Estrogen triggers rapid yet transient activation of the MAPKs, extracellular signal-regulated kinase (Erk)-1 and Erk-2. We have reported that this estrogen action requires the G protein-coupled receptor, GPR30, and occurs via Gbetagamma-subunit protein-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 attenuation of Erk-1/-2 activity by estrogen occurs via GPR30-dependent stimulation of adenylyl cyclase and cAMP-dependent signaling that results in Raf-1 inactivation. We show that 17beta-E2 represses EGF-induced activation of the Raf-to-Erk pathway in human breast carcinoma cells that express GPR30, including MCF-7 and SKBR3 cells which express both or neither, ER, respectively. MDA-MB-231 cells, which express ERbeta, but not ERalpha, 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 Galphas subunit proteins, results in G protein-coupled receptor (GPCR)-independent adenylyl cyclase activity and suppression of EGF-induced Erk-1/-2 activity. Transfection of GPR30 into MDA-MB-231 cells restores their ability to stimulate adenylyl cyclase 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 17alpha-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.

Protein kinases that phosphorylate the hydroxyl group of tyrosine residues of proteins have been implicated in cell transformation by some retroviruses and in regulation of normal cell growth by some polypeptide growth factors. To facilitate the identification of tyrosine kinase substrates, we developed monoclonal antibodies to the hapten azobenzylphosphonate. One of these antibodies, MA-2G8, proved to be especially attractive in that it bound a derivative of aminophenylphosphate, a close phosphotyrosine analog, with higher affinity than it bound the corresponding derivative of aminobenzylphosphonate; however, its affinity for phosphoserine was negligible. In this paper we describe the optimal conditions for using this antibody to isolate phosphotyrosine proteins, emphasizing particularly that its interaction with phosphotyrosyl proteins is sensitive to ionic detergents and to antibody density on the immunosorbent matrix. The antibody also bound ATP citrate lyase; this enzyme lacks phosphotyrosine but contains phosphohistidine, which is similar structurally to phosphotyrosine. By attaching the antibody at high density to Sepharose beads and omitting ionic detergents from the buffers, it was possible by microbatch immunoadsorption (followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to isolate the 120,000-dalton transforming protein and several other phosphotyrosyl proteins from cells transformed by Abelson murine leukemia virus. Under the same conditions, phosphotyrosyl proteins were also isolated from human epidermal carcinoma cells (A431) that had been stimulated with epidermal growth factor; most prominent among these proteins was the 170,000-dalton receptor for epidermal growth factor.

Tyrosine-specific phosphorylation of the epidermal growth factor (EGF) receptor in hormonally stimulated A431 cells is blocked by three chemically distinct classes of tumor promoters. Tumor-promoting esters of the diterpene phorbol (phorbol 12-myristate 13-acetate, P-phorbol 12,13-dibutyrate, and (3-phorbol 12,13-didecanoate), indole alkaloids (teleocidin and lyngbyatoxin A), and polyacetates (aplysiatoxin and debromoaplysiatoxin) all inhibited EGF-stimulated phosphorylation of the receptor. Non-tumor-promoting analogs (,Bphorbol, a-phorbol 12,13-didecanoate, and hydrolyzed teleocidin) had no effect on the levels of receptor phosphorylation.The ED5o values of the inhibitory effect (0.1-3 ng/ml) reflected the relative tumor-promoting abilities of these compounds in vivo. None of the tumor promoters tested significantly de-creased the overall specific binding of 125I-labeled EGF to A431 cells. Scatchard analysis, however, revealed two apparent EGF receptors in this cell type. The dose-responses for tumor-promoter inhibition of EGF receptor tyrosine phosphorylation and high-affinity EGF binding were similar, suggesting that the same initial event is responsible for both effects.This demonstrates a correlation between modulation of EGF receptor binding and phosphorylation of tyrosine by tumor promoters. The data suggest a possible role for protein kinase C, the putative cellular receptor for these tumor promoters, in the mechanism of action.Tumor promoters can modulate the action of epidermal growth factor (EGF) by reducing EGF receptor binding (1-4) and internalization (5, 6) and by potentiating the mitogenic activity of EGF in quiescent cells (7,8). Upon binding to cells, EGF stimulates the tyrosine phosphorylation of its receptors (9, 10) via a receptor-associated kinase activity. The potential regulatory role of tyrosine kinase activity is suggested by its association with the action of other growth factors (insulin and platelet-derived growth factor) (11-13) and its apparent requirement for oncogenic transformation by a number of retroviruses (14,15). This led us to examine whether the effects of tumor promoters on the action of EGF involved changes in receptor phosphorylation.We have found that three chemically distinct classes of tumor promoters [esters of the diterpene phorbol, indole alkaloids (16), and polyacetates (17) 32Pi Labeling and Detergent Extraction of Cells. Low-density cultures (105 cells per 35-mm well) of A431 cells were preincubated with 32p; at 0.5 mCi/ml in phosphate-free DME medium plus dialyzed heat-inactivated fetal calf serum for 3-4 hr at 37°C. Dimethyl sulfoxide (Me2SO; final concentration 1%), tumor promoters, or nonpromoting analogs (dissolved in Me2SO) were added for 10 min. EGF was then added for an additional 40 min. The cultures were placed on ice, the binding media were removed, and the cells were scraped and extracted with Tris-buffered Triton (10 mM Tris'HCl/1% Triton X-100/10 mM NaCl/5 mM EDTA/1 mM phenylmethylsulfonyl fluoride/0.1% bovine serum albumin, pH 8).Phosph...