Fujimori T, Takeichi M
Department of Biophysics, Faculty of Science, Kyoto University, Japan.
Mol Biol Cell. 1993 Jan;4(1):37-47. doi: 10.1091/mbc.4.1.37.
Cadherins, a family of transmembrane cell-cell adhesion receptors, require interactions with the cytoskeleton for normal function. To assess the mechanisms of these interactions, we studied the effect of exogenous expression of a mutant N-cadherin, cN390 delta; on epithelial cell-cell adhesion. The intracellular domain of cN390 delta was intact but its extracellular domain was largely deleted so that this molecule was not functional for cell adhesion. cDNA of cN390 delta was attached to the metallothionein promoter, and introduced into the keratinocyte line PAM212 expressing endogenous E- and P-cadherin. When the expression of cN390 delta was induced by Zn2+, cadherin-dependent adhesion of the transfected cells was inhibited, resulting in the dispersion of cell colonies, although their contacts were maintained under high cell density conditions. In these cultures, cN390 delta was expressed not only on the free surfaces of the cells but also at cell-cell junctions. The endogenous cadherins were concentrated at cell-cell junctions under normal conditions. As a result of cN390 delta expression, however, the endogenous cadherins localizing at the cell-cell junctions were largely diminished, suggesting that these molecules were replaced by the mutant molecules at these sites. As a control, we transfected the same cell line with cDNA of a truncated form of N-cadherin cadherin whose intracellular C terminus had been deleted leaving the extracellular domain intact. This molecule had no effect on cell-cell adhesion, nor did it localize to cell-cell contact sites. We also found that the association of the endogenous cadherins with alpha- and beta-catenins and plakoglobin was not affected by the expression of cN390 delta, which also formed a complex with these molecules, suggesting that no competition occurred between the endogenous and exogenous cadherins for these cytoplasmic proteins. These and other additional results suggest that the nonfunctional cadherins whose intracellular domain is intact occupy the sites where the endogenous cadherins should localize, through interactions with the cytoskeleton, and inhibit the cadherin adhesion system.
钙黏蛋白是一类跨膜细胞间黏附受体，其正常功能需要与细胞骨架相互作用。为了评估这些相互作用的机制，我们研究了突变型N-钙黏蛋白cN390δ的外源表达对上皮细胞间黏附的影响。cN390δ的胞内结构域完整，但其胞外结构域大部分缺失，因此该分子对细胞黏附无功能。将cN390δ的cDNA连接到金属硫蛋白启动子上，并导入表达内源性E-钙黏蛋白和P-钙黏蛋白的角质形成细胞系PAM212中。当通过Zn2+诱导cN390δ表达时，转染细胞的钙黏蛋白依赖性黏附受到抑制，导致细胞集落分散，尽管在高细胞密度条件下它们的接触得以维持。在这些培养物中，cN390δ不仅在细胞的自由表面表达，也在细胞间连接处表达。内源性钙黏蛋白在正常条件下集中在细胞间连接处。然而，由于cN390δ的表达，位于细胞间连接处的内源性钙黏蛋白大量减少，这表明这些分子在这些位点被突变分子取代。作为对照，我们用截短形式的N-钙黏蛋白的cDNA转染同一细胞系，该截短形式的N-钙黏蛋白的胞内C末端已被删除，胞外结构域完整。该分子对细胞间黏附无影响，也不定位到细胞间接触位点。我们还发现，内源性钙黏蛋白与α-连环蛋白、β-连环蛋白和桥粒斑蛋白的结合不受cN390δ表达的影响，cN390δ也与这些分子形成复合物，这表明内源性和外源性钙黏蛋白之间不存在对这些细胞质蛋白的竞争。这些以及其他额外的结果表明，胞内结构域完整的无功能钙黏蛋白通过与细胞骨架相互作用占据内源性钙黏蛋白应定位的位点，并抑制钙黏蛋白黏附系统。
