Inhibition of endocytosis blocks Wnt signalling to β-catenin by promoting dishevelled degradation
Inhibition of endocytosis blocks Wnt signalling to β-catenin by promoting dishevelled degradation
复制标题
DOI：
10.1111/j.1365-201x.2007.01688.x
复制
发表时间：
2007-05-01
影响因子：
6.3
通讯作者：
Schulte, G.
中科院分区：
文献类型：
--
作者：
Bryja, V.;Cajanek, L.;Schulte, G.
目的：Wnt/Frizzleed信号通路在进化过程中高度保守。WNTs的受体FrizzledWNTs具有七个跨膜跨域受体的拓扑结构。调节这些受体的一个重要手段是通过网状蛋白介导的内吞作用进行内化和脱敏。因此，我们研究了内吞抑制对Frizzled4-绿色荧光蛋白(FZD(4)-GFP)定位、乱糟糟的水平和Wnt-3a向β-连环素信号转导的影响。方法：实验在小鼠神经元细胞系SN4741上进行，该细胞系先前被证明对Wnt/Frizzleed信号转导有价值。用激光共聚焦扫描显微镜研究了FZD(4)-GFP的分布。结果：高渗蔗糖和K+耗竭使FZD4-GFP的细胞膜定位增加，并平行触发快速(1~2 h)和几乎完全(约2 h)。95%)降解内源性蓬乱，这与Wnt诱导的、CK1介导的蓬乱的磷酸化无关。此外，即使在渗透状态和内吞作用恢复正常的情况下，由内吞抑制引起的紊乱的耗竭也完全阻止了WNT-3a向β-连环蛋白的典型信号传递。结论：这些数据为分子机制提供了证据，该机制可能是Wnt/Frizzled通路中依赖于蓬乱降解的新的负反馈回路的基础。识别Wnt/Frizzled信号通路调控机制的分子细节有助于我们理解通路调控，这可能对胚胎发育、癌症和神经疾病具有特殊的生理意义。
Aim: 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.Methods: 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.Results: Hyperosmotic sucrose and K+ depletion increased the membrane localization of FZD4-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.Conclusions: 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.