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Division of Endocrinology, Veterans Affairs Medical Center, Long Beach, Long Beach, California 90822; Departments of Medicine (M.R., A.P., E.R.L.) and Pharmacology, and the University of California, Irvine, Irvine, California 92717; Woman’s Health Research Institute (I.M.), Wyeth Research, Collegeville, Pennsylvania 19426; and Ben May Institute (G.L.G.), University of Chicago, Chicago, Illinois 60637
Address all correspondence and requests for reprints to: Ellis R. Levin, M.D., Medical Service (111-I) Long Beach Veterans Affairs Medical Center/University of California-Irvine, 5901 East 7th Street, Long Beach, California 90822. E-mail: ellis.levin{at}med.va.gov.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and -ß) localize at the plasma membrane and rapidly signal to affect cellular physiology. Although nuclear ERs function mainly as homodimers, it is unknown whether membrane-localized ER exists or functions with similar requirements. We report that the endogenous ER isoforms at the plasma membrane of breast cancer or endothelial cells exist predominantly as homodimers in the presence of 17ß-estradiol (E2). Interestingly, in endothelial cells made from ER /ERß homozygous double-knockout mice, membrane ER or ERß are absent, indicating that the endogenous membrane receptors derive from the same gene(s) as the nuclear receptors. In ER-negative breast cancer cells or Chinese hamster ovary cells, we expressed and compared wild-type and dimer mutant mouse ER. Only wild-type ER supported the ability of E2 to rapidly activate ERK, cAMP, and phosphatidylinositol 3-kinase signaling. This resulted from E2 activating Gs and Gq at the membrane in cells expressing the wild-type, but not the dimer mutant, ER. Intact, but not dimer mutant, ER also supported E2-induced epidermal growth factor receptor transactivation and cell survival. We also confirmed the requirement of dimerization for membrane ER function using a second, less extensively mutated, human ER. In summary, endogenous membrane ERs exist as dimers, a structural requirement that supports rapid signal transduction and affects cell physiology. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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One important issue concerning the membrane ER is the structural requirements of this receptor pool to respond to E2 with rapid activation of signaling. Signaling includes the generation of calcium currents, cAMP, inositol phosphate generation, and the stimulation of kinase activity (reviewed in Ref.2). Importantly, activation of ERK MAPK and phosphatidylinositol 3 (PI3) kinase are required for E2 to generate nitric oxide formation in endothelial cells (EC) (8, 9), and this prevents ischemia-reperfusion injury of muscle in mice (8). Estrogen action at the membrane ER has also been reported to salvage multiple cell types from apoptotic cell death (6, 10, 11, 12). This signaling in bone cells may contribute to preventing osteoporosis (10, 13). E2 signaling through ERK also importantly contributes to breast cancer cell proliferation, and this rapid signal results from membrane ER function and not from the nuclear ER (14).
Some aspects of the structure-function relationship of the membrane ER are known. It appears that the ligand binding segment (E domain) of ER, when localized to the membrane, is sufficient to promote rapid signaling in vitro (15). In breast cancer, this stems from the ability of the E domain to activate a cross-talk to the epidermal growth factor receptor (EGFR), resulting in downstream activation of ERK and phosphatidylinositol 3-kinase (PI3K) (15). However, other domains of the membrane receptor might contribute to the magnitude of membrane ER signaling. In this respect, the ability of ER to interact with Shc may augment signaling to cytoskeleton changes resulting in membrane ruffling. The ER-Shc interaction is mediated by the A/B domain of the steroid receptor (16). It is conceivable that in cell context-specific fashion, interactions of other portions of ER with specific signaling molecules results in up-regulation of activity.
One important potential requirement is the dimerization of membrane ER. The nuclear ER functions as a homodimer in the presence of sex steroid (17). However, it is unknown whether the membrane-localized pool of ERs exist as homo or heterodimers, and whether dimerization is required for rapid signaling in response to E2. Here, we investigated this issue and show that liganded, endogenous membrane ER and ERß exist mainly in the homodimer state. Using mouse and human ER that have been mutated to prevent dimerization, we propose that this is an important structural requirement for function. We report that dimerization promotes rapid signals that are generated at the cell surface and that lead to a cell physiological outcome.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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was detected at an apparent size consistent with being a homodimer in the presence of E2 but as a monomer in the absence of sex steroid (Fig. 1A
). Because the molecular weight markers and ER proteins do not always run true to size on separation by native gel electrophoresis, the exact sizes of the complexes cannot be determined. We also incubated three types of EC with/without 10 nM E2 for 5 min, and then isolated the plasma membranes. As seen in Fig. 1B, left, human umbilical EC, bovine aortic EC, and mouse brain capillary EC all contained predominantly an approximately 134-kDa ER band by Western blot, consistent with the expected size of the homodimer. There was also a band compatible with ER/ERß heterodimers (see also Fig. 1B, right), and this is consistent with the reports of both ER and ERß in these cells (19). The heterodimer band was not seen when the samples were first immunoprecipitated for ER, the immunoprecipitate discarded, and the remaining cell lysate blotted for ERß (data not shown). Additionally, a band at approximately 67 kDa was seen, consistent with a small amount of apparent monomer, and a barely detectable band at about 49 kDa was also found. Capillary EC from the brains of ER/ERß double-knockout (KO) mice were also isolated. Here, no ER was detected, using an antiserum directed against the C terminus of this receptor. No detectable receptor protein was found in the nucleus either (data not shown). We also examined the EC for the presence of membrane ERß. Using an antiserum directed against the C terminus, we found evidence for predominantly an ERß homodimer, with lesser amounts of ER/ERß heterodimer and ERß monomer (Fig. 1B, right). Again, no receptor was detected in the mouse brain capillary EC (MBCEC) from double-KO mice. We conclude that the membrane ER and ERß in EC are derived from the same genes that encode the nuclear ER and ERß in EC.
