) are also depicted. The number of counted cells per condition is indicated.

Fig. 3.

XFz7 motifs I and II are essential for Wnt-mediated signaling and XDvl2 recruitment in Xenopus embryos. (A) XFz7 iLoop3 motif I and II mutants display decreased Wnt3a-induced signaling. Indicated motif I and II XFz7 mutants were tested in the TOPFlash luciferase reporter assay as in Fig. 2A. (Left) Shown are the average normalized values of three individual experiments; error bars indicate SDs. Mock, empty vector. (Right) Color-coded activity map of tested XFz7 iLoop3 residues (as in Fig. 2C). (B) XFz7 motif II mutants are defective in secondary axis formation in Xenopus embryos. Ventral injections of combined mRNAs encoding for XFz7 variants and XWnt8 were used to compare β-catenin–mediated secondary axis formation. The primary axis is indicated by straight lines, and (partial) secondary axes are indicated by dotted lines. (C) Quantification of the results presented in B. XFz7 I425A caused severe embryonic lethality and was not quantified. Shown are the average percentages of axis duplication of three independent experiments; error bars depict SEs. The number of counted embryos per condition is indicated. (D) Colocalization of XFz7 motif I and II mutants and XDvl2-GFP in Xenopus animal cap explants is impaired. (Scale bars: 100 μm.) (E) Quantification of the results in D (as in Fig. 2 D and E). The number of counted cells is indicated.

Fig. 4.

Dvl1 DEP-C region binds the discontinuous binding surface in Fz5. (A) Domain structure of Dvl1 with three conserved domains (DIX, PDZ, and DEP), followed by a C-terminal region (C-region). His-tagged recombinant Dvl1 fragments that were generated are shown. (B–E) Fz5 binding motifs of indicated purified recombinant Dvl fragments. Details of best binding Fz peptides are described in Fig. S1. (B) Isolated PDZ (S201–P375) domain binds the Fz C-terminal tail, which comprises the classic PDZ binding sequence (motif III). (C) DEP-C region (R376–M695) binds the entire Fz discontinuous binding surface comprising motifs I, II, and III. (D) Alternative binding mode of DEP-C, involving the classic Fz C-terminal motif but not iLoop3. (E) DEP (S393–N503) domain without the C-region binds motif III in the Fz C-terminal tail and weakly to motif II. (F) Isolated C-region (L500–M695) binds motifs I and II in Fz5 iLoop3. (G) In vitro fluorescence polarization binding assay between a concentration range of recombinant DEP-C (filled) or DEP (open) and a cysteine-maleimide–linked fluorescein-labeled peptide (CKLMIRIGIFGTLESWRRFTG, 100 nM) combining motifs II and III. The titration data are fitted with a binding saturation curve. One representative experiment of two is shown. (H) In vitro fluorescence polarization binding assay (as in G) using recombinant Dvl C-tail.

Fig. 5.

Isolated DEP and DEP-C domains colocalize with Fz5 in cells and interfere with Wnt/β-catenin signaling. (A) Set of N-terminally Flag-tagged Dvl1 fragments. (B) HEK293T cells were transfected with indicated Dvl fragments and the TOPFlash /FOPFlash reporter constructs for 24 h and treated overnight with L-cell medium (L-CM) or Wnt3a-conditioned medium (Wnt3a-CM). Ectopic expression of ΔDIX, DEP-C, and DEP, but not mutated DEP or PDZ fragments, inhibits Wnt3a-induced β-catenin signaling. Data are represented as normalized averages of three individual experiments; error bars depict SDs. Mock, empty vector; KM, K438M; DIDI, D449I/D452I. (C) WT Fz5-mediated recruitment of Dvl1 constructs in HEK293T cells, analyzed by confocal immunofluorescence microscopy and quantified as in Fig. 2E. (D) Recruitment of Dvl1–DEP-C to Fz5 iLoop3 variants is partially and strongly impaired for motif I and motif II mutants, respectively. (E) DEP domain structure (28), indicating the membrane binding positively charged residues (green), as well as the K438 (red), D449 and D452 (blue) residues. (F) Recruitment of K438M and DIDI Dvl1 to mFz5 variants at the PM is strongly impaired. (G) DEP domain residues K438 and D449/D452 participate in Wnt/β-catenin signaling. Dvl variants were expressed in HEK293T cells, together with the TOPFlash luciferase reporter constructs, and analyzed for reporter activity after 24 h. (H) Set of Flag-tagged C-terminal truncations was generated in both full-length (FL) Dvl1 and the DEP-C fragment. (I) Most C-terminal region of Dvl1 significantly contributes to Dvl-mediated β-catenin activation, as analyzed by the TOPFlash reporter assay. (J) Extreme C-terminal Dvl1 region plays a role in Fz5-mediated PM recruitment of Dvl1. In addition, a second, more upstream region (spanning residues H526–S628) in the Dvl C terminus contributes to Fz-mediated PM recruitment. (C, D, F, and J) Number of counted cells per condition is indicated.

Fig. 6.

Fz-Dvl interaction model. GPCR-like representation of Fz as a heptahelical TM protein. The discontinuous Dvl binding surface in the Fz iLoop3 and C-terminal tail is indicated. Dvl comprises three conserved, folded domains of which the PDZ, DEP domain, and extended C terminus are able to interact with the Fz interface. We propose that the DEP domain interacts with negatively charged lipid head groups to stabilize the Fz-Dvl interaction further. The DIX domain does not bind Fz and is free to multimerize and form receptor clusters.

Fig. P1.

Model for Wnt-induced formation of functional Fz–Dvl complexes at the plasma membrane. (Left) Fz receptor in an unbound state. Three Dvl binding motifs in the third loop and cytoplasmic tail of the Fz protein are indicated in red. Dvl resides in the cytoplasm. PM, plasma membrane. (Right) Wnt binding to Fz induces recruitment of Dvl to bind the three-segmented motif in the Fz receptor. Binding involves both the Dvl DEP domain (DEP) and the C terminus (C).