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Updated: Jun 18, 2026
Modeling Paracrine Noncanonical Wnt Signaling In Vitro
Published on: December 10, 2021
Wnt5a regulates distinct signalling pathways by binding to Frizzled2
Akira Sato1, Hideki Yamamoto, Hiroshi Sakane
1Department of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan.
Abstract:
Wnt5a regulates multiple intracellular signalling cascades, but how Wnt5a determines the specificity of these pathways is not well understood. This study examined whether the internalization of Wnt receptors affects the ability of Wnt5a to regulate its signalling pathways. Wnt5a activated Rac in the beta-catenin-independent pathway, and Frizzled2 (Fz2) and Ror1 or Ror2 were required for this action. Fz2 was internalized through a clathrin-mediated route in response to Wnt5a, and inhibition of clathrin-dependent internalization suppressed the ability of Wnt5a to activate Rac. As another action of Wnt5a, it inhibited Wnt3a-dependent lipoprotein receptor-related protein 6 (LRP6) phosphorylation and beta-catenin accumulation. Wnt3a-dependent phosphorylation of LRP6 was enhanced in Wnt5a knockout embryonic fibroblasts. Fz2 was also required for the Wnt3a-dependent accumulation of beta-catenin, and Wnt5a competed with Wnt3a for binding to Fz2 in vitro and in intact cells, thereby inhibiting the beta-catenin pathway. This inhibitory action of Wnt5a was not affected by the impairment of clathrin-dependent internalization. These results suggest that Wnt5a regulates distinct pathways through receptor internalization-dependent and -independent mechanisms.
Insights
Wnt5a signaling specificity is determined by receptor internalization. Clathrin-mediated endocytosis of Frizzled2 (Fz2) mediates Wnt5a-induced Rac activation, while Wnt5a inhibits beta-catenin signaling independently of internalization.
Area of Science:
- Cell Biology
- Molecular Biology
- Signal Transduction
Background:
- Wnt5a is a key regulator of diverse intracellular signaling pathways.
- The mechanisms underlying Wnt5a's pathway specificity remain incompletely understood.
- Receptor internalization is a known modulator of Wnt signaling.
Purpose of the Study:
- To investigate the role of Wnt receptor internalization in Wnt5a-mediated signaling specificity.
- To determine if Wnt5a utilizes distinct mechanisms for activating beta-catenin-independent and -dependent pathways.
Main Methods:
- Utilized Wnt5a knockout embryonic fibroblasts.
- Assessed Rac activation and LRP6 phosphorylation.
- Investigated Frizzled2 (Fz2) internalization via clathrin-mediated pathways.
- Performed in vitro and cell-based binding assays.
Main Results:
- Wnt5a activated Rac in a beta-catenin-independent manner, requiring Frizzled2 (Fz2) and Ror1/Ror2 receptors.
- Wnt5a-induced Fz2 internalization occurred via clathrin-dependent endocytosis, essential for Rac activation.
- Wnt5a inhibited Wnt3a-induced LRP6 phosphorylation and beta-catenin accumulation.
- Wnt5a competed with Wnt3a for Fz2 binding, inhibiting beta-catenin signaling independently of Fz2 internalization.
Conclusions:
- Wnt5a employs distinct mechanisms for pathway regulation.
- Receptor internalization is critical for Wnt5a's beta-catenin-independent signaling.
- Wnt5a inhibits beta-catenin pathways through Fz2 binding competition, independent of internalization.
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