Bahiru Gametchu

Rapid actions of estrogens in GH3/B6 pituitary tumor cells via a plasma membrane version of estrogen receptor-α

The focus of our work on rapid actions of estrogens has been on the immuno-identification of a membrane version of the estrogen receptor-alpha (mERalpha) and the correlation of the presence of this receptor to the rapid secretion of prolactin in pituitary tumor cells. We demonstrated the mERalpha by both fluorescence and immuno-enzyme-cytochemistry and with both conventional and confocal microscopy in the cell line GH3/B6 and its sublines. Its presence on cells (including recently subcloned ones) is very heterogenous, unlike the nuclear ERalpha, which is present in every cell. An impeded ligand (estradiol covalently linked to BSA) binds to mERalpha and elicits the same response. A total of eight antibodies to ERalpha recognize mERalpha, making it likely that the membrane and nuclear proteins are highly related. Immuno-identification techniques have also been used to identify mERalpha on the MCF-7 human breast cancer cell line. Estradiol at very low concentrations elicits prolactin release from GH3/B6 cells within a few minutes of application. This response is bimodal, with effective concentrations in both the picomolar and nanomolar ranges. Prolactin release is also elicited or inhibited by ERalpha-specific antibodies. The characteristics of mERalpha and the membrane receptor for glucocorticoids have many similarities, suggesting that this mode of subcellular location/function alternative might be used by other members of the gene family.

Estrogen Receptor-α Detected on the Plasma Membrane of Aldehyde-Fixed GH3/B6/F10 Rat Pituitary Tumor Cells by Enzyme-Linked Immunocytochemistry1

A population of estrogen receptor-α (ERα) proteins, located at the plasma membrane, is postulated to mediate the rapid, nongenomic responses of GH3/B6/F10 pituitary cells to estrogen. To demonstrate the presence of ERα at the plasma membrane and to distinguish this receptor population from that in the nucleus, GH3/B6/F10 cells were first prepared in 2% paraformaldehyde/0.1% glutaraldehyde in PBS (P/G) without detergent, then exposed to one of several antibodies (Abs) raised against nuclear ERα. Ab binding was visualized as a fluorescent/chromagenic reaction product catalyzed by avidin-biotin-complexed alkaline phosphatase. With P/G fixation, Abs could only access antigens at the cell surface, as evidenced by the inability of 70K mol wt dextrans to permeate cells and the absence of intracellular staining by Abs to cytoplasmic or nuclear antigens. ERα Abs generated membrane, but not nuclear, staining in P/G-fixed cells; nuclear receptor labeling could only be detected in detergent-treated cells. Specificity...

Antibodies to the estrogen receptor-α modulate rapid prolactin release from rat pituitary tumor cells through plasma membrane estrogen receptors

Antibodies (Abs) raised against the estrogen receptor‐α (ERα) were used to investigate the role of ERα proteins located at the plasma membrane in mediating the rapid, estrogen‐stimulated secretion of prolactin (PRL) from rat pituitary GH3/B6/F10 cells. Exposure of the cells to 1 nM 17 β‐estradiol (E2) significantly increased PRL release after 3 or 6 min. When ERα Abs that bind specifically to ERα but are too large to diffuse into cells were tested for activity at the cell membrane, Ab R4, targeted to an ERα hinge region sequence, increased PRL release in a time‐ and concentration‐dependent fashion. Ab H151, directed against a different hinge region epitope, decreased PRL release and blocked the stimulatory action of E2. Abs raised against the DNA binding domain (H226) or the carboxyl terminus (C542) were not biologically active. When each Ab was examined for recognition of ERα on the cell surface by immunocytochemistry, all except H151 generated immunostaining in aldehyde‐fixed cells. In live cells, however, Ab H151 but not Ab R4 blocked the membrane binding of fluorescently tagged E2‐BSA. Overall, the data indicate that plasma membrane ERα proteins mediate estrogen‐stimulated PRL release from GH3/B6/F10 cells. These results may also convey information about conformationally sensitive areas of the membrane form of ERα involved in rapid, nongenomic responses to estrogens.—Norfleet, A. M., Clarke, C. H., Gametchu, B., Watson, C. S. Antibodies to the estrogen receptor‐α modulate rapid prolactin release from rat pituitary tumor cells through plasma membrane estrogen receptors. FASEB J. 14, 157–165 (2000)

