Proteomic /Genetic Approaches to Monoamine Transporters

描述(由申请人提供)： 生物胺转运体通过控制特定分子靶点上递质作用的持续时间和强度，已经成为调节大脑中单胺传递的不可或缺的分子。三个不同的基因编码密切相关的质膜转运蛋白：多巴胺转运蛋白(DAT)、去甲肾上腺素转运蛋白(NET)和5-羟色胺转运蛋白(SEPT)。这些转运蛋白的主要作用是将释放的递质从突触间隙移回突触前终末，以便随后的囊泡存储和释放。编码生物胺转运蛋白的基因缺失会导致小鼠神经化学和行为的深刻变化，从而说明这些转运蛋白在控制突触前单胺稳态中的作用。广泛的神经和精神障碍，包括药物滥用、情感障碍和帕金森氏病，被认为与单胺传递和单胺转运体有关。事实上，众所周知，生物胺转运体是有效的精神刺激剂，如可卡因、苯丙胺和(+)-3，4-亚甲基二氧基甲基苯丙胺(MDMA或摇头丸)的生物靶标，以及用于治疗精神疾病的治疗药物，包括抑郁症、注意力缺陷多动障碍、强迫症和饮食障碍。最近，我们和其他实验室的结果表明，单胺转运蛋白是高度受调控的蛋白质，表明这些转运蛋白的组织比之前预期的更复杂。研究结果包括阐明DAT的寡聚体性质，鉴定参与组装和运输的结构域，以及鉴定和初步鉴定调节单胺转运体的靶向、运输和功能的相互作用蛋白。基于这些结果，我假设单胺转运体在神经元中以高度受调控的大分子蛋白复合体的形式存在。因此，这项研究的总体目标是通过识别相互作用的蛋白质并确定这种蛋白质-蛋白质相互作用在转运蛋白功能中的作用，来更清楚地了解与单胺转运蛋白相关的细胞调控机制。具体目标如下：目的I：利用蛋白质组学方法确定生物胺转运蛋白复合体中蛋白质-蛋白质相互作用的总补体/网络。目的II：用遗传学方法阐明PICK1和Hic-5在DAT功能调节中的生理作用。目的：从生化和功能上验证以前在酵母双杂交筛选中发现的可能的相互作用蛋白是否为真正的DAT相互作用蛋白。这些研究应该为大脑中单胺转运体的调节和治疗方法的新分子靶点提供前所未有的见解。这项研究提案还旨在进一步发展P.I.的科学事业，通过从国际公认的科学家那里获得蛋白质组和遗传方法方面的专业知识，以更好地解决生物胺转运体在正常和异常大脑功能中的作用，为获得独立的教职员工职位做准备。

DESCRIPTION (provided by applicant): Biogenic amine transporters have emerged as the indispensable molecules regulating monoamine transmission in the brain by controlling the duration and intensity of transmitter actions at specific molecular targets. Three distinct genes encode closely related plasma membrane transporter proteins: the dopamine transporter (DAT), the norepinephrine transporter (NET), and the serotonin transporter (SEPT). The primary role of these transporter proteins is to remove released transmitters from the synaptic cleft back into presynaptic terminals for subsequent vesicular storage and release. Deletion of the genes encoding biogenic amine transporters in mice results in profound neurochemical and behavioral changes, thereby, illustrating the impact of these transporters in controlling presynaptic monoamine homeostasis. A wide spectrum of neurological and psychiatric disorders, including drug abuse, affective disorders, and Parkinson's disease is thought to involve monoamine transmission and monoamine transporters. Indeed, it has been well established that biogenic amine transporters are the biological targets for potent psychostimulants such as cocaine, amphetamine, and (+)-3,4-methylenedioxymethamphetamine (MDMA or ecstasy) as well as therapeutic agents used to treat mental disorders including depression, attention deficit hyperactivity disorder, obsessive-compulsive disorder, and eating disorders. Recently, results from our and other laboratories suggest that monoamine transporters are highly regulated proteins and indicate a more complicated degree of organization for these transporters than previously anticipated. Findings include the elucidation of the oligomeric nature of DAT, the identification of domains involved in assembly and trafficking, and the identification and preliminary characterization of interacting proteins that regulate the targeting, trafficking, and function of monoamine transporters. Based on these results, I hypothesize that monoamine transporters exist as highly regulated macromolecular protein complexes in neurons. Thus, the overall goal of this research proposal is to provide a clearer understanding of the cellular regulatory mechanisms associated with monoamine transporters by identifying interacting proteins and establishing the role of such protein-protein interactions in transporter function. The following specific aims are proposed: Aim I: To determine the total complement/network of protein-protein interactions involved in biogenic amine transporter complexes in vivo using proteomic approaches. Aim II: To elucidate the physiological role of PICK1 and Hic-5 in the regulation of DAT function using genetic approaches. Aim IlI: To examine biochemically and functionally if putative interacting proteins identified previously in a yeast two-hybrid screen are real DAT interacting proteins. These studies should provide unprecedented insights into the regulation of monoamine transporters in brain and novel molecular targets for therapeutic approaches. This research proposal has also been designed to further develop the scientific career of the P.I. in preparation for an independent faculty position by acquiring expertise in proteomic and genetic approaches from internationally recognized scientists to better address the role of biogenic amine transporters in normal and abnormal brain function.