SITE-DIRECTED MUTAGENESIS OF THE CELL-BINDING DOMAIN OF HUMAN FIBRONECTIN - SEPARABLE, SYNERGISTIC SITES MEDIATE ADHESIVE FUNCTION
SITE-DIRECTED MUTAGENESIS OF THE CELL-BINDING DOMAIN OF HUMAN FIBRONECTIN - SEPARABLE, SYNERGISTIC SITES MEDIATE ADHESIVE FUNCTION
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DOI：
10.1016/0092-8674(88)90580-6
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发表时间：
1988-05-20
期刊：
影响因子：
64.5
通讯作者：
YAMADA, KM
中科院分区：
文献类型：
--
作者：
OBARA, M;KANG, MS;YAMADA, KM
通过定点突变分析人纤连蛋白的细胞结合结构域的功能所需的多肽序列。推定识别序列Arg-Gly-Asp-Ser的位点特异性缺失或Asp-to-Glu突变使在大肠杆菌中表达的纤连蛋白融合蛋白的粘附活性降低≥百分之九十七第二个功能位点超过0.5 kb的距离，通过缺失诱变鉴定。这些突变体还显示出≥96%的活性丧失，表明位点之间的协同性。这两类突变蛋白在反式互补测定中显示出协同活性。有效的肌动蛋白微丝束组织也依赖于这两个网站的组合功能。因此，成纤维细胞粘附和细胞内反应的纤连蛋白细胞结合域涉及两个协同位点，每个主要的定量重要性。
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 Escherichia coli by .gtoreq. 97%. A second functional site over 0.5 kb away was identified by deletion mutagenesis. These mutants also showed a .gtoreq. 96% loss of acivity, 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.