National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, China.
J Biol Chem. 2010 Mar 19;285(12):8953-66. doi: 10.1074/jbc.M109.070680. Epub 2010 Jan 27.
Tumor necrosis factor-related apoptosis-inducing ligand receptors death receptor (DR) 4 and DR5 are potential targets for antibody-based cancer therapy. Activation of the proapoptotic DR5 in various cancer cells triggers the extrinsic and/or intrinsic pathway of apoptosis. It has been shown that there are several functional domains in the DR5 extracellular domain. The cysteine-rich domains of DR5 have a conservative role in tumor necrosis factor-related apoptosis-inducing ligand-DR5-mediated apoptosis, and the pre-ligand assembly domain within the N1-cap contributes to the ligand-independent formation of receptor complexes. However, the role of the N-terminal region (NTR) preceding the N1-cap of DR5 remains unclear. In this study, we demonstrate that NTR could mediate DR5 activation that transmits an apoptotic signal when bound to a specific agonistic monoclonal antibody. A novel epitope in the NTR of DR5 was identified by peptide array. Antibodies against the antigenic determinant showed high affinities for DR5 and triggered caspase activation in a time-dependent manner, suggesting the NTR of DR5 might function as a potential death-inducing region. Moreover, permutation analysis showed that Leu(6) was pivotal for the interaction of DR5 and the agonistic antibody. Synthetic wild-type epitopes eliminated the cytotoxicity of all three agonistic monoclonal antibodies, AD5-10, Adie-1, and Adie-2. These results indicate that the NTR of DR5 could be a potential target site for the development of new strategies for cancer immunotherapy. Also, our findings expand the current knowledge about DR5 extracellular functional domains and provide insights into the mechanism of DR5-mediated cell death.
肿瘤坏死因子相关凋亡诱导配体受体死亡受体 (DR)4 和 DR5 是抗体为基础的癌症治疗的潜在靶点。在各种癌细胞中,促凋亡 DR5 的激活触发了外在和/或内在的凋亡途径。已经表明,DR5 的细胞外结构域中有几个功能域。DR5 富含半胱氨酸的结构域在肿瘤坏死因子相关凋亡诱导配体-DR5 介导的凋亡中具有保守作用,而 N1 帽内的前配体组装结构域有助于配体独立的受体复合物形成。然而,DR5 的 N 端区域 (NTR) 在前 N1 帽之前的作用尚不清楚。在这项研究中,我们证明了 NTR 可以介导 DR5 的激活,当与特异性激动性单克隆抗体结合时,它会传递凋亡信号。通过肽阵列鉴定了 DR5 的 NTR 中的一个新表位。针对抗原决定簇的抗体与 DR5 具有高亲和力,并以时间依赖性方式触发半胱天冬酶的激活,这表明 DR5 的 NTR 可能作为潜在的死亡诱导区域发挥作用。此外,置换分析表明,亮氨酸 (Leu)6 对于 DR5 和激动性抗体的相互作用至关重要。合成的野生型表位消除了所有三种激动性单克隆抗体 AD5-10、Adie-1 和 Adie-2 的细胞毒性。这些结果表明,DR5 的 NTR 可能是开发癌症免疫治疗新策略的潜在靶点。此外,我们的发现扩展了目前对 DR5 细胞外功能域的认识,并为 DR5 介导的细胞死亡机制提供了深入了解。