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. 2004 Jun 17;23(28):4873-84.
doi: 10.1038/sj.onc.1207642.

Truncated mutants of the putative Wnt receptor LRP6/Arrow can stabilize beta-catenin independently of Frizzled proteins

Keith Brennan  1 , José M Gonzalez-Sancho, Leslie A Castelo-Soccio, Louise R Howe, Anthony M C Brown
Affiliations

Truncated mutants of the putative Wnt receptor LRP6/Arrow can stabilize beta-catenin independently of Frizzled proteins

Keith Brennan et al. Oncogene. .

Abstract

Secreted signaling proteins of the Wnt family are known to regulate a diverse range of developmental processes, and their signaling pathway through beta-catenin is frequently activated in cancer. The identification of both Frizzled and LRP5/6 (LRP: low-density lipoprotein receptor-related protein) proteins as components of cell-surface receptors for Wnt proteins has raised questions about their individual functions. We have investigated this issue through a structure-function analysis of Frizzled and LRP proteins that have been implicated in Wnt1 signaling. Consistent with other reports, we find that LRP6/Arrow proteins deleted for their extracellular domain are able to activate the Wnt/beta-catenin signaling pathway. Importantly, our results demonstrate that this signaling from LRP6/Arrow derivatives can occur in a Frizzled- and ligand-independent manner. Furthermore, we show that the PPSP motifs within the intracellular domain of LRP6 are required for signaling. In contrast to results with LRP6, overexpression of Frizzled proteins did not activate the pathway. Based on evidence of ligand binding to both Frizzled and LRP6, current models suggest that both proteins are components of a Wnt receptor complex that signals to beta-catenin. In light of these models, our data imply that LRP5/6/Arrow proteins constitute the distal signal-initiating component of these receptors. The results also support the notion that LRP5/6 are candidate oncogenes.

