Dynamics of cadherin/catenin complex formation: novel protein interactions and pathways of complex assembly.
Dynamics of cadherin/catenin complex formation: novel protein interactions and pathways of complex assembly.
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DOI：
10.1083/jcb.125.6.1327
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发表时间：
1994-06
期刊：
影响因子：
--
通讯作者：
Nelson WJ
中科院分区：
文献类型：
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作者：
Hinck L;Näthke IS;Papkoff J;Nelson WJ
钙依赖的细胞间黏附是由细胞黏附蛋白中的钙粘附素家族介导的。钙粘附素黏附到细胞重组的转导是由胞浆蛋白调节的，这些蛋白被称为α-、β-和伽马-连环蛋白(Plakhemin)，它们结合到钙粘蛋白的细胞质区域，并将它们连接到细胞骨架上。以前对钙粘蛋白/连环蛋白复合体的组装和组织的研究依赖于该复合体与钙粘附素抗体的免疫共沉淀，并且局限于对这些蛋白质的Triton X-100(TX-100)可溶部分的分析。这些研究得出的结论是，只有一种复合体存在，它含有钙粘附素和所有的连环蛋白。我们提出了针对每个连接素的抗体，以独立于其与E-钙粘附素的关联来分析每个蛋白质。将狗肾上皮细胞提取物依次进行免疫沉淀和免疫印迹，结果表明TX-100可溶性部分中存在E-钙粘蛋白/α-连环蛋白、β-连环蛋白或白蛋白的复合体。我们用[35S]蛋氨酸脉冲追逐标记和蔗糖密度梯度分离分析了钙粘附素/连环蛋白复合体在TX-100可溶组分中的组装。合成后，E-钙粘附素、β-连环素和白蛋白立即以复合体的形式合成。α-连环蛋白在合成后不与这些复合体结合，但一个亚群的α-连环蛋白在E-钙粘蛋白到达质膜时加入复合体。E-钙粘蛋白到达质膜的同时，它在TX-100中的不溶性增加，但用1%的十二烷基硫酸钠提取这个不溶性的池破坏了钙粘附素/连环蛋白复合体。因此，为了检测TX-100可溶组分和不可溶组分中的蛋白质复合体组装，我们使用了[35S]蛋氨酸标记，然后在细胞提取之前进行化学交联。从细胞标记到稳定状态的交联复合体的分析表明，除了钙粘附素/连环蛋白复合体外，在TX-100可溶和不溶组分中都存在不依赖于钙粘连蛋白的连环素池。代谢标记和Chase显示，合成后立即在TX-100可溶部分中出现钙粘蛋白/β-连环蛋白和钙粘蛋白/白蛋白复合体。大约50%的络合物被滴定到TX-100不溶部分，这与络合物到达质膜和α-连环蛋白的组装相一致。随后，90%的标记钙粘蛋白进入TX-100不溶性部分，但没有额外的标记连环蛋白复合体。
Calcium-dependent cell-cell adhesion is mediated by the cadherin family of cell adhesion proteins. Transduction of cadherin adhesion into cellular reorganization is regulated by cytosolic proteins, termed alpha-, beta-, and gamma-catenin (plakoglobin), that bind to the cytoplasmic domain of cadherins and link them to the cytoskeleton. Previous studies of cadherin/catenin complex assembly and organization relied on the coimmunoprecipitation of the complex with cadherin antibodies, and were limited to the analysis of the Triton X-100 (TX- 100)-soluble fraction of these proteins. These studies concluded that only one complex exists which contains cadherin and all of the catenins. We raised antibodies specific for each catenin to analyze each protein independent of its association with E-cadherin. Extracts of Madin-Darby canine kidney epithelial cells were sequentially immunoprecipitated and immunoblotted with each antibody, and the results showed that there were complexes of E-cadherin/alpha-catenin, and either beta-catenin or plakoglobin in the TX-100-soluble fraction. We analyzed the assembly of cadherin/catenin complexes in the TX-100- soluble fraction by [35S]methionine pulse-chase labeling, followed by sucrose density gradient fractionation of proteins. Immediately after synthesis, E-cadherin, beta-catenin, and plakoglobin cosedimented as complexes. alpha-Catenin was not associated with these complexes after synthesis, but a subpopulation of alpha-catenin joined the complex at a time coincident with the arrival of E-cadherin at the plasma membrane. The arrival of E-cadherin at the plasma membrane coincided with an increase in its insolubility in TX-100, but extraction of this insoluble pool with 1% SDS disrupted the cadherin/catenin complex. Therefore, to examine protein complex assembly in both the TX-100- soluble and -insoluble fractions, we used [35S]methionine labeling followed by chemical cross-linking before cell extraction. Analysis of cross-linked complexes from cells labeled to steady state indicates that, in addition to cadherin/catenin complexes, there were cadherin- independent pools of catenins present in both the TX-100-soluble and - insoluble fractions. Metabolic labeling followed by chase showed that immediately after synthesis, cadherin/beta-catenin, and cadherin/plakoglobin complexes were present in the TX-100-soluble fraction. Approximately 50% of complexes were titrated into the TX-100- insoluble fraction coincident with the arrival of the complexes at the plasma membrane and the assembly of alpha-catenin. Subsequently, > 90% of labeled cadherin, but no additional labeled catenin complexes, entered the TX-100-insoluble fraction.(ABSTRACT TRUNCATED AT 400 WORDS)