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Domains with Known Nuclear Functions
Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75390
Address all correspondence and requests for reprints to: Philip W. Shaul, Department of Pediatrics, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75390. E-mail: philip.shaul{at}utsouthwestern.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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. The process requires Src and erk signaling and eNOS phosphorylation by phosphoinositide 3-kinase (PI3 kinase)-Akt kinase, with Src and PI3 kinase associating with ER upon ligand activation. To delineate the basis of nongenomic eNOS stimulation, the potential roles of ER domains necessary for classical nuclear function were investigated in COS-7 cells. In cross-linking studies, estradiol-17ß (E2) caused PM-associated ER to form dimers. However, eNOS activation by E2 was unaltered for a dimerization-deficient mutant ER (ERL511R). In contrast, ER mutants lacking the nuclear localization signals (NLS), NLS2,3 (ER
250–274) or the DNA binding domain (ER185–251), which targeted normally to PM and caveolae/rafts, were incapable of activating eNOS. The loss of NLS2/NLS3 prevented Src and erk activation, and it altered ligand-induced PI3 kinase-ER interaction and prevented eNOS phosphorylation. Loss of the DNA binding domain did not change E2 activation of Src or erk, but ligand-induced PI3 kinase-ER binding and eNOS phosphorylation did not occur. Thus, dimerization is not required for ER coupling to eNOS; however, NLS2/NLS3 plays a role in Src activation, and the DNA binding region is involved in the dynamic interaction between ER and PI3 kinase. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and ERß, mediate nongenomic eNOS activation (4, 5). In the classical model of nuclear ER action, the binding of estrogen releases receptors from inactive complexes containing heat shock proteins and immunophilins, and it induces receptor dimerization and binding to estrogen response elements (ERE) on target genes, thereby altering rates of gene transcription (6, 7, 8). ERs can also modulate gene expression in the absence of direct DNA binding via protein-protein interactions with other transcription factors (8, 9). However, there are also subpopulations of the receptors that are localized to the plasma membrane (PM) of numerous cell types, including endothelial cells, where they mediate membrane-initiated signaling (10, 11). On the PM of endothelial cells, both ER and ERß are coupled to eNOS in caveolae, which are a subset of lipid rafts known to compartmentalize signaling molecules (5, 12).
Although the processes linking estrogen binding to ER and eNOS stimulation are complex, a degree of clarity has been afforded by prior knowledge of the multiple mechanisms mediating the activity of the enzyme. Numerous stimuli impact upon eNOS by activating kinases that alter the phosphorylation of the enzyme. Akt kinase stimulates eNOS by directly phosphorylating the enzyme at Ser-1179, and Akt itself is phosphorylated and activated by phosphoinositide 3-kinase (PI3 kinase), which is activated by both receptor and nonreceptor tyrosine kinases. In addition, eNOS is modulated by erk MAPKs, which can have either positive or negative effects on enzyme function (13, 14). As for estrogen-induced stimulation of eNOS, studies have shown that it entails signaling by erks and PI3 kinase/Akt kinase, and there is also evidence of an upstream role for Src kinase (4, 15, 16, 17, 18). Direct interaction has been reported between ER and the p85 subunit of PI3 kinase with ligand activation of the receptor (16), there is evidence that c-Src, ER, and PI3 kinase form a complex upon ligand activation (18), and it has been demonstrated that ER modulation of eNOS also involves receptor interaction with Gi (19). Thus, certain aspects of the proximal mechanisms by which ER are coupled to eNOS have been elucidated. However, the functional roles of ER domains in signaling to eNOS are yet to be determined.
