Institute of Cardiovascular Sciences, Albrechtsen Research Centre, Winnipeg, Canada.
Departments of Medicine, Physiology and Pharmacology, Robarts Research Institute, London, Canada.
Pharmacol Res Perspect. 2022 Oct;10(5):e00995. doi: 10.1002/prp2.995.
Aldosterone exerts some of its effects not by binding to mineralocorticoid receptors, but rather by acting via G protein-coupled estrogen receptors (GPER). To determine if aldosterone binds directly to GPER, we studied the ability of aldosterone to compete for the binding of [ H] 2-methoxyestradiol ([ H] 2-ME), a high potency GPER-selective agonist. We used GPER gene transfer to engineer Sf9-cultured insect cells to express GPER. We chose insect cells to avoid interactions with any intrinsic mammalian receptors for aldosterone. [ H] 2-ME binding was saturable and reversible to a high-affinity population of receptors with K = 3.7 nM and B = 2.2 pmol/mg. Consistent with agonist binding to G Protein-coupled receptors, [ H] 2-ME high-affinity state binding was reduced in the presence of the hydrolysis-resistant GTP analog, GppNHp. [ H] 2-ME binding was competed for by the GPER agonist G1, the GPER antagonist G15, estradiol (E2), as well as aldosterone (Aldo). The order of potency for competing for [ H] 2-ME binding, namely 2ME > Aldo > E2 ≥ G1, paralleled the orders of potency for inhibition of cell proliferation and inhibition of ERK phosphorylation by ligands acting at GPER. These data confirm the ability of aldosterone to interact with the GPER, consistent with the interpretation that aldosterone likely mediates its GPER-dependent effects by direct binding to the GPER. SIGNIFICANCE STATEMENT: Despite the growing evidence for aldosterone's actions via G protein-coupled estrogen receptors (GPER), there remains significant skepticism that aldosterone can directly interact with GPER. The current studies are the first to demonstrate directly that aldosterone indeed is capable of binding to the GPER and thus likely mediates its GPER-dependent effects by direct binding to the receptor.
醛固酮通过与盐皮质激素受体结合发挥其部分作用，而是通过与 G 蛋白偶联雌激素受体 (GPER) 相互作用来发挥作用。为了确定醛固酮是否直接与 GPER 结合，我们研究了醛固酮竞争 [ H] 2-甲氧基雌二醇 ([ H] 2-ME) 结合的能力，[ H] 2-ME 是一种高活性的 GPER 选择性激动剂。我们使用 GPER 基因转染工程 Sf9 培养的昆虫细胞来表达 GPER。我们选择昆虫细胞来避免与任何内在的哺乳动物醛固酮受体相互作用。[ H] 2-ME 结合是可饱和的和可还原的，具有高亲和力的受体，K d = 3.7 nM，B = 2.2 pmol/mg。与激动剂与 G 蛋白偶联受体结合一致，[ H] 2-ME 高亲和力状态结合在存在非水解的 GTP 类似物 GppNHp 时减少。GPER 激动剂 G1、GPER 拮抗剂 G15、雌二醇 (E2) 以及醛固酮 (Aldo) 均可竞争 [ H] 2-ME 结合。竞争 [ H] 2-ME 结合的效力顺序，即 2ME > Aldo > E2 ≥ G1，与配体在 GPER 上作用时抑制细胞增殖和抑制 ERK 磷酸化的效力顺序平行。这些数据证实了醛固酮与 GPER 相互作用的能力，这与醛固酮通过直接与 GPER 结合来介导其 GPER 依赖性作用的解释一致。意义声明：尽管越来越多的证据表明醛固酮通过 G 蛋白偶联雌激素受体 (GPER) 发挥作用，但仍有很大的怀疑认为醛固酮可以直接与 GPER 相互作用。目前的研究首次直接证明醛固酮确实能够与 GPER 结合，因此可能通过直接与受体结合来介导其 GPER 依赖性作用。
