BINDING OF AN ANTAGONISTIC MONOCLONAL-ANTIBODY TO AN INTACT AND FRAGMENTED EGF-RECEPTOR POLYPEPTIDE
BINDING OF AN ANTAGONISTIC MONOCLONAL-ANTIBODY TO AN INTACT AND FRAGMENTED EGF-RECEPTOR POLYPEPTIDE
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DOI：
10.1016/0003-9861(87)90062-2
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发表时间：
1987-02-01
影响因子：
3.9
通讯作者：
DAS, M
中科院分区：
文献类型：
--
作者：
MURTHY, U;BASU, A;DAS, M
一种针对人A431癌细胞的鼠单抗(425号)特异性地免疫沉淀A431细胞提取物以及人胎盘和培养的成纤维细胞提取物中的17万分子量表皮生长因子(EGF)受体，但不识别该小鼠受体。通过固定的非通透性细胞的间接免疫荧光染色，表明与人EGF受体的外区结合。该抗体与糖基和糖基受体结合，表明该表位是多肽链的一部分。抗体与受体的结合是构象依赖的，即缺乏EGF结合活性的变性受体不被抗体识别。抗体结合研究的结果表明，该表位与EGF结合活性部位紧密相连，在高亲和力和低亲和力的EGF受体中都是共同的。该表位与抗体的相互作用抑制了EGF的结合和生物活性，并触发了受体下调，但在体外或体内都不能产生EGF样的激酶刺激或有丝分裂反应。该抗体被测试其与170 kDa EGF受体的结构域大小片段结合的能力。它既能识别蛋白水解物产生的110 kDa EGF结合肽，又能识别A431细胞分泌的可溶性100 kDa EGF受体。这表明，在蛋白水解性将EGF结合域从受体分子的其余部分分离后，识别该抗体的表位保持其构象。
A murine monoclonal antibody (No. 425) raised against human A431 carcinoma cells specifically immunoprecipitates the 170,000 molecular weight epidermal growth factor (EGF)-receptor from extracts of A431 cells as well as from extracts of human placenta and cultured fibroblasts, but does not recognize the murine receptor. Binding to the external domain of the human EGF-receptor was indicated by indirect immunofluorescent staining of fixed nonpermeable cells. The antibody binds to both glyco- and aglycoreceptor forms, indicating that the epitope is a part of the polypeptide chain. Binding of the antibody to the receptor is conformation dependent; i.e., denatured receptors lacking EGF-binding activity are not recognized by the antibody. The results of antibody binding studies indicate that the epitope is closely linked to the EGF binding active site, and is common to both high- and low-affinity EGF-receptors. Interaction of this epitope with the antibody inhibits EGF binding and bioactivity, and triggers receptor down-regulation, but does not generate EGFlike kinase-stimulatory or mitogenic responses either or in vitro or in vivo. The antibody was tested for its ability to bind to domain-sized fragments of the 170-kDa EGF-receptor. It can recognize both the proteolytic generated 110-kDa EGF binding peptide, and a soluble 100-kDa EGF-receptor secreted by A431 cells. This indicates that the epitope recognized this antibody retains its conformation after proteolytic separation of the EGF binding domain from the rest of the receptor molecule.