Modulatory roles of NHERF1 and NHERF2 in cell surface expression of the glutamate transporter GLAST
Modulatory roles of NHERF1 and NHERF2 in cell surface expression of the glutamate transporter GLAST
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NHERF1 和 NHERF2 在谷氨酸转运蛋白 GLAST 细胞表面表达中的调节作用
DOI：
10.1016/j.bbrc.2012.11.059
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发表时间：
2013
影响因子：
3.1
通讯作者：
M.
中科院分区：
文献类型：
--
作者：
Sato;K.;Otsu;W.;Otsuka;Y.;Inaba;M.
含有PDZ（PSD-95/果蝇盘大蛋白/闭合小带蛋白）结构域的蛋白质Na+/H+交换调节因子1（NHERF 1）和NHERF 2与谷氨酸转运蛋白GLAST相互作用。为了表征这些NHERF蛋白在GLAST质膜靶向中的作用，我们检测了绿色荧光蛋白（EGFP）标记的GLAST与人胚肾（HEK）293 T细胞中表位标记的NHERF蛋白的相互作用。任一NHERF蛋白的共表达增加了EGFP-GLAST的细胞表面表达。C-末端PDZ结构域结合基序的缺失导致EGFP-GLAST与不成熟的糖苷内切酶H-敏感的N-连接寡糖的增加，表明EGFP-GLAST从内质网（ER）的退出受损。免疫沉淀实验显示，NHERF 1主要结合含有未成熟N-聚糖的EGFP-GLAST，而NHERF 2与成熟N-聚糖共沉淀EGFP-GLAST。GTSAr 1的显性负突变体的表达增加了EGFP-GLAST与ER中NHERF 1的相互作用。相比之下，免疫荧光显微镜显示NHERF 2与EGFP-GLAST共定位于内质网高尔基体中间室（ERGIC）、质膜和早期内体，但不在内质网中。这些结果表明，NHERF 1在ER输出过程中与GLAST相互作用，而NHERF 2在从ERGIC到质膜的分泌途径中与GLAST相互作用，从而调节GLAST的细胞表面表达。
The PDZ (PSD-95/Drosophila discs-large protein/zonula occludens protein) domain-containing proteins Na+/H+exchanger regulatory factor 1 (NHERF1) and NHERF2 interact with the glutamate transporter GLAST. To characterize the roles of these NHERF proteins in the plasma membrane targeting of GLAST, we examined the interaction of green fluorescent protein (EGFP)-tagged GLAST with epitope-tagged NHERF proteins in human embryonic kidney (HEK) 293T cells. Co-expression of either NHERF protein increased the cell surface expression of EGFP-GLAST. Deletion of the C-terminal PDZ domain-binding motif caused an increase in EGFP-GLAST with immature endoglycosidase H-sensitive N-linked oligosaccharides, suggesting impaired exit of EGFP-GLAST from the endoplasmic reticulum (ER). Immunoprecipitation experiments revealed that NHERF1 predominantly bound EGFP-GLAST containing immature N-glycans, whereas NHERF2 co-precipitated EGFP-GLAST with mature N-glycans. Expression of a dominant-negative mutant of the GTPase Sar1 increased the interaction of EGFP-GLAST with NHERF1 in the ER. By contrast, immunofluorescence microscopy showed that NHERF2 co-localized with EGFP-GLAST in ER–Golgi intermediate compartments (ERGICs), at the plasma membrane and in early endosomes, but not in the ER. These results suggest that NHERF1 interacts with GLAST during ER export, while NHERF2 interacts with GLAST in the secretory pathway from the ERGIC to the plasma membrane, thereby modulating the cell surface expression of GLAST.