RNA interference screen reveals an essential role of Nedd4-2 in dopamine transporter ubiquitination and endocytosis
RNA interference screen reveals an essential role of Nedd4-2 in dopamine transporter ubiquitination and endocytosis
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DOI：
10.1523/jneurosci.1301-06.2006
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发表时间：
2006-08-02
影响因子：
5.3
通讯作者：
Sorkin, Alexander
中科院分区：
文献类型：
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作者：
Sorkina, Tatiana;Miranda, Manuel;Sorkin, Alexander
多巴胺转运体（DAT）终止多巴胺神经传递的功能是由DAT的内吞转运调节的。为了阐明DAT内吞作用的机制，我们生成了一个全功能的人DAT突变体，其中一个血凝素表位（HA）被纳入第二个细胞外环。基于HA抗体摄取的内吞试验，旨在研究HA- dat在非神经元细胞和大鼠多巴胺能神经元中表达的组成型和蛋白激酶C（PKC）依赖性内化。对HA-DAT的pkc依赖性内吞作用的大规模RNA干扰分析揭示了E3泛素连接酶Nedd4-2（神经前体细胞表达，发育下调4-2）的必要和特异性作用，以及存在于网格蛋白包被窝中的衔接蛋白的参与，如epsin， Eps15（表皮生长因子途径底物克隆15）和Eps15R （Eps15相关蛋白）。Nedd4-2的缺失导致pkc依赖性DAT泛素化的显著降低。内源性Nedd4-2、epsin和Eps15与异源表达的人HA-DAT和从大鼠纹状体分离的内源性DAT共免疫沉淀。提出了一种新的DAT内吞机制模型，即pkc诱导的由Nedd4-2介导的DAT泛素化导致DAT与包被坑中的接头蛋白相互作用并加速DAT内吞。
The function of the dopamine transporter (DAT) to terminate dopamine neurotransmission is regulated by endocytic trafficking of DAT. To elucidate the mechanisms of DAT endocytosis, we generated a fully functional mutant of the human DAT in which a hemagglutinin epitope ( HA) was incorporated into the second extracellular loop. The endocytosis assay, based on the uptake of an HA antibody, was designed to study constitutive-and protein kinase C(PKC)-dependent internalization of HA-DAT expressed in non-neuronal cells and rat dopaminergic neurons. Large-scale RNA interference analysis of PKC-dependent endocytosis of HA-DAT revealed the essential and specific role of an E3 ubiquitin ligase, Nedd4-2 (neural precursor cell expressed, developmentally downregulated 4-2), as well as the involvement of adaptor proteins present in clathrin-coated pits, such as epsin, Eps15 ( epidermal growth factor pathway substrate clone 15), and Eps15R (Eps15-related protein). Depletion of Nedd4-2 resulted in a dramatic reduction of PKC-dependent ubiquitination of DAT. Endogenous Nedd4-2, epsin, and Eps15 were coimmunoprecipitated with heterologously expressed human HA-DAT and endogenous DAT isolated from rat striatum. A new mechanistic model of DAT endocytosis is proposed whereby the PKC-induced ubiquitination of DAT mediated by Nedd4-2 leads to interaction of DAT with adaptor proteins in coated pits and acceleration of DAT endocytosis.