Komagata Sayaka, Nakajima Miki, Tsuchiya Yuki, Katoh Miki, Kizu Ryoichi, Kyo Satoru, Yokoi Tsuyoshi
Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan.
J Steroid Biochem Mol Biol. 2006 Jul;100(1-3):79-86. doi: 10.1016/j.jsbmb.2006.02.006. Epub 2006 May 18.
Estrogen receptor alpha (ERalpha) is a ligand-inducible transcription factor that mediates the biological effects of estrogens and antiestrogens. Many point mutations in the human ERalpha gene have been reported to be associated with breast cancer, endometrial cancer, and psychiatric diseases. However, functional analyses for most mutants with amino acid changes are still lacking. In the present study, to investigate the effects of point mutations on the function, gel-shift assays and luciferase assays were performed for eight kinds of mutated ERalpha proteins, including a single nucleotide change of C207G (N69K), G478T (G160C), T887C (L296P), A908G (K303R), C926T (S309F), A1058T (E353V), A1186G (M396V), and G1231deletion (411fsX7). The mutated ERalpha expression plasmids were constructed by site-directed mutagenesis. With gel-shift assays using in vitro translated ERalpha proteins, binding to the consensus estrogen response element (ERE) was observed for the mutated ERalpha proteins except ERalpha (G160C) and ERalpha (411fsX7), the binding of which was comparable with that of the wild type. Western blot analyses showed that ERalpha (G160C) could not be efficiently translated with the in vitro transcription/translation system and that ERalpha (411fsX7) produced a truncated protein. To investigate the transactivation potency, wild-type or mutated ERalpha expression plasmids were co-transfected with pGL3-3EREc38 reporter plasmid into human breast adenocarcinoma MDA-MB-435 cells. The concentration-response curves (10pM-100nM E2) of the mutant ERalpha proteins except ERalpha (E353V) and ERalpha (411fsX7) were similar to that of wild-type ERalpha. However, at a low level of E2 (100pM), the mutants ERalpha (N69K), ERalpha (L296P), ERalpha (S309F), and ERalpha (M396V) showed a significant decrease of transactivation compared with that of the wild-type ERalpha. The mutants ERalpha (E353V) and ERalpha (411fsX7) did not show responsiveness to E2 and antiestrogens, 4-hydroxytamoxifen (4OHT) and ICI 182,780. The mutant ERalpha (S309F) showed decreased responsiveness for the antiestrogenicity of 4OHT. In conclusion, we found that some of the naturally occurring human ERalpha mutants with amino acid changes may have an altered responsiveness to estrogen and antiestrogens.
雌激素受体α（ERα）是一种配体诱导型转录因子，介导雌激素和抗雌激素的生物学效应。据报道，人类ERα基因中的许多点突变与乳腺癌、子宫内膜癌和精神疾病有关。然而，大多数氨基酸发生变化的突变体的功能分析仍然缺乏。在本研究中，为了研究点突变对功能的影响，对8种突变的ERα蛋白进行了凝胶迁移试验和荧光素酶试验，包括C207G（N69K）、G478T（G160C）、T887C（L296P）、A908G（K303R）、C926T（S309F）、A1058T（E353V）、A1186G（M396V）和G1231缺失（411fsX7）的单核苷酸变化。通过定点诱变构建了突变的ERα表达质粒。使用体外翻译的ERα蛋白进行凝胶迁移试验，除了ERα（G160C）和ERα（411fsX7）外，突变的ERα蛋白与雌激素反应元件（ERE）共有序列结合，其结合能力与野生型相当。蛋白质印迹分析表明，ERα（G160C）不能通过体外转录/翻译系统有效翻译，而ERα（411fsX7）产生截短蛋白。为了研究反式激活能力，将野生型或突变的ERα表达质粒与pGL3-3EREc38报告质粒共转染到人乳腺腺癌MDA-MB-435细胞中。除了ERα（E353V）和ERα（411fsX7）外，突变的ERα蛋白的浓度-反应曲线（10pM-100nM E2）与野生型ERα相似。然而，在低水平的E2（100pM）时，与野生型ERα相比，突变体ERα（N69K）、ERα（L296P）、ERα（S309F）和ERα（M396V）的反式激活显著降低。突变体ERα（E353V）和ERα（411fsX7）对E2和抗雌激素4-羟基他莫昔芬（4OHT）和ICI 182,780无反应。突变体ERα（S309F）对4OHT的抗雌激素作用反应性降低。总之，我们发现一些天然存在的氨基酸发生变化的人类ERα突变体可能对雌激素和抗雌激素的反应性发生改变。
