Frizzled-4 C-terminus Distal to KTXXXW Motif is Essential for Normal Dishevelled Recruitment and Norrin-stimulated Activation of Lef/Tcf-dependent Transcriptional Activation

Abstract

The carboxy (C)-termini of G protein coupled receptors (GPCR) dictate essential functions. The KTXXXW motif C-terminus of Frizzleds (FZD) has been implicated in recruitment of Dishevelled (DVL). Through study of FZD4 and its associated ligand Norrin, we report that a minimum of three residues distal to the KTXXXW motif in the C-terminal tail of Frizzled-4 are essential for DVL recruitment and robust Lef/Tcf-dependent transcriptional activation in response to Norrin.

Keywords: Dishevelled; Frizzled; Frizzled-4; Norrin; carboxy-terminus; helix VIII.

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 (*).