The sequence Pro295–Thr311 of the hinge region of oestrogen receptor α is involved in ERK1/2 activation via GPR30 in leiomyoma cells

Abstract

The ERα (oestrogen receptor α)-derived peptide ERα17p activates rapid signalling events in breast carcinoma cells under steroid-deprived conditions. In the present study, we investigated its effects in ELT3 leiomyoma cells under similar conditions. We show that it activates ERK1/2 (extracellular-signal-regulated kinase 1/2), the Gαi protein, the trans-activation of EGFR (epidermal growth factor receptor) and, finally, cell proliferation. It is partially internalized in cells and induces membrane translocation of β-arrestins. The activation of ERK1/2 is abolished by the GPR30 (G-protein-coupled receptor 30) antagonist G15 and GPR30 siRNA. When ERα is down-regulated by prolonged treatment with E2 (oestradiol) or specific ERα siRNA, the peptide response is blunted. Thus the simultaneous presence of GPR30 and ERα is required for the action of ERα17p. In addition, its PLM sequence, which interferes with the formation of the ERα-calmodulin complex, appears to be requisite for the phosphorylation of ERK1/2 and cell proliferation. Hence ERα17p is, to our knowledge, the first known peptide targeting ERα-GPR30 membrane cross-talk and the subsequent receptor-mediated biological effects.

Selective activation of estrogen receptors, ERα and GPER-1, rapidly decreases food intake in female rats

“…Additionally, blockade of both ERα and ERβ in an isolated perfused heart attenuated G-1-mediated changes in heart rate and left ventricular pressure (Filice et al, 2009). In another study, blockade of GPER-1 attenuated the ability of the ERα-derived peptide, ERα17p, to activate ERK1/2 in leiomyoma cells (Leiber et al, 2015). Even fewer studies have investigated the potential cross talk between GPER-1 and ERα in the brain.…”

Interaction of the Anti-Proliferative GPER Inverse Agonist ERα17p with the Breast Cancer Cell Plasma Membrane: From Biophysics to Biology

“…Strikingly, ERα17p aggregates induce membrane invaginations, an observation that could be related to an entry mechanism ( Figure 4B) [51]. Accordingly, it has been demonstrated that the peptide ERα17p was internalized, even though weakly, in leiomyoma cells (Elt3) and after 75 min incubation (5.5 ± 1.8 pmol/250,000 cells) [14]. In CHO cells and under similar experimental conditions, it is marginally internalized (0.3 ± 0.1 pmol/10 6 cells) [25].…”

“…5(6)-carboxyfluorescein was purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). The peptide ERα17p was obtained and characterized as previously described [14,25]. MCF-7 breast cancer cells were cultured in growth Dulbecco's Modified Eagle Medium DMEM (ThermoFisher Scientific, Courtaboeuf, France) supplemented with fetal calf serum (FCS), penicillin/streptomycin (100,000 IU/L), and amphotericin B (1 mg/L), in a humidified atmosphere containing 5% CO 2 at 37 • C.…”

“…μM [11,12,14] with POPC/POPS/Chol (7:1:2 mol/mol/mol) induces about 18% membrane leakage at the lipid:peptide ratio of 10:1 and around 10% at the lipid:peptide ratio 20:1 (Figure 3). A two-fold decrease of the percentage of membrane leakage is found, as previously published with vesicles composed of anionic lipids, exclusively [25].…”

“…In the light of these observations, we have synthesized the 17-mer peptide corresponding to the ERα 295-311 region (sequence: H 2 N-PLMIKRSKKNSLALSLT-COOH, peptide ERα17p) and we have tested its action in different experimental conditions and on different breast cancer cell lines. For unknown reasons, this peptide is estrogenic in steroid-deprived conditions [2,[11][12][13][14], whereas it is anti-proliferative in complete (i.e., physiological) serum conditions, as evidenced by the decrease of the Bcl xL/ Bax ratio, the increase of cleaved caspase-9, and changes in cell migration and actin network [15][16][17][18]. Albeit much more weakly than in cells expressing the ERα, apoptosis is also observed in ERα-negative breast cancer cell lines, suggesting the involvement of other estrogen-dependent receptor(s).…”