| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Division of Endocrinology, Veterans Affairs Medical Center, Long Beach, Long Beach, California 90822; and Departments of Medicine (A.P., M.R., E.R.L.) and Pharmacology (E.R.L.), University of California, Irvine, Irvine, California 92717
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.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
/ERß combined-deleted mice, estradiol (E2) failed to specifically bind, and did not activate cAMP, ERK, or phosphatidyinositol 3-kinase or stimulate DNA synthesis. This is in contrast to wild-type ECs, indicating the lack of any functional estrogen-binding proteins in ER/ERß combined-deleted ECs. To directly determine the identity of membrane and nuclear-localized ER, we isolated subcellular receptor pools from MCF7 cells. Putative ER proteins were trypsin digested and subjected to tandem array mass spectrometry. The output analysis identified membrane and nuclear E2-binding proteins as classical human ER. We also determined whether GPR30 plays any role in E2 rapid actions. MCF7 (ER and GPR30 positive) and SKBR-3 (ER negative, GPR30 positive) cells were incubated with E2. Only MCF7 responded with significantly increased signaling. In MCF7, the response to E2 was not different in cells transfected with small interfering RNA to green fluorescent protein or GPR30. In contrast, interfering RNA to ER or ER inhibition prevented rapid signaling and resulting biology in MCF7. In breast cancer and ECs, nuclear and membrane ERs are the same proteins. Furthermore, classical ERs mediate rapid signals induced by E2 in these cells. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
The vast majority of studies have implicated rapid actions of steroids to originate at the cell surface rather than in the nucleus. Estrogen activates ERK and induces the survival of multiple cells that are engineered to express only a membrane-localized E domain of ER (12, 13). In contrast, the same cells that express only a nuclear-localized, ligand-binding domain fail to support these actions of estradiol (E2) (13). Similarly, cells transfected to express a nuclear localization sequence-deficient ER show kinase activation by E2 (14). Finally, membrane-restrained compounds such as E2-BSA or estrogenic compounds that activate only membrane -localized steroid-binding proteins capably activate rapid estrogen signaling (12, 15). These findings make it imperative to understand the structural and functional aspects of endogenous estrogen-binding proteins localized to the cell membrane.
ERs translocate to the plasma membrane where the receptors are probably tethered to the cytoplasmic face of the bilayer and are contained within caveolae rafts (16, 17, 18). The exact nature of these localized proteins remains controversial. Much data support the idea that membrane-localized ERs are very similar to the classical nuclear ERs. Using a series of antibodies to multiple epitopes of the nuclear ER, plasma membrane ERs were identified in several cell types (19, 20), indicating broad similarity of the two receptor pools of this isoform. Introduction of antisense oligomers to ER into pituitary cells diminished detection of ER at the plasma membrane by immunohistochemistry (20). Expression of the cDNAs for either ER or ERß in ER null cells resulted in the codetection of membrane and nuclear ERs, indicating that the two cellular pools could arise from a single transcript (21). More recently, endogenous ER and ERß receptors of 67 and 54 kDa, respectively, have been identified in the caveolae and cell membranes from endothelial cells (ECs). These studies used antibodies against the classical nuclear ER and ERß (22). Finally, ECs from the DERKO mouse (ER and ERß deleted) do not show membrane or nuclear ER or ERß by immunoblot, in contrast to wild-type ECs that endogenously express both ER isoforms at both locations (23).
Alternatively, multiple reports involving several cell types describe nonclassical steroid-binding proteins that functionally mediate rapid actions of estrogen. In neurons, alternative ER or ER-X have been characterized to have slightly altered receptor pharmacologies, compared with classical ERs in breast or uterus (24, 25). It has even been reported that estrogen binds putative ERs to signal in cells that have been traditionally thought to be ER null (26). The latter report, however, has not been the experience of dozens of laboratories that report a lack of estrogen binding or E2 action in Chinese hamster ovary and COS cells. Furthermore, the protein sequences, membrane-localization mechanisms, and structure/functional relationships of these putative ERs have largely not been determined (24, 25, 26). Finally, it has not been established that these receptors function in cells from DERKO mice.
It has been proposed that an orphan GPR, GPR30 (27), is an alternative ER (28). This protein has been reported to respond to E2 at the plasma membrane, or in the endoplasmic reticulum (28, 29). GPR30 has been implicated to mediate E2 signaling, but often these reports used ER-negative cells in which overall E2 biological action is unproven (28, 29, 30, 31). The original reports on this protein indicated an unusual pharmacology, in that 17ß-estradiol (17ß-E2) or ICI 182780 (ER antagonist) each activated GPR30. This interaction occurred through unknown mechanisms to signal downstream kinase activation and second messenger generation via epidermal growth factor receptor (EGFR) transactivation (28, 30). More recent work showed a low-capacity (very modest) binding of GPR30 by sex steroid (31). E2 binding resulted in modest second messenger generation, as shown primarily in GPR30-transfected, overexpressing cells (31). Another study indicated that GPR30-E2 interactions required micromolar (not nanomolar) E2 (32). More definitively, expression of antisense oligonucleotides to GPR30 did not affect E2 activation of ERK or cell proliferation in breast cancer cells (33). However, a more physiological interaction between E2 and GPR30 has been reported in keratinocytes (34). Thus, a role for GPR30 as an ER remains controversial.
In this report we address many of these controversies. We determined the lack of any functional estrogen-binding protein in ECs from DERKO mice. We also isolated and analyzed the membrane and nuclear estrogen-binding proteins in MCF7 cells by mass spectrometry (MS). Finally, we carried out a series of studies to determine whether GPR30 or classical ERs support E2 action in both ER-positive and -negative breast cancer cells.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
and ERß in the plasma membrane, compared with wild-type ECs (23). This led us to conclude that the genes that code for the classical ER isoforms produce the membrane ER proteins. Whether nuclear and membrane ER proteins are identical could not be determined from this approach.
