Signal transduction of the GPCR Smoothened: a key protein in Hedgehog-regulated morphogenesis and oncogenesis

Thesis

The Hedgehog signalling pathway is crucial for embryonic development and tissue homeostasis. Misregulation can result in severe developmental defects or oncogenesis, with an estimated 25% of all cancer deaths exhibiting improper Hedgehog signalling. The G-protein coupled receptor Smoothened plays a vital role in this pathway; it is responsible for transducing the Hedgehog signal across the plasma membrane. This makes Smoothened a highly attractive target for anticancer drug development. Despite the importance of Smoothened signalling and the plethora of structural data now available, the molecular mechanism of signal transduction is poorly understood. Here, we explore the mechanism of Smoothened activation and regulation by a range of small molecule ligands. We show that agonist binding at multiple sites can induce conformational changes required for effector binding. We couple these data with mutational analysis to propose distinct roles for each binding site. We also develop and validate a novel small molecule binding assay and adapt it to study allosteric communication within Smoothened. Finally, we present a strategy for use of the novel assay to identify next generation cancer treatments and address drug resistance.

Authors: Woolley, R

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: G-protein coupled receptor; Hedgehog signalling; nanobodies; FRET
• Subjects: Biochemistry; Structural biology; Cell signalling; Developmental biology