Physical and Functional Interaction between the Dopamine Transporter and the Synaptic Vesicle Protein Synaptogyrin-3
Physical and Functional Interaction between the Dopamine Transporter and the Synaptic Vesicle Protein Synaptogyrin-3
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DOI：
10.1523/jneurosci.4559-08.2009
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发表时间：
2009-04-08
影响因子：
5.3
通讯作者：
Torres, Gonzalo E.
中科院分区：
文献类型：
--
作者：
Egana, Loreto A.;Cuevas, Rolando A.;Torres, Gonzalo E.
通过多巴胺转运体（DAT）的摄取代表了用于终止脑中多巴胺能传递的主要机制。虽然众所周知，多巴胺（DA）所采取的转运蛋白是用来补充突触囊泡存储随后释放，这一机制的分子细节还没有完全理解。在这里，我们确定了突触囊泡蛋白synaptogyrin-3作为DAT相互作用的蛋白质使用的分裂泛素系统。通过使用异源细胞系和小鼠脑的共免疫沉淀实验证实了这种相互作用。DAT和synaptogyrin-3共定位于小鼠纹状体的突触前终末。使用荧光共振能量转移显微镜，我们表明，这两种蛋白质在活神经元中相互作用。GST（谷胱甘肽S-转移酶）蛋白的下拉分析表明，DAT和突触回蛋白-3的细胞质N末端足以进行这种相互作用。此外，DAT的N末端能够结合来自脑组织的纯化的突触囊泡。功能分析显示，突触回蛋白3的表达与DAT活性在PC 12和MN 9D细胞，但不是在非神经元HEK-293细胞。这些变化不归因于转运蛋白细胞表面水平的变化或蛋白质相互作用的直接影响。相反，突触回蛋白-3对DAT活性的影响被废除的存在下，囊泡单胺转运蛋白-2（VMAT 2）抑制剂利血平，这表明依赖于囊泡DA存储系统。最后，我们提供的证据涉及DAT，突触回蛋白-3，VMAT 2的生化复合物。总的来说，我们的数据确定了DAT和突触回蛋白-3之间的一种新的相互作用，并提出了DAT和囊泡DA系统之间的物理和功能联系。
Uptake through the dopamine transporter (DAT) represents the primary mechanism used to terminate dopaminergic transmission in brain. Although it is well known that dopamine (DA) taken up by the transporter is used to replenish synaptic vesicle stores for subsequent release, the molecular details of this mechanism are not completely understood. Here, we identified the synaptic vesicle protein synaptogyrin-3 as a DAT interacting protein using the split ubiquitin system. This interaction was confirmed through coimmunoprecipitation experiments using heterologous cell lines and mouse brain. DAT and synaptogyrin-3 colocalized at presynaptic terminals from mouse striatum. Using fluorescence resonance energy transfer microscopy, we show that both proteins interact in live neurons. Pull-down assays with GST (glutathione S-transferase) proteins revealed that the cytoplasmic N termini of both DAT and synaptogyrin-3 are sufficient for this interaction. Furthermore, the N terminus of DAT is capable of binding purified synaptic vesicles from brain tissue. Functional assays revealed that synaptogyrin-3 expression correlated with DAT activity in PC12 and MN9D cells, but not in the non-neuronal HEK-293 cells. These changes were not attributed to changes in transporter cell surface levels or to direct effect of the protein protein interaction. Instead, the synaptogyrin-3 effect on DAT activity was abolished in the presence of the vesicular monoamine transporter-2(VMAT2) inhibitor reserpine, suggesting a dependence on the vesicular DA storage system. Finally, we provide evidence for a biochemical complex involving DAT, synaptogyrin-3, and VMAT2. Collectively, our data identify a novel interaction between DAT and synaptogyrin-3 and suggest a physical and functional link between DAT and the vesicular DA system.