Dopamine Transporter Regulation by Endocytosis

描述(由申请人提供)：多巴胺转运体(DAT)是关键 用于控制局部细胞外多巴胺(DA)浓度，从而 DA在中枢神经系统的突触传递。表达的DAT分子数量 神经元表面决定了细胞外DA的清除速度。快速 DAT表面表达的变化由胞内转运决定， 也就是说，内化到内体和从内吞体内循环之间的平衡 内小体回到细胞表面。胞质蛋白介导的内化是 主要限速步骤调节DAT的胞内循环。然而， 通过涂层凹坑控制DAT内化的分子机制是 未知。此外，鉴于DAT内吞作用的研究使用了模型 表达系统中，是否存在类似的机制还有待确定 介导DAT在神经元内的内化。 我们的长期目标是了解中南欧对DAT贩运的监管。 这一提议的重点是阐明基本的分子机制。 和DAT胞内转运过程中发生的蛋白质-蛋白质相互作用 以及这些相互作用导致动态变化的具体调控机制 DAT和DA转运的表面表达变化。异源表达 系统将用于开发工具和方法，然后应用这些工具和方法 至大鼠黑质纹状体DA神经元。在目标1中，假设DAT 内化依赖于DAT中特定序列与 将通过定义分子来测试笼状蛋白适配器AP2复合体 DAT-AP2相互作用的机制和功能作用。一种组合 将利用分子、生物物理和生化技术。在AIMS#2 第三，通过突触前受体和蛋白质传递信号的假说 调节DAT的内吞作用的激酶将被测试。具体地说， 酪氨酸磷酸化和多巴胺D2受体(D2R)激活在调节细胞周期中的作用 DAT的内吞作用和/或循环以及控制DAT的数量 在细胞表面表达的蛋白质将被分析。理解分子 涉及DAT贩运的机制将为了解 调节DAT活性，从而调节大脑中的DA神经传递，如 以及DA和D2R药物的作用。

DESCRIPTION (provided by applicant): Dopamine transporters (DATs) are critical for controlling local extracellular concentrations of dopamine (DA) and thereby DA synaptic transmission in the CNS. The number of DAT molecules expressed at the neuronal surface determines the rate of extracellular DA clearance. Rapid changes in surface expression of DAT are determined by endocytic trafficking, i.e., the equilibrium between internalization into endosomes and recycling from endosomes back to the cell surface. Clathrin-mediated internalization is the major rate-limiting step regulating endocytic cycling of DAT. However, the molecular mechanisms that control DAT internalization through coated pits are unknown. Moreover, whereas studies of DAT endocytosis have used model expression systems, it remains to be established whether similar mechanisms mediate internalization of DAT in neurons. Our long-term goal is to understand regulation of DAT trafficking in the CNS. The focus of this proposal is on elucidation of the basic molecular machinery and protein-protein interactions that occur during endocytic trafficking of DAT and on specific regulatory mechanisms of these interactions leading to dynamic changes in surface expression of DAT and DA transport. Heterologous expression systems will be used to develop tools and approaches that will then be applied to rat nigrostriatal DA neurons. In Aim #1, the hypothesis that DAT internalization depends on the interaction of specific sequences in DAT with the clathrin adaptor AP2 complex will be tested by defining the molecular mechanisms and functional role of DAT-AP2 interactions. A combination of molecular, biophysical and biochemical techniques will be utilized. In Aims #2 and #3, the hypothesis that signaling through presynaptic receptors and protein kinases regulates endocytosis of DAT will be tested. In particular, the role of tyrosine phosphorylation and DA D2 receptor (D2R) activation in regulating the endocytosis and/or recycling of DAT and in controlling the number of DATs expressed on the cell surface will be analyzed. Understanding the molecular mechanisms involved in DAT trafficking will provide new insights into the regulation of DAT activity and thereby DA neurotransmission in the brain, as well as into the actions of DA and D2R drugs.