The mechanism of endogenous receptor activation functionally distinguishes prototype canonical and noncanonical Wnts

Abstract

Wnt glycoproteins are developmentally essential signaling molecules, and lesions afflicting Wnt pathways play important roles in human diseases. Some Wnts signal to the canonical pathway by stabilizing beta-catenin, while others lack this activity. Frizzled serpentine receptors mediate distinct signaling pathways by both classes of Wnts. Here, we tandemly linked noncanonical Wnt5a with the C-terminal half of Dickkopf-2 (Dkk2C), a distinct ligand of the Wnt coreceptor LRP5/6. Whereas Wnt5a, Dkk2C, or both together were incapable of stimulating endogenous canonical signaling, the Wnt5a/Dkk2C chimera efficiently activated this pathway in a manner inhibitable by specific antagonists of either frizzled or LRP receptors. Thus, activation of the canonical pathway requires ligand coupling of an endogenous frizzled/LRP coreceptor complex, rather than Wnt triggering each receptor independently. Moreover, fusion of Wnt5a with Dkk2C unmasked its ability to signal to Dishevelled through multiple frizzleds, indicating that the lack of functional interaction with LRP distinguishes noncanonical Wnt5a from canonical Wnts in mammalian cells. These findings provide a novel mechanism by which the same receptor can be switched between distinct signaling pathways depending on the differential recruitment of a coreceptor by members of the same ligand family.

FIG. 1.

Lack of ability of Wnt5a to activate canonical Wnt signaling. (a) Comparison of Wnt3a and Wnt5a effects on β-cateninin stabilization. One microgram of vector, Wnt3a, or Wnt5a construct was transfected into 293T cells, and the uncomplexed β-catenin (β-cat) assay was performed after 48 h. Wnt expressions were detected by anti-Flag (α-Flag) antibody. (b) RT-PCR analysis of Wnt receptors in 293T cells. −RT, amplification in the absence of reverse transcriptase as a negative control. (c) Wnt5a does not activate the canonical β-catenin pathway through Hfz5. One microgram of each construct was transfected into 293T cells, and uncomplexed β-catenin levels were measured 48 h later. Hfz5 functional expression was confirmed by its activity in the TCF reporter assay (see below). (d) Comparison of Wnt3a and Wnt5a in TOPFLASH TCF signaling through different frizzleds. The values represent the means (± standard deviations) of two independent experiments performed in triplicate and are expressed in relative luciferase units (RLU).

FIG. 2.

Differential canonical signaling responses of LRP6 to Wnt3a, Wnt5a, or Dkk2C. (a) Comparison of β-catenin (β-cat) stabilization induced by Wnt3a, Wnt5a, and Dkk2C in response to exogenous LRP6. One microgram of each construct was transfected into 293T cells. Uncomplexed β-catenin was measured after 48 h. Vec, vector; α-Flag, anti-Flag antibody. (b) Differential TCF reporter activation by Wnt3a, Wnt5a, and Dkk2C in response to exogenous LRP6. RLU, relative luciferase units. (c) Effects of Wnt3a, Wnt5a, and Dkk2C on constitutive tyrosine phosphorylation of the LRP6-FGFR chimera. One microgram of Flag-tagged LRP6-FGFR was cotransfected with 4 μg of vector, Wnt3a, Wnt5a, or Dkk2C into 293T cells. At 48 h, 1 mg of each cell lysate was immunoprecipitated (IP) with anti-Flag M2 beads followed by immunoblotting with anti-p-Tyr. The same blot was stripped and reprobed with anti-Flag for detection of LRP6-FGFR protein levels. The results shown are representative of three independent experiments. The relative intensity of each phosphorylated LRP6-FGFR band was normalized against the corresponding LRP6-FGFR protein band following densitometry analysis. Expression of transfected proteins was detected with anti-Flag antibody in a and c.

FIG. 3.

A Wnt5a/Dkk2C chimeric ligand efficiently activates canonical β-catenin signaling. (a) Induction of β-catenin stabilization by expression of Wnt5a/Dkk2C alone (left panel) or with ectopic LRP6 (right panel). One microgram of each construct was transfected into 293T cells followed by uncomplexed β-catenin analysis after 48 h. Vec, vector; α-β-cat, anti-β-catenin antibody; α-Flag, anti-Flag antibody. (b) Detection of secretion of Wnt5a and Wnt5a/Dkk2C in 293T cells though surface biotinylation assay. One microgram of Flag-tagged Wnt5a or Wnt5a/Dkk2C construct was transfected into 293T cells. Cell surface biotinylation assay was performed 48 h later. (c) Specific antagonists of frizzled and LRP receptors inhibit canonical signaling by Wnt5a/Dkk2C. Upper panel, FRP and LRP6ΔC inhibit Wnt5a/Dkk2C-induced β-catenin stabilization. One microgram of Wnt5a/Dkk2C was cotransfected with 1 μg of vector, FRP, or LRP6ΔC in 293T cells. Lower panel, short hairpin RNA against LRP6 (shLRP6) inhibits β-catenin stabilization induced by Wnt5a/Dkk2C. One microgram of Wnt5a/Dkk2C was cotransfected with 2.5 μg of control vector or pSuper-shLRP6. Uncomplexed β-catenin was measured after 48 h. The specificity and effectiveness of the shLRP6 have been proven previously by targeting the exogenously expressed Flag-LRP6 receptors (3). (d) Dvl is required for canonical signaling induced by Wnt5a/Dkk2C. A total of 1.5 μg of Wnt3a, Wnt5a/Dkk2C, or ΔN-LRP6 was cotransfected with 4 μg of an unrelated short hairpin construct or combined short hairpin Dvl (shDvl) constructs. Uncomplexed β-catenin levels were determined at 72 h posttransfection. A pool of antibodies against Dvl-1, Dvl-2, and Dvl-3 were used to detect endogenous Dvl proteins in 50 μg of cell lysate. Tubulin levels served as a loading control. (e) Activation of TOPFLASH TCF luciferase reporter by Wnt5a/Dkk2C through multiple frizzled/LRP6 coreceptors. The expression of transfected proteins in panels a, b, c, and d were detected with anti-Flag antibody.

FIG. 4.

Activation of canonical Wnt signaling requires ligand-induced coupling of endogenous frizzled and LRP6 coreceptors. (a) β-catenin stabilization by the Wnt5a/Dkk2C chimera but not by Wnt5a and Dkk2C together. Each ligand construct as indicated was transfected in 293T cells at 0.5 or 2 μg. Uncomplexed β-catenin levels were measured after 48 h posttransfection. The anti-Flag antibody (α-Flag) was utilized to detect transfected proteins. α-β-cat, anti-β-catenin antibody; Vec, vector. (b) Paracrine TCF reporter assay. Target 293T cells transfected with Topglow TCF reporter (500 ng) and pRL-CMV (5 ng) were cocultured with 293T effector cells transfected with 2 μg of each empty vector, Wnt5a, Dkk2C, Wnt5a plus Dkk2C, or Wnt5a/Dkk2C. Luciferase activities were measured 48 h later. RLU, relative luciferase units.