Department of Biomedical Sciences, Texas Tech University Health Sciences Center, 1300 Coulter Drive, Amarillo, TX 79106, United States.
Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, Amarillo, TX 79106, United States.
Eur J Pharmacol. 2014 Jan 15;723:131-40. doi: 10.1016/j.ejphar.2013.10.064. Epub 2013 Nov 18.
Controlling angiotensin AT1 receptor function has been shown to be protective for many pathophysiological disorders. Although estrogen metabolite, 2-methoxyestradiol (2ME2) can down-regulate angiotensin AT1 receptor expression independently of nuclear receptors, no specific cellular targets have been identified. This study was focused on identification and validation of a cellular target responsible for 2ME2-mediated angiotensin AT1 receptor down-regulation in a continuously passaged rat liver epithelial cell line. Cell membranes were isolated and used to determine 2ME2 specific binding. Cell membranes exposed to [(3)H]2ME2 showed specific saturable binding, which was found to be pertussis toxin (PTx) sensitive. Under similar conditions, G-protein coupled receptor 30 (GPR30) agonist (G1) and antagonist (G15) inhibited 2ME2 specific binding. In these cells GPR30 was found localized to endoplasmic reticulum (ER) membranes. In intact cells, G1 down-regulated angiotensin AT1 receptor expression and this effect was reversed by G15. Furthermore, 2ME2 mediated activation of epidermal growth factor receptor (EGFR) followed by ERK1/2 phosphorylation, an essential signaling step in angiotensin AT1 receptor down-regulation, was abrogated by G15, suggesting that this signal is GPR30 dependent. Additionally, EGF was found to independently down-regulate angiotensin AT1 receptor in an ERK1/2-dependent manner. In summary, our results demonstrate for the first time that 2ME2 down-regulation of angiotensin AT1 receptor is dependent on ER membrane-associated GRP30. Moreover, this effect is facilitated by GPR30 dependent transactivation of EGFR and ERK1/2 phosphorylation. This study provides further understanding of the physiological significance of 2ME2 and its role in modulating angiotensin AT1 receptor expression.
血管紧张素 AT1 受体功能的控制已被证明对许多病理生理紊乱具有保护作用。尽管雌激素代谢物 2-甲氧基雌二醇（2ME2）可以独立于核受体下调血管紧张素 AT1 受体表达，但尚未确定特定的细胞靶标。本研究集中于鉴定和验证负责 2ME2 介导的连续传代大鼠肝上皮细胞系中血管紧张素 AT1 受体下调的细胞靶标。分离细胞膜并用于确定 2ME2 特异性结合。暴露于[（3）H]2ME2 的细胞膜显示出特异性可饱和结合，该结合被发现对百日咳毒素（PTx）敏感。在相似的条件下，G 蛋白偶联受体 30（GPR30）激动剂（G1）和拮抗剂（G15）抑制 2ME2 特异性结合。在这些细胞中，GPR30 被发现定位于内质网（ER）膜。在完整的细胞中，G1 下调血管紧张素 AT1 受体表达，并且该效应被 G15 逆转。此外，2ME2 介导的表皮生长因子受体（EGFR）的激活以及随后的 ERK1/2 磷酸化，这是血管紧张素 AT1 受体下调的必需信号步骤，被 G15 阻断，表明该信号是 GPR30 依赖性的。此外，发现 EGF 以 ERK1/2 依赖性方式独立地下调血管紧张素 AT1 受体。总之，我们的结果首次表明，2ME2 下调血管紧张素 AT1 受体依赖于 ER 膜相关的 GPR30。此外，这种作用是通过 GPR30 依赖性 EGFR 的转激活和 ERK1/2 磷酸化来促进的。本研究进一步了解了 2ME2 的生理意义及其在调节血管紧张素 AT1 受体表达中的作用。