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Figures

Figure 1
Figure 1. Diagram of LRP6 and Frizzled constructs used in this study
(A) Full-length LRP6 and deletion derivatives, showing EGF-like repeats (EGF, gray ovals), LDLR repeats (LDLR, black diamonds) and transmembrane domain (TM, hatched box). (B) Full-length Frizzled 1 and Frizzled 8 (Fzd1, Fzd8), showing the extracellular cysteine-rich domain (CRD) and seven transmembrane domains (black rectangles); the soluble extracellular domain construct Fzd8 Ex; and ΔN-Fzd1, from which the extracellular domain is deleted.
Figure 2
Figure 2. Dominant negative forms of both LRP6 and Frizzled inhibit Wnt/β-catenin signaling
(A) Dominant negative Fzd8 inhibits cytosolic β-catenin stabilization in response to Wnt1 signaling. Western blot analysis of cytosolic β-catenin levels in 293T cells transiently transfected with either empty vector (lanes 1 and 2), or vector encoding Wnt1 (lanes 3 and 4), in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of a Fzd8 Ex expression construct. Analysis of GSK-3β provides a loading control (lower panel). (B) Dominant negative LRP6 inhibits cytosolic β-catenin stabilization. 293T cells were transiently transfected with empty vector (lanes 1 and 3) or a Wnt1 expression vector (lanes 2, 4, and 5) in the absence (lanes 1 and 2) or presence (lanes 3, 4 and 5) of the indicated LRP6 dominant negative constructs. Cytosolic β-catenin levels and GSK-3β were analyzed as in (A).
Figure 3
Figure 3. Overexpression of Frizzled1 does not activate Wnt/β-catenin signaling
(A) Full length Fzd1 and the deletion mutant ΔN-Fzd1 do not stabilize β-catenin. 293T cells were transfected with vectors encoding either Wnt1 (lane 2), full-length Fzd1 (lane 3), ΔN-Fzd1 (lane 4), or with empty vector (lane 1) and cytosolic β-catenin levels were determined by Western blotting. GSK-3β served as a loading control. Expression of the epitope-tagged Fzd proteins was confirmed using anti-Myc and anti-His antibodies (lanes 3 and 4). Only Wnt1 caused significant stabilization of β-catenin. (B) Overexpression of full length Fzd1 or ΔN-Fzd1 does not significantly activate TCF/β-catenin-dependent transcription. 293T cells were transfected with the indicated amounts of plasmid encoding Wnt1, full length Fzd1 or ΔN-Fzd1, together with the TCF/β-catenin responsive reporter pTOPFLASH. The cells were also transfected with the Renilla luciferase expression plasmid pRL-TK as an internal control. Luciferase activities were measured and TOPFLASH values normalized to Renilla values. Results shown are the means + S.D. of six replicates. (C) Overexpression of full length Fzd1 can block Wnt1 signaling. 293T cells were transfected with empty vector (lanes 1 and 4) or Wnt1 (lanes 2 and 3) in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of full-length Fzd1. Cytosolic β-catenin levels were analyzed as in Figure 2 and GSK-3β was used as a loading control.
Figure 4
Figure 4. Overexpression of full-length LRP6, or extracellular domain deletion derivatives, mimics Wnt1 signaling
(A) LRP6 derivatives stabilize β-catenin. Western analysis of cytosolic β-catenin in 293T cells transfected with empty vector (lanes 1, 4 and 8), Wnt1 (lanes 2 and 9), full-length LRP6 (lane 5), the indicated LRP6 constructs (lanes 3, 6, and 7), or ΔN-Arr (lane 10). GSK-3β was used as a loading control. (B) LRP6 derivatives lacking the EGF-like repeat domain potently activate TCF/β-catenin-dependent transcription. 293T cells were transfected with varying amounts of plasmid encoding the indicated LRP6 constructs, together with pTOPFLASH and pRL-TK. Luciferase activities were measured and TOPFLASH values normalized to Renilla values. Results shown are the means + S.D. of six replicates. LRP6 and ΔLDLR-LRP6 have significant activity above background, but ΔN-LRP6 and ΔEGF-LRP6 have markedly stronger activity.
Figure 5
Figure 5. Signaling by ΔN-LRP6 requires intracellular PPSP motifs
(A) Diagram of ΔN-LRP6 and C-terminal deletions thereof. The repeated motifs PPSP and PPTTP are indicated by hatched boxes and the transmembrane domain (TM) with a black rectangle. (B) Effect of ΔN-LRP6 C-terminal deletions on β-catenin stabilization. 293T cells were transfected with empty vector (lane 1) or vectors encoding the indicated ΔN-LRP6 derivatives (lanes 2-6). Cytosolic β-catenin levels were analyzed as in Figure 2 and GSK-3β was used as a loading control. Removal of 105 amino acids does not reduce activity in this assay (lane 5) but removal of 139 residues eliminates signaling (lane 6). (C) Effect of ΔN-LRP6 C-terminal deletions on TCF/β-catenin-dependent transcription. 293T cells were transfected with the indicated amounts of LRP6 or ΔN-LRP6 derivatives, together with pTOPFLASH and pRL-TK. Luciferase assays were performed as in Figure 3. Results are the mean + S.D. of six replicates. Note the progressive loss of activity with increasing C-terminal truncation. (D) Mutations in the sole PPSP motif of ΔN-LRP6-Δ105 abolish signaling to β-catenin. Cells were transfected with His-tagged ΔN-LRP6-Δ105 constructs carrying the indicated point mutations. Cytosolic β-catenin and GSK-3β levels were analyzed by Western blotting and membrane fractions were probed with anti-His antibody to verify expression of the mutant LRP6 proteins. (E) Subcellular localization of inactive ΔN-LRP6-Δ105 mutants by immunofluorescence. Cells transfected with the indicated Myc-tagged ΔN-LRP6 proteins or empty vector were fixed and immunostained (red) with anti-Myc antibody in the absence of detergents. The cells were co-transfected with pEGFP-C3 (Clontech) to identify transfected cells (green) independently of LRP6 expression.
