Geerts D, Fontao L, Nievers M G, Schaapveld R Q, Purkis P E, Wheeler G N, Lane E B, Leigh I M, Sonnenberg A
Division of Cell Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.
J Cell Biol. 1999 Oct 18;147(2):417-34. doi: 10.1083/jcb.147.2.417.
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 alpha6beta4 integrin and have shown that the cytoplasmic domain of the beta4 subunit associates with an NH(2)-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 alpha6beta4 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 beta4 interacts directly with plectin, and identified a major plectin-binding site on the second fibronectin type III repeat of the beta4 cytoplasmic domain. Mapping of the beta4 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 beta4 can compete out the plectin ABD fragment from its association with F-actin. The ability of beta4 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 beta4 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.
半桥粒是基底上皮细胞中稳定的黏附复合体，它在中间丝网络和细胞外基质之间提供连接。我们研究了α6β4整合素介导的网蛋白募集到半桥粒中的情况，并表明β4亚基的胞质结构域与网蛋白的一个包含肌动蛋白结合结构域（ABD）的NH2末端片段相关联。当在患有单纯性大疱性表皮松解症伴肌营养不良（MD-EBS）的人类患者的永生化网蛋白缺陷角质形成细胞中表达时，该片段与α6β4在基底半桥粒样簇中共定位，或与应力纤维或黏着斑中的F-肌动蛋白相关联。我们结合酵母双杂交结合试验和体外斑点印迹覆盖试验，证明β4与网蛋白直接相互作用，并在β4胞质结构域的第二个III型纤连蛋白重复序列上鉴定出一个主要的网蛋白结合位点。β4和网蛋白上肌动蛋白结合位点的定位表明，这些结合位点重叠且都位于网蛋白ABD中。使用体外竞争试验，我们可以证明β4可以从其与F-肌动蛋白的结合中竞争出网蛋白ABD片段。β4阻止F-肌动蛋白与网蛋白结合的能力解释了为什么从未发现F-肌动蛋白与半桥粒相关联，并提供了一种分子机制，说明当β4表达时，网蛋白定位从肌动蛋白丝向基底中间丝锚定的半桥粒的转变。最后，通过使用酵母双杂交试验和MD-EBS角质形成细胞的细胞转染实验，绘制网蛋白上几种不同中间丝蛋白的COOH末端结合位点图谱，我们证实网蛋白与不同的细胞骨架网络相互作用。
