Physial and Functional Link of the Dopamine Transporter with Synaptic Proteins

描述（由申请人提供）：多巴胺（DA）传递涉及重要的脑功能，包括运动控制、神经内分泌、认知、情感和奖励。多巴胺能信号传导主要通过质膜DA转运蛋白（DAT）对递质的再摄取而终止。这种蛋白质也是可卡因和安非他明等广泛滥用的精神兴奋剂的主要目标。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，并在转运蛋白的靶向，运输和功能调节中发挥重要作用。使用酵母交配为基础的分裂泛素系统，我们现在已经确定了突触囊泡蛋白synaptogyrin-3（SG 3）作为这样的DAT相互作用蛋白。我们已经收集了初步的数据，表明DAT和SG 3之间的物理和功能相互作用。基于这些观察结果，我们的中心假设是，SG 3和DAT之间的物理和功能相互作用影响转运功能，并最终DA稳态。这可能是由于质膜DAT介导的摄取和囊泡DA储存系统之间的联系，或者，通过调节DAT靶向，回收和/或内在转运活性。在本申请中，生物化学，分子和功能的方法在体外和体内的组合将用于与细胞培养，突触体制剂，纯化的突触囊泡，和整个动物，以产生详细的结构，功能和亚细胞的DAT/SG 3相互作用的描述。具体而言，我们将确定DAT/SG 3相互作用中涉及的蛋白质残基，并检查这种相互作用的特异性（目标1）;研究SG 3对DAT功能的影响（目标2）;检查DAT/SG 3相互作用的调节（目标3）;并确定DAT/SG 3相互作用的亚细胞位置（目标4）。我们研究计划的长期目标是了解DAT调节DA稳态的机制以及这些机制如何被精神兴奋剂改变。新的DAT相互作用蛋白的发现可能提示与转运体活性相关的新机制，因此，这些机制将对DA稳态的调节产生重要影响。公共卫生声明：这些研究的结果有望有助于推进DA相关疾病（包括药物成瘾）干预的新策略。

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.