Here we find that both nuclear and membrane ER and ERß at the expected sizes are seen in ECs from wild-type littermates but are absent in ECs from the DERKO mouse (Fig. 1A
). This justified asking the question, "Are there any functional E2-binding proteins at the membrane of ER/ERß-deleted cells?" To address this, we first carried out studies in DERKO ECs, and found virtually no binding of labeled E2 in the whole cell. This contrasts to strong binding of E2 in wild-type ECs (Fig. 1B). In addition, no binding was detected specifically in cell membrane or cytoplasmic fractions of DERKO ECs (data not shown).
|
, E2 stimulated cAMP and ERK activity in wild-type EC, but not in DERKO ECs. Similarly, there was no evidence that E2 stimulated phosphatidylinositol 3-kinase (PI3K) activity (AKT phosphorylation) in the DERKO ECs, in contrast to wild-type ECs that support this function (Fig. 1D). Finally, E2 stimulated EC thymidine incorporation, as an index of DNA synthesis, in wild-type ECs (Fig. 1E). This occurred in an ERK-dependent and PI3K-dependent fashion, because the MAPK kinase inhibitor PD98059 (PD) and PI3K inhibitor wortmannin (WTM) blocked this E2 action. It is well documented that E2 stimulates ERK and PI3K in these cells via membrane ERs (17, 18, 35). By contrast, no thymidine incorporation was measured in DERKO ECs incubated with E2 (Fig. 1E). We also found comparable expression of GPR30 in wild-type or DERKO ECs, yet vastly different signaling by E2 (Fig. 1F). We conclude that the ability of E2 to activate rapid signaling and resulting cell biology in EC requires the classical ER or ERß. Furthermore, our results do not support the idea that functional estrogen-binding proteins exist in DERKO cells.
Isolation and Characterization of Membrane ERs from MCF7 Cells
To determine the identity of endogenous estrogen-binding proteins at the plasma membrane of a target cell (breast cancer), we approached this by MS analysis. MCF7 cell lysate fractions from the nucleus and the plasma membrane were isolated by sucrose gradient centrifugation. The lack of contamination of the cell fractions after isolation was previously published (13, 36) and is shown here (Fig 2A).
|
). Only one discrete band appeared, notably at approximately 67 kDa, in both the membrane and nuclear fractions of MCF7 cells. This was comparable to the position on gel of recombinant human ER protein, run in a parallel well. Importantly, no antibodies were used in the isolation of the proteins, including antisera for ER. This prevented bias toward isolating only known ER protein. Approximately eight times more cell lysate was used to isolate membrane receptors, compared with the protein used for nuclear receptor isolation, consistent with the fact that nuclear/membrane ERs exist in an approximately 9:1 ratio (21). Each prominent gel band was cut out.
The bands at approximately 67 kDa were analyzed in the Mass Spectrometry Facility at the Ben May Cancer Institute, University of Chicago. The proteins constituting the prominent nuclear and membrane bands were trypsin cleaved and analyzed by electrospray MS. The output was further interrogated using two rigorous protein analysis programs, Mascot and Spectrum Mill (see Materials and Methods). Thirteen small to moderate size peptides resulted from the trypsin digests in each fraction and were all scored as 100% identical to classical human ER (Table 1). Analysis of the membrane peptides showed coverage of 15% of the classical human ER, and analysis of the nuclear proteins showed 19% coverage, ranging from the A/B domains to the E/F domains of the sex steroid receptor. Overall, the results were interpreted as unlikely to be a protein other than ER, at a significance level of greater than P < 0.00000000001. Furthermore, although some peptides from the nuclear and membrane samples partially overlapped at several portions of ER, there were also unique peptides in each cell fraction, covering differing portions of the receptor protein (representative peptides described in Table 1).
|
To further rule out other E2-binding proteins in the cell membranes of the MCF7 cells, solubilized membrane fractions were subjected to preclearing of potential E2-binding proteins. Excess antibody to ER (H222), ERß, GPR30, or IgG (control) was bound to Protein A sepharose beads and incubated with solubilized membrane protein. This retained the specific proteins from the membrane fractions onto the antibody-conjugated beads. Nonretained membrane protein was then incubated with labeled estradiol, and specific binding was determined.
As seen in Fig. 2B, E2 bound strongly to solubilized membranes that were first incubated with IgG-sepharose (control), indicating one or more E2-binding proteins were not retained on the antibody-conjugated beads. However, ER antibody-bead complexes removed 88% of subsequent E2 binding to the cell membrane. ERß antibody-bead complexes removed the rest of the E2 binding, this being consistent with a very small amount of ERß detected at the membrane by immunoblot (our unpublished data). Importantly, beads complexed with GPR30 antibody failed to remove E2 binding to membranes. Because this latter result was very similar to IgG antibody/bead exposure, little binding could have occurred to the orphan GPR. It is possible that solubilization or immunodepletion of the membranes disrupted ER-GPR30 binding that could have participated in an E2-binding complex. However, we previously reported that binding of small G proteins to ER was readily apparent in membranes solubilized by our procedure (21, 23). These results support the idea that classical ER proteins mediate the binding of E2 at the membrane.
E2 Does Not Function in ER-Negative Breast Cancer Cells
We then investigated the potential role of GPR30 for E2 signaling in breast cancer cells. GPR30 has been described at the plasma membrane or in the endoplasmic reticulum, and some investigations reported that this protein mediates second messenger generation, kinase activation, or calcium signaling by E2 (28, 29, 30). We used ER-positive breast cancer cells that are reported to produce GPR30 (MCF7), and ER-negative breast cancer cells that express this orphan GPR (SKBR-3) (28, 29, 30, 31). Also, we briefly compared HCC-1569 breast cancer cells that lack both ER and GPR30 (data not shown) for binding E2. In membranes prepared from HCC-1569 or SKBR-3 cells, comparably insignificant binding of isotope-labeled E2 was seen (Fig. 3A, left). This supports the lack of any meaningful estrogen-binding protein at the membranes in these cells. In contrast, similar numbers of MCF7 cells showed 2.6 fmol of estrogen-binding protein at the membrane.
|
, right). We also confirmed that GPR30 is expressed in both MCF7 and SKBR-3 cells, with some protein localized to the plasma membrane, but much of the orphan GPR residing in other, undetermined cell locations (Fig. 3B).
