Selective uncoupling of p120(ctn) from E-cadherin disrupts strong adhesion.
Selective uncoupling of p120(ctn) from E-cadherin disrupts strong adhesion.
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DOI：
10.1083/jcb.148.1.189
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发表时间：
2000-01-10
期刊：
影响因子：
--
通讯作者：
Reynolds AB
中科院分区：
文献类型：
--
作者：
Thoreson MA;Anastasiadis PZ;Daniel JM;Ireton RC;Wheelock MJ;Johnson KR;Hummingbird DK;Reynolds AB
P120ctn是一种连接素，它直接与经典钙粘附素的膜旁结构域结合，提示其在调节细胞间黏附中发挥作用。膜旁结构域参与了钙粘附素聚集、细胞运动和神经元突起等多种作用，增加了p120介导这些活动的可能性。我们已经在这个区域产生了最小的突变，使E-钙粘蛋白-p120相互作用解偶联，但不影响与其他连环蛋白的相互作用。通过稳定地转染E-钙粘蛋白缺陷细胞系，我们证明了钙粘附素是p120重新募集到连接的必要条件和充分性。无洗涤剂的亚细胞分离研究表明，与以前的报道相反，相互作用的化学计量比非常高。与α-和β-连环蛋白不同，p120在钙粘附素缺乏的细胞中代谢稳定，并在细胞质中存在高水平。对表达E-钙粘素突变结构的细胞的分析表明，p120是E-钙粘素介导的从弱到强黏附转换所必需的。在聚集实验中，表达p120解偶联E-钙粘附素的细胞只形成弱的细胞聚集体，这些聚集体在吸管后立即分散成单个细胞。一个明显的后果是，肌动蛋白细胞骨架未能正确插入外周E-钙粘素斑块，导致无法在细胞克隆周围形成连续的环状结构。我们的数据表明，p120直接或间接地调节E-钙粘附素介导的细胞间紧密黏附的转变，可能阻止随后的肌动蛋白细胞骨架重组和紧凑所必需的事件。
p120ctn is a catenin whose direct binding to the juxtamembrane domain of classical cadherins suggests a role in regulating cell–cell adhesion. The juxtamembrane domain has been implicated in a variety of roles including cadherin clustering, cell motility, and neuronal outgrowth, raising the possibility that p120 mediates these activities. We have generated minimal mutations in this region that uncouple the E-cadherin–p120 interaction, but do not affect interactions with other catenins. By stable transfection into E-cadherin–deficient cell lines, we show that cadherins are both necessary and sufficient for recruitment of p120 to junctions. Detergent-free subcellular fractionation studies indicated that, in contrast to previous reports, the stoichiometry of the interaction is extremely high. Unlike α- and β-catenins, p120 was metabolically stable in cadherin-deficient cells, and was present at high levels in the cytoplasm. Analysis of cells expressing E-cadherin mutant constructs indicated that p120 is required for the E-cadherin–mediated transition from weak to strong adhesion. In aggregation assays, cells expressing p120-uncoupled E-cadherin formed only weak cell aggregates, which immediately dispersed into single cells upon pipetting. As an apparent consequence, the actin cytoskeleton failed to insert properly into peripheral E-cadherin plaques, resulting in the inability to form a continuous circumferential ring around cell colonies. Our data suggest that p120 directly or indirectly regulates the E-cadherin–mediated transition to tight cell–cell adhesion, possibly blocking subsequent events necessary for reorganization of the actin cytoskeleton and compaction.
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