type: paper
title: WNT-induced association of Frizzled and LRP6 is not sufficient for the initiation of WNT/β-catenin signaling
year: 2025
authors:
- Voss, Jan Hendrik
- Koszegi, Zsombor
- Yan, Yining
- Shorter, Emily
- Grätz, Lukas
- Lanner, Johanna T.
- Calebiro, Davide
- Schulte, Gunnar
journal: Nature Communications
doi: 10.1038/s41467-025-60096-7
url: https://www.nature.com/articles/s41467-025-60096-7
project:
- IGP
system:
- plasma_membrane
- wnt_receptor_complex
- signalosome
- beta_catenin_destruction_complex
disease:
- cancer
- fibrosis
genes:
- fzd5
- fzd4
- fzd7
- lrp6
- dvl
- beta_catenin
processes:
- receptor_association
- receptor_clustering
- membrane_confinement
- signal_initiation
- beta_catenin_stabilization
- receptor_phosphorylation
- transcriptional_activation
methods:
- nanobret
- single_molecule_tracking
- tirf_microscopy
- topflash_reporter_assay
- rna_sequencing
- photobleaching_step_analysis
key_findings:
- ligand_induced_receptor_association
- signaling_independent_complex_formation
- clustering_required_for_signaling
- phosphorylation_downstream_of_clustering
- allosteric_modulation_by_scaffold
limitations:
- cell_line_specific_context
- incomplete_physiological_validation
- unresolved_downstream_specification
relevance:
- challenges_signalosome_dogma
- decouples_binding_from_signaling
- informs_wnt_therapeutic_design
concepts:
- two_step_wnt_signal_initiation
- receptor_clustering_thresholds
- ligand_biased_wnt_signaling
- membrane_nanoscale_organization
tags:
- wnt_signaling
- receptor_dynamics
- signal_initiation
- membrane_biology
The Wingless/Int-1 (WNT) signaling network is essential to orchestrate central physiological processes such as embryonic development and tissue homeostasis. In the currently held tenet, WNT/β-catenin signaling is initiated by WNT-induced recruitment of Frizzleds (FZDs) and LRP5/6 followed by the formation of a multiprotein signalosome complex. Here, we use bioluminescence resonance energy transfer (BRET) to show that different WNT paralogs dynamically trigger FZD-LRP6 association. While WNT-induced receptor interaction was independent of C-terminal LRP6 phosphorylation, it was allosterically modulated by binding of the phosphoprotein Dishevelled (DVL) to FZD. WNT-16B emerged as a ligand of particular interest, as it efficiently promoted FZD-LRP6 association but, unlike WNT-3A, did not lead to WNT/β-catenin signaling. Transcriptomic analysis further revealed distinct transcriptional fingerprints of WNT-3A and WNT-16B stimulation in HEK293 cells. Additionally, single-molecule tracking demonstrated that, despite increasing FZD5 and LRP6 confinement, WNT-16B stimulation did not result in formation of higher-order receptor clusters, in contrast to WNT-3A. Our results suggest that FZD-WNT-LRP5/6 complex formation alone is not sufficient for the initiation of WNT/β-catenin signaling. Instead, we propose a two-step model, where initial ligand-induced FZD-LRP6 association must be followed by receptor clustering into higher-order complexes and subsequent phosphorylation of LRP6 for efficient activation of WNT/β-catenin signaling.
FZD–LRP6 association is ligand-induced but not sufficient to initiate WNT/β-catenin signaling
WNT-16B robustly induces FZD–LRP6 binding without β-catenin stabilization
Higher-order receptor clustering correlates with signaling competence
LRP6 C-terminal phosphorylation occurs downstream of clustering, not binding
DVL binding allosterically alters receptor geometry without enforcing signaling
Direct NanoBRET assay to measure dynamic FZD–LRP6 association
Single-molecule TIRF tracking to quantify confinement, interaction kinetics, and clustering
Photobleaching step analysis to infer receptor stoichiometry
TOPFlash reporter assays to isolate β-catenin–dependent output
Bulk RNA-seq to compare ligand-specific transcriptional programs
Reframes WNT signaling as multi-step spatial organization, not binary receptor binding
Demonstrates ligand bias at the level of receptor nanoscale behavior
Separates physical receptor engagement from signal competence
Undermines assumptions behind WNT surrogate crosslinking strategies
Figure 1: Establishes NanoBRET assay and ligand-dependent FZD–LRP6 association
Figure 2: Shows DVL modulates receptor orientation without enforcing signaling
Figure 3: Demonstrates WNT-16B uncouples association from β-catenin activation
Figure 4: Single-molecule evidence for ligand-specific clustering behavior
Figure 5: LRP6 clustering occurs independently of phosphorylation
Figure 6: Conceptual two-step model of WNT/β-catenin initiation
Heavy reliance on HEK293 and CHO cell models
Transcriptomic outputs may not reflect physiological stem-cell contexts
Downstream determinants of cluster maturation remain undefined