WNT/Frizzled signaling : illuminating the road towards pathway selectivity

Abstract: Cells sense and respond to their environment via receptors embedded in the plasma membrane. Receptors allow flow of information from outside to the inside of the cell and are generally regulated by extracellular molecules and proteins, known as ligands. Receptors are dynamic and – when activated – change conformation to initiate signal transduction. One family of receptors are the G protein-coupled receptors (GPCRs) in which the Class F receptors comprised of Frizzleds (FZDs) and Smoothened (SMO) are found. FZDs bind their endogenous ligands called WNTs, a group of lipoglycoproteins, and interact with multiple intracellular signal transducing proteins, such as the scaffolding-protein Dishevelled (DVL) and heterotrimeric G proteins. WNT/FZD signaling is crucial for proper embryonic development and tissue homeostasis but can when dysregulated lead to diseases such as cancer. This thesis aims to illuminate the molecular mechanisms underlying WNT/FZD signal transduction and signaling specification. The findings will further the understanding of events regulated by these receptors and aid in development of therapeutics to treat FZD-related diseases. This thesis began with the description of a molecular switch present in all Class F receptors that when mutated is a driver of cancer. It was found that the molecular switch opened in the process of receptor activation to accommodate the G protein and to initiate signaling. Mutation of the molecular switch in FZDs inhibited the receptor’s ability to adopt DVL-interacting conformations, leading to increased receptor activity and enhanced WNT-induced signaling towards heterotrimeric G proteins. Furthermore, the molecular switch network was extended to include additional amino acids, including a conserved proline in FZDs. Interestingly, SMO, which binds cholesterol, harbors a phenylalanine in this position. Mutating this phenylalanine in SMO obstructed binding of cholesterol, producing a G protein signal impaired receptor. Surprisingly, mutating the conserved proline in FZDs resulted in heterogeneous signaling behavior, suggesting FZD homologue-specific signaling mechanisms. The thesis further investigated WNT/-catenin signaling, which is a FZD-controlled signaling pathway important for cell proliferation and differentiation. DVL has a critical role in this signaling pathway, but the importance of heterotrimeric G proteins has been a long-standing debate. To that end, a series of experiments in heterotrimeric G protein knockout cells were conducted. It was concluded that heterotrimeric G proteins are not required for efficient WNT/-catenin signaling although they still have an important regulatory role as demonstrated by earlier studies. The final part of this thesis described the development of biosensors to enable the investigation of the poorly explored area of WNT-induced FZD-DVL dynamics. It was discovered that distinctly different conformations could be adopted in WNT-induced FZD-DVL dynamics and that these conformations were WNT- and FZD-dependent. Overall, this thesis has broadened the understanding of molecular mechanisms involved in the initiation and regulation of WNT/FZD signaling. More specifically, some molecular details of the mechanisms that determine how FZDs activate DVL- and heterotrimeric G protein-dependent signaling were clarified and, thus, this thesis has illuminated the road towards pathway selectivity.