Mandai K, Nakanishi H, Satoh A, Obaishi H, Wada M, Nishioka H, Itoh M, Mizoguchi A, Aoki T, Fujimoto T, Matsuda Y, Tsukita S, Takai Y
Takai Biotimer Project, ERATO, Japan Science and Technology Corporation, c/o JCR Pharmaceuticals Co., Ltd., Kobe 651-22, Japan.
J Cell Biol. 1997 Oct 20;139(2):517-28. doi: 10.1083/jcb.139.2.517.
A novel actin filament (F-actin)-binding protein with a molecular mass of approximately 205 kD (p205), which was concentrated at cadherin-based cell-to-cell adherens junction (AJ), was isolated and characterized. p205 was purified from rat brain and its cDNA was cloned from a rat brain cDNA library. p205 was a protein of 1,829 amino acids (aa) with a calculated molecular mass of 207,667 kD. p205 had one F-actin-binding domain at 1,631-1,829 aa residues and one PDZ domain at 1,016- 1,100 aa residues, a domain known to interact with transmembrane proteins. p205 was copurified from rat brain with another protein with a molecular mass of 190 kD (p190). p190 was a protein of 1,663 aa with a calculated molecular mass of 188,971 kD. p190 was a splicing variant of p205 having one PDZ domain at 1,009-1,093 aa residues but lacking the F-actin-binding domain. Homology search analysis revealed that the aa sequence of p190 showed 90% identity over the entire sequence with the product of the AF-6 gene, which was found to be fused to the ALL-1 gene, known to be involved in acute leukemia. p190 is likely to be a rat counterpart of human AF-6 protein. p205 bound along the sides of F-actin but hardly showed the F-actin-cross-linking activity. Northern and Western blot analyses showed that p205 was ubiquitously expressed in all the rat tissues examined, whereas p190 was specifically expressed in brain. Immunofluorescence and immunoelectron microscopic studies revealed that p205 was concentrated at cadherin-based cell-to-cell AJ of various tissues. We named p205 l-afadin (a large splicing variant of AF-6 protein localized at adherens junction) and p190 s-afadin (a small splicing variant of l-afadin). These results suggest that l-afadin serves as a linker of the actin cytoskeleton to the plasma membrane at cell-to-cell AJ.
一种分子量约为205kD（p205）的新型肌动蛋白丝（F-肌动蛋白）结合蛋白被分离并鉴定，该蛋白集中于基于钙黏蛋白的细胞间黏附连接（AJ）处。p205从大鼠脑中纯化得到，其cDNA从大鼠脑cDNA文库中克隆。p205是一种由1829个氨基酸（aa）组成的蛋白质，计算分子量为207,667kD。p205在1631 - 1829个aa残基处有一个F-肌动蛋白结合结构域，在1016 - 1100个aa残基处有一个PDZ结构域，该结构域已知可与跨膜蛋白相互作用。p205与另一种分子量为190kD（p190）的蛋白质从大鼠脑中共同纯化得到。p190是一种由1663个aa组成的蛋白质，计算分子量为188,971kD。p190是p205的剪接变体，在1009 - 1093个aa残基处有一个PDZ结构域，但缺乏F-肌动蛋白结合结构域。同源性搜索分析显示，p190的aa序列与AF-6基因产物在整个序列上有90%的同一性，AF-6基因被发现与已知参与急性白血病的ALL-1基因融合。p190可能是人类AF-6蛋白的大鼠对应物。p205沿F-肌动蛋白的侧面结合，但几乎不表现出F-肌动蛋白交联活性。Northern和Western印迹分析表明，p205在所有检测的大鼠组织中普遍表达，而p190在脑中特异性表达。免疫荧光和免疫电子显微镜研究表明，p205集中于各种组织基于钙黏蛋白的细胞间AJ处。我们将p205命名为l-afadin（定位于黏附连接的AF-6蛋白的大剪接变体），将p190命名为s-afadin（l-afadin的小剪接变体）。这些结果表明，l-afadin在细胞间AJ处作为肌动蛋白细胞骨架与质膜的连接物。