To determine the mediation of the rapid effects of E2, we examined multiple signals. In MCF7 cells, E2 activated ERK and PI3K, cAMP, and calcium. The ER inhibitor, ICI 182780, substantially blocked all these effects (Fig. 3C). These results suggest that ER mediates E2-induced rapid signaling in ER-positive breast cancer cells. In contrast, E2 did not significantly activate ERK, PI3K, or cAMP in SKBR-3 cells. Interestingly, E2 modestly stimulated an increase in calcium flux in these cells, but this was unaffected by ICI 182780, small interfering RNA (siRNA) to GPR30, or control siRNA to green fluorescent protein (GFP) (also see Fig. 4). Furthermore, 10 nM E2 caused a 4-fold greater increase in calcium in MCF7 than in SKBR-3 cells. Thus, potential interactions of the sex steroid with any putative binding protein do not have any recognizable and significant outcome in these ER null breast cancer cells.
|
, left). In SKBR-3 cells, a very modest stimulation of thymidine incorporation in response to E2 was seen, unaffected by ICI 182780. Furthermore, E2-induced a 75% survival of MCF7 breast cancer cells after UV-radiation that was 85% reversed by ICI 182780 (Fig. 3D, right). In SKBR-3 cells, there was a statistically insignificant 17% decrease in cell death in E2-incubated cells. We and others previously showed that the ability of membrane ER/E2 to stimulate ERK and PI3K contributes to survival in ER-positive breast cancer cells (8, 23). This coincides with previous demonstrations of rapid signaling from cell membrane ER that are required for similar E2 actions in a variety of cells (7, 8, 9, 10).
Because only MCF7 cells demonstrated meaningful responses to E2, we asked whether GPR30 might contribute to these functions of the sex steroid in this cell that produces both putative binding proteins. To accomplish this, we silenced GPR30, using interfering RNA. As seen in Fig. 4A, left, approximately 80% knockdown of the GPR30 protein occurred with a specific siRNA (siRNA4) to this orphan GPR, not seen with the GFP control siRNA. Actin expression was unaffected by either siRNA, and transfection efficiency was about 82%, determined by expressing a second control, fluorescent siRNA (latter data not shown). The ability of E2 to activate rapid signaling to cAMP (Fig. 4A, right), ERK (Fig. 4B), PI3K (Fig. 4C), and calcium (Fig. 3C) was not affected by GPR30 knockdown. Furthermore, E2-induced thymidine incorporation in MCF7 was significantly inhibited by ICI 182780, but was unaffected by GPR30 silencing (Fig. 3D, left). A similar lack of GPR30 importance was seen for cell survival induced by E2 in these cells (Fig. 3D, right, lanes 2, 3, and 5 on the left side of the figure).
In contrast, siRNA to ER significantly prevented ERK, PI3K, cAMP, and calcium signaling (Fig. 5). Knockdown of ERß also had a modest effect on ERK activation, an insignificant effect on cAMP generation and calcium signaling, and no effect on PI3K activity, as stimulated by E2: these findings indicate the importance of the ER isoform. We conclude that E2 does not utilize GPR30 for rapid signaling in MCF7 cells but does require mainly classical ER.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Membrane and nuclear-localized estrogen-binding proteins from MCF7 cells were isolated in an unbiased fashion and analyzed by MS. We report that membrane and nuclear ER in these human breast cancer cells are the same proteins, classical ER. In supporting studies, no appreciable specific binding of E2 was found after immunodepleting ER (primarily) and ERß (secondarily) from MCF7-solubilized membrane fractions. This indicates that no residual E2-binding proteins remain in the absence of classical ER. We cannot rule out the presence of a small amount of another E2-binding protein that shares epitope homology to ER. However, we used several different antibodies against N- and C-terminal motifs (data not shown), making it less likely that another protein was inadvertently precipitated. Also, immunodepleting GPR30 did not significantly influence E2 binding at the membrane.
Supporting this theme, we report that in ECs or breast cancer cells, E2 requires a classical ER to rapidly activate multiple signals to downstream cell biology. In ECs, wild-type, and not the combined ER/ERß-deleted cell, shows estrogen binding and activation of multiple rapid signals that are known to originate at the membrane. This is despite the fact that wild-type and DERKO EC contain comparable amounts of GPR30. In ECs, E2/ER have been reported to stimulate Gi-dependent PI3K activity and resulting nitric oxide generation (37).
In ER null breast cancer cells, E2 failed to significantly activate multiple signals or enact cell physiology. A modest cell survival response to E2 was seen in radiated SKBR-3 cells. However, it has been reported previously that E2 directly acts as an antioxidant to prevent cell death, unrelated to ER binding (38). Perhaps such a mechanism underlies this modest steroid action in the SKBR-3 cells, effects not comparable to ER-mediated signaling in MCF7 cells. Consideration of whether E2 functions in ER-negative cells is also relevant to clinical medicine and breast cancer biology. In women, aromatase inhibitors are ineffective in modulating the behavior of ER-negative breast cancer (39). Aromatase inhibitor compounds might be expected to have some therapeutic effects if E2 bound and functioned through alternative ER (such as GPR30) in classical ER-negative tumors.
