Cell Membrane and Nuclear Estrogen Receptors (ERs) Originate from a Single Transcript: Studies of ER{alpha} and ERß Expressed in Chinese Hamster Ovary Cells

Mahnaz Razandi, Ali Pedram, Geoffrey L. Greene and Ellis R. Levin

Division of Endocrinology (E.R.L.) Long Beach Veterans Affairs Medical Center Long Beach, California 90822
Departments of Medicine (M.R., A.P., E.R.L.) and Pharmacology (E.R.L.) University of California, Irvine Irvine, California 92717
The Ben May Institute (G.L.G.) University of Chicago Chicago, Illinois 60637


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The existence of a putative membrane estrogen receptor (ER) has been supported by studies accomplished over the past 20 yr. However, the origin and functions of this receptor are not well defined. To study the membrane receptor, we transiently transfected cDNAs for ER{alpha} or ERß into Chinese hamster ovary (CHO) cells. Transfection of ER{alpha} resulted in a single transcript by Northern blot, specific binding of labeled 17ß-estradiol (E2), and expression of ER in both nuclear and membrane cell fractions. Competitive binding studies in both compartments revealed near identical dissociation constants (Kds) of 0.283 and 0.287 nM, respectively, but the membrane receptor number was only 3% as great as the nuclear receptor density. Transfection of ERß also yielded a single transcript and nuclear and membrane receptors with respective Kd values of 1.23 and 1.14 nM; the membrane receptor number was only 2% compared with expressed nuclear receptors. Estradiol binding to CHO-ER{alpha} or CHO-ERß activated G{alpha}q and G{alpha}s proteins in the membrane and rapidly stimulated corresponding inositol phosphate production and adenylate cyclase activity. Binding by 17-ß-E2 to either expressed receptor comparably enhanced the nuclear incorporation of thymidine, critically dependent upon the activation of the mitogen-activated protein kinase, ERK (extracellular regulated kinase). In contrast, c-Jun N-terminal kinase activity was stimulated by 17-ß-E2 in ERß-expressing CHO, but was inhibited in CHO-ER{alpha} cells. In summary, membrane and nuclear ER can be derived from a single transcript and have near-identical affinities for 17-ß-E2, but there are considerably more nuclear than membrane receptors. This is also the first report that cells can express a membrane ERß. Both membrane ERs activate G proteins, ERK, and cell proliferation, but there is novel differential regulation of c-Jun kinase activity by ERß and ER{alpha}.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The cellular actions of the sex steroid estrogen are hypothesized to be mediated through the transcriptional regulation of target genes (1, 2). These effects mainly occur when estradiol (E2) binds to the nuclear estrogen receptor (ER); this complex then either binds to response elements expressed on various genes (3) or modifies transcription through protein-protein interactions (4). However, it has increasingly been appreciated that multiple ligands for the steroid receptor superfamily can modulate cell functions through nongenomic actions mediated through plasma membrane proteins (5, 6, 7, 8). As examples of nongenomic functions, aldosterone rapidly activates inositol-1,4,5-trisphosphate (IP3) generation in several cell types (8), while progesterone quickly stimulates increased [Ca2+]i in sperm (6). Regarding estrogen, there is evidence that 17-ß-E2 can trigger a variety of signal transduction events in seconds to a few minutes. These events include the stimulation of cAMP (9), calcium flux (10), phospholipase C activation, and inositol phosphate generation (11, 12), as well as the rapid release of PRL (13). Many of these rapid actions have been attributed to the ability of 17-ß-E2 to act at the cell membrane. Cell membrane effects could include 17-ß-E2 indirectly activating tyrosine kinase growth factor receptors, such as epidermal growth factor receptor (EGFR), with subsequent signal transduction initiated through these receptors (14). Alternatively, the existence of a cell membrane ER was reported more than 20 yr ago (15, 16). This putative receptor has been investigated by several laboratories more recently (17, 18) and appears capable of enacting signal transduction.

Recently, we showed that primary cultures of human vascular smooth muscle cells express what appears to be membrane ER (19). The putative membrane ER on vascular smooth muscle cells participates in the inhibition of growth factor-induced cell proliferation. This effect occurs when 17-ß-E2 interferes with the ability of growth factors to enact signal transduction to the nuclear growth program (20). Estrogen can also inhibit growth factor signaling, which enhances gene transcription (21, 22). This novel mechanism by which ER negatively modulates transcription is probably mediated through a putative membrane ER (21). However, the isolation and structural characterization of the membrane ER have not been described, and the derivation, roles, and cell functions of this putative protein are still largely unknown. To begin to address these issues, we transfected Chinese hamster ovary (CHO) cells with the cDNAs for ER{alpha} and ERß and carried out characterization studies in these cells, which normally do not express ER. We found several novel aspects of ER expression and signal transduction and evidence that the membrane ER importantly contributes to 17-ß-E2 action in this model.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Single Transcripts Expressed for Either ER{alpha} or ERß Result in the Expression of Both Cell Membrane and Nuclear Receptors
To determine whether multiple genes or transcripts are needed to produce nuclear and membrane receptors, we transfected CHO cells with a single cDNA for either the mouse ER{alpha} or ERß. In both situations, a single transcript was detected in either ER{alpha}- or ERß-expressing cells, determined by Northern analysis; this was expected from the expression of single cDNAs for each receptor (Fig. 1AGo). Empty vector-transfected (lanes 1) or non transfected CHO (data not shown) did not express ER mRNA.



