Biochemical characterization of Wnt-Frizzled interactions using a soluble,biologically active vertebrate Wnt protein

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 biologicall y active Xenopus Wnt8 (XWnt8). Epitope- or alkaline phosphatase-tagged XWnt 8 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 l iter, 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 fo r studying the interactions between soluble XWnt8 and various Frizzled prot eins, 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 affi nity of the XWnt8-alkaline phosphatase Fusion for the purified mouse Frizzl ed 8-CRD-IgG fusion is approximate to 9 nM.