Sorkin A, Waters C, Overholser K A, Carpenter G
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
J Biol Chem. 1991 May 5;266(13):8355-62.
We have utilized site-directed mutants to study the role of autophosphorylation of the epidermal growth factor (EGF) receptor in the regulation of receptor kinase activity and ligand-induced endocytosis. A single mutation of the major autophosphorylation site, Y1173, and a double mutation of two autophosphorylation sites, Y1173 and Y1148, did not inhibit kinase activity in vivo, using PLC gamma 1 as a specific substrate for the EGF receptor kinase. The simultaneous mutation of three major autophosphorylation sites (Y1173, Y1148, Y1068), however, caused more than a 50% decrease in EGF-induced tyrosine phosphorylation of PLC gamma 1. The triple mutation also resulted in a substantial inhibition of the EGF-receptor endocytic system. We have used three types of experiments to analyze internalization, recycling, and degradation of EGF in cells with these mutants or the wild-type receptor. Using a simple mathematical model we have shown that the internalization rate constant is 2-fold lower in cells expressing the triple mutation receptor (F3 cells) than in cells expressing wild-type EGF receptor (wild-type cells). However, the rate constant for recycling was similar in both cell types. The EGF degradation rate constant was also lower in F3 cells. EGF-induced EGF receptor degradation was slower in F3 cells (t1/2 = 4 h) than in wild-type cells (t1/2 = 1 h). Therefore, our results suggest that multiple autophosphorylations of the carboxyl terminus of the EGF receptor are required for EGF receptor kinase activation, and for the internalization and intracellular processing of the EGF.receptor complex.
我们利用定点突变体研究表皮生长因子（EGF）受体自身磷酸化在调节受体激酶活性和配体诱导的内吞作用中的作用。以PLCγ1作为EGF受体激酶的特异性底物，主要自身磷酸化位点Y1173的单点突变以及两个自身磷酸化位点Y1173和Y1148的双点突变在体内并未抑制激酶活性。然而，三个主要自身磷酸化位点（Y1173、Y1148、Y1068）的同时突变导致EGF诱导的PLCγ1酪氨酸磷酸化降低了50%以上。三重突变还导致EGF受体内吞系统受到显著抑制。我们使用了三种类型的实验来分析携带这些突变体或野生型受体的细胞中EGF的内化、再循环和降解。使用一个简单的数学模型，我们发现表达三重突变受体的细胞（F3细胞）的内化速率常数比表达野生型EGF受体的细胞（野生型细胞）低2倍。然而，两种细胞类型的再循环速率常数相似。F3细胞中的EGF降解速率常数也较低。EGF诱导的EGF受体降解在F3细胞中（t1/2 = 4小时）比在野生型细胞中（t1/2 = 1小时）更慢。因此，我们的结果表明，EGF受体羧基末端的多次自身磷酸化对于EGF受体激酶激活以及EGF-受体复合物的内化和细胞内加工是必需的。
