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

Bertalovitz AC, Pau MS, Gao S, Malbon CC, Wang HY - J Mol Signal (2016)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology & Biophysics, Health Sciences Center, School of Medicine, Stony Brook University, Stony Brook, NY 11794-8661, USA.

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.

No MeSH data available.


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.
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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.

Mentions: We analyzed the effects that truncation and substitution of the mouse Frizzled-4 C-tail (Figure 1) have on the ability of Norrin to induce Lef/Tcf-dependent transcriptional activation (Figure 2A). Mutant mFZD4 (1-503) truncates the C-tail immediately beyond T503. Truncation at this position (mFZD4(1-503)) nearly abolished the ability of Norrin to activate Lef/Tcf- dependent transcription (Figure 2A). Restoring the wild-type sequence of the C-tail beyond T503 to N513 (mFZD4(1-513)) gradually ameliorated the loss of the Lef/Tcf-dependent transcriptional activation in response to Norrin to that of the WT response. We gauged the expression of mFZD4 and the truncations using amount of the surface expression (Figure 2B). All of the FZD4 constructs were expressed. Truncating the FZD4 carboxy-terminus at W504 to S508 resulted in some loss of cell surface expression. Although a positive correlation was observed between C-tail length and surface expression, the loss in activation was far greater than the apparent loss of some surface expression, suggesting that the simple reduction in cell surface mFZD4 could not account for the more profound loss of its function.


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

Bertalovitz AC, Pau MS, Gao S, Malbon CC, Wang HY - J Mol Signal (2016)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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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.
Mentions: We analyzed the effects that truncation and substitution of the mouse Frizzled-4 C-tail (Figure 1) have on the ability of Norrin to induce Lef/Tcf-dependent transcriptional activation (Figure 2A). Mutant mFZD4 (1-503) truncates the C-tail immediately beyond T503. Truncation at this position (mFZD4(1-503)) nearly abolished the ability of Norrin to activate Lef/Tcf- dependent transcription (Figure 2A). Restoring the wild-type sequence of the C-tail beyond T503 to N513 (mFZD4(1-513)) gradually ameliorated the loss of the Lef/Tcf-dependent transcriptional activation in response to Norrin to that of the WT response. We gauged the expression of mFZD4 and the truncations using amount of the surface expression (Figure 2B). All of the FZD4 constructs were expressed. Truncating the FZD4 carboxy-terminus at W504 to S508 resulted in some loss of cell surface expression. Although a positive correlation was observed between C-tail length and surface expression, the loss in activation was far greater than the apparent loss of some surface expression, suggesting that the simple reduction in cell surface mFZD4 could not account for the more profound loss of its function.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology & Biophysics, Health Sciences Center, School of Medicine, Stony Brook University, Stony Brook, NY 11794-8661, USA.

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.

No MeSH data available.