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A novel set of Wnt-Frizzled fusion proteins identifies receptor components that activate beta -catenin-dependent signaling.
Holmen SL
,
Salic A
,
Zylstra CR
,
Kirschner MW
,
Williams BO
.
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Wnt proteins initiate the canonical (beta-catenin-regulated) signaling cascade by binding to seven-transmembrane spanning receptors of the Frizzled (Fz) family together with the coreceptors LRP5 and -6, members of the low density lipoprotein receptor-related protein family (LRP). Several reports have shown physical and functional associations between various Wnt, LRP, and Frizzled molecules; however, the underlying mechanisms for selectivity remain poorly understood. We present data on a novel set of Wnt-Fz fusion constructs that are useful for elucidating mechanisms of Wnt signal transduction specificity in both Xenopus embryos and 293T cells. In 293T cells, coexpression of several Wnt-Fz fusion proteins with LRP6, but not LRP5, significantly activated a Wnt-responsive promoter, Optimized TOPFlash. Interestingly, Wnt proteins from both the Wnt1 and Wnt5A classes, when fused to the same Frizzled, can synergize with LRP6 to activate signaling and induce secondary axes in Xenopus embryos. However, when several Wnt-Fz constructs containing different Frizzled molecules were tested, it was found that all Frizzled molecules are not equivalent in their ability to activate the canonical Wnt pathway in this context. The data suggest that the distinction between the two Wnt classes lies not in intrinsic differences in the molecules but via the Frizzled molecules with which they interact.
Figure 1
. Coexpression of Wnt1 or Wnt3A with LRP5 or LRP6 synergistically transactivates the OT reporter. A, relative transactivation of the OT reporter upon transfection of the indicated plasmids. All values are expressed relative to the levels of luciferase activity seen in cells transfected with empty vectors and are normalized for transfection efficiency by determining levels of β-galactosidase activity expressed from the pCMV-βgal plasmid equally transfected in each experiment. Values represent the average of duplicate experiments, and bars representing the standard error are shown. βcat is S37A β-catenin. B, immunoblot for β-catenin protein (top) and α-tubulin (bottom) in cytosolic fractions from cell lysates transfected with the indicated plasmids. The anti-tubulin blot is included to confirm equivalent protein loading in each lane.C, immunoblot for the V5 epitope (top) on lysates from untransfected (UT) 293T cells and cells transfected with expression vectors encoding versions of LRP5 (5) and LRP6 (6) tagged on the carboxyl terminus with the V5 epitope. An anti-tubulin blot (bottom) is again shown to confirm equivalent loading.
Figure 2
Schematic diagram of constructs. The constructs used in this work are linearly represented.Xenopus Wnt8 (XWnt8) was fused to human Frizzled 5 via a linker containing a single myc tag and a glycine-serine rich region to provide steric flexibility. A dominant negative (DN) form (missing the last 98 amino acids) of XWnt8, as well as full-length XWnt11 and XWnt5A were also fused to hFz5 and produced in a similar manner. Constructs containing DNWnt8 fused to mouse Fz3, -4, -6, and -7 were also produced that contained a glycine-serine rich linker region and also contained five copies of the myc epitope. Finally, fusion proteins containing either full-length XWnt8 or DNWnt8 fused to a form of hFz5 truncated prior to the first transmembrane domain and epitope tagged with the V5 epitope were also created.
Figure 3
Both an XWnt8-Fz5 Fusion and a DNWnt8-Fz5 fusion protein synergize with LRP6 to strongly transactivate the OT reporter and stabilize β-catenin. A, optimized TOPFlash luciferase reporter gene assay. Experiments were performed as described in Fig. 1 A. Values represent the average of duplicate experiments, and bars representing the standard error are shown. Note the difference in the scale of they axis between Fig. 1 and this and the following figures.B, Western blot of cytoplasmic β-catenin (top) and α-tubulin (bottom) on 293T cell lysates transfected with the expression plasmids indicated in A. C, immunoblot for Frizzled 5 protein in whole cell lysates from cells transfected with full-length XWnt8-Fz5 fusion (WT) and the DNWnt8-Fz5 (DN) fusions. An α-tubulin immunoblot is also shown. D, immunoprecipitation analysis performed on cell lysates and media from 293T cells transfected with different combinations of the following expression plasmids: an amino-terminally truncated form of LRP6 fused to the constant region of mouse IgG (N-LRP6-IgG, see text) and either full-length XWnt8 (WT) or a dominant negative form of XWnt8 (DN) fused to hFz5 truncated prior to the first transmembrane domain and tagged with the V5 epitope. Proteins were immunoprecipitated with anti-mouse IgG, and the immunoprecipitate was immunoblotted for the V5 epitope.
