Jiang Houbo, Jiang Qian, Feng Jian
Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, New York 14214, USA.
J Biol Chem. 2004 Dec 24;279(52):54380-6. doi: 10.1074/jbc.M409282200. Epub 2004 Oct 18.
Mutations of parkin, a protein-ubiquitin E3 ligase, are linked to Parkinson's disease (PD). Although a variety of parkin substrates have been identified, none of these is selectively expressed in dopaminergic neurons, whose degeneration plays a critical role in PD. Here we show that parkin significantly increased dopamine uptake in the human dopaminergic neuroblastoma cell line SH-SY5Y. This effect was accompanied by increased V(max) of dopamine uptake and unchanged K(m). Consistent with this, increased binding sites for dopamine transporter (DAT) ligand were observed in SH-SY5Y cells overexpressing parkin. The results were confirmed when parkin was transfected in HEK293 cells stably expressing DAT. In these cells, parkin enhanced the ubiquitination and degradation of DAT, increased its cell surface expression, and augmented dopamine uptake. The effects of parkin were significantly abrogated by its PD-causing mutations. Because the cell surface expression of functional DAT requires its oligomerization, misfolded DAT, induced either by the protein glycosylation inhibitor tunicamycin or by its C-terminal truncation, significantly attenuated cell surface expression of native DAT and reduced dopamine uptake. Expression of parkin, but not its T240R mutant, significantly alleviated these detrimental effects of misfolded DAT. Thus, our studies suggest that parkin increases dopamine uptake by enhancing the ubiquitination and degradation of misfolded DAT, so as to prevent it from interfering with the oligomerization and cell surface expression of native DAT. This function of parkin would enhance the precision of dopaminergic transmission, increase the efficiency of dopamine utilization, and reduce dopamine toxicity on neighboring cells.
泛素蛋白连接酶帕金（Parkin）的突变与帕金森病（PD）相关。尽管已鉴定出多种帕金底物，但这些底物均未在多巴胺能神经元中选择性表达，而多巴胺能神经元的退化在帕金森病中起关键作用。在此我们表明，帕金显著增加了人多巴胺能神经母细胞瘤细胞系SH-SY5Y中的多巴胺摄取。这种作用伴随着多巴胺摄取的V(max)增加而K(m)不变。与此一致的是，在过表达帕金的SH-SY5Y细胞中观察到多巴胺转运体（DAT）配体的结合位点增加。当帕金转染到稳定表达DAT的HEK293细胞中时，结果得到证实。在这些细胞中，帕金增强了DAT的泛素化和降解，增加了其细胞表面表达，并增强了多巴胺摄取。帕金的帕金森病致病突变显著消除了这些作用。由于功能性DAT的细胞表面表达需要其寡聚化，由蛋白质糖基化抑制剂衣霉素或其C末端截短诱导的错误折叠的DAT显著减弱了天然DAT的细胞表面表达并降低了多巴胺摄取。帕金的表达而非其T240R突变体显著减轻了错误折叠的DAT的这些有害作用。因此，我们的研究表明，帕金通过增强错误折叠的DAT的泛素化和降解来增加多巴胺摄取，从而防止其干扰天然DAT的寡聚化和细胞表面表达。帕金的这一功能将提高多巴胺能传递的精确性，增加多巴胺利用效率，并降低多巴胺对邻近细胞的毒性。