To further delineate the basis of nongenomic activation of eNOS by estrogen, the present investigation evaluated the role of ER domains which are critical to classical nuclear function. This approach was chosen in an effort to distinguish domains which are uniquely relevant to membrane vs. nuclear ER actions. Like other steroid hormone receptors, ER has six structural regions designated A-F. The DNA binding domain is in region C, and the ligand binding domain is in region E. The other regions are the hypervariable A/B domain, the hinge region D, and the C-terminal F domain (6). The primary estrogen-inducible dimerization signal is in region E (20). Paralleling the features of ER involved in classical nuclear action, we first tested the hypothesis that receptor dimerization is not required for estrogen-induced stimulation of eNOS. In addition, we determined whether domains within the two key nuclear localization signals (NLS) of ER, which reside within the DNA binding domain and hinge region (21), play a role in eNOS activation. Furthermore, we determined whether domains within the DNA binding region of the receptor are involved in eNOS activation. Based on reports that the ligand binding domain of ER is sufficient to mediate certain nonnuclear actions of estrogen (22, 23), the hypothesis raised was that the NLS and DNA binding domains are not required for estrogen-induced stimulation of eNOS.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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involved in receptor dimerization, nuclear localization, and DNA binding were first confirmed in the COS-7 cell model system. The capacity to mediate estrogen-induced transcriptional transactivation was compared between wild-type ER and ERL511R using the estrogen-responsive reporter plasmid 2ERE-Luc (Fig. 1A
). Whereas 2ERE-Luc reporter activity was increased by 4- to 5-fold with estradiol-17ß (E2) in cells expressing wild-type receptor, and the response was prevented by ER antagonism, reporter activity was not stimulated by the hormone in cells expressing ERL511R. The abilities of ER250–274 and ER185–251 to mediate transcriptional transactivation were evaluated using the 3ERE-Luc reporter plasmid (Fig. 1B). In these experiments, there was a 9-fold increase in reporter activity with E2 in cells expressing wild-type receptor that was attenuated by ICI 182,780; in contrast, reporter activity was not stimulated by E2 in cells expressing either ER mutant.
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dimerization was assessed by immunoblot analysis after cell treatment with E2 and either vehicle or the permeable cross-linking agent EDAC (Fig. 2). With wild-type receptor, a 67-kDa species indicative of ER monomer was principally observed in the absence of cross-linker treatment, and a 134-kDa species of proper size to represent receptor dimer was also evident in cross-linker-treated cells (Fig. 2A). Similar experiments were done with truncated forms of ER to confirm the detection of dimers using this approach. With ER185–251, a 58-kDa band indicative of monomer was detected in vehicle-treated cells, and a 116-kDa band of proper size to be dimerized mutant was also observed after cross-linker treatment. To provide another ER construct of different size, the dimerization of an N-terminal deletion mutant (ER1–175) was evaluated. In the absence of EDAC, a 47-kDa monomeric form was detected by immunoblot analysis; after cross-linker treatment, a 94-kDa species was also detected of proper size to represent dimer. The attenuated capacity of ERL511R to dimerize was then evaluated in a similar manner (Fig. 2B). Whereas wild-type receptor displayed a 134-kDa species upon cross-linker treatment, such a band was absent in studies of ERL511R.
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Dimerization and eNOS Activation undergo dimerization upon ligand activation. COS-7 cells transfected with wild-type receptor were treated with E2 for 0–24 h and also with either vehicle or EDAC, whole cell lysates and purified PM fractions were obtained, and dimerization was assessed by immunoblot analysis. In samples of whole cell lysates from cross-linker-treated cells, a 134-kDa species was apparent after E2 treatment for 20 min or longer (Fig. 2C
, upper panel). In a similar manner, PM fractions from EDAC-treated cells displayed a 134-kDa band after E2 exposure for 20 min or longer (Fig. 2C, lower panel). The capacity of wild-type ER and ERL511R to form dimers on the PM was also compared. Transfected COS-7 cells were treated with vehicle or E2 for 1 h followed by EDAC treatment. Whereas wild-type ER displayed a 134-kDa species greatest in abundance with E2 in whole cell lysate and PM, such bands were negligible for both whole cell lysate and PM in studies of ERL511R (Fig. 2D).
The role of dimerization in estrogen-induced activation of eNOS was determined in studies of COS-7 cells expressing eNOS and either wild-type ER or ERL511R (Fig. 3). To evaluate the localization of wild-type or mutant receptor coupling to eNOS (12), the activation of the enzyme was assessed in both intact cells and in isolated PMs obtained from the transfected cells. In intact cells expressing wild-type receptor, E2 caused a 2-fold increase in NOS activity, and the stimulation was completely prevented by concomitant treatment with ICI 182,780 (Fig. 3A). Similarly, in intact cells expressing ERL511R, E2 treatment resulted in a 2.6-fold stimulation of eNOS that was blocked by ER antagonism. In a parallel manner, E2 caused a doubling in eNOS activity in PMs isolated from cells expressing wild-type receptor, and 2.4-fold stimulation was evident in PMs from cells expressing ERL511R (Fig. 3B).
