Laboratory of Molecular Medicine, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
Biochem Biophys Res Commun. 2013 Jan 11;430(2):839-45. doi: 10.1016/j.bbrc.2012.11.059. Epub 2012 Nov 27.
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.
PDZ（PSD-95/果蝇 discs-large 蛋白/闭合蛋白）结构域含有蛋白 Na(+)/H(+) 交换调节因子 1（NHERF1）和 NHERF2 与谷氨酸转运体 GLAST 相互作用。为了研究这些 NHERF 蛋白在 GLAST 质膜靶向中的作用，我们在人胚肾（HEK）293T 细胞中检测了绿色荧光蛋白（EGFP）标记的 GLAST 与表位标记的 NHERF 蛋白的相互作用。NHERF 蛋白的共表达增加了 EGFP-GLAST 的细胞表面表达。C 端 PDZ 结构域结合基序的缺失导致 EGFP-GLAST 与不成熟的内切糖苷酶 H 敏感的 N-连接寡糖一起增加，这表明 EGFP-GLAST 从内质网（ER）中出口受损。免疫沉淀实验表明，NHERF1 主要与含有不成熟 N-糖基化的 EGFP-GLAST 结合，而 NHERF2 则与成熟 N-糖基化的 EGFP-GLAST 共沉淀。Sar1 鸟嘌呤核苷酸交换因子的显性失活突变体的表达增加了 EGFP-GLAST 与 NHERF1 在 ER 中的相互作用。相比之下，免疫荧光显微镜显示 NHERF2 与 EGFP-GLAST 在 ER-Golgi 中间区（ERGIC）、质膜和早期内体中共定位，但不在 ER 中。这些结果表明，NHERF1 在 ER 出口过程中与 GLAST 相互作用，而 NHERF2 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。
