Wang Hai-Bo, Guan Ji-Song, Bao Lan, Zhang Xu
Institute of Biochemistry and Cell Biology and Laboratory of Molecular Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
Neurochem Res. 2008 Oct;33(10):2028-34. doi: 10.1007/s11064-008-9678-9. Epub 2008 Mar 26.
In small dorsal root ganglion neurons, delta-opioid receptors (DORs) have been found to be mainly distributed in the cytoplasm and often associated with the membrane of large dense-core vesicles (LDCVs) that contain neuropeptides. To study the distribution of DORs under various physiological or pharmacological conditions, the receptors fused with different tags are constructed, transfected into cells or animals, and examined with microscopy. In this study, we show that DOR with different tags have distinct patterns of subcellular distribution in neuroendocrine cells, PC12 cells. Both immunostaining and vesicle fraction analysis showed that the native DORs expressed in PC12 cells were mainly associated with LDCVs. In transfected PC12 cells, DOR tagged with Myc or hemagglutinin exhibited LDCV localization. However, DOR fused with GFP at N- or C-terminus was found to be mainly localized on the cell surface, and mediated the function of DOR agonist. Therefore, the distribution of DOR fused with GFP differs from the native DORs. These results suggest that the subcellular distribution of the receptor could be better presented by the fused tag with smaller molecular size.
在小的背根神经节神经元中,已发现δ阿片受体(DORs)主要分布在细胞质中,且常与含有神经肽的大致密核心囊泡(LDCVs)的膜相关联。为了研究在各种生理或药理条件下DORs的分布情况,构建了与不同标签融合的受体,将其转染到细胞或动物中,并用显微镜进行检测。在本研究中,我们表明带有不同标签的DOR在神经内分泌细胞PC12细胞中具有不同的亚细胞分布模式。免疫染色和囊泡分级分析均表明,PC12细胞中表达的天然DORs主要与LDCVs相关联。在转染的PC12细胞中,用Myc或血凝素标记的DOR表现出LDCV定位。然而,在N端或C端与绿色荧光蛋白(GFP)融合的DOR主要定位于细胞表面,并介导DOR激动剂的功能。因此,与GFP融合的DOR的分布与天然DORs不同。这些结果表明,受体的亚细胞分布可以通过分子尺寸较小的融合标签更好地呈现。