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Molecular Endocrinology, doi:10.1210/me.2003-0262
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Molecular Endocrinology 18 (1): 86-96
Copyright © 2004 by The Endocrine Society

Membrane Restraint of Estrogen Receptor {alpha} Enhances Estrogen-Dependent Nuclear Localization and Genomic Function

Yun Xu, Richard J. Traystman, Patricia D. Hurn and Michael M. Wang

Departments of Anesthesiology/Critical Care Medicine (Y.X., R.J.T., P.D.H., M.M.W.) and Neurology, Johns Hopkins University, Ross 364, Baltimore, Maryland 21287; and Department of Neurology (Y.X.), The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, People’s Republic of China

Address all correspondence and requests for reprints to: Michael M. Wang, University of Michigan 7629 Medical Science II, Box 0622, Ann Arbor, Michigan 48109-0622. E-mail: micwang{at}umich.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen receptor (ER) {alpha} localizes to both the nucleus and the plasma membrane, mediating estrogen-dependent genomic and nongenomic signaling, respectively. In some cells, ER{alpha} appears to be excluded from the nucleus, and it is unclear whether genomic signaling takes place. The purpose of this study was to determine whether membrane-associated ER{alpha} is capable of genomic signaling, or whether this pool of receptors strictly serves membrane-mediated signaling. ER{alpha} fused to the C-terminal cytoplasmic tail of bovine rhodopsin (Rh-ER{alpha}) activates ER response element-dependent transcription only in the presence of estrogen; the activity is antagonized by the estrogen antagonist ICI 182,780 and by the dominant-negative mutant of ER{alpha} and is unaffected by inhibitors of MAPKs and Akt signaling, indicating that this was due to direct genomic action. The activity of Rh-ER{alpha} containing the activating Y537S mutation was also estrogen dependent, suggesting that estrogen gated the entry of Rh-ER{alpha} into the nucleus. Indeed, cell fractionation studies demonstrated that Rh-ER{alpha} protein, in contrast to ER{alpha} that was nuclear at baseline, was excluded from the nucleus in the absence of hormone, and localized to the inner nuclear membrane on incubation with estrogen. These data demonstrate that membrane tethered ER{alpha} is capable of nuclear function and that its transcriptional activity is regulated by hormone-dependent entry into the inner nuclear membrane. Furthermore, these experiments provide evidence that under certain circumstances, membrane proteins are capable of nuclear function without detectable nucleoplasmic localization.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IN THE CLASSICAL model of gene regulation, nuclear estrogen receptor (ER) {alpha} drives transcription by binding estrogen that enables DNA binding and nuclear cofactor assembly. However, mounting evidence suggests that ER{alpha} also localizes to the plasma membrane (1, 2, 3, 4), where it functionally regulates kinase cascades in response to estrogen (1, 2, 5, 6, 7, 8). These nonnuclear effects are linked to diverse estrogen-dependent processes such neuroprotection (9), vascular protection (10), and neoplastic proliferation (11). In cultured neurons, ER{alpha} is localized to both the nucleus and the neurites, where it functions to signal to MAPK, an integral component of the neuroprotective effects of estrogen (9). Remarkably, in transfected neurons, a significant fraction of neurons contain large amounts of nonnuclear and very little nuclear ER{alpha} (12). In vivo, CA1 hippocampal neurons rapidly modulate dendritic spine density in response to estrogen (13, 14); although mRNA for ER{alpha} is easily detected, conventional immunocytochemistry failed to reveal significant nuclear ER{alpha} in CA1; however, immunoelectron microscopy revealed abundant ER{alpha} in the axons and dendrites of neurons (15, 16). It is not known whether potential modifications that cause extranuclear ER{alpha} to associate with plasma membranes perturb the ability of ER{alpha} to directly modulate gene transcription. Therefore, the major objective of this report was to understand whether nonnuclear, membrane-associated ER{alpha} could function to activate transcription.

