Washburn T, Hocutt A, Brautigan D L, Korach K S
Receptor Biology Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
Mol Endocrinol. 1991 Feb;5(2):235-42. doi: 10.1210/mend-5-2-235.
We have characterized further the heterogeneous nuclear-specific doublet forms of the mouse uterine estrogen receptor (ER). Estrogen treatment produced the multiple nuclear ER forms of 65 and 66.5 kDa, which were isolated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Soluble ER preparations exhibited only a single 65-kDa form. Isolation of the individual nuclear ER forms and reanalysis demonstrated that formation of the multiple bands was not due to artifacts of nuclear sample preparation or the presence of contaminating proteins. Analysis of individual uterine cell types (epithelial and stromal/myometrium) indicated that both ER forms were present in both cell fractions. Fractionation of nuclear components with low salt showed that both ER forms were found in the salt-resistant fraction. Extraction of nuclei with high salt (0.6 M KCl) solubilized both ER forms. Phosphorylation was studied as a protein modification to account for the multiple forms. Incorporation of 32P into uterine protein both in vivo and in intact tissue incubation indicated 32P labeling of uterine nuclear ER after hormone treatment. Both nuclear ER forms are labeled, although the 66.5-kDa form appears to be more heavily labeled. Phosphoamino acid analysis of the immunopurified 32P-labeled ER from intact uterine tissue indicated phosphoserine as the only phospholabeled residue. These data suggest that phosphorylation is associated with the physiological functioning of the ER in response to hormone and produces the heterogeneous ER forms in the nucleus.
我们进一步对小鼠子宫雌激素受体（ER）的异质性核特异性双峰形式进行了表征。雌激素处理产生了65 kDa和66.5 kDa的多种核ER形式，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳对其进行分离和分析。可溶性ER制剂仅显示单一的65 kDa形式。分离单个核ER形式并重新分析表明，多条带的形成不是由于核样品制备的假象或污染蛋白的存在。对单个子宫细胞类型（上皮细胞和基质/肌层）的分析表明，两种ER形式均存在于两种细胞组分中。用低盐对核成分进行分级分离表明，两种ER形式均存在于耐盐级分中。用高盐（0.6 M KCl）提取细胞核可溶解两种ER形式。研究了磷酸化作为一种蛋白质修饰来解释多种形式。体内和完整组织孵育中32P掺入子宫蛋白表明，激素处理后子宫核ER有32P标记。两种核ER形式均被标记，尽管66.5 kDa形式似乎标记更重。对来自完整子宫组织的免疫纯化的32P标记的ER进行磷酸氨基酸分析表明，磷酸丝氨酸是唯一的磷酸标记残基。这些数据表明，磷酸化与ER对激素的生理功能相关，并在细胞核中产生异质性ER形式。
