2-methoxyestradiol binding of GPR30 down-regulates angiotensin AT(1) receptor.

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.