TI - Membrane estrogen receptor alpha levels predict estrogen -induced ERK1/2 activation in MCF-7 cells . AB - Introduction We examined the participation of a membrane form of estrogen receptor ( mER ) -alpha in the activation of mitogen -activated protein kinases ( extracellular signal-regulated kinase [ERK] 1 and ERK2 ) related to cell growth responses in MCF-7 cells . Methods We immunopanned and subsequently separated MCF-7 cells ( using fluorescence-activated cell sorting ) into mER-alpha-enriched ( mERhigh ) and mER-alpha-depleted ( mERlow ) populations . We then measured the expression levels of mER-alpha on the surface of these separated cell populations by immunocytochemical analysis and by a quantitative 96-well plate immunoassay that distinguished between mER-alpha and intracellular ER-alpha . Western analysis was used to determine colocalized estrogen receptor ( ER ) -alpha and caveolins in membrane subfractions . The levels of activated ERK1 and ERK2 were determined using a fixed cell -based enzyme -linked immunosorbent assay developed in our laboratory . Results Immunocytochemical studies revealed punctate ER-alpha antibody staining of the surface of nonpermeabilized mERhigh cells , whereas the majority of mERlow cells exhibited little or no staining . Western analysis demonstrated that mERhigh cells expressed caveolin-1 and caveolin-2 , and that ER-alpha was contained in the same gradient-separated membrane fractions . The quantitative immunoassay for ER-alpha detected a significant difference in mER-alpha levels between mERhigh and mERlow cells when cells were grown at a sufficiently low cell density , but equivalent levels of total ER-alpha ( membrane plus intracellular receptors ) . These two separated cell subpopulations also exhibited different kinetics of ERK1/2 activation with 1 pmol/l 17beta-estradiol ( E2 ) , as well as different patterns of E2 dose -dependent responsiveness . The maximal kinase activation was achieved after 10 min versus 6 min in mERhigh versus mERlow cells , respectively . After a decline in the level of PHOSphorylated ERKs , a reactivation was seen at 60 min in mERhigh cells but not in mERlow cells . Both 1A and 2B protein phosphatases participated in dePHOSphorylation of ERKs , as demonstrated by efficient reversal of ERK1/2 inactivation with okadaic/ acid and cyclosporin A . Conclusion Our results suggest that the levels of mER-alpha play a role in the temporal coordination of phosphorylaTION/dePHOSphorylation events for the ERKs in breast cancer cells , and that these signaling differences can be correlated to previously demonstrated differences in E2 -induced cell proliferation outcomes in these cell types .