In contrast, E2 up-regulated cAMP, ERK, PI3K, and calcium flux in MCF7 cells, all inhibited by ICI 182780. Additionally, the ability of E2 to stimulate DNA synthesis or cell survival in these breast cancer cells required an ER that was inhibited by ICI 182780. Finally, silencing ER in MCF7 cells significantly inhibited rapid signaling by E2. We also asked whether there is functional cooperation between ER and GPR30 in MCF7 cells. In cells expressing a control siRNA, E2 rapidly and strongly activated multiple signals that led to cell cycle progression and cell survival. Comparable signaling and outcome response was found in MCF7 that showed approximately 80% GPR30 protein knockdown. We conclude that ER, and not GPR30, is required for rapid E2 actions in these cells.
When estrogen-binding proteins other than ER or ERß are further considered, previous reports of alternative ERs failed to show 1) the presence of functional receptors in cells from DERKO mice, and 2) the isolation and characterization of such receptors from the membrane. The receptor pharmacology of classical ER at the membrane may be somewhat different in various cell types (40, 41), depending on differences in receptor orientation, membrane domain localization, or unique binding/scaffolding partners. Importantly, it is largely unproven that E2 carries out any cell physiological actions mediated through alternative ERs. By contrast, rapid signaling and cell actions in vivo result from classical ER isoforms (42, 43).
More recent studies of nonclassical estrogen-binding proteins implicate GPR30. As recently reported, E2 stimulates calcium mobilization and PI3K activation in the endoplasmic reticulum though binding this orphan GPR (29). The location of GPR30 is in dispute: many earlier studies indicated a plasma membrane localization of this typical heptahelical protein (27, 28, 30), but this was notably absent in the more recent report (29). Furthermore, initial studies indicated that 17-ß-E2 or ICI 182780 each activated GPR30 by unknown mechanisms and transmitted a downstream signal to the activation of the EGFR (28, 30). EGFR transactivation then resulted in rapid kinase activation.
Subsequent studies of E2 engagement of GPR30 at the membrane revealed an extremely low-capacity binding interaction and quite modest generation of cAMP, the latter determined mainly in cells transfected to overexpress GPR30 (31). In GPR30 overexpressing-cells, 100 nM E2 generated approximately 30% increase over baseline cAMP (31). This is compared with a 200% increase above basal levels, generated by endogenous ER responding to a 1 logarithm lower concentration of E2 as shown here in MCF7 cells.
Many of the GPR30 studies did not show a cell physiological function resulting from the interaction of E2 and GPR30 in cells expressing endogenous GPR30 (28, 29, 30, 31). We confirmed that native SKBR-3 cells (ER negative) produce abundant GPR30, found at the plasma membrane and other locations in the cell. Despite this, E2 could not signal to cell biology. Similarly, we and others have reported in many ER null cells that E2 does not stimulate rapid signaling: However, this does occur after transfection of classical ER (13, 14, 22, 33). Thus, endogenous GPR30 supports no discernible functions of E2 in these cells.
Our results contrast to Revankar et al. (29), who showed that COS7 cells transfected to overexpress either ER or GPR30 responded to E2 with a strong activation of intracellular calcium mobilization. These investigators did not determine calcium signaling in SKBR-3 cells that produce abundant endogenous GPR30 protein. We did find an insignificant activation of calcium by E2, unrelated to ER or GPR30 in SKBR-3 cells. Previously, direct activation of the Maxi-K calcium channel was triggered by pharmacological concentrations of E2 (44). Perhaps such a mechanism accounts for the modest stimulation of calcium reported here in SKBR-3 cells.
However, recent studies in endometrial cancer cells have implicated both ER and GPR30 in E2-induced c-Fos transcription, and cell proliferation (45) and previous studies have implicated this orphan GPR in rapid signaling by E2 to transcription in keratinocytes (34). Thus, in some cell contexts, GPR30 may play a contributory role. It remains to be demonstrated whether GPR30 acts independently of ER in transmitting rapid E2 signaling (45).
In summary, E2 fails to activate multiple pathways in cells that lack classical ER even when GPR30 is present. Consistent with these findings, we report that the membrane and nuclear receptors in MCF7 are the classical ER. We suggest that future reports of alternative estrogen-binding proteins should 1) isolate and analyze the proteins, 2) establish physiological functions of estrogen mediated through the endogenous putative receptors, and 3) define whether endogenous classical ER are also required for E2 action.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
109) of MCF7 cells were grown to near confluence in DMEM/F12 medium. The cells were lysed, and nuclear and membrane fractions were isolated by sucrose gradient centrifugation. Cell fractions were dialyzed and the membrane receptors were solubilized with 0.2% Nonidet P-40. The purity and lack of contamination of the two cell fractions have been extensively validated previously (13, 36) and were further validated by the presence of integral proteins in cell fractions, determined by Western immunoblotting. Antibody to ER (MC-20, directed against the C terminus of this protein) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) for this purpose. Membrane and nuclear fractions were then passed through an affinity column composed of E2-sephadex beads (kindly provided by Dr. Geoffrey Greene, University of Chicago) (46). The proteins were released from the E2-sephadex by sodium dodecyl sulfate (2%)-containing buffer and subjected to denaturing by boiling. Proteins were separated on 8% gels by SDS-PAGE, and the resulting bands were visualized using Gel-Code Blue staining reagents (Pierce Chemical Co., Madison, WI). The excised membrane and nuclear protein bands were further analyzed.
Electrospray MS of Proteins
Each protein band was destained, and the gel slice was washed with water and then with 0.5% formic acid in acetonitrile to dehydrate the gel. The gel slice was digested (50 mM ammonium carbonate, pH 9.0; 250 mM CaCl2; and 12.5 ng/ml of modified Trypsin enzyme (final enzyme-protein ratio, 1:5). The gel in tube was rehydrated and placed in a clean tube with 100 ml of an extracting solution containing 5% formic acid. Equal volume of 5% formic acid-acetonitrile (50:50) was added to solubilize any hydrophobic material remaining in the gel.
