Beguinot L, Werth D, Ito S, Richert N, Willingham M C, Pastan I
J Biol Chem. 1986 Feb 5;261(4):1801-7.
An antibody against the human epidermal growth factor receptor (EGF), capable of activating its tyrosine kinase has been produced. Antibody 2913 recognizes only the cytoplasmic portion of the EGF receptor in A431 carcinoma cells, in normal human fibroblasts, and in a variety of other human tumor cell lines (Xu, Y.-A., Richert, N., Ito, S., Merlino, G. T., and Pastan, I. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 7308-7313). Indirect immunofluorescence and electron microscopy show that the antibody binds to intact cells only after membrane permeabilization. Moreover the antibody immunoprecipitates the v-erb-B gene product in avian myeloblastosis virus-infected cells but does not recognize the secreted form (105 kDa) of the A431 cell EGF receptor which lacks the cytoplasmic domain. Antibody 2913 activates the EGF receptor kinase in solubilized A431 membranes causing autophosphorylation on tyrosine residues only. Tryptic peptide maps suggest that antibody 2913 and EGF stimulate phosphorylation of the same amino acid residues. By electron microscopy, the cytoplasmic portion of the receptor was followed throughout its endocytotic pathway. The results show that the kinase domain is rapidly degraded in lysosomes with no accumulation in the cytoplasm or in the nucleus.
已制备出一种针对人表皮生长因子受体（EGF）的抗体，它能够激活其酪氨酸激酶。抗体2913仅识别A431癌细胞、正常人成纤维细胞以及多种其他人类肿瘤细胞系中EGF受体的胞质部分（徐，Y.-A.，里希特，N.，伊藤，S.，梅林诺，G. T.，和帕斯坦，I.（1984年）美国国家科学院院刊81，7308 - 7313）。间接免疫荧光和电子显微镜显示，该抗体仅在细胞膜通透后才与完整细胞结合。此外，该抗体能免疫沉淀禽成髓细胞瘤病毒感染细胞中的v-erb-B基因产物，但不识别缺乏胞质结构域的A431细胞EGF受体的分泌形式（105 kDa）。抗体2913能激活溶解的A431细胞膜中的EGF受体激酶，仅导致酪氨酸残基的自磷酸化。胰蛋白酶肽图谱表明，抗体2913和EGF刺激相同氨基酸残基的磷酸化。通过电子显微镜，追踪了受体的胞质部分在其整个内吞途径中的情况。结果表明，激酶结构域在溶酶体中迅速降解，在细胞质或细胞核中无积累。
