Inhibition of endocytosis blocks Wnt signalling to ?-catenin by promoting dishevelled degradation

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Abstract
The Wnt/Frizzled signalling pathway is highly conserved through evolution. Frizzled, the receptors for Wnts, have the topology of seven transmembrane spanning domain receptors. An important means of regulation of these receptors is internalization and desensitization through clathrin-mediated endocytosis. Therefore, we investigated the effects of endocytosis inhibition on Frizzled4-green fluorescent protein (FZD(4)-GFP) localization, dishevelled levels and Wnt-3a signalling to beta-catenin. Experiments were performed in the mouse neuronal cell line SN4741 that has previously proven to be valuable for the investigation of Wnt/Frizzled signalling. FZD(4)-GFP distribution has been examined using confocal laser scanning microscopy. Dishevelled protein expression levels and the activation of beta-catenin upon treatment with endocytosis inhibitors (hyperosmolaric sucrose and K(+) depletion), kinase inhibitors and Wnt-3a were analysed by immunoblotting. Hyperosmotic sucrose and K(+) depletion increased the membrane localization of FZD(4)-GFP, and in parallel triggered fast (1-2 h) and almost complete (approx. 95%) degradation of endogenous dishevelled, which was independent of Wnt-induced, CK1-mediated phosphorylation of dishevelled. In addition, dishevelled depletion induced by endocytosis inhibition completely prevented canonical signalling by Wnt-3a to beta-catenin even when osmotic conditions and endocytosis were reverted to normal. The data provide evidence for a molecular mechanism that could be a basis for a novel negative feedback loop within the Wnt/Frizzled pathway depending on dishevelled degradation. The identification of molecular details of regulatory mechanisms for the Wnt/Frizzled signalling pathway increases our understanding of pathway regulation, which might be of special physiological significance for embryonic development, cancer and neurological disorders.