Wnt5a promotes Frizzled-4 signalosome assembly by stabilizing cysteine-rich domain dimerization

Abstract

Wnt/β-catenin signaling is activated when extracellular Wnt ligands bind Frizzled (FZD) receptors at the cell membrane. Wnts bind FZD cysteine-rich domains (CRDs) with high affinity through a palmitoylated N-terminal "thumb" and a disulfide-stabilized C-terminal "index finger," yet how these binding events trigger receptor activation and intracellular signaling remains unclear. Here we report the crystal structure of the Frizzled-4 (FZD₄) CRD in complex with palmitoleic acid, which reveals a CRD tetramer consisting of two cross-braced CRD dimers. Each dimer is stabilized by interactions of one hydrophobic palmitoleic acid tail with two CRD palmitoleoyl-binding grooves oriented end to end, suggesting that the Wnt palmitoleoyl group stimulates CRD-CRD interaction. Using bioluminescence resonance energy transfer (BRET) in live cells, we show that WNT5A stimulates dimerization of membrane-anchored FZD₄ CRDs and oligomerization of full-length FZD₄, which requires the integrity of CRD palmitoleoyl-binding residues. These results suggest that FZD receptors may form signalosomes in response to Wnt binding through the CRDs and that the Wnt palmitoleoyl group is important in promoting these interactions. These results complement our understanding of lipoprotein receptor-related proteins 5 and 6 (LRP5/6), Dishevelled, and Axin signalosome assembly and provide a more complete model for Wnt signalosome assembly both intracellularly and at the membrane.

Keywords: Frizzled; Frizzled-4; WNT5A; Wnt; cysteine-rich domain; signalosome.

Figure 1.

Crystal structure of the FZD₄ CRD dimer with palmitoleic acid. (A) Electrostatic surface rendering of FZD₄ dimers showing end-to-end orientation of palmitoleoyl-binding grooves and chemical complementarity with palmitoleic acid (white stick model). Based on the palmitoleic acid orientation, Wnts would make residue–residue contact with only FZD₄ CRD #1, while FZD₄ CRD #2 contacts only palmitoleic acid. (B) Cartoon of FZD₄ CRD dimers (orange and blue), with hydrophobic residues in the palmitoleoyl-binding groove shown as sticks. (C) Electron density map showing hydrophobic residues in palmitoleoyl-binding grooves of both CRDs that coordinate with palmitoleic acid. (D) Distances (in angstroms) between hydrophobic residues in the palmitoleoyl-binding grooves of FZD₄ CRD dimers. (E) Schematic of shared hydrophobic coordination networks in both FZD₄ CRDs, which facilitate bimolecular palmitoleic acid docking (here modeled onto Xenopus Wnt8; PDB 4F0A).

Figure 2.

Structure of FZD₄ CRD tetrameric assembly. (A) Tetrameric complex of four FZD₄ CRDs, with two palmitoleic acids (sticks; cyan) shown as a dimer of dimers (one dimer is colored with electrostatic surface, and the other dimer is in teal). (B) Surface rendering of residues on one dimer within 5 Å of the other dimer (shown in teal). (C) The tetrameric complex of four FZD₄ CRDs (dimers in blue and orange) with two palmitoleic acids (sticks; cyan). (D) Rotated inset from A. Palmitoleic acid molecules are oriented within the tetramer with heads facing the opening of the pocket.

Figure 3.

WNT5A and WNT8A induce FZD₄ CRD interactions. (A) BRET ratios for two-hybrid and three-hybrid experiments in which FZD₄ CRDs were fused to a single-transmembrane helix tagged with either Rlu, YFP, or split YFP. Wnts were cotransfected. (*) P < 0.05; (***) P < 0.001. n ≥ 6. Error bars indicate SEM. (B) Structure of XWnt8 (PDB 4F0A) aligned to the FZD₄ dimer showing that Wnt can bind this dimer form by contacting a second CRD through the palmitoleoyl modification but without making direct amino acid–amino acid contact.

Figure 4.

WNT5A increases FZD₄ homodimer interactions through palmitoleoyl-binding residues. (A) Cartoon of two-hybrid BRET assay design in which FZD₄:Rlu and FZD₄:YFP C-terminal fluorophore fusion constructs were cotransfected. (B) Titration of WNT5A recombinant protein (in micrograms) or cotransfection of WNT5A (1 µg of DNA plasmid) with FZD₄ BRET constructs shows that FZD₄ autodimerizes in the absence of Wnt and that Wnt further increases FZD₄–FZD₄ interactions. Recombinant protein was added 15 min before analysis, and plasmid was added 24 h before analysis. (C) WNT5A cannot increase FZD₄–FZD₄ interactions when hydrophobic palmitoleoyl-binding FZD₄ residues are replaced with alanine residues. FZD₄ expression plasmids were cotransfected with or without WNT5A expression plasmid; both YFP and Rlu-tagged plasmids contained FZD₄ mutations as indicated. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001; (ns) not significant. n ≥ 6. Error bars indicate SEM for all graphs.

Figure 5.

WNT5A induces FZD₄ oligomerization through palmitoleoyl-binding residues. (A) Cartoon of three-hybrid BRET assay design in which FZD₄:Rlu and split FZD₄:YFP C-terminal fluorophore fusion constructs were cotransfected. (B) Titration of WNT5A recombinant protein (in micrograms) or cotransfection of WNT5A (1 µg of DNA plasmid) with FZD₄ BRET constructs shows that WNT5A induces FZD₄ oligomerization in a dose-dependent manner and that FZD₄ does not auto-oligomerize. Recombinant protein was added 15 min before analysis, and plasmid was added 24 h before analysis. (C) WNT5A cannot induce FZD₄ oligomerization when alanine residues are substituted for hydrophobic palmitoleoyl-binding FZD₄ residues. FZD₄ expression plasmids were cotransfected with or without WNT5A expression plasmid; both YFP^N/YFP^C and Rlu-tagged plasmids contained FZD₄ mutations as indicated. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001; (ns) not significant. n ≥ 6. Error bars indicate SEM for all graphs.

Figure 6.

FZD₄ palmitoleoyl-binding residues are required for WNT5A signaling. (A) TCF-luciferase reporter gene assay performed with cotransfection of WNT5A, an ROR2:LRP5 chimeric coreceptor (ROR2 extracellular domain fused to the LRP5 TMD and intracellular domain), and FZD₄ palmitoleoyl-binding groove mutants. n = 4. Error bars indicate SEM. (B) AP1-luciferase reporter gene assay performed with cotransfection of WNT5A, ROR2 coreceptor, and FZD₄ palmitoleoyl-binding groove mutants followed by PMA treatment. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. n = 3. Error bars indicate SEM for all graphs.

Figure 7.

The palmitoleoyl-binding groove in FZD CRDs is highly conserved. (A) Proposed model in which FZD autodimerizes, and WNT5A induces FZD oligomerization by mediating CRD dimerization through end-to-end palmitoleoyl-binding grooves. (B) Sequence conservation of FZD₄ CRD palmitoleoyl-binding groove residues among human FZD family members. (C) Structural conservation of FZD CRDs revealing lipid- and sterol-binding compatibility.