Gandini O, Kohno H, Curtis S, Korach K S
Receptor Biology Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Steroids. 1997 Jul;62(7):508-15. doi: 10.1016/s0039-128x(97)00001-9.
To determine the characteristics of the N-terminal transactivation domain (AF-1) of the mouse estrogen receptor (ER), we constructed a number of deletion mutants. Wild-type and mutant receptors were expressed in yeast cells and assayed for their ability to transactivate an estrogen-responsive reporter plasmid (ERE-CYCl-LacZ) that contained a single estrogen response element of the vitellogenin A2 gene promoter. Deletion of the N-terminal 121 amino acids from the mouse ER resulted in a 50% reduction in transactivation activity compared with the full-length wild-type ER. Deletion of the first 150 amino acids resulted in loss of 90% transactivation activity. An ER deletion mutant lacking residues 121-154 retained full transcriptional activity, suggesting that this region plays a significant transacting role only when the first portion is deleted. A point mutation was introduced in the C-terminal region at Met-521 in order to study the possible interaction between the C-terminal ligand-binding domain and the N-terminal AF-1 region. This mutant ER, M521G, exhibited 150% of the transcriptional activity of the wild-type ER. An M521G mutant lacking the N-terminal 121 amino acids retained full transactivation activity, whereas, M521G lacking 150 amino acids resulted in only 10% of wild-type activity. These results suggest that residues 121-154 might interact with the C terminus to affect transcription. In summary, multiple N-terminal regions in the ER were identified that function in transactivation. Furthermore, a point mutation in the C-terminal portion of the ER may change the conformation of the ER ligand-binding domain, producing a more stable receptor/ligand complex that increases transcriptional activity. These data suggest that the N- and C-terminal portions of the ER interact in a cooperative manner to activate transcription from target genes.
为了确定小鼠雌激素受体（ER）的N端反式激活结构域（AF-1）的特征，我们构建了许多缺失突变体。野生型和突变型受体在酵母细胞中表达，并检测它们反式激活雌激素反应报告质粒（ERE-CYCl-LacZ）的能力，该质粒含有卵黄蛋白原A2基因启动子的单个雌激素反应元件。从小鼠ER中缺失N端121个氨基酸导致反式激活活性与全长野生型ER相比降低了50%。缺失前150个氨基酸导致90%的反式激活活性丧失。一个缺少121-154位残基的ER缺失突变体保留了完全的转录活性，这表明该区域仅在第一部分缺失时才发挥重要的反式作用。为了研究C端配体结合结构域与N端AF-1区域之间可能的相互作用，在C端区域的Met-521处引入了一个点突变。这个突变型ER，M521G，表现出野生型ER转录活性的150%。一个缺少N端121个氨基酸的M521G突变体保留了完全的反式激活活性，而缺少150个氨基酸的M521G仅导致野生型活性的10%。这些结果表明121-154位残基可能与C端相互作用以影响转录。总之，在ER中鉴定出多个在反式激活中起作用的N端区域。此外，ER C端部分的一个点突变可能改变ER配体结合结构域的构象，产生一个更稳定的受体/配体复合物，从而增加转录活性。这些数据表明ER的N端和C端部分以协同方式相互作用以激活靶基因的转录。
