Molecular Basis of the Recognition of Nephronectin by Integrin α8β1
Molecular Basis of the Recognition of Nephronectin by Integrin α8β1
复制标题
DOI：
10.1074/jbc.m900200200
复制
发表时间：
2009-05-22
影响因子：
4.8
通讯作者：
Sekiguchi, Kiyotoshi
中科院分区：
文献类型：
--
作者：
Sato, Yuya;Uemura, Toshihiko;Sekiguchi, Kiyotoshi
整合素α 8 β 1与细胞外基质中多种含Arg-Gly-Asp（RGD）的配体相互作用。在这里，我们研究了α 8 β 1整合素对一组含RGD配体的结合活性。整合素α 8 β 1特异性结合肾结合蛋白，表观解离常数为0.28 +/- 0.01 nM，但对纤连蛋白和其他含RGD的配体仅显示出边缘亲和力。与α 8 β 1整联蛋白的高亲和力结合用来自含RGD的中心“接头”区段的重组肾连蛋白片段完全再现。重组片段的一系列缺失突变体鉴定了RGD基序C-末端侧的LFEIFEIER序列作为高亲和力结合α 8 β 1整联蛋白所需的辅助位点。LFEIFEIER序列内的丙氨酸扫描诱变将EIE序列定义为确保高亲和力整联蛋白-配体相互作用的关键基序。虽然合成的LFEIFEIER肽不能抑制α 8 β 1整联蛋白与肾连蛋白的结合，但含有RGD基序和LFEIFEIER序列的较长肽具有强烈的抑制作用，并且比仅含有RGD基序的肽的效力强2，000倍。此外，使用含有RGD基序或LFEIFEIER序列的重组片段的反式互补测定揭示了与α 8 β 1整联蛋白结合的明显协同作用。总之，这些结果表明，肾连蛋白与α 8 β 1整联蛋白的特异性高亲和力结合是通过整联蛋白与RGD基序和LFEIFEIER序列的双向相互作用实现的，后者作为协同位点，极大地增强了RGD驱动的整联蛋白-配体相互作用，但仅具有边缘活性以确保其自身的相互作用。
Integrin alpha 8 beta 1 interacts with a variety of Arg-Gly-Asp (RGD)-containing ligands in the extracellular matrix. Here, we examined the binding activities of alpha 8 beta 1 integrin toward a panel of RGD-containing ligands. Integrin alpha 8 beta 1 bound specifically to nephronectin with an apparent dissociation constant of 0.28 +/- 0.01 nM, but showed only marginal affinities for fibronectin and other RGD-containing ligands. The high-affinity binding to alpha 8 beta 1 integrin was fully reproduced with a recombinant nephronectin fragment derived from the RGD-containing central "linker" segment. A series of deletion mutants of the recombinant fragment identified the LFEIFEIER sequence on the C-terminal side of the RGD motif as an auxiliary site required for high-affinity binding to alpha 8 beta 1 integrin. Alanine scanning mutagenesis within the LFEIFEIER sequence defined the EIE sequence as a critical motif ensuring the high-affinity integrin-ligand interaction. Although a synthetic LFEIFEIER peptide failed to inhibit the binding of alpha 8 beta 1 integrin to nephronectin, a longer peptide containing both the RGD motif and the LFEIFEIER sequence was strongly inhibitory, and was similar to 2,000-fold more potent than a peptide containing only the RGD motif. Furthermore, trans-complementation assays using recombinant fragments containing either the RGD motif or LFEIFEIER sequence revealed a clear synergism in the binding to alpha 8 beta 1 integrin. Taken together, these results indicate that the specific high-affinity binding of nephronectin to alpha 8 beta 1 integrin is achieved by bipartite interaction of the integrin with the RGD motif and LFEIFEIER sequence, with the latter serving as a synergy site that greatly potentiates the RGD-driven integrin-ligand interaction but has only marginal activity to secure the interaction by itself.