Binding of integrin alpha6beta4 to plectin prevents plectin association with F-actin but does not interfere with intermediate filament binding.
Binding of integrin alpha6beta4 to plectin prevents plectin association with F-actin but does not interfere with intermediate filament binding.
复制标题
整联蛋白α6BETA4与原蛋白的结合可预防与F-肌动蛋白的蛋白质结合，但不会干扰中间丝结合。
DOI：
10.1083/jcb.147.2.417
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发表时间：
1999-10-18
影响因子：
7.8
通讯作者：
Sonnenberg, A
中科院分区：
文献类型：
--
作者：
Geerts, D;Fontao, L;Nievers, M G;Schaapveld, R Q;Purkis, P E;Wheeler, G N;Lane, E B;Leigh, I M;Sonnenberg, A
半桥粒是基底上皮细胞中的稳定粘附复合物，其提供中间丝网络和细胞外基质之间的联系。我们研究了α6β4整联蛋白将凝集素募集到半桥粒中，并表明β4亚基的胞质结构域与含有肌动蛋白结合结构域（ABD）的凝集素NH 2端片段相关。当在来自患有单纯性大疱性表皮松解症（EB）伴肌营养不良症（MD-EBS）的人类患者的永生化的plectin缺陷型角质形成细胞中表达时，该片段与基底半桥粒样簇中的α6β4共定位或与应力纤维或局灶性接触中的F-肌动蛋白相关。我们使用酵母双杂交结合试验结合体外斑点印迹重叠试验来证明β4与纤维连接蛋白直接相互作用，并在β4胞质结构域的第二个纤连蛋白III型重复序列上鉴定了一个主要的纤维连接蛋白结合位点。对纤维素上β4和肌动蛋白结合位点的定位表明，结合位点重叠，并且都位于纤维素ABD中。使用体外竞争测定，我们可以证明β4可以从与F-肌动蛋白的结合中竞争出plectin ABD片段。β4阻止F-肌动蛋白与纤维素结合的能力解释了为什么F-肌动蛋白从未被发现与半桥粒结合，并提供了当β4表达时纤维素从肌动蛋白丝定位到基底中间粘附锚定半桥粒的分子机制。最后，通过映射的COOH-末端定位的几个不同的中间丝蛋白的结合位点的果胶使用酵母双杂交试验和细胞转染实验与MD-EBS角质形成细胞，我们证实，果胶与不同的细胞骨架网络相互作用。
Hemidesmosomes are stable adhesion complexes in basal epithelial cells that provide a link between the intermediate filament network and the extracellular matrix. We have investigated the recruitment of plectin into hemidesmosomes by the α6β4 integrin and have shown that the cytoplasmic domain of the β4 subunit associates with an NH2-terminal fragment of plectin that contains the actin-binding domain (ABD). When expressed in immortalized plectin-deficient keratinocytes from human patients with epidermol- ysis bullosa (EB) simplex with muscular dystrophy (MD-EBS), this fragment is colocalized with α6β4 in basal hemidesmosome-like clusters or associated with F-actin in stress fibers or focal contacts. We used a yeast two-hybrid binding assay in combination with an in vitro dot blot overlay assay to demonstrate that β4 interacts directly with plectin, and identified a major plectin-binding site on the second fibronectin type III repeat of the β4 cytoplasmic domain. Mapping of the β4 and actin-binding sites on plectin showed that the binding sites overlap and are both located in the plectin ABD. Using an in vitro competition assay, we could show that β4 can compete out the plectin ABD fragment from its association with F-actin. The ability of β4 to prevent binding of F-actin to plectin explains why F-actin has never been found in association with hemidesmosomes, and provides a molecular mechanism for a switch in plectin localization from actin filaments to basal intermediate filament–anchoring hemidesmosomes when β4 is expressed. Finally, by mapping of the COOH-terminally located binding site for several different intermediate filament proteins on plectin using yeast two-hybrid assays and cell transfection experiments with MD-EBS keratinocytes, we confirm that plectin interacts with different cytoskeletal networks.