3T3-L1脂肪细胞膜FGF-21结合蛋白的初步鉴定  

Identification of Binding Partners of Fibroblast Growth Factor-21 in Cell Membrane of 3T3-L1 Cells

作　　者：王文飞[1] 任桂萍[1] 侯玉婷[1] 李德山[1] 

机构地区：[1]东北农业大学生命科学学院生物制药教研室,哈尔滨150030

出　　处：《中国生物化学与分子生物学报》2008年第7期667-673,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：黑龙江省科技厅重点攻关项目(No2006G0461-00)~~

摘　　要：成纤维细胞生长因子(fibroblast growth factor,FGF)-21是最近发现的1个可以独立调节血糖的细胞因子,有望成为治疗2型糖尿病的备选药物.但是,FGF-21调解血糖的机理尚不十分清楚.为探讨该因子功能受体,应用偶联方法,以3T3-L1脂肪细胞为靶标,以FGF-21为诱饵,在3T3-L1脂肪细胞膜上寻找结合蛋白.结果表明,生物素标记的FGF-21可与脂肪细胞膜蛋白形成分子质量大小约300kD以上两组复合物.竞争试验显示,非标记的FGF-21可与生物素标记的FGF-21竞争、抑制标记的FGF-21参入复合物;应用非标记FGF-21剂量越大,抑制后者参入复合物的程度越强.结果提示,该复合物是FGF-21特异性的.此外,随着生物素标记的FGF-21剂量增加,观察到的标记复合物越多;但是,当FGF-21剂量达12.5mg/L以上时,观察到的复合物数量不再增加.实验结果提示,复合物形成与FGF-21剂量相关;FGF-21特异结合的蛋白质结合位点饱和后,复合物形成量最大.同时,采用FGF受体特异性抑制剂SU5402可特异性抑制FGF-21在3T3-L1脂肪细胞中的促进葡萄糖吸收作用,提示本实验所观察到的FGF-21-膜蛋白复合物可能就是FGF-21-FGF受体.Fibroblast growth factor （FGF）-21 is a novel regulator of insulin-independent glucose transport, and could be a potential therapeutic agent for treatment of type Ⅱ diabetes. However, the precise mechanisms whereby FGF-21 regulates blood glucose metabolism remain unknown. In order to study the function of FGF-21 receptor, cross-link method was utilized; 3T3-L1 adipocytes were used as target cells and FGF-21 was used as a bait to search for binding partners from 3T3-L1 cell membrane. Biotinylated FGF-21 could form two complexes with cell membrane proteins of 3T3-L1 adipocytes. The size of the complexes was more than 300 kD. Competition experiment showed that the cold FGF-21 could compete with labeled FGF-21 in forming the complexes, the higher dose of the cold FGF-21 was used, the stronger inhibition of the complex formation was observed. These results suggest that the complexes are FGF-21-specific. In addition, the yield of the complexes increased with increment of the biotinylated FGF-21 added to the reaction. However, the complexes became saturated when FGF-21 concentration reached to 12.5 mg/L or more. The result suggests that the formation of the complexes is FGF-21-dependent. After the binding sites of the FGF-21-associated proteins were saturated, the quantity of complexes reached a maximum. Furthermore, FGF receptor inhibitor SU5402 could restrain FGF-21-induced glucose up-take in 3T3-L1 adipocytes, suggesting that the complexes might be the FGF-21-FGF receptor.

关 键 词：成纤维细胞生长因子-21(FGF-21) 脂肪细胞 复合物鉴定 

分 类 号：Q71[生物学—分子生物学]