Intrinsic Selectivity of Notch 1 for Delta-like 4 Over Delta-like 1
Intrinsic Selectivity of Notch 1 for Delta-like 4 Over Delta-like 1
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DOI：
10.1074/jbc.m113.454850
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发表时间：
2013-08-30
影响因子：
4.8
通讯作者：
Blacklow, Stephen C.
中科院分区：
文献类型：
--
作者：
Andrawes, Marie Blanke;Xu, Xiang;Blacklow, Stephen C.
Notch 信号传导对所有后生动物生物体的细胞命运决定做出了关键贡献，但人们对四种哺乳动物 Notch 受体与其三种 Delta 样和两种 Jagged 家族配体的结合亲和力知之甚少。在这里，我们利用纯化的重组配体和受体分子的信号传导测定和生化研究来研究Notch1-Dll1和Notch1-Dll4复合物之间信号传导行为和内在亲和力的差异。人Notch1胞外域的系统缺失诱变表明，表皮生长因子（EGF）重复序列6-15足以在报告测定中将信号传导维持在与全长受体相当的水平，并确定了EGF重复序列8-10在传递响应Dll1或Dll4的激活信号方面的重要贡献。 Dll1 和 Dll4 胞外域的截断研究表明，MNNL-EGF3 区域对于完全激活来说是必要且充分的。基于板的细胞结合测定揭示了细胞表面与重组Notch受体和配体分子之间的特异性、钙依赖性相互作用。最后，直接测量Notch1 EGF重复序列6-15对Dll1和Dll4的结合亲和力表明，Dll4的结合亲和力至少比Dll1高一个数量级。总之，这些研究为 Notch1 配体识别的特征提供了新的见解，并强调了受体-配体复合物的生化行为的内在差异如何影响发育信号通路的受体介导的反应。
Notch signaling makes critical contributions to cell fate determination in all metazoan organisms, yet remarkably little is known about the binding affinity of the four mammalian Notch receptors for their three Delta-like and two Jagged family ligands. Here, we utilized signaling assays and biochemical studies of purified recombinant ligand and receptor molecules to investigate the differences in signaling behavior and intrinsic affinity between Notch1-Dll1 and Notch1-Dll4 complexes. Systematic deletion mutagenesis of the human Notch1 ectodomain revealed that epidermal growth factor (EGF) repeats 6-15 are sufficient to maintain signaling in a reporter assay at levels comparable with the full-length receptor, and identified important contributions from EGF repeats 8-10 in conveying an activating signal in response to either Dll1 or Dll4. Truncation studies of the Dll1 and Dll4 ectodomains showed that the MNNL-EGF3 region was both necessary and sufficient for full activation. Plate-based and cell binding assays revealed a specific, calcium-dependent interaction between cell-surface and recombinant Notch receptors and ligand molecules. Finally, direct measurement of the binding affinity of Notch1 EGF repeats 6-15 for Dll1 and Dll4 revealed that Dll4 binds with at least an order of magnitude higher affinity than Dll1. Together, these studies give new insights into the features of ligand recognition by Notch1, and highlight how intrinsic differences in the biochemical behavior of receptor-ligand complexes can influence receptor-mediated responses of developmental signaling pathways.