Physial and Functional Link of the Dopamine Transporter with Synaptic Proteins

描述(申请人提供)：多巴胺(DA)传递涉及重要的大脑功能，包括运动控制、神经内分泌、认知、情感和奖励。多巴胺能信号主要通过质膜DA转运体(DAT)重新摄取递质而终止。这种蛋白质也是被广泛滥用的精神刺激剂，如可卡因和苯丙胺的主要靶标。最近，我们的团队和其他人已经确定了几种与DAT相互作用的蛋白质，并在靶向、运输和转运蛋白的功能调节中发挥重要作用。利用酵母交配的分裂泛素系统，我们现在已经鉴定出突触小泡蛋白Synaptogyrin-3(SG3)就是这样一种DAT相互作用蛋白。我们已经收集了初步数据，表明DAT和SG3之间存在物理和功能上的相互作用。基于这些观察，我们的中心假设是SG3和DAT之间的物理和功能相互作用影响转运蛋白功能，最终影响DA动态平衡。这可能是由于质膜DAT介导的摄取和囊泡DA存储系统之间的联系，或者通过调节DAT的靶向、循环和/或内在转运蛋白活性。在这项应用中，结合体外和体内的生化、分子和功能方法，将与培养细胞、突触小体制备、纯化的突触小泡和整个动物一起使用，以产生对DAT/SG3相互作用的详细的结构、功能和亚细胞描述。具体地说，我们将确定DAT/SG3相互作用中涉及的蛋白质残基并研究这种相互作用的特异性(AIM 1)；调查SG3对DAT功能的影响(AIM 2)；研究DAT/SG3相互作用的调节(AIM 3)；以及确定DAT/SG3相互作用的亚细胞位置(AIM 4)。我们研究计划的长期目标是了解DAT调节DA动态平衡的机制，以及这些机制如何被精神刺激剂改变。新的DAT相互作用蛋白的发现可能提示与转运蛋白活性相关的新机制，因此，这些机制将在DA动态平衡的调节中产生重要影响。公共卫生声明：这些研究的结果预计将有助于发展新的干预策略，以干预包括药物成瘾在内的DA相关疾病。

DESCRIPTION (provided by applicant): Dopamine (DA) transmission is involved in important brain functions including locomotor control, neuroendocrine secretion, cognition, emotion, and reward. Dopaminergic signaling is terminated primarily by the re-uptake of the transmitter via a plasma membrane DA transporter (DAT). This protein is also the main target of widely abused psychostimulants, such as cocaine and amphetamine. Recently, our group and others have identified several proteins that interact with DAT and play important roles in targeting, trafficking, and functional regulation of the transporter. Using the yeast mating-based split ubiquitin system, we have now identified the synaptic vesicle protein synaptogyrin-3 (SG3) as such a DAT interacting protein. We have gathered preliminary data suggesting a physical and functional interaction between DAT and SG3. Based on these observations, our central hypothesis is that a physical and functional interaction between SG3 and DAT influences transporter function and ultimately DA homeostasis. This may be due to a link between the plasma membrane DAT-mediated uptake and the vesicular DA storage system, or alternatively, through regulation of DAT targeting, recycling, and/or intrinsic transporter activity. In this application, a combination of biochemical, molecular, and functional approaches in vitro and in vivo will be used with cells in culture, synaptosomal preparations, purified synaptic vesicles, and whole animals to generate a detailed structural, functional, and subcellular description of the DAT/SG3 interaction. Specifically, we will identify the protein residues involved in the DAT/SG3 interaction and examine the specificity of this interaction (Aim 1); investigate the impact of SG3 on DAT function (Aim 2); examine the regulation of the DAT/SG3 interaction (Aim 3); and determine the subcellular location for the DAT/SG3 interaction (Aim 4). The long-term goal of our research program is to understand the mechanisms involved in the regulation of DA homeostasis by DAT and how these mechanisms are altered by psychostimulants. The discovery of novel DAT interacting proteins may suggest novel mechanisms associated with the activity of the transporter, and as a consequence, these mechanisms will have an important impact in the regulation of DA homeostasis. Public Health Statement: Findings from these studies are expected to aid in the advancements of new strategies for intervention in DA-related disorders including drug addiction.