Anatomical location and redistribution of G protein-coupled estrogen receptor-1 during the estrus cycle in mouse kidney and specific binding to estrogens but not aldosterone

https://doi.org/10.1016/j.mce.2013.11.005Get rights and content

Highlights

Abstract

Prior studies have linked renoprotective effects of estrogens to G-protein-coupled estrogen receptor-1 (GPER-1) and suggest that aldosterone may also activate GPER-1. Here, the role of GPER-1 in murine renal tissue was further evaluated by examining its anatomical distribution, subcellular distribution and steroid binding specificity. Dual immunofluorescent staining using position-specific markers showed that GPER-1 immunoreactivity primarily resides in distal convoluted tubules and the Loop of Henle (stained with Tamm-Horsfall Protein-1). Lower GPER-1 expression was observed in proximal convoluted tubules marked with megalin, and GPER-1 was not detected in collecting ducts. Plasma membrane fractions prepared from whole kidney tissue or HEK293 cells expressing recombinant human GPER-1 (HEK-GPER-1) displayed high-affinity, specific [3H]-17β-estradiol ([3H]-E2) binding, but no specific [3H]-aldosterone binding. In contrast, cytosolic preparations exhibited specific binding to [3H]-aldosterone but not to [3H]-E2, consistent with the subcellular distribution of GPER-1 and mineralocorticoid receptor (MR) in these preparations. Aldosterone and MR antagonists, spironolactone and eplerenone, failed to compete for specific [3H]-E2 binding to membranes of HEK-GPER-1 cells. Furthermore, aldosterone did not increase [35S]-GTP-γS binding to membranes of HEK-GPER-1 cells, indicating that it is not involved in G protein signaling mediated through GPER-1. During the secretory phases of the estrus cycle, GPER-1 is upregulated on cortical epithelia and localized to the basolateral surface during proestrus and redistributed intracellularly during estrus. GPER-1 is down-modulated during luteal phases of the estrus cycle with significantly less receptor on the surface of renal epithelia. Our results demonstrate that GPER-1 is associated with specific estrogen binding and not aldosterone binding and that GPER-1 expression is modulated during the estrus cycle which may suggest a physiological role for GPER-1 in the kidney during reproduction.

Introduction

Estrogens exert their effects on a broad spectrum of target tissues. Estrogens regulate the growth and development of female reproductive epithelia through mechanisms mediated via the nuclear estrogen receptors (ERs), ERα and ERβ (Hewitt et al., 2005, Terasawa and Kenealy, 2012). Estrogens also promote homeostatic and protective effects in numerous estrogen responsive tissues including bone (Nelson et al., 2002, Gennari et al., 2007, Ren and Wu, 2012), brain (Wise, 2005, Carroll and Rosario, 2012, Liu et al., 2012), heart (Grodstein et al., 2000, Vogelvang et al., 2006), liver (Holm et al., 2011), pancreas (Ropero et al., 2012) and kidney (Christy and Shaver, 1974, Hutchens et al., 2010, Hutchens et al., 2012, Lindsey et al., 2011). However, the precise molecular mechanism by which estrogens promote homeostatic and protective actions remains unclear. ERs are expressed at low levels in these tissues, but their roles as intermediaries in these biological effects have not been clearly established in studies employing ER null mice or selective ER ligands (Korach, 2000, Hutchens et al., 2010).

Recent evidence suggests that nonproliferative effects of estrogens in responsive tissues are manifested by the G-protein-coupled estrogen receptor, GPER-1 (Hsieh et al., 2007, Gros et al., 2011, Gros et al., 2013, Lindsey et al., 2011, Hutchens et al., 2012, Liu et al., 2012). GPER-1 is a specific, high affinity (2–6 nM, Thomas et al., 2005, Revankar et al., 2005) estrogen membrane receptor coupled to a stimulatory G protein in vertebrates that is activated by naturally occurring and synthetic estrogens and antiestrogens including estradiol-17β, G-1, tamoxifen, ICI182,780, genestein and bisphenol A, but not by cortisol, progesterone or testosterone in both mammals and fish (Thomas et al., 2005, Bologa et al., 2006, Thomas and Dong, 2006, Pang et al., 2008, Filardo and Thomas, 2012). Genetic manipulation and/or selective ligand studies have revealed that GPER-1 plays a spectrum of physiological roles in the cell cycle, meiotic arrest (Pang et al., 2008, Holm et al., 2011), thymic development (Wang et al., 2008), insulin release (Ropero et al., 2012), glucose metabolism, bone growth, cardiovascular activity (Martensson et al., 2009), and neuroprotection (Liu et al., 2012). GPER-1 activates plasma membrane-associated enzymes and transactivates integral plasma membrane receptors, including integrin α5β1 and the epidermal growth factor receptor, erbB1 in response to estrogen treatment (Filardo et al., 2002, Quinn et al., 2009).