To address this issue, we targeted ER{alpha} to the membrane by genetic fusion to rhodopsin. Rhodopsin-tethered ER{alpha} (Rh-ER{alpha}) has lower transcriptional activity than wild-type ER{alpha} in the absence of ligand; however, unexpectedly, in the presence of estrogen, Rh-ER{alpha} is highly active. The membrane protein localizes to the plasma membrane and intracellular membranes at baseline and shifts to the inner nuclear membrane on ligand stimulation. These results demonstrate that membrane forms of ER{alpha} are able to function in the nucleus, and supports the more general concept that membrane proteins could function directly in the nucleus by translocation within nuclear membranes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To determine whether membrane targeted ER{alpha} is still capable of activating transcription directly, we developed a membrane-anchored Rh-ER{alpha}, expressed in cultured cells. Prior studies demonstrated that fusions to the C terminus of the seven-transmembrane integral membrane protein bovine rhodopsin result in topologically homogeneous protein facing the cytoplasm (17). To generate membrane-anchored ER{alpha}, we created an expression plasmid (Rh-ER{alpha}) directing the fusion of human ER{alpha} to the C terminus of bovine rhodopsin (Fig. 1AGo). This plasmid is expected to direct the expression of ER{alpha} anchored to the cytoplasmic face of the plasma membrane. Immunocytochemical analysis revealed that in transfected 293 cells (Fig. 1BGo, upper panels), Rh-ER{alpha} is localized in a punctate distribution within the plasma membrane and in a perinuclear distribution that is consistent with the endoplasmic reticulum. Scant, reticular nucleoplasm staining was seen, but the pattern of expression was identical after addition of estrogen, indicating that the protein did not enter the nucleoplasm with hormone treatment. In contrast, wild-type ER{alpha} localized heavily to the nucleus under both conditions (Fig. 1BGo, lower panels). Immunoblot analysis revealed that although Rh-ER{alpha} was consistently expressed at lower levels than ER{alpha} in parallel experiments, Rh-ER{alpha} protein was of the expected molecular mass, and there was no evidence of proteolysis of the protein to smaller forms (Fig. 1CGo). We concluded that Rh-ER{alpha} was expressed in cell membranes and was not expressed in detectable levels within the nucleoplasm, even in the presence of estrogen. Rh-ER{alpha} also functionally mediated activation of MAPK, as cells transfected with Rh-ER{alpha} contained significantly higher levels of phosphorylated p44/42 MAPK when stimulated with estrogen (Fig. 1DGo). Similar findings were made in ER{alpha}-transfected cells, whereas vector-transfected cells did not stimulate MAPK phosphorylation in response to estrogen.



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  		Fig. 1. Rh-ER{alpha} Expression in 293 Cells

A, Rh-ER{alpha} construct was generated by fusing the open reading frames encoding bovine rhodopsin and human ER{alpha} into a cytomegalovirus (CMV) expression vector. B, The 293 cells transiently transfected with 100 ng of either Rh-ER{alpha} or human ER{alpha} in the presence or absence of estrogen were fixed and immunostained with antibodies against ER{alpha}. Rh-ER{alpha} appeared largely outside the nucleus consistent with endoplasmic reticulum localization. ER{alpha} was mostly nuclear with faint diffuse cytoplasmic staining. Estrogen did not affect the distribution of the proteins. C, Immunoblot analysis of total cell lysates of transiently transfected 293 cells in the presence of absence of estrogen demonstrated strong expression of ER{alpha} and Rh-ER{alpha} at the expected molecular masses. Rhodopsin-transfected cells showed no endogenous expression of ER{alpha}. Estrogen did not affect the protein yields. Loading was normalized by probing the blot for actin, shown below. D, Immunoblot analysis of p44/42 activation in cells transfected with membrane-restrained ER{alpha}. ER{alpha}- and Rh-ER{alpha}-expressing cells stimulate significant amounts of activated MAPK in response to estrogen. Control cells fail to demonstrate estrogen sensitivity.

 
To determine whether this membrane-anchored ER{alpha} was capable of nuclear function, we cotransfected Rh-ER{alpha} with an ER response element (ERE) reporter plasmid driving luciferase. Transcriptional potency of Rh-ER{alpha} would be expected to drive luciferase transcription that was quantitated by luminometry. Rh-ER{alpha} did indeed stimulate ERE-dependent transcriptional activity in the presence of estrogen. The levels of transcriptional activity were dependent on the amount of expression plasmid transfected (Fig. 2AGo). We compared the transcriptional potency of Rh-ER{alpha} to wild-type, soluble ER{alpha} evaluated in parallel transfections. Although wild-type ER{alpha} had higher baseline activation of ERE-dependent transcription than Rh-ER{alpha} (Fig. 2BGo), Rh-ER{alpha} had significantly higher estrogen stimulated activity. The ratios of estrogen-stimulated to vehicle-treated activity were 3 and 30 for wild-type and Rh-ER{alpha}, respectively. It thus appeared that Rh-ER{alpha} was capable of activating transcription from an ERE reporter plasmid, and that it displayed much greater estrogen stimulation that soluble ER{alpha}. The activity of Rh-ER{alpha} was fully inhibited by the estrogen antagonist ICI 182,780 (Fig. 2CGo), demonstrating the dependency of ER{alpha} on activation of transcription.



