The Wnt signaling pathway contributes to many developmental decisions in a variety of cell types and animal models. Wnt signaling is involved in cell fate determination, cell polarity, tissue patterning, control of cell proliferation, and development of neoplasia.1,2 Wnt proteins are secreted glycoproteins with a conserved pattern of 23-24 cysteine residues. There are 19 Wnt family members in the human genome and multiple Wnt proteins have been identified in laboratory model organisms, including 7 Wnt signals in Drosophila and 18 in mice [Please refer to the Wnt homepage at http://web.stanford.edu/~rnusse/wntwindow.html]. Wnt ligands bind to receptors of the Frizzled family, sometimes in conjunction with other membrane-associated proteins. Downstream effects of Wnt signaling occur through different intracellular components, depending on which pathway is activated. Three pathways have been characterized: the canonical Wnt/beta-catenin pathway, Wnt/Ca2+ pathway, and planar cell polarity (see Figure 1).3,4
One mode of signaling pathway regulation occurs at the level of extracellular antagonists that interact with ligands and prevent signaling. The secreted Frizzled-related proteins (sFRP) were the first Wnt antagonists to be identified. These proteins consist of approximately 300 amino acids (aa) containing a signal sequence, a Frizzled-like cysteine-rich domain (CRD), and a small hydrophilic C-terminal domain.5 The fact that these proteins had significant similarity to the extracellular domain of Frizzled, including a series of ten diagnostic cysteine residues that make up the CRD region of the protein, suggested that they would bind Wnts and likely inhibit signaling. Five mammalian sFRPs have been identified to date with additional family members characterized in Xenopus and chick. These proteins have many different names, but for this review they will be referred to as sFRPs (see Table 1). As a group, sFRPs are expressed in a variety of embryonic and adult tissues, indicating that they may provide a common mechanism for inhibiting Wnt signaling in diverse tissues and cell types. Individual family members, however, have highly specific spatial and temporal expression patterns thus suggesting that they may play distinct roles in developmental processes.5,6
Early studies with sFRPs indicate that these proteins bind Wnts directly and functionally inhibit Wnt signaling in Xenopus embryos and cell lines.7 More recently, deletion analysis indicates that the Frizzled CRD domain of sFRP-3 is necessary and sufficient for both binding and functional inhibition of Wnt-1 signaling.8 Some partial deletions of CRD maintain Wnt-1 binding while functional inhibition is lost, however, suggesting that more than mere binding of the ligand is necessary for antagonizing Wnt signaling.8 Using recombinant sFRP-1 protein, Uren et al.2 demonstrate that this protein directly binds Wingless (Wg) in ELISA and co-precipitation assays.2 Surprisingly, the CRD is not required for binding as deletions of CRD retain the ability to bind Wg. This suggests that other sites within the protein are also involved in Wg binding. It remains to be seen if the differences between sFRP-1 and -3 or differences between Wg and Wnt-1 can explain this discrepancy of whether the CRD of sFRP is necessary for binding Wnt ligands.
The simple model of binding between a sFRP molecule and Wnt ligand to inhibit signaling is being further revised. Crystallographic analysis reveals a high degree of similarity in how the CRDs of sFRP-3 and Frizzled-8 bind Wnts, and that the CRDs exhibit a conserved dimer interface that may be a feature of Wnt signaling.9 This agrees with biochemical evidence that the stoichiometry of sFRP and Wg occurs at 2:1 in addition to a 1:1 ratio in the cross-linked complex.2 In addition, sFRP-1 exhibits a biphasic effect on Wg activity as measured by an armadillo stabilization assay: increasing activity at low concentrations and decreasing activity at high concentrations. These results suggest that sFRP-1 may only act as a Wnt antagonist at regions of high, localized concentrations and as an agonist at regions of lower Wnt concentrations.2 sFRPs may have another way to inhibit Wnt signaling, besides direct binding. sFRP-1 forms complexes with Frizzled-6, and the interaction is mediated by their homologous CRD regions.10 Thus, sFRPs may inhibit Wnt signaling through interactions directly with Wnts and/or through formation of non-functional complexes with the Frizzled receptor.10
||SARP-2, FrzA, Frp or FRP-1
||SARP-1, SDF-5, FRP-2
||FrzB or Frzb-1, Fritz, FRP-3
||FRP-4, FrpAP, frpHE
||(found in Xenopus and chick)
||(found in Xenopus)
||(found in Xenopus)
|Table 1. Five mammalian sFRPs have been identified to date with additional family members characterized in Xenopus and chick (i.e. proteins identified in italics within the Table).
- Cadigan, K.M. and R. Nusse (1997) Genes Dev. 11:3286.
- Uren, A. et al. (2000) J. Biol. Chem. 275:4374.
- Kuhl, M. et al. (2000) Trends Genet. 16:279.
- Wallingford, J.B. et al. (2000) Nature 405:81.
- Rattner, A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2859.
- Leimeister, C. et al. (1998) Mech. Dev. 75:29.
- Moon, R.T. et al. (1997) Cell 88:725.
- Lin, K. et al. (1997) Proc. Natl. Acad. Sci. USA 94:11196.
- Dann, C.E. et al. (2001) Nature 412:86.
- Bafico, A. et al. (1999) J. Biol. Chem. 274:16180.