TI - Discussion . AB - In the late 1970s , Pietras and Szego [33] reported the presence of high-affinity binding sites for E2 associated with the plasma membranes of the MCF-7 human breast cancer cell line . Since that time few laboratories have followed up on this finding [34,35] until very recently , when more detailed studies started to emerge . However , none of these studies established a correlation between the level of identified mER-alpha expression and its functions . To address this issue we used immunopanning followed by FACS to separate MCF-7 cells into two subpopulations that were enriched and depleted for mER-alpha expression . We then used several approaches to assess the appearance and levels of mER-alpha in these two subpopulations . These studies demonstrated that MCF-7 cells are heterogeneous with respect to mER-alpha expression , and that difficulties in reproducibly demonstrating nongenomic estrogenic effects in these cells could in part be due to the dilution of responding cells in the largely nonresponsive total cell population . We were able to obtain two distinct cell subpopulations without applying plasmid-based transfection manipulations to over-express receptor ( with probable accompanying altered regulatory parameters ) . Our experimental model avoids changes in stoichiometry of the multiple interacting proteins that are involved in steroid actions . Because our mERhigh and mERlow cells were not clonally derived ( from a single cell ) , the observed differences between these subpopulations cannot be attributed to clonal variations . A characteristic punctate staining for mER ( earlier reported for GH3/B6/F10 cells [22] ) was also detected on the surface of most of our mERhigh MCF-7 cells . MCF-7 mERlow cells also had occasional punctuate staining , but at a much lower level . Because it is difficult and time-consuming to quantify this staining via immunocytochemistry , we modified our previously developed 96-well plate immunoassay [23] to measure both membrane and intracellular receptors in breast cancer cells , and to quantitate the relative amounts in these two receptor subpopulations . This assay must be tailored to specific cell types to ensure preservation of the membranes ( which have quite different compositions in different cell types ) and to optimize for different antibody labeling systems . Although mER-alpha levels differed between the two cell types named for these differences ( high versus low ) , we discovered that the two subpopulations had the same quantity of total receptor ( measured in permeabilized cells ) . This finding is consistent with intracellular and membrane fractions of ER-alpha being from the same ER pool , but with a different balance of subcellular distribution . There is disagreement in the literature on the expression of caveolin-1 and -2 in MCF-7 cells . To test whether mER is localized in caveolar membranes , we had first to resolve this uncertainty . Some have reported that in MCF-7 cells caveolin-1 is downregulated and only caveolin-2 is expressed [36,37] . Others reported that caveolin-1 could be upregulated and downregulated in MCF-7 cells in concert with the cells' ability to develop drug resistance [38] . Some studies used transient transfection to re-express the missing caveolin-1 and test for its function in signaling [7,39] . Apparently , there are several different subpopulations of MCF-7 cells growing in different laboratories [40,41] . Our MCF-7 cells expressed both caveolin-1 and -2 , and ER-alpha was associated with the same gradient fractions in which most of the caveolin proteins were detected . A 67 kDa ER and a variant of lower molecular weight ( 52 kDa ) were detected in both the cell lysate and the caveolar membrane gradient fractions . Until recently the lower molecular weight ER variants were thought to be proteolytic fragments , but evidence that some of those molecules are truncated products of the full-length ER-alpha was recently presented . It is well documented that in MCF-7 breast cancer cells ER-alpha mRNA can undergo alternative splicing , generating transcripts with single , double , or multiple exon deletions [42] , and that a 52 kDa protein is translated from the predominant splice variant mRNA that is missing exon 7 [43] . Others have identified a 46 kDa variant in human endothelial cells [44] and in MCF-7 cells [45,46] . Some evidence suggests that the 46 kDa receptor is the major functional membrane form of ER [44] ; however , the precise functions of the truncated ER-alphas are still under investigation . The traditional detection and quantitation of PHOSphorylated MAPKs via Western blot analysis is laborious and somewhat arbitrary in the designation of appropriate densitometric backgrounds for comparison , especially in situations where the significant activation response is not large . We developed an enzyme -linked immunosorbent assay to deal with these experimental problems using fixed cells [47] , which allowed convenient testing of large numbers of different conditions . Ours is the first report that different levels of mER in MCF-7 cells influence the different temporal and dose -dependent estrogen -induced PHOSphorylations of ERKs . The subpopulation of cells with high mER levels exhibited early and more robust activation , peaking at 10 min with a reactivation at 60 min , whereas the subpopulation of cells with low mER levels were only capable of weakly activating ERKs at one early time point ( 6 min ) . Furthermore , in the ER-alpha-negative cell line MDA-MB-231 , E2 could not activate these kinases . The physiologic significance of early ERK1/2 activation was confirmed by showing that short pulse E2 treatment also stimulated cell proliferation . We and others [46] have confirmed that mER-alpha in breast cancer cells is responsible for this effect by showing that E2-peroxidase can stimulate proliferation and that this effect can be abolished by prior blocking of ER-alpha with ligand -binding domain -specific antibody . The inability to activate ERKs with E2 in an ER-alpha-negative cell line , and the ability to do so in MCF-7 ER-alpha-positive cells , was assigned by others to the orphan G-protein-coupled receptor GPR30 [48] . However , recent studies with antisense GPR30 knockdowns revealed that E2 -stimulated MCF-7 cells proliferate as well as cells with normal levels of GPR30 [49,50] . ERK activation associated with the ability of these cells to respond to estrogens by proliferating thus does not appear to be a function of GPR30 levels . Different levels of mER also determined