MOESIN, EZRIN, AND P205 ARE ACTIN-BINDING PROTEINS ASSOCIATED WITH NEUTROPHIL PLASMA-MEMBRANES
MOESIN, EZRIN, AND P205 ARE ACTIN-BINDING PROTEINS ASSOCIATED WITH NEUTROPHIL PLASMA-MEMBRANES
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DOI：
10.1091/mbc.6.3.247
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发表时间：
1995-03-01
影响因子：
3.3
通讯作者：
LUNA, EJ
中科院分区：
文献类型：
--
作者：
PESTONJAMASP, K;AMIEVA, MR;LUNA, EJ
用I-125标记的F-肌动蛋白印迹分析牛中性粒细胞质膜上的肌动蛋白结合蛋白。与表面生物素化的蛋白质一起，膜上富含78、81和205 kDa的主要肌动蛋白结合多肽。F-肌动蛋白的结合是特异的，因为G-肌动蛋白不结合。此外，未标记的F-肌动蛋白阻断了I-125标记的F-肌动蛋白的结合，而其他酸性生物聚合物相对无效。结合也被肌球蛋白亚片段1特异性地抑制，但不被CAPZ或血浆明胶蛋白所抑制，这表明膜蛋白与肌球蛋白一样，沿着肌动蛋白细丝的两侧结合。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法和免疫沉淀法分别鉴定78 kDa和81 kDa的多肽分别为Moesin和Ezrin。虽然在牛中性粒细胞中不存在可检测到的量，Radixin(该基因家族的第三个密切相关成员)也会将I-125标记的F-肌动蛋白结合在印迹覆盖层上。用全长和截短的细菌融合蛋白进行的实验将Moesin中的肌动蛋白结合部位定位到极端的羧基末端，这是一个高度保守的序列。免疫荧光显微照片显示，渗透细胞和细胞足迹显示moesin与肌动蛋白共同定位在质膜的细胞质表面，这与作为膜-肌动蛋白连接蛋白的作用一致。
Actin-binding proteins in bovine neutrophil plasma membranes were identified using blot overlays with I-125-labeled F-actin. Along with surface-biotinylated proteins, membranes were enriched in major actin-binding polypeptides of 78, 81, and 205 kDa. Binding was specific for F-actin because G-actin did not bind. Further, unlabeled F-actin blocked the binding of I-125-labeled F-actin whereas other acidic biopolymers were relatively ineffective. Binding also was specifically inhibited by myosin subfragment 1, but not by CapZ or plasma gelsolin, suggesting that the membrane proteins, like myosin, bind along the sides of the actin filaments. The 78- and 81-kDa polypeptides were identified as moesin and ezrin, respectively, by co-migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoprecipitation with antibodies specific for moesin and ezrin. Although not present in detectable amounts in bovine neutrophils, radixin (a third and closely related member of this gene family) also bound I-125-labeled F-actin on blot overlays. Experiments with full-length and truncated bacterial fusion proteins localized the actin-binding site in moesin to the extreme carboxy terminus, a highly conserved sequence. Immunofluorescence micrographs of permeabilized cells and cell ''footprints'' showed moesin co-localization with actin at the cytoplasmic surface of the plasma membrane, consistent with a role as a membrane-actin-linking protein.