Muñoz M, Serrador J, Sánchez-Madrid F, Teixidó J
Centro de Investigaciones Biológicas, Departamento de Inmunología, Madrid, Spain.
J Biol Chem. 1996 Feb 2;271(5):2696-702. doi: 10.1074/jbc.271.5.2696.
The VLA-4 (alpha 4 beta 1) integrin is involved in the adhesion of cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In order to study alpha 4 structure-function relationships, we have expressed mutated alpha 4 subunit by transfection into VLA-4-negative K562 cells. Substitutions at alpha 4 residues Arg89-Asp90, which show the highest surface probability indexes inside the N-terminal alpha 4/80 fragment, resulted in a reduction in the reactivity of all anti-alpha 4 epitope A monoclonal antibodies (mAbs) tested, compared with the reactivity with anti-alpha 4 epitopes B1, B2, and C mAb, both by transfectant flow cytometry, and by immunoprecipitation and SDS-polyacrylamide gel electrophoresis analysis of transfectant surface-iodinated proteins. In contrast, substitutions at nearby residues, Gln101, Pro102, and Ile105 did not affect the reactivity of any anti-alpha 4 mAb representing the known alpha 4 epitopes. Homotypic cell aggregation triggered by anti-alpha 4 epitope A mAb was prevented in the transfectants expressing mutated alpha 4 Arg89-Asp90Asp residues, while cell aggregation was fully achieved with either anti-alpha 4 epitope B2 or anti-beta 1 mAb. Mutations at alpha 4 residues Gln101, Pro102, and Ile108 did not affect the homotypic cell aggregation of the transfectants expressing these mutations. In addition, the adhesion of mutant Arg89-Asp90 alpha 4 transfectants to the connecting segment-1-containing fibronectin-40 (FN-40) fragment of fibronectin was diminished compared to wild type alpha 4 transfectants, as well as to other mutant alpha 4 transfectants. This adhesion to FN-40 was restored when the activating anti-beta 1 TS2/16 mAb was present in the adhesion assays. In contrast, adhesion to VCAM-1 was not affected by mutations at Arg89-Asp90, nor at Gln101, Pro102, and Ile108 alpha 4 residues. Altogether, these results indicate that alpha 4 residues Arg89 and Asp90 are included in a region involved in homotypic cell aggregation, as well as in adhesion to FN-40, but not to VCAM-1.
VLA - 4（α4β1）整合素参与细胞与纤连蛋白及血管细胞黏附分子 - 1（VCAM - 1）的黏附。为了研究α4的结构 - 功能关系，我们通过转染VLA - 4阴性的K562细胞来表达突变的α4亚基。在α4残基Arg89 - Asp90处进行替换，这些残基在N端α4/80片段内显示出最高的表面概率指数，与用抗α4表位B1、B2和C单克隆抗体（mAb）检测的反应性相比，通过转染细胞流式细胞术以及对转染细胞表面碘化蛋白进行免疫沉淀和SDS - 聚丙烯酰胺凝胶电泳分析，所有测试的抗α4表位A单克隆抗体的反应性均降低。相比之下，在附近残基Gln101、Pro102和Ile105处的替换并不影响代表已知α4表位的任何抗α4 mAb的反应性。在表达突变的α4 Arg89 - Asp90Asp残基的转染细胞中，由抗α4表位A mAb触发的同型细胞聚集受到抑制，而用抗α4表位B2或抗β1 mAb则能完全实现细胞聚集。在α4残基Gln101、Pro102和Ile108处的突变并不影响表达这些突变的转染细胞的同型细胞聚集。此外，与野生型α4转染细胞以及其他突变型α4转染细胞相比，突变的Arg89 - Asp90α4转染细胞与纤连蛋白含连接段 - 1的纤连蛋白 - 40（FN - 40）片段的黏附减少。当在黏附试验中存在激活的抗β1 TS2/16 mAb时，对FN - 40的这种黏附得以恢复。相比之下，对VCAM - 1的黏附不受Arg89 - Asp90处突变的影响，也不受α4残基Gln101、Pro102和Ile108处突变的影响。总之，这些结果表明α4残基Arg89和Asp90包含在一个与同型细胞聚集以及与FN - 40黏附有关的区域中，但与VCAM - 1的黏附无关。
