Yanai S, Sugiyama Y, Kim D C, Iga T, Fuwa T, Hanano M
Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Am J Physiol. 1991 Mar;260(3 Pt 1):C457-67. doi: 10.1152/ajpcell.1991.260.3.C457.
The interaction of epidermal growth factor (EGF) with cell surface receptors and their subsequent endocytosis in isolated rat hepatocytes were analyzed by measuring changes in the concentrations of cell surface-bound, internalized, and degraded EGF. The kinetic model proposed by Wiley and Cunningham (Cell 25: 433-440, 1981) and Gex-Fabry and Delisi [Am. J. Physiol. 247 (Regulatory Integrative Comp. Physiol. 16): R768-R779, 1984] was basically utilized for the model analysis. The following kinetic parameters were obtained: association and dissociation rate constants for EGF-receptor interaction, internalization rate constant for EGF-receptor complex (kappa e), internalization rate constant for free receptor (kappa t), sequestration rate constant (kappa s) of the complex from shallow (exchangeable) to deep (nonexchangeable) membraneous compartment, intracellular degradation rate constant and initial cell-surface receptor density. The kappa s value, which was obtained by analyzing the time profiles of EGF association with cells, was approximately 5-10 times larger than the kappa e value determined by directly measuring internalized EGF with the acid-washing technique. This suggests the necessary presence of deep (nonexchanging) compartment of the complex in the plasma membrane. The calculated kappa e value is at least several times larger than the kappa t value, yielding the kinetic basis for the occurrence of receptor downregulation induced by excess EGF. We conclude that, in the overall receptor-mediated processing of EGF after bound to the cell surface receptors, the dissociation process is rapid [half-time (t1/2) less than 1 min], the degradation process is much slower (t1/2 approximately equal to 3 h), and the receptor internalization process is intermediate (t1/2 approximately equal to 6-7 min). In addition, two pools for EGF-receptor complex in the plasma membrane seem to be present, although their identification cannot be made.
通过测量细胞表面结合、内化和降解的表皮生长因子（EGF）浓度的变化，分析了EGF与分离的大鼠肝细胞表面受体的相互作用及其随后的内吞作用。基本采用了Wiley和Cunningham（《细胞》25: 433 - 440, 1981）以及Gex - Fabry和Delisi [《美国生理学杂志》247（调节整合与比较生理学16）: R768 - R779, 1984]提出的动力学模型进行模型分析。获得了以下动力学参数：EGF - 受体相互作用的结合和解离速率常数、EGF - 受体复合物的内化速率常数（κe）、游离受体的内化速率常数（κt）、复合物从浅（可交换）到深（不可交换）膜区室的隔离速率常数（κs）、细胞内降解速率常数和初始细胞表面受体密度。通过分析EGF与细胞结合的时间曲线获得的κs值，比用酸洗技术直接测量内化EGF所确定的κe值大约大5 - 10倍。这表明质膜中复合物存在深（不可交换）区室的必要性。计算得到的κe值至少比κt值大几倍，为过量EGF诱导受体下调的发生提供了动力学基础。我们得出结论，在EGF与细胞表面受体结合后受体介导的整个过程中，解离过程很快[半衰期（t1/2）小于1分钟]，降解过程慢得多（t1/2约等于3小时），受体内化过程处于中间水平（t1/2约等于6 - 7分钟）。此外，质膜中似乎存在两个EGF - 受体复合物池，尽管无法对它们进行鉴定。
