Visualization of serotonin 1A receptor trafficking in neuron-like PC12 cells
- VernacularTitle:5-HT_(1A)受体蛋白在PC12细胞膜转运的动态变化
- Author:
Yanyan JIN
;
Qiong LU
;
Zhuqing YAN
;
Erjing GAO
;
Chunli ZHAO
;
Jinlu ZHANG
;
Zhiqing XU
- Publication Type:Journal Article
- Keywords:
serotonin 1 A receptor(5-HT_(1A));
G protein-coupled receptors(GPCRs);
green fluorescent protein(GFP);
PC 12 cell;
fluorescence recovery after photobleaching(FRAP)
- From:
Basic & Clinical Medicine
2010;30(5):487-491
- CountryChina
- Language:Chinese
-
Abstract:
Objective To explore the mechanisms of trafficking and signaling of serotonin 1A receptor(5-HT_(1A))and its spatiotemporal distribution in living cells.Methods The mouse 5-HT_(1A) gene amplified by RT-PCR was recombined into pEGFP-N1 vector and the EGFP coding sequence was located in-frame at the C-terminal end of the 5-HT_(1A) receptor.The 5-HT_(1A)-EGFP was transfected into neuron-like PC12 cells as well as HEK293.The transfected cells were visualized using confocal microscopy,the mobility of 5-HT_(1A)-EGFP was monitored by live measurements and fluorescence recovery after photobleaching.Results The 5-HT_(1A) gene was identitical with the published gene sequence NM_008308.4 and a 5-HT_(1A)-EGFP fusion construct was created.After stable transfection of the plasimd into a PC12 cell line and analysis with a confocal laser scanning microscopy,the EGFP-tagged 5-HT_(1A) was predominantly associated with the plasma membrane,but some intracellular vesicles in the perinuclear region also contained the fusion protein.The predominant localization of 5-HT_(1A)-EGFP at the plasma membrane was confirmed in transiently transfected HEK293 cells.Bleached fluorescence was partialy recovered in 100 seconds,indicating that the 5-HT_(1A)-EGFP was mobiled on the membrane.Conclusion Spatiotemporal distribution and mobility of 5-HT_(1A) tagged with EGFP can be monitored in the 5-HT_(1A)-EGFP stable PC12 cell line,which could be an excellent neuron-like experimental cell model for research of 5-HT_(1A) trafficking and signaling.
