Wu D G, Wang L H, Sato G H, West K A, Harris W R, Crabb J W, Sato J D
W. Alton Jones Cell Science Center, Inc., Lake Placid, New York 12946.
J Biol Chem. 1989 Oct 15;264(29):17469-75.
Epitopes recognized by three epidermal growth factor (EGF) competitive monoclonal antibodies, LA22, LA58, and LA90, have been localized to a 14-amino acid region in the extracellular domain of the human EGF receptor. The binding of each of these mutually competitive antibodies to A431 epidermoid carcinoma cells was inhibited up to 87% by EGF. Furthermore, binding to A431 cells was inhibited 100% by the EGF competitive monoclonal antibody 528 IgG. The EGF receptor monoclonal antibody 455 IgG, which recognizes a blood group A-related carbohydrate modification of A431 receptors and does not inhibit EGF binding, did not inhibit the binding of these three antibodies to A431 cells. Antibodies LA22, LA58, and LA90 were unusual in that they bound to recognized denatured and endoglycosidase F-treated antigenic determinants in Western blots. This suggested that the antibodies recognized continuous peptide epitopes. The epitopes for these antibodies were first localized in cyanogen bromide- and V8 protease-generated fragments of a truncated form of the EGF receptor secreted by A431 cells. In experiments with synthetic peptides, all three antibodies were found to bind to the 14 amino acids from Ala-351 to Asp-364 of the mature human EGF receptor. These amino acids are located between the two Cys-rich regions of the extracellular domain of the receptor, and they include an Arg-Gly-Asp-Ser recognition site for adhesion molecule receptors. The homologous sequence in the chicken EGF receptor, which binds mouse EGF with a 100-fold lower affinity than the human EGF receptor, contains four amino acid differences including two in the Arg-Gly-Asp-Ser tetramer. The mutually competitive binding of EGF and antibodies LA22, LA58, and LA90 implied that the amino acids between Ala-351 and Asp-364 participated in the formation of the EGF-binding site of the human EGF receptor.
三种表皮生长因子（EGF）竞争性单克隆抗体LA22、LA58和LA90所识别的表位，已定位到人EGF受体胞外域的一个14个氨基酸的区域。这些相互竞争的抗体与A431表皮样癌细胞的结合，被EGF抑制高达87%。此外，EGF竞争性单克隆抗体528 IgG可100%抑制与A431细胞的结合。识别A431受体上与血型A相关碳水化合物修饰且不抑制EGF结合的EGF受体单克隆抗体455 IgG，不抑制这三种抗体与A431细胞的结合。抗体LA22、LA58和LA90不同寻常之处在于，它们在蛋白质印迹中能与已识别的变性和经内切糖苷酶F处理的抗原决定簇结合。这表明这些抗体识别连续的肽表位。这些抗体的表位最初定位在A431细胞分泌的截短形式的EGF受体经溴化氰和V8蛋白酶产生的片段中。在合成肽实验中，发现所有三种抗体都能与成熟人EGF受体从Ala-351到Asp-364的14个氨基酸结合。这些氨基酸位于受体胞外域的两个富含半胱氨酸区域之间，且包含一个黏附分子受体的Arg-Gly-Asp-Ser识别位点。鸡EGF受体中的同源序列，与小鼠EGF的结合亲和力比人EGF受体低100倍，包含四个氨基酸差异，其中包括Arg-Gly-Asp-Ser四聚体中的两个差异。EGF与抗体LA22、LA58和LA90的相互竞争性结合表明，Ala-351和Asp-364之间的氨基酸参与了人EGF受体EGF结合位点的形成。
