McLean Sarah, Di Guglielmo Gianni M
Department of Anatomy and Cell Biology, Western University, London, Ontario, Canada; Department of Physiology and Pharmacology, Western University, London, Ontario, Canada.
Department of Physiology and Pharmacology, Western University, London, Ontario, Canada.
Methods Enzymol. 2014;535:39-54. doi: 10.1016/B978-0-12-397925-4.00003-1.
The transforming growth factor beta (TGFβ) signaling pathway is important for normal cell homeostasis and has critical roles in apoptosis, cell-cycle arrest, and cellular differentiation (reviewed in Massague, 2008). In the classical TGFβ pathway, the endosomal trafficking of receptors has a direct outcome on signal transduction-receptors internalized via clathrin-mediated endocytosis enter the early endosome and propagate signaling, while those internalized via membrane rafts are targeted for degradation. Recently, there have been a number of articles that have identified TGFβ receptor-binding proteins that direct receptor endocytosis and/or intracellular trafficking and affect signal output (Atfi et al., 2007; Bauge, Girard, Leclercq, Galera, & Boumediene, 2012; Bizet et al., 2011, 2012; Chen et al., 2007; Gunaratne, Benchabane, & Di Guglielmo, 2012; Hao et al., 2011; McLean, Bhattacharya, & Di Guglielmo, 2013; Zhao et al., 2012). Given the importance of TGFβ receptor trafficking to signaling outcome, this chapter will focus on strategies to isolate membrane rafts and techniques to follow the trafficking of cell-surface TGFβ receptors and provide examples of functional readouts to assess TGFβ signal transduction.
转化生长因子β（TGFβ）信号通路对正常细胞稳态至关重要，在细胞凋亡、细胞周期停滞和细胞分化中发挥关键作用（Massague，2008年综述）。在经典的TGFβ通路中，受体的内体运输对信号转导有直接影响——通过网格蛋白介导的内吞作用内化的受体进入早期内体并传播信号，而通过膜筏内化的受体则被靶向降解。最近，有许多文章鉴定出了TGFβ受体结合蛋白，这些蛋白指导受体内吞作用和/或细胞内运输并影响信号输出（Atfi等人，2007年；Bauge、Girard、Leclercq、Galera和Boumediene，2012年；Bizet等人，2011年、2012年；Chen等人，2007年；Gunaratne、Benchabane和Di Guglielmo，2012年；Hao等人，2011年；McLean、Bhattacharya和Di Guglielmo，2013年；Zhao等人，2日）。鉴于TGFβ受体运输对信号转导结果的重要性，本章将重点介绍分离膜筏的策略、追踪细胞表面TGFβ受体运输的技术，并提供评估TGFβ信号转导的功能读数示例。