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Figure 1. Expression of cDNAs for ER{alpha} or ERß in CHO Cells

A, Northern blot of CHO cells transfected with mER{alpha} and mERß constructs. A single transcript is expressed for each receptor. Lanes 1 are transfected empty vectors (pcDNA3) for mER{alpha} (left) and (pSG5) for mERß (right). Results are representative of three experiments. The location of 28S and 18S ribosomal RNA is shown on the right. B, Cross-linking of [125I]-17-ß-E2 to cell membrane and nuclear ER in CHO-ER{alpha} (lanes 1 and 2) and CHO-ERß (lanes 3 and 4). Molecular mass markers in kilodaltons are shown on the right side of the figure. Approximately 10 times more cell membrane protein was loaded, compared with nuclear protein, to detect the membrane ER after separation by SDS-PAGE. A representative experiment is shown, repeated twice. Cross-linked binding (see Materials and Methods) was detected by autoradiography.

 
We then carried out cross-linking studies to determine the presence and size of ER proteins. Cross-linking of labeled 17-ß-E2 to the transfected CHO subcellular fractions identified both membrane and nuclear receptors of near-identical mass, approximately 69 kDa for ER{alpha} and 60 kDa for ERß (Fig. 1BGo). This is comparable to what is reported for the size of the native ER{alpha} (22) or for ERß expressed after transfection (23). Thus, both cell membrane and nuclear receptor proteins result from expression of a single cDNA for either ER subtype.

Competition binding studies on transiently transfected CHO cells were then carried out and analyzed by Scatchard analysis. In initial studies, high efficiency expression of ER{alpha} or ERß after transient transfection was revealed in that 60–70% of added [3H]17-ß-E2 bound specifically to the CHO cells. We estimated that the total number of ER expressed was approximately 4 million/transfected CHO cell. In contrast, nontransfected CHO failed to show specific binding of labeled 17-ß-E2. The ER{alpha} nuclear and membrane (Fig. 1Go, inset) receptors had nearly identical affinities for 17-ß-E2, of 0.283 ± 0.017 and 0.287 ± 0.011 nM, respectively (Fig. 2AGo), while the receptor densities in these two compartments were greatly disparate, there being almost 40-fold more nuclear than membrane receptors (nuclear Bmax is 362 ± 26 pM; membrane Bmax is 9.7 ± 0.6 pM). For ERß, nuclear and membrane receptors demonstrated near-equal estradiol affinities of 1.23 ± 0.08 and 1.14 ± 0.06 nM, respectively, and a receptor density ratio of 62:1 (nuclear Bmax is 1.62 ± 0.07 nM; membrane Bmax is 21 ± 0.8 pM) (Fig. 2BGo). The lower affinity of ERß compared with ER{alpha} for 17-ß-E2 has been reported previously (24), confirming the utility of our model. These results indicate that the receptors in the two compartments have near-identical affinities for 17-ß-E2, but many more nuclear than membrane receptors are expressed.



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Figure 2. Binding Studies and Labeling of Expressed ER in CHO Cells

A, Competition binding of [3H]-17ß-E2 to nuclear and cell membrane (inset) receptors of CHO cells transfected to express ER{alpha}. Dissociation constants (Kds) for nuclear and cell membrane receptors were 0.283 and 0.287 nM, respectively. B, Competition binding to expressed ERß receptors. Kd values were 1.23 and 1.14 nM. Scatchard plots are shown below each binding figure, and the studies were repeated twice. C, E2-BSA conjugated to FITC labels a cell membrane ER in CHO-ER{alpha} (panel A), competed off by unlabeled E2, 10 nM (B), or ICI 182,780, 1 µM (C). Additionally, an antibody directed against the LBD of ER{alpha} (D), but not against the N terminus of ER{alpha} (E), competes off labeling of the cell membrane. Permeabilization of the cells with 0.2% Triton X-100 resulted in dense labeling of the cell nucleus (F). The representative study shown was repeated twice.

 
To further support the existence of a cell membrane receptor, whole CHO-ER{alpha} cells were incubated with fluorescein isothiocyanate (FITC)-conjugated E2-BSA (Fig. 2CGo). E2-BSA has been shown to not enter the cell and has been found by several laboratories, including our own, to bind putative membrane ER, although with lower affinity than 17-ß-E2 (18, 19). The lower affinity may be due to the BSA protein physically hindering 17-ß-E2 binding to its receptor. Labeling/binding by E2-BSA-FITC of the outer cell membrane of most transfected cells was evident (panel A), consistent with the expression of membrane ER{alpha}. No labeling of nontransfected CHO-K1 cells was seen with this compound (data not shown). The binding was competed off by unlabeled 17-ß-E2 (panel B), the ER receptor antagonist, ICI 182,780 (C), or H222 (D), an antibody directed against the ligand-binding domain (LBD) of ER{alpha} (25). Inhibition by the H222 ER{alpha} antibody is especially important, since it is extremely unlikely that this antibody can enter the cell and bind cytoplasmic/nuclear ER, especially after a short incubation with the CHO-ER{alpha}. The specificity of this well characterized antibody also strongly supports the idea that E2-BSA is specifically binding a membrane ER, one whose LBD is similar to that of the nuclear ER. In contrast, ER-21, an antibody directed against the N-terminal first 21 amino acids of ER (26), did not compete off binding/labeling (panel E). Because antibody specific to the LBD of ERß was not available to us for these studies, we were not able to carry out comparable studies in CHO-ERß.

To demonstrate the nuclear pool of ER, we permeabilized the CHO-ER{alpha} cells by incubation with detergent; this resulted in a dense nuclear labeling (panel F). This finding is consistent with the binding studies, which showed ER in both membrane and nuclear compartments and that the number of nuclear receptors greatly exceeds those localized to the cell membrane. The results also support our previous ER{alpha} labeling studies of a membrane receptor expressed in primary cultures of nontransfected human vascular smooth muscle cells (19).

