Identification of a talin binding site in the cytoskeletal protein vinculin.
Identification of a talin binding site in the cytoskeletal protein vinculin.
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鉴定细胞骨架蛋白质体蛋白中的塔林结合位点。
DOI：
10.1083/jcb.109.6.2917
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发表时间：
1989-12
影响因子：
7.8
通讯作者：
Critchley, D R
中科院分区：
文献类型：
--
作者：
Jones, P;Jackson, P;Price, G J;Patel, B;Ohanion, V;Lear, A L;Critchley, D R
细胞骨架蛋白黏着斑蛋白与塔林蛋白的结合是将F-肌动蛋白连接到细胞-基质连接处的许多相互作用之一。为了确定黏着斑蛋白中的塔林结合域，我们使用体外转录翻译系统表达了由两个非常相似但不同的黏着斑蛋白cDNA编码的分子的NH 2-末端区域。黏着斑蛋白cDNA的5' Eco RI-Bam HI片段编码一个45 kD的多肽，该多肽含有黏着斑蛋白分子的前398个氨基酸。第二黏着斑蛋白cDNA（cVin 5）的等效限制酶片段缺少核苷酸746- 867，并编码缺少氨基酸167-207的41-kD多肽。放射性标记的45-kD黏着斑蛋白多肽结合到用talin包被的微量威尔斯孔上，但不结合到BSA上，并且结合被未标记的黏着斑蛋白抑制。相比之下，41-kD黏着斑蛋白多肽缺乏talin结合活性。残基167-207在talin结合中的作用通过在该区域进行一系列缺失来进一步分析，每个缺失7个氨基酸与下一个相邻。残基167-173、174-180、181-187、188-194或195-201的缺失导致talin结合活性的显著降低，尽管残基202-208的缺失具有小得多的影响。当45-kD黏着斑蛋白多肽在Cos细胞中表达时，其定位于细胞基质连接处，而缺乏残基167-207的41-kD多肽不能这样做。有趣的是，一些缺失突变体在体外结合塔林的能力降低，仍然能够定位于细胞基质连接处。
Binding of the cytoskeletal protein vinculin to talin is one of a number of interactions involved in linking F-actin to cell-matrix junctions. To identify the talin binding domain in vinculin, we expressed the NH2-terminal region of the molecule encoded by two closely similar, but distinct vinculin cDNAs, using an in vitro transcription translation system. The 5' Eco RI-Bam HI fragment of a partial 2.89-kb vinculin cDNA encodes a 45-kD polypeptide containing the first 398 amino acids of the molecule. The equivalent restriction enzyme fragment of a second vinculin cDNA (cVin5) lacks nucleotides 746- 867, and encodes a 41-kD polypeptide missing amino acids 167-207. The radiolabeled 45-kD vinculin polypeptide bound to microtiter wells coated with talin, but not BSA, and binding was inhibited by unlabeled vinculin. In contrast, the 41-kD vinculin polypeptide was devoid of talin binding activity. The role of residues 167-207 in talin binding was further analyzed by making a series of deletions spanning this region, each deletion of seven amino acids contiguous with the next. Loss of residues 167-173, 174-180, 181-187, 188-194, or 195-201 resulted in a marked reduction in talin binding activity, although loss of residues 202-208 had much less effect. When the 45-kD vinculin polypeptide was expressed in Cos cells, it localized to cell matrix junctions, whereas the 41-kD polypeptide, lacking residues 167-207, was unable to do so. Interestingly, some deletion mutants with reduced ability to bind talin in vitro, were still able to localize to cell matrix junctions.