Phosphorylation and Regulation of Dopamine Transporters

描述（由申请人提供）：多巴胺转运蛋白（DAT）是细胞外多巴胺（DA）从突触间隙清除的主要机制。因此，它在终止多巴胺能神经传递和调节可用于结合突触前和突触后受体的DA浓度方面发挥关键作用。DAT是许多精神活性药物的靶标，包括滥用的化合物如可卡因和安非他明（AMPH），以及治疗剂如利他林和Wellbutrin。DAT的调节障碍，这将导致DA水平异常，被推测发生在多巴胺能疾病，如帕金森病和精神分裂症，虽然机制尚不清楚。DAT是高度调节的蛋白质，目前的证据表明涉及许多蛋白激酶，包括蛋白激酶C和钙-钙调蛋白依赖性蛋白激酶。虽然我们知道DAT是代谢磷酸化的，但我们不知道蛋白质上被修饰的精确位点，催化磷酸盐添加或去除的激酶或磷酸酶，或者磷酸化影响功能调节的分子基础。对这一过程与滥用药物或治疗药物的关系也只有很少的了解。本项目的长期目标是阐明DAT受磷酸化调节的确切机制。具体的目的是阐明激酶和磷酸酶参与，氨基酸修饰，和这些过程发生的亚细胞区域;确定在特定位点的磷酸化如何影响AMPH刺激的外排;并确定磷酸化如何受到DAT阻断剂的影响。这些研究的完成将提供对DAT磷酸化的更精确的理解，以及这一过程如何与多巴胺能疾病和药物滥用中DAT的失调有关。公共卫生相关性：这些研究的完成将提供一个更精确的理解DAT磷酸化及其在苯丙胺和多巴胺转运阻滞剂诱导的过程中的作用。因此，这些信息可能有助于阐明治疗药物成瘾的新治疗靶点，并有助于澄清多巴胺能疾病（如帕金森病和抑郁症）与DAT失调的潜在联系。

DESCRIPTION (provided by applicant): The dopamine transporter (DAT) is the primary mechanism by which extracellular dopamine (DA) is cleared from the synaptic space. As such it performs a key role in terminating dopaminergic neurotransmission and in regulating the concentration of DA available for binding to pre- and post-synaptic receptors. DATs are targets for numerous psychoactive drugs including abused compounds such as cocaine, and amphetamine (AMPH), and therapeutic agents such as Ritalin and Wellbutrin. Dysrgulation of DAT, which would lead to abnormal levels of DA, is speculated to occur in dopaminergic diseases such as Parkinson's disease and schizophrenia, although mechanisms are not known. DATs are highly regulated proteins, with current evidence implicating the involvement of numerous protein kinases including Protein Kinase C and Calcium- Calmodulin Dependent Protein Kinase. While we know that DATs are metabolically phosphorylated, we do not know the precise sites on the protein that are modified, the kinases or phosphatases that catalyze phosphate addition or removal, or the molecular basis by which phosphorylation effects functional regulation. There is also only marginal understanding of the relationship of this process to abused or therapeutic drugs. The long term goal of this project is to clarify the precise mechanisms by which DAT is regulated by phosphorylation. The specific aims are to elucidate the kinases and phosphatases involved, the amino acids modified, and the subcellular regions where these processes occur; determine how phosphorylation at specific sites impacts AMPH-stimulated efflux; and to determine how phosphorylation is impacted by DAT blockers. The completion of these studies will provide a much more precise understanding of DAT phosphorylation and how this process could be related to dysregulation of DAT in dopaminergic diseases and drug abuse. PUBLIC HELATH RELEVANCE: The completion of these studies will provide a much more precise understanding of DAT phosphorylation and its role in processes induced by amphetamine and dopamine transport blockers. This information may thus be useful in elucidating novel therapeutic targets for treating drug addiction, and help to clarify potential links to DAT dysregulation in dopaminergic disorders such as Parkinson's disease and depression.