Involvement of estrogen receptor variant ER-alpha36, not GPR30, in nongenomic estrogen signaling

Abstract

Accumulating evidence suggested that an orphan G protein-coupled receptor (GPR)30, mediates nongenomic responses to estrogen. The present study was performed to investigate the molecular mechanisms underlying GPR30 function. We found that knockdown of GPR30 expression in breast cancer SK-BR-3 cells down-regulated the expression levels of estrogen receptor (ER)-alpha36, a variant of ER-alpha. Introduction of a GPR30 expression vector into GPR30 nonexpressing cells induced endogenous ER-alpha36 expression, and cotransfection assay demonstrated that GPR30 activated the promoter activity of ER-alpha36 via an activator protein 1 binding site. Both 17beta-estradiol (E2) and G1, a compound reported to be a selective GPR30 agonist, increased the phosphorylation levels of the MAPK/ERK1/2 in SK-BR-3 cells, which could be blocked by an anti-ER-alpha36-specific antibody against its ligand-binding domain. G1 induced activities mediated by ER-alpha36, such as transcription activation activity of a VP16-ER-alpha36 fusion protein and activation of the MAPK/ERK1/2 in ER-alpha36-expressing cells. ER-alpha36-expressing cells, but not the nonexpressing cells, displayed high-affinity, specific E2 and G1 binding, and E2- and G1-induced intracellular Ca(2+) mobilization only in ER-alpha36 expressing cells. Taken together, our results demonstrated that previously reported activities of GPR30 in response to estrogen were through its ability to induce ER-alpha36 expression. The selective G protein-coupled receptor (GPR)30 agonist G1 actually interacts with ER-alpha36. Thus, the ER-alpha variant ER-alpha36, not GPR30, is involved in nongenomic estrogen signaling.

Figure 1

GPR30 signaling induces ER-α36 expression. A, GPR30 transfection induced endogenous ER-α36 expression in HEK293 and COS-7 cells. An HA-GPR30 expression vector or an empty expression vector was transiently transfected into HEK293 and COS-7 cells. Western blot and RT-PCR analysis were used to examine the expression levels of GPR30 and endogenous ER-α36 or ER-α66 in transfected cells. B, Knockdown of GPR30 expression in SK-BR-3 cells resulted in down-regulation of endogenous ER-α36 expression. SK-BR-3 cells were transiently transfected with GPR30 siRNA or control siRNA for 48 h. The expression levels of endogenous ER-α36 protein and mRNA in transfected cells were assessed with Western blot and RT-PCR analysis.

Figure 2

GPR30 signaling activates ER-α36 promoter activity. A, Schematic structures of luciferase reporter plasmid driven by different 5′-truncated promoters of ER-α36. The −736, −584, −513, and −296 indicate residues upstream of the transcription initiation site, respectively. An AP-1-binding site, which was mutated in pER36-mAP-1, is also indicated. B, HEK293 cells were transfected with different reporter plasmids together with an empty expression vector or the expression vector for HA-GPR30, and luciferase activities were assayed and normalized using a cytomegalovirus-driven Renilla luciferase plasmid. Columns, Means of four independent experiments; bars, se. *, P < 0.05, for cells transfected with GPR30 vs. without GPR30. C, HEK293 cells were transfected with the pER36–736 reporter plasmid with the empty expression vector or the expression vector for GPR30 and then treated with vehicle, 10 μm U0126, 10 μm PP2, or 10 μm LY294002 for 24 h after which luciferase activities were analyzed. Results shown in the graph were mean ± se, and experiments were repeated four times. *, P < 0.05 for cells cotransfected with an empty expression vector and treated with vehicle vs. different conditions. D, SK-BR-3 cells maintained in DMEM supplemented with 10% FBS were treated with vehicle, 10 μm U0126, 10 μm PP2, or 10 μm LY294002 for 24 h. Cells were then harvested and analyzed by Western blot analysis. RLU, Relative light units.

Figure 3

G1 specifically induces ER-α36 functions. A, Schematic drawing of the expression vectors for VP16-ER-α36, ER-α66, and ER-β, and of a luciferase reporter gene driven by two EREs and a minimal promoter, 2×ERE-Luc. B, G1 activates transcriptional activity mediated by a VP-16-ER-α36 fusion protein. HEK293 cells were cotransfected with expression vectors for ER-α66, ER-β, or VP16-ER-α36 together with 2×ERE-Luc and then treated with indicated concentrations of E2 or G1. After normalization, luciferase activities were assessed. The columns represent means ± se of three experiments. *, P < 0.05 for vehicle control vs. different treatments. C and D, G1 induces ERK1/2 phosphorylation in 293/ER-α36 cells. The 293/Vector, 293/ER-α36, and 293/ER-α66 cells in serum-free medium were treated with vehicle and indicated concentrations of E2, G1, or EGF for 10 min. Western blot analysis was performed to assess induction of ERK1/2 phosphorylation. The columns represent three studies combined. *, P < 0.05 for vehicle control vs. different treatments. The representative results are shown in panel C. E, The GPR30, ER-α36, and ER-α66 expression status in HEK293/Vector (HEK293/V), HEK293/ER-α36 (HEK293/36), and HEK293/ER-α66 (HEK293/66) cells.