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Nuclear Localization Signals and eNOS Activation mediating the coupling of the receptor to eNOS are yet to be clarified. To determine whether residues within the principal nuclear localization signals, NLS 2 and 3, are involved, the PM association of ER250–274 was first evaluated. COS-7 cells were transfected with either wild-type ER or the mutant form, and the relative abundance of ER protein in the PM fraction vs. whole cell lysate was determined by immunoblot analysis using equal protein loads. Per unit of protein, wild-type receptor was approximately one third as prevalent in the PM as in whole cell lysate (Fig. 4A). Similar findings were obtained for ER250–274. When repeated studies were performed, the PM abundance per unit of protein relative to that in whole cell lysate was found to be similar for wild-type ER and ER250–274 (Fig. 4B).
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). As already shown in Fig. 3, E2 caused a 2-fold increase in NOS activity in intact cells expressing wild-type receptor, and the response was completely prevented by concomitant treatment with ICI 182,780 (Fig. 5A). In contrast, in intact cells expressing ER250–274, E2 did not stimulate eNOS activity. In a parallel manner, E2 caused a doubling in eNOS activity in PMs from cells expressing wild-type receptor, but no stimulation was evident in PMs from cells expressing the NLS2,3-deficient mutant receptor (Fig. 5B).
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DNA Binding Domain and eNOS Activation is critical to many nuclear actions of the receptor (6). However, it is not known whether residues within the DNA binding domain are involved in ER mediation of eNOS activity. This question was first approached by evaluating the PM localization of ER185–251. After transient transfection in COS-7 cells, the mutant receptor was approximately one third as prevalent per unit of protein in the PM fraction as in the whole cell lysate, mimicking the relative distribution of wild-type receptor (Fig. 4). Because PM localization was retained by this mutant, the ability to mediate estrogen-induced activation of eNOS was determined in studies of COS-7 cells coexpressing eNOS (Fig. 5). Whereas E2 caused a 2-fold increase in NOS activity in intact cells expressing wild-type receptor that was blocked by ICI 182,780, cells expressing ER185–251 were unresponsive (Fig. 5A). Similarly, E2 stimulated eNOS activity in PMs from cells expressing wild-type ER, but no activation occurred in PMs from cells expressing the mutant receptor lacking the DNA binding domain (Fig. 5B).
Bases for ER Mutant Inactivity
Further experiments were performed to determine the bases by which ER250–274 and ER185–251 are incapable of eNOS activation. Having previously demonstrated that ER coupling to eNOS occurs in caveolae, which are a subset of lipid rafts (12), the possibility was tested that the deletions perturb the targeting of the receptor to this specific microdomain. COS-7 cells were transfected with wild-type or mutant forms of ER, whole cell lysates and purified PMs were obtained, caveolae subfractions of the PM were isolated, and the distribution of receptor protein was evaluated by immunoblot analysis. Wild-type ER was detected in both whole PM and caveolae membranes (CM) (Fig. 6). ER250–274 and ER185–251 displayed a distribution within the PM that was comparable with that of wild-type receptor. ERL511R showed a similar distribution (data not shown). After correction for the efficiency of membrane subfraction recovery, the distribution of wild-type and mutant receptors were quantitated (Table 1). A total of 7.3–11.0% of total cellular receptors were localized to the PM, 0.12–0.28% were localized to CMs, and there were no differences in the distribution of wild-type and mutant receptors.
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250–274 and ER185–251 to stimulate eNOS, the capacities of the mutants to activate these processes were evaluated. As expected, erk activation and eNOS phosphorylation occurred within minutes of E2 treatment of cells expressing wild-type receptor (Fig. 7A). In contrast, negligible erk activation or eNOS phosphorylation was observed with E2 exposure of cells expressing ER250–274. However, the mutant of ER lacking the DNA binding region (ER185–251) was capable of mediating erk activation, but eNOS phosphorylation was not detected. These findings were confirmed in additional quantitative studies of erk phosphorylation (Fig. 7B) and eNOS phosphorylation (Fig. 7C) at 10 min of E2 treatment. In additional experiments in cells expressing ER250–274 or ER185–251, the coexpression of a constitutively active mutant form of Akt resulted in levels of eNOS phosphorylation, which were at least comparable with those seen with E2 activation of wild-type receptor (Fig. 8A). In contrast, the coexpression of constitutively active Akt did not alter the level of erk activation in cells expressing either wild-type or mutant receptor forms (Fig. 8B).
