Structure and functional properties of Norrin mimic Wnt for signalling with Frizzled4, Lrp5/6, and proteoglycan

Abstract

Wnt signalling regulates multiple processes including angiogenesis, inflammation, and tumorigenesis. Norrin (Norrie Disease Protein) is a cystine-knot like growth factor. Although unrelated to Wnt, Norrin activates the Wnt/β-catenin pathway. Signal complex formation involves Frizzled4 (Fz4), low-density lipoprotein receptor related protein 5/6 (Lrp5/6), Tetraspanin-12 and glycosaminoglycans (GAGs). Here, we report crystallographic and small-angle X-ray scattering analyses of Norrin in complex with Fz4 cysteine-rich domain (Fz4 _CRD), of this complex bound with GAG analogues, and of unliganded Norrin and Fz4 _CRD. Our structural, biophysical and cellular data, map Fz4 and putative Lrp5/6 binding sites to distinct patches on Norrin, and reveal a GAG binding site spanning Norrin and Fz4 _CRD. These results explain numerous disease-associated mutations. Comparison with the Xenopus Wnt8–mouse Fz8 _CRD complex reveals Norrin mimics Wnt for Frizzled recognition. The production and characterization of wild-type and mutant Norrins reported here open new avenues for the development of therapeutics to combat abnormal Norrin/Wnt signalling.

DOI: http://dx.doi.org/10.7554/eLife.06554.001

eLife digest

The cells within an animal need to be able to communicate with each other to coordinate many complex processes in the body, such as the formation of tissues and organs. One way in which the cells can communicate is through a pathway called Wnt signalling. Generally, one cell releases a protein called Wnt, which binds to a receptor protein called Frizzled that sits on the surface of the same or another cell. This activates a series of events in the cells that can change the activity of particular genes. Wnt signalling has many roles in animals, and defects in it can contribute to cancer and other devastating diseases.

Another protein called Norrin can also activate Wnt signalling by binding to Frizzled and another receptor protein called Lrp5/6. This group or ‘complex’ also includes molecules called glycosaminoglycans. In humans, mutations in the gene that encodes Norrin can cause a disease in which blood vessels in the eye fail to form correctly, which can result in blindness. However, it is not clear how Norrin activates Wnt signalling.

Chang et al. developed a method to produce large quantities of Norrin protein to allow them to study the structure of the protein. Then, a technique called X-ray crystallography was used to reveal the three-dimensional structure of Norrin when it is bound to Frizzled. The model reveals that a pair of Norrin proteins form a complex with two Frizzled proteins and highlights particular areas of the Norrin protein that interact with Frizzled. Molecules of glycosaminoglycan bind to a site in the complex that spans both Norrin and Frizzled. The model also predicts that other areas of the Norrin protein may be involved in binding Lrp5/6.

Chang et al. compared the model to the structure of a Wnt protein bound to Frizzled, which revealed that Norrin and Wnt show some fundamental similarities in the way they bind to Frizzled. These findings move us closer to defining the essential features of the protein complexes that modify Wnt signalling, and may aid the development of new therapies for diseases that affect the development of the eye.

DOI: http://dx.doi.org/10.7554/eLife.06554.002