Figure 5
Figure 5. Signaling by ΔN-LRP6 requires intracellular PPSP motifs
(A) Diagram of ΔN-LRP6 and C-terminal deletions thereof. The repeated motifs PPSP and PPTTP are indicated by hatched boxes and the transmembrane domain (TM) with a black rectangle. (B) Effect of ΔN-LRP6 C-terminal deletions on β-catenin stabilization. 293T cells were transfected with empty vector (lane 1) or vectors encoding the indicated ΔN-LRP6 derivatives (lanes 2-6). Cytosolic β-catenin levels were analyzed as in Figure 2 and GSK-3β was used as a loading control. Removal of 105 amino acids does not reduce activity in this assay (lane 5) but removal of 139 residues eliminates signaling (lane 6). (C) Effect of ΔN-LRP6 C-terminal deletions on TCF/β-catenin-dependent transcription. 293T cells were transfected with the indicated amounts of LRP6 or ΔN-LRP6 derivatives, together with pTOPFLASH and pRL-TK. Luciferase assays were performed as in Figure 3. Results are the mean + S.D. of six replicates. Note the progressive loss of activity with increasing C-terminal truncation. (D) Mutations in the sole PPSP motif of ΔN-LRP6-Δ105 abolish signaling to β-catenin. Cells were transfected with His-tagged ΔN-LRP6-Δ105 constructs carrying the indicated point mutations. Cytosolic β-catenin and GSK-3β levels were analyzed by Western blotting and membrane fractions were probed with anti-His antibody to verify expression of the mutant LRP6 proteins. (E) Subcellular localization of inactive ΔN-LRP6-Δ105 mutants by immunofluorescence. Cells transfected with the indicated Myc-tagged ΔN-LRP6 proteins or empty vector were fixed and immunostained (red) with anti-Myc antibody in the absence of detergents. The cells were co-transfected with pEGFP-C3 (Clontech) to identify transfected cells (green) independently of LRP6 expression.
Figure 6
Figure 6. LRP6 can signal in a ligand-independent manner
(A) Fzd8 Ex and LRP6-Δ173 both inhibit Wnt-mediated accumulation of β-catenin, as in Figure 2. (B) The same Fzd8 Ex and LRP-Δ173 constructs fail to inhibit β-catenin stabilization mediated by LRP6 or ΔN-LRP6, implying that signaling by the LRP6 cytoplasmic domain in these experiments is ligand-independent. Cytosolic β-catenin levels in co-transfected 293T cells were analyzed by Western blotting as in Figure 2. (C) Effect of Fzd8 Ex and LRP6-Δ173 expression on TCF/β-catenin-dependent transcription induced by LRP6 or ΔN-LRP6. 293T cells were transfected with the empty vector, LRP6 or ΔN-LRP6, together with pTOPFLASH and pRL-TK, in the presence or absence of Fzd8 Ex and LRP6-Δ173. Luciferase assays were performed as in Figure 3. Results are the mean + S.D. of six replicates. The expression of either dominant negative protein does not significantly alter LRP6 or ΔN-LRP6 signaling.
Figure 7
Figure 7. A truncated form of Drosophila Arrow can signal independently of Frizzled proteins
(A) Drosophila S2 cells do not respond to Wingless unless transfected with Dfz2. S2 cells were transfected with empty vector (lanes 1 and 3) or vector encoding the Wnt1 homolog Wingless (Wg; lanes 2 and 4) in the presence (lanes 3 and 4) or absence (lanes 1 and 2) of Dfrizzled2 (Dfz2). Cytosolic levels of Armadillo (the Drosophila homolog of β-catenin) were analyzed by Western blot and β-tubulin was used as a loading control. (B) Quantitative RT-PCR analysis of Dfz2 and Dfz3 transcripts in S2 cells. Analysis of S2 cells (lane 6 and 7) indicated that Dfz2 and Dfz3 transcripts were present, but comparison to reactions containing known numbers of template molecules (lanes 2-5) demonstrated that the transcripts were at very low abundance. From this comparison it is possible to estimate the number of transcripts per cell. Given an average yield of 40μg of total RNA from 8×106 S2 cells and the use of 0.5μg of total RNA in each RT-PCR, these results indicated that there were approximately 0.16 and 0.04 transcripts per cell for Dfz2 and Dfz3 respectively. (C) Expression of ΔN-Arr is sufficient to cause stabilization of Armadillo, independently of Dfz2 expression. S2 cells were transfected with empty vector (lane 1), or vectors encoding ΔN-Arr, an N-terminally truncated form of the LRP6 homolog Arrow (lane 2), Dfz2 (lane 3), or both ΔN-Arr and Dfz2 (lane 4). Armadillo and β-tubulin levels were analyzed by Western blot as in (A). (D) Overexpression of ΔN-Arr in S2 cells does not induce fz or Dfz2 expression. RT-PCR analysis of S2 cells (lanes 2 and 4) and S2 cells expressing ΔN-Arr (lanes 3 and 5) demonstrated that the numbers of fz and Dfz2 transcripts were the same or similar in the two cell types.
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