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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.


Related in: MedlinePlus

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.
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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.

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)

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

License
Show All Figures
getmorefigures.php?uid=PMC4834752&req=5

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.
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.


Related in: MedlinePlus