Structural and functional characterisation of hedgehog ligand-receptor complexes

Thesis

Members of the Hedgehog (HH) family of morphogenic signalling molecules are key mediators of many fundamental processes in embryonic development. A relatively small change in HH concentration results in the specification of distinct cell types. Such fine-tuning necessitates a range of regulatory cell-surface proteins to control the concentration of HH to which responding cells are exposed. This thesis focuses on the structural and functional characterisation of three human extracellular modulators of the HH pathway, namely the hedgehog-interacting protein HHIP, the glypican GPC3 and the Growth Arrest Specific 1 (GAS1). Cell culture robot protocols were developed to allow large-scale expression of these targets in mammalian cells (Chapter 3). In Chapter 4, the structure of the HH antagonist HHIP alone and in complex with HH ligands is described. These studies combined with functional experiments reveal a binding mode distinct from previously defined ligand interaction sites, with the HH metal-binding sites playing key roles. The structural and functional analysis of GPC3, another negative HH regulator, is described in Chapter 5. Binding studies suggest that the GPC3 core domain does not bind directly to SHH and that the GPC3-attched heparan sulphate chains play an important role in HH regulation. Chapter 6 reveals crystal structures of the GAS1 ectodomain, a HH agonist, allowing comparison to glial cell line-derived neurotrophic factor receptors, the identification of an unexpected ligand and mapping of the HH binding site. In summary, this work provides insights into the extracellular modulation of HH signalling and extends our current knowledge of this fundamental signalling pathway. It also offers a model to explain how both agonists and antagonists can adopt similar mechanisms of HH binding.

Authors: Bishop, BF

• DOI: 10.5287/ora-9ejo6dkaz
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: HHIP; Hedgehog
• Subjects: Structural Biology