Study of Dopamine Transporter, a Direct Downstream Effector of Goalpha Signaling

描述(由申请人提供)： 多巴胺转运体(DAT)是一种在多巴胺能神经元中表达的跨膜蛋白，它是细胞外多巴胺(DA)重新摄取回到DA神经元中导致DA神经传递终止所必需的。由于DAT的主要作用是清除DA，因此DAT对许多涉及DA神经传递的生理功能如认知、运动、动机和奖赏相关行为都是至关重要的。另一方面，DAT还与几种DA神经传递异常的神经疾病有关，如帕金森氏症、精神分裂症、注意力缺陷多动障碍和药物成瘾。只有当DAT存在于质膜上时，它才参与DA清除。DAT在细胞膜上的水平受到内吞作用的严格调控，但对DAT内吞转运的机制却知之甚少。目前关于DAT转运调控的大部分知识都是基于非神经元表达系统的研究，因此DAT在DA神经元中转运的机制尚不清楚。更好地理解DAT内吞转运的分子机制不仅对于了解正常的DA神经传递是至关重要的，而且对于设计预防和治疗神经系统疾病中异常DA信号的策略也是重要的。最近，我们开发了表达带有HA标签的功能性DAT的敲入小鼠(HA-DAT)。HA-Tag为我们提供了一种新的工具来监测活的DA神经元中的HA-DAT，以研究内吞过程。在这些小鼠的DA神经元中，我们发现HA-DAT经历了结构性内吞作用。我们在这些小鼠身上的初步研究也强烈表明DAT和G蛋白的1个亚单位GO之间存在直接的相互作用。我们发现在敲入小鼠的DA神经元中存在DAT和GO的相互免疫共沉淀和共定位。此外，百日咳毒素抑制GO可增加这些小鼠DA神经元DAT的内吞作用。已知像GO这样的G蛋白是大脑中信号的中心媒介。在小鼠体内缺乏GO会导致严重的神经异常，但其机制尚不清楚。DAT和GO之间的相互作用是一个新的发现，进一步的研究将为深入了解这两个重要蛋白在DA神经元正常功能中的重要性提供依据。根据我们的数据，我们假设DAT是GO的直接下游效应者，从而GO直接与DAT相互作用并调节其内吞运输。本项目的总体目标是利用HA-DAT小鼠来确定Go调节的DAT转运的分子机制，并确定这两种蛋白在DA神经元中相互作用所必需的因素。众所周知，安非他明等精神刺激性药物通过调节DAT运输来调节DA神经传递，但分子机制尚不清楚。从长远来看，我们希望研究GO介导的DAT贩运在苯丙胺活动中的作用，并对药物成瘾的病理机制有更深入的了解。 公共卫生相关性： 多巴胺转运体(DAT)是存在于脑多巴胺能神经元中的一种蛋白质，它负责将神经递质多巴胺(DA)从细胞外间隙重新摄取回多巴胺能神经元，导致DA神经传递的终止。DAT功能的异常从而导致DA重新摄取会导致阿尔茨海默氏症、精神分裂症、注意力缺陷多动障碍和药物成瘾等神经疾病。彻底了解DAT转运的潜在分子机制决定了DA的再摄取量，这对于制定预防和治疗神经疾病的策略至关重要。

DESCRIPTION (provided by applicant): Dopamine transporter (DAT) is a transmembrane protein expressed in dopaminergic neurons where it is necessary for reuptake of extracellular dopamine (DA) back into DA neurons leading to termination of DA neurotransmission. Since the main role of DAT is DA clearance, DAT is vital for numerous physiological functions involving DA neurotransmission such as cognition, locomotion, motivation and reward-related behaviors. On the other hand, DAT is also linked to several neurological disorders with abnormal DA neurotransmission such as Parkinson's disease, schizophrenia, attention deficit hyperactivity disorder and drug addiction. DAT participates in DA clearance only when it is present on the plasma membrane. The levels of DAT at the plasma membrane are tightly regulated by the process of endocytosis but mechanisms of DAT endocytic trafficking are poorly understood. Most of the current knowledge on regulation of DAT trafficking is based on studies done in non-neuronal expression systems and therefore the mechanism of DAT trafficking in DA neurons remains unclear. A better comprehension of molecular mechanisms of DAT endocytic trafficking is not only crucial for understanding normal DA neurotransmission but is also important for designing strategies for prevention and treatment of abnormal DA signaling in neurological disorders. Recently, we have developed knock-in mice that express functional DAT with a HA-tag (HA-DAT). The HA-tag provides us with a novel tool to monitor HA-DAT in live DA neurons to study the process of endocytosis. In DA neurons of these mice, we found that HA-DAT undergoes constitutive endocytosis. Our preliminary studies in these mice also strongly suggest a direct interaction between DAT and an 1-subunit of G protein, Go. We found reciprocal co-immunoprecipitation and co-localization of DAT and Go in DA neurons of the knock-in mice. Additionally, inhibition of Go by pertussis toxin increased endocytosis of DAT in DA neurons of these mice. G proteins like Go are known to be the central mediators of signaling in the brain. Absence of Go in mice results in severe neurological abnormalities but the mechanisms are poorly understood. Interaction between DAT and Go is a novel finding and further studies will provide insights into the importance of these two vital proteins in normal function of DA neurons. Based on our data, we hypothesize that DAT is a direct downstream effector of Go whereby Go directly interacts with DAT and modulates its endocytic trafficking. The overall goal of this project is to employ HA-DAT mice to define molecular mechanisms of Go-regulated DAT trafficking and to identify the factors necessary for interaction between these two proteins in DA neurons. Psychostimulant drugs like amphetamine are known to modulate DA neurotransmission by regulating DAT trafficking but the molecular mechanisms are unknown. In the long run, we hope to investigate the role of Go- mediated DAT trafficking in action of amphetamine and gain insights into the pathology of drug addiction. PUBLIC HEALTH RELEVANCE: Dopamine transporter (DAT) is a protein found in the dopaminergic neurons of the brain where is it responsible for re-uptake of the neurotransmitter dopamine (DA) from extracellular space back into the dopaminergic neurons causing termination of DA neurotransmission. Abnormalities in DAT function and hence DA re-uptake leads to neurological disorders like Alzheimer's, schizophrenia, attention deficit hyperactivity disorder and drug addiction. A thorough understanding of the underlying molecular mechanisms of DAT trafficking which dictates the amount of DA re-uptake is crucial for designing strategies for prevention and treatment of neurological disorders.