The secreted glycoproteins in the Wnt/Wingless (Wg) family serve diverse functions in developmental processes, ranging from cell fate specification and cell proliferation to cell migration and cell polarity [1, 2], and deregulation of the Wnt signaling pathway can lead to cancer [3, 4]. In a simplified model of the canonical Wnt/β-catenin signaling pathway, the binding of Wnts to their receptors activates a downstream component, Dishevelled (Dsh). Dsh in turn inhibits glycogen synthase kinase (GSK)-3β in the β-catenin destruction complex, which mainly consists of Axin, GSK-3β, adenomatous polyposis coli (APC) and β-catenin. Consequently, the level of cytoplasmic β-catenin rises, and stabilized β-catenin, together with the transcriptional factor LEF/TCF, regulates the transcription of Wnt target genes.
The mechanism by which the Wnt signal is transduced through its transmembrane receptors is not clear. Wnt proteins have been shown to bind to Frizzleds (Fzs), which are seven-transmembrane receptors [5, 6]. The binding of Wnts occurs within an aminoterminal cysteine-rich-domain (CRD) of Fzs. At least 11 vertebrate and 4 Drosophila Fz genes http://www.stanford.edu/~rnusse/wntwindow.html have been identified. However, their functions and ligand specificities remain to be understood. Recently, it was reported that two single-transmembrane proteins of the LDL-receptor-related proteins (LRP) family, LRP5 and LRP6, are also involved in receiving the Wnt signal [7–9]. Drosophila Arrow (Arr), which is homologous to murine and human LRP5 and LRP6, was shown to be essential for Wg signaling. Genetic data from flies and mice indicate that Arr and LRP6 play a positive role in Wnt signaling [7, 9]. A genetic epistasis experiment placed Arr between Wg and Dsh , acting in parallel or downstream of DFz. In addition, LRP6 has been reported to bind Wnt-1 and to associate with Fz in a Wnt-dependent manner . Taken together, these results support a co-receptor model: upon exposure to Wnts, LRP5 or LRP6 forms a complex with Wnt and Fzs, transducing the Wnt signal downstream to stabilize cytoplasmic β-catenin. Consistent with this, it has been demonstrated that Dickkopf-1 inhibits Wnt/β-catenin signaling by binding to LRP5 or LRP6 to prevent Wnt-receptor complex formation [10–12].
Alternatively, however, it is possible that Wnt/Wg can signal through LRP/Arr in the absence of Fzs. In agreement with this possibility, an intracellular domain of LRP5 was reported to interact directly with Axin , and Wnt signaling appears to stimulate the recruitment of Axin to LRP5 at the membrane, where Axin is degraded. Furthermore, it was reported that LRP signaling can be activated in a Dsh – independent fashion . These data suggest that Wnt/Wg could signal through LRP directly to the β-catenin destruction complex in a Fz- and Dsh-independent fashion.
To evaluate whether Fzs are required for the transduction of Wnt/Wg signaling in the presence of LRPs, we have established a sensitive method to measure Wg signaling activities using a LEF-luciferase reporter in Drosophila S2 cells. We show that DFz2 and LRP/Arr cooperate with Wg to activate the signaling pathway. Using a double-strand RNA interference (dsRNAi) technique designed to eliminate specific proteins [15, 16], such as Arr, DFz2 and Dsh, we have developed strong evidence that Wg requires both types of receptors to transduce a signal efficiently through Dsh to stabilize β-catenin.