Norepinephrine Transport Regulation By Phosphorylation

描述(申请人提供)：儿茶酚胺，去甲肾上腺素(NE)控制从血管收缩和心率到注意力和动力的一系列生理过程。NE信号受多种大分子的动态调节，其中包括NE转运体(Net)。在中枢和外周表达的Net是三环类抗抑郁药和精神刺激剂的重要靶点。在心血管疾病和大脑疾病中，NE运输的改变是有文献记载的。第二，在某些心血管表型中，信使连接激酶介导的人类Net基因变异的调节发生了改变，这意味着寻找NE运输调节的潜在机制。Net含有蛋白激酶C(PKC)等几种蛋白的可能的磷酸化位点。最近，我们证明，在大鼠胎盘滋养层细胞中，PKC的激活通过脂筏增强天然净内化，并刺激转运蛋白的磷酸化。在对天然网络调节的研究之后，我们的研究方向是探索网络磷酸化在去甲肾上腺素运输调节中的作用。使用表达hNET的人胎盘滋养层细胞系(HTR-hNET细胞)，我们证明了预测的hNET的PKC磷酸化基序的双突变阻止了神经激肽1受体(NK1R)/PKC介导的转运体调节和磷酸化。过多的神经激肽分泌与子痫前期有关，因此，NK1R激活对胎盘网的调节表明这种调节在维持正常妊娠方面具有生理学意义。基于这些观察，我们建议检验一个特定的假设，即在两个不同的特定目的中，去甲肾上腺素运输调节需要净磷酸化。在特定的目标I中，我们将确定NK1R/PKC调节的净磷酸化和表达所涉及的信号、特定位点和基序，以探索净磷酸化与NE转运的关系。特殊目的II将检验这样的假设，即NK1R/PKC调控的净磷酸化和与转运体相关蛋白的相互作用发生在脂筏中，并且这种关联以一种活性依赖的方式建立转运体的分布和功能，以应对传入信号环境中的需求。这项研究的结果将为开发新的治疗策略提供有用的信息，目的是在心血管疾病和脑部疾病的治疗中调节NE的运输。

DESCRIPTION (provided by applicant): The catecholamine, norepinephrine (NE) governs a spectrum of physiologic processes from vasoconstriction and heart rate to attention and motivation. NE signaling is dynamically regulated by a diverse set of macromolecules including NE transporters (NETs). NETs expressed in the CNS and periphery are important targets for tricyclic antidepressants and psychostimulants. Altered NE transport is documented in cardiovascular diseases and brain disorders. Second messenger linked kinase mediated regulation of human genetic variants of NET is altered in some cardiovascular phenotypes signifying the search for underlying mechanisms of NE transport regulation. NET contains putative phosphorylation sites for several kinases including protein kinase C (PKC). Recently, we demonstrated that, in rat placental trophoblasts, PKC activation enhances native NET internalization via lipid rafts and stimulates transporter phosphorylation. Following the studies on native NET regulation, we directed our studies to explore the role of NET phosphorylation in NE transport regulation. Using a human placental trophoblast cell line expressing hNET (HTR-hNET cells), we demonstrate that a double mutation at a predicted PKC phosphorylation motif of hNET prevents neurokinin 1 receptor (NK1R)/PKC-mediated transporter regulation and phosphorylation. Excessive neurokinin secretion is linked to pre-eclampsia, and thus, the regulation of placental NET by NK1R activation suggests physiological relevance of such regulation in the maintenance of a normal pregnancy. Based on these observations, we propose to test a specific hypothesis that NET phosphorylation is required for NE transport regulation in two separate Specific Aims. In Specific Aim I, we will identify the signals, specific sites and motifs involved in NK1R/PKC regulated NET phosphorylation and expression to explore the relationship between NET phosphorylation and NE transport. Specific Aim II will test the hypothesis that NK1R/PKC regulated NET phosphorylation and interaction with transporter-associated proteins occur in lipid rafts, and that this association establishes transporter distribution and function in an activity-dependent manner to cope with the demands in the milieu of incoming signals. The results from this research will provide information that could be of use in the development of new therapeutic strategies aimed at NE transport regulation in the treatment of both cardiovascular diseases and brain disorders.