Dopamine Transporter Regulation by Endocytosis

描述（由申请人提供）：多巴胺转运蛋白（DAT）至关重要 用于控制多巴胺（DA）的局部细胞外浓度， 中枢神经系统中的DA突触传递。DAT分子的数量在 神经元表面决定细胞外DA清除的速率。快速 DAT表面表达的变化由内吞运输决定， 也就是说，内化到内体和再循环之间的平衡， 内体回到细胞表面。网格蛋白介导的内化是 调节DAT内吞循环的主要限速步骤。然而， 通过被膜小窝控制DAT内化的分子机制是 未知此外，尽管DAT内吞作用的研究使用了模型 表达系统，仍有待确定是否类似的机制， 介导神经元中DAT的内化。 我们的长期目标是了解中枢神经系统中DAT运输的调节。 这项建议的重点是阐明基本的分子机制 以及DAT内吞运输过程中发生的蛋白质-蛋白质相互作用 以及这些相互作用的特定调节机制， DAT和DA转运的表面表达的变化。异源表达 系统将用于开发工具和方法，然后应用于 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 将通过定义分子量来测试网格蛋白衔接子AP 2复合物， DAT-AP 2相互作用的机制和功能作用。的组合 将利用分子、生物物理和生物化学技术。目标#2 第三，突触前受体和蛋白质的信号传递 激酶调节DAT的内吞作用。特别是， 酪氨酸磷酸化和DA D2受体（D2 R）活化在调节 DAT的内吞和/或再循环以及DAT数量的控制 将分析在细胞表面上表达的蛋白。了解分子 DAT贩运所涉及的机制将为 调节DAT活性，从而调节大脑中的DA神经传递， 以及DA和D2 R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.