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  		Fig. 2. Activation of Estrogen-Dependent Transcription in 293 Cells Expressing Rh-ER{alpha}

A, The 293 cells were transiently transfected with a combination of pERE-luc (900 ng shown above; an indicator of ER{alpha} transcriptional activity) and varying amounts of Rh-ER{alpha}, phRG-TK (100 ng directing the constitutive expression of renilla luciferase for normalization of transfections) and empty vector such that the DNA concentration was 2 µg per transfection. Black and white bars represent normalized luciferase activities with and without 10 nM estrogen, respectively. B, Comparison of activities of ER{alpha} and Rh-ER{alpha}. The 293 cells were transiently transfected with 100 ng of one of four expression vectors shown, 900 ng pERE-luc, and 100 ng phRG-TK for normalization. Both ER{alpha} and Rh-ER{alpha} activated transcription in response to estrogen. However, Rh-ER{alpha} had a much higher hormone stimulated response. Both rhodopsin and vector-transfected cells had baseline transcriptional responses. ERE activation by Rh-ER{alpha} was not markedly affected by incubation with the p44/42 inhibitor PD98059 (50 µM). ER{alpha} continued to work in the presence of this inhibitor, although estrogen stimulated transcription was more markedly attenuated. Transfection efficiency was normalized by inclusion of phRG-TK. C, Inhibition of Rh-ER{alpha} by ICI 182,780. Cotransfections were performed as described above with and without ICI 182,780 (100 nM). This estrogen inhibitor alone had no effect on ER{alpha} and Rh-ER{alpha} and when incubated with estrogen complete blocked transcriptional activity. D, Rh-ER{alpha} activates a chromatin encoded gene. The 293 cells transfected with Rh-ER{alpha} or ER{alpha} were stimulated with control or estrogen-containing media. RNA from these cells was analyzed by Northern blotting using a radiolabeled VEGF probe. VEGF is markedly induced by estrogen in both Rh-ER{alpha}- and ER{alpha}-transfected cells. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.

 
We next asked whether Rh-ER{alpha} was also capable of activating a chromosomally encoded, native gene (Fig. 2DGo). Cells transfected with Rh-ER{alpha} were either treated with control media or estrogen and expression of an estrogen-regulated gene, vascular endothelial growth factor (VEGF) (18, 19, 20), was determined by Northern blot hybridization. Estrogen stimulated VEGF mRNA levels in Rh-ER{alpha}-transfected cells to a similar degree as in ER{alpha}-transfected cells. Vector-transfected cells were not estrogen responsive. Together, these experiments show that Rh-ER{alpha} is able to activate transcription of both episomally and chromosomally encoded genes in a ligand-dependent fashion.

To address the potential mechanism of Rh-ER{alpha} activation of transcription, we first investigated the possibility that estrogen stimulated transcriptional activation of the ERE reporter through Rh-ER{alpha}-mediated phosphorylation events. We cotransfected Rh-ER{alpha} and the ERE reporter and determined transcriptional activation by estrogen in the presence of specific kinase inhibitors. Estrogen continued to stimulate reporter transcription in Rh-ER{alpha}-transfected cells in the presence of MAPK and Akt inhibitors (Fig. 2BGo shows results with p44/42 inhibitor; other data not shown). PD98059, an inhibitor of p44/p42 MAPK, appeared to decrease the estrogen stimulated activity of ER{alpha}; however, the Rh-ER{alpha} stimulation was less sensitive to the inhibitor and Rh-ER{alpha} remained robustly active in presence of PD98059. Cotransfection with dominant-negative kinases linked to the ER function failed to affect the activity of Rh-ER{alpha} (data not shown). Thus, we excluded the possibility of indirect stimulation of ERE-mediated transcription by Rh-ER{alpha}, acting through established kinase cascades. It was more likely that Rh-ER{alpha} was activating transcription by conventional means, i.e. by stimulating transcription in the nucleus.

To further confirm that ERE activation was dependent upon conventional nuclear function of the ER domain of Rh-ER{alpha}, we used a dominant-negative ER{alpha} [pCMV5-ER{alpha}-554FS (21)] in cotransfection assays (Fig. 3AGo). Estrogen-stimulated activation of Rh-ER{alpha} was markedly reduced by cotransfection of the dominant-negative construct (Fig. 3AGo, bars at far right). In addition, mutation of Rh-ER{alpha} to a dominant-negative (Rh-ER{alpha}-FS) diminished both baseline and estrogen stimulated transcription (Fig. 3AGo, second set from the left), consistent with Rh-ER{alpha} acting directly in the nucleus. That the mutation has a similar effect on both ER{alpha} and Rh-ER{alpha} suggests that Rh-ER{alpha} is working by conventional nuclear ER{alpha} mechanisms.



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  		Fig. 3. Effect of Dominant-Negative Mutations of ER{alpha} in trans and in cis on Rh-ER{alpha} Transcriptional Action

A, The 293 cells were transiently transfected with a total of 100 ng of expression vectors shown below, 900 ng pERE-luc, and 100 ng phRG-TK for normalization. Black and white bars represent the activity of the ERE reporter in the presence or absence of estrogen, respectively. Cotransfection (expression in trans) of the dominant-negative pCVM5-ER{alpha}-FS results in a drastic decrement in estrogen-stimulated transcriptional action of Rh-ER{alpha}. Creation of the corresponding dominant-negative mutation (in cis) within Rh-ER{alpha} completely ablates its transcriptional activity. B, In the same transfection/reporter paradigm, cotransfection of Rh-ER{alpha}-FS fails to attenuate transcriptional activation of ER{alpha}.

