Role of Calcium in Activation of Estrogen Receptor-Alpha

Abstract

ABSTRACT Growth factors are thought to activate estrogen receptor-alpha ERα by phosphorylating the serines in the N-terminus of the receptor. However, this mechanism does not account for the conformational changes that occur in the ligand binding domain (LBD) of receptor to render the receptor active. It is hypothesized that epidermal growth factor (EGF) activates ERα through the PLC--calcium-calmodulin pathway by increasing intracellular calcium which, upon binding to the LBD, induces a conformational change that activates the receptor. In support of this hypothesis, treatment of MCF-7 cells with EGF (150 ng/ml) lead to an increase in intracellular calcium from 150 nM to 350 nM (+/-70 nM) and the induction of the ERα responsive genes, progesterone receptor (PgR) and pS2. The ability of EGF to induce PgR and pS2 mRNA was blocked by inhibitors of phospholipase C-, calmodulin, and calmodulin kinase II, the intracellular calcium chelator BAPTA-AM, and by an antiestrogen. Treatment with high concentrations of extracellular calcium also increased intracellular calcium and induced PgR and pS2. This induction was blocked by chelating intracellular calcium providing additional support that intracellular calcium activates ERα. Treatment with calcium also resulted in dose dependent growth of MCF-7 cells suggesting that calcium has estrogen-like effects on cell proliferation. To determine whether EGF activates the C-terminus of the ERα, transient transfections were done with wild type ERα, N-terminus and C-terminus mutants in. As expected, EGF activated the wt ERα and the N-terminus mutant of the receptor. In addition EGF activated the C-terminus mutant suggesting that EGF could activate ERα through the C-terminus. To test the hypothesis that calcium activates ERα by directly binding to the ligand binding domain of the receptor, the ability of ERα to bind to calcium was tested. Calcium bound to the receptor with an affinity of 0.5 µM (+/- 0.6 μM) and four moles of calcium bound per mole of the receptor. Similar results were obtained with the LBD of the receptor suggesting that calcium bound in the LBD of the receptor. When the LBD is activated, the major movements involve helices 3, 4, 10, 11 & 12. To determine the amino acids involved in calcium activation, wild type ERα, mutants D351A, C381A, E380Q, C447A (helix 3 & 4), mutants E419A, E423A (helix 7 & loop between helix 7 &8), mutants H516A, N519A, E523A (helix 10 & 11) and mutants N532A, D538A, E542A, D545N and H547A (loop between helix 11&12 and helix 12) were tested in transient transfection assays. Of all the mutants tested E380 (H4), H516A (H10), E523A (H11) and D538A (H12) significantly blocked the effect of EGF and calcium suggesting that these amino acids are involved in calcium activation. Thus, EGF activates ERα by increasing intracellular calcium which binds to the LBD, thereby activating the receptor.