Figure 4

G1 is a selective ER-α36 agonist. A, Western blot and RT-PCR analysis of SK-BR-3/V and SK-BR-3/36Sh cells for GPR30 and ER-α36 expression. SK-BR-3/V, SK-BR-3 cells transfected the empty expression vector. SK-BR-3/36Sh, SK-BR-3 cells transfected with an ER-α36-specific shRNA expression vector. B, SK-BR-3/V and SK-BR-3/36Sh cells maintained in serum-free medium were treated with vehicle and indicated concentrations of E2, G1, or EGF for 10 min. Cells were then harvested and lysed for Western blot analysis to assess phosphorylation levels of ERK1/2. The columns represent three experiments combined. *, P < 0.05 for vehicle control vs. different treatments. The representative results are also shown. C, Both E2 and G1 failed to induce ERK phosphorylation in GPR30 expression knocked down SK-BR-3 cells. SK-BR-3+Csi, SK-BR-3 cells transfected with control siRNA. SK-BR-3+Gsi, SK-BR-3 cells transfected with GPR30 siRNA. The columns represent three combined experiments. *, P < 0.05 for vehicle control vs. different treatments. The representative results are also shown. D, An anti-ER-α36-specific antibody blocked induction of ERK1/2 phosphorylation by both E2 and G1. SK-BR-3 cells maintained in serum-free medium were treated with PBS, anti-ER-α36 antibody at 0.1 or 1 μg/ml, or 1 μg/ml of rabbit IgG 1 h before being treated with vehicle and indicated concentrations of E2, G1, or EGF for 10 min. Western blot analysis was used to assess the levels of ERK1/2 phosphorylation. The columns represent the mean ± se from three experiments. The representative results are also shown. *, P < 0.05 for cells treated with vehicle vs. different treatments; Δ, P < 0.05, for cells treated with antibody vs. without antibody; the representative results are also shown. E, The expression levels of GPR30, ER-α36, and ER-α66 in MCF7/V and MCF7/36 cells transiently transfected with control siRNA or GPR30 siRNA, respectively. F, The phosphorylation levels of the MAPK/ERK1/2 after the treatment of G1 (0.1 nm) in MCF7/V and MCF7/36 cells transfected with control siRNA or GPR30 siRNA. Ab, Antibody; Ctrl, control.

Figure 5

Saturation and competitor binding assay of E2 and G1. A and B, Saturation and competitor binding assay in HEK293/vector (293/V) and HEK293/ER-α36 (293/36) cells. C and D, Saturation and competitor binding assay in SK-BR-3/V (SK/V) and SK-BR-3/36Sh (SK/36S) cells. The spots represent the means from three experiments. E, The difference of B_max between 293/V and 293/36, and SK/V and SK/36S. F, Both ER-α36 (a) and ER-α66 (b) are colored green for side-by-side comparison. The ligand E2 is colored blue. The conformation is derived from cocrystallization data for ER-α66 agonist conformation (Protein Data Bank Code: 1ERE). c, Docking of G-1 into the ER-α36 homology model. The optimal ER-α36 docked structures of G1 based on protein interactions as computed using AutoDock is shown in yellow, and complementary residues on ER-α36 receptor model are shown in green.

Figure 6

ER-α36 mediates intracellular Ca²⁺ mobilization induced by E2 and G1. A, The expression status of ER-α36 and GPR30 in MDA-MB-231 cell variants: MDA-MB-231 cells transfected with an empty expression vector (231/V) and with an ER-α36 shRNA expression vector (231/36Sh). B and C, Intracellular Ca²⁺ mobilization was measured in SK-BR-3cell variants (B) and MDA-MB-231 cell variants (C). Experiments were performed as described in detail in Materials and Methods after being treated with vehicle or indicated concentrations of E2 or G1. D, Intracellular Ca²⁺ mobilization was measured in COS-7 cells transiently transfected with the GPR30 expression vector together with the empty expression vector or the ER-α36 shRNA expression vector. Results shown are means of 20 cells emission. Experiments were repeated three times and the representative data are shown.