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250–274 and ER185–251 was then investigated. Prior works indicate that E2 binding to membrane ER leads to direct interaction between ER, Src, and the p85 subunit of PI3 kinase, Src activation and Src-dependent PI3 kinase activation (16, 18). In studies evaluating Src activation (Fig. 9, A and B), E2 treatment of cells expressing wild-type ER caused Src phosphorylation as expected. Src activation was also evident with E2 in cells expressing ER185–251. However, ER250–274 was incapable of inducing Src activation by E2. Coimmunoprecipitation studies of the interaction between wild-type or mutant ER and the p85 subunit of PI3 kinase were also performed (Fig. 9, C and D). As anticipated, p85 was minimally associated with wild-type receptor in the absence of ligand and E2 promoted the interaction. In contrast, there was marked binding of p85 to ER250–274 in the absence of ligand, and the addition of E2 actually caused a decrease in the interaction between the two proteins. Cells expressing ER185–251 also displayed binding of the mutant receptor to p85 in the absence of ligand, and the addition of E2 had no effect.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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domains that are known to be critically involved in the classical nuclear functions of the receptor. We have shown that although certain domains mediating nuclear actions are most likely not involved in nongenomic activation of eNOS, other ER domains relevant to nuclear function also modulate specific components of the rapid signaling events upstream of the enzyme.
The classical nuclear actions of ER entail receptor dimerization, which is essential for stable binding of the receptor to EREs (6, 8). In the past, this process has been evaluated primarily from the context of DNA binding (20, 24). To study the potential dimerization of PM-associated ER, we developed a strategy involving cross-linking and immunoblot analysis. Using forms of ER with intact dimerization signals but different molecular weights, antibodies to the receptor-detected protein species, which were twice the size of monomers in all instances when cells were exposed to estrogen and cross-linker. Effective detection of dimer formation was further confirmed by loss of the dimer signal in studies of ERL511R. Although the proportion of receptors undergoing dimerization is most likely not reliably quantitated in this manner, the presence or absence of the process is demonstrable. With this approach, we showed that estrogen causes the dimerization of PM-associated ER. The requirement for dimerization in ER modulation of eNOS activity was then tested, and we found that eNOS stimulation in either intact cells or in isolated PMs was induced by both wild-type ER and ERL511R, which has attenuated dimerization. In fact, there was modestly greater eNOS activation by ERL511R vs. wild-type receptor that was equivalently evident in intact cells and isolated PMs. These observations indicate that dimerization does not play a significant role in ER modulation of eNOS activity. The parallel findings in cells and PMs further suggest that E2-induced eNOS stimulation occurs primarily at the PM and not in the Golgi apparatus (25).
Our finding of E2-induced dimerization of membrane ER is consistent with the recently reported observations of Razandi et al. (26). However, our finding of a complete lack of dependence on dimerization for eNOS activation by ER in COS-7 cells contrasts with their observations in CHO cells of dependence on dimerization for Akt phosphorylation, which is upstream of eNOS phosphorylation (26). This disparity may be related to the differing cellular contexts of the two investigations, including the differences in ER coupling to Gi, which is required for eNOS activation by ER in endothelial cells and which is demonstrable in the COS-7 but not in the CHO reconstitution system (19, 26).
Another critical aspect of classical genomic action of ER is the localization of the receptor in the nucleus, which is mediated by three NLS domains (21). Because the short-term actions of ER are characterized instead by either the activation of receptors already localized to the cell surface or the recruitment of receptors to the PM (12, 17, 27), we originally hypothesized that the principal NLS domains, NLS2 and NLS3, would not be required for ER targeting and coupling to eNOS on the membrane. The NLS2,3-deficient mutant form of ER, ER250–274, was localized to the PM in a manner comparable with wild-type receptor. This finding indicates that protein folding occurred well enough to pass the quality control mechanisms of the endoplasmic reticulum. However, in marked contrast to wild-type ER, ER250–274 did not mediate eNOS activation in either intact cells or isolated PMs. One potential explanation was that ER250–274 may not be targeted to caveolae to reside within the domain to be coupled to eNOS. This possibility was ruled out in experiments assessing the distribution of the mutant receptor within the PM, which revealed targeting to caveolae that was comparable to that of wild-type receptor. We then determined whether either of the two key phosphorylation events coupling ER activation to eNOS were disrupted by the deletion of NLS2 and NLS3. Whereas wild-type ER mediated both E2-induced erk activation and eNOS phosphorylation at serine 1179, which is critical to enzyme activation (3), both of these processes were absent in cells expressing ER250–274. The level of phosphorylation of serine 1179 is positively regulated by Akt, and it is negatively regulated by the phosphatase PP2A (28). To differentiate between potential alterations in Akt and PP2A action, additional studies were performed in cells coexpressing a constitutively active form of Akt. In this context, eNOS phosphorylation at least comparable with that observed with E2 activation of wild-type receptor was observed in ER250–274-expressing cells. This finding makes an alteration in PP2A activity less likely, and instead suggests that ER250–274 is incapable of activating PI3kinase-Akt kinase signaling. Constitutively, active Akt did not impact upon erk phosphorylation, suggesting that erk signaling is independent of Akt in this paradigm. In ongoing studies of ER modulation of the cyclooxygenase-1 promoter in the same model system, we have observed that ER250–274 has equivalent potency as wild-type receptor (Gibson, L. L., L. Hahner, S. Osborne-Lawrence, Z. German, K. K. Wu, K. L. Chambliss, and P. W. Shaul, unpublished observation). Thus, the mutant receptor is indeed functional in another context, and a subpopulation is effectively localized in caveolae, but it is entirely incapable of erk and PI3 kinase/Akt kinase-mediated signaling on the cell surface.
