eGFP-tagged Wnt-3a enables functional analysis of Wnt trafficking and signaling and kinetic assessment of Wnt binding to full-length Frizzled

• Verfasst von: Wesslowski, Janine [VerfasserIn]
• Verfasst von: Kozielewicz, Pawel [VerfasserIn]
• Verfasst von: Wang, Xianxian [VerfasserIn]
• Verfasst von: Cui, Haijun [VerfasserIn]
• Verfasst von: Schihada, Hannes [VerfasserIn]
• Verfasst von: Kranz, Dominique [VerfasserIn]
• Verfasst von: Karuna, Pradhipa M. [VerfasserIn]
• Verfasst von: Levkin, Pavel [VerfasserIn]
• Verfasst von: Gross, Julia Christina [VerfasserIn]
• Verfasst von: Boutros, Michael [VerfasserIn]
• Verfasst von: Schulte, Gunnar [VerfasserIn]
• Verfasst von: Davidson, Gary [VerfasserIn]
• Titel: eGFP-tagged Wnt-3a enables functional analysis of Wnt trafficking and signaling and kinetic assessment of Wnt binding to full-length Frizzled
• Verf.angabe: Janine Wesslowski, Pawel Kozielewicz, Xianxian Wang, Haijun Cui, Hannes Schihada, Dominique Kranz, Pradhipa M. Karuna, Pavel Levkin, Julia Christina Gross, Michael Boutros, Gunnar Schulte, and Gary Davidson
• E-Jahr: 2020
• Jahr: May 7, 2020
• Umfang: 16 S.
• Fussnoten: Gesehen am 26.08.2020
• Titel Quelle: Enthalten in: The journal of biological chemistry
• Ort Quelle: Bethesda, Md. : ASBMB Publications, 1905
• Jahr Quelle: 2020
• Band/Heft Quelle: 295(2020), 26, Seite 8759-8774
• ISSN Quelle: 1083-351X
• DOI: doi:10.1074/jbc.RA120.012892
• Datenträger: Online-Ressource
• Sprache: eng
• Sach-SW: bioluminescence resonance energy transfer (BRET)
• Sach-SW: complex
• Sach-SW: expression
• Sach-SW: family
• Sach-SW: Frizzled
• Sach-SW: fusion protein
• Sach-SW: G protein?coupled receptor (GPCR)
• Sach-SW: gradient
• Sach-SW: ligand binding
• Sach-SW: membrane protein
• Sach-SW: NanoBiT
• Sach-SW: NanoBRET
• Sach-SW: organizer
• Sach-SW: pathway
• Sach-SW: platform
• Sach-SW: proteins
• Sach-SW: recognition
• Sach-SW: secretion
• Sach-SW: trafficking
• Sach-SW: Wnt signaling
• K10plus-PPN: 1727824504

Abstract

The Wingless/Int1 (Wnt) signaling system plays multiple, essential roles in embryonic development, tissue homeostasis, and human diseases. Although many of the underlying signaling mechanisms are becoming clearer, the binding mode, kinetics, and selectivity of 19 mammalian WNTs to their receptors of the class Frizzled (FZD(1?10)) remain obscure. Attempts to investigate Wnt-FZD interactions are hampered by the difficulties in working with Wnt proteins and their recalcitrance to epitope tagging. Here, we used a fluorescently tagged version of mouse Wnt-3a for studying Wnt-FZD interactions. We observed that the enhanced GFP (eGFP)-tagged Wnt-3a maintains properties akin to wild-type (WT) Wnt-3a in several biologically relevant contexts. The eGFP-tagged Wnt-3a was secreted in an evenness interrupted (EVI)/Wntless-dependent manner, activated Wnt/?-catenin signaling in 2D and 3D cell culture experiments, promoted axis duplication inXenopusembryos, stimulated low-density lipoprotein receptor-related protein 6 (LRP6) phosphorylation in cells, and associated with exosomes. Further, we used conditioned medium containing eGFP-Wnt-3a to visualize its binding to FZD and to quantify Wnt-FZD interactions in real time in live cells, utilizing a recently established NanoBRET-based ligand binding assay. In summary, the development of a biologically active, fluorescent Wnt-3a reported here opens up the technical possibilities to unravel the intricate biology of Wnt signaling and Wnt-receptor selectivity.