Membrane ERs Are G Protein-Coupled Receptors
The ability of 17-ß-E2 to rapidly stimulate cAMP generation or Ca++i mobilization has been reported by others (9, 10). This led us to hypothesize that the membrane ER could stimulate G protein-induced signal transduction, qualifying this receptor as G protein linked (directly or indirectly). To support this idea, we determined the ability of 17-ß-E2 to stimulate the production of IP3. In ER{alpha} or ERß expressing CHO cells, 10 nM 17-ß-E2 stimulated a respective 210% and 122% increase in IP3 generation above control (ER{alpha} or ERß expressed in the absence of 17-ß-E2 incubation) (Fig. 3AGo). The respective stimulations were prevented 56% and 53% by ICI 182,780, while results with ICI alone were similar to control. These data suggest that the membrane ER may couple to G{alpha}q activation, which enacts inositol phosphate hydrolysis through the activation of phospholipase C in many cells (27).



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Figure 3. Membrane ERs Activate G Proteins and Resulting Signal Transduction

A, Generation of IP3 by 17-ß-E2 in CHO-ER. CHO-ER{alpha} or ERß were incubated with or without 17-ß-E2, 10 nM, for 15 sec, in some conditions preceded by ICI 182,780 for 15 min. Lysate was processed and IP3 was measured by RIA kit. Data represent the mean ± SEM from two experiments combined, each condition in each experiment done in triplicate. *, P < 0.05 for 17-ß-E2 vs. control by ANOVA plus Schefe’s test. +, P < 0.05 for 17-ß-E2vs. E2 plus ICI 182,780, 1 µM. B, Stimulation of adenylate cyclase activity from membranes in these cells. Equal amounts of membrane protein from CHO-ER were incubated with 17-ß-E2, 10 nM, for 20 min, with or without preincubation with ICI 182,780, or tamoxifen, 1 µM. *, P < 0.05 for condition vs. control by ANOVA plus Schefe’s test. Data are combined from two separate experiments, each condition in triplicate per experiment, and represent the mean ± SEM (n = 6 observations). G protein activation by 17-ß-E2 in CHO-ER{alpha} or ERß. Binding of GTP{gamma}s to G{alpha}s (panel C) or G{alpha}q (panel D) in CHO cell membranes are shown. Bar graph data are the mean ± SEM from three experiments combined. In both ER-expressing CHO, increased Gs and Gq protein activation is seen (lanes 1 or 10 vs. 3 or 11 in both figures), inhibited by the ER antagonist ICI 182,780, 1 µM (lanes 5 and 13). Also, ER antibody to the ligand binding domain (H222) inhibited G protein activation in CHO-ER{alpha} (lane 6 in both figures). *, P < 0.05 for 17-ß-E2 (lanes 3 or 11) vs. control (lanes 1 or 2) by ANOVA plus Schefe’s test. +, P < 0.05 for 17-ß-E2vs. E2 plus ICI 182,780 (lanes 5 or 13) or an antibody to ER{alpha} (lane 6).

 
We also found that 17-ß-E2 stimulated greater than a 100% increase in adenylate cyclase activity from membranes prepared from either CHO-ER{alpha} or ERß; this was determined by measuring cAMP as a readout (Fig. 3BGo). Interestingly, neither ICI 182,780 nor tamoxifen significantly inhibited 17-ß-E2-activation of adenylate cyclase and actually significantly stimulated enzyme activity in the absence of E2. These latter findings are very similar to those reported by Aronica et al. (9), concerning cAMP generation in MCF-7 cells. Collectively, these results could indicate that the membrane ER is linked to G{alpha}s.

To directly show that the membrane ER activates G proteins, we again used membrane preparations from ER{alpha}- and ERß-expressing CHO cells. Increased binding of guanosine 5'-3-O-(thio) triphosphate (GTP{gamma}S) to G{alpha}s or G{alpha}q in response to 10 nM 17-ß-E2 in CHO-ER{alpha} was seen at 5 min; 17-ß-E2 increased the respective binding to G{alpha}s and G{alpha}q by 93% and 70% above control (Fig. 3Go, C and D, lanes 2 vs. 3). Preincubation with either ICI 182,780 (lane 5 in both figures) or the ER{alpha} H222 antibody (lane 6), reduced the augmented binding by 70–85%. By contrast, incubation with 17-{alpha}-E2 (lane 4) had no effect on GTP{gamma}S binding to the G proteins in the CHO-ER{alpha} membrane. Similarly, in CHO-ERß cells, 17-ß-E2 (but not 17-{alpha}-E2) stimulated GTP{gamma}s binding to G{alpha}s and G{alpha}q above basal levels by 83% and 61% (Fig. 3Go, C and D, lanes 10 vs. 11), only slightly less than in cells expressing ER{alpha}. ICI 182,780 inhibited G protein activation by 50–60% in each of the ERß studies (lane 13). These data indicate that both membrane ERs have the capacity to stimulate signal transduction through the activation of Gs and Gq proteins, accounting for the generation of cAMP (Gs) and IP3 (Gq).

Stimulation of Extracellular Regulated Kinase (ERK) Activity through the Membrane Receptor Is Necessary for the Stimulation of Cell Proliferation
Expression of either ER{alpha} or ERß in the CHO cells resulted in the significant stimulation of ERK activity by 17-ß-E2 (Fig. 4AGo). Stimulation of ERK activity occurred after 10 min incubation with 17-ß-E2 in the ER- transfected CHO (lanes 3 and 9), compared with the control vector-transfected (lane 1) or ER-transfected CHO incubated in the absence of 17-ß-E2 (lane 2). Nontransfected CHO also did not respond to 17-ß-E2 (data not shown).



