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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 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 (*).
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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 (*).

Mentions: To determine if the specific side chain properties of either K506 or C507 are required for receptor activation a double mutant was generated in which K506 and C507 were concurrently substituted with alanine residues (Figure 1). This mFZD4KC506-507AA mutant exhibited normal Norrin-induced Lef/Tcf-dependent transcriptional activation (similar to WT, see Figure 3). The mFZD4KC506-507AA double mutant also stabilized DVL2 at the cell surface (data not shown).


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

License
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getmorefigures.php?uid=PMC4834752&req=5

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 (*).
Mentions: To determine if the specific side chain properties of either K506 or C507 are required for receptor activation a double mutant was generated in which K506 and C507 were concurrently substituted with alanine residues (Figure 1). This mFZD4KC506-507AA mutant exhibited normal Norrin-induced Lef/Tcf-dependent transcriptional activation (similar to WT, see Figure 3). The mFZD4KC506-507AA double mutant also stabilized DVL2 at the cell surface (data not shown).

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.