2 SEPARATE MECHANISMS FOR LIGAND-INDEPENDENT ACTIVATION OF THE ESTROGEN-RECEPTOR

Transient transfection experiments in which three different estrogen r esponse element-containing reporter genes were cotransfected into HeLa cells, together with constitutively expressed estrogen receptor (ER) constructs, demonstrate that activation of the transcription of the re porter genes by epidermal growth factor (EGF) and by cholera toxin wit h 3-isobutyl-1-methyl-xanthine, which elevate cellular cAMP, is depend ent upon the presence of functional ER, Cotransfection of the reporter genes with truncated versions of the ER shows that the two non-ligand activators of ER require different regions of the receptor to produce their effects on transcription, EGF acts primarily by means of transa ctivation domain AF-1, whereas cAMP acts via transactivation domain AF -2 of the ER. A point mutation that removes a major site of inducible phosphorylation within the AF-1 domain of the ER abolishes the respons e to EGF, but the response to estradiol and cAMP is retained, Specific inhibition of cAMP-activated protein kinase (protein kinase A) preven ts the response to elevated cAMP but does not affect EGF or estradiol responses. Overexpression of the protein kinase A catalytic subunit in HeLa cells results in an amplified response to estradiol, similar to that induced by cholera toxin with 3-isobutyl-1-methyl-xanthine. Compa rable experiments performed using COS-1 cells produce similar results but also reveal cell type- and promoter-specific aspects of the activa tion mechanisms. Apparently, the ER may be activated by three differen t signal molecules, estradiol, EGF, and cAMP, each using a mechanism t hat is distinguishable from that of the others.