Psychoactive substrates stimulate dopamine transporter phosphorylation and down-regulation by cocaine-sensitive and protein kinase C-dependent mechanisms
Psychoactive substrates stimulate dopamine transporter phosphorylation and down-regulation by cocaine-sensitive and protein kinase C-dependent mechanisms
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DOI：
10.1074/jbc.m501969200
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发表时间：
2005-12-09
影响因子：
4.8
通讯作者：
Vaughan, RA
中科院分区：
文献类型：
--
作者：
Cervinski, MA;Foster, JD;Vaughan, RA
多巴胺转运蛋白（DAT）在接触精神兴奋剂底物后会发生细胞内隔离和功能下调。为了研究这些反应背后的潜在机制，我们使用野生型和 N 端截短突变体检查了苯丙胺和甲基苯丙胺 (METH) 对大鼠 DAT 磷酸化和下调的急性体外和体内影响。通过用安非他明或 METH 体外处理 rDAT LLC-PK1 细胞，通过 (PO4)-P-32 代谢标记评估的 DAT 磷酸化增加高达 2 倍，并且通过体外应用或体内注射 METH 在大鼠纹状体组织中也同样增加。多巴胺转运阻滞剂 (-)-可卡因不影响 DAT 磷酸化，但阻止 METH 诱导的磷酸化增加。 METH 诱导的 DAT 磷酸化也被蛋白激酶 C 阻断剂双吲哚酰马来酰亚胺 I 所阻止，并且在 N 末端截短的蛋白质中不存在，该蛋白质缺乏前 21 个残基，包括 6 个丝氨酸，这也代表佛波酯诱导的磷酸化位点。 METH 诱导的转运下调也依赖于可卡因和蛋白激酶 C，但在 N 端截短突变体中保留了下来。这些结果表明，METH 的转运或结合通过需要蛋白激酶 C 的机制刺激 DAT 磷酸化和下调，但 METH 诱导的下调可以独立于直接转运蛋白磷酸化而发生。 DAT 磷酸化受到安非他明刺激的发现揭示了这些药物以前未知的作用，这种作用不是由可卡因产生的，可能与强化有关。
Dopamine transporters (DATs) undergo intracellular sequestration and functional down-regulation upon exposure to psychostimulant substrates. To investigate the potential mechanism underlying these responses, we examined the acute in vitro and in vivo effects of amphetamine and methamphetamine ( METH) on phosphorylation and down-regulation of rat DAT using wild type and N-terminal truncation mutants. Phosphorylation of DAT assessed by (PO4)-P-32 metabolic labeling was increased up to 2-fold by in vitro treatment of rDAT LLC-PK1 cells with amphetamine or METH and was similarly increased in rat striatal tissue by in vitro application or in vivo injection of METH. The dopamine transport blocker (-)-cocaine did not affect DAT phosphorylation but prevented the phosphorylation increase induced by METH. Phosphorylation of DAT induced by METH was also prevented by the protein kinase C blocker bisindoylmaleimide I and was absent in an N-terminally truncated protein that lacks the first 21 residues including 6 serines that also represent the site of phorbol ester induced phosphorylation. Down-regulation of transport induced by METH was also cocaine- and protein kinase C-dependent but was retained in the N-terminal truncation mutant. These results demonstrate that transport or binding of METH stimulates DAT phosphorylation and down-regulation by a mechanism that requires protein kinase C but that METH-induced down-regulation can occur independently of direct transporter phosphorylation. The finding that DAT phosphorylation is stimulated by amphetamines reveals a previously unknown effect of these drugs that is not produced by cocaine and may be related to reinforcement.