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, the predominant form of ER is the size expected of the homodimer in the presence of E2. This was found for both the nuclear and membrane-localized receptors. A small band consistent with protein monomer was also seen. The monomer was corroborated when we immunoprecipitated ER from a membrane sample, added buffer containing sodium dodecyl sulfate (SDS) and 6-mercaptoethanol, and boiled the sample before gel electrophoresis (reducing conditions). We also found that endogenous membrane ERß exists predominantly as a band that is consistent with apparent homodimer-sized protein in these cells. However, much less receptor in general is seen, compared with ER (Fig. 1C). There was no membrane ER/ERß heterodimer detected, probably because of the small amount of ERß present in these cells.
It is possible that monomeric ER at the membrane associates with another membrane-localized protein, and this complex is the same size as the ER dimer. To examine this possibility, we individually expressed human wild-type ER and an A/B domain-truncated ER in Chinese hamster ovary (CHO) cells. After isolating the plasma membrane fraction and separating the membrane proteins by native gel electrophoresis, we determined the size of the resulting complex by Western blot. We found that under nonreducing conditions, both receptors migrated at a size that is consistent with homodimerization (Fig. 1D). This includes the A/B domain-truncated receptor, where the detected complex is consistent with the predicted apparent size of a homodimer (
80,000 kDa), especially when the cells are incubated with E2. This result makes it unlikely that an ER-associating protein accounts for the size of the ER complex at the membrane and strongly supports the notion that ER is a homodimer in the membranes of EC and breast cancer cells.
Dimerization of Membrane ER Is Required for Signaling Functions
These studies justified our asking the important question: is homodimerization of membrane ER necessary for signaling to affect cellular physiology? Dimerization is required for the ability of the nuclear ER to transactivate genes (17) but it is unknown whether dimerization is needed for the function of ER at the membrane. To determine this, we made a mutant mouse ER construct by site-directed mutagenesis (Stratagene, La Jolla, CA). Leu512/Leu513/Leu514 were all changed to alanine and alanine 509 changed to glutamine, based upon the known requirements for the mouse ER to dimerize (20). The mutations were subsequently confirmed by sequencing. We first expressed the wt or dimer mutant (DM) ER in CHO cells. Expression of wt or DM ER resulted in monomer formation at the membrane, but upon addition of E2, only the wt receptor dimerized (Fig. 2A). It is important to note that the protein expression levels of the wt and DM ER were comparable.
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, we then compared the two constructs in their ability to support rapid signaling from the membrane. The ability of E2 to rapidly activate the ERK member of the MAPK family occurs only through the membrane ER (14, 18). Exposure of CHO-wt ER cells to 10 nM E2 for 8 min resulted in up-regulation of ERK MAPK activity (Fig. 2B, lanes 3 vs. 4). In contrast, E2 did not stimulate kinase activity in CHO-DM ER cells (lanes 5 vs. 6). As a further control, E2 did not stimulate ERK activity in CHO-pcDNA3 cells (lanes 1 and 2). We then examined another rapid signal, the generation of cAMP after 5 min exposure of the cells to E2. As seen in Fig. 2C, there was modest stimulation of this cyclic nucleotide/second messenger in DM ER-expressing CHO. However, this was approximately 67% less than the stimulation seen in CHO-wt ER cells. Thus, the generation of both rapid signals is highly dependent on ER dimerization.
We then determined another important signal, the activation of PI3K. This signal molecule underlies the ability of E2 to stimulate NO formation in EC (8, 9) and to rescue mice from ischemia-reperfusion injury of muscle in vivo (8). Furthermore, PI3K is activated by E2 in breast cancer cells (21) and plays a significant role in the induction of cell proliferative genes, such as cyclin D1 (22). We therefore expressed wt and DM ER in both CHO and ER-negative breast cancer cells (HCC-1569). In CHO-wt ER cells, E2 caused the PI3K-dependent phosphorylation of AKT at Ser 473, detected with phospho-specific antibodies (15) (Fig. 2D, left; lanes 3 and 4). In contrast, CHO-DM ER or CHO-pcDNA3 cells did not significantly respond to E2 with AKT phosphorylation. Similar results were seen in transfected HCC-1569 cells (Fig. 2D, right).
