par Sharma, Shivam 
Président du jury Baeyens, Nicolas
Promoteur Vanhollebeke, Benoît
Publication Non publié, 2026-09-09
Président du jury Baeyens, Nicolas
Promoteur Vanhollebeke, Benoît
Publication Non publié, 2026-09-09
Thèse de doctorat
| Résumé : | Wnt signaling is a conserved pathway that evolved in multicellular organisms. A classical Wnt transduction pathway is the β-catenin-dependent pathway, in which Wnt ligand binding to receptor complexes leads to cytoplasmic stabilization and nuclear accumulation of β-catenin, followed by transcriptional activation of Wnt target genes. In humans, 19 Wnt genes encode secreted ligands with substantial sequence and structural conservation. To distinguish between overlapping Wnt signals, cells rely on spatial and temporal control of ligand and receptor expression, diversification of receptor and co-receptor repertoires, and cooperation between receptors and co-receptors at the cell surface. One specialized example is Wnt7a/b-dependent signaling in the central nervous system. Wnt7a and Wnt7b activate β-catenin signaling in CNS endothelial cells, promote CNS angiogenesis, and support acquisition of blood-brain barrier (BBB) properties. In addition to Frizzled receptors and LRP5/6 co-receptors, this pathway requires Gpr124, an adhesion G-protein-coupled receptor, and Reck, a GPI-anchored protein, which together enable efficient Wnt7a/b-specific signaling. In this study, we asked whether other known Wnt co-receptors could also contribute to Gpr124/Reck-dependent Wnt7a/b signaling.During this study, we identified RYK, an atypical Wnt co-receptor, and MIB1, an E3 ubiquitin ligase, as regulators of Gpr124/Reck-dependent Wnt7a/b signaling. Functional cell-based reporter assays and zebrafish genetic-interaction analyses showed that RYK contributes to Gpr124/Reck-dependent Wnt/β-catenin signaling. We next assessed the interaction between RYK and Gpr124 to determine how RYK could contribute to this specialized signaling module. These interaction analyses revealed a direct association between RYK and the extracellular region of Gpr124 containing the LRR, Ig-like, and HRM subdomains, with an affinity of 58 µM as assessed by atomic force microscopy. This interaction was not altered by Frizzled expression.To explore how the RYK-Gpr124 interaction could influence signaling output, proximity-dependent biotinylation mass-spectrometry analysis was used to identify proteins associated with the intracellular region of Gpr124. This analysis identified MIB1, which has previously been shown to internalize RYK and positively regulate Wnt/β-catenin signaling, as a candidate Gpr124-associated protein. Additional cell-based experiments confirmed that MIB1 associates with Gpr124 at the plasma membrane in a Gpr124 intracellular domain-dependent manner, independently of the extracellular Gpr124-RYK interaction. Mapping experiments further showed that Gpr124-MIB1 association depends on a conserved motif (amino acids 82-88 of ICD) within the Gpr124 intracellular domain. However, reporter assays demonstrated that this direct interaction is not essential for the Wnt/β-catenin signaling activity of Gpr124. In contrast, MIB1 knockdown or knockout reduced Gpr124/Reck-dependent Wnt7a/b signaling, indicating that MIB1 contributes to this pathway through a mechanism that remains to be identified.Overall, this work supports a model in which RYK would act as an additional component of the Wnt7a/b/Gpr124/Reck/Fzd/Lrp5/6 ligand-receptor complex through its interaction with Gpr124. In this model, RYK may help fine-tune receptor-complex activity by functioning as an accessory co-receptor that can engage Wnt ligands through its WIF domain while also interacting directly with Gpr124 to support the activity of Gpr124/Reck-based signalosome. Due to its ubiquitination activity, MIB1 would influence signaling by regulating the internalization and control of recycling or degradation of Wnt-activated signalosomes, including its known target, RYK. Thus, this study expands the current understanding of Wnt7a/b-specific receptor complex organization and provides a basis for further investigation of how co-receptors with distinct signaling roles can coordinate during CNS angiogenesis and barrier formation. |
