eGFP-tagged Wnt-3a enables functional analysis of Wnt trafficking and signaling and kinetic assessment of Wnt binding to full-length Frizzled
eGFP-tagged Wnt-3a enables functional analysis of Wnt trafficking and signaling and kinetic assessment of Wnt binding to full-length Frizzled
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eGFP 标记的 Wnt-3a 能够对 Wnt 运输和信号传导进行功能分析，并对 Wnt 与全长卷曲蛋白结合进行动力学评估
DOI：
10.1074/jbc.ra120.012892
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发表时间：
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影响因子：
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通讯作者：
Davidson G
中科院分区：
文献类型：
--
作者：
Wesslowski J;Kozielewicz P;Wang X;Schihada H;Kranz D;Karuna M P;Levkin P;Gross JC;Boutros M;Schulte G;Davidson G
无翼/Int1 （Wnt）信号系统在胚胎发育、组织稳态和人类疾病中起着多种重要作用。尽管许多潜在的信号机制正变得越来越清晰，但19种哺乳动物WNTs与其frzzled类受体（FZD1-10）的结合模式、动力学和选择性仍然不清楚。研究Wnt- fzd相互作用的尝试受到与Wnt蛋白合作的困难及其对表位标记的抵抗的阻碍。在这里，我们使用荧光标记的小鼠Wnt-3a来研究Wnt-FZD的相互作用。我们观察到，增强的GFP （eGFP）标记的Wnt-3a在几种生物学相关环境中保持了与野生型（WT） Wnt-3a相似的特性。egfp标记的Wnt-3a以EVI /Wnt依赖性的方式分泌，在2D和3D细胞培养实验中激活Wnt/β-catenin信号，促进xenopusembryo的轴复制，刺激细胞中的低密度脂蛋白受体相关蛋白6 （LRP6）磷酸化，并与外泌体相关。此外，我们使用含有eGFP-Wnt-3a的条件培养基来观察其与FZD的结合，并利用最近建立的基于nanobret的配体结合试验，在活细胞中实时量化Wnt-FZD的相互作用。总之，本文报道的具有生物活性的荧光Wnt-3a的开发为揭示Wnt信号传导和Wnt受体选择性的复杂生物学提供了技术可能性。
The Wingless/Int1 (Wnt) signaling system plays multiple, essential roles in embryonic development, tissue homeostasis, and human diseases. Although many of the underlying signaling mechanisms are becoming clearer, the binding mode, kinetics, and selectivity of 19 mammalian WNTs to their receptors of the class Frizzled (FZD1–10) remain obscure. Attempts to investigate Wnt-FZD interactions are hampered by the difficulties in working with Wnt proteins and their recalcitrance to epitope tagging. Here, we used a fluorescently tagged version of mouse Wnt-3a for studying Wnt-FZD interactions. We observed that the enhanced GFP (eGFP)-tagged Wnt-3a maintains properties akin to wild-type (WT) Wnt-3a in several biologically relevant contexts. The eGFP-tagged Wnt-3a was secreted in an evenness interrupted (EVI)/Wntless-dependent manner, activated Wnt/β-catenin signaling in 2D and 3D cell culture experiments, promoted axis duplication inXenopusembryos, stimulated low-density lipoprotein receptor-related protein 6 (LRP6) phosphorylation in cells, and associated with exosomes. Further, we used conditioned medium containing eGFP-Wnt-3a to visualize its binding to FZD and to quantify Wnt-FZD interactions in real time in live cells, utilizing a recently established NanoBRET-based ligand binding assay. In summary, the development of a biologically active, fluorescent Wnt-3a reported here opens up the technical possibilities to unravel the intricate biology of Wnt signaling and Wnt-receptor selectivity.
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影响因子：
11.8
作者：
Chen, Shuo;Bubeck, Doryen;MacDonald, Bryan T.;Liang, Wen-Xue;Mao, Jian-Hua;Malinauskas, Tomas;Llorca, Oscar;Aricescu, A. Radu;Siebold, Christian;He, Xi;Jones, E. Yvonne
通讯作者：
Jones, E. Yvonne
DOI：
10.1073/pnas.96.7.3546
发表时间：
1999-03-30
影响因子：
11.1
作者：
Hsieh, JC;Rattner, A;Nathans, J
通讯作者：
Nathans, J
影响因子：
64.5
作者：
MCMAHON, AP;MOON, RT
通讯作者：
MOON, RT
荧光和光活性蛋白的生长和发光工具箱。
影响因子：
13.8
作者：
Rodriguez EA;Campbell RE;Lin JY;Lin MZ;Miyawaki A;Palmer AE;Shu X;Zhang J;Tsien RY
通讯作者：
Tsien RY
影响因子：
16
作者：
Menck K;Sönmezer C;Worst TS;Schulz M;Dihazi GH;Streit F;Erdmann G;Kling S;Boutros M;Binder C;Gross JC
通讯作者：
Gross JC