Membrane estrogen receptor-α levels in MCF-7 breast cancer cells predict cAMP and proliferation responses

Introduction17β-estradiol (E2) can rapidly induce cAMP production, but the conditions under which these cAMP levels are best measured and the signaling pathways responsible for the consequent proliferative effects on breast cancer cells are not fully understood. To help resolve these issues, we compared cAMP mechanistic responses in MCF-7 cell lines selected for low (mERlow) and high (mERhigh) expression of the membrane form of estrogen receptor (mER)-α, and thus addressed the receptor subform involved in cAMP signaling.MethodsMCF-7 cells were immunopanned and subsequently separated by fluorescence activated cell sorting into mERhigh (mER-α-enriched) and mERlow (mER-α-depleted) populations. Unique (compared with previously reported) incubation conditions at 4°C were found to be optimal for demonstrating E2-induced cAMP production. Time-dependent and dose-dependent effects of E2 on cAMP production were determined for both cell subpopulations. The effects of forskolin, 8-CPT cAMP, protein kinase A inhibitor (H-89), and adenylyl cyclase inhibitor (SQ 22,536) on E2-induced cell proliferation were assessed using the crystal violet assay.ResultsWe demonstrated a rapid and transient cAMP increase after 1 pmol/l E2 stimulation in mERhigh cells; at 4°C these responses were much more reliable and robust than at 37°C (the condition most often used). The loss of cAMP at 37°C was not due to export. 3-Isobutyl-1-methylxanthine (IBMX; 1 mmol/l) only partially preserved cAMP, suggesting that multiple phosphodiesterases modulate its level. The accumulated cAMP was consistently much higher in mERhigh cells than in mERlow cells, implicating mER-α levels in the process. ICI172,780 blocked the E2-induced response and 17α-estradiol did not elicit the response, also suggesting activity through an estrogen receptor. E2 dose-dependent cAMP production, although biphasic in both cell types, was responsive to 50-fold higher E2 concentrations in mERhigh cells. Proliferation of mERlow cells was stimulated over the whole range of E2concentrations, whereas the number of mERhigh cells was greatly decreased at concentrations above 1 nmol/l, suggesting that estrogen over-stimulation can lead to cell death, as has previously been reported, and that mER-α participates. E2-mediated activation of adenylyl cyclase and downstream participation of protein kinase A were shown to be involved in these responses.ConclusionRapid mER-α-mediated nongenomic signaling cascades generate cAMP and downstream signaling events, which contribute to the regulation of breast cancer cell number.

Use of receptor antibodies to demonstrate membrane glucocorticoid receptor in cells from human leukemic patients.

Anti‐peptide antibody to the human glucocorticoid receptor (GR) was produced and used to demonstrate that a subset of the GR population resides in the plasma membrane of human leukemic cells. Characterization of the antibody with intracellular GR (iGR) showed its ability to shift [3H]triamcinolone acetonide‐labeled GR (4S protein) from two human leukemic cell lines to a higher density in sucrose gradients; Western and autoradiographic analysis of affinity‐labeled ([3H]dexamethasone 21‐mesylate) receptor revealed an immunoreactive and competitively labeled band of 94 kDa. CCRF‐CEM cell membrane GR (mGR) resolved as a > 7S protein on density gradients and immunoselected cell surface protein labeled by whole cell biotinylation or affinity‐labeling with [3H]dexamethasone 21‐mesylate was ~145 kDa, demonstrating that mGR was larger in size than iGR, as has been shown previously for the mGR of mouse lymphoma cells. Analysis of mGR in lymphocytes of leukemic patients and the CCRF‐CEM cell line indicated differences in levels of expression as shown by FACS and immunocytochemical analyses. We arc currently using this system to study the correlation between the quantity of membrane‐resident GRs and the glucocorticoid‐induced lytic response, a relationship previously shown in the murine (S‐49 cell) system.—Gametchu, B., Watson, C. S., Wu, S. Use of receptor antibodies to demonstrate membrane glucocorticoid receptor in cells from human leukemic patients. FASEB J. 7: 1283‐1292; 1993.

