Brown Ashley C, Dysart Marilyn M, Clarke Kimberly C, Stabenfeldt Sarah E, Barker Thomas H
From the Department of Biomedical Engineering, North Carolina State University and the University of North Carolina, Raleigh, North Carolina 27606.
the Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta Georgia 30332.
J Biol Chem. 2015 Oct 16;290(42):25534-47. doi: 10.1074/jbc.M115.656702. Epub 2015 Aug 28.
Fibronectin (Fn) is a promiscuous ligand for numerous cell adhesion receptors or integrins. The vast majority of Fn-integrin interactions are mediated through the Fn Arg-Gly-Asp (RGD) motif located within the tenth type III repeat. In the case of integrins αIIbβ3 and α5β1, the integrin binds RGD and the synergy site (PHSRN) located within the adjacent ninth type III repeat. Prior work has shown that these synergy-dependent integrins are exquisitely sensitive to perturbations in the Fn integrin binding domain conformation. Our own prior studies of epithelial cell responses to recombinant fragments of the Fn integrin binding domain led us to hypothesize that integrin α3β1 binding may also be modulated by the synergy site. To explore this hypothesis, we created a variety of recombinant variants of the Fn integrin binding domain: (i) a previously reported (Leu → Pro) stabilizing mutant (FnIII9'10), (ii) an Arg to Ala synergy site mutation (FnIII9(R)→(A)10), (iii) a two-Gly (FnIII9(2G)10) insertion, and (iv) a four-Gly (FNIII9(4G)10) insertion in the interdomain linker region and used surface plasmon resonance to determine binding kinetics of integrin α3β1 to the Fn fragments. Integrin α3β1 had the highest affinity for FnIII9'10 and FnIII9(2G)10. Mutation within the synergy site decreased integrin α3β1 binding 17-fold, and the four-Gly insertion decreased binding 39-fold compared with FnIII9'10. Cell attachment studies demonstrate that α3β1-mediated epithelial cell binding is greater on FnIII9'10 compared with the other fragments. These studies suggest that the presence and spacing of the RGD and synergy sites modulate integrin α3β1 binding to Fn.
纤连蛋白（Fn）是众多细胞黏附受体或整合素的一种多配体。绝大多数Fn-整合素相互作用是通过位于第十个III型重复序列内的Fn精氨酸-甘氨酸-天冬氨酸（RGD）基序介导的。就整合素αIIbβ3和α5β1而言，整合素结合RGD以及位于相邻第九个III型重复序列内的协同位点（PHSRN）。先前的研究表明，这些依赖协同作用的整合素对Fn整合素结合域构象的扰动极为敏感。我们自己先前对上皮细胞对Fn整合素结合域重组片段反应的研究使我们推测整合素α3β1的结合也可能受到协同位点的调节。为了探究这一假设，我们创建了多种Fn整合素结合域的重组变体：（i）先前报道的（Leu→Pro）稳定突变体（FnIII9'10），（ii）精氨酸到丙氨酸的协同位点突变（FnIII9（R）→（A）10），（iii）在结构域间连接区插入两个甘氨酸（FnIII9（2G）10），以及（iv）在结构域间连接区插入四个甘氨酸（FNIII9（4G）10），并使用表面等离子体共振来确定整合素α3β1与Fn片段的结合动力学。整合素α3β1对FnIII9'10和FnIII9（2G）10具有最高亲和力。与FnIII9'10相比，协同位点内的突变使整合素α3β1结合减少了17倍，四个甘氨酸插入使结合减少了39倍。细胞黏附研究表明，与其他片段相比，α3β1介导上皮细胞在FnIII9'10上的黏附更强。这些研究表明，RGD和协同位点的存在及间距调节整合素α3β1与Fn的结合。
