Correlation of functional and radioligand binding characteristics of GPER ligands confirming aldosterone as a GPER agonist

Abstract

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 [3H] 2‐methoxyestradiol ([3H] 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. [3H] 2‐ME binding was saturable and reversible to a high‐affinity population of receptors with Kd = 3.7 nM and Bmax = 2.2 pmol/mg. Consistent with agonist binding to G Protein‐coupled receptors, [3H] 2‐ME high‐affinity state binding was reduced in the presence of the hydrolysis‐resistant GTP analog, GppNHp. [3H] 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 [3H] 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.

Hypertension in Primary Aldosteronism Is Initiated by Salt-Induced Increases in Vascular Resistance With Reductions in Cardiac Output

“…It should be noted that a significant reduction in heart rate and increases in stroke volume occurred in aldosterone-treated rats even on the low-salt diet, that is, before initiation of salt-induced elevations in blood pressure. The heart rate changes are consistent with studies showing that aldosterone binds to GPERs (G-protein–coupled estrogen receptors) 39 and decreases heart rate by activating GPER in cardiac vagal neurons of nucleus ambiguus. 40 Other investigators using continuous telemetry recordings have shown similar reductions in heart rate during initiation of hypertension induced by deoxycorticosterone (DOC) and salt.…”

G protein–coupled estrogen receptor: a promising therapeutic target for aldosterone-induced hypertension

“…Unexpectedly, accumulating evidence is revealing that, in addition to MR, aldosterone can bind to GPER to exert its non-genomic actions ( 9 , 18 , 19 ). Furthermore, Ang II can influence aldosterone synthesis through the interaction of Ang II receptor type 1 with GPER (8), providing a mechanism to link GPER overexpression with the elevation in aldosterone in patients with aldosterone-producing adenomas.…”

“…A subsequent study showed that GPER can interact with aldosterone and mediate non-genomic pathways in a manner distinct from the classic MR-mediated activation mechanism. A recent study suggested that the GPER-dependent effects of aldosterone likely occur by direct binding, with a stronger binding potency of GPER to aldosterone than to estrogen; however, the binding of GPER is the strongest to [ 3 H] 2‐ME, a high-potency GPER-selective agonist in insect cells without any of the intrinsic mammalian receptors for aldosterone ( 19 ).…”

The Role of G Protein-Coupled Estrogen Receptor (GPER) in Vascular Pathology and Physiology

“…Despite accumulating evidence for aldosterone’s actions through GPER, there remains significant speculation that aldosterone directly binds to GPER [ 97 , 98 , 99 ]. However, a recent work has demonstrated a direct interaction of aldosterone with GPER through the data showing that aldosterone competitively binds GPER with other GPER ligands [ 100 ]. Meanwhile, in a reperfusion injury study of the heart, using a polymer analogue of aldosterone to selectively activate the non-genomic receptors of aldosterone, it was found that the GPER antagonist G-36 but not spironolactone, a MR blocker, could block the action of aldosterone [ 101 ].…”