KINETICS OF BINDING, ENDOCYTOSIS, AND RECYCLING OF EGF RECEPTOR MUTANTS
KINETICS OF BINDING, ENDOCYTOSIS, AND RECYCLING OF EGF RECEPTOR MUTANTS
复制标题
DOI：
10.1083/jcb.117.1.203
复制
发表时间：
1992-04-01
影响因子：
7.8
通讯作者：
SCHLESSINGER, J
中科院分区：
文献类型：
--
作者：
FELDER, S;LAVIN, J;SCHLESSINGER, J
本报告分析了调节EGF与EGF受体结合、受体内化和受体再循环的因素。三种不同的方法被用来抑制高亲和力的EGF结合，在平衡状态下测量：用活性磷酯（PMA）处理细胞，结合针对EGF受体（mAb108）的单抗，截断受体的大部分细胞质区域。这些处理降低了低浓度EGF与细胞结合的速度，但不影响EGF解离的速度。我们得出结论，EGF与活细胞的高亲和力结合是由于EGF结合速率的明显差异。然后，我们使用这些条件和细胞系来测试不同浓度EGF下EGF的内化率。我们证明，在饱和浓度的EGF下，EGF受体的内化被刺激了大约50倍，但在低浓度（< 1 nM）下，被刺激了2到3倍。四种处理将低浓度EGF的内在化速率降低到EGF饱和浓度时的速率。phobol酯处理和mAb108与“野生型”受体的结合降低了这一比率（并降低了高亲和力结合）。Lys721（激酶阴性EGF受体）点突变和Thr654点突变（去除蛋白激酶C磷酸化的一个主要位点）降低了内化率，但不影响高亲和力结合。我们认为，虽然EGF刺激所有受体的内吞作用，但高亲和力受体比低亲和力受体结合和内化得更快。酪氨酸激酶活性和Thr654区域似乎是这种反应的必要条件。
This report describes analysis of factors which regulate the binding of EGF to EGF receptor, receptor internalization, and receptor recycling. Three different methods were used to inhibit high-affinity EGF binding as measured at equilibrium: treatment of cells with an active phorbol ester (PMA), binding of a mAb directed against the EGF receptor (mAb108), and truncation of most of the cytoplasmic domain of the receptor. These treatments reduced the rate at which low concentrations of EGF bound to cells, but did not affect the rate of EGF dissociation. We conclude that high-affinity EGF binding on living cells results from a difference in the apparent on rate of EGF binding. We then used these conditions and cell lines to test for the rate of EGF internalization at different concentrations of EGF We demonstrate that internalization of the EGF receptor is stimulated roughly 50-fold at saturating concentrations of EGF, but is stimulated an additional two- to threefold at low concentrations (< 1 nM). Four treatments reduce the rate of internalization of low concentrations of EGF to the rate seen at saturating EGF concentrations. Phorbol ester treatment and mAb108 binding to "wild type" receptor reduce this rate (and reduce high-affinity binding). Point mutation at Lys721 (kinase negative EGF receptor) and point mutation at Thr654 (removing a major site of protein kinase C phosphorylation) reduce the internalization rate, without affecting high-affinity binding. We suggest that while EGF stimulates endocytosis for all receptors, high-affinity receptors bind and are internalized more quickly than low-affinity receptors. Tyrosine kinase activity and the Thr654 region appear necessary for this response.