Figure 1
Schematic representation of the FZD4 C-tail variants investigated in this study. The constructs were generated using a template containing a V5 tag inserted in the amino- terminus of the protein following the signal peptide. The residues of the highly conserved KTXXXW domain are colored red. The alanine residues substituted for the lysine and cysteine residues in the mFZD4KC506-507AA receptor are colored blue. The mFZD4/C-tailmFZD1, mFZD4/C-tailmFZD3, and mFZD4/C-tailmFZD7 constructs differ from the mFZD4 WT construct. In these chimera, the region of the C-tail distal to the KTXXXW domain of mFZD4 has been substituted by the corresponding region of mFZD1 (mFZD1 residues R626 to V642), mFZD3 (mFZD3 residues A508 to A666 of mFZD3) or mFZD7 (mFZD7 residues R556 to V572), respectively. Note that for simplicity the mFZD4/mFZD3 C-tail is not shown in its entirety.
Figure 2
The FZD4 C-tail modulates surface expression and receptor-DVL interaction. (A) V5mFZD4 variants were overexpressed in HEK293 cells with hLRP5 and M50 reporter plasmid, stimulated with Norrin and Lef/Tcf-dependent luciferase activity was assayed as described. (B) Cell surface receptor expression detected by IFA in HEK293 cells transfected with the V5mFZD4 variant shown and hLRP5. Following fixation the non-permeabilized cells were incubated with V5 primary antibody followed by incubation with a compatible Alexafluor594 secondary antibody and subsequent fluorescence quantification on a multimode plate reader. Statistically significant differences compared to WT as determined by an ANOVA analysis followed by the Dunnett’s post hoc test is indicated with an asterisk (*). (C) Confocal images of non-permeabilized HeLa cells co-transfected with GFP-tagged DVL2 and V5mFZD4 WT or truncation variants using a V5 primary antibody.
Figure 3
The FZD4 C-tail distal to the KTXXXW domain requires three additional amino acid residues for substantial activation of Lef/Tcf-dependent transcriptional activation by Norrin. V5mFZD4 variants were overexpressed in HEK293 cells with hLRP5 and M50 reporter plasmid, stimulated with Norrin. Lef/Tcf-dependent transcriptional activation was measured using a luciferase-based assay, as described in the Materials and Methods section. An experiment comparing the Norrin-induced mFZD4 WT activation to the Norrin-induced activation of the mFZD4/C-tailmFZD1, mFZD4/C-tailmFZD3, mFZD4/C-tailmFZD7 and mFZD4 KC506-507AA constructs. Statistically significant differences compared to WT as determined by an ANOVA analysis followed by the Dunnett’s post hoc test are indicated with an asterisk (*).