Razandi Mahnaz, Alton Gordon, Pedram Ali, Ghonshani Sanjiv, Webb Paul, Levin Ellis R
Division of Endocrinology, Veterans Affairs Medical Center, Long Beach, Long Beach, California 90822, USA.
Mol Cell Biol. 2003 Mar;23(5):1633-46. doi: 10.1128/MCB.23.5.1633-1646.2003.
Estrogen receptors (ER) have been localized to the cell plasma membrane (PM), where signal transduction mediates some estradiol (E2) actions. However, the precise structural features of ER that result in membrane localization have not been determined. We obtained a partial tryptic peptide/mass spectrometry analysis of membrane mouse ERalpha protein. Based on this, we substituted alanine for the determined serine at amino acid 522 within the E domain of wild-type (wt) ERalpha. Upon transfection in CHO cells, the S522A mutant ERalpha resulted in a 62% decrease in membrane receptor number and reduced colocalization with caveolin 1 relative to those with expression of wt ERalpha. E2 was significantly less effective in stimulating multiple rapid signals from the membranes of CHO cells expressing ERalpha S522A than from those of CHO cells expressing wt ERalpha. In contrast, nuclear receptor expression and transcriptional function were very similar. The S522A mutant was also 60% less effective than wt ERalpha in binding caveolin 1, which facilitates ER transport to the PM. All functions of ERalpha mutants with other S-to-A substitutions were comparable to those of wt ER, and deletion of the A/B or C domain had little consequence for membrane localization or function. Transfection of ERalpha S522A into breast cancer cells that express native ER downregulated E2 binding at the membrane, signaling to ERK, and G1/S cell cycle events and progression. However, there was no effect on the E2 transactivation of an ERE-luciferase reporter. In summary, serine 522 is necessary for the efficient translocation and function of ERalpha at the PM. The S522A mutant also serves as a dominant-negative construct, identifying important functions of E2 that originate from activating PM ER.
雌激素受体（ER）已定位到细胞质膜（PM），在那里信号转导介导一些雌二醇（E2）的作用。然而，导致膜定位的ER的确切结构特征尚未确定。我们对膜结合型小鼠ERα蛋白进行了部分胰蛋白酶肽段/质谱分析。基于此，我们将野生型（wt）ERα的E结构域中第522位氨基酸处确定的丝氨酸替换为丙氨酸。在CHO细胞中进行转染后，与表达wt ERα相比，S522A突变型ERα导致膜受体数量减少62%，并且与小窝蛋白1的共定位减少。与表达wt ERα的CHO细胞膜相比，E2在刺激表达ERα S522A的CHO细胞膜产生多种快速信号方面的效果明显较差。相比之下，核受体表达和转录功能非常相似。S522A突变体与小窝蛋白1结合的能力也比wt ERα低60%，而小窝蛋白1有助于ER转运到PM。其他丝氨酸到丙氨酸替换的ERα突变体的所有功能与wt ER相当，并且A/B或C结构域的缺失对膜定位或功能影响很小。将ERα S522A转染到表达天然ER的乳腺癌细胞中，可下调膜上的E2结合、向ERK的信号传导以及G1/S细胞周期事件和进程。然而，对ERE - 荧光素酶报告基因的E2反式激活没有影响。总之，丝氨酸522对于ERα在PM处的有效转运和功能是必需的。S522A突变体还可作为显性负性构建体，确定源自激活PM ER的E2的重要功能。
