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信号转导受包括NE转运蛋白（NET）在内的多种大分子的动态调节。在CNS和外周中表达的NET是三环类抗抑郁药和精神兴奋剂的重要靶点。在心血管疾病和脑部疾病中记录了NE转运的改变。在某些心血管表型中，第二信使连接激酶介导的人类NET遗传变异体的调节发生改变，这意味着寻找NE转运调节的潜在机制。NET含有几种激酶的假定磷酸化位点，包括蛋白激酶C（PKC）。最近，我们证明，在大鼠胎盘滋养层细胞，PKC激活增强天然NET通过脂筏内化和刺激转运蛋白磷酸化。在对天然NET调节的研究之后，我们将我们的研究导向探索NET磷酸化在NE转运调节中的作用。使用人胎盘滋养层细胞系表达hNET（HTR-hNET细胞），我们证明了在预测的PKC磷酸化基序的hNET的双突变防止神经激肽1受体（NK 1 R）/PKC介导的转运调控和磷酸化。过度的神经激肽分泌与先兆子痫有关，因此，NK 1 R激活对胎盘NET的调节表明这种调节在维持正常妊娠中的生理相关性。基于这些观察结果，我们建议测试一个特定的假设，即NET磷酸化需要NE运输调节在两个单独的特定目的。在第一部分中，我们将鉴定参与NK 1 R/PKC调控NET磷酸化和表达的信号、特异位点和模体，以探讨NET磷酸化和NE转运之间的关系。具体目标II将测试的假设，即NK 1 R/PKC调节NET磷酸化和相互作用与转运相关蛋白发生在脂筏，这种关联建立转运蛋白的分布和功能，以活性依赖的方式，以科普在传入信号的环境中的需求。这项研究的结果将提供信息，可以用于开发新的治疗策略，旨在NE转运调节治疗心血管疾病和脑疾病。

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.