A third important feature of classical nuclear action of ER is direct binding of the receptor, via the DNA binding domain, to EREs on target genes (6). We originally hypothesized that the DNA binding domain would not be needed for cell surface ER localization and coupling to eNOS, and found that wild-type receptor and the ER185–251 mutant displayed equivalent targeting to PM. As for ER250–274, this observation indicates that the folding of ER185–251 was sufficient enough to allow the protein to be released from the endoplasmic reticulum. However, whereas wild-type ER invoked eNOS activation in both intact cells and isolated PMs, ER185–251was entirely inactive. This was not explained by ineffective targeting of the mutant to the caveolae fraction of the PM. We then determined whether erk and eNOS phosphorylation were stimulated by ER185–251. In contrast with wild-type receptor, which mediated both processes, and ER250–274, which stimulated neither, ER185–251 caused erk activation equivalent to that seen with wild-type receptor but eNOS phosphorylation was completely absent. However, eNOS phosphorylation at least comparable with that observed with E2 activation of wild-type receptor was evident in ER185–251-expressing cells after coexpression of constitutively active Akt kinase. These cumulative observations indicate that components within the DNA binding domain of ER impact uniquely upon processes regulating PI3 kinase-Akt kinase activation.
To further determine how deletion of NLS2/NLS3 or the DNA binding region attenuate PI3 kinase-Akt kinase activation by ER, experiments were performed to assess how these domains impact on the association or activation of Src and PI3 kinase. We found that loss of the DNA binding domain does not alter the capacity for E2 to activate Src in parallel with the retained capacity to activate erk, whereas ligand-induced association of PI3 kinase with the receptor and eNOS phosphorylation do not occur. In fact, there was ligand-independent association of p85 with the ER mutant lacking the DNA binding domain that was unchanged with E2. These findings indicate that E2-ER-induced Src activation alone is not sufficient to promote dynamic PI3 kinase recruitment to a complex with ER and its activation and resultant downstream phosphorylation events. They also indicate that sequences within the DNA binding domain are important to the modulation of PI3 kinase, possibly by binding an accessory protein that prevents PI3 kinase interaction with the receptor until ligand activation occurs. We also found that loss of NLS2/NLS3 prevents Src and erk activation and it perturbs ligand-induced PI3 kinase recruitment, causing an interaction that is E2 independent and actually markedly attenuated by the ligand. Src association with ER entails the A/B domain, which binds MNAR (modulator of nongenomic activity of ER), which interacts with the SH3 domain of Src, and phosphotyrosine 537 in the E region, which interacts with the SH2 domain of Src (29). As such, the loss of NLS2/NLS3 is not likely to disrupt Src activation by preventing its interaction with the receptor. It is most likely through other mechanisms that NLS2/NLS3 of ER are critical to the regulation of Src and PI3 kinase, which play important roles not only in eNOS regulation on the membrane (3), but in ER nuclear actions (30). Based on these detailed new findings, additional studies are now warranted to identify membrane-associated or cytosolic proteins interacting with NLS2/NLS3 and the DNA binding domain, and to further delineate the discrete domains of ER that are involved.
