Phosphorylation and Regulation of Dopamine Transporters

描述(申请人提供)：多巴胺转运体(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与突触前和突触后受体结合的DA浓度方面发挥关键作用。DATS是许多精神活性药物的目标，包括滥用的化合物，如可卡因和苯丙胺，以及治疗剂，如利他林和韦布曲林。据推测，多巴胺能疾病如帕金森氏病和精神分裂症都会引起DAT的异常，这会导致DA水平的异常，尽管机制尚不清楚。DATS是高度受调控的蛋白质，目前的证据表明，包括蛋白激酶C和钙-钙调蛋白依赖的蛋白激酶在内的许多蛋白激酶都参与其中。虽然我们知道DAT是代谢性的磷酸化的，但我们不知道蛋白质上被修饰的确切位置，催化磷酸盐加成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。对这一过程与滥用或治疗药物的关系也只有微乎其微的理解。该项目的长期目标是阐明DAT通过磷酸化进行调节的精确机制。其具体目的是阐明涉及的激酶和磷酸酶、修饰的氨基酸以及这些过程发生的亚细胞区域；确定特定位置的磷酸化如何影响AMPH刺激的外排；以及确定DAT阻断剂如何影响磷酸化。这些研究的完成将提供对DAT磷酸化的更准确的理解，以及这一过程如何与多巴胺能疾病和药物滥用中的DAT调节失调有关。公共卫生相关性：这些研究的完成将对DAT的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提供更准确的理解。因此，这些信息可能有助于阐明治疗药物成瘾的新的治疗靶点，并有助于澄清与帕金森氏病和抑郁症等多巴胺能障碍的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.