雌激素受体β潜在磷酸化位点突变体的构建及其活性测定

Construction and Activity Detection of Potent Phosphorylation Sites Mutants of Estrogen Receptor β

目的:构建雌激素受体(ER)β潜在磷酸化位点突变体,并在人胚肾细胞293T中检测其对ERβ下游基因转录的影响。方法:通过ERα和ERβ序列同源性比对,寻找ERβ的潜在磷酸化位点;以pcDNA3-ERβ-FLAG为模板,通过重组PCR,将ERβ264位、469位Ser编码基因突变为Ala编码基因,将突变片段连接到同样双酶切的pcDNA3-FLAG载体中,Western blot检测其表达;将重组质粒转入293T细胞中,检测突变体对含雌激素应答元件(ERE)的报告基因转录活性的影响。结果:ERα和ERβ序列同源性比对发现ERβ的264位Ser和469位Ser可能是其潜在的磷酸化位点;构建了ERβ264位和469位2个点突变体载体pcDNA3-FLAG-ERβ(S264A)和pcDNA3-FLAG-ERβ(S469A),Western blot可检测到ERβ突变体融合蛋白在293T细胞中表达。萤光素酶活性检测表明,在没有雌激素刺激的情况下,2个突变体的活性较野生型没有变化;加入雌激素后,突变体的活性较野生型略有升高。结论:ERβ的264位和469位Ser位点的磷酸化可能不是ERβ调节下游基因转录所必需的,活性升高可能是由于ERβ构象变化造成的。

Objective： To construct the potent phosphorylation sites mutants of estrogen reeeptor（ER）β and to detect the effect of the mutants on ERβ transcriptional activity in 293T cell. Methods： Fingding the potent phosphorylation sites mutants of ERβ through comparing homologous sequences of ERα and ERβ. Using recombinant PCR, the coden of Ser^264 and Ser^469 of ERβ were mutated to one of Ala, and the mutated fragments were inserted into the vector pcDNA3-FLAG. The recombinants were transfected into 293T cells, to determine the effects of the mutants on ERβ transcriptional activity. Results： The Ser^264 and Ser^469 of ERβ might be phosphorylation sites of ERβ. The recombinant plasmids, pcDNA3-FLAG-ERβ （S264A） and pcDNA3-FLAG-ERβ （S469A）, were constructed. Western blot assay showed that the mutants were expressed in 293T cell. Compared with wild type, the mutants had no effect on the ERE-LUC reporter when estrogen E2 was absent. When E2 was present, the transcriptional activity of mutants was slightly higher than that of the wild type. Conclusion： Ser^264 and Ser^469 might not be the necessary phosphorylation sites and the higher activity of mutants might be due to the changed conformation of ERβ.