Integration of two concepts in WNT signalling: Signalosome formation and Frizzled conformational dynamics
Biochemistry
Final Report Abstract
Frizzleds (FZD1-10) constitute the class F of the G protein-coupled receptor superfamily. They act as receptors for WNT morphogens and play vital roles in embryonic development and stem cell maintenance. However, dysregulation of WNT signaling manifests in pathologies such as diverse cancers and fibrosis. This work aims to improve our understanding of the initiation of WNT signaling. Here, WNTs bind to their cognate membrane receptors – FZDs – but also to diverse unrelated co-receptors, such as Low-Density Lipoprotein Receptor-Related Proteins 5 and 6 (LRP5/6), receptor tyrosine kinase-like orphan receptor 1/2 (ROR1/2), Tyrosine-Protein Kinase 7 (PTK7), and Related to Receptor Tyrosine Kinase (RYK). The current dogma for the initiation of WNT/β-catenin signaling, the best-described WNT signaling pathway, claims that dimerization of FZDs with LRP5/6 is both necessary and sufficient for effective β-catenin signaling. However, my postdoctoral work conducted during the fellowship clearly demonstrated that WNT-induced association of FZDs with LRP6 is not sufficient to initiate signaling. We used bioluminescence resonance energy transfer (BRET) and single molecule fluorescence microscopy to establish that both WNT-3A and WNT-16B induce association of FZD5 and LRP6, however, only WNT-3A displayed productive activation of WNT/β-catenin signaling. In other objectives, we have investigated the interactions between FZDs and putative coreceptors in β-catenin-independent signaling, where we detected broad low-affinity interactions between FZDs and ROR1/2, PTK7, and RYK. Stimulation with diverse WNTs led to association between FZD and ROR1/2, analogously to FZD and LRP5/6. Interestingly, only very specific combinations of WNT, FZD, and ROR led to productive signaling, which curiously resembles findings from our first objective. Further functional analyses are pending. Lastly, we profiled constitutive interactions between FZDs and Receptor Activity-Modifying Proteins (RAMP1-3). Wide interactions between GPCRs and RAMPs have recently been established and our experiments revealed several specific interactions between FZDs and RAMPs, but were not prioritized as we could not confirm a functional effect of RAMP coexpression on FZD expression or signaling until now. In summary, the work performed during the course of this program has challenged longstanding dogmas in the field of WNT signaling. Extending our methodology to less well understood putative co-receptors, we hope to paint a holistic image of the initiation of WNT signaling.
Publications
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A Putative Frizzled 7-Targeting Compound Acts as a Firefly Luciferase Inhibitor. Journal of Medicinal Chemistry, 67(24), 22332-22341.
Kinsolving, Julia; Grätz, Lukas; Voss, Jan Hendrik; Löw, Bente; Shorter, Emily; Jude, Baptiste; Lanner, Johanna T.; Löber, Stefan; Gmeiner, Peter & Schulte, Gunnar
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Class-Wide Analysis of Frizzled-Dishevelled Interactions Using BRET Biosensors Reveals Functional Differences among Receptor Paralogs. ACS Sensors, 9(9), 4626-4636.
Grätz, Lukas; Voss, Jan H. & Schulte, Gunnar
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Frizzleds act as dynamic pharmacological entities. Trends in Pharmacological Sciences, 45(5), 419-429.
Schulte, Gunnar; Scharf, Magdalena M.; Bous, Julien; Voss, Jan Hendrik; Grätz, Lukas & Kozielewicz, Pawel
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Structural basis of frizzled 7 activation and allosteric regulation. Nature Communications, 15(1).
Bous, Julien; Kinsolving, Julia; Grätz, Lukas; Scharf, Magdalena M.; Voss, Jan Hendrik; Selcuk, Berkay; Adebali, Ogün & Schulte, Gunnar
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WNT-induced association of Frizzled and LRP6 is not sufficient for the initiation of WNT/β-catenin signaling. Springer Science and Business Media LLC.
Schulte, Gunnar; Voss, Jan; Koszegi, Zsombor; Yan, Yining; Shorter, Emily; Grätz, Lukas; Lanner, Johanna & Calebiro, Davide
