3T3-L1脂肪细胞膜FGF-21结合蛋白的初步鉴定

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300 kD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12.5 mg/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

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

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.