If the mutant receptor cannot dimerize with itself or wt receptor, then expression of this mutant is predicted to not influence wt ER signaling from the membrane. To determine this, we transiently transfected equal amounts of cDNA for wtER with either equal amounts of cDNA for pcDNA3 or dimer mutant ER into CHO cells. Activation of ERK by E2 was subsequently determined. As seen in Fig. 2E, the expression of the dimer mutant had a modest, insignificant effect on the ability of E2 to activate ERK, mediated by wt ER expression. These data further support the idea that dimerization of the wt receptor is required for full signaling activation.
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DM Studies may impact signaling, unrelated to dimerization. This consideration is underscored by the facts that 1) the dimerization motifs are within the E domain of ER, and 2) we previously showed that by expressing only the E domain at the cell membrane, full signaling occurs equivalent to that seen with full-length receptor. The critical requirements for human dimerization are less extensive (17), and we recently made a simple human ER DM (leucine to glutamine at residue 504), confirmed by sequencing. This also allowed us to determine the requirement of dimerization for signaling in a second mammalian species.
We first expressed the human DM ER in CHO cells and established that the wt ER, but not the mutant human ER, forms a dimer in the presence of E2 (Fig. 3A). This also occurred in the nucleus of the transfected cells, and equal expression of the wt and mutant proteins was seen in each cell compartment. We then compared the abilities of human wt and DM ER to support signaling. In transfected CHO cells, only the human wt ER responded to E2 with increased ERK activation (Fig. 3B). We also directly compared mouse and human wt and DM ER in their ability to activate PI3K/AKT signaling. As seen in Fig. 3C, it was the two wt receptors that resulted in strong E2 phosphorylation of AKT. In contrast, CHO cells expressing pcDNA3 or either mouse or human DM ER did not show significantly increased AKT activation in response to E2. Finally, we determined the effects on cAMP generation (Fig. 3D). We found that the human wt ER significantly supported cAMP generation to a much greater extent than the DM ER. However, because there was some stimulation by E2 in cells expressing the human DM receptor, additional residues probably augment the action of the dimerized receptor to activate this specific signal.
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necessary for G protein activation, as an early step in signaling? To determine this, we transfected CHO cells with either mouse wt or DM ER and determined the incorporation of GTP
s into several membrane-associated G protein -subunits that associate with ER. This serves as a standard measure of G protein activation (23). We found that in CHO cells expressing either pcDNA3 or the DM ER, E2 was not able to significantly activate various G protein -subunits (Fig. 4). In contrast, the wt ER showed strong GTPS incorporation into ER-associated GS and Gq. Showing specificity, neither ER was capable of supporting Gi association and activation in this cell type. These data support the idea that wt, but not DM, ER supports a very early event, crucial to generating multiple downstream signals.
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supports 1) the ability of E2 to activate the EGFR and 2) prevents radiation-induced cell death. HCC1569 cells (ER-negative breast cancer) were transiently transfected with the wt or DM ER and recovered. After 5 min exposure of the cells to 10 nM E2, cell lysate was processed to detect phosphorylation of the EGFR at tyrosine 1138, indicating activation. We found that it was the wt ER that promoted the crucial cross-talk to the autophosphorylation of EGFR (Fig. 5). In contrast, the DM ER was not capable of supporting activation of the EGFR by E2.
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in HCC-1569 cells (ER negative) and then briefly exposed the cells to UV irradiation (20 J/cm2) for 1 min. The cells were examined for cell death 4 h later by TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) staining (Fig. 6). In control cells (no UV) that expressed wt ER, there was a small amount of spontaneous cell death that was up-regulated 7-fold by UV exposure. E2 significantly prevented the apoptotic death of the cells, but this was reversed by soluble inhibitors of ERK [PD98059 (Parke-Davis, Ann Arbor, MI)] and PI3K (wortmannin). In DM ER-expressing cells, there was comparable cell death in response to UV exposure, but E2 had no survival effect in these cells. In control DM ER, or wt ER-expressing cells exposed to PD98059 or wortmannin alone, no cell death above control was seen. We also confirmed the significant conditions by another measure of cell death, Annexin V staining at the cell periphery determined by fluorescence-activated cell sorting (FACS) analysis (data not shown). Thus, E2 signaling from the membrane wt, but not the DM, ER to ERK and PI3K contributes to the survival effect of the sex steroid in breast cancer.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We first report that endogenous ER and ERß at the membranes of endothelial and breast cancer cells are predominantly homodimers. In addition, there is heterodimerization between ER and ERß, and this is seen most clearly where both receptors are greatly produced as in EC. It is unclear what role the heterodimers play, but when ERß is overexpressed in MCF-7 cells, ER-mediated transcription and proliferation are down-regulated (30, 31). Localization and signaling at the membrane are complex processes that are probably facilitated by ER complexing directly or indirectly with various scaffold proteins (caveolin), adapter proteins (shc and modulator of nongenomic activity of ER), tyrosine kinases (EGF, IGF receptors, and src), and G proteins (14, 16, 24, 28, 32, 33, 34). Here, we found that the ability of ER to associate with G proteins occurs with wt but not DM receptors.