 
Finally, although immunocytochemical and Western experiments above indicated otherwise, we wanted to ensure that activation of transcription was not due to small amounts of soluble ER{alpha} released by proteolysis of Rh-ER{alpha}-FS, which could migrate into the nucleoplasm. If this were the case, transfection of Rh-ER{alpha}-FS fusions containing the dominant-negative mutation would release inhibitory fragments of mutant ER{alpha}-554FS, which would diminish ERE-driven transcription. However, cotransfection of dominant-negative Rh-ER{alpha} did not inhibit activity of transfected wild-type ER{alpha} (Fig. 3BGo), confirming that estrogen does not cause the proteolytic release of ER{alpha} from the membrane fusion protein.

To account for the high estrogen inducibility of Rh-ER{alpha} and evidence that it was acting directly in the nucleus, we hypothesized that estrogen could strictly regulate nuclear localization of Rh-ER{alpha}. In the absence of estrogen, we surmised that no protein could translocate to the nucleus, resulting in a low basal transcription. We reasoned that when estrogen was added, Rh-ER{alpha} could then localize to the nucleus in a fully active state. To address this possibility, we examined the activity of a mutant Rh-ER{alpha}-Y [rhodopsin fused to the mutant ER{alpha}-Y537S (22)], which contains a well-characterized activating mutation. This mutation in soluble ER{alpha} results in a protein that is constitutively active and hormone independent. If nuclear access of Rh-ER{alpha} is strictly regulated by estrogen, the Rh-ER{alpha}-Y mutant should exhibit basal activity equivalent to unstimulated Rh-ER{alpha} rather than constitutive activity. Indeed, cells transfected with Rh-ER{alpha} displayed baseline transcriptional activity in the absence of estrogen, indicating that the protein was absent from the nucleus (Fig. 4AGo). However, when exposed to estrogen, cells transfected with Rh-ER{alpha}-Y efficiently stimulated ERE-dependent transcription. In addition, cells transfected with Rh-ER{alpha}-Y regulated the chromosomally encoded VEGF gene in response to estrogen (Fig. 4BGo). These results strongly support estrogen-dependent, regulated transport of Rh-ER{alpha} to the nucleus.



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  		Fig. 4. Effect of Activating Mutations on Rh-ER{alpha} Activity

A, The 293 cells were transiently transfected with a total of 100 ng of expression vectors shown below, 900 ng pERE-luc, and 100 ng phRG-TK for normalization. Black and white bars represent the activity of the ERE reporter in the presence or absence of estrogen, respectively. The transcriptional activity of Rh-ER{alpha}-Y, like Rh-ER{alpha}, is regulated by estrogen. B, Rh-ER{alpha}-Y mediates estrogen regulation of chromosomally encoded VEGF. 293 cells transiently transfected with Rh-ER{alpha}-Y were exposed to media with and without estrogen (10 nM) for 24 h. RNA was collected and analyzed by Northern blotting with a VEGF radiolabeled probe. Normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown to demonstrate equivalent RNA loading.

 
Although these functional studies suggest that membrane-anchored Rh-ER{alpha} is stimulated to localize to the nucleus, we needed to explain our immunofluorescence data demonstrating lack of estrogen-stimulated nucleoplasmic ER{alpha}. One possibility to account for this was that Rh-ER{alpha} accesses the nucleus by estrogen-regulated movement from sites of synthesis in the endoplasmic reticulum to the inner nuclear membrane. Immunostaining of inner nuclear membrane localized proteins cannot be distinguished in the presence of excess outer nuclear and endoplasmic reticulum staining. To confirm this mechanism of Rh-ER{alpha} protein regulation, cells expressing ER{alpha} or ER{alpha}-fusions were fractionated (Fig. 5Go) to separate endoplasmic reticulum and outer nuclear membrane proteins (postnuclear fraction, P) from inner nuclear and nucleoplasmic proteins (INM); nuclear proteins were further extracted with high salt to separate soluble and insoluble protein (S). We verified that nuclear preparations were free of endoplasmic reticulum and plasma membrane proteins by immunoblot analysis with antibodies to binding protein (BiP)/glucose-regulated protein (GRP) 78 and very late activation protein-2 (VLA-2)/{alpha}2ß1integrin, respectively. In addition, we demonstrated that the soluble protein actin was completely extracted from nuclear preparation.



