甘丙肽2型受体在HEK293细胞中的表达及内化过程  

Expression and Internalization of myc-Tagged Galanin R2 Receptor in HEK293 Cells

作　　者：吕琼[1] 金艳燕[1] 吴波[2] 路雅静[1] 李晓晓[1] 赵春礼[1] 徐志卿[1,3] 

机构地区：[1]首都医科大学神经生物学系,北京神经科学研究所,北京神经再生修复研究重点实验室,北京100069 [2]首都医科大学组胚教研室,北京100069 [3]Departments of Neuroscience,Karolinska Institutet

出　　处：《生物物理学报》2010年第5期373-379,共7页Acta Biophysica Sinica

基　　金：国家自然科学基金(30870815);北京市自然科学基金重点项目(09G0013);人事部留学人员科技活动择优资助项目;Swedish Research Council(3106);北京市人才强教深化高层次人才计划(PHR20100510)~~

摘　　要：构建了在甘丙肽2型受体(GalR2)N端带myc表位标签的真核表达载体,并将其转染到HEK293细胞,进而应用免疫荧光细胞化学方法结合激光扫描共聚焦显微技术观察GalR2蛋白的亚细胞分布和膜转运过程。Western blot结果发现转染组myc-GalR2蛋白水平呈高表达。myc-GalR2主要分布在细胞膜上,少量分布于胞浆中。当给予甘丙肽(10-7mol/L)刺激后,5min时可见细胞膜上myc-GalR2明显减少,胞浆内myc-GalR2增多,15min时膜上myc-GalR2几乎全部消失。表明GalR2受体与配体结合后发生内化,且受体蛋白内化为时间依赖性的转运过程,证实在HEK293细胞GalR2会出现配体依赖性内化。同时,以已知膜蛋白5-HT1AR的内化作为对照,可见在给予甘丙肽刺激以后,5-HT1AR没有发生内化,受体蛋白仍然表达在膜上。此外,通过测定胞内钙水平激活情况来检测其信号通路激活情况,表明myc对GalR2功能没有明显影响。Galanin,a neuropeptide,is involved in numerous physiological and pathological neuronal functions,including learning and memory,mood and pain control,feeding behavior and neuronal survival and regeneration.So far,three galanin receptor subtypes have been identified,termed as GalR1,GalR2 and GalR3,all belonging to the G protein coupled receptor superfamily.The present study was undertaken to address membrane expression and internalization of GalR2 in HEK293 cells by transfecting the receptor tagged with myc at its N-terminal into HEK293 cells.To study the internalization of Galanin R2 receptor（GalR2） after different treatment time of galanin in HEK293 cells.The protein level of myc-tagged GalR2 was measured with Western blot,and the cellular distribution of myc-GalR2 was analyzed with confocal laser scanning fluorescence microscopy（Leica SP5） after immunofluorescence staining.The myc-tagged GalR2 was predominantly localized on the plasma membrane with some intracellular fluorescent structures,mainly in the perinuclear region.Incubation with galanin induced a concentration-dependent increase in [Ca 2＋]i levels in myc-GalR2 transfected cells,suggesting that the myc-GalR2 is functional.The myc-GalR2 on the plasma membrane was internalized within 5～15 min after treatment of galanin at 10-7 mol/L and 37℃,with a dramatic reduction in plasma membrane localization and appearance in intracellular vesicles.This is in agreement with our previous study on PC12 cells transfected with a GalR2–EGFP construct.Taken together these findings indicate that GalR2 is expressed and undergoes ligand-dependent internalization in HEK293 cells.

关 键 词：表位标签 甘丙肽2型受体(GalR2) G蛋白耦联受体(GPCRs) HEK293细胞 膜转运 

分 类 号：Q753[生物学—分子生物学]