Opresko L K, Chang C P, Will B H, Burke P M, Gill G N, Wiley H S
Department of Pathology, University of Utah School of Medicine, Salt Lake City 84132.
J Biol Chem. 1995 Mar 3;270(9):4325-33. doi: 10.1074/jbc.270.9.4325.
Ligand-induced internalization of the epidermal growth factor receptor (EGFR) leads to accelerated receptor degradation. Two models have been proposed to explain this. In the first model, induced internalization expands the intracellular pool of receptors, leading to enhanced lysosomal targeting. The second model proposes that activation of intrinsic receptor kinase activity induces inward vesiculation of endosomes, thus interrupting receptor recycling. To test these models, we created EGFR mutants that lack the conserved tyrosine kinase domain, but retain different parts of the distal carboxyl terminus regulatory region. Mutants lacking all distal regulatory sequences underwent slow internalization (0.02 min-1) and turnover (t1/2 approximately 24 h), similar to unoccupied, holo-EGFR. Mutant receptors that lacked the kinase domain, but retained the entire distal regulatory domain, were constitutively internalized and targeted to lysosomes, even in the absence of EGF. The turnover of these receptors (t1/2 approximately 11 h) was similar to that of occupied, kinase-active holo-EGFR (t1/2 approximately 9.5 h). These results show that receptor tyrosine kinase activity is not required for the targeting of EGFR to lysosomes. Receptor mutants which expressed previously identified endocytic sequences underwent rapid internalization. Unexpectedly, enhanced turnover of EGFR mutants required additional sequences located between residues 945 and 991 in the holo-EGFR. Thus, internalization and lysosomal targeting of EGFR are separate processes mediated by distinct sequences. Our results indicate that induced internalization is necessary, but not sufficient, for enhanced EGFR degradation. Instead, down-regulation requires exposure of previously cryptic internalization and lysosomal targeting sequences. Occupied EGFR thus appear to be handled by the endocytic machinery in the same fashion as other constitutively internalized or lysosomally targeted receptors.
配体诱导的表皮生长因子受体（EGFR）内化导致受体降解加速。已提出两种模型来解释这一现象。在第一个模型中，诱导内化扩大了细胞内受体池，导致溶酶体靶向增强。第二个模型提出，内在受体激酶活性的激活诱导内体向内囊泡化，从而中断受体再循环。为了验证这些模型，我们构建了缺乏保守酪氨酸激酶结构域但保留远端羧基末端调节区域不同部分的EGFR突变体。缺乏所有远端调节序列的突变体经历缓慢内化（0.02分钟-1）和周转（半衰期约24小时），类似于未占据的全EGFR。缺乏激酶结构域但保留整个远端调节结构域的突变受体即使在没有表皮生长因子（EGF）的情况下也会持续内化并靶向溶酶体。这些受体的周转（半衰期约11小时）与被占据的、激酶活性的全EGFR（半衰期约9.5小时）相似。这些结果表明，EGFR靶向溶酶体不需要受体酪氨酸激酶活性。表达先前鉴定的内吞序列的受体突变体经历快速内化。出乎意料的是，EGFR突变体周转增强需要全EGFR中位于945和991位残基之间的额外序列。因此，EGFR的内化和溶酶体靶向是由不同序列介导的独立过程。我们的结果表明，诱导内化对于增强EGFR降解是必要的，但不是充分的。相反，下调需要暴露先前隐蔽的内化和溶酶体靶向序列。因此，被占据的EGFR似乎以与其他持续内化或靶向溶酶体的受体相同的方式被内吞机制处理。
