The eighth FIII domain of human fibronectin promotes integrin α5β1 binding via stabilization of the ninth FIII domain
The eighth FIII domain of human fibronectin promotes integrin α5β1 binding via stabilization of the ninth FIII domain
复制标题
DOI：
10.1074/jbc.m105868200
复制
发表时间：
2001-10-19
影响因子：
4.8
通讯作者：
Mardon, HJ
中科院分区：
文献类型：
--
作者：
Altroff, H;van der Walle, CF;Mardon, HJ
细胞外基质分子纤维连接蛋白与整合素受体α(5)β(1)的结合引发了调节细胞功能的下游信号通路。纤维连接蛋白与α(5)β(1)的相互作用是通过纤维连接蛋白第10 FII(FIII10)结构域中保守的RGD序列发生的。在邻近的FIII9结构域中也发现了一个包含PHSRN序列的协同位点。在这里，我们使用多种方法来研究第八个FiII结构域在整合素介导的细胞黏附中的作用，包括生化、生物学和生物物理方法，包括整合素结合、细胞黏附和蛋白质变性的检测。FIII9-10中FIII9协同位点(PHSRN到PHAAA)的突变使整合素α(5)β(1)的结合活性降低到仅对FIII10观察到的水平，但相应的FIII8-9-10突变体没有表现出结合活性的丧失。细胞黏附实验也显示含有FIII8的构建体具有更强的功能活性。平衡化学变性研究表明，FIII8在FIII9上具有构象稳定性，但只有在FIII9和FIII8上暴露的环PHSRN和VKNEED完好无损的情况下才能提供构象稳定性。这些结果表明，在FIII9-10的PHSRN协同位点改变时观察到的整合素结合活性的丧失，部分是由于FIII9的构象稳定性的丧失。我们的数据提示了整合素α(5)β(1)-纤维连接蛋白相互作用的机制，除了主要的RGD结合事件外，还包括整合素对配体上可访问位点的构象敏感扫描，从而发生下游信号的完全激活。
Binding of the extracellular matrix molecule fibronectin to the integrin receptor alpha (5)beta (1) elicits downstream signaling pathways that modulate cell function. Fibronectin-alpha (5)beta (1) interaction occurs via the conserved RGD sequence in the tenth FIII (FIII10) domain of fibronectin. A synergistic site containing the sequence PHSRN in the adjacent FIII9 domain has also been identified. Here we investigate the function of the eighth FIII domain in integrin-mediated cell adhesion using a wide range of methods, including biochemical, biological, and biophysical assays of integrin binding, cell adhesion, and protein denaturation. Mutation of the FIII9 synergistic site (PHSRN to PHAAA) in FIII9-10 reduced the binding activity for integrin alpha (5)beta (1) to levels observed for FIII10 alone, but the corresponding mutant in FIII8-9-10 showed no loss of binding activity. Cell adhesion assays also demonstrated enhanced functional activity of constructs containing FIII8. Equilibrium chemical denaturation studies indicated that FIII8 confers conformational stability upon FIII9, but only if the exposed loops, PHSRN and VKNEED on FIII9 and FIII8, respectively, are intact. These results demonstrate that the loss of integrin binding activity, observed upon alteration of the PHSRN synergistic site of FIII9-10, results partly from a loss of conformational stability of FIII9. Our data suggest a mechanism for integrin alpha (5)beta (1)-fibronectin interaction, which in addition to the primary RGD binding event, involves a conformation-sensitive scanning by the integrin for accessible sites on the ligand, whereupon full activation of downstream signaling occurs.