Hsieh, J. C., Rattner, A., Smallwood, P. M. & Nathans, J. Biochemical characterization of Wnt-frizzled interactions using a soluble, biologically active vertebrate Wnt protein. Proc. Natl. Acad. Sci. USA 96, 3546-3551

Johns Hopkins University, Baltimore, Maryland, United States
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 04/1999; 96(7):3546-51. DOI: 10.1073/pnas.96.7.3546
Source: PubMed


Biochemical studies of Wnt signaling have been hampered by difficulties in obtaining large quantities of soluble, biologically active Wnt proteins. In this paper, we report the production in Drosophila S2 cells of biologically active Xenopus Wnt8 (XWnt8). Epitope- or alkaline phosphatase-tagged XWnt8 proteins are secreted by concentrated S2 cells in a form that is suitable for quantitative biochemical experiments with yields of 5 and 0.5 mg per liter, respectively. Conditions also are described for the production in 293 cells of an IgG fusion of the cysteine-rich domain (CRD) of mouse Frizzled 8 with a yield of 20 mg/liter. We demonstrate the use of these proteins for studying the interactions between soluble XWnt8 and various Frizzled proteins, membrane anchored or secreted CRDs, and a set of insertion mutants in the CRD of Drosophila Frizzled 2. In a solid phase binding assay, the affinity of the XWnt8-alkaline phosphatase fusion for the purified mouse Frizzled 8-CRD-IgG fusion is approximately 9 nM.

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Available from: Amir Rattner, Jun 27, 2015
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    • "Wnt signals have been suggested to be important for the generation of RPE cells (Fujimura et al., 2009; Steinfeld et al., 2013), but whether Wnt activity plays any role in the specification of neural retina cells has not been determined. We therefore first tested whether Wnt activity is required for the specification of neural retina cells, by culturing stage 10 OVL explants and stage 13 OV explants together with a soluble Frizzled receptor (Frizzled-conditioned medium) to inhibit Wnt activity (Gunhaga et al., 2003; Hsieh et al., 1999). Stage 10 OVL explants cultured alone or in the presence of Frizzled generated Rax2 + and Vsx2 + neural retina cells, whereas no FoxG1 + cells were detected (supplementary material Fig. S8A,B). "
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    • "The Wnt ligand binds immediately to its receptor gene, Frizzled, and this binding site is divided into the canonical and non-canonical pathways (1,2). Among them, the canonical pathway is dependent on β-catenin and affects cancer cell adhesion. "
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    • "Fzd and LRP are transmembrane proteins. The extracellular domain of Fzd contains a cysteine-rich domain, involved in Wnt binding [84] followed by a seven-pass transmembrane region. The extracellular domain of LRP is composed by EGF-like repeats involved in the interaction with Fzd receptor [85]. "
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