An important finding regarding the membrane-localized ER is that neither ER nor ERß was detected in EC membranes made from double-KO ER mice. This provides the strongest genetic evidence to date that the endogenous membrane receptor(s) (ER or ERß) is derived from the same gene(s) as the nuclear receptor isoforms, and that the cell-localized proteins are probably the same. This is important because Thomas and colleagues (35) recently described heptahelical membrane progesterone receptors in mammals. These proteins are products of genes distinct from the genes encoding nuclear PR.
It was reported that in some organs a 55-kDa receptor can be produced in the ER KO mouse constructed by Lubahn et al. (36). This is the same mouse that was used for breeding the double ERKO mice used for EC preparation here, but at least in this cell type, we did not identify a receptor of this length in the EC from the double ERKO mice. Also, the laboratory of Jeff Bender (37) has previously described a 46-kDa membrane ER that is the dominant receptor in an immortalized, human EC-derived cell line (EA.hy926). However, this might be a receptor that has evolved as the result of immortalization or repetitive cell passage. We examined three different species and types of EC in our study, including two cells that were primary/nonpassaged cells. We found a low-molecular mass band in the wt but not KO cells that could be the 46-kDa receptor described. However, this receptor clearly is a minor portion of the overall membrane receptor pool, and so it is unlikely that it significantly contributes to the function of E2/ER in these ECs. Other laboratories extensively working with membrane ER in vascular cells have also not reported this low-molecular mass receptor (9, 19).
Most importantly, we asked whether dimerization of the membrane ER is necessary for rapid signaling to affect cellular physiology. Using human and mouse ER wt and DM expression constructs, we found that only the wt ER significantly supports rapid E2 signaling. These results indicate that 1) the dimer conformation is the preferred and necessary structure for full E2 signaling at membrane-localized ER and 2) this is conserved across species. It is possible that other motifs may contribute to additional signaling or augment the dominant influence of the dimer conformation. We previously showed that only the membrane, and not the nuclear ER, supports rapid signaling (14, 18).
Interestingly, the identified structural elements for dimerization of the nuclear receptor are mainly within the ligand binding (E domain) of ER (20, 38). This allows us to postulate that the E domain is critical for signaling from the membrane. In support of this idea, we previously showed that serine 522 in the E domain is important for membrane localization and optimal binding to caveloin-1, a protein that provides membrane transport and localization for membrane ER (14). Mutating serine 522 to alanine and expressing this mutant receptor in MCF-7 leads to down-regulation of ERK activation, cyclin D1 production, and cell proliferation, while not affecting nuclear ER function (14). Migliaccio et al. (39) showed that Src complexes with E2/ER. The tyrosine 537 within the E domain mediates the interaction of Src with ER that results in Src activation. The relationship between the E domain, dimerization, and ER function also is relevant to the nuclear pool of ER. In one model, nuclear ER transactivation of genes requires dimer formation, both for functional ER homodimers and the ER/ERß heterodimer, and the interface for either dimer involves elements of the ligand-binding domain (38).
We also found that dimerization promotes selective G protein activation, a very early event that results from concurrent association of ER with specific G proteins. This indicates that the E domain elements that mediate dimerization also mediate this role. G protein-coupled receptors, as a class of receptors, dimerize and associate with/activate multiple G proteins, but there are no conserved structural elements that predictably mediate these actions by various receptor family members. Our findings provide the first insights as to the specific structural elements that allow steroid receptors to associate with and activate G proteins at the membrane.
One important issue is whether our creation of a multimotif mutation of mouse ER interfered with signaling because of the critical nature of the amino acids involved, unrelated to dimerization. To determine this, we used a single-base pair change to create a human DM ER. This mutant also did not support signaling by E2 nearly as well as the wt human ER. Thus, it is likely to be dimerization per se that accounts for the results shown in these studies. We speculate that the loss of the dimer fails to provide the structural contact points for G proteins to bind in an active conformation, thus impairing signaling to adenylate cyclase and EGFR activation. The latter is required for ERK and PI3K signaling in breast cancer (15, 24), although other receptor tyrosine kinases [ErbB2 and IGF type 1 receptor (IGF1R)] also play similar roles (32, 34, 40, 41). Dimer formation was found here to be required for EGFR transphosphorylation and the physiological outcome of cell survival in response to the sex steroid. The latter effect depended upon both ERK (6) and PI3K signaling, and we now report the novel participation of PI3K. Activation of PI3K/AKT is well known to contribute to the survival of many cells (42).