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Figure 4. ERK Activation by Estradiol Leads to CHO-ER{alpha} and CHO-ERß Proliferation

A, ERK activation in CHO-ER{alpha} and CHO-ERß. Transfected cells were exposed for 10 min to 17-ß-E2, 10 nM, or E2-BSA, 100 nM, without or with ER{alpha} antibody (in CHO-ER{alpha}), or after ICI 182,780 (1 µM) preincubation in CHO-ERß. The representative study shown here was repeated twice; combined data are reflected in the bar graph. Protein immunoblots of ERK are shown below the kinase activities. Myelin basic protein (1 µg) is myelin basic protein, used as substrate for ERK activity. *, P < 0.05 for 17-ß-E2 (lanes 3 or 9) or E2-BSA (lanes 4 or 10) vs. control (lanes 1 or 2 and 7 or 8) by ANOVA plus Schefe’s test. +, P < 0.05 for 17-ß-E2 or E2-BSA vs. estrogen plus ER{alpha} antibody (lanes 5 and 6), or vs. estrogen plus ICI 182,780 (lanes 11 or 12). B, Nuclear thymidine incorporation in CHO-ER{alpha} (left) or CHO-ERß (right) is dependent on ERK activation by 17-ß-E2. Activation of nuclear [3H]thymidine incorporation by 17-ß-E2 or E2-BSA is inhibited by ICI 182,780, ER{alpha} antibody, and the specific MEK inhibitor, PD98059 (20 µM). *, P < 0.05 for 17-ß-E2 or E2-BSA vs. control. +, P < 0.05 for 17-ß-E2 or E2-BSA vs. estrogen plus ER antagonists, ER{alpha} antibody, or the MEK inhibitor, PD98059. Data are the mean ± SEM and are combined from three experiments, triplicate determinations for each condition.

 
To provide support for a membrane ER mediating this effect, cells were also incubated with E2-BSA. E2-BSA at a higher concentration than 17-ß-E2 alone significantly activated ERK activity in either ER{alpha}- or ERß-expressing CHO cells (Fig. 4AGo). ERK activation by either 17-ß-E2 or E2-BSA was approximately 3- to 4-fold above basal activity (lane 2 vs. lanes 3 and 4, or lane 8 vs. lanes 9 and 10) and was not appreciably different for ER{alpha} or ERß. In ER{alpha}-expressing CHO cells, approximately 70% of 17-ß-E2 or E2-BSA-enhanced ERK activity was inhibited by coincubation of the cells with ER{alpha} antibody (lanes 5 and 6). Since the antibody is highly unlikely to enter the cell, these results support the idea that 17-ß-E2 (and E2-BSA) activate ERK through the membrane receptor. In the ERß expressing CHO, we found that ICI 182,780 inhibited 17-ß-E2 or E2-BSA activation of ERK by 60 and 70%, respectively (lanes 9 and 10 vs. lanes 11 and 12). BSA itself was found not to affect ERK activity in our previous studies (19). It is possible that the E2-BSA compound could have dissociated, allowing E2 to enter the cell, or that E2-BSA was internalized. In either situation, activation of the nuclear ER could contribute to the results shown here. To validate the use of this compound to activate only the membrane ER, CHO-ER{alpha} cells were cotransfected with an ERE-luciferase reporter, and then incubated with either 17-ß-E2, 10 nM, or E2-BSA, 100 nM, for 10 min or 24 h. Luciferase activity at 10 min was not affected by either of the treatments. At 24 h, 17-ß-E2 stimulated a 2.5-fold increase above baseline in luciferase activity, but E2-BSA had no effect on reporter function (data not shown). These results corroborate similar findings from Watters et al. (20), and indicate that E2-BSA probably does not bind the nuclear ER.

It is widely accepted that growth factors stimulate cell proliferation in part through the activation and subsequent translocation of ERK to the nucleus (28). In target cells, ER has been shown to activate ERK (29), presumably contributing to induced cell division in which 17-ß-E2 is mitogenic. As an index of cell proliferation, we determined that 17-ß-E2 or E2-BSA could induce a modest but consistent 42% and 27% increase in nuclear thymidine incorporation in CHO-ER{alpha}, respectively (Fig. 4BGo, left). Comparable stimulation was also seen in CHO-ERß (Fig. 4BGo, right). It must be appreciated that CHO cells do not normally respond to 17-ß-E2, and hence the endogenous systems (e.g. signal protein scaffold mechanisms) that mediate growth effects of the sex steroid in target cells, such as breast or uterus, are probably not comparable in CHO. Nevertheless, CHO-ER respond to 17-ß-E2 in a qualitatively comparable way to MCF-7 cells (29), and the effects shown here are mediated through ER since ICI 182,780 significantly inhibits this stimulation. The ability of 17-ß-E2 or E2-BSA to enhance DNA synthesis was reversed 77% and 85%, respectively, in CHO-ER{alpha}, and 64% and 75% in CHO-ERß by PD98059, a soluble MAP kinase kinase (MEK) inhibitor (30). This indicates that the ability of 17-ß-E2 to stimulate ERK activity is required for the stimulation of thymidine incorporation, and that E2-induced MEK activation of ERK is involved. The substantial reversal of E2-BSA-stimulated thymidine incorporation by PD98059 is of additional interest. This finding particularly supports the idea that stimulation of ERK activity through the cell membrane ER is crucial to the proliferative action of the sex steroid. Further supporting this role of the membrane ER, the ER{alpha} antibody prevented 17-ß-E2 or E2-BSA stimulation of thymidine incorporation by 74% and 86%, respectively, in CHO-ER{alpha} cells (Fig. 4BGo, left). Progesterone or 17-{alpha}-E2 had no effect on thymidine incorporation in either ER-expressing CHO cell.

ERß, but Not ER{alpha}, Stimulates c-Jun Kinase (JNK) Activity
To further investigate the effects of the two ERs in signal transduction, the modulation of the activity of the important mitogen-activated protein (MAP) kinase, JNK, was determined. This kinase was activated by 17-ß-E2 but, surprisingly, only in ERß-expressing cells (Fig. 5AGo). In dose-related fashion, 17-ß-E2 stimulated a maximum 2-fold increase above basal activity (lane 9), inhibited 85% by ICI 182,780. No effect on JNK activity was observed in response to 17-{alpha}-E2. By contrast, in ER{alpha}-expressing CHO cells, JNK activity was inhibited by 17-ß-E2. Maximal inhibition was 55% of basal JNK activity at 10 nM 17-ß-E2 (lane 3), again prevented by ICI 182,780. Activation or inhibition was most prominent at 15 min; based upon preliminary time course studies, these same effects were seen by 10 min exposure to 17-ß-E2 (data not shown).