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  		Fig. 5. Distribution of ER{alpha} Expression Proteins within Subcellular Fractionations of Transfected 293 Cells

The 293 cells grown with or without estrogen were transiently transfected with ER{alpha}, Rh-ER{alpha}, or Rh-ER{alpha}-Y. One day after transfection, cells were harvested and a portion of these cells lysed and designated the whole homogenate (H). The remainder of the cells were Dounce homogenized (Kontes, Vineland, NJ) in the presence of NP-40 and subjected to a low speed spin to separate the postnuclear (P) components from nuclear components. The nuclear components were further fractionated by high salt extraction and the insoluble components (INM) separated from the soluble nuclear fraction (N). The extracts were resolve by SDS-PAGE onto several identical filters and probed with the indicated antibodies. The top four panels show the distribution of ER{alpha} proteins from respective transfections. The remainder of the figure displays the distribution of marker proteins within fractions that demonstrate that the effectiveness of the cell fractiontionation. These control panels are representative of fractions of cells containing rhodopsin and each of the three ER{alpha} constructs; the controls were virtually identical. This study demonstrates that estrogen regulates the localization of Rh-ER{alpha} to the insoluble fractions of the nucleus, whereas ER{alpha} is constitutively localized to the nucleus and is present in both the insoluble and soluble fractions.

 
The subcellular distribution of ER{alpha} or ER{alpha}-fusions was analyzed by immunoblotting for expression in these protein fractions (Fig. 5Go). Rh-ER{alpha}, Rh-ER{alpha}-Y, and ER{alpha} protein were readily detected in the postnuclear and nuclear fractions of Rh-ER{alpha}-, Rh-ER{alpha}-Y-, or ER{alpha}-transfected cells. Estrogen did not cause a shift in the postnuclear levels of Rh-ER{alpha}, Rh-ER{alpha}-Y, or ER{alpha}. However, estrogen markedly enhanced the amount of Rh-ER{alpha} and Rh-ER{alpha}-Y present in detergent-stripped nuclear fractions containing inner nuclear membrane proteins. In accordance with immunofluorescence data showing constitutive nucleoplasmic ER{alpha}, estrogen did not modulate ER{alpha} levels in the INM preparation. High salt extraction of the detergent-stripped nuclei failed to solubilize Rh-ER{alpha} or Rh-ER{alpha}-Y, confirming their integral association with the inner nuclear membrane. In contrast, a modest but detectable proportion of ER{alpha} could be solubilized. The residual ER{alpha} in the nucleus is consistent with reports that ER{alpha} is associated with components of the nuclear matrix and under certain circumstances becomes very tightly bound (23, 24, 25, 26). In sum, in contrast to soluble ER, Rh-ER{alpha} requires estrogen for localization to the nucleus where it is found in the inner nuclear membrane. From its inner nuclear membrane location, Rh-ER{alpha} stimulates transcription.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Our experiments demonstrate that 1) membrane-tethered ER{alpha} maintains estrogen responsive classical function; and 2) nuclear localization of ER{alpha} tethered to membranes is mediated by estrogen.

Membrane ER{alpha} Can Activate Transcription
ER{alpha} is capable of mediating both nuclear and nonnuclear actions, although the mechanism by which ER{alpha} signals at the membrane has been elusive because it contains neither a predicted membrane spanning domain nor a potential lipid modification motif (27). It has thus been hypothesized that ER{alpha} is closely associated with another protein that anchors it to the membrane (28). Alternatively, a splice product of ER{alpha} or a closely related protein (29) could contain sequences that enable membrane localization. Recent data suggest that, in neurons, ER{alpha} expressed in axons and dendrites are stable in the presence of estrogen, suggesting that they are scaffolded to the fixed structures (12). To answer the question of whether membrane-bound ER{alpha} is capable of signaling in the nucleus, we created a situation where ER{alpha} was irreversibly linked to the membrane using a well-characterized integral membrane protein. It should be noted that, in previous studies, membrane-tethered ER{alpha} has been mutated (5) or truncated (30) to deliberately inactivate genomic function; thus, our studies provide a novel window into the potential function of the full-length, intact protein localized to the membrane. Our data indicated that even in the context of tight and irreversible membrane association, ER{alpha} can still function in the nucleus.

This study characterizes the nuclear function of ER{alpha} expressed at the cytoplasmic face of the membrane. However, the topology of membrane ER{alpha} remains controversial; immunological, protease sensitivity, and tethered ligand studies suggest that the membrane form of ER{alpha} is exposed to the extracellular space (1, 8, 31, 32). On the other hand, the protein has also been shown to interact with caveolins and directed targeting of the hormone binding domain by lipid tagging also suggest that the protein functions when tethered to the cytoplasmic face of the plasma membrane (5, 30). Although full characterization of the topology of membrane forms of ER{alpha} was not the goal of this study, the activity of Rh-ER{alpha} on MAPK does imply that cytoplasmic ER{alpha} is sufficient for nongenomic function and is a reasonable model for membrane-associated ER{alpha}.

