title: Membrane estrogen receptor-alpha levels predict estrogen-induced ERK1/2 activation in MCF-7 cells.
abstract: BACKGROUND
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas, USA. ddzivadi@utmb.edu
authors.forename: Dragoslava
authors.initials: D
authors.lastname: Zivadinovic
authors.forename: Cheryl S
authors.initials: CS
authors.lastname: Watson
mesh_terms: D015153:Blotting, Western; D001943:Breast Neoplasms / Q000201:enzymology* / Q000473:pathology*; D002462:Cell Membrane; D049109:Cell Proliferation; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D004958:Estradiol / Q000502:physiology; D047628:Estrogen Receptor alpha / Q000032:analysis / Q000096:biosynthesis / Q000502:physiology*; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D007150:Immunohistochemistry; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D010766:Phosphorylation; D014407:Tumor Cells, Cultured
chemical_list: D047628:Estrogen Receptor alpha; D004958:Estradiol; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3