Biophysical and functional characterization of Norrin signaling through Frizzled4

Abstract

Wnt signaling is initiated by Wnt ligand binding to the extracellular ligand binding domain, called the cysteine-rich domain (CRD), of a Frizzled (Fzd) receptor. Norrin, an atypical Fzd ligand, specifically interacts with Fzd4 to activate β-catenin-dependent canonical Wnt signaling. Much of the molecular basis that confers Norrin selectivity in binding to Fzd4 was revealed through the structural study of the Fzd4_CRD-Norrin complex. However, how the ligand interaction, seemingly localized at the CRD, is transmitted across full-length Fzd4 to the cytoplasm remains largely unknown. Here, we show that a flexible linker domain, which connects the CRD to the transmembrane domain, plays an important role in Norrin signaling. The linker domain directly contributes to the high-affinity interaction between Fzd4 and Norrin as shown by ∼10-fold higher binding affinity of Fzd4_CRD to Norrin in the presence of the linker. Swapping the Fzd4 linker with the Fzd5 linker resulted in the loss of Norrin signaling, suggesting the importance of the linker in ligand-specific cellular response. In addition, structural dynamics of Fzd4 associated with Norrin binding investigated by hydrogen/deuterium exchange MS revealed Norrin-induced conformational changes on the linker domain and the intracellular loop 3 (ICL3) region of Fzd4. Cell-based functional assays showed that linker deletion, L430A and L433A mutations at ICL3, and C-terminal tail truncation displayed reduced β-catenin-dependent signaling activity, indicating the functional significance of these sites. Together, our results provide functional and biochemical dissection of Fzd4 in Norrin signaling.

Keywords: Dishevelled; Frizzled 4; Norrin; linker domain; signaling.

Fig. 1.

Fzd4 oligomerization in vivo observed with BRET. All measurements were done with at least four repeats, and the error bars are drawn with SEM. (A) Each ΔBRET ratio was calculated by taking the BRET ratio of a structurally unrelated muscarinic receptor 2 (M2R) and Fzd4 pair as the background ratio. Transfected receptor pair is labeled below in RLuc8-/YFP-tagged order. (B) Kinetic measurement of the BRET ratio is plotted with a black arrow marking the injection time point of MBP–Norrin. For M2R and Fzd4ΔN₂₀₀, only the data at the highest MBP–Norrin concentration (5 μg/mL) are plotted because similar results were obtained at all three concentrations.

Fig. 2.

Two representative computational models of Fzd4–Norrin complex. Two molecules of Norrin are shown in green and yellow-green. One Fzd4 chain is in magenta and the other is in cyan. For clarity, both CRDs are colored in darker shades. Both models were aligned against the TMDs and are shown in the same orientation. Eight disulfide bonds in each Fzd4 are shown in yellow sticks, and two molecules of GlcNAc, which were identified in the crystal structure of Fzd4_CRD (PDB ID code 5BQC), are shown in orange sticks. Schematic drawings were made for each top view.

Fig. 3.

HDX-MS result for Fzd4. (A) Differences in deuterium uptake observed for Fzd4 upon Norrin binding is noted on model 7. Whereas two Fzd4 molecules are in light green and light blue, the uncolored white patches represent the regions with no peptides identified by MS. Disulfide bonds are shown as yellow sticks. Norrin dimer is colored with light and dark gray. The peptides with altered HDX upon Norrin binding are color-coded (red, magenta, green, blue, and orange) and marked on the model. Next to the model, the deuterium uptake plots of the color-coded peptides are shown. Error bars represent SEM of three to six independent experiments. (*Significant at P < 0.05.) (B) Mass spectra of peptides from ICL1 (Left) and helix 8 (Right) showing a binomial and a bimodal isotropic distribution, respectively, are presented.

Fig. 4.

In vitro binding assays between Norrin and Fzd4. (A) The size exclusion chromatography profile (Top), Coomassie-stained SDS/PAGE gel (Middle), and Western blot (Bottom) of each complex are shown. MBP–Norrin is plotted in black for all three profiles. Fzd4_CRD is in sky blue, Fzd4_CRDlinker in yellow, and full-length Fzd4 in green. The complexes are in a darker corresponding shade. Fractions marked by the black bar below the x-axis were loaded on SDS/PAGE gel. Fzd4_CRD and Fzd4_CRDlinker were detectable only with a Western blot. Although Fzd4 bands were visible with Coomassie staining, Fzd4 was verified with an anti-Flag antibody in a Western blot. (B) MST experiments were done to measure the binding affinity of Fzd4_CRD and Fzd4_CRDlinker for MBP–Norrin, and their binding curves are shown in red and blue, respectively. Error bars represent SD of three independent experiments.

Fig. 5.

Functional assessment of various Fzd4 mutations. (A) Schematic view of all Fzd4 constructs used for functional and binding assays. The conserved KTXXXW motif is colored blue. Based on our mutational studies, functionally significant residues at the cytoplasmic region are colored red, whereas insignificant ones are in green. FEVR-associated mutations tested in our assays are colored pink. Orange colored region represents the Fzd4 linker domain. (B–D) The effects of Fzd4 mutations on Norrin signaling were investigated. The bar graphs are plotted with SEM of at least four repeats. Luciferase activities of the linker deletion and the linker swap mutations are shown in B, and those of FEVR-associated mutations and point mutations at ICL regions are shown in C and D, respectively.

Fig. 6.

BRET assays showing the interaction between YFP-Dvl2 and various Fzd4-RLuc mutations. All graphs are plotted with SEM of five repeats. (A) Measurements were made in the absence of Norrin to show basal level interaction. (**Significant at P < 0.01; ns, not significant.) (B) The effect of Norrin on BRET between each Fzd4 construct and Dvl2 was investigated by adding an increasing amount of Norrin (0, 0.1, 1, 5, 8 μg/mL) in each BRET assay. (***Significant at P < 0.001.)