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-α (mERα). 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 mERα, and smaller amounts of mERβ and GPR30. A combination of microarrays, immunoblots, and quantitative immunoassays demonstrated the expression of members of all α, β, and γ G protein classes in these cells. Use of selective inhibitors showed that the G(αi) subtype was the primary initiator of downstream ERK signaling. Using antibodies against the GTP-bound form of G(α) protein subtypes i and s, we showed that xenoestrogens (bisphenol A, nonylphenol) activated G(αi) at 15-30s; all alkylphenols examined subsequently suppressed activation by 5min. GTP-activation of G(αi) for all estrogens was enhanced by irreversible cumulative binding to GTPγS. In contrast, G(αs) was neither activated nor deactivated by these treatments with estrogens. ERα and G(αi) co-localized outside nuclei and could be immuno-captured together. Interactions of ERα with G(αi) and caveolin I were demonstrated by epitope proximity ligation assays. An ERα/β 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 ERα with G(αi) and caveolin I, but with some different characteristics, which could explain their disruptive actions.
Dept. of Biochemistry & Molecular Biology, Univ. of Texas Medical Branch, Galveston, TX 77555-0645, USA. cswatson@utmb.edu
Cheryl S
CS
Watson
Yow-Jiun
YJ
Jeng
Guangzhen
G
Hu
Ann
A
Wozniak
Nataliya
N
Bulayeva
Jutatip
J
Guptarak
D000818:Animals; D001559:Benzhydryl Compounds; D021941:Caveolae / Q000378:metabolism; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism*; D004967:Estrogens / Q000494:pharmacology*; D004968:Estrogens, Non-Steroidal / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D015151:Immunoblotting; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D008856:Microscopy, Fluorescence; D020411:Oligonucleotide Array Sequence Analysis; D010636:Phenols / Q000494:pharmacology*; D010911:Pituitary Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D000098826:Bisphenol A Compounds
D001559:Benzhydryl Compounds; D051242:Caveolin 1; D047628:Estrogen Receptor alpha; D004967:Estrogens; D004968:Estrogens, Non-Steroidal; D048049:Extracellular Signal-Regulated MAP Kinases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D006160:Guanosine Triphosphate; D010636:Phenols; C025256:nonylphenol; C006780:bisphenol A; D000098826:Bisphenol A Compounds