Recently, Song et al. (16, 32) showed that the adaptor protein Shc contributes to membrane ER localization and signaling to ERK. Shc binds receptor tyrosine kinases (EGFR, IGF1R, insulin receptor), often to link Src or Grb2/SOS to ras activation (43). As a linker protein, Shc may be part of the multiprotein complex at the membrane that recruits and retains ER in an active conformation, resulting in E2-induced rapid signaling. A similar role has been proposed for modulator of nongenomic activity of ER to mediate the interactions between Src and ER, as part of a larger signal complex (33). It was reported that ER-Shc interactions occur through the N terminus of ER (16). However, an A/B domain-deleted ER supports rapid ERK activation comparably to wt ER (14). Thus, there may be indirect connections between ER and Shc involving binding to a common scaffold such as caveolin, EGFR, or IGF1R. Alternatively, another part of ER might directly complex with Shc, and the E domain is a candidate for either possibility.
In summary, plasma membrane ERs exist as functional dimers when bound by steroid ligand. It will be important to determine how the receptor dimer selectively associates/activates with some G proteins and not others, promoting selective signaling pathways in cell context-specific fashion. Other steroid receptors that rapidly signal in response to androgens, mineralocorticoids, progesterone, or thyroid hormone have been proposed to also localize to the cell membrane (3). It is certainly possible that these receptors exist in functional dimer conformation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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ER at the Membrane
Human umbilical vein endothelial cells (HUVEC) were purchased from Clonetics, San Diego, CA), whereas bovine aortic EC and MBCEC were isolated and cultured as previously described (12, 44). Mice that were genetically deleted for both ER and ERß were constructed at Wyeth Research as previously described (45). EC were cultured in the absence of serum or phenol red for 24 h before experimentation. The cells were incubated with 10 nM E2 for 5 min, and plasma membrane fractions were obtained as previously noted (14, 18); the lack of contamination with nuclear material was extensively determined as previously described (14, 18). In some experiments, low-speed centrifugation was used to isolate nuclear cell fractions for comparison. Membrane proteins were then separated by gel electrophoresis under nonreducing conditions, and then subjected to Western blot. Antibodies included a C-terminal antiserum (catalog no. sc-542, Santa Cruz Biotechnology) and H222 (raised against the ligand-binding domain) for ER, and a C-terminal antibody to ERß (catalog no. 51–7700, Zymed Laboratories, Inc., South San Francisco, CA).
Plasmids
Mouse ER in pcDNA3 was a kind gift from the laboratory of Dr. Ken Korach (National Institutes of Health, Research Triangle, North Carolina). DM mouse ER was created by site-directed mutagenesis (Stratagene, La Jolla, CA). This PCR-based method involved changing Leu512/Leu513/Leu514 to alanine and alanine 509 to glutamine, based upon the known requirements for the mouse ER to dimerize (20). The mutations were verified by sequencing the entire ER. Similarly, a human ER dimer mutant (leucine to glutamine at residue 504) was created by Jim Radek and sequenced. Transient expression in CHO cells or HCC-1569 cells (ER negative) was carried out as previously described (15, 23). The cells were grown to 60–70% confluence in DMEM-F12 medium without phenol red but with 10% FBS. The cells were then washed and transiently transfected with 5–10 µg of fusion plasmids, depending on the plate size and the amount of cells. Transfections were done with Lipofectamine Reagent (Invitrogen, San Diego, CA; cells were incubated with liposome-DNA complexes at 37 C for approximately 5 h followed by overnight recovery in DMEM-F12 medium containing 10% FBS. Then, before experimental treatment, cells were synchronized in serum-free DME-F12 for 24 h and then treated with 17-ß-E2. Cotransfections with a green fluorescent protein expression vector (Promega Corp., Madison, WI) indicated approximately 70–85% efficiency of transfection in CHO and HCC-1569 cells.
Electrophoresis of ER
Membrane or nuclear cell fractions were isolated, and then subjected to nonreducing (native) conditions and gel, or reducing conditions for electrophoretic separation (PAGE). The components of the reagents used were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). For native PAGE, samples were mixed with Tris-glycine buffer without reducing agents and were separated on a 10% Tris-glycine gel with Tris-glycine native running buffer (5 mM Tris and 38 mM glycine), according to the manufacturer’s instructions. For SDS-PAGE, samples were mixed with reducing buffer containing SDS and 2-mercaptoethanol (2%), incubated 5 min at 95 C, and then separated on a 10% gel using 0.5% SDS in the running buffer. Under both native and SDS-PAGE conditions, protein samples were separated at a constant 35 mAmp for 3 h. Equal quantities of protein were loaded in each gel lane for each experimental condition. Apparent molecular masses were determined by running the "Broad Range" protein markers in parallel (Bio-Rad).
Signaling Studies
Adenylate cyclase activity in the membrane was determined by measuring cAMP generation after 5 min incubation of the cells with 10 nM E2. This was determined in CHO-K1 cells expressing wt or DM ER, by methods previously described (15, 23). ERK MAPK activation and AKT activation in the CHO or HCC-1569 cells was determined at 9 and 15 min exposure to 10 nM E2, as previously described in detail (15, 23). EGFR phosphorylation in HCC1569 cells was determined by Western blot, as previously described (15). Briefly, the cells were transfected to express pcDNA3 (control) or wt or DM ER. Antibodies to EGFR (tyrosine 1138) (Santa Cruz Biotechnology) (1:50 dilution) were conjugated to Sepharose beads, and then added to the control or E2-treated cell lysates for 2 h at 4 C. After pelleting and washing, samples were electrophoretically separated on a 7% SDS gel, transferred to nylon, and immunoblotted. Detection used the ECL kit (Amersham Pharmacia Biotech, Arlington Heights, IL).