The sample was injected onto a trapping column (Zorbax Stablebond C18, 300 A pore) connected to a reversed-phase column (75 mm id Zorbax Stablebond C18 (300 A pore), connected to a 1100 nano/capillary LC/MSD XCT system (Agilent Technologies, Palo Alto, CA), equipped with an orthogonal nanospray interface and a nanopump. The samples were chromatographed using a solvent system of A) 0.1% formic acid and 10% acetonitrile and B) 0.1% formic acid and 100% acetonitrile at a flow rate of 300 nl/min, using a 15–55% gradient over 60 min. The instrument completed a mass scan from 400-2200 daltons in 1 sec and optimized the acquisition of quantitative data in MS mode as well as qualitative data in MS/MS mode.
Data analysis was carried out using both Mascot (Matrix Science, Boston, MA) (47) and SpectrumMill software platforms (Agilent Technologies, Palo Alto, CA). Raw data from the MS data files were first subjected to protein library search and spectral interpretation by the Sherenga (48) module. The results for all proteins detected from both platforms received a search score. A general requirement for a positive identification (at P < 0.01 or greater) is that the library matches agree between both search platforms and that at least four distinct peptides for the tentatively identified protein are present.
Cells
ECs from the brain capillaries of wild-type and DERKO mice were prepared as previously described (23). MCF7, SKBR-3, and HCC-1569 cells were from American Type Culture Collection (Manassas, VA). DNA synthesis (thymidine incorporation) and apoptosis assays (TUNEL) were conducted as previously described (21, 23). Cells were transfected to express siRNAs to GFP, or to GPR30 (duplex 4, antisense strand, 5'-PCUUCAGCGAAUCUCACUCCUU-3') (Dharmacon, Lafayette, CO). ER or ERß siRNAs (QIAGEN, Chatsworth, CA) were reported to be directed against the following DNA sequences, respectively; human esr1, 5'-AAGCCCAAATGTGTTGTGGCC-3', and esr2, 5'-AAGGTGGGATACGAAAAGACC-3'. siRNA (2–3 µg) was used in each well of six-well plates or 100-mm dishes of cells. Fluorescent control siRNA (QIAGEN) was used to determine transfection efficiency over three experiments.
Signaling Assays
The activities of ERK, PI3K, and cAMP were determined as previously described (23, 35), all within 5–15 min exposure of cells to E2. In some experiments, the cells were pretreated with 1 µM ICI 182780 for 20 min before addition of E2. For some signaling studies, data from three experiments were combined and analyzed for statistical significance by calculating a mean ± SEM and comparing conditions by ANOVA plus Schefe’s test, at a significance level of P < 0.05. In additional experiments, siRNAs to GPR30, ER or ERß, or GFP (control) were introduced into cells with oligofectamine and recovered overnight, and GPR30 or ER protein knockdown was determined by Western blot after 48 h, as we described previously (36). Transfection efficiency was determined using an Alexa-fluor 488-tagged, scrambled double-stranded RNA oligonucleotide (QIAGEN), with Hoechst nuclear stain. Antibody to GPR30 was kindly provided by Dr. Eric Prossnitz (29) and was used at a dilution of 1:2500. For cell signaling assays, the assays proceeded 48 h after siRNA transfection.
Calcium activity reflected both extracellular calcium influx through membrane channels and intracellular calcium mobilization from stores and was determined as follows. Calcium was measured in MCF7 and SKBR-3 cells by loading with Fluo-4 NW (Molecular Probes, Inc., Eugene, OR). The cells were cultured in 96-well microplates (poly-D-lysine-coated) to subconfluence (40,000–50,000 cells per well) and grown overnight. The next day, some cells were transfected with siRNA as described. After 24 h, the cells were synchronized overnight in medium lacking fetal bovine serum and phenol red. The medium was removed to eliminate sources of baseline fluorescence, particularly esterase activity. Fluo-4 NW (100 µl) in loading solution was carefully added to each well, and the cells were incubated at 37 C for 30 min. The microplate was then transferred into a 37 C prewarmed, Nowastar Spectrofluorometer (BMG Labtech, Offenburg, Germany). The instrument was preset and loaded with test solutions at 2x concentration, subsequently injecting 50 µl of control or test substances to each well, followed immediately by 250 cycles of reading over 60 sec. Each condition was done in triplicate, and the study was repeated a second time. Fluorescence was measured at excitation, 494 nm, and emission at 516 nm.
E2 Binding Studies
Competition binding studies on whole ECs or on the membranes of MCF7 cells were conducted as follows: 1 nM [3H]E2 was added to tubes containing 104 ECs or membranes fractionated from 106 MCF7 cells, each tube also containing 0.01 nM-1 µM unlabeled E2. Binding to equilibrium (21) was carried out for 1 h at 37 C. Saturation binding on MCF-7 cell membranes was done using a set of tubes containing [3H]E2 at 0.1–1.5 nM (total binding). Another set of identical tubes also contained 100-fold excess unlabeled E2 (nonspecific binding). Binding was carried out at 1 h and 37 C, and specific binding was determined after passing the mixture through GF/C Whatman filters under mild vacuum. Labeled E2 bound to membrane protein was quantified by ß-scintillation counting of the washed filters, subtracting nonspecific binding from total binding. Binding data were subjected to Scatchard analysis using the LIGAND computer program. Each point was done in triplicate and the study was repeated a second time.
For the preclearing studies, antibodies to ER, ERß (L-20, directed against the C terminus of this protein, Santa Cruz Biotechnology) GPR30, or IgG (control) were linked to sepharose beads for 2 h, and then added to protein-normalized, separate aliquots of solubilized membrane fractions from MCF7 cells. After 2 h, the membrane proteins bound to the antibody-bead complexes were removed by pelleting through centrifugation. The supernatants (unbound membrane proteins) were then incubated with 1 nM [3H]E2 for 1 h, and binding was determined after passing the mixture through filters as described above.
|
FOOTNOTES |
|---|
All authors report that they have no disclosures to make, as they have nothing to declare.
