Monoamine Transporter Phosphorylation and Trafficking

描述：（申请人提供） 单胺重摄取是神经细胞凋亡终止的决定性步骤。 通过Na+-/C1-依赖性质膜家族介导 运输机单胺转运蛋白是免疫调节的分子靶点。 精神兴奋剂可卡因，安非他明和MDMA（“摇头丸”），它们与 抑制转运蛋白，从而增加细胞外单胺浓度 增强突触后反应。作为主要的精神兴奋剂靶点， 在细胞表面的转运体呈递对于精神兴奋剂是至关重要的 约束和行动。大量调查人员提供的证据 证明了细胞信号通路的动态激活 调节单胺转运蛋白细胞表面呈递。此外，数据来自 我们的实验室证明，运输动态交通下稳定 状态条件。转运蛋白调节和运输与变化相关 处于转运蛋白磷酸化状态。然而， 转运蛋白运输和磷酸化没有很好的定义。主要 本项目的目标是了解磷酸化在 运输功能和贩运。我们会继续调查这一领域 通过检验以下假设：（1）转运蛋白是磷酸化的， 去磷酸化作为其组成性运输过程的一部分，和（2） 蛋白激酶C（PKC）激活阻断基础转运蛋白去磷酸化， 从而干扰内吞转运蛋白运输。拟议的研究将 集中在一个代表性的单胺转运蛋白，多巴胺转运蛋白 （DAT）。我们将通过直接分析基础的和调节的 转运蛋白磷酸化和细胞系中的运输。本征域 将使用分子截短鉴定介导DAT磷酸化， 诱变方法。预计这些方法将提供一个 更清楚地了解转运蛋白运输和磷酸化是如何结合的。 这些结果有望对未来的治疗产生重大影响。 针对单胺相关精神兴奋剂药物滥用的战略。而且 结果将提高我们对造成这些问题的因素的理解， 单胺的可用性和大脑中的信号传导。

DESCRIPTION: (provided by applicant) Monoamine reuptake is a definitive step in the termination of neural transmission and is mediated by a family of Na+-/C1-dependent plasma membrane transporters. Monoamine transporters are the molecular targets for the psychostimulants cocaine, amphetamines, and MDMA ("ecstasy"), which bind to and inhibit transporters, thereby increasing extracellular monoamine concentrations and enhancing post-synaptic responses. As the major psychostimulant targets, transporter presentation at the cell surface is paramount to psychostimulant binding and action. Mounting evidence from a number of investigators demonstrates that activation of cellular signaling pathways dynamically modulates monoamine transporter cell surface presentation. Moreover, data from our laboratory demonstrates that transporters dynamically traffic under steady state conditions. Transporter regulation and trafficking correlate with changes in transporter phosphorylation states. However, the relationship between transporter trafficking and phosphorylation is not well defined. The major goals of this project are to understand the role of phosphorylation in transporter function and trafficking. We will pursue this area of investigation by testing the following hypotheses: (1) transporters are phosphorylated and dephosphorylated as part of their constitutive trafficking process, and (2) protein kinase C (PKC) activation blocks basal transporter dephosphorylation, thereby perturbing endocytic transporter trafficking. The proposed studies will focus on a representative monoamine transporter, the dopamine transporter (DAT). We will test our hypotheses by directly analyzing basal and regulated transporter phosphorylation and trafficking in cell lines. Intrinsic domains mediating DAT phosphorylation will be identified using molecular truncation and mutagenesis approaches. It is expected that these approaches will provide a clearer picture of how transporter trafficking and phosphorylation are coupled. Such results are expected to have a significant impact on future therapeutic strategies aimed at monoamine-related psychostimulant drug abuse. moreover, the outcomes will enhance our understanding of the factors contributing to monoamine availability and signaling in the brain.