G Protein Association with ER and Activation Studies
We transiently transfected CHO cells with wt ER or DM receptor, and then isolated the plasma membrane from cells expressing the two receptors by sucrose gradient centrifugation. G protein activation by membrane ER was determined as follows. Aliquots (20 µg) containing immunoprecipitated mouse wt ER or DM ER at the membrane were resuspended in buffer containing 30 nM [35S]GTPS (Sigma Chemical Co., St. Louis, MO), with or without 10 nM 17-ß-E2 for 5 min at 30 C (23). The incubation was stopped by adding 600 µl ice-cold 50 mM Tris-HCl, 20 mM MgCl2, Nonidet P-40 0.5%, and 100 µM GTP, and then the extract was placed into microfuge tubes containing 2 µl of nonimmune serum preincubated with 10% suspension of pansorbin cells (18). Nonspecifically bound proteins were removed by centrifugation after 20 min. The supernatant was then incubated with Gq, Gs, and Gi-subunit antibodies, preincubated with 5% protein A sepharose. Immunoprecipitants were washed in buffer without detergent and boiled with SDS, and equal protein aliquots from each condition were separated by gel electrophoresis. This yielded a gel band of the activated/nonactivated G protein that associates with membrane wt or mutant ER.
Cell Survival Studies
CHO-transfected cells were grown on 18-mm cover slips in 12-well culture dishes in DMEM-F12 medium without phenol red but with added charcoal-stripped serum. For apoptosis studies, the cells were subjected to 1 min of UV irradiation (20 J/cm2), and then incubated for 4 h at 37 C in the presence or absence of 10 nM E2 ± 1 µM PD98059 or 100 nM wortmannin. At the end of incubation, the cells were washed with PBS and fixed with 1% freshly prepared paraformaldehyde in PBS, pH 7.4, at 4 C overnight. Apoptosis was then determined by the terminal deoxynucleotidyl transferase-stimulated incorporation of nucleotides into the 3-OH end of damaged DNA in the cell, detected by fluorescent antibodies to the nucleotides (TUNEL), using a kit from Intergen (Purchase, NY). From each experimental condition, 400 cells were visually scored for apoptosis and viewed by fluorescence microscopy using standard fluorescein excitation and emission filters. In addition, FACS-based cell counting for apoptosis was carried out after bromodeoxyuridine labeling. Apoptosis in both these cell lines was also determined by FACS detection of Annexin-V binding using a kit (Annexin-V; Becton-Dickinson, Franklin Lakes, NJ). In early apoptosis, the plasma membrane phospholipid, phosphatidylserine, translocates from the inner to the outer membrane leaflet. In cells undergoing apoptosis, phosphatidylserine is then available to bind phospholipid-binding proteins, such as Annexin V.
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FOOTNOTES |
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Abbreviations: CHO, Chinese hamster ovary; E2, estradiol; DM, dimer mutant; EC, endothelial cells; EFGR, epidermal growth factor receptor; ER, estrogen receptor; FACS, fluorescence-activated cell sorting; HUVEC, human umbilical vein EC; IGF1R, type 1 IGF receptor; KO, knockout; MBCEC, mouse brain capillary EC; SDS, sodium dodecyl sulfate; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling; wt, wild type.
