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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
- Source :
- Journal of Biological Chemistry, 295 (26), 8759–8774, The Journal of Biological Chemistry
- Publication Year :
- 2020
- Publisher :
- American Society for Biochemistry and Molecular Biology, 2020.
-
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 (FZD1–10) 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/β-catenin signaling in 2D and 3D cell culture experiments, promoted axis duplication in Xenopus embryos, 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.
- Subjects :
- 0301 basic medicine
Life sciences
biology
Frizzled
ligand binding
Xenopus
Green Fluorescent Proteins
Biochemistry
Green fluorescent protein
03 medical and health sciences
Mice
trafficking
Wnt3A Protein
ddc:570
fusion protein
Animals
Humans
membrane protein
Protein Interaction Maps
Molecular Biology
Wnt Signaling Pathway
Tissue homeostasis
bioluminescence resonance energy transfer (BRET)
Microscopy, Confocal
030102 biochemistry & molecular biology
Chemistry
NanoBRET
Wnt signaling pathway
LRP6
G protein–coupled receptor (GPCR)
Cell Biology
biology.organism_classification
Fusion protein
Wnt signaling
Frizzled Receptors
3. Good health
Cell biology
NanoBiT
Protein Transport
030104 developmental biology
HEK293 Cells
Phosphorylation
Subjects
Details
- Language :
- English
- ISSN :
- 00219258 and 1083351X
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry, 295 (26), 8759–8774, The Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....5084d0a3a5bb8f44031f8b79904d7fbd