Regulation of epidermal growth factor receptor signaling by endocytosis and intracellular trafficking
Regulation of epidermal growth factor receptor signaling by endocytosis and intracellular trafficking
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DOI：
10.1091/mbc.12.6.1897
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发表时间：
2001-06-01
影响因子：
3.3
通讯作者：
Wiley, HS
中科院分区：
文献类型：
--
作者：
Burke, P;Schooler, K;Wiley, HS
表皮生长因子受体（EGFR）的配体活化导致其快速内化并最终递送至溶酶体。该过程被认为是减弱信号传导的机制，但信号可能在胞吞作用后产生。为了直接评估受体运输过程中的EGFR信号传导，我们开发了一种技术，使用可逆生物素化的抗EGFR抗体快速选择性分离内化的EGFR和相关分子。此外，我们开发了酪氨酸磷酸化EGFR特异性抗体。结合使用荧光成像和亲和沉淀的方法，我们评估了表皮生长因子受体的激活状态和底物协会在上皮细胞的运输。我们发现，在内化后，EGFR在早期内体中保持活性。然而，受体在降解前失活，显然是由于配体从内体中去除。衔接分子，如Shc，与EGFR在细胞表面和内体。一些分子，如Grb 2，主要与表面EGFR相关，而其他分子，如Eps 8，仅与细胞内受体相关。在失活阶段，c-Cbl与EGFR相关，与其在受体衰减中的假定作用一致。我们的结论是，协会的EGFR与不同的蛋白质是区室特异性。此外，配体丢失是EGFR失活的近端原因。因此，受管制的贩运可能会影响信号转导的模式和持续时间。
Ligand activation of the epidermal growth factor receptor (EGFR) leads to its rapid internalization and eventual delivery to lysosomes. This process is thought to be a mechanism to attenuate signaling, but signals could potentially be generated after endocytosis. To directly evaluate EGFR signaling during receptor trafficking, we developed a technique to rapidly and selectively isolate internalized EGFR and associated molecules with the use of reversibly biotinylated anti-EGFR antibodies. In addition, we developed antibodies specific to tyrosine-phosphorylated EGFR. With the use of a combination of fluorescence imaging and affinity precipitation approaches, we evaluated the state of EGFR activation and substrate association during trafficking in epithelial cells. We found that after internalization, EGFR remained active in the early endosomes. However, receptors were inactivated before degradation, apparently due to ligand removal from endosomes. Adapter molecules, such as Shc, were associated with EGFR both at the cell surface and within endosomes. Some molecules, such as Grb2, were primarily found associated with surface EGFR, whereas others, such as Eps8, were found only with intracellular receptors. During the inactivation phase, c-Cbl became EGFR associated, consistent with its postulated role in receptor attenuation. We conclude that the association of the EGFR with different proteins is compartment specific. In addition, ligand loss is the proximal cause of EGFR inactivation. Thus, regulated trafficking could potentially influence the pattern as well as the duration of signal transduction.