Srinivas U, Tagliabue E, Campiglio M, Ménard S, Colnaghi M I
Division of Experimental Oncology E, Istituto Nazionale Tumori, Milan, Italy.
Cancer Immunol Immunother. 1993 Jun;36(6):397-402. doi: 10.1007/BF01742256.
In the present study we utilized two previously described monoclonal antibodies (mAb), and their respective Fab portions, directed against the extracellular domain of p185HER2, a transmembrane glycoprotein with intrinsic tyrosine kinase activity coded by the HER2/neu oncogene, to study the mechanism of mAb-induced receptor internalization and phosphorylation. Fluorescence scan analysis and direct binding of radiolabelled mAb and their Fab fragments showed that entire MGR2 and MGR3 mAb were reactive with similar binding affinity on two cell lines (Calu-3 and Sk-Br-3) overexpressing the p185HER2 receptor, and unreactive on unrelated cells. The corresponding Fab fragments were positive on the related cells, but bound with diminished intensity and affinity. Entire MGR2 and MGR3 induced internalization in both Calu-3 and Sk-Br-3 cells, whereas their Fab portions were not internalized. When the bivalency of the MGR2 Fab fragment was artificially reconstituted by incubation with rabbit anti-(mouse IgG), internalization was obtained. Monovalent binding of the entire labelled antibodies, obtained in the presence of a saturating amont of unlabelled antibody, decreased both the rate and the final amount of internalized antibody. Metabolic labelling and immunoblotting experiments showed that incubation with entire MGR3 amplified the basal phosphorylation of the p185HER2 receptor in Calu-3 and Sk-Br-3 cells, whereas MGR3 Fab decreased the signal. Taken together, our data indicate that antibody-mediated activation of p185HER2 in Calu-3 and Sk-Br-3 cells occurs through the dimerization of receptor molecules and that bivalency of the activating antibody is mandatory for induction of internalization and phosphorylation of the receptor. Our data support an allosteric model of activation for the p185HER2 receptor.
在本研究中，我们使用了两种先前描述的单克隆抗体（mAb）及其各自的Fab片段，它们针对p185HER2的细胞外结构域，p185HER2是一种具有内在酪氨酸激酶活性的跨膜糖蛋白，由HER2/neu癌基因编码，以研究mAb诱导的受体内化和磷酸化机制。荧光扫描分析以及放射性标记的mAb及其Fab片段的直接结合表明，完整的MGR2和MGR3 mAb在两种过表达p185HER2受体的细胞系（Calu-3和Sk-Br-3）上具有相似的结合亲和力且有反应性，而在不相关的细胞上无反应性。相应的Fab片段在相关细胞上呈阳性，但结合强度和亲和力降低。完整的MGR2和MGR3均在Calu-3和Sk-Br-3细胞中诱导内化，而它们的Fab部分未被内化。当通过与兔抗（小鼠IgG）孵育人工重建MGR2 Fab片段的二价性时，可实现内化。在存在饱和量未标记抗体的情况下获得的完整标记抗体的单价结合，降低了内化抗体的速率和最终量。代谢标记和免疫印迹实验表明，用完整的MGR3孵育可增强Calu-3和Sk-Br-3细胞中p185HER2受体的基础磷酸化，而MGR3 Fab则降低了信号。综上所述，我们的数据表明，在Calu-3和Sk-Br-3细胞中，抗体介导的p185HER2激活是通过受体分子的二聚化发生的，并且激活抗体的二价性对于诱导受体的内化和磷酸化是必不可少的。我们的数据支持p185HER2受体的变构激活模型。