First Published Online April 27, 2006
Abbreviations: DERKO, ER and ERß deleted; E2, Estradiol; EC, endothelial cell; EGFR, epidermal growth factor receptor; ER, estrogen receptor; GFP, green fluorescent protein; GPR, G protein-coupled receptor; MS, mass spectrometry; PD, PD98059; PI3K, phosphatidylinositol 3-kinase; WTM, wortmannin.
Received for publication December 21, 2005. Accepted for publication April 17, 2006.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
. Mol Cell Biol 23:1633–1646 mediates estrogen effect on MAPK activation. Biochem Biophys Res Commun 294:926–933[CrossRef][Medline] localized in caveolae. Biochem Biophys Res Commun 263:257–262[CrossRef][Medline] and endothelial nitric oxide synthase are organized into a functional signaling module in caveolae. Circ Res 87:E44–E52 detected on the plasma membrane of aldehyde-fixed GH3/B6/F10 rat pituitary tumor cells by enzyme-linked immunocytochemistry. Endocrinology 140:3805–3814 domains with known nuclear functions. Mol Endocrinol 19:277–289 and estrogen receptor ß. Proc Natl Acad Sci USA 97:11603–11608 and ß. J Biol Chem 280:19704–19710NURSA Molecule Pages Link:
This article has been cited by other articles:
 |
|
 |
|
  E. R. Levin Membrane oestrogen receptor \#945; signalling to cell functions J. Physiol., November 1, 2009; 587(21): 5019 - 5023. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Chen, K. Breen, and M. E Pepling Estrogen can signal through multiple pathways to regulate oocyte cyst breakdown and primordial follicle assembly in the neonatal mouse ovary J. Endocrinol., September 1, 2009; 202(3): 407 - 417. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Iqbal, O. Latchoumanin, I. P. Sari, R. J. Lang, H. A. Coleman, H. C. Parkington, and I. J. Clarke Estradiol-17{beta} Inhibits Gonadotropin-Releasing Hormone-Induced Ca2+ in Gonadotropes to Regulate Negative Feedback on Luteinizing Hormone Release Endocrinology, September 1, 2009; 150(9): 4213 - 4220. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Grassi, A. Frondaroli, C. Dieni, M. Scarduzio, and V. E. Pettorossi Long-Term Potentiation in the Rat Medial Vestibular Nuclei Depends on Locally Synthesized 17{beta}-Estradiol J. Neurosci., August 26, 2009; 29(34): 10779 - 10783. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. A. Kazi, K. H. Molitoris, and R. D. Koos Estrogen Rapidly Activates the PI3K/AKT Pathway and Hypoxia-Inducible Factor 1 and Induces Vascular Endothelial Growth Factor A Expression in Luminal Epithelial Cells of the Rat Uterus Biol Reprod, August 1, 2009; 81(2): 378 - 387. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. M. Popov, J. Zhou, L. A. Shirley, J. Quong, W.-S. Yeow, J. A. Wright, K. Wu, H. Rui, R. K. Vadlamudi, J. Jiang, et al. The Cell Fate Determination Factor DACH1 Is Expressed in Estrogen Receptor-{alpha}-Positive Breast Cancer and Represses Estrogen Receptor-{alpha} Signaling Cancer Res., July 15, 2009; 69(14): 5752 - 5760. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Pedram, M. Razandi, A. J. Evinger, E. Lee, and E. R. Levin Estrogen Inhibits ATR Signaling to Cell Cycle Checkpoints and DNA Repair Mol. Biol. Cell, July 15, 2009; 20(14): 3374 - 3389. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. C. Lin, M. Suzawa, R. D. Blind, S. C. Tobias, S. E. Bulun, T. S. Scanlan, and H. A. Ingraham Stimulating the GPR30 Estrogen Receptor with a Novel Tamoxifen Analogue Activates SF-1 and Promotes Endometrial Cell Proliferation Cancer Res., July 1, 2009; 69(13): 5415 - 5423. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. R. Levin G Protein-Coupled Receptor 30: Estrogen Receptor or Collaborator? Endocrinology, April 1, 2009; 150(4): 1563 - 1565. [Full Text] [PDF]
|
|
|
|
|
|
R. Dominguez, E. Hu, M. Zhou, and M. Baudry 17{beta}-Estradiol-Mediated Neuroprotection and ERK Activation Require a Pertussis Toxin-Sensitive Mechanism Involving GRK2 and {beta}-Arrestin-1 J. Neurosci., April 1, 2009; 29(13): 4228 - 4238. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Isensee, L. Meoli, V. Zazzu, C. Nabzdyk, H. Witt, D. Soewarto, K. Effertz, H. Fuchs, V. Gailus-Durner, D. Busch, et al. Expression Pattern of G Protein-Coupled Receptor 30 in LacZ Reporter Mice Endocrinology, April 1, 2009; 150(4): 1722 - 1730. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Wang, G. T. Wijewickrama, K.-w. Peng, B. M. Dietz, L. Yuan, R. B. van Breemen, J. L. Bolton, and G. R. J. Thatcher Estrogen Receptor {alpha} Enhances the Rate of Oxidative DNA Damage by Targeting an Equine Estrogen Catechol Metabolite to the Nucleus J. Biol. Chem., March 27, 2009; 284(13): 8633 - 8642. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. D. Noel, K. L. Keen, D. I. Baumann, E. J. Filardo, and E. Terasawa Involvement of G Protein-Coupled Receptor 30 (GPR30) in Rapid Action of Estrogen in Primate LHRH Neurons Mol. Endocrinol., March 1, 2009; 23(3): 349 - 359. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. N. Schultz, S. A. von Esenwein, M. Hu, A. L. Bennett, R. T. Kennedy, S. Musatov, C. D. Toran-Allerand, M. G. Kaplitt, L. J. Young, and J. B. Becker Viral Vector-Mediated Overexpression of Estrogen Receptor-{alpha} in Striatum Enhances the Estradiol-Induced Motor Activity in Female Rats and Estradiol-Modulated GABA Release J. Neurosci., February 11, 2009; 29(6): 1897 - 1903. