Obara M, Kang M S, Yamada K M
Membrane Biochemistry Section, National Cancer Institute, Bethesda, Maryland 20892.
Cell. 1988 May 20;53(4):649-57. doi: 10.1016/0092-8674(88)90580-6.
Polypeptide sequences required for function of the cell-binding domain of human fibronectin were analyzed by site-directed mutagenesis. Site-specific deletion of the putative recognition sequence Arg-Gly-Asp-Ser or an Asp-to-Glu mutation decreased the adhesive activity of fibronectin fusion proteins expressed in E. coli by greater than or equal to 97%. A second functional site over 0.5 kb away was identified by deletion mutagenesis. These mutants also showed a greater than or equal to 96% loss of activity, indicating cooperativity between sites. The two classes of mutant protein displayed synergism of activity in a trans complementation assay. Effective actin microfilament bundle organization was also dependent on the combined function of both sites. Thus, fibroblast adhesion and intracellular response to the fibronectin cell-binding domain involve two synergistic sites, each of major quantitative importance.
通过定点诱变分析了人纤连蛋白细胞结合域功能所需的多肽序列。假定识别序列Arg-Gly-Asp-Ser的位点特异性缺失或Asp到Glu的突变使大肠杆菌中表达的纤连蛋白融合蛋白的粘附活性降低了97%或更多。通过缺失诱变鉴定出一个距离超过0.5 kb的第二个功能位点。这些突变体也表现出96%或更多的活性丧失，表明位点之间存在协同作用。在反式互补试验中，两类突变蛋白表现出活性协同作用。有效的肌动蛋白微丝束组织也依赖于两个位点的联合功能。因此，成纤维细胞粘附和对纤连蛋白细胞结合域的细胞内反应涉及两个协同位点，每个位点在数量上都具有重要意义。