Interestingly, Rh-ER{alpha} appears to be excluded from the nucleoplasm by cell staining, even in the presence of estrogen; however, it is still capable of performing nuclear functions in an estrogen-dependent fashion. This is relevant to cells, such as specific subsets of neurons, which appear to express ER{alpha} in nonnuclear distributions. Our data imply that even in cells that fail to express significant amounts of nucleoplasmic ER{alpha}, the protein can still signal to the nucleus.

Increased Gain in Estrogen Signaling with Membrane-Bound ER{alpha}
If genomic responses can be carried out by both membrane-bound and soluble ER{alpha}, are there advantages of having both forms? One potential advantage is that the two forms appear to differ in transcriptional potency, with the soluble form showing higher baseline levels and the membrane-bound receptor demonstrating higher inducibility. The ratio of the two forms could alter both basal ER{alpha} potency and the overall estrogen responsiveness of a cell. For example, an increase in membrane localization of ER{alpha} could decrease baseline ERE function and increase estrogen-dependent ERE function, effectively increasing the signal to noise ratio of estrogen signaling. It is known that ERE responses can vary greatly between cell lines (33), and it remains to be determined if the ratio of soluble to membrane ER{alpha} is linked to these cell-type differences in estrogen responsiveness.

In addition, membrane-tethered ER{alpha}, which is diffusion restricted, could have preferential access to a subset of genes that are scaffolded to the inner nuclear membrane, whereas soluble ER{alpha} could have a wider spectrum of targets. Furthermore, membrane-bound ER{alpha} acting at the nucleus could potentially have extremely rapid novel functions at the nuclear membrane. For example, prior studies (34, 35, 36, 37) have shown acute changes in nuclear membrane and membrane scaffolded heterochromatin structure induced by estrogen, which appear too rapidly to be explained by genomic activation.

Mechanisms of Rh-ER{alpha} Action
What is the mechanism by which Rh-ER{alpha} translocates to the nucleus and activates transcription? Prior studies from our lab have shown that fusions of GAL4 and GFP to the N terminus of ER{alpha} do not affect baseline and estrogen stimulated function of in 293 cells; therefore, our findings are not a consequence of blockade of the N terminus of ER{alpha}. It is also unlikely that the transcriptional effects seen are due to proteolysis of the protein into functional fragments because the rhodopsin fusions Rh-ER{alpha}-Y and Rh-ER{alpha}-FS failed to activate or inhibit (respectively) the ERE promoter, and we found no evidence by immunoblotting of estrogen-dependent proteolytic degradation.

The most plausible mechanism (Fig. 6Go) is that membrane tethering makes the protein unavailable to the normal nuclear transport mechanisms, which allow transit of soluble proteins through the center of the nuclear pore complex. These mechanisms allow soluble ER{alpha} to localize to the nucleus, resulting in large quantities of nuclear ER{alpha} seen in cell types such as 293 cells. In contrast, Rh-ER{alpha}, like inner nuclear membrane proteins, likely enter the nucleus not through the center but through the small side ports of the nuclear pore complex. At 66 kDa, ER{alpha} is at the upper limit in size of cytoplasmic domains that can traverse the side ports of the nuclear pore complex by diffusion (38). Without ligand, chaperone complexes (e.g. heat shock proteins) bind ER{alpha} fusions and increase the size of the complex, restricting their movement into the nucleus. When ligand is added, the large molecular weight complex dissociates, and the membrane-tethered protein is then capable of diffusing into the inner nuclear membrane. Rh-ER{alpha} gains access to transcriptional machinery by localization to the inner nuclear membrane or to membranous channels that form a tubular structure deep within nuclei of many cell types (39). Strict nuclear exclusion of the fusion protein in the absence of hormone contributes, in part, to the dramatic estrogen inducibility of the protein compared with soluble ER{alpha}.



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  		Fig. 6. Model for the Nuclear Action of Membrane-Bound Rh-ER{alpha}

This schematic demonstrates the topological relationship between the endoplasmic reticulum and the outer and inner nuclear membranes. The inner nuclear membrane is functionally separated from the outer nuclear membrane by the nuclear pore complex, which facilitates transport of soluble proteins between the nucleus and the cytoplasm. It is thought that membrane proteins synthesized in the endoplasmic reticulum diffuse to the outer nuclear membrane and then through the side ports of the nuclear pore complex to reach the inner nuclear membrane, where specific proteins are scaffolded and concentrated. In the absence of estrogen of Rh-ER{alpha} is likely associated with multiple chaperone proteins (crescents bound to ER{alpha}), which are released when ligand is added. This allows Rh-ER{alpha} to travel through the nuclear pore complex side port into the inner nuclear membrane where transcriptional activation can take place. Alternatively, transit through membranous tubules could carry Rh-ER{alpha} deep into the nucleus to activate genes from a membranous position. Soluble ER{alpha}, in contrast, diffuses through the nuclear pore complex in the absence of ligand, where it has constitutive activity.

