Role of C-terminal region in the functional regulation of rat serotonin transporter (SERT)
Role of C-terminal region in the functional regulation of rat serotonin transporter (SERT)
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DOI：
10.1016/j.neuint.2004.08.008
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发表时间：
2005-01-01
影响因子：
4.2
通讯作者：
Sakai, N
中科院分区：
文献类型：
--
作者：
Mochizuki, H;Amano, T;Sakai, N
以前，我们发现，肌动蛋白细胞骨架的状态影响5-羟色胺转运蛋白（SERT）的摄取活性。最近，有报道称SERT的C-末端与MacMARCKS相互作用，MacMARCKS是PKC的底物，可以与肌动蛋白细胞骨架结合。为了阐明C-末端区域在调节SERT活性和与肌动蛋白细胞骨架相互作用中的重要性，我们研究了C-末端的过表达是否影响SERT的转运活性。为此，我们在稳定表达FLAG标记的SERT的HEK 293细胞（FL-SERT-HEK 293细胞）中过表达SERT C末端的GFP融合的30个氨基酸构建体（GFP-SERT-CT）。与表达GFP的FL-SERT-HEK 293细胞相比，表达GFP-SERT-CT的FL-SERT-HEK 293细胞中的SERT摄取活性和转运体电流减弱。Eadie-Hofstee分析显示GFP-SERT-CT过表达通过降低V-max而减弱SERT摄取活性，但不改变K-m，这与使用生物素化/免疫印迹分析对SET的细胞表面表达的实验结果一致。免疫细胞化学分析表明GFP-SERT-CT与FLAG-SERT和皮质肌动蛋白共定位于质膜上。此外，SERT C-末端不影响多巴胺转运蛋白的活性。这些发现表明C端区域对SERT的功能调节具有重要意义，表明GFP-SERT-CT作为分子诱饵破坏SERT与肌动蛋白细胞骨架之间的相互作用。(C)2004 Elsevier Ltd.保留所有权利。
Previously, we revealed that the state of the actin cytoskeleton affects the uptake activity of the serotonin transporter (SERT). Recently, it was reported that the C-terminus of SERT interacts with MacMARCKS, a substrate of PKC that can bind to the actin cytoskeleton. To elucidate the importance of the C-terminal region in the regulation of SERT activity and the interaction with the actin cytoskeleton, we examined whether the overexpression of the C-terminus affects the transport activity of SERT. To this end, we overexpressed a GFP-fused 30-amino acid construct of the SERT C-terminus (GFP-SERT-CT) in HEK293 cells stably expressing FLAG-tagged SERT (FL-SERT-HEK293 cells). The SERT uptake activity and transporter current were attenuated in GFP-SERT-CT-expressing FL-SERT-HEK293 cells, as compared with GFP-expressing FL-SERT-HEK293 cells. Eadie-Hofstee analysis revealed that GFP-SERT-CT overexpression attenuated the SERT uptake activity by reducing the V-max, but not changing the K-m, which was consistent with the results of experiments on the cell-surface expression of SET using biotinylation/immunoblot analysis. Immunocytochemical analysis demonstrated that GFP-SERT-CT was colocalized with FLAG-SERT and cortical actin at the plasma membrane. In addition, the SERT C-terminus did not affect dopamine transporter activity. These findings showed the significance of the C-terminal region to the functional regulation of SERT, suggesting that GFP-SERT-CT acts as a molecular decoy to disrupt the interaction between SERT and the actin cytoskeleton. (C) 2004 Elsevier Ltd. All rights reserved.