Figure 4
XWnt11-Fz5 induced ectopic secondary axis in Xenopus embryos. 0.1–1 ng of a given fusion mRNA was injected into one ventralblastomere of four cell-stage Xenopus embryos. Embryos were scored at stages 18–24 for the presence of a duplicated axis. A, uninjected;B, XWnt11 injected; C, hFz5 injected;D, XWnt11-Fz5 injected.
Figure 5
Fusion proteins containing either class of Wnt molecules fused to Frizzled 5 synergize with LRP6 to activate the OT reporter in 293T cells. A, OT reporter assays performed on cell lysates transfected with the indicated expression vectors. Values are determined and represented as described in Fig. 1.B, β-catenin and tubulin immunoblots performed on cell lysates transfected with the expression plasmids indicated inA. C, immunoblot with anti-hFz5 antibody of lysates from cells transfected with Xwnt11-Fz5 (11), XWnt5A-Fz5 (5A), or XWnt8-Fz5 (8) fusion protein expression vectors.
Figure 6
Fusion of different Frizzled genes to DNWnt8 reveals differences in their ability to activate the canonical pathway. A, OT reporter gene assay on lysates from 293T cells transfected with the indicated expression plasmids. Assays are represented as described in Fig. 1. B, Western blot of cytoplasmic β-catenin and α-tubulin levels in cells transfected with the plasmids indicated in A. C, immunoblot with antibody to the myc epitope on cell lysates from 293T cells either untransfected (UT) or transfected with expression vectors encoding fusions of DNWnt8 to mouse Frizzled 3 (3), mouse Frizzled 4 (4), human Frizzled 5 (5), mouse Frizzled 6 (6), or mouse Frizzled (7). Note that five copies of the myc epitope have been inserted between the Wnt and Frizzled portions of the fusion protein (see Fig. 2).
Figure 7
Transactivation of the OT reporter induced by the XWnt8-Fz5 expression vector is inhibited by dominant negative forms of LRP6 and TCF4 in a dose-dependent manner. A, OT reporter gene assay on cells transfected with the indicated expression plasmids. Note that the shaded trianglerepresents increasing amounts of N-LRP6-IgG fusion or ΔN-TCF4 protein transfected into the cells. B, Western blot of LRP6-V5 and N-LRP6-mIgG expression in the absence or presence of increasing amounts of the inhibitors. C, Western blot of XWnt8-Fz5 expression in the absence or presence of increasing amounts of the inhibitors.
Figure 8
Model of Wnt-Fz fusion protein function. The four epidermal growth factor repeat (EGFR) regions of LRP5/6 are represented as gray boxes, with EGFR regions 1 and 2 interacting with Wnt. The CRD of Frizzled is represented by ablack box. Wnt is fused to the N terminus of Frizzled and functions to bring LRP5/6 into a physical complex with the Frizzled member of the fusion, which then signals to stabilize β-catenin and activate the canonical Wnt pathway. The following Wnt-Fz fusions are capable of signaling in synergy with LRP5/6 to activate the canonical Wnt pathway XWnt8-Fz5, DNWnt8-Fz5, XWnt5A-Fz5, XWnt11-Fz5, XWnt8-Fz4, and XWnt8-Fz7. XWnt8-Fz3 and XWnt8-Fz6 do not strongly activate the canonical Wnt pathway. A dominant negative form of LRP6 (N-LRP6-IgG), consisting of only the first two EGFR regions, as well as Frizzled related protein 1 (FRP1), block the synergistic signaling activity of XWnt8-Fz5 with LRP5/6, presumably by interfering with formation of the Wnt-Fz·LRP5/6 complex.
