Laboratory of Neurobiology, Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 845 51, Bratislava, Slovakia.
Laboratory of Neurobiology, Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 845 51, Bratislava, Slovakia.
Neurochem Int. 2023 May;165:105522. doi: 10.1016/j.neuint.2023.105522. Epub 2023 Mar 25.
The GABA transporter GAT1 regulates brain inhibitory neurotransmission and it is considered a potential therapeutic target for the treatment of wide spectrum of neurological diseases including epilepsy, stroke and autism. Syntenin-1 binds to syntaxin 1A, which is known to regulate the plasma membrane insertion of several neurotransmitter transporters. Previously, a direct interaction of syntenin-1 with the glycine transporter GlyT2 was reported. Here, we show that the GABA transporter GAT1 also directly interacts with syntenin-1, involving both unidentified protein interaction interface and the GAT1 C-terminal PDZ binding motif interacting mainly with syntenin-1 PDZ domain 1. The PDZ interaction was eliminated by the mutation of GAT1 isoleucine 599 and tyrosine 598 located in PDZ positions 0 and -1, respectively. This indicates an unconventional PDZ interaction and possible regulation of the transporter PDZ motif via tyrosine phosphorylation. Whole syntenin-1 protein fused to GST protein and immobilised on glutathione resin coprecipitated intact GAT1 transporter from an extract of GAT1 transfected neuroblastoma N2a cells. This coprecipitation was inhibited by tyrosine phosphatases inhibitor pervanadate. The fluorescence tagged GAT1 and syntenin-1 colocalized upon coexpression in N2a cells. The above results show that syntenin-1 might be, in addition to GlyT2, directly involved in the trafficking of GAT1 transporter.
GABA 转运体 GAT1 调节脑抑制性神经递质传递，被认为是治疗包括癫痫、中风和自闭症在内的广泛神经疾病的潜在治疗靶点。Syntenin-1 与突触融合蛋白 1A（Syntaxin 1A）结合，已知后者调节多种神经递质转运体的质膜插入。先前有报道称 syntenin-1 与甘氨酸转运体 GlyT2 直接相互作用。在这里，我们表明 GABA 转运体 GAT1 也与 syntenin-1 直接相互作用，涉及未鉴定的蛋白质相互作用界面和 GAT1 C 末端 PDZ 结合基序，主要与 syntenin-1 PDZ 结构域 1 相互作用。PDZ 相互作用被突变的 GAT1 异亮氨酸 599 和酪氨酸 598 消除，它们分别位于 PDZ 位置 0 和 -1。这表明存在非传统的 PDZ 相互作用，并且可能通过酪氨酸磷酸化来调节转运体 PDZ 基序。完整的 syntenin-1 蛋白与 GST 蛋白融合，并固定在谷胱甘肽树脂上，从 GAT1 转染的神经母细胞瘤 N2a 细胞提取物中共沉淀完整的 GAT1 转运体。这种共沉淀被酪氨酸磷酸酶抑制剂过钒酸钠抑制。荧光标记的 GAT1 和 syntenin-1 在共表达于 N2a 细胞时发生共定位。上述结果表明，syntenin-1 可能除了 GlyT2 之外，还直接参与 GAT1 转运体的运输。
