A 220-kD undercoat-constitutive protein: its specific localization at cadherin-based cell-cell adhesion sites.
A 220-kD undercoat-constitutive protein: its specific localization at cadherin-based cell-cell adhesion sites.
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DOI：
10.1083/jcb.115.5.1449
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发表时间：
1991-12
期刊：
影响因子：
--
通讯作者：
Tsukita S
中科院分区：
文献类型：
--
作者：
Itoh M;Yonemura S;Nagafuchi A;Tsukita S;Tsukita S
最近，我们开发了一种从大鼠肝脏中分离细胞-细胞粘附连接（AJ;基于钙粘蛋白的连接）的方法（Tsukita，Sh.和Sa。月田1989. 108：31-41）。在这项研究中，使用分离的AJ，我们已经获得了两个单克隆抗体特异性的220 kD的undercoat组成蛋白。免疫荧光和免疫电子显微镜与这些单克隆抗体表明，这220 kD的蛋白质是高度集中在底层的细胞到细胞AJ在各种类型的组织，这种蛋白质位于紧邻的质膜在底层的AJ。在细胞缺乏典型的细胞间AJ，如成纤维细胞，220 kD的蛋白质免疫荧光显示与钙粘蛋白分子在细胞间粘附位点共浓缩。这些定位分析似乎表明，220-kD蛋白与钙粘蛋白分子的可能的直接或间接协会。此外，它揭示了220-kD蛋白和α-血影蛋白与上述单克隆抗体在分离的AJ和脑中的共免疫沉淀。亲和纯化的220-kD蛋白分子看起来像一个球形颗粒，其在血影蛋白分子上的结合位点通过低角旋转阴影电子显微镜显示在距离血影蛋白四聚体中点约10-20 nm的位置。将所有这些结果与生物化学和免疫学比较一起，我们被说服推测220-kD蛋白是锚蛋白家族的新成员。然而，不能排除220-kD蛋白是β-血影蛋白的同种型的可能性。这220 kD的蛋白质在协会的钙粘蛋白分子与血影蛋白为基础的膜骨架在钙粘蛋白为基础的细胞-细胞粘附位点的可能作用进行了讨论。
Recently we developed an isolation procedure for the cell-to-cell adherens junctions (AJ; cadherin-based junctions) from rat liver (Tsukita, Sh. and Sa. Tsukita. 1989. J. Cell Biol. 108:31-41). In this study, using the isolated AJ, we have obtained two mAbs specific to the 220-kD undercoat-constitutive protein. Immunofluorescence and immunoelectron microscopy with these mAbs showed that this 220-kD protein was highly concentrated at the undercoat of cell-to-cell AJ in various types of tissues and that this protein was located in the immediate vicinity of the plasma membrane in the undercoat of AJ. In the cells lacking typical cell-to-cell AJ, such as fibroblasts, the 220- kD protein was immunofluorescently shown to be coconcentrated with cadherin molecules at cell-cell adhesion sites. These localization analyses appeared to indicate the possible direct or indirect association of the 220-kD protein with cadherin molecules. Furthermore, it was revealed that the 220-kD protein and alpha-spectrin were coimmunoprecipitated with the above mAbs in both the isolated AJ and the brain. The affinity-purified 220-kD protein molecule looked like a spherical particle, and its binding site on the spectrin molecule was shown to be in the position approximately 10-20 nm from the midpoint of spectrin tetramer by low-angle rotary-shadowing electron microscopy. Taking all these results together with biochemical and immunological comparisons, we are persuaded to speculate that the 220-kD protein is a novel member of the ankyrin family. However, the possibility cannot be excluded that the 220-kD protein is an isoform of beta-spectrin. The possible roles of this 220-kD protein in the association of cadherin molecules with the spectrin-based membrane skeletons at the cadherin- based cell-cell adhesion sites are discussed.