The kidney is composed of distinct tubules that function differentially to regulate fluid and electrolyte homeostasis. Prior studies have demonstrated a high level of GPER-1 expression in the kidney, albeit with differences regarding its subcellular distribution, which may in part be due to differences in methodological approaches in measuring its expression and activity (Grimont et al., 2009, Hazell et al., 2009, Cheng et al., 2011a, Lindsey et al., 2011, Hofmeister et al., 2012). For example, the specific GPER-1 agonist, G-1 (Bologa et al., 2006), estradiol-17β (E2), and the ER antagonist and GPER-1 agonist, ICI 182,780 (Thomas et al., 2005) have been reported to induce intracellular calcium signals in microdissected renal tubule segments and isolated intercalated cells not detected in similar explant and cell cultures isolated from GPER-1-deleted mice (Hofmeister et al., 2012). Chappell and co-workers have shown that GPER-1 colocalizes with megalin in renal proximal tubules and that G1 ameliorates salt-induced renal injury in female mRen2.Lewis mice independently of changes in systolic blood pressure (Lindsey et al., 2011). GPER-1 has also been suggested to function as an aldosterone receptor, based on the observation that GPER-1 is required to promote an increase in phosphorylation of ERK and myosin light chain in vascular smooth muscle cells and aortic ring fragments that is antagonized by both G-15, a GPER-1 antagonist (Dennis et al., 2009), and the mineralocorticoid receptor (MR) antagonist, eplerenone via a MR-independent mechanism (Feldman and Gros, 2011, Gros et al., 2011, Gros et al., 2013). G-15 has also been shown to antagonize the potentiating effects of aldosterone on angiotensin II-induced contractions of human coronary microarteries (Batenburg et al., 2012). These findings have potentially important implications for the role of GPER-1 in the regulation of kidney function. Moreover, the suggestion that GPER-1 is an aldosterone receptor (Funder, 2011) challenges the earlier proposals that GPER-1 is a specific estrogen membrane receptor (Thomas et al., 2005, Revankar et al., 2005). However, despite claims that GPER-1 functions as an aldosterone receptor, specific binding of aldosterone and GPER-1 has not yet been demonstrated.

In order to determine the localization and ligand specificity of GPER-1 in the kidney, we explored the topographic distribution of GPER-1 in renal tubules and investigated whether aldosterone directly binds to GPER-1. Finally, GPER-1 expression was evaluated in renal epithelia during the estrus cycle. Our results indicate that GPER-1 is not expressed in glomeruli or collecting tubules but preferentially expressed within, or in close proximity to the basolateral membrane of epithelia that comprise proximal and distal convoluted tubules and the loop of Henle, and that its expression is modulated during estrus cycle. We also demonstrate that ligand binding to GPER-1 is specific for estrogens and that aldosterone and several MR antagonists do not bind to GPER-1 or cause G protein activation through the receptor.

Section snippets

Animals, cell lines and antibodies

Male and female mice were purchased from Charles River Laboratories (Shrewsbury, MA). Animals were kept in climate-controlled colonies (23.3 ± 2 °C; humidity 30–70%) with a 12 h light/dark cycle. All procedures involving animals were performed in accordance with the guidelines of the Institutional Animal Care and Use committee of Brown University in compliance with the guidelines established by the National Institute of Health. Estrus cycles were determined via vaginal lavage cytology as previously

Anatomic distribution of GPER-1 in murine renal tubules

Our prior study revealed that GPER-1 is predominantly localized to the basolateral membrane of epithelia in the cortex and medulla but not glomeruli of adult male rats by immunohistochemistry (Cheng et al., 2011a). Here, the topographic localization of GPER-1 was further mapped in mouse male and female renal tubules using distinct position-specific markers, including aquaporin-2 for collecting tubules, megalin for proximal convoluted tubules, and Tamms-Horsfeld Protein (THP) for the Loop of

Discussion

Recent studies have begun to address a potential role of GPER-1 in estrogen responsiveness in the kidney (Lindsey et al., 2011, Hutchens et al., 2012). Other work has suggested that GPER-1 may promote aldosterone signaling in vascular smooth muscle (Gros et al., 2011, Gros et al., 2013), a result with potentially important implications for understanding the physiological roles of GPER-1in kidney tissue. Here, we have addressed the anatomical distribution pattern of GPER-1 in the kidney and its

Conclusions

Our findings reveal that GPER-1 is expressed on the basolateral surfaces in proximal convoluted tubules and both at the plasma membrane and intracellularly in distal convoluted tubules and in the Loop of Henle, but not in the glomerulus. Moreover, we show that plasma membrane fractions prepared from kidney tissue exhibit specific binding activity for 17β-estradiol but not aldosterone, although aldosterone-binding activity is easily measured in soluble cytoplasmic fractions where

Acknowledgements

This work was supported by National Institutes of Health Grants RO1 CA119165 (to E.J.F.) and R01 ESO 12961 (to P.T.). The sponsors had no involvement in any aspect of this project.

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      Although the general location of GPCRs is practically exclusive to the plasma membrane, in the case of GPER there are studies that demonstrate its location in the plasma membrane (Filardo, 2002; Filardo et al., 2002) but also in intracellular membranes such as the endoplasmic reticulum (Revankar et al., 2005) and in the nucleus (Pupo et al., 2013; Smith et al., 2009). Recent studies have shown that the cellular localization of GPER depends on a complex trafficking in which clathrin vesicles are involved and new studies are necessary to clarify the differential signaling that may be regulating this trafficking (Cheng et al., 2014). This receptor contains specific binding site for E2 and the affinity of this binding it has been reported in 3–6 mM range, this range is higher compare with ERα and ERβ that binds in the range of 0.1–0.5 nM (Revankar et al., 2005; Thomas et al., 2005).

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