DISRUPTION OF EPITHELIAL CELL-CELL ADHESION BY EXOGENOUS EXPRESSION OF A MUTATED NONFUNCTIONAL N-CADHERIN
DISRUPTION OF EPITHELIAL CELL-CELL ADHESION BY EXOGENOUS EXPRESSION OF A MUTATED NONFUNCTIONAL N-CADHERIN
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DOI：
10.1091/mbc.4.1.37
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发表时间：
1993-01-01
影响因子：
3.3
通讯作者：
TAKEICHI, M
中科院分区：
文献类型：
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作者：
FUJIMORI, T;TAKEICHI, M
钙粘蛋白是跨膜细胞间粘附受体家族，需要与细胞骨架相互作用才能发挥正常功能。为了评估这些相互作用的机制，我们研究了外源性表达的突变型N-钙粘蛋白，cN 390 DELTA，上皮细胞-细胞粘附的影响。cN 390 DELTA的胞内结构域是完整的，但其胞外结构域大部分缺失，使得该分子对于细胞粘附没有功能。将cN 390 DELTA的cDNA连接到金属硫蛋白启动子上，并引入表达内源性E-和P-钙粘蛋白的角质形成细胞系PAM 212中。当cN 390 DELTA的表达由Zn 2+诱导时，转染细胞的钙粘蛋白依赖性粘附被抑制，导致细胞集落分散，尽管它们的接触在高细胞密度条件下保持。在这些培养物中，cN 390 DELTA不仅在细胞的游离表面上表达，而且在细胞-细胞连接处也表达。在正常条件下，内源性钙粘蛋白集中在细胞-细胞连接处。然而，作为cN 390 DELTA表达的结果，定位于细胞-细胞连接处的内源性钙粘蛋白大大减少，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-钙粘蛋白的cDNA转染相同的细胞系，其细胞内C末端已缺失，而细胞外结构域保持完整。该分子对细胞间粘附没有影响，也不定位于细胞间接触部位。我们还发现内源性钙粘蛋白与α-和β-连环蛋白和斑珠蛋白的结合不受cN 390 DELTA表达的影响，cN 390 DELTA也与这些分子形成复合物，这表明内源性和外源性钙粘蛋白之间没有竞争这些细胞质蛋白。这些和其他额外的结果表明，非功能性钙粘蛋白的细胞内结构域是完整的占据的网站，内源性钙粘蛋白应该本地化，通过与细胞骨架的相互作用，并抑制钙粘蛋白粘附系统。
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, cN390DELTA, on epithelial cell-cell adhesion. The intracellular domain of cN390DELTA was intact but its extracellular domain was largely deleted so that this molecule was not functional for cell adhesion. cDNA of cN390DELTA was attached to the metallothionein promoter, and introduced into the keratinocyte line PAM212 expressing endogenous E- and P-cadherin. When the expression of cN390DELTA 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, cN390DELTA 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 cN390DELTA 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 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 cN390DELTA, 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.