Trafficking and Regulation of Monoamine Transporters

描述(申请人提供)：突触前再摄取是限制细胞外单胺水平和终止突触信号的主要手段。SLC6载体基因家族的生物胺转运体促进再摄取，是成瘾性和治疗性精神刺激剂以及抗抑郁药的主要靶点。这些药物有效地抑制单胺再摄取，从而增加细胞外单胺浓度，增强神经元信号转导，并显著调节单胺相关行为。因此，转运蛋白的活性和可用性是正常生物胺神经传递和精神活性药物疗效的关键决定因素。大量数据支持生物胺转运体的表面呈现不是静态的。相反，转运体受到强大的结构性内吞贩运的影响。多巴胺转运体(DAT)受蛋白激酶C(PKC)的激活和精神刺激剂可卡因和苯丙胺(AMPH)的刺激，调节DAT的内化和循环速率，最终降低DAT的表面可获得性。这些研究的主要目标是阐明控制基础和管制生物胺贩运的机制。在这一更新应用中，我们建立在我们以前在细胞和分子方法方面的优势以及我们之前的发现，即Rin GTP酶与DAT结合，并且是PKC介导的DAT内化所必需的。具体地说，我们的目标是(1)阐明控制Rin依赖的DAT内吞作用的分子决定因素，并确定是否需要Rin GTPase来进行amph介导的神经元细胞系的DAT运输，(2)确定PKC和amph介导的DAT运输是否具有区域依赖性，以及是否需要Rin GTPase来进行PKC和Amph介导的原位DAT运输，以及(3)测试是否需要Rin依赖的DAT运输来获得精神刺激奖赏。这些假说基于强大的初步数据，这些数据表明Rin与DAT而不是其他SLC6转运蛋白具有特定的相互作用，以及钙调蛋白在Rin下游信号转导中的潜在作用。我们将使用嵌合蛋白来定义赋予DAT/Rin相互作用特异性的DAT结构域。我们将使用生化和药理学方法，结合GTP酶突变体和shRNA介导的Rin敲除来确定Rin下游的信号通路，这些信号通路是神经元细胞系和小鼠纹状体中调节DAT内化所必需的和充分的。我们将进一步测试PKC和Amph介导的DAT运输是否在纹状体以区域特异性的方式发生，并在体内使用Rin敲除来测试Rin在原位调节DAT内吞中的作用。最后，我们将使用体内shRNA方法敲除小鼠VTA中的Rin GTP酶，以测试DAT交易是否需要精神刺激奖励。这些努力的结果将使我们更清楚地了解转运蛋白表面表达的控制机制，以及DAT运输在奖励行为中的作用。我们预计，我们的发现将极大地影响未来旨在治疗情感障碍和药物成瘾的策略。此外，这些结果无疑将加深我们对影响大脑中单胺利用率的分子因素的理解。

DESCRIPTION (provided by applicant): Presynaptic reuptake is the primary means to limit extracellular monoamine levels and terminate synaptic signaling. Biogenic amine transporters in SLC6 carrier gene family facilitate reuptake and are the major targets for addictive and therapeutic psychostimulants, as well as for antidepressants. These drugs potently inhibit monoamine reuptake, and thereby increase extracellular monoamine concentrations, enhance neuronal signaling and significantly modulate monoamine-related behaviors. Thus, transporter activity and availability are critical determinants of normal biogenic amine neurotransmission and psychoactive drug efficacy. A wealth of data supports that biogenic amine transporter surface presentation is not static. Rather, transporters are subject to robust constitutive endocytic trafficking. The dopamine transporter (DAT) is acutely modulated by protein kinase C (PKC) activation and exposure to psychostimulants cocaine and amphetamine (AMPH), which modulate DAT internalization and recycling rates, ultimately decreasing DAT surface availability. The major goal of these studies is to elucidate the mechanisms that control both basal and regulated biogenic amine trafficking. In this renewal application we build on our previous strengths in cellular and molecular approaches and our previous findings that Rin GTPase binds to DAT and is required for PKC-mediated DAT internalization. Specifically, we aim to (1) Elucidate the molecular determinants governing Rin-dependent DAT endocytosis and determine whether Rin GTPase is required for AMPH-mediated DAT trafficking in neuronal cell lines, (2) Determine whether PKC- and AMPH-mediated DAT trafficking are region-dependent and whether Rin GTPase is required for PKC- and AMPH-mediated DAT trafficking in situ, and (3) test whether Rin-dependent DAT trafficking is required for psychostimulant reward. These hypotheses are based on strong preliminary data that demonstrate specific Rin interactions with DAT, but not other SLC6 transporters, and a potential role for calmodulin in Rin downstream signaling. We will use chimeric proteins to define the DAT domains that confer specificity of the DAT/Rin interaction. We will use biochemical and pharmacological approaches in combination with GTPase mutants and shRNA-mediated Rin knockdown to determine the signaling pathways downstream of Rin that are necessary and sufficient for regulated DAT internalization in neuronal cell lines and mouse striatum. We will further test whether PKC- and AMPH-mediated DAT trafficking occur in a region-specific manner in the striatum, and use in vivo Rin knockdown to test the role Rin in regulated DAT endocytosis in situ. Finally, we will use in vivo shRNA approaches to knockdown Rin GTPase in mouse VTA to test whether DAT trafficking is required for psychostimulant reward. The results obtained from these endeavors will provide a clearer understanding of the mechanisms controlling transporter surface expression and the role of DAT trafficking in rewarding behaviors. We anticipate that our findings will greatly impact future strategies aimed at treating affective disorders and drug addiction. Moreover, the results will undoubtedly enhance our understanding of the molecular factors influencing monoamine availability in the brain.