Rin GTPase And Dopamine Transporter Trafficking: Linking Mechanisms To Behavior

 描述(由申请人提供)：中枢神经系统中的多巴胺(DA)信号对于调节复杂的行为，如运动、认知、奖励和动机是必不可少的。异常的多巴能传递与帕金森氏症、注意力缺陷多动障碍、精神分裂症和成瘾直接相关。质膜多巴胺转运体(DAT)是DA能信号转运体的中枢，既限制细胞外DA的可获得性，又维持突触前DA的储存。重要的是，DAT是成瘾和治疗精神活性药物的主要靶点，如可卡因、苯丙胺、哌甲酸甲酯(利他林)和抗抑郁剂安非他酮(Wellbutrin)，所有这些都竞争性地抑制DAT的活性。一大笔钱 数据支持这一假设，即DAT在细胞表面的往返运输是结构性的，而蛋白激酶C(PKC)的激活通过剧烈地调节DAT的运输速率而迅速减少DAT细胞表面的表达。虽然已在研究管理受管制的DAT贩运的分子机制方面取得了一些进展，但对这一复杂过程的详细了解尚未实现。此外，DAT贩运对DAR能功能施加的生理相关性还知之甚少。我们的实验室报道了神经元GTP酶Rin(RIT2)特异性地结合到DAT的羧基末端，并且是PKC刺激的DAT内化所必需的。在拟议的研究中，我计划研究DAT/Rin相互作用所需的分子基础，以及这些相互作用如何潜在地调节胞质DAT结构域之间的协同作用。目的1研究将使用免疫共沉淀和FRET显微镜方法与嵌合的DAT蛋白来确定细胞内的DAT结构域，这些结构域是实现DAT/Rin相互作用的必要条件和充分条件。嵌合和点突变转运蛋白将进一步揭示DAT氨基和羧基末端对PKC刺激的DAT内化的潜在协同需求。目的2项研究将利用细胞特异性的、AAV2介导的DAR能终末区域敲除Rin，直接测试Rin介导的DAT转运在体内DAR能行为中的潜在作用。综上所述，这些研究将提供第一次调查，直接检查DAT贩运如何潜在地影响奖励行为。此外，这条研究路线的完成将为我提供分子和行为神经科学方面的出色培训。

 DESCRIPTION (provided by applicant): Dopamine (DA) signaling in the CNS is essential for modulating complex behaviors such as movement, cognition, reward, and motivation. Aberrant DAergic transmission is directly linked to Parkinson's disease, attention-deficit hyperactivity disorder, schizophrenia and addiction. The plasma membrane dopamine transporter (DAT) is central to DAergic signaling, and both limits extracellular DA availability and maintains presynaptic DA stores. Importantly, DAT is the primary target for both addictive and therapeutic psychoactive drugs, such as cocaine, amphetamine, methylphenidate (Ritalin), and the antidepressant bupropion (Wellbutrin), all of which competitively inhibit DAT activity. A wealth of data supports the premise that DAT constitutively traffics to and from the cell surface, and that protein kinase C (PKC) activation rapidly decreases DAT cell surface expression by acutely modulating DAT trafficking rates. While some progress has been made investigating the molecular mechanisms that govern regulated DAT trafficking, a detailed understanding of this complex process has yet to be achieved. Further, the physiological relevance that DAT trafficking imposes onto DAergic function is poorly understood. Our laboratory reported that neuronal GTPase, Rin (RIT2), specifically binds to the DAT carboxy terminus and is required for PKC-stimulated DAT internalization. In the proposed studies, I plan to investigate the molecular underpinnings required for DAT/Rin interactions, and how these interactions potentially mediate synergy between cytosolic DAT domains. Aim 1 studies will use co-immunoprecipitations and FRET microscopy approaches with chimeric DAT proteins to define the intracellular DAT domains that are necessary and sufficient to confer DAT/Rin interactions. Chimeric and point mutant transporters will further reveal the potential synergistic requirement of DAT amino- and carboxy-termini for PKC-stimulated DAT internalization. Aim 2 studies will directly test the potential role of Rin-mediated DAT trafficking in DAergic behaviors in vivo, using cell-specific, AAV2-mediated Rin knockdown in DAergic terminal regions. Taken together, these studies will provide the first investigations that directly examine how DAT trafficking potentially impacts rewarding behavior. Moreover, completion of this investigative line will provide me with outstanding training in molecular and behavioral neuroscience.