HEK293细胞中细丝蛋白-C对α_(1A)-肾上腺素受体介导细胞外信号调节激酶磷酸化作用的影响  

Effect of filamin-C on extracellular signal-regulated kinase phosphorylation mediated by α_(1A)-adrenergic receptor in HEK293 cell

作　　者：张坦[1] 徐琦[1] 宋峣[1] 徐明[1] 陈凤荣[1] 韩启德[1] 张幼怡[1] 

机构地区：[1]北京大学第三医院血管医学研究所分子心血管学教育部重点实验室,北京100083

出　　处：《中国药理学与毒理学杂志》2004年第3期178-183,共6页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金资助项目( 30 2 70 5 4 0 );国家重点基础研究发展规划( 973)基金资助项目 (G2 0 0 0 5 6 90 6 )~~

摘　　要：目的　寻找与α1A 肾上腺素受体 (α1A AR)相互作用的胞浆内非G蛋白 ,研究蛋白对受体信号转导的影响。方法　应用酵母双杂交技术 ,以人α1A AR胞浆内C末端 (α1A AR CT)为诱饵 ,筛选人脑cDNA文库 ,用 5 溴 4 氯 3 吲哚半乳糖苷 (X Gal)定性分析及邻硝基苯基 β D 半乳糖苷 (ONPG)定量分析对筛选出的阳性克隆细丝蛋白 C(FLNC)进行确证。采用脂质体转染及蛋白免疫印迹技术探讨人胚胎肾 2 93(HEK2 93)细胞中FLNC对α1A AR信号转导通路的影响。结果　①缺陷培养基筛选出FLNC的C末端部分片段可以与α1A AR CT在酵母中结合。②X Gal定性分析中 ,FLNC与α1A AR CT的共转子菌落 6h即呈现蓝色 ,而对照菌落颜色无变化 ;经ONPG定量分析发现 ,FLNC与α1A AR CT的共转子中 β 半乳糖苷酶的活性大约是对照的 2～ 3倍(P <0 .0 1)。③编码FLNC的C末端片段的质粒瞬时转染于稳定表达全长α1A AR的HEK2 93细胞中 ,可以明显增强去氧肾上腺素 ( 10 μmol·L- 1,30min)激动α1A AR介导的细胞外信号调节激酶 (ERK1/ 2 )磷酸化作用。结论　FLNC的C末端部分片段可以与α1A AR CT在酵母中结合 ,并且增强HEK2 93细胞中α1A AR介导ERK1/AIM To find novel non G proteins that may interact with the α1A adrenergic receptor(α1A AR) and study the effects of the proteins on receptor cell signaling. METHODS Yeast two hybrid assay was performed, using the C terminal tail of α1A AR(α1A AR CT) as a bait protein, to screen human brain cDNA library. 5 Bromo 4 chloro 3 indolyl β D galactopyranoside(X Gal) and o nitrophenyl β D galactopyranoside(ONPG) assay were subsequently carried out to confirm the positive interactions. Transfection with lipofactamine and immunoblotting assay were used to observe the effects of filamin C(FLN C) on α1A AR cell signaling. RESULTS ① The C terminal part of FLN C was found to interact with α1A AR CT on selection medium in yeast. ② In X Gal assay, co transformants of FLN C and α1A AR CT turned blue at 6 h, while control yeast clone did not; The results of ONPG assay indicated the β galactosidase activity in co transformants were about 2-3 fold higher than control (P<0.01). ③ Level of extracellular signal regulated kinase(ERK1/2) phosphorylation induced by phenylephrine(10 μmol·L-1, 30 min) increased greatly when C terminal part of FLN C protein was expressed instantly in the human embryonic kidney 293(HEK293) cells expressing full length α1A AR stably. CONCLUSION Fragments of FLN C could bind to α1A AR CT in yeast cells and enhance the level of ERK1/2 phophorylation induced by α1A AR in HEK293 cells.

关 键 词：酵母茵 杂交 受体 肾上腺素α1 细丝蛋白 信号转导 

分 类 号：R341[医药卫生—基础医学]