Methods to study endocytic trafficking of the EGF receptor.
Methods to study endocytic trafficking of the EGF receptor.
复制标题
研究EGF受体的内吞运输的方法。
DOI：
10.1016/bs.mcb.2015.05.008
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发表时间：
2015
影响因子：
--
通讯作者：
Sorkin A
中科院分区：
文献类型：
--
作者：
Pinilla-Macua I;Sorkin A
表皮生长因子受体(EGFR)的内吞和胞后分选是EGFR信号转导的主要调节因子。EGFR内吞作用和泛素依赖的溶酶体靶向也被认为是研究刺激诱导和结构性内吞转运分子机制的典型实验系统。因此，阐明EGFR内吞作用的机制及其对信号网络的调控，不仅对于更好地理解EGFR生物学，而且对于确定内吞作用系统中的一般调控原则都是至关重要的。对这些机制的全面分析需要定量的和生理上相关的方法学方法来衡量EGFR的内化、降解和再循环的速度。本章介绍的基本实验方案包括单细胞显微镜和生化方法的结合，用于实时跟踪EGF诱导的EGFR内吞作用，测量EGFR内化和循环的动力学速率参数，并分析EGF依赖的泛素化和EGFR的降解。
Endocytosis and postendocytic sorting of epidermal growth factor (EGF) receptor (EGFR) are the major regulators of EGFR signaling. EGFR endocytosis and ubiquitin-dependent lysosomal targeting are also considered to be the prototypic experimental system for studying the molecular mechanisms of stimulus-induced and constitutive endocytic trafficking. Therefore, elucidation of the mechanisms of EGFR endocytosis and its regulation of the signaling network is essential not only for better understanding of the EGFR biology but also for defining general regulatory principles in the endocytosis system. Comprehensive analysis of these mechanisms requires quantitative and physiologically relevant methodological approaches for measuring the rates of EGFR internalization, degradation, and recycling. Basic experimental protocols described in this chapter cover a combination of single-cell microscopy and biochemical methods that are used to follow EGF-induced endocytosis of EGFR in real time, measure the kinetic rate parameters of EGFR internalization and recycling, and analyze EGF-dependent ubiquitination and degradation of EGFR.