The present findings that domains within regions C and D of ER are required for effective signaling to erk, PI3 kinase/Akt kinase, and eNOS should be interpreted in the context of previously reported observations related both directly to eNOS and to other membrane-initiated actions of estrogen. It has been found that certain endothelial cells express an ER variant designated ER46 that is a truncated form of the receptor lacking the N-terminal A and B regions, and that ER46 is capable of modulating eNOS activation (31). ER46 contains the C, D, and E regions, and the present results indicate that ligand binding and coupling to erk and PI3 kinase would likely be conserved in such a construct. Consistent with the current observations, it has also been found in studies limited to the activation of erk that such function is retained in an A/B deletion mutant of ER as well as in a mutant lacking the C region (32). Furthermore, the present finding that ER mutants lacking domains within the C and D regions are properly targeted to PM caveolae is in agreement with the recent report that serine 533 within the remaining E region is important to membrane localization of the receptor (32). However, despite all of these consistencies, the currently observed inability of the mutant of ER deficient in NLS2 and NLS3 in regions C and D to activate erk is inconsistent with previous reports that the E domain alone is capable of doing so. It may be relevant that the latter observations were made with directed targeting of the E domain to PM (22, 23, 33). At any rate, paralleling the previous investigations that revealed functional domains of ER mediating various aspects of nuclear responses (6), information is now emerging regarding the domains which are required for nongenomic, membrane initiated actions of the receptor. Further dissection of the nongenomic functional domains will be critical to our ultimate understanding of the diverse processes regulated by estrogen in multiple cell types.
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MATERIALS AND METHODS |
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The PM distribution and function of wild-type human ER or mutant forms of the receptor was tested 48 h after transient transfection using previously reported approaches (5). Studies focused on a mutant of ER with a substitution within the dimerization domain (ERL511R) (20), a deletion mutant lacking the two primary NLS, NLS 2 and 3 (ER250–274), and a deletion mutant lacking the DNA binding domain (ER185–251). The ERL511R mutant was generated by PCR amplification of human ER cDNA with primers 5'-GGTCCACCTTCTAGAATGTG-3' and 5'-TGTGCCTGATGTGGGAGCGG-3' in one reaction and primers 5'-CCGCTCCCACATCAGGCACA-3' and 5'-TAGAAGGCACAGTCGAGGCTG-3' in a separate reaction. The two PCR products were combined and reamplified with 5'-GGTCCACCTTCTAGAATGTG-3' and 5'-TAGAAGGCACAGTCGAGGCTG-3'. The resulting product was ligated into wild-type ER cDNA in pCDNA3.1 using the XbaI and EcoRV restriction sites. The ER250–274 mutant was constructed by ligating two PCR products generated from wild-type human ER cDNA template. The upstream PCR product was amplified using primers both directed to ER (sense primer 5'-GCCGCCTACGAGTTCAACGC-3' and antisense primer 5'-CCATCGATTCCCACTTCGTAGCATTTGC-3'), whereas the downstream PCR product was made using sense primer 5'-GGATCGATGAGGGCAGGGGTGAAGTGGG-3' directed to the ER D domain and antisense primer 5-TAGAAGGCACAGTCGAGGCTG-3' anchored in the pCDNA3.1 vector. The PCR products were ligated to each other using the ClaI site, which was engineered into the primers, and into the ER wild-type cDNA construct in pCDNA3.1 at the SacII restriction site upstream and the EcoRV site located downstream in the vector. The resulting construct is comparable with HE257G (21). ER185–251, which is equivalent to HE11 (21), was kindly provided by Dr. Michael Mendelsohn (Molecular Cardiology Research Institute, New England Medical Center Hospitals, Inc., Tufts University School of Medicine, Boston, MA). All mutants were sequenced to verify structure.
To assess the nuclear function of the constructs, E2-induced transcriptional transactivation was evaluated by cotransfection with a luciferase reporter plasmid that contained either three exact copies of the Xenopus vitellogenin ERE, designated 3ERE-Luc, as previously reported (5), or two copies of ERE within the native context of the vitellogenin promoter (2ERE-Luc) (34). The latter reporter plasmid, which was necessary to effectively assess the requirement for receptor dimerization, was kindly provided by Dr. David Shapiro in the Department of Biochemistry, University of Illinois at Urbana-Champaign. After transfection, cells were placed in either phenol red-free, estrogen-free media, phenol red-free media containing 10–8 M E2, or phenol red-free media containing E2 plus 10–5 M ICI 182,780 for 48 h, and reporter activity was measured. Cells were cotransfected with a plasmid containing simian virus 40-driven ß-galactosidase to normalize for transfection efficiency (35).
