Moritz Amy E, Rastedt Danielle E, Stanislowski Daniel J, Shetty Madhur, Smith Margaret A, Vaughan Roxanne A, Foster James D
From the Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58202-9061.
From the Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58202-9061
J Biol Chem. 2015 Nov 27;290(48):29095-105. doi: 10.1074/jbc.M115.667055. Epub 2015 Sep 30.
The dopamine transporter is a neuronal protein that drives the presynaptic reuptake of dopamine (DA) and is the major determinant of transmitter availability in the brain. Dopamine transporter function is regulated by protein kinase C (PKC) and other signaling pathways through mechanisms that are complex and poorly understood. Here we investigate the role of Ser-7 phosphorylation and Cys-580 palmitoylation in mediating steady-state transport kinetics and PKC-stimulated transport down-regulation. Using both mutational and pharmacological approaches, we demonstrate that these post-translational modifications are reciprocally regulated, leading to transporter populations that display high phosphorylation-low palmitoylation or low phosphorylation-high palmitoylation. The balance between the modifications dictates transport capacity, as conditions that promote high phosphorylation or low palmitoylation reduce transport Vmax and enhance PKC-stimulated down-regulation, whereas conditions that promote low phosphorylation or high palmitoylation increase transport Vmax and suppress PKC-stimulated down-regulation. Transitions between these functional states occur when endocytosis is blocked or undetectable, indicating that the modifications kinetically regulate the velocity of surface transporters. These findings reveal a novel mechanism for control of DA reuptake that may represent a point of dysregulation in DA imbalance disorders.
多巴胺转运体是一种神经元蛋白，它驱动多巴胺（DA）的突触前再摄取，并且是大脑中递质可用性的主要决定因素。多巴胺转运体功能受蛋白激酶C（PKC）和其他信号通路调控，其机制复杂且了解甚少。在此，我们研究丝氨酸-7磷酸化和半胱氨酸-580棕榈酰化在介导稳态转运动力学和PKC刺激的转运下调中的作用。使用突变和药理学方法，我们证明这些翻译后修饰相互调节，导致转运体群体呈现高磷酸化-低棕榈酰化或低磷酸化-高棕榈酰化。修饰之间的平衡决定了转运能力，因为促进高磷酸化或低棕榈酰化的条件会降低转运最大速度（Vmax）并增强PKC刺激的下调，而促进低磷酸化或高棕榈酰化的条件会增加转运Vmax并抑制PKC刺激的下调。当内吞作用被阻断或无法检测到时，这些功能状态之间会发生转变，表明这些修饰在动力学上调节表面转运体的速度。这些发现揭示了一种控制DA再摄取的新机制，这可能是DA失衡疾病中失调的一个点。