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Figure 5. N-Terminal JNK Activity in CHO-ER{alpha} and ERß

A, Transfected cells were incubated for 15 min with 17-ß-E2, 0.1–10 nM, with or without ICI 182,780, 1 µM. Controls (lane 1) are empty vector-transfected CHO, and (lane 2) activity from ERß vector-transfected cells, incubated in the absence of 17-ß-E2. After incubation, cells were lysed and processed for JNK activity directed against the glutathione-S-transferase-c-jun substrate protein, as described in Materials and Methods. The study shown is representative of three experiments, combined for the bar graph. *, P < 0.05 for 17-ß-E2 (lanes 3–4 or 9–10) vs. control (lanes 1 and 2, or 8) by ANOVA plus Schefe’s test. Lane 1 is nontransfected CHO incubated with 17-ß-E2. +, P < 0.05 for 17-ß-E2vs. E2 plus ICI 182,780 (lanes 6 or 12). Protein immunoblots are shown below the kinase activities. B, Comparison of E2, 10 nM, and E2-BSA, 100 nM, stimulation of JNK activity in CHO-ERß cells, with and without preincubation of ICI 182,780. *, P < 0.05 for 17-ß-E2 or E2-BSA vs. control; +, P < 0.05 for 17-ß-E2 or E2-BSA vs. either plus ICI 182,780

 
We also compared the effects of 17-ß-E2 and E2-BSA in the CHO-ERß (Fig. 5BGo). Both compounds caused a significant and comparable increase in JNK activity above control (basal) levels, and the stimulations were approximately 65% inhibited by ICI 182,780. These data support the ERß-expressing cell membrane as mediating 17-ß-E2-activation of JNK.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A series of emerging data supports the existence and importance of membrane ER in the cell biological actions of the sex steroid (9, 10, 11, 12, 13, 19, 20, 21). However, since this putative receptor has not been isolated, its derivation and structural and functional characteristics are unknown. To begin to understand these issues, we transfected CHO cells (which normally do not produce ER) with expression vectors for either ER{alpha} or ERß. We found that expression of cDNAs for either receptor results in the production of a single transcript that gives rise to both membrane and nuclear receptors. Membrane and nuclear receptors are similarly sized and have nearly identical affinities for 17-ß-E2. We further report for the first time that ERß can exist in the cell membrane. Both receptors enact a variety of signal transduction events, some of which contribute to cell proliferation. Our results indicate that the membrane ER is G protein linked, and that this receptor should be considered to possibly contribute to the cell biological functions of 17-ß-E2 on normal target cells (9, 10, 11, 12, 13, 19, 21).

What is the structural nature of the membrane ER? Although we did not experimentally examine this complicated issue, we speculate that the protein must be very similar to the classical nuclear ER. Supporting this position, both membrane and nuclear proteins are derived from the same transcript. In target cells, alternatively spliced transcripts for ER{alpha} have been identified that give rise to ER proteins of different lengths. However, here we demonstrate that a single cDNA and RNA are capable of producing both membrane and nuclear receptors, although the great majority of receptors are nuclear. Posttranslational modification of some ER protein must occur to ensure targeting to the membrane. Targeting modifications would likely include the addition of lipid anchors such as glycosylphosphatidylinositol (31) and/or other lipid modifications such as palmitoylation or myristolation; these alterations would promote movement to the cell membrane. However, in analyzing the structure of ER{alpha}, there are no obvious palmitoylation sites and only a few candidate myristolation sites. It is also not obvious from a structural analysis how the ER would insert into the cell membrane. Insertion would require a core hydrophobic region of the receptor, and one potential region has been previously noted (18). There is precedent for a subpopulation of ER to be posttranslationally modified, glycosylated by N-acetylglucosamine (32). Cytoskeletal and oncoproteins are substrates for this enzyme, and several of these proteins protrude from the plasma membrane, suggesting that this modification might play a role in the translocation of ER to the membrane. However, our cross-linking studies show that the size of both membrane and nuclear receptors is approximately the same. This indicates that there can not be extensive modification of the membrane ER protein, such as by the addition of bulky glycosyl groups which substantially increase the receptor size and alter gel migration. A subtle glycosylation of the membrane ER is more plausible, and this analysis is ongoing.

Additional proof of similarity between membrane and nuclear ER was provided by our studies. An antibody raised against the LBD of the nuclear ER{alpha} competes for binding of labeled 17-ß-E2 to the putative ER{alpha} cell membrane (shown here and in Ref. 19). Pappas et al. (18) previously showed that antibodies raised against the classical ER identify a membrane ER. Thus, by ligand affinity, receptor protein size, and immunological/shared epitope criteria, the membrane and nuclear receptors are similar. These findings presuppose that our membrane preparations are not contaminated with cytosolic ER (our isolation procedure precludes nuclear ER). Although we previously established the lack of cytosol in our typical membrane preparations (33), we cannot exclude very small amounts of cytosolic membrane (e.g. endoplasmic reticulum) in our samples. However, here we show that G proteins and adenylate cyclase are activated by 17-ß-E2 in membrane preparations and, according to current knowledge, this cannot result from cytosolic membrane binding.

