Towards a structure-based understanding of WNT receptor function

Thesis

The highly conserved WNT signalling pathways are vital during development and throughout the lives of animals, while aberrant WNT signalling is implicated in a range of degenerative diseases, as well as cancers. Cells detect WNT signals through members of the Frizzled (FZD1- 10) family of cell-surface receptor proteins, resulting in multiple possible outcomes including changes in gene expression and cell polarity. However, how WNT-FZD specificity is achieved, and how different WNT signalling modules are differentially activated by FZDs, remains unclear. It is also uncertain how the overall structure of FZD compares to other similar proteins and contributes to WNT signalling regulation.

In this thesis, work towards the determination of the first multidomain FZD structure is presented. The expression levels of a representative range of human FZDs are compared. The optimisation of expression and thermostability of candidate receptors for structural studies, through protein engineering, leading to poorly diffracting crystals, is detailed.

The crystal structure of the FZD3 extracellular domain in complex with a highly specific nanobody (Nb) is described, suggesting a role for the latter in inhibiting signalling. Conversely, a synthetic WNT signalling agonist has been created by fusing this Nb to a WNT-inhibitory protein domain that binds to the WNT coreceptor, LRP6. Functional dissection of FZD3 facilitated by this agonist confirms that the long C-terminal tail of FZD3 is dispensable for ‘canonical’ WNT signalling, whereas disruption of the intracellular loops abolishes this activity. Moreover, the mutation of glycine5.64f, perfectly conserved amongst FZD receptors and implicated in a developmental disorder, is shown to have no effect on ‘canonical’ signalling, and instead this residue may form part of a previously overlooked molecular ‘switch’ required for G protein-dependent signalling.

Finally, the crystal structures of the FZD10 and FZD3 extracellular domains contribute to a more complete understanding of the structural biology of these enigmatic receptors.

Authors: Hillier, J

• DOI: 10.5287/ora-dqz6ey0e6
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English