Direct identification of residues of the epidermal growth factor receptor in close proximity to the amino terminus of bound epidermal growth factor.
Direct identification of residues of the epidermal growth factor receptor in close proximity to the amino terminus of bound epidermal growth factor.
复制标题
直接鉴定紧邻结合的表皮生长因子的氨基末端的表皮生长因子受体的残基。
DOI：
10.1073/pnas.89.16.7801
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发表时间：
1992
影响因子：
11.1
通讯作者：
Staros,JV
中科院分区：
文献类型：
--
作者：
Woltjer,RL;Lukas,TJ;Staros,JV
我们最近开发了一种动力学控制的逐步亲和交联技术，通过其N端与EGF受体特异性地、高产量地共价连接小鼠表皮生长因子(MEGF)。利用该技术将A431细胞的EGF受体与放射性标记的mEGF(125I-mEGF)进行交联，并对125I-mEGF-受体复合体进行纯化和变性。该制剂经胰酶消化后，在SDS/Tricine凝胶中不与经胰酶处理的125I-mEGF结合，但可与mEGF抗体免疫共沉淀。免疫沉淀肽经SDS/Tricine凝胶电泳法分离、洗脱、测序。该序列对应于EGF受体以Gly-85开头的胰酶解肽的序列，该序列位于受体第一个富含半胱氨酸的区域N端的结构域I。第17个测序周期信号的选择性丢失表明，N末端修饰的125I-mEGF与受体的结合点是Tyr-101。本文提供的数据提供了通过直接蛋白质微测序鉴定EGF和EGF受体相互作用的位置。
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.