We report that both expressed ERs are capable of stimulating a modest but consistent increase in nuclear thymidine incorporation. Since the cells are not normally responsive to 17-ß-E2, it is perhaps remarkable to see this effect. However, it is known that many signal transduction molecules are present in CHO, and hence these cells can serve as a model to understand some of the effects of ER. We found that ER{alpha} and ERß comparably activate ERK activity, and that this is necessary for their proliferative effects, based upon reversal by a soluble MEK inhibitor, PD98059; MEK is the consensus upstream activator of ERK (30). The stimulation of ERK activity by 17-ß-E2 has been previously shown in MCF-7 cells (29), mediated through undetermined subtype or membrane/nuclear receptors. In these same cells, 17-ß-E2 was shown to trigger a signal cascade including the activation of the Src and Ras tyrosine kinases, and the phosphorylation of the adapter protein, Shc. Similar signal transduction in response to progesterone has been reported to be mediated through ER (34). The activation of ERK by 17-ß-E2 has been attributed previously to signaling through a membrane ER in a neuroblastoma cell line (20), and we showed that when 17-ß-E2 is an antigrowth factor, this effect is mediated through membrane ER-induced inhibition of ERK activation by growth factors (19). Furthermore, inhibition of transcription by ER can be, in part, mediated through interrupting stimulatory signaling through ERK in endothelial cells (21).

Thus, although ER stimulates signal transduction in some cells, the receptor inhibits these same molecules in other cells. This likely requires the assembly of different intermediary signal molecules in different cell types, activated by the membrane receptor. Understanding the mechanisms involved should provide insight into the differential effects of 17-ß-E2, including transcriptional modulation. Negative transcriptional regulation may be an important function of the membrane ER (21).

As a positive effector of growth-related genes, the membrane ER might activate growth factor receptor tyrosine kinases (TK). Most notably, ER might activate the EGFR, which then enacts signal transduction to stimulate ERK activity (35). ERK nuclear translocation and activation of targets such as c-fos (36) or egr-1 (37) would then activate cell proliferation. Here, we found that added EGF could not activate ERK in the transfected CHO-ER{alpha} cells, consistent with the absence of EGFR in these cells. Further, when the CHO-ER{alpha} cells were incubated with 17-ß-E2 in the presence (or absence) of the specific EGFR-TK antagonist, tyrphostin AG127 (38), there was no difference in ERK activation (data not shown). We therefore propose that G{alpha}q activation by 17-ß-E2 in transfected CHO cells leads to ERK activation, as has been demonstrated for a variety of G protein-coupled transmembrane receptors (39).

By investigating additional signal transduction effects, we found that ERß is capable of activating JNK, while ER{alpha} inhibits basal JNK activity. To our knowledge, this is the first report that ER modulates this proline-directed serine/threonine kinase in any model. Further, this is one of the few differential effects of the two ERs reported to date. It was recently found that ER{alpha} activates AP-1-dependent transcription, while ERß inhibits this action of AP-1 (40). It is unlikely that activation of JNK by ERß (which we show here) contributes to the previously described inhibition of AP-1-induced transcription (40): AP-1 is composed of fos/jun family heterodimers, and c-jun is a natural substrate for JNK activity, which usually enhances AP-1 activity. On the other hand, stimulation of this MAP kinase by ERß could contribute to cell proliferation, because it is now believed that activation of JNK by growth factors contributes to this function (41). These findings may be especially relevant to natural target cells expressing predominantly ERß.

Based upon signal transduction events triggered by ER in a variety of cell types (9, 10, 11, 12, 13), it can be speculated that 17-ß-E2 activates Gs and Gq by binding to a cell membrane receptor. Here, we provide the first direct evidence that this occurs. In membrane preparations, we showed that each expressed ER stimulates the activation of both G{alpha}s and G{alpha}q, as well as adenylate cyclase in the membrane and increased production of IP3 determined from whole cells. The stimulation of most of these events was significantly inhibited by the specific ER antagonist ICI 182,780, and in CHO-ER{alpha} cells, H222 antibody directed against the LBD prevented G protein activation. Interestingly, we showed that 17-ß-E2 activates G{alpha}s, and that this is reversed by ICI 182,780; however, we also found that this ER antagonist independently stimulates adenylate cyclase activity in ER-expressing CHO cells. These seemingly dichotomous findings indicate that ICI 182,780 is capable of inhibiting ER-mediated G protein activation but can also stimulate adenylate cyclase, perhaps by reducing the Michaelis-Menton constant (Km) of the enzyme. Alternatively, ICI might prevent cAMP degradation. However, we think that this is unlikely since Aronica et al. (9) also showed that ER antagonists stimulate cAMP production but have no effect on phosphodiesterase activity.

These data strongly support the idea that membrane ER couple in some way to G proteins and transduce intracellular signals. The structure of the membrane ER that potentially allows contact with G proteins awaits isolation of the protein, but it is very unlikely that the protein is structurally similar to a typical, heptahelical G protein-coupled receptor. We (19, 21) and others (9, 10, 13, 20, 29) have previously implicated intracellular signaling by ER as contributing significantly to the cellular effects of the sex steroid. The present demonstration of ER-induced G protein activation supports and lends mechanistic details to those previous studies.

One caveat to our results is that we have used an artificial system. However, cells expressing only ER{alpha} or ERß are not readily available for culture, nor are there available discrete receptor antagonists or ligands for each individual receptor. Therefore, it is difficult to separate out the independent effects of the two receptor isoforms in native cells at this time. Studies of cells from ER knockout animals will be of use in further defining the relative contributions of the two receptors. However, our model lends itself to identifying and implicating the membrane form of each ER to signal and thereby potentially contribute to the cell physiology modulated by ER.

In summary, we present the first direct evidence that membrane and nuclear ER arise from a single transcript, implying that novel posttranslational processing occurs on a minority of these proteins. How this occurs, and how the ER translocates and inserts into the cell membrane requires further study. We also show that a membrane ERß can exist, that it can signal, and that there are both common and unique signal transduction pathways enacted by membrane ER{alpha} and ERß. The intracellular actions of ER and other members of the steroid receptor superfamily arise from both genomic and nongenomic effects. Direct interaction between progesterone and G protein-coupled cell membrane receptors has been shown recently (42). Signal transduction through cell membrane receptors may regulate discrete actions of steroid ligands and may play a significant role in the cross-talk between tyrosine kinase receptors, such as EGF, and ERs (35). Just as several signal transduction systems target and amplify nuclear events (such as c-fos expression), there is likely to be cooperation between both signal transduction and direct transcriptional effects of the membrane and nuclear ERs, respectively (21). The challenge is to identify and define the unique and interactive contributions of each mechanism.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
CHO-K1 cells (ATCC, Manassas, VA) were cultured and maintained at 37 C in a 5% CO2 humidified atmosphere in DMEM-F12 medium with 10% FBS. Cells were passaged every 5 days. For transfection, cells were subcultured in cell culture dishes 24 h before transfection. Serum was removed at least 12 h before experimentation.

