CD44-ERM-ACTIN SYSTEM,SIGNAL TRANSDUCTION,AND APOPTOSIS

ERM蛋白ezrin、radixin和moesin作为交联剂参与肌动蛋白丝/质膜相互作用。CD44被确定为ERM蛋白的主要膜结合伙伴之一。为了研究CD44/ERM蛋白在体外的相互作用，我们通过重组杆状病毒感染制备了小鼠ezrin、radixin、moesin和GST/CD44细胞质域融合蛋白（GST- cd44cyt），并构建了ERM蛋白与CD44细胞质域结合的体外实验。在该体系中，在低离子强度下，ERM蛋白与GST-CD44cyt结合具有高亲和力（moesin的Kd为9.3(]SY.+- [)1.6nM），但在生理离子强度下具有低亲和力。然而，在磷酸肌苷（PI，4- pip和4,5- pip <@D22@>D2）存在时，即使在生理离子强度下，与GST-CD44cyt结合的ERM蛋白也具有相对高的亲和力：4,5- pip <@D22@>D2显示出最显著的作用（moesin在4,5- pipp <@D22@>D2存在时的Kd为9.3(]SY…More .+- . [)4.8nM）。接下来，为了在体内研究CD44/ERM相互作用的调节机制，我们重新检查了来自BHK细胞的免疫沉淀CD44/ERM复合物，发现它含有Rho GTPase的调节剂Rho- gdi。然后，我们评估了Rho参与CD44/ERM复合物形成的调控。加入重组ERM蛋白，与培养的BHK细胞裂解物孵育，离心后，部分重组ERM蛋白在不溶性部分中被回收。这种结合被GTPgammaS增强，并被C3毒素（Rho的一种特异性抑制剂）再次显著抑制，这表明裂解物中gtp形式的Rho是这种结合所必需的。CD44细胞质域特异性单克隆抗体也显著抑制了这种结合，识别出不溶性部分中外源性ERM蛋白的大多数结合伙伴为CD44。与这一结合分析相一致的是，当活的BHK细胞用C3毒素处理时，大多数不溶性ERM蛋白移动到细胞质中的可溶性区室，使CD44脱离ERM。这些发现表明Rho在体内调控CD44/ERM复合物的形成，磷脂酰肌醇的转换可能参与了这一调控机制。少

The ERM proteins, ezrin, radixin and moesin, are involved in actin filament/plasma membrane interaction as crosslinkers. CD44 was identified as one of the major membrane binding partners for ERM proteins. To examine the CD44/ERM protein interaction in vitro, we produced mouse ezrin, radixin, moesin, and the GST/CD44 cytoplasmic domain fusion protein (GST-CD44cyt) by means of recombinant baculovirus infection, and constructed an in vitro assay for the binding between ERM proteins and the cytoplasmic domain of CD44. In this system, at low ionic strength ERM proteins bound to GST-CD44cyt with high affinity (Kd of moesin was 9.3(]SY.+-。[)1.6nM), but with low affinity at physiological ionic strength. However, in the presence of phosphoinositides (PI,4-PIP,and 4,5-PIP<@D22@>D2), ERM proteins bound with relatively high affinity to GST-CD44cyt even at physiological ionic strength : 4,5-PIP<@D22@>D2 showed the most remarkable effect (Kd of moesin in the presence of 4,5-PIPP<@D22@>D2 was 9.3(]SY … More .+-。[)4.8nM). Next, to examine the regulation mechanism of CD44/ERM interaction in vivo, we reexamined the immunoprecipitated CD44/ERM complex from BHK cells and found that it contains Rho-GDI,a regulator of Rho GTPase. We then evaluated the involvement of Rho in the regulation of the CD44/ERM complex formation. When recombinant ERM proteins were added and incubated with lysates of cultured BHK cells followed by centrifugation, a portion of the recombinant ERM proteins was recovered in the insoluble fraction. This binding was enhanced by GTPgammaS,and remarkedly suppressed by C3 toxin, a specific inhibitor of Rho, indicating that the GTP-form of Rho in the lysate is required for this binding. A mAb specific for the cytoplasmic domain of CD44 also remarkably suppressed this binding, identifying most of the binding partners for exogenous ERM proteins in the insoluble fraction as CD44. Consistent with this binding analysis, when living BHK cells were treated with C3 toxin, most insoluble ERM proteins moved to soluble compartments in the cytoplasm, leaving CD44 free from ERM.These findings indicate that Rho regulates the CD44/ERM complex formation in vivo and that the phosphatidylinositol turnover is possibly involved in this regulation mechanism. Less