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Pedram, M. Razandi, J. K Kim, F. O'Mahony, E. Y. Lee, U. Luderer, and E. R. Levin Developmental Phenotype of a Membrane Only Estrogen Receptor {alpha} (MOER) Mouse J. Biol. Chem., February 6, 2009; 284(6): 3488 - 3495. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Zhang, X. Liu, A. M. Farkas, A. V. Parwani, K. L. Lathrop, D. Lenzner, S. R. Land, and H. Srinivas Estrogen Receptor {beta} Functions through Nongenomic Mechanisms in Lung Cancer Cells Mol. Endocrinol., February 1, 2009; 23(2): 146 - 156. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Otto, I. Fuchs, G. Kauselmann, H. Kern, B. Zevnik, P. Andreasen, G. Schwarz, H. Altmann, M. Klewer, M. Schoor, et al. GPR30 Does Not Mediate Estrogenic Responses in Reproductive Organs in Mice Biol Reprod, January 1, 2009; 80(1): 34 - 41. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Xu, Y. Cheng, J. R. Keast, and P. B. Osborne 17{beta}-Estradiol Activates Estrogen Receptor {beta}-Signalling and Inhibits Transient Receptor Potential Vanilloid Receptor 1 Activation by Capsaicin in Adult Rat Nociceptor Neurons Endocrinology, November 1, 2008; 149(11): 5540 - 5548. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Christian, C. Glidewell-Kenney, J. L. Jameson, and S. M. Moenter Classical Estrogen Receptor {alpha} Signaling Mediates Negative and Positive Feedback on Gonadotropin-Releasing Hormone Neuron Firing Endocrinology, November 1, 2008; 149(11): 5328 - 5334. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. R. Levin Rapid signaling by steroid receptors Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2008; 295(5): R1425 - R1430. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. John, I. Nayvelt, H.-C. Hsu, P. Yang, W. Liu, G. M. Das, T. Thomas, and T.J. Thomas Regulation of Estrogenic Effects by Beclin 1 in Breast Cancer Cells Cancer Res., October 1, 2008; 68(19): 7855 - 7863. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Otto, B. Rohde-Schulz, G. Schwarz, I. Fuchs, M. Klewer, D. Brittain, G. Langer, B. Bader, K. Prelle, R. Nubbemeyer, et al. G Protein-Coupled Receptor 30 Localizes to the Endoplasmic Reticulum and Is Not Activated by Estradiol Endocrinology, October 1, 2008; 149(10): 4846 - 4856. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Madak-Erdogan, K. J. Kieser, S. H. Kim, B. Komm, J. A. Katzenellenbogen, and B. S. Katzenellenbogen Nuclear and Extranuclear Pathway Inputs in the Regulation of Global Gene Expression by Estrogen Receptors Mol. Endocrinol., September 1, 2008; 22(9): 2116 - 2127. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Pang, J. Dong, and P. Thomas Estrogen Signaling Characteristics of Atlantic Croaker G Protein-Coupled Receptor 30 (GPR30) and Evidence It Is Involved in Maintenance of Oocyte Meiotic Arrest Endocrinology, July 1, 2008; 149(7): 3410 - 3426. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Klinge, N. S. Wickramasinghe, M. M. Ivanova, and S. M. Dougherty Resveratrol stimulates nitric oxide production by increasing estrogen receptor {alpha}-Src-caveolin-1 interaction and phosphorylation in human umbilical vein endothelial cells FASEB J, July 1, 2008; 22(7): 2185 - 2197. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S Gorjestani, V Rider, B. Kimler, C Greenwell, and N. Abdou Extracellular signal-regulated kinase 1/2 signalling in SLE T cells is influenced by oestrogen and disease activity Lupus, June 1, 2008; 17(6): 548 - 554. [Abstract] [PDF]
|
|
|
|
 |
|
 A. Razmara, L. Sunday, C. Stirone, X. B. Wang, D. N. Krause, S. P. Duckles, and V. Procaccio Mitochondrial Effects of Estrogen Are Mediated by Estrogen Receptor {alpha} in Brain Endothelial Cells J. Pharmacol. Exp. Ther., June 1, 2008; 325(3): 782 - 790. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. M. Miller and S. P. Duckles Vascular Actions of Estrogens: Functional Implications Pharmacol. Rev., June 1, 2008; 60(2): 210 - 241. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Arpino, L. Wiechmann, C. K. Osborne, and R. Schiff Crosstalk between the Estrogen Receptor and the HER Tyrosine Kinase Receptor Family: Molecular Mechanism and Clinical Implications for Endocrine Therapy Resistance Endocr. Rev., April 1, 2008; 29(2): 217 - 233. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. L. Yanes, J. C. Sartori-Valinotti, and J. F. Reckelhoff Sex Steroids and Renal Disease: Lessons From Animal Studies Hypertension, April 1, 2008; 51(4): 976 - 981. [Full Text] [PDF]
|
|
|
|
 |
|
 V. Bourdeau, J. Deschenes, D. Laperriere, M. Aid, J. H. White, and S. Mader Mechanisms of primary and secondary estrogen target gene regulation in breast cancer cells Nucleic Acids Res., January 17, 2008; 36(1): 76 - 93. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. R. Hammes and E. R. Levin Extranuclear Steroid Receptors: Nature and Actions Endocr. Rev., December 1, 2007; 28(7): 726 - 741. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Iqbal, O. Latchoumanin, and I. J. Clarke Rapid in Vivo Effects of Estradiol-17{beta} in Ovine Pituitary Gonadotropes Are Displayed by Phosphorylation of Extracellularly Regulated Kinase, Serine/Threonine Kinase, and 3',5'-Cyclic Adenosine 5'-Monophosphate-Responsive Element-Binding Protein Endocrinology, December 1, 2007; 148(12): 5794 - 5802. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Alzamora, L. R. Brown, and B. J. Harvey Direct Binding and Activation of Protein Kinase C Isoforms by Aldosterone and 17{beta}-Estradiol Mol. Endocrinol., November 1, 2007; 21(11): 2637 - 2650. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. McDevitt, C. Glidewell-Kenney, J. Weiss, P. Chambon, J. L. Jameson, and J. E. Levine Estrogen Response Element-Independent Estrogen Receptor (ER)-{alpha} Signaling Does Not Rescue Sexual Behavior but Restores Normal Testosterone Secretion in Male ER{alpha} Knockout Mice Endocrinology, November 1, 2007; 148(11): 5288 - 5294. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
