The role of receptor complex composition and dynamics for signal specification and initiation in the WNT/Frizzled pathway
Anatomy and Physiology
Final Report Abstract
Class Frizzleds (FZDs) belong to the superfamily of seven transmembrane spanning G protein-coupled receptors. These receptors bind the lipoglycoproteins of the WNT family and WNT/FZD signaling is crucial during embryonic development and stem cell regulation in the embryo and the adult. Dysfunction, resulting in an exaggerated or reduced signaling can lead to devastating human diseases, such as neurodegenerative disorders or diverse forms of cancer. One of the FZDs, FZD6, has been found involved in breast, ovarian, and prostate cancer. Not surprisingly, the WNT/FZD signaling system represents an attractive target for pharmacological treatment. Within this project a many-faceted characterization of FZD6 was achieved. We have identified a novel mechanism of FZD6 phosphorylation through Dishevelled, one of the central key players for WNT/FZD signaling, and the kinase CK1 with implications for receptor polarization and cell surface expression. Furthermore, we have shown that the CRD, an extracellular part of the receptor, known as the main binding site for WNT, is not essential for receptor expression and interaction with intracellular signaling partners. FZD6 lacking the CRD is still expressed at the cell surface, and thus we asked, how a ligand binding mode in a CRD-lacking receptor will look like. By applying the new technology of amber codon suppression accociated with a fluorescent labeling step we were able to present - for the first time - an extracellular FZD sensor resolving a potential movement of the CRD upon ligand binding. We hope that the results supporting the existence of an additional WNT interaction site at the receptor core will be published soon after finishing supplementary experiments. A detailed knowledge about the FZDs ligand binding site and the ligand interaction mode will facilitate the de novo design and screening of FZD-targeting small molecule compounds based on the binding mechanism. There are not only extracellular conformational changes upon WNT binding. Intrastructural changes as a consequence of receptor activation remain so far unexplored. To this end, we combined in Wright et al. (2019) cancer genomics data, structural analyses, mutagenesis and resonance energy transfer-based approaches and identified a conserved amino acid in FZD’s transmembrane domain 6 that acts as a molecular switch to mediate receptor activation. In conclusion, we have used state-of-the-art methodology to better understand FZDs and in detail FZD6’ role as WNT receptors, and that might open new routes for the therapy of receptor mediated diseases. The general importance might lie in the idea to create a new class of drugs targeting the alternative (core) binding site of WNTs instead of the primary CRD site. This is a potentially more versatile site regarding its pharmacology. Also the identification of a common activation mode conserved in all FZDs might establish a basis which facilitates assay development and future discovery of FZD-targeting drugs.
Publications
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2018. Dishevelled enables casein kinase 1–mediated phosphorylation of Frizzled 6 required for cell membrane localization. J. Biol. Chem. 293, 18477–18493
Strakova, K., Kowalski-Jahn, M., Gybel, T., Valnohova, J., Dhople, V.M., Harnos, J., Bernatik, O., Ganji, R.S., Zdrahal, Z., Mulder, J., Lindskog, C., Bryja, V., Schulte, G.
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2018. Functional dissection of the N-terminal extracellular domains of Frizzled 6 reveals their roles for receptor localization and Dishevelled recruitment. J. Biol. Chem. 293, 17875–17887
Valnohova, J., Kowalski-Jahn, M., Sunahara, R.K., Schulte, G.
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2019. A conserved molecular switch in Class F receptors regulates receptor activation and pathway selection. Nat. Commun. 10, 667
Wright, S.C., Kozielewicz, P., Kowalski-Jahn, M., Petersen, J., Bowin, C.-F., Slodkowicz, G., Marti-Solano, M., Rodríguez, D., Hot, B., Okashah, N., Strakova, K., Valnohova, J., Babu, M.M., Lambert, N.A., Carlsson, J., Schulte, G.
