Serotonin Transporter Phosphorylation

描述(申请人提供)：在5-羟色胺能突触，5-羟色胺(5-羟色胺)转运体(SERTS)通过重新摄取释放的5-羟色胺进入突触前终末来决定突触5-羟色胺的浓度和持续时间。因此，突触前质膜中SERT的活性是决定5-羟色胺能神经传递强度从而决定行为和生理功能的关键因素。SERTS是几种成瘾和治疗药物的高亲和力分子靶点，包括可卡因、安非他明、亚甲二氧基甲基安非他明“摇头丸”、三环抗抑郁药和SSRIs。在我们之前的资助期间，我们和合作者确定了几种蛋白激酶调节急性SERT的信号机制，并证明了脂筏介导的胺转运体在天然和体外模型系统中的重新分布。然而，SERT磷酸化在5-羟色胺转运调节中的生理相关性以及相关的部位或结构域仍有待研究。这一新应用的主要目的是确定SERT磷酸化在正常的5-羟色胺转运调节以及与人类强迫症(OCD)和其他疾病表型的SERT编码变体相关的失调中的生理作用。特定目的1将验证这样一种假设，即蛋白激酶PKG和p38Mark在组胺受体(HSR)激活后，在特定的磷酸位点/基序上磷酸化SERT，并调节5HT的运输。特定目的2将检查hSERT编码变体是否存在于改变的磷酸化状态，以及是否对H3R/PKG/p38 Mark调节不敏感。具体目标3将验证以下假设：在体内，HSR的调节改变了SERT的磷酸化，调节了5-羟色胺的运输，从而改变了突触5-羟色胺水平。本申请中建议的研究将：(I)确定影响5-羟色胺转运的生理相关信号/受体；(Ii)确定转运体磷酸化和5-羟色胺转运之间的机制联系；(Iii)建立负责SERT分布的细胞机制；以及(Iv)提供与hSERT变体相关的失调调节信息，这些信息可用于开发新的治疗策略，旨在调节SERT在治疗强迫症、抑郁症和药物滥用等精神疾病中的功能。

DESCRIPTION (provided by applicant): At serotonergic synapse, serotonin (5-HT) transporters (SERTs) dictate the concentration and duration of synaptic 5-HT by re-uptake of released 5-HT into the presynaptic terminals. Therefore, the activity of SERT in the presynaptic plasma membrane is a critical factor that determines the strength of serotonergic neurotransmission and hence behavioral and physiological functions. SERTs are high-affinity molecular targets for several addictive and therapeutic drugs including cocaine, amphetamine, MDMA "ecstasy", tricyclic antidepressants and SSRIs. In our previous funding period, we, and collaborators identified the signaling mechanisms of acute SERT regulation by several protein kinases, and documented lipid raft-mediated redistribution of amine transporters in native and in vitro model systems. However, the physiological relevance of SERT phosphorylation in 5-HT transport regulation, and the sites or domains responsible have yet to be examined. The major goal of this renewal application is to determine the physiological role of SERT phosphorylation in normal 5-HT transport regulation and in dysregulations associated with human SERT coding variants of obsessive-compulsive disorder (OCD) and other disease phenotypes. Specific Aim 1 will test the hypothesis that protein kinases PKG and p38 MARK phosphorylate SERT on specific phospho-sites/motifs following histamine receptor (HSR) activation and regulate 5HT transport. Specific Aim 2 will examine whether hSERT coding variants exist in altered phosphorylation state and are insensitive to H3R/PKG/p38 MARK modulation. Specific Aim 3 will test the hypotheses that in vivo modulation of HSR alters SERT phosphorylation regulating 5HT transport and hence synaptic 5HT levels. The studies proposed in this application will (i) determine the physiologically relevant signals/receptors influencing 5-HT transport; (ii) define the mechanistic link between transporter phosphorylation and 5-HT transport; (iii) establish the cellular machinery responsible for SERT distribution; and (iv) provide information on dysregulations associated with hSERT variants that could be of use in the development of new therapeutic strategies aimed at regulating SERT function in the treatment of mental illnesses such as OCD, depression and drug abuse.