Plasma membrane-Actin filament Association through ERM Family Members

ERM家族成员，ezrin， radixin和moesin，定位于质膜下方，被认为参与肌动蛋白丝/质膜结合。为了鉴定与ERM家族成员直接相关的整体膜蛋白，我们使用抗moesin单抗进行了免疫沉淀研究，并培养了用[^<35>S]蛋氨酸代谢标记或生物素表面标记的BHK细胞。结果表明，moesin与一个140kD的完整膜蛋白直接相关。以BHK细胞为抗原，获得了识别140kD膜蛋白的单抗。接下来，我们克隆了一个编码140kD膜蛋白的cDNA，并将其鉴定为CD44，一种广泛分布的细胞表面糖蛋白。多种抗CD44单克隆抗体免疫沉淀表明，不仅在BHK细胞中，在小鼠L成纤维细胞中，ezrin和radixin以及moesin都与CD44相关。此外，免疫荧光显微镜显示，在BHK和L细胞中，Triton x -100不溶性CD44与ERM家族成员精确地共定位。我们得出结论，ERM家族成员作为CD44细胞质结构域和基于肌动蛋白的细胞骨架之间的分子连接体。

The ERM family members, ezrin, radixin, and moesin, localizing just beneath the plasma membranes are thought to be involved in the actin filament/plasma membrane association. To identify the integral membrane protein directly associated with ERM family members, we performed immunoPrecipitaion studies using anti-moesin mAb and cultured BHK cells metabol-ically-labeled with [^<35>S] methionine or surface-labeled with biotin. The results indicated that moesin is directly associated with a 140kD integral membrane protein. Using BHK cells as antigens, we obtained a mAb which recognized the 140kD membrane protein. We next cloned a cDNA encoding the 140kD membrane protein and identified it as CD44, a broadly-distributed cell surface glycoprotein. Immunoprecipitation with various anti-CD44 mAbs showed that ezrin and radixin as well as moesin are associated with CD44 not only in BHK cells but also in mouse L fibroblasts. Furthermore, immunofluorescence microscopy revealed that in both BHK and L cells, the Triton X-100-insoluble CD44 is precisely colocalized with ERM family members. We concluded that ERM family members work as molecular linkers between the cytoplasmic domain of CD44 and actin-based cytoskeletons.