Bryja V, Cajánek L, Grahn A, Schulte G
Department of Medical Biochemistry & Biophysics, Molecular Neurobiology, Karolinska Institutet, Stockholm, Sweden.
Acta Physiol (Oxf). 2007 May;190(1):55-61. doi: 10.1111/j.1365-201X.2007.01688.x.
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.
Wnt/Frizzled信号通路在进化过程中高度保守。Frizzled作为Wnts的受体，具有七次跨膜结构域受体的拓扑结构。调节这些受体的一个重要方式是通过网格蛋白介导的内吞作用实现内化和脱敏。因此，我们研究了内吞抑制对Frizzled4-绿色荧光蛋白（FZD(4)-GFP）定位、散乱蛋白水平以及Wnt-3a向β-连环蛋白信号传导的影响。
实验在小鼠神经元细胞系SN4741中进行，该细胞系先前已被证明对研究Wnt/Frizzled信号传导很有价值。使用共聚焦激光扫描显微镜检查FZD(4)-GFP的分布。通过免疫印迹分析内吞抑制剂（高渗蔗糖和钾离子耗竭）、激酶抑制剂和Wnt-3a处理后散乱蛋白的表达水平以及β-连环蛋白的激活情况。
高渗蔗糖和钾离子耗竭增加了FZD(4)-GFP的膜定位，同时引发了内源性散乱蛋白快速（1 - 2小时）且几乎完全（约95%）的降解，这与Wnt诱导的、CK1介导的散乱蛋白磷酸化无关。此外，即使渗透条件和内吞作用恢复正常，内吞抑制诱导的散乱蛋白耗竭也完全阻止了Wnt-3a向β-连环蛋白的经典信号传导。
这些数据为一种分子机制提供了证据，该机制可能是Wnt/Frizzled通路中依赖于散乱蛋白降解的新型负反馈环的基础。Wnt/Frizzled信号通路调节机制分子细节的确定增进了我们对通路调节的理解，这可能对胚胎发育、癌症和神经疾病具有特殊的生理意义。
