Selectivity of commonly used inhibitors of clathrin-mediated and caveolae-dependent endocytosis of G protein-coupled receptors
Selectivity of commonly used inhibitors of clathrin-mediated and caveolae-dependent endocytosis of G protein-coupled receptors
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DOI：
10.1016/j.bbamem.2015.05.024
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发表时间：
2015-10-01
影响因子：
3.4
通讯作者：
Kim, Kyeong-Man
中科院分区：
文献类型：
--
作者：
Guo, Shuohan;Zhang, Xiaohan;Kim, Kyeong-Man
在多种G蛋白偶联受体(GPCRs)的内吞途径中，笼蛋白介导的内吞作用(CME)和空泡内吞作用比其他内吞途径具有更广泛的特征。许多内吞抑制物已被用于阻断CME；然而，需要进行系统研究以确定这些抑制物的选择性。网织蛋白重链或小窝蛋白1敲除细胞已被用来确定各种化学和分子生物学工具对CME和小窝内吞作用的特异性。除CME外，蔗糖、刀豆蛋白A和动力蛋白的显性负性突变体还阻断了其他内吞途径。特别是，刀豆蛋白A非特异性地干扰了研究中测试的几个GPCR的信号。与其他处理相比，降低pH、单丹尼西林和显性负突变体对CME具有更高的特异性。最近推出的CME抑制剂Pitstop2(TM)对CME只有边际选择性，并干扰细胞表面受体的表达。表观蛋白突变体阻断受体的内吞作用和敲除蛋白重链可增强MR介导的ERK激活。总体而言，我们的研究表明，如果之前的实验结果包括使用以前被认为是CME选择性的内吞抑制药，那么应该谨慎地解释这些结果。此外，我们的研究表明，通过网状蛋白介导的132肾上腺素受体内吞作用对ERK的激活有负面影响。(C)2015爱思唯尔B.V.保留所有权利。
Among the multiple G protein-coupled receptor (GPCR) endocytic pathways, clathrin-mediated endocytosis (CME) and caveolar endocytosis are more extensively characterized than other endocytic pathways. A number of endocytic inhibitors have been used to block CME; however, systemic studies to determine the selectivity of these inhibitors are needed. Clathrin heavy chain or caveolin1-knockdown cells have been employed to determine the specificity of various chemical and molecular biological tools for CME and caveolar endocytosis. Sucrose, concanavalin A, and dominant negative mutants of dynamin blocked other endocytic pathways, in addition to CME. In particular, concanavalin A nonspecifically interfered with the signaling of several GPCRs tested in the study. Decreased pH, monodansylcadaverine, and dominant negative mutants of epsin were more specific for CME than other treatments were. A recently introduced CME inhibitor, Pitstop2 (TM), showed only marginal selectivity for CME and interfered with receptor expression on the cell surface. Blockade of receptor endocytosis by epsin mutants and knockdown of the clathrin heavy chain enhanced the MR-mediated ERK activation. Overall, our studies show that previous experimental results should be interpreted with discretion if they included the use of endocytic inhibitors that were previously thought to be CME-selective. In addition, our study shows that endocytosis of 132 adrenoceptor through clathrin-mediated pathway has negative effects on ERK activation. (C) 2015 Elsevier B.V. All rights reserved.