Rapid trafficking of the neuronal glutamate transporter, EAAC1 -: Evidence for distinct trafficking pathways differentially regulated by protein kinase C and platelet-derived growth factor
Rapid trafficking of the neuronal glutamate transporter, EAAC1 -: Evidence for distinct trafficking pathways differentially regulated by protein kinase C and platelet-derived growth factor
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DOI：
10.1074/jbc.m404032200
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发表时间：
2004-08-13
影响因子：
4.8
通讯作者：
Robinson, MB
中科院分区：
文献类型：
--
作者：
Fournier, KM;González, MI;Robinson, MB
神经元谷氨酸转运蛋白EAAC 1似乎既限制兴奋性突触之间的溢出，又为抑制性神经递质γ-氨基丁酸的合成提供前体。有证据表明，EAAC 1的细胞内库很大，在蛋白激酶C（PKC）或血小板衍生生长因子（PDGF）受体通过看似独立的途径活化后，转运蛋白被重新分布到细胞表面。采用多种生物素化策略来测量EAAC 1进出质膜的运输，并检查佛波酯和PDGF对这些事件的影响。在允许运输的条件（37 ℃）下，细胞表面蛋白的生物素化导致C6神经胶质瘤和原代神经元培养物中生物素化的EAAC 1的量在15分钟内增加2倍，这表明EAAC 1在质膜上停留的半衰期类似于5 - 7分钟。佛波酯和PDGF都增加了在这些条件下标记的转运蛋白的量。使用可逆的生物素化策略，在C6胶质瘤中观察到EAAC 1的类似快速内化。佛波酯，但不是PDGF，阻止这种措施的内化。在18 degreesC下孵育，阻断了某些形式的细胞内膜运输，抑制了EAAC 1的PKC和PDGF依赖性再分布，但对EAAC 1的基础运输没有影响。这些研究表明，PKC和PDGF都加速EAAC 1向细胞表面的递送，并且PKC对内吞作用具有额外的作用。这些数据还表明，基础和调节池的EAAC 1存在于不同的隔室。
The neuronal glutamate transporter, EAAC1, appears to both limit spillover between excitatory synapses and provide precursor for the synthesis of the inhibitory neurotransmitter, gamma-aminobutyric acid. There is evidence for a large intracellular pool of EAAC1 from which transporter is redistributed to the cell surface following activation of protein kinase C ( PKC) or platelet-derived growth factor ( PDGF) receptor by seemingly independent pathways. A variety of biotinylation strategies were employed to measure trafficking of EAAC1 to and from the plasma membrane and to examine the effects of phorbol ester and PDGF on these events. Biotinylation of cell surface protein under trafficking-permissive conditions ( 37 degreesC) resulted in a 2-fold increase in the amount of biotinylated EAAC1 within 15 min in C6 glioma and in primary neuronal cultures, suggesting that EAAC1 has a half-life of similar to 5 - 7 min for residence at the plasma membrane. Both phorbol ester and PDGF increased the amount of transporter labeled under these conditions. Using a reversible biotinylation strategy, a similarly rapid internalization of EAAC1 was observed in C6 glioma. Phorbol ester, but not PDGF, blocked this measure of internalization. Incubation at 18 degreesC, which blocks some forms of intracellular membrane trafficking, inhibited PKC- and PDGF-dependent redistribution of EAAC1 but had no effect on basal trafficking of EAAC1. These studies suggest that both PKC and PDGF accelerate delivery of EAAC1 to the cell surface and that PKC has an additional effect on endocytosis. The data also suggest that basal and regulated pools of EAAC1 exist in distinct compartments.