Vogelmann Roger, Nelson W James
Department of Molecular and Cellular Physiology, Beckman Center for Molecular and Genetic Medicine, Stanford University of Medicine, Stanford, CA, USA.
Methods Mol Biol. 2007;370:11-22. doi: 10.1007/978-1-59745-353-0_2.
The number of proteins found associated with cell-cell adhesion substructures is growing rapidly. Based on potential protein-protein interactions, complex protein networks at cell-cell contacts can be modeled. Traditional studies to examine protein-protein interactions include co-immunoprecipitation or pull-down experiments of tagged proteins. These studies provide valuable information that proteins can associate directly or indirectly through other proteins in a complex. However, they do not clarify if a given protein is part of other protein complexes or inform about the specificity of those interactions in the context of adhesion substructures. Thus, it is not clear if models compiled from these types of studies reflect the combination of protein interactions in the adhesion complex in vivo for a specific cell type. Therefore, we present here a method to separate cell-cell contact membrane substructures with their associated protein complexes based on their buoyant behavior in iodixanol density gradients. Analysis of 16 proteins of the apical junctional complex (AJC) in epithelial Madin-Darby canine kidney cells revealed a more simple organization of the AJC adhesion complex than that predicted from the combination of all possible protein-protein interactions defined from co-immunoprecipitation and pull-down experiments.
与细胞间粘附亚结构相关的蛋白质数量正在迅速增加。基于潜在的蛋白质-蛋白质相互作用，可以构建细胞间接触处的复杂蛋白质网络模型。传统研究蛋白质-蛋白质相互作用的方法包括对标记蛋白质进行共免疫沉淀或下拉实验。这些研究提供了有价值的信息，即蛋白质可以通过复合物中的其他蛋白质直接或间接结合。然而，它们并未阐明给定蛋白质是否是其他蛋白质复合物的一部分，也未告知这些相互作用在粘附亚结构背景下的特异性。因此，尚不清楚从这类研究中汇编的模型是否反映了特定细胞类型在体内粘附复合物中蛋白质相互作用的组合。因此，我们在此提出一种基于其在碘克沙醇密度梯度中的浮力行为来分离细胞间接触膜亚结构及其相关蛋白质复合物的方法。对上皮性犬肾细胞中顶端连接复合体（AJC）的16种蛋白质的分析表明，AJC粘附复合物的组织比根据共免疫沉淀和下拉实验定义的所有可能蛋白质-蛋白质相互作用的组合所预测的更为简单。