 
Implications for the Action of Transmembrane Proteins in the Nucleus
The receptors for FGF and EGF and other transmembrane proteins (40, 41) find their way into the nucleus and are thought to stimulate nuclear processes, though the mechanisms of nuclear translocation remains obscure. For example, EGFR, a transmembrane tyrosine kinase receptor, has been shown to localize to the nucleus where it could potentially phosphorylate novel nuclear substrates. The Notch family of transmembrane receptors contain a cytoplasmic domain that, upon regulated proteolytic release, migrates to the nucleus and modulates transcription. Overexpression of the Epstein Barr virus protein BARF0, causes accumulation of the full-length receptor in the nucleus (42). This is mediated by the nuclear localization signal of Notch.

The behavior of Rh-ER{alpha} suggests that some transmembrane proteins migrate through the endoplasmic reticulum into the inner nuclear membrane, where they can act in the nucleus. Our findings represent the first known example of regulated protein translocation from the outer to the inner nuclear membrane and a novel use of hormone binding domains of nuclear receptors. The translocation of Rh-ER{alpha} described here can be measured using a simple luciferase assay, offering a convenient method to study the mechanisms of membrane protein access to the nucleus.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Drugs and Antibodies
Drugs were obtained from Sigma (St. Louis, MO). Antibodies were purchased as follows: ER{alpha} sc-542 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), actin A 2066 (Sigma), organelle sampler kit catalog no. 611436 (nucleoporin p62, VLA-2{alpha}, Bip/GRP78; from Becton Dickinson, Franklin Lakes, NJ) and goat antirabbit Alexa-conjugated A-11004 (Molecular Probes, Eugene, OR).

Cell Lines and Plasmids
ER{alpha}-negative human embryonic kidney cell line 293A (Quantum Biotechnologies, Carlsbad, CA) were grown in 24-well plates in DMEM (Invitrogen, Carlsbad, CA), supplemented with 5% fetal bovine serum, 2 mM glutamine, and 1% penicillin/streptomycin at 37 C as above. Before transfection, cells were split into phenol red-free media supplemented with charcoal dextran-stripped serum (Hyclone, Logan, UT). The reporter gene pERE-Luc contains two tandem estrogen response elements followed by a TATA box, respectively and was created by inserting a double-stranded oligonucleotide containing two consensus EREs flanked by a 5' overhang compatible with HindIII and a blunt end (top strand 5'agc ttg gtc aca gtg acc tag gtc aca gtg acc tag atc t3') into the HindIII and SmaI sites of pFR-luc (Stratagene, La Jolla, CA). This results in the replacement of the Gal4 binding sites of pFR-luc with two EREs linked to a TATA box. pCMV5-hER{alpha}2 directs the expression of human ER{alpha} and is essentially the same as pCMV5-ER{alpha} (43) (a gift from BK) except the full 5' end of the pCMV5 polylinker has been reconstructed, enabling the full-length coding sequence of the former to be excised with BamHI. phRG-thymidine kinase (TK) directs the expression of a humanized renilla luciferase gene under the control of the TK promoter (Promega, Madison, WI). Myristolation tagged ER{alpha} was generated by PCR amplifying ER{alpha} with oligonucleotides encoding the c-src myristoylation tag (first 11 amino acids) fused to the 5' end of human ER{alpha} (5' CGG AAT TCC GGA CC A TGG GGA GCA GCA AGA GCA AGC CCA AGT CTA GA A CCA TGA CCC TCC ACA CC) and to ER{alpha} (5' GGG GAA TTC GAG GGT CAA ATC CAC AAA GCC). The PCR fragment was digested with EcoRI and NotI and used to replace the corresponding fragment in pCMV5-hER{alpha}2. Rh-ER{alpha} and mutants were generated by inserting the coding sequence for ER{alpha} and mutants into a mutant rhodopsin which results in a fusion of human ER{alpha} clone to amino acid 332 of bovine rhodopsin, controlled by the pCIS vector backbone (17). Expression constructs used (except some plasmids which used the very similar pCMV5 backbone noted specifically in Figs. 1DGo and 3AGo) were constructed in the same vector backbone (17).

Immunostaining and Microscopy
Samples were fixed in 4% formalin/PBS for 30 min, washed with TBST, blocked with 5% goat serum in TBST, and incubated with primary antibodies overnight at 4 C. Secondary antibodies were applied for one hour at room temperature. Samples were then washed and mounted. Standard immunofluorescence was imaged using a Nikon (Melville, NY) TE200 with a SPOT RT digital camera. Confocal imaging was performed using a Leica (Bannockburn, IL) NCS NT, and merged using Leica NCS NT software or Adobe Photoshop (San Jose, CA).

