Serotonin Transporter Phosphorylation

描述（由申请人提供）：神经递质血清素 5-羟色胺（5-hydroxytryptamine或5 HT）在许多行为和 生理功能包括情绪、睡眠、食欲、记忆， 血管收缩和成瘾。突触前5-羟色胺转运蛋白（SERT）是 负责有效清除突触间隙中的5-HT， 参与终止5 HT突触信号传导的主要分子。SERT是 多种抗抑郁药的高亲和力分子靶点，以及 滥用包括可卡因、安非他明和摇头丸。“更改SERT功能 SERT基因多态性与焦虑，抑郁， 自杀自闭症和药物滥用尽管几十年来的外源操纵 对于临床目的的SERT，我们对内源性 调节SERT活性的机制。最近，我开始识别 SERT的内源性调节机制。我发现SERT 活性受蛋白激酶C（PKC）和蛋白磷酸酶的剧烈调节 2A（PP 2A）相关途径，这种调节主要通过 膜运输，并伴随着快速转运蛋白磷酸化。 此外，PKC依赖性调节受到转运蛋白自身的影响。 内在活性，并通过5-HT，抗抑郁药，可卡因和 安非他明该提案的重点是彻底表征SERT PKC和Ca 2 +/钙调蛋白依赖性激酶II介导的磷酸化 （CaMKII）与功能性转运蛋白调节的关系， 分子生物学和生物化学方法。要实现的具体目标 这些目标是：1）表征PKC和CaMKII介导的SERT 磷酸化，并确定突触前受体和药理学 调节SERT功能的刺激和2）表征SERT的作用 PKC和CaMKII介导的磷酸化和SERT中的去磷酸化 （a）SERT磷酸化的可逆性和特异性 和去磷酸化与SERT活性和螯合的关系，（B） SERT磷酸化的结构域和位点与调节的 螯合和5 HT易位，和（c）SERT底物的相互作用 和拮抗剂调节SERT磷酸化和表达。这些 研究将为PKC和CaMKII的作用提供重要见解， 磷酸酶在SERT功能调节中可能提供新的机制 与理解药物作用和改变的调节有关， 会导致精神疾病

DESCRIPTION (provided by applicant): The neurotransmitter serotonin (5-hydroxytryptamine or 5HT) plays an important role in numerous behavioral and physiological functions including mood, sleep, appetite, memory, vasoconstriction and addiction. Presynaptic serotonin transporters (SERTs) are responsible for efficient clearance of 5HT from synaptic spaces and are the primary molecules involved in terminating 5HT synaptic signaling. SERTs are high-affinity molecular targets for multiple antidepressants, and drugs of abuse including cocaine, amphetamine and MDMA "ecstasy." Altered SERT function and SERT gene polymorphisms have been implicated in anxiety, depression, suicide, autism and substance abuse. Despite decades of exogenous manipulation of SERTs for clinical ends, we have little knowledge of the endogenous mechanisms that regulate SERT activity. Recently, I have begun to identify mechanisms by which SERTs are regulated endogenously. I found that SERT activity is acutely regulated by protein kinase C (PKC) and protein phosphatase 2A (PP2A) linked pathways, that this regulation occurs primarily through membrane trafficking, and is accompanied by rapid transporter phosphorylation. Moreover, PKC-dependent regulation is impacted by the transporter's own intrinsic activity and is modulated by 5HT, antidepressants, cocaine and amphetamines. The focus of this proposal is to characterize thoroughly the SERT phosphorylation mediated by PKC and Ca2+/calmodulin-dependent kinase II (CaMKII) in relation to functional transporter regulation by combining molecular biologic and biochemical approaches. The specific aims to achieve these goals are: 1) to characterize the PKC- and CaMKII- mediated SERT phosphorylation, and to identify presynaptic receptors and pharmacological stimuli that regulate SERT function and 2) to characterize the role SERT phosphorylation mediated by PKC and CaMKII and dephosphorylation in SERT function, in terms of (a) reversibility and specificity of SERT phosphorylation and dephosphorylation in relationship to SERT activity and sequestration, (b) the domains and sites of SERT phosphorylation in relationship to regulated sequestration and 5HT translocation, and (c) the interaction of SERT substrates and antagonists in the regulation of SERT phosphorylation and expression. These studies will provide critical insights to the role(s) of PKC and CaMKII, and phosphatases in SERT functional regulation and may provide new mechanisms relevant for the understanding of drug action and altered regulation that contribute to mental illness.