Activity dependent internalization of the glutamate transporter GLT-1 mediated by β-arrestin 1 and ubiquitination
Activity dependent internalization of the glutamate transporter GLT-1 mediated by β-arrestin 1 and ubiquitination
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DOI：
10.1016/j.neuropharm.2016.03.042
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发表时间：
2016-08-01
影响因子：
4.7
通讯作者：
Zafra, Francisco
中科院分区：
文献类型：
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作者：
Ibanez, Ignacio;Javier Diez-Guerra, F.;Zafra, Francisco
GLT-1是脑中主要的谷氨酸转运蛋白，并经历控制其在细胞表面上的浓度的运输过程，从而形成谷氨酸能神经传递。我们已经研究了GLT-1的交通是如何通过转运蛋白活性调节的。我们报告说，从细胞表面的GLT-1的内化是由可转运的底物，如谷氨酸或天冬氨酸，以及由可转运的抑制剂L-反式-2，4-PDC加速，但不是由非底物抑制剂WAY 213613在原代混合培养物和瞬时转染HEK 293细胞。对HEK 293细胞内吞作用机制的分析表明，谷氨酸促进了与衔接蛋白β-抑制蛋白和泛素连接酶Nedd 4 -2的转运蛋白的结合。谷氨酸的加入伴随着转运蛋白泛素化的增加，并且内化被泛素化抑制剂（PYR 41）抑制，并且在C-末端赖氨酸缺陷的突变体中。谷氨酸触发的内吞作用也被β-抑制蛋白的siRNA抑制。这种调节机制可能与在缺血或创伤性脑损伤等条件下控制细胞表面转运蛋白的量有关，其中细胞外谷氨酸浓度持续升高。(C)2016爱思唯尔有限公司版权所有
GLT-1 is the main glutamate transporter in the brain and undergoes trafficking processes that control its concentration on the cell surface thereby shaping glutamatergic neurotransmission. We have investigated how the traffic of GLT-1 is regulated by transporter activity. We report that internalization of GLT-1 from the cell surface is accelerated by transportable substrates like glutamate or aspartate, as well as by the transportable inhibitor L-trans-2,4-PDC, but not by the non-substrate inhibitor WAY 213613 in primary mixed cultures and in transiently transfected HEK293 cells. Analysis of the mechanism of endocytosis in HEK293 cells revealed that glutamate promoted the association with the transporter of the adaptor protein beta-arrestin and the ubiquitin ligase Nedd4-2. The addition of glutamate is accompanied by an increase in the transporter ubiquitination, and the internalization is suppressed by an ubiquitination inhibitor (PYR41), and in a mutant defective in C-terminal lysines. The glutamate triggered endocytosis was also suppressed by siRNA for beta-arrestin. This regulatory mechanism might be relevant in controlling the amount of transporter on the cell surface in conditions such as ischemia or traumatic brain injury, where extracellular concentrations of glutamate are persistently elevated. (C) 2016 Elsevier Ltd. All rights reserved.