the E2 dose -dependent PHOSphorylation of ERKs . Cells with low levels of mER responded to a limited range of concentrations ( 10-13 to 10-12 mol/l ) . However , mERhigh cells responded to a much wider range of E2 concentrations ( 10-13 to 10-7 mol/l ) with a declining ( but still significant ) ERK activation at 10-100 nmol/l E2 . This finding corresponds to our observation that 10 nmol/l and higher E2 decreases cell proliferation in mERhigh MCF-7 cells , and is consistent with the idea that E2 induces cAMP -activated protein kinase A inhibition of MAPK pathways at these higher concentrations ( see accompanying paper [28] ) . Activation of ERKs has been linked to the proliferative cellular response [51] . Our accompanying report [28] addresses other rapid estrogen-elicited signaling responses that , in concert with ERK activation , can affect and perhaps balance cell proliferation responses . The classical antiestrogen ICI182,780 has been well defined as an antagonist of the action of estrogen at the transcriptional level . In our studies this compound was also a potent and rapid ERK activator . With different kinetics , the transcriptionally inactive 17alpha-estradiol also activated ERKs . It has been demonstrated that pure antiestrogen-ER complex can bind to estrogen response elements , but that the resultant transcriptional unit is inactive [52] . This binding and inactivation is generally used for pharmacological identification of ER-alpha participation in gene transcription . However , in a yeast reporter system antiestrogens ( ICI182,780 and tamoxifen ) induce ER dimerization and transcriptional activity [53] . Thus , although the published data disagree , it remains possible that antiestrogen-ER complexes can exert rapid effects such as induction of ERK PHOSphorylation and other signaling effects [54,55] . Other differences between findings may stem from effects assessed at different time points [56-58] . The length of time between estrogen or antiestrogen administration and kinase assessment can clearly influence outcomes , as evidenced by our time - and compound sequencing -dependent changes in ERK activation . It is also known that lengthy exposures to ICI182,780 can dramatically decrease ER-alpha protein levels [47,52] , which could explain the decreased ERK activation observed in some studies . Long-term estrogen-deprived MCF-7 cells express higher levels of ER than do wild-type MCF-7 cells and are hypersensitive to E2 ; ICI182,780 did not alter the pattern of response to E2 -stimulated growth in these cells [59,60] . To reconcile this observation with the expected inhibitory effect of ICI182,780 , the authors suggested that ICI182,780 blocked only the effect of the residual E2 coming from plastic tissue culture flasks , without affecting the added E2 . We can speculate that the observed effect of ICI182,780 was genuine and resulted from potentially increased levels of mER in long-term estrogen deprived MCF-7 cells , because our previous studies suggested that serum deprivation elevates mER-alpha levels [61] . In other cell types ICI182,780 behaved as a potent agonist of ERK1/2 activation ( eg in rat cerebellar neurons expressing plasmid-generated ERs [60] ) , or as a partial ER agonist ( in the superficial stroma and glandular epithelium of the sheep uterine endometrium [62] ) . It is likely that subtle differences in the shape of estrogen - and antiestrogen-liganded ERs , whether nuclear or membrane , present different interaction platforms for a variety of co-modulators , and that each signaling pathway must be considered in the context of a particular cell type's repertoire of partnering proteins . In addition to having positive effects by itself , ICI182,780 was able to alter the E2 response differently , dependent on the timing of its administration . If the cells were pretreated for only 30 min ( the time point when ICI182,780 alone did not change basal ERK activation ) , then the initial E2 activation and the late re-activation at 60 min were preserved , whereas the after-peak deactivation was enhanced . If ICI182 , 780 and E2 were added simultaneously , then the activation was delayed to 20 min , but weakly persisted up to 1 hour . Similar observations that ICI182,780 can change the time of ERK activation were reported in the case of a human thyroid carcinoma cell line , in which it reduced the 30 min to 1 hour activation , but enhanced later sustained activation at 6 hours [63] . The same authors showed that , in differentiated thyroid gland carcinoma cells (XTC133) , addition of ICI182,780 induces a small decrease in the sustained ERK PHOSphorylation . Thus , in addition to demonstrating that ICI182,780 is not just an antagonist for this response , these studies and ours may point to cell -specific differences in ERK regulation . PHOSphorylations leading to activation and subsequent inactivation of proteins are important regulatory mechanisms for control of cell growth and differentiation [64 , 65] . Although ERKs are thought to play a key role in cell proliferation , it has been suggested that persistent activation of ERKs might result in cell cycle arrest and differentiation in PC12 cells or cell growth inhibition and apoptosis in normal rat hepatocytes and several human tumor cell lines [65] . However , a detailed time course and transient ERK activations , interspersed with phosphatase-mediated inactivations , are rarely addressed . We ( in the present study ) and others [66] showed that the serine/threonine phosphatases PP1 and PP2A participate in the dePHOSphorylation of ERKs . It is interesting that OA ( a PP1 and PP2A inhibitor ) was more effective in mERhigh cells , whereas cyclosporin A ( a PP2B inhibitor ) was almost equally efficient in both cell populations . These data suggest that the level of mER-alpha expression/activation can be associated with activation of different types of ERK-modulating phosphatases . Other phosphatases , such as tyrosine phosphatase and dual specificity phosphatases ( which dephosphorylaTE tyrosine , threonine and serine residues ) , have been implicated in ERK dePHOSphorylation [67,68] and will be the subject of our future studies . Additional studies of ERK activations , deactivations and stability will be needed before we can formulate a more global picture of the post-translational modifications that lead to function of this important group of regulators in proliferation and differentiation . However , it is clear that nongenomic estrogen actions and the membrane receptors through which they act participate in this regulation .