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Frizzleds (FZD1-10) belong to the class F of the G protein-coupled receptor (GPCR) superfamily and play a key role in the signal transduction of secreted lipoglycoproteins of the Wingless-Int1 (WNT) family. In embryonal development, WNT signaling governs essential processes such as cellular differentiation, polarization, and migration, while in the adult, WNT signaling is involved in tissue homeostasis. Abnormal WNT and FZD signaling is related to cancer, fibrosis, arthritis, and Alzheimer’s disease. Upon WNT binding, FZDs pair with their co-receptors low-density lipoprotein receptor-related protein (LRP) 5/6 and initiate the formation of a large protein complex at the membrane, the signalosome. Signalosome formation ultimately leads to stabilization of the transcriptional cofactor β-catenin by disruption of the β-catenin-destruction complex. Due to the complexity of WNT signaling pathways, many questions remain unanswered on a molecular level, e.g. the stoichiometry of the signalosome or interaction parameters and surfaces between FZDs and their coreceptors. Furthermore, FZDs were shown to exhibit large conformational changes, typical to other GPCRs, upon ligand binding and it is currently not clear, whether and how these FZD dynamics are involved in FZD signaling. The proposed project is divided into three work packages. The first and main objective aims to form a coherent theory of FZDs in WNT signaling by integrating the concepts of signalosome formation and FZD dynamics. To this end, we will develop BRET biosensors to interrogate the protein-protein interactions between FZDs and LRPs in presence and absence of WNTs. For these experiments, we will create CRISPR-Cas9 edited cells lacking endogenous expression of respective proteins. By using additional tools, such as oncogenic FZD mutants, surrogate WNT peptides, conformational biosensors, and positive allosteric FZD modulators, we intend to characterize the FZD-LRP complex formation that initiates signalosome formation and to unravel the role of FZD dynamics in WNT signal transduction. In another work package, we intend to characterize the interaction between FZDs and other WNT-binding co-receptors, such as RO1/2, RYK, and PTK7 with similar methods. These proteins are known to play a role in WNT signaling, but currently, direct evidence for interactions between these proteins and FZDs is lacking due to unsuitable assay readouts. Lastly, we will investigate if FZDs interact with receptor-activity modifying proteins (RAMPs) and whether a possible interaction affects FZD signaling either directly or e.g. by modulating FZD trafficking in the cell.