Rin GTPase And Dopamine Transporter Trafficking: Linking Mechanisms To Behavior

Rin GTP 酶和多巴胺转运蛋白贩运：将机制与行为联系起来

基本信息

批准号：
9250610
负责人：
Carolyn Gray Sweeney
金额：
$ 3.02万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-04-10 至 2018-04-09
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9250610
关键词：
Acute Address Amphetamines Antidepressive Agents Attention deficit hyperactivity disorder Behavior Behavioral Binding Binding Sites Brain Bupropion Carrier Proteins Cell Line Cell Surface Proteins Cell membrane Cell surface Cells Chimera organism Chimeric Proteins Chronic Co-Immunoprecipitations Cocaine Cognition Complex Corpus striatum structure County Data Disease Dopamine Dopaminergic Cell Down-Regulation Drug Prescriptions Endocytosis Energy Transfer Extracellular Space Foundations Guanosine Triphosphate Phosphohydrolases In Situ Investigation Laboratories Lead Link LoxP-flanked allele Mediating Methylphenidate Microscopy Molecular Molecular Target Motivation Motor Activity Movement Mus N-terminal Neurobiology Neurons Neurosciences Parkinson Disease Pharmaceutical Preparations Physiological Preparation Process Property Protein Kinase C Psychotropic Drugs Regulation Reporting Rewards Ritalin Role Schizophrenia Scientist Signal Transduction Slice Surface Synapses Testing Therapeutic Therapeutic Agents Time Training Ubiquitin Ubiquitination United States addiction career dopamine transporter drug of abuse extracellular functional loss in vivo knock-down mutant novel therapeutic intervention presynaptic psychostimulant public health relevance response small hairpin RNA stimulant abuse synergism trafficking transmission process

项目摘要

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.

描述（由申请人提供）：中枢神经系统中的多巴胺 (DA) 信号对于调节运动、认知、奖励和动机等复杂行为至关重要，异常的 DA 信号传输与帕金森病、注意力缺陷多动障碍、精神分裂症和精神分裂症直接相关。质膜多巴胺转运蛋白 (DAT) 是 DA 信号传导的核心，既限制细胞外 DA 的可用性，又维持突触前 DA。重要的是，DAT 是成瘾性和治疗性精神活性药物的主要目标，例如可卡因、安非他明、哌醋甲酯（利他林）和抗抑郁药安非他酮（维布特林），所有这些药物都会竞争性抑制 DAT 活性。数据支持这样的前提：DAT 持续往返于细胞表面，并且蛋白激酶 C (PKC) 激活可通过急剧调节 DAT 运输速率来快速降低 DAT 细胞表面表达，同时在研究控制受调节的分子机制方面已取得了一些进展。 DAT 运输，对这一复杂过程的详细了解尚未实现。此外，我们的实验室报告称，DAT 运输对 DAergic 功能的生理相关性知之甚少。 (RIT2)，特异性结合 DAT 羧基末端，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介导胞质 DAT 之间的协同作用。目标 1 研究将使用免疫共沉淀和 FRET 显微镜方法与嵌合 DAT 蛋白来定义必要且充分的细胞内 DAT 结构域。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 氨基和羧基末端对于 PKC 刺激的 DAT 内化的潜在协同需求。目标 2 研究将直接测试 Rin 介导的 DAT 运输在 DAergic 中的潜在作用。总而言之，这些研究将提供第一个直接研究 DAT 运输如何潜在影响奖励的研究。此外，完成这项研究将为我提供分子和行为神经科学方面的出色培训。