Figure 1
Frizzled-4 Mediates Norrin induced Lef/Tcf-dependent transcriptional activation. (A) Increasing concentrations of mouse Frizzled-4 (mFz4) were cotransfected into HEK293 cells with 1 ng of human hLRP5 construct (hLRP5) and stimulated with 200 ng/mL of recombinant Norrin and the Lef/Tcf-dependent luciferase enzyme induction was quantified as described in Materials and Methods with the exception that the basal values were determined for each condition in duplicate. (B) Increasing concentrations of hLRP5 coreceptor were cotransfected into HEK293 cells with 2 ng of mFz4 and stimulated with 200 ng/mL of recombinant Norrin and the Lef/Tcf-dependent luciferase enzyme induction was quantified with the exception that the basal values were determined for each condition in duplicate. (C) HEK293 cells cotransfected with 0.25 ng of mFz4 and 1 ng hLRP5 were stimulated with increasing concentrations of recombinant Norrin and the Lef/Tcf-dependent luciferase enzyme induction was quantified. (D) Transiently transfected V5-tagged mFz4 (V5mFz4) in HeLa cells were detected by means of indirect immunofluorescence (IIF) in the absence of or following a cell permeabilization step using a V5 primary antibody.
Figure 2
Characterization of Frizzled-4 variants implicated in FEVR. (A) A snake diagram of the V5-tagged mouse Frizzled-4 (V5mFz4) used in this study. The V5 epitope cloned into the amino terminus after the signal peptide is represented by a red shading. The putative CRD domain or ligand binding site is colored blue. The general localization of amino acids found to be mutated in certain FEVR patients that were investigated in this study are shown inside stars. F328 of iloop2 is displayed in an orange star. (B) V5mFz4 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. (C) IIF of nonpermeabilized HeLa cells transfected with V5mFz4 variants using a V5 primary antibody.
Figure 3
F328S Frizzled-4 exhibits reduced Norrin-stimulation and Dvl2 recruitment. (A) Cell surface receptor expression detected by IFA in HEK293 cells contransfected with the amount in ng of V5mFz4 variant shown in parentheses and 1 ng of hLRP5 per well. Following fixation the nonpermeabilized cells were incubated with V5 primary antibody followed by an incubation with a compatible alexaflour594 secondary antibody and subsequent fluorescence quantification on a multimode plate reader. The dotted line represents the background fluorescence level of the wells transfected with hLRP alone. (B) V5mFz4 WT and F328S were overexpressed in HEK293 cells and their ability to induce Lef/Tcf-dependent luciferase enzyme induction following the addition of recombinant Norrin was performed as described in the Materials and Methods with an exception being that the wells for the “5X” hLRP5 condition were transfected with 5 ng of hLRP5 instead of 1 ng. (C) Confocal images of permeabilized HeLa cells cotransfected with GFP-tagged Dvl2 and V5mFz4WT or F328S using a V5 primary antibody.
Figure 4
Aromaticity is required at position F328 for normal Dvl2 recruitment and canonical activation. (A) A series of V5mFz4 variants with substitutions at F328 were overexpressed in HEK293 cells with hLRP5 and M50 reporter plasmid, stimulated with Norrin and Lef/Tcf-dependent luciferase activity assayed. Statistically significant differences between basal value transcriptional activation of the variants compared to that of the WT receptors as determined by one-way analysis of variance (ANOVA) followed by the Dunnett’s post hoc test is indicated with an asterisk (*). (B) Confocal images of permeabilized HeLa cells cotransfected with GFP-tagged Dvl2 and V5mFz4WT or F328S using a V5 primary antibody.
Figure 5
Intracellular Loop 2 may have a conserved role in enabling a Frizzled-Dvl2 Interaction. (A) A sequence alignment (adapted from an alignment generated at http://www.uniprot.org/) shows the aligned sequences of the intracellular loop 2 (IC2) regions of the 10 Frizzleds and the flanking transmembrane domain three (TM3) and four (TM4) displayed in blue. The phenylalanine residues at the corresponding position to F328 of mouse Frizzled-4 are indicated in red. (B) Frizzled 1 variants with mutations corresponding to Frizzled-4 F328S and F328W were overexpressed in HEK293 cells with hLRP5 and M50 reporter plasmid, and Lef/Tcf-dependent luciferase activity assay was performed as described in the Materials and Methods with an exception being that the wells were stimulated with recombinant Wnt3a in lieu of Norrin. Statistically significant differences between basal value transcriptional activation of the variants compared to that of the WT receptors as determined by an ANOVA analysis followed by the Dunnett’s post hoc test is indicated with an asterisk (*). (C) Confocal images of HeLa cells transfected with 10 ng GFP-tagged Dvl2 with or without Fz1 variants were obtained as described in the Materials and Methods.