Transient Transfections and Reporter Assays
All cells were switched to estrogen-free media 24 h before transfection. Cells were transfected in the presence or absence of 10 nM ß-estradiol. The 293A cells in 24-well plates were transiently transfected using 2 µl of Lipofectamine 2000 (Invitrogen) mixed with 2 µg plasmid DNA in 100 µl of OptiMEM (Invitrogen). Unless noted, the following DNA mixtures were used to determine the classical ERE activity of transfected ER{alpha} constructs: 100 ng of ER construct or empty vector, 900 ng of pERE-luc, and 100 ng phRG-TK. The transfection mixture was left on the cells until harvesting unless otherwise noted. Cell lysates were prepared 24 h after transfection and luciferase assays were performed using the dual luciferase protocol enabling normalization of firefly luciferase to cotransfected renilla luciferase (Promega). All reporter assays were performed in at least three independent experiments in triplicate.

Subcellular Distribution Analysis
We fractionated transfected cells (2 x 106) using the protocol described by Meyer and Radsak (44). All manipulations were performed at 4 C, and all buffers were supplemented with proteinase inhibitors (cocktail) and 0.3 mM phenylmethylsulfonyl fluoride. Cells were washed twice with cold PBS, scraped off, sedimented, and resuspended in 1 ml TKM [25 mM Tris-HCl (pH 7.4), 5 mM KCl, 1 mM MgCl2] (the homogenate fraction). To strip the outer membrane and endoplasmic reticulum, 1 ml TKM with 1% Nonidet P-40 (NP-40) was added on ice with occasional vortexing for 5 min. The mixture was sedimented at 1000 x g, and the supernatant containing the postnuclear fraction was separated from the stripped nuclei. The pellet containing nuclei was resuspended in 3 ml TKM containing 0.5% NP-40 and 100 mM NaCl and 6 ml of 2.3 M sucrose were added to bring sucrose concentration to 1.62 M. This mixture was layered over a 3-ml cushion of 2.3 M sucrose in TKM and centrifuged at 130,000 x g for 1 h. The pellet was washed sequentially with PBS, PBS + 0.5% Triton X-100, and PBS. Finally, the pellet was extracted with 50 µl of a solution of 1.6 M NaCl, 1 M Tris-HCl (pH 7.4), 1 M HEPES, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin overnight, resedimented, and the pellet used as the insoluble nuclear preparation with the supernatant as the nuclear extract.

Western Blotting
For Fig. 1Go, whole cell lysates in sample buffer were prepared from transfected cells treated with and without estrogen (10 nM) for 1 d. For Fig. 5Go, transfected cells were exposed to 10 nM estrogen or vehicle before harvest and subcellular fractionation. All protein samples were boiled for 5 min was resolved on a 4–20% gradient polyacrylamide gel, transferred to polyvinylidene difluoride, blocked for 1 h with 5% milk/TBST at room temperature, and incubated overnight at 4 C with primary antibodies used at the following dilutions: ER{alpha} (1:500); nucleoporin p62 (1:1000); VLA-2{alpha} (1:250); Bip/GRP78 (1:250); antiactin (1:2500). Horseradish peroxidase-conjugated secondary antibodies (1:5000) were applied in TBST for 1 h and detected by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ).

Northern Blotting
The 293 cells in estrogen-free media grown in 15-mm dishes were transfected with 10 µg of vector DNA and 1 µg of either Rh-ER{alpha} or ER{alpha} using Lipofectamine 2000. After 24 h, estrogen was added where indicated. Total RNA was harvested after an additional 24 h period by using the RNEasy kit (QIAGEN, Valencia, CA). Five micrograms of RNA were fractionated on formaldehyde gels and blotted onto Nytran (Schleicher and Schuell, Keene, NH). Filters were UV cross-linked, probed with 32P-labeled rat VEGF probe, washed, and phosphorimaged (Molecular Dyamics, Sunnyvale, CA). The filters were then stripped and reprobed with glyceraldehyde-3-phosphate dehydrogenase probe to ensure equal RNA loading.


   ACKNOWLEDGMENTS
 
Sokhon Pin provided outstanding technical assistance. We thank Jimo Borjigin and for very helpful discussions and to Jimo Borjigin and Benita Katzenellenbogen for cDNA clones.


   FOOTNOTES
 
This work was supported by NIH Grants (NS20020, NS33668, NR03521, and NS41342), the Richard Ross Clinician Scientist Award, and a Burroughs Wellcome Fund Career Award in Biomedical Sciences.

Abbreviations: BIP, Binding protein; ER, estrogen receptor; ERE, ER response element; GRP, glucose-regulated protein; INM, inner nuclear and nucleoplasmic proteins; NP-40, Nonidet P-40; Rh-ER{alpha}, ER{alpha} fused to the C-terminal cytoplasmic tail of bovine rhodopsin; TK, thymidine kinase; VEGF, vascular endothelial growth factor; VLA-2, very late activation protein-2.

Received for publication July 7, 2003. Accepted for publication October 16, 2003.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   ERα
Ligands:   17β-Estradiol



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