PKC-beta Regulation of the Dopmaine Transporter Trafficking

摘要 突触中的多巴胺能传递主要是通过将多巴胺(DA)移入 通过多巴胺转运蛋白(DAT)的突触前神经末梢，这是Na+/Cl-依赖的超家族之一 神经递质转运体。DAT是安非他明和安非他明等精神刺激剂的主要目标 可卡因。DAT功能障碍与神经退行性疾病和神经精神疾病有关，如 帕金森病，精神分裂症，注意力缺陷和多动障碍，以及毒瘾。我们有 蛋白激酶C(PKC)在基础和底物诱导的DAT调节中的新作用 贩运/活动。有趣的是，PKC对DAT贩运/活动的管制似乎涉及到另外两个DAT 直接相互作用的蛋白质RACK1和D2多巴胺受体(D2R)。RACK1增强Amph诱导的DA 在稳定表达人DAT(HDAT)的小鼠中脑细胞中与PKC？II共表达时的外流 多巴胺能细胞系(MN9D)。D2R激活增加PKC？WT(PKC？+/+)小鼠的DAT转运/活性 但在PKC基因敲除(PKC-/-)小鼠中不存在。这些数据表明RACK1和D2R可能是重要的 促进PKC在DAT调控中的中间作用。该提案的总体目标是 鉴定RACK1和D2R在PKC基础和诱导的DAT运输和活性调节中的作用。 该建议的具体目的是检验以下假设：1)RACK1促进PKC与DAT的偶联 及其对基础和底物诱导的DAT转运和活性的调节作用；(2)D2R诱导的DAT 贩运需要PKC？，涉及在D2R激活时贩运D2R-DAT复合体。这些 假说将通过在MN9D细胞中过度表达、敲除或敲除靶蛋白来检验 和/或PKC？+/+和PKC？-/-小鼠。该项目将提供一条新的分子和细胞途径，以实现 靶向RACK1，D2R，RACK1，D2R，RACK1，D2R， 和PKC？作为多巴胺能动态平衡的调节剂。

Abstract Dopaminergic transmission in the synapse is primarily terminated by removal of dopamine (DA) into presynaptic nerve terminals via the dopamine transporter (DAT), one of a superfamily of Na+/Cl--dependent neurotransmitter transporters. DAT is a major target of psychostimulants such as amphetamine (AMPH) and cocaine. Dysfunctional DAT has been implicated in neurodegenerative and neuropsychiatric disorders such as Parkinson disease, schizophrenia, attention deficit and hyperactivity disorder, and drug addiction. We have demonstrated a novel role of protein kinase C¿ (PKC¿) in regulation of basal and substrate-induced DAT trafficking/activity. Interestingly, PKC¿ regulation of DAT trafficking/activity seems to involve two other DAT directly interacting proteins RACK1 and D2 dopamine receptor (D2R). RACK1 enhances AMPH-induced DA efflux when co-expressed with PKC¿II in a human DAT (hDAT)-stably expressing mouse mesencephalic dopaminergic cell line (MN9D). Activation of D2R increases DAT trafficking/activity in PKC¿ WT (PKC¿+/+) mice but not in PKC¿ knockout (PKC¿-/-)mice. These data suggest that RACK1 and D2R may be important intermediate players for facilitation of PKC¿ effect on DAT regulation. The overall objective of the proposal is to characterize roles of RACK1 and D2R in PKC¿ regulation of basal and induced DAT trafficking and activity. The specific aims of this proposal are to test the hypothesis that 1) RACK1 facilitates PKC¿ coupling to DAT and its regulatory effect on basal and substrate-induced DAT trafficking and activity; (2) D2R-induced DAT trafficking requires PKC¿ and involves trafficking of the D2R-DAT complex upon D2R activation. These hypotheses will be tested using overexpression, knockdown, or knockout of targeted proteins in MN9D cells and/or PKC¿+/+ and PKC¿-/- mice. This project will provide a new molecular and cellular avenue for potential drug intervention to drug addiction, psychiatric, and neurodegenerative diseases by targeting RACK1, D2R, and PKC¿ as modulators of dopaminergic homeostasis.