Woltjer R L, Lukas T J, Staros J V
Department of Biochemistry, Vanderbilt University, Nashville, TN 37235.
Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7801-5. doi: 10.1073/pnas.89.16.7801.
We have recently developed a kinetically controlled, step-wise affinity cross-linking technique for specific, high-yield, covalent linkage of murine epidermal growth factor (mEGF) via its N terminus to the EGF receptor. EGF receptor from A431 cells was cross-linked to radiolabeled mEGF (125I-mEGF) by this technique and the 125I-mEGF-receptor complex was purified and denatured. Tryptic digestion of this preparation gave rise to a unique radiolabeled peptide that did not comigrate with trypsin-treated 125I-mEGF in SDS/Tricine gels but that could be immunoprecipitated with antibodies to mEGF. The immunoprecipitated peptide was isolated by electrophoresis in SDS/Tricine gels, eluted, and sequenced. The sequence was found to correspond to that of a tryptic peptide of the EGF receptor beginning with Gly-85, which is in domain I, a region N terminal to the first cysteine-rich region of the receptor. Selective loss of signal in the 17th sequencing cycle suggests that the point of attachment of N-terminally modified 125I-mEGF to the receptor is Tyr-101. The data presented here provide identification by direct protein microsequencing of a site of interaction of EGF and the EGF receptor.
我们最近开发了一种动力学控制的逐步亲和交联技术，用于将小鼠表皮生长因子（mEGF）通过其N端与EGF受体进行特异性、高产率的共价连接。通过该技术将A431细胞中的EGF受体与放射性标记的mEGF（125I-mEGF）交联，然后纯化并变性125I-mEGF-受体复合物。对该制剂进行胰蛋白酶消化后产生了一种独特的放射性标记肽，在SDS/三羟甲基甘氨酸凝胶中，它与经胰蛋白酶处理的125I-mEGF迁移率不同，但可以用抗mEGF抗体进行免疫沉淀。通过在SDS/三羟甲基甘氨酸凝胶中电泳分离、洗脱并测序免疫沉淀的肽。发现该序列与EGF受体的一种胰蛋白酶肽的序列相对应，起始于Gly-85，位于结构域I，该区域在受体第一个富含半胱氨酸区域的N端。第17个测序循环中信号的选择性丢失表明，N端修饰的125I-mEGF与受体的连接点是Tyr-101。本文提供的数据通过直接蛋白质微测序鉴定了EGF与EGF受体的相互作用位点。