Received for publication March 17, 2004. Accepted for publication June 28, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(i). J Biol Chem 276:27071–27076 mediates the nongenomic activation of endothelial nitric oxide synthase by estrogen. J Clin Invest 103:401–406[Medline]
. Mol Cell Biol 23:1633–1646-Shc association and Shc pathway activation. Mol Endocrinol 16:116–127, ß (double) knockout mice. Circ Res 89:534–549 and ERß expressed in CHO cells. Mol Endocrinol 13:307–319 to the plasma membrane. Proc Natl Acad Sci USA 101:2076–2081 rapidly activates the IGF-1 receptor pathway. J Biol Chem 275:18447–18453 and ß form heterodimers on DNA. J Biol Chem 272:19858–198621. J Biol Chem 277:22558–22565NURSA Molecule Pages Link:
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 S. Khan, F. Wu, S. Liu, Q. Wu, and S. Safe Role of specificity protein transcription factors in estrogen-induced gene expression in MCF-7 breast cancer cells J. Mol. Endocrinol., October 1, 2007; 39(4): 289 - 304. [Abstract] [Full Text] [PDF]
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 S. Masia, S. Alvarez, A. R. de Lera, and D. Barettino Rapid, Nongenomic Actions of Retinoic Acid on Phosphatidylinositol-3-Kinase Signaling Pathway Mediated by the Retinoic Acid Receptor Mol. Endocrinol., October 1, 2007; 21(10): 2391 - 2402. [Abstract] [Full Text] [PDF]
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C. Wang, E. R. Prossnitz, and S. K. Roy Expression of G Protein-Coupled Receptor 30 in the Hamster Ovary: Differential Regulation by Gonadotropins and Steroid Hormones Endocrinology, October 1, 2007; 148(10): 4853 - 4864. [Abstract] [Full Text] [PDF]
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 A. Pedram, M. Razandi, R. C. A. Sainson, J. K. Kim, C. C. Hughes, and E. R. Levin A Conserved Mechanism for Steroid Receptor Translocation to the Plasma Membrane J. Biol. Chem., August 3, 2007; 282(31): 22278 - 22288. [Abstract] [Full Text] [PDF]
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 A. Morales, M. Gonzalez, R. Marin, M. Diaz, and R. Alonso Estrogen inhibition of norepinephrine responsiveness is initiated at the plasma membrane of GnRH-producing GT1-7 cells J. Endocrinol., July 1, 2007; 194(1): 193 - 200. [Abstract] [Full Text] [PDF]
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 N. Heldring, A. Pike, S. Andersson, J. Matthews, G. Cheng, J. Hartman, M. Tujague, A. Strom, E. Treuter, M. Warner, et al. Estrogen Receptors: How Do They Signal and What Are Their Targets Physiol Rev, July 1, 2007; 87(3): 905 - 931. [Abstract] [Full Text] [PDF]
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A. Zheng, A. Kallio, and P. Harkonen Tamoxifen-Induced Rapid Death of MCF-7 Breast Cancer Cells Is Mediated via Extracellularly Signal-Regulated Kinase Signaling and Can Be Abrogated by Estrogen Endocrinology, June 1, 2007; 148(6): 2764 - 2777. [Abstract] [Full Text] [PDF]
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A. A. Kazi and R. D. Koos Estrogen-Induced Activation of Hypoxia-Inducible Factor-1{alpha}, Vascular Endothelial Growth Factor Expression, and Edema in the Uterus Are Mediated by the Phosphatidylinositol 3-Kinase/Akt Pathway Endocrinology, May 1, 2007; 148(5): 2363 - 2374. [Abstract] [Full Text] [PDF]
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 S. A. Hart, M. A. Snyder, T. Smejkalova, and C. S. Woolley Estrogen Mobilizes a Subset of Estrogen Receptor-{alpha}-Immunoreactive Vesicles in Inhibitory Presynaptic Boutons in Hippocampal CA1 J. Neurosci., February 21, 2007; 27(8): 2102 - 2111. [Abstract] [Full Text] [PDF]
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N. Vasudevan and D. W. Pfaff Membrane-Initiated Actions of Estrogens in Neuroendocrinology: Emerging Principles Endocr. Rev., February 1, 2007; 28(1): 1 - 19. [Abstract] [Full Text] [PDF]
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 S. Rice and S. A Whitehead Phytoestrogens and breast cancer -promoters or protectors? Endocr. Relat. Cancer, December 1, 2006; 13(4): 995 - 1015. [Abstract] [Full Text] [PDF]
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R. X-D Song, P. Fan, W. Yue, Y. Chen, and R. J Santen Role of receptor complexes in the extranuclear actions of estrogen receptor {alpha} in breast cancer Endocr. Relat. Cancer, December 1, 2006; 13(Supplement_1): S3 - S13. [Abstract] [Full Text] [PDF]
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A. Pedram, M. Razandi, and E. R. Levin Nature of Functional Estrogen Receptors at the Plasma Membrane Mol. Endocrinol., September 1, 2006; 20(9): 1996 - 2009. [Abstract] [Full Text] [PDF]
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 C. D. Bushnell, P. Hurn, C. Colton, V. M. Miller, G. del Zoppo, M. S.V. Elkind, B. Stern, D. Herrington, G. Ford-Lynch, P. Gorelick, et al. Advancing the Study of Stroke in Women: Summary and Recommendations for Future Research From an NINDS-Sponsored Multidisciplinary Working Group Stroke, September 1, 2006; 37(9): 2387 - 2399. [Abstract] [Full Text] [PDF]
