Involvement of Estrogen Receptor Variant ER-α36, Not GPR30, in Nongenomic Estrogen Signaling

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)-alpha 36, a variant of ER-alpha. Introduction of a GPR30 expression vector into GPR30 nonexpressing cells induced endogenous ER-alpha 36 expression, and cotransfection assay demonstrated that GPR30 activated the promoter activity of ER-alpha 36 via an activator protein 1 binding site. Both 17 beta-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-alpha 36-specific antibody against its ligand-binding domain. G1 induced activities mediated by ER-alpha 36, such as transcription activation activity of a VP16-ER-alpha 36 fusion protein and activation of the MAPK/ERK1/2 in ER-alpha 36 expressing cells. ER-alpha 36-expressing cells, but not the nonexpressing cells, displayed high-affinity, specific E2 and G1 binding, and E2- and G1-induced intracellular Ca2+ mobilization only in ER-alpha 36 expressing cells. Taken together, our results demonstrated that previously reported activities of GPR30 in response to estrogen were through its ability to induce ER-alpha 36 expression. The selective G protein-coupled receptor (GPR) 30 agonist G1 actually interacts with ER-alpha 36. Thus, the ER-alpha variant ER-alpha 36, not GPR30, is involved in nongenomic estrogen signaling. (Molecular Endocrinology 24: 709-721, 2010)