甘丙肽2型受体在HEK293A细胞中的同源二聚化研究  

Homodimerization of Galanin Receptor 2 in HEK293A Cells

作　　者：闫竹青[1] 吕琼[1] 金艳燕[1] 李杰[1] 马克乾[1] 杨予涛[1] 徐志卿[1,2] 张进禄[1] 

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]Departments of Neuroscience,Karolinska Institutet

出　　处：《生物物理学报》2011年第5期410-416,共7页Acta Biophysica Sinica

基　　金："973"计划项目(2010CB912003);国家自然科学基金项目(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：G蛋白偶联受体二聚化在受体的转运和信号转导中起着重要的调节作用。为了验证甘丙肽2型受体(GalR2)是否形成同源二聚体,本实验分别构建了N端带FLAG和HA标签的GalR2融合蛋白,并转染到HEK293A细胞中。Western blot发现,除了相当于GalR2分子量的位置有条带之外,在大约2倍于GalR2分子量的位置也有条带,提示GalR2可能是以二聚体的形式存在的。共转染HA-GalR2和FLAG-GalR2,通过免疫共沉淀方法,在抗HA的免疫片段中检测到了FLAG的免疫活性,且在相当于单倍分子量和二倍分子量上均有条带;反过来,在FLAG的免疫片段中检测HA的免疫活性,条带同样出现在单倍分子量和二倍分子量上。以上结果表明甘丙肽2型受体之间形成了同源二聚体。Galanin is a neuropeptide with a wide range of effects in the nervous and endocrine systems,mediated through three G protein-coupled receptor（GPCR） subtypes termed as galanin receptor 1（GalR1）,galanin receptor 2（GalR2） and galanin receptor 3（GalR3）.The GPCRs have been shown to function as monomers,homodimers,heterodimers and oligomers.In the study presented here,biochemical techniques were used to determine whether GalR2 receptors exist as homodimers in HEK293A cells.HA-tagged GalR2 and FLAG-tagged GalR2 were created and each fusion protein was evaluated for functionality following expression in HEK293A cells using ERK phosphorylation assays.Immunoreactive bands of the approximate size of HA-GalR2 or FLAG-GalR2 monomers were visible,along with higher molecular weight bands which were the approximate predicted size of HA-GalR2 or FLAG-GalR2 dimers,indicating the existence of dimeric species of the GalR2.To determine whether the higher-MW immunoreactive bands observed might be the result of protein-protein interaction between GalR2 receptors,co-immunoprecipitation studies were performed.Thus,immunoreactive bands of the approximate predicted size of HA-GalR2：FLAG-GalR2 dimers were visible in samples prepared from cells cotransfected with HA-GalR2 and FLAG-GalR2,but not HA-GalR2 with pCDNA3-FLAG or FLAG-GalR2 with pCMV-HA.These data indicate that GalR2 form homodimer in HEK293A cells.

关 键 词：甘丙肽2型受体 G蛋白偶联受体 同源二聚体 免疫共沉淀 

分 类 号：Q291[生物学—细胞生物学]