ERR1与核受体辅活化子相互作用位点研究

目的分析ERR1与核受体辅活化子相互作用的位点.方法用常规PCR方法和重叠延伸PCR构建ERR1的各种突变体到表达质粒pGBT9上,通过酵母双杂交实验分析其与已知核受体辅活化子PNRC的相互作用.结果成功构建了ERR1的缺失突变体pGBT9/ERR1 1-412、pGBT9/ERR1 1-144、pGBT9/ERR1 190-412 、pGBT9/ERR1 145-423、 pGBT9/ERR1 190-423、 pGBT9/ERR1 190-423 F232A、 pGBT9/ERR1 190-423 K244A.酵母双杂交实验显示ERR1的氨基酸145-423和190-423 可分别与PNRC相互作用;ERR1 190-423 K244A与PNRC的相互作用明显低于ERR1 190-423;其它突变体与PNRC的相互作用未检测到.结论 ERR1与核受体辅活化子相互作用的位点位于ERR1的配体结合结构域(LBD,aa190-423),ERR1与核受体辅活化子相互作用依赖于其LBD的AF2结构域,AF2结构域外的其它氨基酸F232、K244也对ERR1与核受体辅活化子相互作用起关键作用.

Study on binding site of estrogen-related receptor 1 interacting with coactivator

Objective To identify the binding site of estrogen-related receptor 1 (ERR1) interacting with nuclear receptor coactivaor. Methods Deletants and mutants of ERR1, obtained by normal PCR and overlap extension PCR, were inserted into MCS of yeast expression plasmid pGBT9. After identification of the positive clone by digestion of restriction endonuclease and sequencing, the interaction between each of the recombinant mutations and PNRC was detected by yeast-two hybrid. Results pGBT9/ERR1 1-412, pGBT9/ERR1 1-144, pGBT9/ERR1 190-412, pGBT9/ERR1 145-423, pGBT9/ERR1 190-423, pGBT9/ERR1 190-423 F232A, pGBT9/ERR1 190-423 K244A, the mutants or deletants of ERR1, were successfully constructed. GalDBD-ERR1/aa145-423 or aa190-423 fusion protein could interact with the known coactivator PNRC. Yeast-two hybrid assay revealed that the interacting effect of ERR1 190-423 K244A with PNRC was significantly lower than that of ERR1 190-423 with PNRC, but no interaction between the other mutations and PNRC could be found. Conclusion By a combination of mutagenesis and yeast two-hybrid assay, the coactivator-interacting domain on ERR1 has been mapped to a region encompassing amino acids 190-423 which is the ligand-binding domain (LBD) of ERR1. ERR1 can interact with coactivator in an AF2-dependent manner and the other amino acids F232 or K244 not involved in AF2 domain are the key amino acids for ERR1 interacting with coactivator.