Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF

Estrogen rapidly activates the mitogen-activated protein kinases, Erk-1 and Erk-2, via an as yet unknown mechanism. Here, evidence is provided that es trogen-induced Erk-1/-2 activation occurs independently of known estrogen r eceptors, but requires the expression of the G protein-coupled receptor hom olog, GPR30. We show that 17 beta-estradiol activates Erk-1/-2 not only in MCF-7 cells, which express both estrogen receptor alpha (ER alpha) and ER b eta, but also in SKBR3 breast cancer cells, which fail to express either re ceptor. Immunoblot analysis using GPR30 peptide antibodies showed that this estrogen response was associated with the presence of GPR30 protein in the se cells. MDA-MB-231 breast cancer cells (ER alpha(-), ER beta(+)) are GPR3 0 deficient and insensitive to Erk-1/-2 activation by 17 beta-estradiol. Tr ansfection of MDA-MB-231 cells with a GPR30 complementary DNA resulted in o verexpression of GPR30 protein and conversion to an estrogen-responsive phe notype. In addition, GPR30-dependent Erk-1/-2 activation was triggered by E R antagonists, including ICI 182,780, yet not by 17 alpha-estradiol or prog esterone. Consistent with acting through a G protein-coupled receptor, estr adiol signaling to Erk-1/-2 occurred via a G beta gamma-dependent, pertussi s toxin-sensitive pathway that required Src-related tyrosine kinase activit y and tyrosine phosphorylation of tyrosine 317 of the Shc adapter protein. Reinforcing this idea, estradiol signaling to Erk-1/-2 was dependent upon t rans-activation of the epidermal growth factor (EGF) receptor via release o f heparan-bound EGF (HB-EGF). Estradiol signaling to Erk-1/-2 could be bloc ked by: 1) inhibiting EGF-receptor tyrosine kinase activity, 2) neutralizin g HB-EGF with antibodies, or 3) down-modulating HB-EGF from the cell surfac e with the diphtheria toxin mutant, CRM-197. Our data imply that ER-negativ e breast tumors that continue to express GPR30 may use estrogen to drive gr owth factor-dependent cellular responses.