Estrogen- and xenoestrogen-induced ERK signaling in pituitary tumor cells involves estrogen receptor-α interactions with G protein-αi and caveolin I

Multiple physiologic estrogens (estradiol, estriol, and estrone), as well as xenoestrogenic compounds (including alkylphenols and bisphenol A), can act via nongenomic signaling initiated by liganding of the plasma membrane estrogen receptor-alpha (mER alpha). We examined heterotrimeric G protein involvement leading to extracellular-regulated kinase (ERK) activation in GH3/B6/F10 rat anterior pituitary tumor cells that express abundant mER alpha, and smaller amounts of mER beta and GPR30. A combination of microarrays, immunoblots, and quantitative immunoassays demonstrated the expression of members of all alpha, beta, and gamma G protein classes in these cells. Use of selective inhibitors showed that the G(alpha i) subtype was the primary initiator of downstream ERK signaling. Using antibodies against the GTP-bound form of G(alpha) protein subtypes i and s, we showed that xenoestrogens (bisphenol A. nonylphenol) activated G(alpha i) at 15-30 s; all alkylphenols examined subsequently suppressed activation by 5 min. GTP-activation of G(alpha i) for all estrogens was enhanced by irreversible cumulative binding to GTP gamma S. In contrast, G(alpha s) was neither activated nor deactivated by these treatments with estrogens. ER alpha and G(alpha i) co-localized outside nuclei and could be immuno-captured together. Interactions of ER alpha with G(alpha i) and caveolin I were demonstrated by epitope proximity ligation assays. An ER alpha/beta antagonist (ICI182780) and a selective disruptor of caveolar structures (nystatin) blocked estrogen-induced ERK activation. Conclusions: Xenoestrogens, like physiologic estrogens, can evoke downstream kinase signaling involving selective interactions of ER alpha with G(alpha i) and caveolin I, but with some different characteristics, which could explain their disruptive actions. (C) 2012 Elsevier Inc. All rights reserved.