Liu Yiting, Vidensky Svetlana, Ruggiero Alicia M, Maier Susanne, Sitte Harald H, Rothstein Jeffrey D
Departments of Neurology and Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, MD 21287, USA.
J Biol Chem. 2008 Mar 7;283(10):6561-71. doi: 10.1074/jbc.M708096200. Epub 2007 Dec 20.
Excitatory amino acid transporters (EAATs) are the primary regulators of extracellular glutamate concentrations in the central nervous system. Their dysfunction may contribute to several neurological diseases. To date, five distinct mammalian glutamate transporters have been cloned. In brain, EAAC1 (excitatory amino acid carrier 1) is the primary neuronal glutamate transporter, localized on the perisynaptic membranes that are near release sites. Despite its potential importance in synaptic actions, little is known concerning the regulation of EAAC1 trafficking from the endoplasmic reticulum (ER) to the cell surface. Previously, we identified an EAAC1-associated protein, GTRAP3-18, an ER protein that prevents ER exit of EAAC1 when induced. Here we show that RTN2B, a member of the reticulon protein family that mainly localizes in the ER and ER exit sites interacts with EAAC1 and GTRAP3-18. EAAC1 and GTRAP3-18 bind to different regions of RTN2B. Each protein can separately and independently form complexes with EAAC1. RTN2B enhances ER exit and the cell surface composition of EAAC1 in heterologous cells. Expression of short interfering RNA-mediated knockdown of RTN2B decreases the EAAC1 protein level in neurons. Overall, our results suggest that RTN2B functions as a positive regulator in the delivery of EAAC1 from the ER to the cell surface. These studies indicate that transporter exit from the ER controlled by the interaction with its ER binding partner represents a critical regulatory step in glutamate transporter trafficking to the cell surface.
兴奋性氨基酸转运体（EAATs）是中枢神经系统细胞外谷氨酸浓度的主要调节因子。它们的功能障碍可能导致多种神经疾病。迄今为止，已克隆出五种不同的哺乳动物谷氨酸转运体。在大脑中，EAAC1（兴奋性氨基酸载体1）是主要的神经元谷氨酸转运体，定位于释放位点附近的突触周围膜上。尽管其在突触作用中具有潜在重要性，但关于EAAC1从内质网（ER）向细胞表面运输的调节机制却知之甚少。此前，我们鉴定出一种与EAAC1相关的蛋白GTRAP3-18，它是一种内质网蛋白，诱导时可阻止EAAC1从内质网输出。在此我们表明，网质蛋白家族成员RTN2B主要定位于内质网和内质网出口位点，它与EAAC1和GTRAP3-18相互作用。EAAC1和GTRAP3-18与RTN2B的不同区域结合。每种蛋白都能分别独立地与EAAC1形成复合物。RTN2B可增强异源细胞中EAAC1的内质网输出和细胞表面组成。短发夹RNA介导的RTN2B敲低表达可降低神经元中EAAC1蛋白水平。总体而言，我们的结果表明RTN2B在EAAC1从内质网向细胞表面的运输过程中起正向调节作用。这些研究表明，转运体通过与其内质网结合伴侣的相互作用而从内质网输出，这是谷氨酸转运体向细胞表面运输过程中的一个关键调节步骤。