Transient Transfections
CHO-K1 cells were grown to 40–50% confluence and then transiently transfected with 0.5–10 µg of fusion plasmids, depending on the plate size and the amount of cells; the plasmids included pcDNA3mER{alpha} encoding nucleotides 17–2001 (43) and pSG5mERß encoding nucleotides 12–1469 (23) (kindly provided by Dr. K. Korach and Dr. J. A. Gustafsson via Dr. Korach, respectively) or respective backbone vectors. Transfections were done with Lipofectamine Reagent (GIBCO-BRL, Grand Island, NY); cells were incubated with liposome-DNA complexes at 37 C for 5 h, followed by overnight recovery in DMEM-F12 medium containing 10% FBS. Then, before experimental treatment, cells were synchronized in serum-free DMEM-F12 for 24 h and then treated with 17-ß-E2 and/or related compounds. Cotransfections with a green fluorescent protein expression vector (Promega, Madison, WI) indicated 80–90% efficiency of transfection, corroborated by the labeling of cells with FITC-E2-BSA. In other studies, CHO cells were cotransfected with pcDNA3mER{alpha} and ERE-SV40 Luciferase (kindly provided by Dr. B. Gehm) and then incubated with 10 nM 17-ß-E2 or 100 nM E2-BSA (Sigma Chemical Co., St Louis, MO) for 10 min and 24 h, and luciferase activity was determined as previously described (44). The concentration of E2-BSA was calculated from the number of E2 molecules attached to each BSA molecule.

Northern Analysis
Total RNA was extracted from the CHO cells transfected with ER{alpha} or ERß plasmids, using Tri-Reagent-LS (Molecular Research Center, Inc., Cincinnati, OH). RNA (25 µg) from each CHO-ER type was separated by electrophoresis on a denaturing 1% agarose gel, transferred to nitrocellulose, and prehybridized, as we previously described (19, 45). The blots were hybridized for 12 h at 65 C with 32P-labeled, antisense cRNAs for ER{alpha} or ERß. The probes were transcribed from a rat cDNA template (ER{alpha}), nucleotides 81–398 (46) (kindly provided by Dr. E. Spreafico) and a mouse cDNA template (ERß) (pCRSK+, nucleotides 49–310) (47) (kindly provided by Dr. K. Korach), using T7 and T3 RNA polymerases, respectively. Hybridization bands were quantified by laser densitometry, after autoradiography. Sense probes produced no hybridization.

Cross-Linking
For cross-linking studies, [125I]17ß-E2 (2200 Ci/mmol) was bound to CHO-ER{alpha} or CHO-ERß. After binding, nuclear pellets and cell membrane pellets were washed twice with PBS and then cross-linked by incubating with 4% formaldehyde (Mannich Reaction) (48) at 57 C for 24 h. After washing, the samples were solubilized in sample buffer (80 mM Tris-HCl, pH 6.8, 10% glycerol, 1% SDS, 0.025% bromphenol blue). Proteins were denatured at 95 C for 5 min and then loaded onto 8% polyacrylamide gel and electrophoresed. Gels were dried and then subjected to autoradiography.

Binding Studies
Cells were grown on 100-mm petri dishes in DMEM-F12 without phenol red. Twenty four hours after transfection with ER{alpha} or ERß constructs, the cultures were washed three times with PBS; cells were then lysed in buffer A (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 100 nM NaCl, 50 mM NaF, 100 µM phenylmethylsulfonyl fluoride, protease inhibitor cocktail, and 0.2% Triton X-100). Nuclear pellets were collected through low-speed centrifugation. The supernatants were centrifuged at 100,000 x g for 30 min to pellet cell membranes. Both pellets were washed twice, once with buffer A and once without detergent. Fifty microliters of membrane proteins from CHO-ER{alpha} or CHO-ERß cells were incubated in buffer A without Triton X-100 and with 0.5% BSA, increasing concentrations of unlabeled 17-ß-E2 (10-11 M to 10-7 M), and [3H]-17-ß-E2 (specific activity, 80 Ci/mmol, pH 7.5) (New England Nuclear, Boston, MA) at 37 C for 45 min, similar to binding studies previously described by us (49, 50). The nuclear and cell membrane pellets were washed three times by centrifugation to remove unbound isotope and then quantified by ß-scintillation counting. Binding studies were repeated three times, and data were combined for Scatchard analysis.

Membrane ER Labeling Studies
Nontransfected CHO and CHO-ER{alpha} cells were grown on coverslips. The cells were incubated at 4 C for 30 min with FITC-conjugated E2-BSA (Sigma). The E2-BSA-FITC compound has previously been shown to label a membrane ER in several cell types (18, 19). For competition studies, cells were preincubated for 5 min with 17-ß-E2, 10 nM, or ICI 182,780 1 µM, or antibodies to the LBD of the ER (H-222) (18) or N terminus of the receptor protein (ER-21); this was followed by the addition of E2-BSA-FITC. After labeling, the cells were fixed for 3 min in freshly prepared 4% paraformaldehyde and mounted for microscopic evaluations. Some cells were also permeabilized with 0.2% Triton X-100 to allow the labeling of the nuclear pool of ER. Antibody was diluted 1:100 from an original concentration of 0.56 mg/ml and was used for labeling and functional studies as noted.

