The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines
The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines
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DOI：
10.1074/jbc.m301845200
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发表时间：
2003-06-13
影响因子：
4.8
通讯作者：
Melikian, HE
中科院分区：
文献类型：
--
作者：
Loder, MK;Melikian, HE
多巴胺转运蛋白 (DAT) 从细胞外环境中去除多巴胺，并受到许多精神活性药物的有效抑制，包括可卡因、安非他明和哌醋甲酯（利他林）。多种证据表明，蛋白激酶 C (PKC) 下调多巴胺转运，主要是通过将 DAT 从质膜重新分配到内体区室，尽管促进转运蛋白隔离的机制尚未确定。在这里，我们证明 DAT 在大鼠嗜铬细胞瘤 (PC12) 细胞中组成性内化和循环。温度封锁证明了基础内化和依赖循环来维持 DAT 细胞表面水平。相反，巴弗洛霉素 A(1) 的再循环阻断显着降低了转铁蛋白受体 (TfR) 表面表达，但对 DAT 表面水平没有影响，表明 DAT 和 TfR 通过不同的内体机制进行运输。动力学分析揭示了往返于质膜的强大的组成型 DAT 循环，与转运蛋白表达水平无关。相反，佛波酯介导的 PKC 激活以对 DAT 表达水平敏感的方式加速 DAT 内吞作用并减弱转运蛋白循环。这些数据证明了 DAT 的组成型运输以及 PKC 介导的 DAT 隔离是通过加速内化和减少回收相结合来实现的。此外，组成型和调节型 DAT 运输对表达水平的敏感性不同，表明这两个过程是由独立的细胞机制介导的。
The dopamine transporter (DAT) removes dopamine from the extracellular milieu and is potently inhibited by number of psychoactive drugs, including cocaine, amphetamines, and methylphenidate ( Ritalin). Multiple lines of evidence demonstrate that protein kinase C (PKC) down-regulates dopamine transport, primarily by redistributing DAT from the plasma membrane to endosomal compartments, although the mechanisms facilitating transporter sequestration are not defined. Here, we demonstrate that DAT constitutively internalizes and recycles in rat pheochromocytoma (PC12) cells. Temperature blockades demonstrated basal internalization and reliance on recycling to maintain DAT cell surface levels. In contrast, recycling blockade with bafilomycin A(1) significantly decreased transferrin receptor (TfR) surface expression but had no effect on DAT surface levels, suggesting that DAT and TfR traffic via distinct endosomal mechanisms. Kinetic analyses reveal robust constitutive DAT cycling to and from the plasma membrane, independent of transporter expression levels. In contrast, phorbol ester-mediated PKC activation accelerated DAT endocytosis and attenuated transporter recycling in a manner sensitive to DAT expression levels. These data demonstrate constitutive DAT trafficking and that PKC-mediated DAT sequestration is achieved by a combination of accelerated internalization and reduced recycling. Additionally, the differential sensitivity to expression level exhibited by constitutive and regulated DAT trafficking suggests that these two processes are mediated by independent cellular mechanisms.