应用酵母双杂交系统筛选FGFR3相互作用的蛋白质

目的通过酵母双杂交系统从小鼠17.5 d全胚胎cDNA质粒文库中筛选与成纤维细胞生长因子受体3(FGFR3)相互作用的蛋白质,为研究FGFR3信号通路提供新的线索.方法构建FGFR3胞内区的真核表达载体R3-PI,转化酵母菌AH109.毒性实验及自激活实验证实FGFR3胞内区蛋白对酵母细胞无毒性,不存在自激活现象.将小鼠17.5 d全胚胎cDNA质粒文库转入AH109/R3-PI酵母菌中,能够与FGFR3胞内区相互作用的蛋白质.结果通过初步筛选得到3种能够与FGFR3胞内区相互作用的蛋白质,经回复性实验进一步验证CLK4与FGFR3胞内区之间存在相互作用.结论通过筛选发现CLK4与FGFR3胞内区之间存在相互作用,提示FGFR3可能通过CLK4参与能量代谢调节,而CLK4可能通过对FGFR3的磷酸化或去磷酸化调节FGFR3的活性.

Screening for protein interacting with FGFR3 in mouse 17.5-day embryo cDNA library by yeast two-hybrid system

Objective FGFR3 is important to bone development and some epithelial carcinoma.To screen proteins interacting with FGFR3 by the yeast two-hybrid system.Methods The intracellular domain of FGFR3 was fused to a DNA-binding domain as "BAIT" and the yeast strain AH109 was transformed with FGFR3.Then the yeast strain AH109/R3-PI was transformed with mouse 17.5-day embryo cDNA library.Results FGFR3 had no self-activation and toxicity to the yeast strain AH109.CLK4 was identified interacted with FGFR3.Conclusion The interaction between CLK4 and FGFR3 suggests that FGFR3 can accommodate energy metabolism through CLK4.The activation of FGFR3 may be regulated by CLK4 through phosphorylation and dephosphorylation.