Kinase Assays
ERK and JUN kinase assays were carried out as previously described (51, 52). CHO-ER{alpha} or -ERß was incubated with various steroids alone, or coincubated with ER antagonist for 10 or 15 min, based upon our preliminary time course studies. Cell lysate (200 µl) was then added to Erk2 or Jnk-1 antiserum (Santa Cruz Biotechnology, Santa Cruz, CA) (10 µl) conjugated to prewashed protein A-Sepharose; the mixture was then incubated for 2 h at 4 C in microfuge tubes. After washing, each bead-antibody-antigen complex was then incubated for 30 min at 30 C with 40 µl kinase buffer (25 mM HEPES, 10 mM MgAc, 40 µM ATP, 2 mM dithiothreitol), 10 µCi 32P-ATP, and myelin basic protein or glutathione-S-transferase-c-jun (Santa Cruz), as substrate for kinase activity. Reactions were terminated by adding SDS reducing buffer, samples were boiled, and the proteins were separated by SDS-PAGE. After autoradiography, the bands were compared by laser densitometry. Western blot documented similar amounts of kinase protein loaded under each condition.

Nuclear Thymidine Incorporation
Subconfluent CHO-ER were synchronized for 24 h in serum-free media. All cells were then incubated for 20 h in the absence or presence of 17-ß-E2 or E2-BSA, with or without ICI 182,780 or the H222 antibody. In some conditions, the MEK inhibitor, PD 98059, 20 µM (kindly provided by Dr. A. Saltiel) was added to the incubation mixture 30 min before the steroid. After 20 h, 0.5 µCi of [3H]thymidine was added for 4 more hours, as previously described (19). Cells were then washed in cold HBSS, incubated for 10 min with 10% trichloroacetic acid at 4 C to precipitate the nuclear incorporated thymidine, washed, and lysed with 0.2 N NaOH overnight, after which the lysates were counted in a liquid scintillation ß-counter. Experiments were repeated three to four times, and data were combined for analysis by ANOVA plus post hoc test (Scheffe’s).

G Protein Activation Assays
Cell membranes from CHO-ER were prepared and analyzed for lack of cytosol as we previously reported (32). Membrane aliquots (20 µg) were resuspended in 50 mM Tris-HCl buffer, with 1 µMGTP, 100 µM Mg++ and incubated with 30 nM [S35] GTP{gamma}S (Sigma), in the presence of 10 nM 17-ß-E2 for 5 min at 30 C. The incubation was terminated by adding 600 µl ice-cold 50 mM Tris-HCl, 20 mM MgCl2, 0.5% Nonidet P-40, and 100 µM GTP. After 30 min, the extract was placed into microfuge tubes containing 2 µl of nonimmune serum preincubated with 150 µl of a 10% suspension of pansorbin cells (Calbiochem, San Diego, CA.). Nonspecifically bound proteins were removed by centrifugation after 20 min. The supernatant was then incubated with Gs or Gq {alpha}-subunit antibody (Calbiochem), preincubated with 5% protein A Sepharose. Immunoprecipitants were washed in extraction buffer without detergent and boiled with SDS, and equal protein aliquots from each condition were separated by gel electrophoresis. Other aliquots were quantified by scintillation counting; each condition was prepared in duplicate, and the data from three to four separate experiments were combined.

IP3 and Adenylate Cyclase Activity
The generation of IP3 was assayed as follows. ER-expressing CHO cells were cultured in 100-mm petri dishes followed by synchronization in the absence of serum for 24 h. The cells were washed in PBS (pH 7.4) and incubated in DMEM-F12 (with 20 mM HEPES, 0.1% BSA, and 10 mM LiCl) at 37 C for 10 min. 17-ß-E2, 10 nM, was added to the cells for 15 sec, with or without ICI 182,780 or H222 (for CHO-ER{alpha}), which were added 10 min before 17-ß-E2. The reaction was stopped by adding cold 5% perchloric acid and kept on ice for 20 min. The media were centrifuged at 15,000 x g for 1 min, and the supernatant was neutralized with 60 mM HEPES/1.5 M KOH for 60 min on ice. Insoluble KClO4 was removed by centrifugation at 12,000 x g for 15 min at 4 C. The IP3 content in the supernatant was measured by kit (New England Nuclear, Boston, MA).

Adenylate cyclase activity in the membrane was determined as follows. CHO-ER{alpha} or ERß membranes were prepared (32), and cAMP generation as a function of cyclase activity was measured. For the cAMP assay, equal amounts of cell membrane suspension (containing the adenylate cyclase) and 17-ß-E2, 10 nM, with or without ER antagonists, were added to assay buffer to a final total volume of 200 µl. The buffer contained 100 mM KCl, 20 mM MgCl, 4 mM isobutylmethylxanthine, 0.8 mM EDTA, 1 mM GTP, 1 mM ATP, 20 mM phosphocreatine, and 1 mg/ml creatine phosphokinase in 0.2 M Tris-HCl, pH 7.5. After vortexing, the mixture was incubated in a 37 C shaking water bath for 20 min, and then the enzyme activity was stopped by boiling for 3 min at 95 C. The mixtures were centrifuged at 6500 rpm for 10 min, and from each sample, 100 µl of the supernatant were diluted with acetate buffer, pH 6.2, for the RIA. The inter- and intraassay coefficients of variation of the RIA were always less than 10%.


    FOOTNOTES
 
Address requests for reprints to: Ellis R. Levin M.D., Medical Service (111-I), Long Beach Veterans Affairs Medical Center, 5901 East 7th Street, Long Beach, California 90822. E-mail: elevin{at}pop.long-beach.va.gov

This work was supported by a Merit Review Grant from the Veterans Administration, NIH Grant NS-30521 (E.R.L.), National Cancer Institute Grant CA-02897, and United States Army Research Medical Corp. Grant DAMD-17–04-J4228 (G.L.G.).

Received for publication September 11, 1998. Revision received October 30, 1998. Accepted for publication November 5, 1998.


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