Hocking D C, Smith R K, McKeown-Longo P J
Department of Physiology and Cell Biology, Albany Medical College, NY 12208, USA.
J Cell Biol. 1996 Apr;133(2):431-44. doi: 10.1083/jcb.133.2.431.
Fibronectin matrix assembly is a cell-dependent process which is upregulated in tissues at various times during development and wound repair to support the functions of cell adhesion, migration, and differentiation. Previous studies have demonstrated that the alpha 5 beta 1 integrin and fibronectin's amino terminus and III-1 module are important in fibronectin polymerization. We have recently shown that fibronectin's III-1 module contains a conformationally sensitive binding site for fibronectin's amino terminus (Hocking, D.C., J. Sottile, and P.J. McKeown-Longo. 1994. J. Biol. Chem. 269: 19183-19191). The present study was undertaken to define the relationship between the alpha 5 beta 1 integrin and fibronectin polymerization. Solid phase binding assays using recombinant III-10 and III-1 modules of human plasma fibronectin indicated that the III-10 module contains a conformation-dependent binding site for the III-1 module of fibronectin. Unfolded III-10 could support the formation of a ternary complex containing both III-1 and the amino-terminal 70-kD fragment, suggesting that the III-1 module can support the simultaneous binding of III-10 and 70 kD. Both unfolded III-10 and unfolded III-1 could support fibronectin binding, but only III-10 could promote the formation of disulfide-bonded multimers of fibronectin in the absence of cells. III-10-dependent multimer formation was inhibited by both the anti-III-1 monoclonal antibody, 9D2, and amino-terminal fragments of fibronectin. A fragment of III-10, termed III-10/A, was able to block matrix assembly in fibroblast monolayers. Similar results were obtained using the III-10A/RGE fragment, in which the RGD site had been mutated to RGE, indicating that III-I0/A was blocking matrix assembly by a mechanism distinct from disruption of integrin binding. Texas red-conjugated recombinant III-1,2 localized to beta 1-containing sites of focal adhesions on cells plated on fibronectin or the III-9,10 modules of fibronectin. Monoclonal antibodies against the III-1 or the III-9,10 modules of fibronectin blocked binding of III-1,2 to cells without disrupting focal adhesions. These data suggest that a role of the alpha 5 beta 1 integrin in matrix assembly is to regulate a series of sequential self-interactions which result in the polymerization of fibronectin.
纤连蛋白基质组装是一个细胞依赖的过程，在发育和伤口修复的不同时期，组织中的该过程会被上调，以支持细胞黏附、迁移和分化的功能。先前的研究表明，α5β1整合素、纤连蛋白的氨基末端和III-1模块在纤连蛋白聚合中起重要作用。我们最近发现，纤连蛋白的III-1模块含有一个对纤连蛋白氨基末端构象敏感的结合位点（霍金，D.C.，J.索蒂尔，和P.J.麦基翁-隆戈。1994。《生物化学杂志》269:19183 - 19191）。本研究旨在确定α5β1整合素与纤连蛋白聚合之间的关系。使用重组人血浆纤连蛋白的III-10和III-1模块进行的固相结合试验表明，III-10模块含有一个对纤连蛋白III-1模块构象依赖的结合位点。未折叠的III-10能够支持包含III-1和氨基末端70-kD片段的三元复合物的形成，这表明III-1模块能够支持III-10和70 kD的同时结合。未折叠的III-10和未折叠的III-1都能支持纤连蛋白结合，但只有III-10能够在无细胞的情况下促进纤连蛋白二硫键连接的多聚体的形成。III-10依赖的多聚体形成受到抗III-1单克隆抗体9D2和纤连蛋白氨基末端片段的抑制。III-10的一个片段，称为III-10/A，能够阻断成纤维细胞单层中的基质组装。使用III-10A/RGE片段（其中RGD位点已突变为RGE）也获得了类似的结果，这表明III-10/A通过一种不同于破坏整合素结合的机制阻断基质组装。德克萨斯红偶联的重组III-1,2定位于铺有纤连蛋白或纤连蛋白III-9,10模块的细胞上含β1的粘着斑位点。针对纤连蛋白III-1或III-9,10模块的单克隆抗体阻断III-1,2与细胞的结合，而不破坏粘着斑。这些数据表明，α5β1整合素在基质组装中的作用是调节一系列导致纤连蛋白聚合的顺序性自我相互作用。
