Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India.
Biochem J. 2013 Jan 15;449(2):519-30. doi: 10.1042/BJ20121153.
The Notch signalling pathway is implicated in a wide variety of cellular processes throughout metazoan development. Although the downstream mechanism of Notch signalling has been extensively studied, the details of its ligand-mediated receptor activation are not clearly understood. Although the role of Notch ELRs [EGF (epidermal growth factor)-like-repeats] 11-12 in ligand binding is known, recent studies have suggested interactions within different ELRs of the Notch receptor whose significance remains to be understood. Here, we report critical inter-domain interactions between human Notch1 ELRs 21-30 and the ELRs 11-15 that are modulated by calcium. Surface plasmon resonance analysis revealed that the interaction between ELRs 21-30 and ELRs 11-15 is ~10-fold stronger than that between ELRs 11-15 and the ligands. Although there was no interaction between Notch1 ELRs 21-30 and the ligands in vitro, addition of pre-clustered Jagged1Fc resulted in the dissociation of the preformed complex between ELRs 21-30 and 11-15, suggesting that inter-domain interactions compete for ligand binding. Furthermore, the antibodies against ELRs 21-30 inhibited ligand binding to the full-length Notch1 and subsequent receptor activation, with the antibodies against ELRs 25-26 being the most effective. These results suggest that the ELRs 25-26 represent a cryptic ligand-binding site which becomes exposed only upon the presence of the ligand. Thus, using specific antibodies against various domains of the Notch1 receptor, we demonstrate that, although ELRs 11-12 are the principal ligand-binding site, the ELRs 25-26 serve as a secondary binding site and play an important role in receptor activation.
Notch 信号通路在后生动物发育过程中的各种细胞过程中都有涉及。尽管 Notch 信号的下游机制已经得到了广泛的研究,但配体介导的受体激活的细节还不是很清楚。虽然 Notch ELRs [EGF(表皮生长因子)样重复]11-12 在配体结合中的作用是已知的,但最近的研究表明,Notch 受体的不同 ELRs 之间存在相互作用,其意义仍有待理解。在这里,我们报告了人 Notch1 ELRs 21-30 与 ELRs 11-15 之间的关键域间相互作用,这些相互作用受钙调节。表面等离子体共振分析显示,ELRs 21-30 与 ELRs 11-15 之间的相互作用比 ELRs 11-15 与配体之间的相互作用强约 10 倍。虽然在体外 Notch1 ELRs 21-30 与配体之间没有相互作用,但添加预先聚集的 Jagged1Fc 会导致 ELRs 21-30 和 11-15 之间预先形成的复合物解离,表明域间相互作用竞争配体结合。此外,针对 ELRs 21-30 的抗体抑制了配体与全长 Notch1 的结合及其随后的受体激活,针对 ELRs 25-26 的抗体最有效。这些结果表明,ELRs 25-26 代表一个隐蔽的配体结合位点,只有在存在配体的情况下才会暴露。因此,使用针对 Notch1 受体的不同结构域的特异性抗体,我们证明,虽然 ELRs 11-12 是主要的配体结合位点,但 ELRs 25-26 作为次要结合位点,在受体激活中发挥重要作用。