Koster J, van Wilpe S, Kuikman I, Litjens S H M, Sonnenberg A
The Netherlands Cancer Institute, Division of Cell Biology, 1066 CX Amsterdam, The Netherlands.
Mol Biol Cell. 2004 Mar;15(3):1211-23. doi: 10.1091/mbc.e03-09-0697. Epub 2003 Dec 10.
We have previously shown that plectin is recruited into hemidesmosomes through association of its actin-binding domain (ABD) with the first pair of fibronectin type III (FNIII) repeats and a small part of the connecting segment (residues 1328-1355) of the integrin beta4 subunit. Here, we show that two proline residues (P1330 and P1333) in this region of the connecting segment are critical for supporting beta4-mediated recruitment of plectin. Additional binding sites for the plakin domain of plectin on beta4 were identified in biochemical and yeast two-hybrid assays. These sites are located at the end of the connecting segment (residues 1383-1436) and in the region containing the fourth FNIII repeat and the C-tail (residues 1570-1752). However, in cells, these additional binding sites cannot induce the assembly of hemidesmosomes without the interaction of the plectin-ABD with beta4. Because the additional plectin binding sites overlap with sequences that mediate an intramolecular association of the beta4 cytoplasmic domain, we propose that they are not accessible for binding and need to become exposed as the result of the binding of the plectin-ABD to beta4. Furthermore, these additional binding sites might be necessary to position the beta4 cytoplasmic domain for an optimal interaction with other hemidesmosomal components, thereby increasing the efficiency of hemidesmosome assembly.
我们之前已经表明，网蛋白通过其肌动蛋白结合结构域（ABD）与整合素β4亚基的第一对III型纤连蛋白（FNIII）重复序列以及连接片段的一小部分（残基1328 - 1355）结合，被招募到半桥粒中。在此，我们表明连接片段的这一区域中的两个脯氨酸残基（P1330和P1333）对于支持β4介导的网蛋白招募至关重要。在生化和酵母双杂交实验中鉴定出了网蛋白的plakin结构域在β4上的其他结合位点。这些位点位于连接片段的末端（残基1383 - 1436）以及包含第四个FNIII重复序列和C末端的区域（残基1570 - 1752）。然而，在细胞中，如果没有网蛋白 - ABD与β4的相互作用，这些额外的结合位点无法诱导半桥粒的组装。由于额外的网蛋白结合位点与介导β4胞质结构域分子内缔合的序列重叠，我们提出它们无法用于结合，需要在网蛋白 - ABD与β4结合后暴露出来。此外，这些额外的结合位点对于将β4胞质结构域定位以与其他半桥粒成分进行最佳相互作用可能是必要的，从而提高半桥粒组装的效率。
