Physial and Functional Link of the Dopamine Transporter with Synaptic Proteins

多巴胺转运蛋白与突触蛋白的物理和功能联系

• 批准号: 7475170
• 负责人: Gonzalo E. Torres
• 金额: $ 32.74万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-15 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7475170
• 关键词: Amphetamines; Animals; Binding; Binding Sites; Biochemical; Biological Assay; Brain; Cell Line; Cell membrane; Cells; Cocaine; Cognition; Data; Disease; Dopamine; Drug Addiction; Emotions; Goals; Homeostasis; In Vitro; Intervention; Link; Location; Mediating; Molecular; Neurosecretory Systems; Norepinephrine; PC12 Cells; Partner in relationship; Play; Preparation; Process; Proteins; Public Health; Recombinant Proteins; Recycling; Regulation; Research; Research Personnel; Reserpine; Rewards; Role; Signal Transduction; Specificity; Synapses; Synaptic Vesicles; System; Testing; Tetrabenazine; Ubiquitin; Vesicle; Yeasts; base; dopamine transporter; in vivo; neurotransmitter release; novel; programs; psychostimulant; serotonin transporter; stimulant abuse; symporter; synaptogyrin; trafficking; transmission process; uptake; vesicular monoamine transporter 2; yeast two hybrid system

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.

描述（由申请人提供）：多巴胺（DA）传递涉及重要的大脑功能，包括运动控制、神经内分泌分泌、认知、情绪和奖励。多巴胺能信号传导主要通过质膜 DA 转运蛋白 (DAT) 重新摄取递质而终止。这种蛋白质也是广泛滥用的精神兴奋剂（如可卡因和安非他明）的主要目标。最近，我们的团队和其他人鉴定了几种与 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。使用基于酵母交配的分裂泛素系统，我们现已鉴定出突触小泡蛋白 synaptogyrin-3 (SG3) 作为 DAT 相互作用蛋白。我们收集了初步数据，表明 DAT 和 SG3 之间存在物理和功能相互作用。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并最终影响 DA 稳态。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 靶向、回收和/或内在转运蛋白活性。在本申请中，体外和体内的生化、分子和功能方法的组合将与培养的细胞、突触体制剂、纯化的突触小泡和整个动物一起使用，以生成 DAT/SG3 相互作用的详细结构、功能和亚细胞描述。具体来说，我们将鉴定参与 DAT/SG3 相互作用的蛋白质残基并检查这种相互作用的特异性（目标 1）；研究 SG3 对 DAT 功能的影响（目标 2）；检查 DAT/SG3 相互作用的调节（目标 3）；并确定 DAT/SG3 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 稳态的机制以及精神兴奋剂如何改变这些机制。新型 DAT 相互作用蛋白的发现可能表明与转运蛋白活性相关的新机制，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：这些研究的结果预计将有助于推进干预 DA 相关疾病（包括毒瘾）的新策略。