Membrane oestrogen receptors on rat pituitary tumour cells: immuno-identification and responses to oestradiol and xenoestrogens.

Our laboratory has identified plasma membrane oestrogen receptors on a GH3/B6 rat pituitary tumour cell line and several sublines which produce rapid (within minutes), non‐genomic responses to oestrogens. Oestrogen receptors have been identified by their binding to nine different antibodies (Abs) which together recognize at least seven epitopes on the oestrogen receptor‐α. GH3/B6/F10 cells, a membrane oestrogen receptor‐enriched subline, elevate intracellular calcium levels in response to 10 nM oestradiol. Prolactin release in these cells is triggered by both 1 pM and 1 nM oestradiol and diethylstilbestrol (DES). A membrane oestrogen receptor‐α immunocyto‐chemical signal rapidly disappears (at 3 min) and reappears (at 12‐15 min) when 1 nM oestradiol, 10 nM diethylstilbestrol, or 10 nM nonylphenol is applied to the cells. This suggests that both oestrogens and xenoestrogens can utilize this alternative pathway for oestrogenic action. Xenoestrogens, which have so far shown weak effects in genomic assay systems, should now be retested for activity in eliciting membrane‐initiated oestrogenic responses.

The dynamic and elusive membrane estrogen receptor-α

Many studies have demonstrated the nuclear forms of steroid receptors and their activities, while fewer investigators have identified and described the membrane forms of these receptors. Our immuno-identification approaches for the qualitative and quantitative comparison of the membrane form of the estrogen receptor-alpha (mER alpha) to its nuclear counterpart now allow us to address questions about the comparative levels and regulation of these receptor forms. ER alpha-specific antisense oligonucleotides eliminate mER alpha expression, while only mildly reducing the nuclear ER alpha. Success of immuno-identification for the mER alpha is very sensitive to different fixation protocols, affecting cell permeability (and thus distinction from the intracellular form) and differential epitope preservation. All such identifications must be accompanied by proof of cell membrane integrity and focal plane assessments. The mER alpha expression on selected cells declines rapidly with cell passage number and cell density. Expression of mER alpha is enhanced by serum starvation and selection for specific phases of the cell cycle. The hinge region of the protein is sensitive to ligand-induced epitope masking and to antibody-induced changes in receptor-mediated responses. Responsive cells are often diluted within cell populations by loss of the membrane receptor form. The bimodality of the rapid estrogen action, with inhibitory doses between picomolar and nanomolar stimulatory concentrations, requires detailed dose-response curves. Finally, responsive cells can be lost from assays, as upon estrogen treatment they rapidly round up and leave the substrates to which they are attached. These regulatory phenomena demonstrate that levels of the membrane form of the estrogen receptor are very dynamic.

Proteins of Multiple Classes May Participate in Nongenomic Steroid Actions

Responses to steroids initiated from non-nuclear receptors Impinge on a wide variety of cellular responses and utilize nearly all known signal transduction webs. While the mechanisms by which steroid receptors localize in the membrane are still unclear, it is apparent that this alternative localization allows steroid receptors to participate in a wide range of complex functions influencing cell proliferation, death, and differentiation. The central debate still remains the identity of the protein class or classes that mediate membrane-initiated (nongenomic) responses. The data thus far have supported several possibilities, including: nuclear steroid receptor-like forms in non-nuclear locations; other known (nonsteroid) membrane receptors or channels with additional steroid-binding sites; enzymes; transporters; receptors for serum steroid-binding proteins; unique and previously undescribed proteins; or chimeras of typical steroid receptor domains with other unique or known protein domains. Categorizing membrane steroid receptor proteins based exclusively on the actions of antagonists and agonists, without considering cell context and protein partnering Issues, may mislead us into predicting more receptor subtypes than really exist. However, the plethora of signaling and functional outcomes may indicate the participation of more than one kind of steroid-binding protein. Resolving such unanswered questions will require future investigative focus on this alternative arm of steroid action, which is likely to yield as many therapeutic opportunities as have nuclear steroid mechanisms.

