Norepinephrine Transport Regulation By Phosphorylation

描述（由申请人提供）：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机的一系列生理过程。NE信号是由包括NE转运蛋白（NETs）在内的多种大分子动态调控的。中枢神经系统和外周表达的神经网络是三环类抗抑郁药和精神兴奋剂的重要靶点。NE转运改变在心血管疾病和脑部疾病中有文献记载。第二信使激酶介导的人类NET遗传变异的调节在一些心血管表型中发生改变，这意味着对NE运输调节的潜在机制的探索。NET含有几种激酶的推定磷酸化位点，包括蛋白激酶C （PKC）。最近，我们证明，在大鼠胎盘滋养层细胞中，PKC激活通过脂筏增强天然NET内化，并刺激转运蛋白磷酸化。在对天然NET调控的研究之后，我们的研究方向是探索NET磷酸化在NE转运调控中的作用。利用表达hNET的人胎盘滋养细胞系（HTR-hNET细胞），我们证明了hNET预测的PKC磷酸化基序的双重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。过量的神经激肽分泌与先兆子痫有关，因此，通过NK1R激活对胎盘NET的调节表明这种调节与维持正常妊娠的生理相关性。基于这些观察结果，我们建议在两个单独的特定目标中测试一个特定的假设，即NET磷酸化是NE运输调节所必需的。在Specific Aim I中，我们将识别NK1R/PKC调控NET磷酸化和表达的信号、特定位点和基序，以探索NET磷酸化与NE转运之间的关系。特异性Aim II将检验NK1R/PKC调节NET磷酸化和与转运蛋白相关蛋白在脂筏中发生的相互作用的假设，以及这种关联以一种依赖于活性的方式建立转运蛋白分布和功能，以应对输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信息，这些策略旨在调节NE转运，以治疗心血管疾病和脑部疾病。

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.