Gilmore A P, Jackson P, Waites G T, Critchley D R
Department of Biochemistry, University of Leicester, UK.
J Cell Sci. 1992 Nov;103 ( Pt 3):719-31. doi: 10.1242/jcs.103.3.719.
The cytoskeletal protein vinculin is a component of adherens-type junctions where it is one of a number of interacting proteins thought to link the cytoplasmic domain of adhesion receptors to F-actin. Vinculin has been shown to bind to at least three other cytoskeletal proteins, talin, paxillin and alpha-actinin. In this study, we further characterise the talin-binding domain in vinculin using a series of chick vinculin polypeptides expressed as glutathione-S-transferase fusion proteins in Escherichia coli. Thus 125I-talin bound to a fusion protein spanning residues 1-398, but not to those spanning residues 399-881 or 881-1066 in an SDS-PAGE gel-blot assay. We have previously characterised two chick vinculin cDNAs (2.89 kb cDNA and cVin5) which are identical in the region of overlap except that cVin5 lacks coding sequence for residues 167-207. Interestingly, a fusion protein spanning residues 1-398, but lacking residues 167-207, was unable to bind talin. However, further analysis showed that residues 167-207 are insufficient to support binding, and deletion of as few as 31 N-terminal residues abolished binding activity. The results of the gel-blot assay were essentially confirmed using purified fusion proteins adsorbed to glutathione-agarose beads. The smallest vinculin fusion protein able to bind talin contained residues 1-258. This fusion protein was as effective as whole vinculin in inhibiting the binding of 125I-vinculin to talin-coated microtitre wells. Interestingly, mutations which altered the charge characteristics of the highly conserved residues 178 and 181 abolished binding, whereas conservative substitutions were without effect. However, such mutations did not abolish the ability of mutant polypeptides spanning residues 1-398 to target to cell-matrix junctions in Cos cells. We have investigated the possible origin of the cDNA clone cVin5 by defining the structure of a 5' portion of the chicken vinculin gene, and by analysing vinculin transcripts in a variety of adult tissues and embryonic fibroblasts using reverse transcriptase and polymerase chain reaction. Although residues 167-207 are encoded on a separate exon, we have been unable to identify a tissue where this exon is alternatively spliced.
细胞骨架蛋白纽蛋白是黏附连接的一个组成部分，在黏附连接中，它是众多相互作用蛋白之一，这些蛋白被认为可将黏附受体的胞质结构域与F-肌动蛋白相连。纽蛋白已被证明能与至少三种其他细胞骨架蛋白——踝蛋白、桩蛋白和α-辅肌动蛋白结合。在本研究中，我们利用一系列在大肠杆菌中表达为谷胱甘肽-S-转移酶融合蛋白的鸡纽蛋白多肽，进一步对纽蛋白中与踝蛋白结合的结构域进行了表征。因此，在SDS-聚丙烯酰胺凝胶印迹分析中，125I-踝蛋白能与跨越第1至398位残基的融合蛋白结合，但不能与跨越第399至881位或第881至1066位残基的融合蛋白结合。我们之前鉴定了两个鸡纽蛋白cDNA（2.89 kb cDNA和cVin5），它们在重叠区域是相同的，只是cVin5缺少第167至207位残基的编码序列。有趣的是，一个跨越第1至398位残基但缺少第167至207位残基的融合蛋白无法结合踝蛋白。然而，进一步分析表明，第167至207位残基不足以支持结合，仅缺失31个N端残基就消除了结合活性。凝胶印迹分析的结果在很大程度上通过吸附在谷胱甘肽琼脂糖珠上的纯化融合蛋白得到了证实。能够结合踝蛋白的最小纽蛋白融合蛋白包含第1至258位残基。这种融合蛋白在抑制125I-纽蛋白与包被有踝蛋白的微量滴定孔结合方面与完整的纽蛋白一样有效。有趣的是，改变高度保守残基第178和181位电荷特性的突变消除了结合，而保守性替换则没有影响。然而，此类突变并未消除跨越第1至398位残基的突变多肽靶向Cos细胞中细胞-基质连接的能力。我们通过确定鸡纽蛋白基因5'端部分的结构，并利用逆转录酶和聚合酶链反应分析各种成年组织和胚胎成纤维细胞中的纽蛋白转录本，研究了cDNA克隆cVin5可能的来源。尽管第167至207位残基由一个单独的外显子编码，但我们未能鉴定出该外显子发生可变剪接的组织。
