Dopamine Transporter Regulation by Endocytosis

描述（由申请人提供）：多巴胺（DA）参与物种生存的重要行为，包括动机，运动规划，感觉运动整合，习惯形成，奖励预测/评估和神经内分泌调节，以及帕金森病，精神分裂症，ADHD和药物成瘾。多巴胺能神经元的质膜DA转运蛋白（DATs）是通过清除细胞外DA来终止DA神经传递的关键。由于DAT在神经元质膜的突触区域附近起作用，因此调节亚细胞靶向和DAT的表面水平对DA系统的整体活性至关重要。然而，控制神经元数据定位及其运输/内吞作用的机制尚不清楚。在本项目的前一个周期中，我们定义了DAT蛋白激酶c依赖性内吞作用的机制，发现了DAT的泛素化，并揭示了DAT氨基末端在调节其组成性内吞作用中的作用。许多这些进展是可能的，因为我们产生了一个功能表位标记的DAT与血凝素（HA）表位插入到DAT的细胞外环2。然而，总的来说，一个重要的限制是，我们对DAT内吞作用的大部分机制理解是基于使用非神经元细胞的模型表达系统的研究。为了研究DA神经元中的DAT内吞作用，我们最近开发了一种转基因敲入小鼠，其中内源性DAT被HA-DAT取代。HA-DAT小鼠使我们能够开始对天然表达的DAT的内吞作用进行定量分析，并对DAT相互作用蛋白进行大规模分析。我们实验室开发的这种新型小鼠模型和新的显微镜方法，结合对转运体结构的快速理解，使得首次全面表征神经元中DAT运动和交通的动态，并确定这些动态是如何被滥用的精神兴奋剂药物如可卡因、哌甲酯（MPH）和安非他明（AMPH）调节的成为可能。该提案的具体目标是：1)定义确定神经元中DAT的亚细胞定位和构成内吞运输的机制，以及竞争性DAT抑制剂可卡因和MPH对这些过程的影响；2)定义细胞表面数据保留的机制；3)阐明信号过程调节神经元DAT内吞作用的机制。拟议的研究将结合使用最先进的定量细胞成像技术，包括电子显微镜和质谱分析。

DESCRIPTION (provided by applicant): Dopamine (DA) is involved in behaviors important for species survival including motivation, motor planning, sensorimotor integration, habit formation, reward prediction/valuation and neuroendocrine regulation, as well as in Parkinson's disease, schizophrenia, ADHD and drug addiction. Plasmalemma DA transporters (DATs) of dopaminergic neurons are crucial for terminating DA neurotransmission by removing extracellular DA. Because DAT' functions near synaptic areas of the neuronal plasma membrane, regulation of subcellular targeting and surface levels of DAT is critical for overall activity of DA systems. However, mechanisms controlling neuronal DAT localization and its trafficking/endocytosis are not well understood. In the previous cycle of this project, we defined mechanisms of protein kinase C-dependent endocytosis of DAT, discovered ubiquitination of DAT and revealed the role of the DAT amino-terminus in regulating its constitutive endocytosis. Many of these advances were made possible because we generated a functional epitope-tagged DAT with the hemagglutinin (HA) epitope inserted in the extracellular loop 2 of DAT. Overall, however, a significant limitation has been that most of our mechanistic understanding about DAT endocytosis is based on studies in model expression systems using non-neuronal cells. To study DAT endocytosis in DA neurons, we recently developed a transgenic knock-in mouse in which endogenous DAT was replaced by an HA-DAT. The HA-DAT mice have allowed us to begin quantitative analysis of endocytosis of the natively-expressed DAT and large-scale analysis of DAT interacting proteins. Development of this novel mouse model and new microscopy methodologies in our laboratory, combined with rapid advances in understanding the transporter structure, make it feasible - for the first time - to comprehensively characterize the dynamics of DAT movement and traffic in neurons and to determine how these dynamics are regulated by psychostimulant drugs of abuse such as cocaine, methylphenidate (MPH) and amphetamine (AMPH). The specific aims of the proposal are to: 1) define mechanisms that determine the subcellular localization and constitutive endocytic trafficking of DAT in neurons and the effects of the competitive DAT inhibitors cocaine and MPH on these processes; 2) define the mechanisms of DAT retention at the cell surface; and 3) elucidate the mechanisms by which signaling processes regulate neuronal DAT endocytosis. The proposed research will use a combination of the state-of- the-art quantitative cellular imaging, including electron microscopy, and mass-spectrometry.