Serotonin Transporter Phosphorylation

描述(申请人提供)：神经递质5-羟色胺 (5-羟色胺或5-羟色胺)在许多行为和行为中发挥重要作用 生理功能包括情绪，睡眠，食欲，记忆， 血管收缩和上瘾。突触前5-羟色胺转运体(SERT)是 负责有效清除突触间隙中的5-羟色胺 参与终止5-羟色胺突触信号的主要分子。Sserts是 多种抗抑郁药物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。更改的SERT函数 SERT基因的多态与焦虑、抑郁、 自杀、自闭症和药物滥用。尽管有几十年的外源操纵 对于临床用途的SERS，我们对内源性 调节SERT活性的机制。最近，我开始意识到 丝氨酸被内源性调控的机制。我发现SERT 活性受蛋白激酶C(PKC)和蛋白磷酸酶(PKC)的强烈调控 2A(PP2A)连接的通路，这种调节主要通过 膜转运，并伴随着转运体的快速磷酸化。 此外，依赖于PKC的调节受到转运体自身的影响 5-羟色胺、抗抑郁药、可卡因和 安非他明。这项建议的重点是彻底描述SERT的特征 PKC和钙/钙调素依赖的激酶II介导的磷酸化 (CaMKII)与功能性转运体调控有关 分子生物学和生化方法。具体目标是实现 这些目标是：1)表征PKC和CaMKII介导的SERT 磷酸化，并鉴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 SERT中PKC和CaMKII介导的磷酸化和去磷酸化 功能，根据(A)SERT磷酸化的可逆性和特异性 和去磷酸化与SERT活性和封存的关系，(B) SERT磷酸化的结构域和位点与调控的关系 隔离和5-羟色胺转位，以及(C)SERT底物的相互作用 和拮抗剂在调节SERT的磷酸化和表达。这些 研究将为PKC和CAMKII的作用(S)提供批判性见解，以及 磷酸酶在SERT功能调控中的作用及其可能提供的新机制 与了解药物作用和改变的法规有关 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.