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Insights into Wnt binding and signalling from the structures of two Frizzled cysteine-rich domains

Abstract

Members of the Frizzled family of seven-pass transmembrane proteins serve as receptors for Wnt signalling proteins1. Wnt proteins have important roles in the differentiation and patterning of diverse tissues during animal development2,3, and inappropriate activation of Wnt signalling pathways is a key feature of many cancers4. An extracellular cysteine-rich domain (CRD) at the amino terminus of Frizzled proteins binds Wnt proteins1, as do homologous domains in soluble proteins—termed secreted Frizzled-related proteins5—that function as antagonists of Wnt signalling6,7,8. Recently, an LDL-receptor-related protein has been shown to function as a co-receptor for Wnt proteins and to bind to a Frizzled CRD in a Wnt-dependent manner9,10,11. To investigate the molecular nature of the Wnt signalling complex, we determined the crystal structures of the CRDs from mouse Frizzled 8 and secreted Frizzled-related protein 3. Here we show a previously unknown protein fold, and the design and interpretation of CRD mutations that identify a Wnt-binding site. CRDs exhibit a conserved dimer interface that may be a feature of Wnt signalling. This work provides a framework for studies of homologous CRDs in proteins including muscle-specific kinase and Smoothened, a component of the Hedgehog signalling pathway12,13.

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Figure 1: Crystal structure of the sFRP-3 and mFz8 CRDs.
Figure 2: Binding of XWnt8–AP to mFz8 CRD–Myc–GPI displayed on the surface of transfected COS cells.
Figure 3: Structure-based alignment of CRD sequences indicating sites altered by mutagenesis.
Figure 4: Surfaces involved in Wnt–CRD interactions modelled on the mFz8 CRD structure.

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Acknowledgements

We thank J. Beneken, K. Ramyar, C. Ogata and the staff at X4A for help with data collection; Z. Dauter for advice and technical assistance with phase determination using solvent halides; and D. Andrew, P. Beachy, J. Berg, J. Taipale and W. Yang for comments on the manuscript. Structures reported are accession numbers 1IJX for sFRP-3 and 1IJY for mFz8. Supported by the Howard Hughes Medical Institute.

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Correspondence to Daniel J. Leahy.

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Table 1 Data processing, MAD phasing, and model refinement statistics

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Dann, C., Hsieh, JC., Rattner, A. et al. Insights into Wnt binding and signalling from the structures of two Frizzled cysteine-rich domains. Nature 412, 86–90 (2001). https://doi.org/10.1038/35083601

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