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

Wesslowski, Janine; Kozielewicz, Pawel; Wang, Xianxian; Cui, Haijun; Schihada, Hannes; Kranz, Dominique; Karuna M, Pradhipa; Levkin, Pavel; Gross, Julia Christina; Boutros, Michael; Schulte, Gunnar; Davidson, Gary

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 Wnt-3a in several biologically relevant contexts. The eGFP-tagged Wnt-3a was secreted in an evenness interrupted (EVI)/Wntless-dependent manner, activated Wnt/β-catenin signaling in 2D and 3D cell culture experiments, promoted axis duplication in Xenopus embryos, stimulated LDL 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. ... mehr

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Originalveröffentlichung
DOI: 10.1074/jbc.RA120.012892
Scopus
Zitationen: 4
Web of Science
Zitationen: 2
Dimensions
Zitationen: 4
Zugehörige Institution(en) am KIT Institut für Biologische und Chemische Systeme (IBCS)
Publikationstyp Zeitschriftenaufsatz
Publikationsmonat/-jahr 06.2020
Sprache Englisch
Identifikator ISSN: 0021-9258, 1083-351X
KITopen-ID: 1000122720
HGF-Programm 47.01.01 (POF III, LK 01) Biol.Netzwerke u.Synth.Regulat. ITG+ITC
Erschienen in Journal of Biological Chemistry
Verlag American Society for Biochemistry and Molecular Biology
Band 295
Heft 26
Seiten 8759–8774
Vorab online veröffentlicht am 07.05.2020
Nachgewiesen in Dimensions
Web of Science
Scopus
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