Association of the glucocorticoid receptor alternatively-spliced transcript 1A with the presence of the high molecular weight membrane glucocorticoid receptor in mouse lymphoma cells

Using the combination of a cDNA library prepared from membrane glucocorticoid (mGR)‐enriched S‐49 cells and a mouse leukocyte genomic library, we have cloned a 7.3 kb full‐length glucocorticoid receptor 1A cDNA. Primer extension, 5′RACE, and long distance PCR identified the transcription start site as being located at 1026 bp from the ATG codon. The first 1,013 nucleotides (nts) of the full length sequence constitute 5′ UTR sequence (exon 1), the next 2349 bp, the coding region, and the last 3,907 bp, the 3′UTR. The entire 5′UTR sequence is unique to transcript 1A. The 3′UTR sequence is ∼88.5 % conserved with the rat 3′UTR. Western blot analysis compared the molecular weight of in vitro translation products from the cloned 1A cDNA with partially purified cellular mGR. Both preparations contained the novel 150 KD and the 94 KD classical GR peptides, suggesting that transcript 1A encodes both receptor forms. Transfection of mGR‐less and glucocorticoid lysis‐resistant AtT‐20 and HL‐60 cells with full‐length GR 1A cDNA imparted both mGR expression and glucocorticoid lysis‐sensitivity to these cells. J. Cell. Biochem. 74:430–446, 1999.

5′UTR sequences of the glucocorticoid receptor 1A transcript encode a peptide associated with translational regulation of the glucocorticoid receptor

We have recently reported that glucocorticoid receptor (GR) transcript 1A, one of the five mouse GR splice variants (1A–1E), encodes membrane GR (mGR), which subsequently participates in mediating the apoptotic effects of glucocorticoids (GCs); all transcripts vary at their 5′UTR. Computer analysis of the entire1026 bp comprising the 5′UTR of transcript 1A identified five putative translation start sites at positions 85, 217, 478, 628, and 892 with the potential to encode peptides of 33, 93, 6, 18, and 41 amino acids, respectively. We then separately generated point mutations at these five upstream AUG codons of the GR 1A cDNA and performed in vitro transcription/translation experiments to investigate the regulatory effects of these sites on GR synthesis. GR translation products were immuno‐captured with BUGR‐2 antibody (Ab), then subjected to Western blot analysis. Mutation of the uAUG codon‐2 completely inhibited GR synthesis, while mutations at the other four uAUG codons had no significant effect on the translation of transcript 1A. Antibodies (Abs) against the uORF‐2 and uORF‐5 protein products were used to perform Western blot analysis on cytosolic proteins from S‐49 cells (which express GR transcript 1A), U937 cells transfected with GR 1A cDNA, or in vitro translation products from this cDNA. This assay identified an intense immunoreactive band of ∼8.5 kDa recognized only with Ab to the uORF‐2 peptide; this size is consistent with the computer‐predicted size of the uORF‐2 product, suggesting that the uORF‐2 product is indeed synthesized in cells. No peptide was identified with Ab to uORF‐5 peptide. Indirect fluorescent Ab staining, confocal microscopy and FACS analysis all showed that the ORF‐2 peptide is localized both in the interior of the cell and at the plasma membrane. Using Ab to ORF‐2 peptide for immunoadsorption we then asked whether cellular factors interact with the product of uORF‐2. Immuno‐captured uORF‐2 peptide levels correlated with the concentrations of several salt‐wash‐sensitive cellular proteins, suggesting that protein–protein interactions occur between this upstream open reading frame (uORF) product and other factors. The uORF‐2 product, however, does not appear to directly interact with GR, since there was no reciprocal immuno‐capture between these two proteins. In summary, our results show that cells can synthesize the uORF‐2 peptide, blocking of the synthesis of the uORF‐2 peptide product abolishes translation of GR from the GR 1A transcript, and the peptide product of uORF‐2 interacts with other cellular factors which might be involved in translation of GR. J. Cell. Biochem. 81:149–161, 2001.