The role of receptor complex composition and dynamics for signal specification and initiation in the WNT/Frizzled pathway

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Receptors of the Frizzled (FZD) family transduce important signals during embryonic development and in adult mammals, and disturbed FZD signaling can cause severe diseases ranging from cancer and inflammatory diseases to metabolic and neurological disorders. Thus, FZDs present a promising though yet unexplored target for pharmaceutical intervention.FZD receptors (FZD1 to FZD10) belong to the superfamily of G protein-coupled receptors and are activated mainly by the secreted wingless/int1 (WNT) family lipoglycoproteins. Several distinct WNT/FZD-signaling pathways have been identified and they are distinguished as ß-catenin-dependent and ß-catenin-independent signaling routes. So far, it is unclear how WNT/FZD selectivity or other underlying mechanisms define pathway selectivity, and the question remains open, if receptor complex composition or the functional selectivity of the WNT protein that is binding the receptor determines downstream signaling. It is hypothesized, but yet unproven, that FZD is placed at the centre of ligand recognition, while the recruitment of co-receptors might push signaling towards either ß-catenin-dependent (LRP5/6) or ß-catenin-independent (ROR/RYK) signaling. Thus, this project aims to analyze receptor complex composition and dynamics, and their importance for signal specification and initiation in the WNT/FZD pathway. Two different FZD model proteins (FZD4 and FZD6) will be used to understand signal initiation in the ß-catenin-dependent (FZD4) and -independent (FZD6) pathway, with special attention to differential activation of heterotrimeric G proteins.To understand underlying mechanisms of signal specification, this project focuses primarily on gaining precise information of receptor composition and stoichiometry. Second, it is intended to analyze ligand-induced receptor complex dynamics and signaling initiated in the ß-catenin-independent pathway by using the FZD6 receptor. And third, the role of the co-receptor LRP6 as a potential gate keeper switching WNT-induced signaling between the ß-catenin- or G protein-dependent pathways will be evaluated employing FZD4 as a model. Protein-protein interactions within the FZD/co-receptor complex will be analyzed biochemically, as well as by unnatural amino acid mutagesesis, live cell imaging in combination with Förster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and by an affinity-based dual-color fluorescence after photobleaching (FRAP) approach.The outlined project aims at significantly expanding the knowledge on FZD and GPCR receptor biology in general. A thorough understanding of the mechanisms that lead to receptor complex activation and selective signal initiation in this pathologically important communication system will open novel possibilities for the development of drugs targeting class Frizzled receptors.