Further characterization of the interaction between the cytoskeletal proteins talin and vinculin
Further characterization of the interaction between the cytoskeletal proteins talin and vinculin
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DOI：
10.1042/0264-6021:3620761
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发表时间：
2002-03-15
影响因子：
4.1
通讯作者：
Critchley, DR
中科院分区：
文献类型：
--
作者：
Bass, MD;Patel, B;Critchley, DR
细胞骨架蛋白tal in被认为将整合素与f -肌动蛋白结合。含有三个结合位点（VBS1-VBS3）的血管蛋白，参与细胞运动的负调控，其活性是由血管蛋白头部（Vh）和血管蛋白尾部（Vt）之间的分子内相互作用调节。在目前的研究中，我们发现含有三个vbs的重组talin多肽（VBS1，残基498-636，VBS2，残基727-965和VBS3，残基1943-2157）每个都紧密结合到vinculin内相同或重叠的位点上（1-258）。合成的短talin VBS3肽（残基1944-1969）足以抑制I-125标记的talin VBS3多肽与vinculin（1-258）的结合，核磁共振证实该肽与vinculin（1-258）缓慢交换形成I: I复合物。I-125标记的VBS3多肽的结合明显依赖于温度，但不受I- M盐或10% (v/v) 2-甲基-2-丙醇的抑制。试图用凝胶印迹法进一步确定血管蛋白（1-258）内的talin结合位点是不成功的，但在酵母双杂交实验中，一个跨越血管蛋白残基1-131的结构保留了接近最大的talin结合活性。有趣的是，talin VBS3多肽是Vh-Vt相互作用的有效抑制剂，并且VBS3合成肽能够暴露完整的血管蛋白中肌动蛋白结合位点，否则被Vh-Vt相互作用掩盖。结果表明，在一定条件下。Talin可能是一种有效的激活剂。
The cytoskeletal protein tal in, which is thought to couple integrins to F-actin. contains three binding sites (VBS1-VBS3) for vinculin, a protein implicated in the negative regulation of cell motility and whose activity is modulated by an intramolecular interaction between the vinculin head (Vh) and vinculin tail (Vt) domains. In the present study we show that recombinant talin polypeptides containing the three VBSs (VBS1, residues 498-636, VBS2, residues 727-965, and VBS3, residues 1943-2157) each bind tightly to the same or overlapping sites within vinculin(1-258). A short synthetic talin VBS3 peptide (residues 1944-1969) was sufficient to inhibit binding of a I-125-labelled talin VBS3 polypeptide to vinculin(1-258), and NMR spectroscopy confirmed that this peptide forms a I : I complex in slow exchange with vinculin(1-258). Binding of the I-125-labelled VBS3 polypeptide was markedly temperature dependent, but was not inhibited by I M salt or 10% (v/v) 2-methyl-2-propanol. Attempts to further define the talin-binding site within vinculin(1-258) using a gel-blot assay were unsuccessful, but near maximal talin-binding activity was retained by a construct spanning vinculin residues 1-131 in a yeast two-hybrid assay. Interestingly, the talin VBS3 polypeptide was a potent inhibitor of the Vh-Vt interaction, and the VBS3 synthetic peptide was able to expose the actin-binding site in intact vinculin, which is otherwise masked by the Vh-Vt interaction. The results suggest that under certain conditions. talin may be an effective activator of vinculin.