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 R. X.-D. Song and R. J. Santen Membrane Initiated Estrogen Signaling in Breast Cancer Biol Reprod, July 1, 2006; 75(1): 9 - 16. [Abstract] [Full Text] [PDF]
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S. Mahmoodzadeh, S. Eder, J. Nordmeyer, E. Ehler, O. Huber, P. Martus, J. Weiske, R. Pregla, R. Hetzer, and V. Regitz-Zagrosek Estrogen receptor alpha up-regulation and redistribution in human heart failure FASEB J, May 1, 2006; 20(7): 926 - 934. [Abstract] [Full Text] [PDF]
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 A. Pedram, M. Razandi, D. C. Wallace, and E. R. Levin Functional Estrogen Receptors in the Mitochondria of Breast Cancer Cells Mol. Biol. Cell, May 1, 2006; 17(5): 2125 - 2137. [Abstract] [Full Text] [PDF]
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W. R. Harrington, S. H. Kim, C. C. Funk, Z. Madak-Erdogan, R. Schiff, J. A. Katzenellenbogen, and B. S. Katzenellenbogen Estrogen Dendrimer Conjugates that Preferentially Activate Extranuclear, Nongenomic Versus Genomic Pathways of Estrogen Action Mol. Endocrinol., March 1, 2006; 20(3): 491 - 502. [Abstract] [Full Text] [PDF]
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F. Acconcia, C. J. Barnes, and R. Kumar Estrogen and Tamoxifen Induce Cytoskeletal Remodeling and Migration in Endometrial Cancer Cells Endocrinology, March 1, 2006; 147(3): 1203 - 1212. [Abstract] [Full Text] [PDF]
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J. L. Boerner, M. A. Gibson, E. M. Fox, E. D. Posner, S. J. Parsons, C. M. Silva, and M. A. Shupnik Estrogen Negatively Regulates Epidermal Growth Factor (EGF)-Mediated Signal Transducer and Activator of Transcription 5 Signaling in Human EGF Family Receptor-Overexpressing Breast Cancer Cells Mol. Endocrinol., November 1, 2005; 19(11): 2660 - 2670. [Abstract] [Full Text] [PDF]
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E. R. Levin Integration of the Extranuclear and Nuclear Actions of Estrogen Mol. Endocrinol., August 1, 2005; 19(8): 1951 - 1959. [Abstract] [Full Text] [PDF]
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D. Haas, S. N. White, L. B. Lutz, M. Rasar, and S. R. Hammes The Modulator of Nongenomic Actions of the Estrogen Receptor (MNAR) Regulates Transcription-Independent Androgen Receptor-Mediated Signaling: Evidence that MNAR Participates in G Protein-Regulated Meiosis in Xenopus laevis Oocytes Mol. Endocrinol., August 1, 2005; 19(8): 2035 - 2046. [Abstract] [Full Text] [PDF]
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A. Pedram, M. Razandi, M. Aitkenhead, and E. R. Levin Estrogen Inhibits Cardiomyocyte Hypertrophy in Vitro: ANTAGONISM OF CALCINEURIN-RELATED HYPERTROPHY THROUGH INDUCTION OF MCIP1 J. Biol. Chem., July 15, 2005; 280(28): 26339 - 26348. [Abstract] [Full Text] [PDF]
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 E. Rzewuska-Lech, M. Jayachandran, L. A. Fitzpatrick, and V. M. Miller Differential effects of 17{beta}-estradiol and raloxifene on VSMC phenotype and expression of osteoblast-associated proteins Am J Physiol Endocrinol Metab, July 1, 2005; 289(1): E105 - E112. [Abstract] [Full Text] [PDF]
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 C. Bolego, A. Cignarella, P. Sanvito, V. Pelosi, F. Pellegatta, L. Puglisi, and C. Pinna The Acute Estrogenic Dilation of Rat Aorta Is Mediated Solely by Selective Estrogen Receptor-{alpha} Agonists and Is Abolished by Estrogen Deprivation J. Pharmacol. Exp. Ther., June 1, 2005; 313(3): 1203 - 1208. [Abstract] [Full Text] [PDF]
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X. Guo, M. Razandi, A. Pedram, G. Kassab, and E. R. Levin Estrogen Induces Vascular Wall Dilation: MEDIATION THROUGH KINASE SIGNALING TO NITRIC OXIDE AND ESTROGEN RECEPTORS {alpha} AND {beta} J. Biol. Chem., May 20, 2005; 280(20): 19704 - 19710. [Abstract] [Full Text] [PDF]
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M. I. Boulware, J. P. Weick, B. R. Becklund, S. P. Kuo, R. D. Groth, and P. G. Mermelstein Estradiol Activates Group I and II Metabotropic Glutamate Receptor Signaling, Leading to Opposing Influences on cAMP Response Element-Binding Protein J. Neurosci., May 18, 2005; 25(20): 5066 - 5078. [Abstract] [Full Text] [PDF]
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L. Bjornstrom and M. Sjoberg Mechanisms of Estrogen Receptor Signaling: Convergence of Genomic and Nongenomic Actions on Target Genes Mol. Endocrinol., April 1, 2005; 19(4): 833 - 842. [Abstract] [Full Text] [PDF]
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 N. Kanda and S. Watanabe 17{beta}-Estradiol enhances heparin-binding epidermal growth factor-like growth factor production in human keratinocytes Am J Physiol Cell Physiol, April 1, 2005; 288(4): C813 - C823. [Abstract] [Full Text] [PDF]
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K. L. Chambliss, L. Simon, I. S. Yuhanna, C. Mineo, and P. W. Shaul Dissecting the Basis of Nongenomic Activation of Endothelial Nitric Oxide Synthase by Estradiol: Role of ER{alpha} Domains with Known Nuclear Functions Mol. Endocrinol., February 1, 2005; 19(2): 277 - 289. [Abstract] [Full Text] [PDF]
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