Granas Charlotta, Ferrer Jasmine, Loland Claus Juul, Javitch Jonathan A, Gether Ulrik
Molecular Neuropharmacology Group, Department of Pharmacology, Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark.
J Biol Chem. 2003 Feb 14;278(7):4990-5000. doi: 10.1074/jbc.M205058200. Epub 2002 Dec 2.
The structural basis of phosphorylation and its putative role in internalization were investigated in the human dopamine transporter (hDAT). Activation of protein kinase C (PKC) was achieved either directly by treatment with 4-alpha-phorbol 12-myristate 13-acetate (PMA) or by activating the Galpha(q)-coupled human substance P receptor (hNK-1) co-expressed with hDAT in HEK293 cells and in N2A neuroblastoma cells. In both cell lines, activation of the hNK-1 receptor by substance P reduced the V(max) for [(3)H]dopamine uptake to the same degree as did PMA ( approximately 50 and approximately 20% in HEK293 and N2A cells, respectively). In HEK293 cells, the reduction in transport capacity could be accounted for by internalization of the transporter, as assessed by cell surface biotinylation experiments, and by fluorescence microscopy using enhanced green fluorescent protein-tagged hDAT. In HEK293 cells, hNK-1 receptor activation, as well as direct PKC activation by PMA, was accompanied by a marked increase in transporter phosphorylation. However, truncation of the first 22 N-terminal residues almost abolished detectable phosphorylation without affecting the SP- or PMA-induced reduction in transport capacity and internalization. In this background truncation construct, systematic mutation of all the phosphorylation consensus serines and threonines in hDAT, alone and in various combinations, did also not alter the effect of hNK-1 receptor activation or PMA treatment in either HEK293 or N2A cells. Mutation of a dileucine and of two tyrosine-based motifs in hDAT was similarly without effect. We conclude that the major phosphorylation sites in hDAT are within the distal N terminus, which contains several serines. Moreover, the present data strongly suggest that neither this phosphorylation, nor the phosphorylation of any other sites within hDAT, is required for either receptor-mediated or direct PKC-mediated internalization of the hDAT.
我们研究了人多巴胺转运体（hDAT）磷酸化的结构基础及其在内在化中的假定作用。通过用4-α-佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）直接处理，或通过激活与hDAT共表达于HEK293细胞和N2A神经母细胞瘤细胞中的Gαq偶联的人P物质受体（hNK-1）来实现蛋白激酶C（PKC）的激活。在这两种细胞系中，P物质激活hNK-1受体使[³H]多巴胺摄取的Vmax降低程度与PMA相同（在HEK293细胞和N2A细胞中分别约为50%和约20%）。在HEK293细胞中，通过细胞表面生物素化实验以及使用增强型绿色荧光蛋白标记的hDAT的荧光显微镜评估，转运体的内在化可以解释转运能力的降低。在HEK293细胞中，hNK-1受体激活以及PMA直接激活PKC均伴随着转运体磷酸化的显著增加。然而，截短前22个N端残基几乎消除了可检测到的磷酸化，而不影响SP或PMA诱导的转运能力降低和内在化。在这种背景截短构建体中，单独或各种组合地对hDAT中所有磷酸化共有丝氨酸和苏氨酸进行系统性突变，也未改变hNK-1受体激活或PMA处理在HEK293或N2A细胞中的作用。hDAT中双亮氨酸基序和两个基于酪氨酸的基序的突变同样没有效果。我们得出结论，hDAT中的主要磷酸化位点在远端N端内，该区域包含几个丝氨酸。此外，目前的数据强烈表明，hDAT的这种磷酸化以及hDAT内任何其他位点的磷酸化，对于hDAT的受体介导或直接PKC介导的内在化均非必需。
