Abstract
Estrogen triggers rapid yet transient activation of the MAPKs, extracellular signal-regulated kinase (Erk)-1 and Erk-2. We have reported that this es- trogen action requires the G protein-coupled re- ceptor, GPR30, and occurs via G-subunit pro- tein-dependent transactivation of the epidermal growth factor (EGF) receptor through the release of pro-heparan-bound EGF from the cell surface. Here we investigate the mechanism by which Erk- 1/-2 activity is rapidly restored to basal levels after estrogen stimulation. Evidence is provided that at- tenuation of Erk-1/-2 activity by estrogen occurs via GPR30-dependent stimulation of adenylyl cy- clase and cAMP-dependent signaling that results in Raf-1 inactivation. We show that 17-E2 re- presses EGF-induced activation of the Raf-to-Erk pathway in human breast carcinoma cells that ex- press GPR30, including MCF-7 and SKBR3 cells which express both or neither, ER, respectively. MDA-MB-231 cells, which express ER, but not ER, and low levels of GPR30 protein, are unable to stimulate adenylyl cyclase or promote estrogen- mediated blockade of EGF-induced activation of Erk-1/-2. Pretreatment of MDA-MB-231 cells with cholera toxin, which ADP-ribosylates and activates Gs subunit proteins, results in G protein-coupled receptor (GPCR)-independent adenylyl cyclase ac- tivity and suppression of EGF-induced Erk-1/-2 ac- tivity. Transfection of GPR30 into MDA-MB-231 cells restores their ability to stimulate adenylyl cy- clase and attenuate EGF-induced activation of Erk- 1/-2 by estrogen. Moreover, GPR30-dependent, cAMP-mediated attenuation of EGF-induced Erk- 1/-2 activity was achieved by ER antagonists such as tamoxifen or ICI 182, 780; yet not by 17-E2 or progesterone. Thus, our data delineate a novel mechanism, requiring GPR30 and estrogen, that acts to regulate Erk-1/-2 activity via an inhibitory signal mediated by cAMP. Coupled with our prior findings, these current data imply that estrogen balances Erk-1/-2 activity through a single GPCR via two distinct G protein-dependent signaling pathways that have opposing effects on the EGF receptor-to-MAPK pathway. (Molecular Endocrin- ology 16: 70-84, 2002)