摘要
Objective To explore the possible differential trafficking properties of the dopamine D 1-like receptor subtypes, D 1 receptor and D5 receptor. Methods To visualize distributions of dopamine D 1-like receptor subtypes at subcellular level, the yellow and cyan variants of green fluorescent protein （GFP） were used to tag D1 and D5 receptors. After transfection with the tagged dopamine receptors, the neuroblastoma cells NG108-15 were treated with D1 agonist SKF38393 or acetylcholine （ACh）. Then we observed the subcellular distributions of the tagged receptors under the confocal microscopy and tried to determine trafficking properties by comparing their distribution patterns before and after the drug treatment. Results In resting conditions, D 1 receptors located in the plasma membrane of NG108-15 cells, while D5 receptors located in both plasma membrane and cytosol. With the pre-treatment of SKF38393, the subcellular distribution of D1 receptors was changed. The yellow particle-like fluorescence of tagged D 1 receptors appeared in the cytosol, indicating that D 1 receptors were internalized into cytosol from the cell surface. Same situation also occurred in ACh pre-treatment. In contrast, the subcellular distribution of D5 receptors was not changed after SKF38393 or ACh treatment, indicating that D5R was not translocated to cell surface. Interestingly, when D1 and D5 receptors were co-expressed in the same cell, both kept their distinct subcellular distribution patterns and the trafficking properties. Conclusion Our present study reveals that in NG108-15 nerve cells, dopamine D1 and D5 receptors exhibit differential subcellular distribution patterns, and only D1 receptor has a marked trafficking response to the drug stimulation. We further discuss the potential role of the differential trafficking properties of D1-like receptors in complex modulation of DA signaling.
目的探索多巴胺D1样受体家族的D1、D5亚型,可能有的不同的胞内转运(trafficking)特性。方法为了直接观察D1样受体的亚细胞分布,用绿色荧光蛋白(GFP)变体分别标记D1和D5受体,转染进神经瘤细胞系NG108-15中,并对它们进行定位,然后比较在D1受体激动剂SKF38393或乙酰胆碱(ACh)处理前后D1、D5受体亚细胞分布的异同,以研究它们胞内的转运特性。结果在静息条件下,D1受体特异分布在NG108-15的细胞膜上,而D5受体分布于细胞膜和胞浆内。以D1受体激动剂SKF38393孵育处理后,D1受体亚细胞分布发生改变,胞浆中出现代表D1受体的黄色荧光信号,提示D1受体被内吞;ACh的孵育处理也能引起D1受体异源内吞。与D1受体不同,经SKF38393或ACh处理后,D5受体亚细胞分布与静息条件下比较,未显示明显变化。这提示药物处理不引起D5受体由胞浆向细胞膜的转位(translocation)。当D1和D5受体共同表达在同一个NG108-15细胞中时,它们仍然保持各自的亚细胞分布模式。而且,SKF38393和ACh引起的D1或D5受体的细胞内定位状态,也并未因另一种亚型的存在而改变。结论当分别表达在神经瘤细胞系NG108-15上时,D1、D5两个受体亚型显示出不同的亚细胞定位和药物引起的不同胞内转运特性。当两者共同表达时,D1和D5又互不干扰。SKF38393和ACh都可以引起D1受体的内吞。我们对D1样受体家族这种不同的胞内转运特性在DA信号复杂调控中的可能作用,进行了讨论。