M. Chang, K.-w. Peng, I. Kastrati, C. R. Overk, Z.-H. Qin, P. Yao, J. L. Bolton, and G. R. J. Thatcher Activation of Estrogen Receptor-Mediated Gene Transcription by the Equine Estrogen Metabolite, 4-Methoxyequilenin, in Human Breast Cancer Cells Endocrinology, October 1, 2007; 148(10): 4793 - 4802. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Cheng, C. Zhang, and D. J. Shapiro A Functional Serine 118 Phosphorylation Site in Estrogen Receptor-{alpha} Is Required for Down-Regulation of Gene Expression by 17{beta}-Estradiol and 4-Hydroxytamoxifen Endocrinology, October 1, 2007; 148(10): 4634 - 4641. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 A. H. Eid, K. Maiti, S. Mitra, M. A. Chotani, S. Flavahan, S. R. Bailey, C. S. Thompson-Torgerson, and N. A. Flavahan Estrogen increases smooth muscle expression of {alpha}2C-adrenoceptors and cold-induced constriction of cutaneous arteries Am J Physiol Heart Circ Physiol, September 1, 2007; 293(3): H1955 - H1961. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. J. Pietras and D. C. Marquez-Garban Membrane-Associated Estrogen Receptor Signaling Pathways in Human Cancers Clin. Cancer Res., August 15, 2007; 13(16): 4672 - 4676. [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 H. Ish, T. Tsurugizawa, M. Ogiue-Ikeda, M. Asashima, H. Mukai, G. Murakami, Y. Hojo, T. Kimoto, and S. Kawato Local Production of Sex Hormones and Their Modulation of Hippocampal Synaptic Plasticity Neuroscientist, August 1, 2007; 13(4): 323 - 334. [Abstract] [PDF]
|
|
|
|
|
|
R. X.-D. Song, Z. Zhang, Y. Chen, Y. Bao, and R. J. Santen Estrogen Signaling via a Linear Pathway Involving Insulin-Like Growth Factor I Receptor, Matrix Metalloproteinases, and Epidermal Growth Factor Receptor to Activate Mitogen-Activated Protein Kinase in MCF-7 Breast Cancer Cells Endocrinology, August 1, 2007; 148(8): 4091 - 4101. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Murphy and C. Steenbergen Gender-based differences in mechanisms of protection in myocardial ischemia-reperfusion injury Cardiovasc Res, August 1, 2007; 75(3): 478 - 486. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
C. M. Silva and M. A. Shupnik Integration of Steroid and Growth Factor Pathways in Breast Cancer: Focus on Signal Transducers and Activators of Transcription and Their Potential Role in Resistance Mol. Endocrinol., July 1, 2007; 21(7): 1499 - 1512. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Schumacher, R. Guennoun, A. Ghoumari, C. Massaad, F. Robert, M. El-Etr, Y. Akwa, K. Rajkowski, and E.-E. Baulieu Novel Perspectives for Progesterone in Hormone Replacement Therapy, with Special Reference to the Nervous System Endocr. Rev., June 1, 2007; 28(4): 387 - 439. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
E. Haas, M. R. Meyer, U. Schurr, I. Bhattacharya, R. Minotti, H. H. Nguyen, A. Heigl, M. Lachat, M. Genoni, and M. Barton Differential Effects of 17{beta}-Estradiol on Function and Expression of Estrogen Receptor {alpha}, Estrogen Receptor {beta}, and GPR30 in Arteries and Veins of Patients With Atherosclerosis Hypertension, June 1, 2007; 49(6): 1358 - 1363. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Traupe, C. D. Stettler, H. Li, E. Haas, I. Bhattacharya, R. Minotti, and M. Barton Distinct Roles of Estrogen Receptors {alpha} and {beta} Mediating Acute Vasodilation of Epicardial Coronary Arteries Hypertension, June 1, 2007; 49(6): 1364 - 1370. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. D. Feldman and R. Gros Choreographing the Rapid Vascular Effects of Estrogen: Sorting Out the Partners and the Steps Hypertension, June 1, 2007; 49(6): 1222 - 1224. [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
T. Krietsch, M. S. Fernandes, J. Kero, R. Losel, M. Heyens, E. W.-F. Lam, I. Huhtaniemi, J. J. Brosens, and B. Gellersen Human Homologs of the Putative G Protein-Coupled Membrane Progestin Receptors (mPR{alpha}, {beta}, and {gamma}) Localize to the Endoplasmic Reticulum and Are Not Activated by Progesterone Mol. Endocrinol., December 1, 2006; 20(12): 3146 - 3164. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. G. Tasker, S. Di, and R. Malcher-Lopes Rapid Glucocorticoid Signaling via Membrane-Associated Receptors Endocrinology, December 1, 2006; 147(12): 5549 - 5556. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 B. Manavathi, F. Acconcia, S. K. Rayala, and R. Kumar An inherent role of microtubule network in the action of nuclear receptor PNAS, October 24, 2006; 103(43): 15981 - 15986. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
