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）信号传导对于调节复杂行为（如运动、认知、奖励和动机）至关重要。异常DA能传递与帕金森病、注意力缺陷多动障碍、精神分裂症和成瘾直接相关。质膜多巴胺转运蛋白（DAT）是DA能信号传导的中心，既限制细胞外DA的可用性，又维持突触前DA的储存。重要的是，DAT是成瘾性和治疗性精神活性药物的主要靶点，如可卡因，安非他明，哌甲酯（利他林）和抗抑郁药安非他酮（Wellbutrin），所有这些药物都竞争性抑制DAT活性。丰富的 数据支持这样的前提，即DAT组成性地运输到细胞表面和从细胞表面运输，并且蛋白激酶C（PKC）活化通过剧烈调节DAT运输速率而迅速降低DAT细胞表面表达。虽然已经取得了一些进展，调查的分子机制，规范DAT贩运，详细了解这一复杂的过程尚未实现。此外，DAT运输对DA能功能的生理相关性知之甚少。我们的实验室报道，神经元GTdR，Rin（RIT 2），特异性结合到DAT的羧基末端，并需要PKC刺激的DAT内化。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介导胞质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DAT蛋白，以确定细胞内的DAT域是必要的和足够的赋予DAT/Rin相互作用。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研究将直接测试Rin介导的DAT运输在体内DA能行为中的潜在作用，使用DA能末端区域中的细胞特异性AAV 2介导的Rin敲低。总之，这些研究将提供直接研究DAT贩运如何潜在地影响奖励行为的第一次调查。此外，完成这一调查工作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.