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

Abstract

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 mg͞liter. 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 Ϸ9 nM.

Wnt signaling in disease and in development

“…Frizzled receptors are 7 transmembrane molecules with a long amino-terminal extension called CRD (cysteine-rich domain). Wnt proteins bind directly to the CRD of Fz [2][3][4]. Wnt signaling requires not only a functional Fz, but also the presence of a long single pass transmembrane molecule of the LRP (LDL receptor related protein) class, identified as the gene arrow in Drosophila [5] and as LRP5 or 6 in vertebrates.…”

Frizzled and LRP5/6 Receptors for Wnt/ -Catenin Signaling

“…(Continued) (C ) Crystal structure of FZD8CRD. Residues in red (and to a lesser degree those in orange) are suggested to be a Wnt-binding interface from an alanine scanning mutagenesis (Hsieh et al 1999;Dann et al 2001), which partially overlaps with site 2 indentified in the Wnt8 -FZD8CRD co-crystal structure. Residues in green when altered did not affect Wnt activity and areas in gray were not tested.…”

“…1B). Wnt/Wg ligands bind to CRD with high affinity (K d of 1 -10 nM) (Hsieh et al 1999;Rulifson et al 2000;Wu and Nusse 2002), although fz/Dfz1 PCP signaling may not involve any Wnt ligands. Deletion of the CRD prevents Wnt/Wg binding, but the CRD can be replaced with other heterologous Wnt-binding domains to generate a functional FZD receptor Mulligan et al 2012).…”

“…This constitutes Wnt8-FZD8CRD interaction "site 2," which roughly corresponds to a Wnt-binding surface encompassing residues near the second cysteine (C2) of FZD8CRD as suggested by scanning mutagenesis (Fig. 1B,C) (Hsieh et al 1999;Dann et al 2001). Both site 1 and site 2 are dominated by hydrophobic contacts (lipid-in-groove and knob-inhole, respectively), which are mostly mediated by conserved residues of Wnt8 and FZD8CRD, suggesting an explanation for broad or relatively promiscuous specificity of Wnt -FZD relationships, i.e., a single Wnt can often engage multiple FZD proteins and vice versa.…”

Expression Profiles and Functional Analyses of Wnt-Related Genes in Human Joint Disorders

“…Wnt-1, -3a, -4, -7a, and -7b also inhibit early differentiation of chondrocytes in the chick limb. [13][14][15][16][17][18][19][20] In addition, these Wnt genes are associated with BMP signaling or synovial joint formation and the maintenance of joint integrity. 17,20 Recently, the importance of the Wnt pathway in normal bone accrual and in fibroblast-like cells in RA patients was reported.…”