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 Shi-Bin Cheng a , Jing Dong c , Yefei Pang c , Jessica LaRocca b , Mary Hixon b , Peter Thomas c,⇑ , Edward J. Filardo a,⇑ a Division of Hematology & Oncology, Rhode Island Hospital, Alpert Medical School of Brown University, RI, United States b Department of Pathology and Laboratory Medicine, Rhode Island Hospital, Alpert Medical School of Brown University, RI, United States c Marine Science Institute, University of Texas at Austin, Port Aransas, TX, United States article info Article history: Received 11 July 2013 Received in revised form 1 November 2013 Accepted 6 November 2013 Available online 13 November 2013 Keywords: GPER-1 Aldosterone binding Estradiol binding Distal convoluted tubules Estrus cycle Mineralocorticoid receptor 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 ste- roid 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 pre- pared from whole kidney tissue or HEK293 cells expressing recombinant human GPER-1 (HEK-GPER-1) displayed high-affinity, specific [ 3 H]-17b-estradiol ([ 3 H]-E2) binding, but no specific [ 3 H]-aldosterone binding. In contrast, cytosolic preparations exhibited specific binding to [ 3 H]-aldosterone but not to [ 3 H]-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 [ 3 H]-E2 binding to membranes of HEK-GPER-1 cells. Furthermore, aldosterone did not increase [ 35 S]-GTP-cS 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 redis- tributed 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 dur- ing reproduction. Ó 2013 Elsevier Ireland Ltd. All rights reserved. 1. 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), ERa and ERb (Hewitt et al., 2005; Terasawa and Kenealy, 2012). Estrogens also promote homeostatic and protective effects in numerous estrogen respon- sive 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, 2012; Lindsey et al., 2011). However, the precise molecular mechanism by which estrogens promote homeostatic and protec- tive 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 estro- gens in responsive tissues are manifested by the G-protein-coupled 0303-7207/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mce.2013.11.005 ⇑ Corresponding authors. Address: Marine Science Institute, University of Texas at Austin, 750 Channel View Drive, Port Aransas, TX 78373, United States (P. Thomas). Address: Radix BioSolutions, Inc., 111 Cooperative Way, Georgetown, TX 78626, United States. Tel.: +1 512 869 8000 (E.J. Filardo) E-mail addresses: peter.thomas@utexas.edu (P. Thomas), ed@radixbiosolutions. com (E.J. Filardo). Molecular and Cellular Endocrinology 382 (2014) 950–959 Contents lists available at ScienceDirect Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce