Itoh M, Yonemura S, Nagafuchi A, Tsukita S, Tsukita S
Department of Information Physiology, National Institute for Physiological Sciences, Aichi, Japan.
J Cell Biol. 1991 Dec;115(5):1449-62. doi: 10.1083/jcb.115.5.1449.
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.
最近我们开发了一种从大鼠肝脏中分离细胞间黏附连接（AJ；基于钙黏蛋白的连接）的方法（筑田诚和筑田佐知子，1989年，《细胞生物学杂志》108卷：31 - 41页）。在本研究中，利用分离得到的AJ，我们获得了两种对220-kD底层组成蛋白具有特异性的单克隆抗体。用这些单克隆抗体进行免疫荧光和免疫电子显微镜观察表明，这种220-kD蛋白高度集中在各种组织中细胞间AJ的底层，并且该蛋白位于AJ底层质膜的紧邻区域。在缺乏典型细胞间AJ的细胞中，如成纤维细胞，免疫荧光显示220-kD蛋白与钙黏蛋白分子在细胞 - 细胞黏附位点共浓缩。这些定位分析似乎表明220-kD蛋白与钙黏蛋白分子可能存在直接或间接的关联。此外，还发现220-kD蛋白和α - 血影蛋白在分离的AJ和大脑中都能被上述单克隆抗体共免疫沉淀。亲和纯化的220-kD蛋白分子看起来像球形颗粒，通过低角度旋转阴影电子显微镜显示，其在血影蛋白分子上的结合位点位于距血影蛋白四聚体中点约10 - 20 nm处。综合所有这些结果以及生化和免疫学比较，我们推测220-kD蛋白是锚蛋白家族的一个新成员。然而，不能排除220-kD蛋白是β - 血影蛋白同工型的可能性。本文讨论了这种220-kD蛋白在基于钙黏蛋白的细胞 - 细胞黏附位点上钙黏蛋白分子与基于血影蛋白的膜骨架关联中的可能作用。
