Castagnola J, MacLeod C, Sunada H, Mendelsohn J, Taetle R
Department of Medicine, University of California, San Diego 92103.
J Cell Physiol. 1987 Sep;132(3):492-500. doi: 10.1002/jcp.1041320311.
The transferrin (Tf) receptor is a major transmembrane protein which provides iron for normal and malignant cell growth. Epidermal growth factor (EGF) has been reported to rapidly and transiently alter the number of surface Tf receptors in normal and transformed epithelial cells. To investigate mechanisms of EGF-induced changes in surface Tf display, EGF effects on surface Tf receptors were compared in two cell lines which differ in their number of EGF receptors and growth responses to EGF. In cloned A431 cells with high receptor numbers which are growth-inhibited by EGF, EGF caused a 50% decrease in Tf receptor expression after 30 min. In contrast, EGF induced a rapid, transitory increase (within 5 min) in the number of surface Tf receptors on KB carcinoma cells which returned to basal levels by 15 min. The observed changes in Tf receptor display were due to altered receptor distribution and not changes in ligand affinity or total cellular transferrin receptor pools. Anti-EGF receptor monoclonal antibody blocked effects of EGF on transferrin receptor expression. Since the antibody is internalized and causes EGF receptor down-regulation, effects on transferrin receptor expression were independent of these events. EGF-induced alterations in Tf receptor display occurred even when cells were pretreated with colchicine, suggesting that changes in surface Tf binding were not mediated by cytoskeletal components. Na orthovanadate, which mimics some early cellular effects of EGF, duplicated EGF's effects on A431 Tf receptors, but had no effect on KB cells, suggesting these responses occur by differing mechanisms. To determine whether EGF caused changes in Tf receptor phosphorylation, 32P-labelled Tf receptors were immunoprecipitated after EGF treatment. After exposure to EGF, A431 cells showed no change in Tf phosphorylation, but KB cells showed a transient, 6-fold increase in transferrin receptor phosphorylation on serine residues. In both A431 and KB cells, phorbol ester (PMA) also increased phosphorylation on transferrin receptors, but had little effect on surface Tf receptor expression. In malignant cell lines, EGE induces rapid, variable changes in transferrin receptor expression and phosphorylation which differ from the effects of PMA. These early responses to EGF appear to differ with the cell type and correlate poorly with alterations in Tf receptor phosphorylation. These results suggest Tf receptor phosphorylation does not regulate Tf receptor display in all cells.
转铁蛋白（Tf）受体是一种主要的跨膜蛋白，为正常细胞和恶性细胞的生长提供铁。据报道，表皮生长因子（EGF）可快速、短暂地改变正常上皮细胞和转化上皮细胞表面Tf受体的数量。为了研究EGF诱导的表面Tf展示变化的机制，在两种细胞系中比较了EGF对表面Tf受体的影响，这两种细胞系在EGF受体数量和对EGF的生长反应方面存在差异。在具有高受体数量且被EGF抑制生长的克隆A431细胞中，EGF在30分钟后导致Tf受体表达下降50%。相比之下，EGF在KB癌细胞上诱导表面Tf受体数量迅速、短暂增加（5分钟内），并在15分钟时恢复到基础水平。观察到的Tf受体展示变化是由于受体分布改变，而非配体亲和力或总细胞转铁蛋白受体库的变化。抗EGF受体单克隆抗体阻断了EGF对转铁蛋白受体表达的影响。由于该抗体被内化并导致EGF受体下调，因此对转铁蛋白受体表达的影响与这些事件无关。即使细胞先用秋水仙碱预处理，EGF诱导的Tf受体展示变化仍会发生，这表明表面Tf结合的变化不是由细胞骨架成分介导的。正钒酸钠模拟了EGF的一些早期细胞效应，重复了EGF对A431 Tf受体的作用，但对KB细胞没有影响，这表明这些反应通过不同机制发生。为了确定EGF是否导致Tf受体磷酸化的变化，在EGF处理后对32P标记的Tf受体进行免疫沉淀。暴露于EGF后，A431细胞的Tf磷酸化没有变化，但KB细胞的转铁蛋白受体丝氨酸残基磷酸化短暂增加了6倍。在A431和KB细胞中，佛波酯（PMA）也增加了转铁蛋白受体的磷酸化，但对表面Tf受体表达影响很小。在恶性细胞系中，EGE诱导转铁蛋白受体表达和磷酸化的快速、可变变化，这与PMA的作用不同。这些对EGF的早期反应似乎因细胞类型而异，与Tf受体磷酸化的改变相关性较差。这些结果表明，Tf受体磷酸化并非在所有细胞中都调节Tf受体展示。
