关键词: 5-羟色胺1A受体（5-HT1A）, G蛋白耦联受体（GPCRs）, 绿色荧光蛋白（GFP）, PC12细胞

Abstract: Objective In order to get a better understanding of the mechanisms of trafficking and signaling of serotonin 1A receptor （5-HT1A），we fused it with the enhanced green fluorescent protein （EGFP） to study its spatiotemporal distribution in living cells. Methods The mouse 5-HT1 A 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-HT1A receptor. The 5-HT1A-EGFP was transfected into neuron-like PC12 cells as well as HEK293 using cationic liposomes. A stable 5-HT1A-EGFP expressing PC12 cell clone was isolated under fluorescence microscopy. The transfected cells were visualized using a live cell confocal microscopy system , meanwhile the mobility of 5-HT1A-EGFP was monitored by live measurements and fluorescence recovery after photobleaching. Results The 5-HT1A gene was identity with the published gene sequence NM_008308.4 and a 5-HT1A-EGFP fusion construct was created. After stable transfection of the plasimd into a PC12 cell line and analysis with a confocal laser scanning microscope, the EGFP-tagged 5-HT1A 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-HT1A-EGFP at the plasma membrane was confirmed in transiently transfected HEK293 cells. Bleached fluorescence was partialy recovered in 100 seconds, indicating that the 5-HT1A-EGFP was mobiled on the membrane. Conclusion Spatiotemporal distribution and mobility of 5-HT1A tagged with EGFP can be monitored in the 5-HT1A-EGFP stable PC12 cell line, which could be an excellent neuron-like experimental cell model in research on 5-HT1A trafficking and signaling.

Key words: Serotonin 1A receptor (5-HT1A), G protein-coupled receptors (GPCRs), green fluorescent protein (GFP), PC12 cell