To evaluate the capacity of the constructs to activate eNOS, COS-7 cells were cotransfected with bovine eNOS cDNA (36) and either wild-type or mutant forms of ER. Comparable expression of the constructs was confirmed by immunoblot analysis. Coimmunofluorescence experiments revealed that transfection efficiency was approximately 20% for either ER or eNOS, and that the majority of transfected cells expressed both the receptor and enzyme. In selected experiments, the cells were also cotransfected with an Akt mutant generated by fusing a myrisoylation signal to its amino terminus (AktMyr), which is membrane bound and constitutively active (37). AktMyr was kindly provided by Dr. Michael Quon (Diabetes Unit, Laboratory of Clinical Investigation, National Center for Complementary and Alternative Medicine, National Institutes of Health, Bethesda, MD).
Subcellular Fractionation
To study the subcellular distribution of wild-type and mutant forms of ER and to localize their function, subfractionation was performed on transfected COS-7 cells as previously described (12). The purity of the PM fraction obtained has been previously confirmed by measurements of alkaline phosphatase (PM), galactosyl transferase (Golgi) and reduced nicotinamide adenine dinucleotide phosphate cytochrome C reductase (endoplasmic reticulum) activity (38). All fractionation steps were done in the absence of exogenous calcium. Successful isolation of the caveolae subfraction of the PM was confirmed by immunoblot analyses for the caveolae marker protein caveolin-1 and the noncaveolae protein, receptor for activated C-kinase (RACK1) (39). The protein contents of all samples were determined by the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA) or by the method of Bradford (40). In experiments assessing the targeting of wild-type and mutant receptors to PM and CM, immunoblot analyses were quantitated using LabWorks Image Acquisition and Analysis Software (BioImaging Systems, Upland, CA). Receptor distribution was adjusted for the efficiency of recovery of PM and CM calculated by comparing the amount of caveolin in the membrane fractions vs. the whole cell lysate. Quantitation experiments were performed on three separate preparations of each construct.
Immunoblot Analyses
Immunoblot analyses were performed using standard procedures to evaluate the abundance and distribution of wild-type and mutant forms of ER, eNOS, caveolin-1, and RACK1 (41). Equivalence of protein loads was confirmed by amido black staining (Sigma Chemical Co., St. Louis, MO). The analyses of wild-type or mutant ER used a mouse monoclonal antibody directed against amino acids 495–595 of ER (2.5 µg/ml, AER320; Labvision, Fremont, CA,) or antibody AER611 (2.5 mg/ml, Labvision), which we have determined recognizes an epitope in the N-terminal 78 amino acids of the receptor. Monoclonal antiserum to eNOS (0.25 µg/ml) and polyclonal antiserum to caveolin-1 (0.05 mg/ml) or RACK1 (0.1 µg/ml) were from Transduction Laboratories (Lexington, KY). To assess erk activation in response to E2, COS-7 cells were exposed to the hormone (10–8 M) for 0–20 min 48 h after transfection, whole cell lysates were obtained, and immunoblot analysis was performed with anti-ERK2 monoclonal antibody (Upstate Biotechnology, Inc., Lake Placid, NY) and anti-perk polyclonal antibody (Promega Corp., Madison, WI). In a similar manner, eNOS phosphorylation was evaluated after 0–20 min exposure to 10–8 M E2 by immunoblot analysis using anti-phospho-serine-1179 eNOS polyclonal antibody (Cell Signaling Technology, Beverly, MA) (37). Src phosphorylation was also assessed after 0–10 min exposure to 10–8 M E2 by immunoblot analysis using anti-phospho-tyrosine-416 Src polyclonal antibody (Cell Signaling Technology) and anti-Src monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
Assessment of Receptor Dimerization
ER dimerization was evaluated in COS-7 cells expressing ER constructs using the cell-permeable cross-linking agent l-ethyl-3-[3-(dimethylamino) propyl] carbodiimide (EDAC, Pierce Biochemicals, Rockford, IL). Experiments were performed with wild-type receptor, ERL511R, ER185–251, or an N-terminal deletion mutant lacking residues 1–175 (ER1–175) kindly provided by Dr. Michael Mendelsohn (42, 43). Forty-eight hours after transfection, cells were exposed to buffer or E2 (10–8 M) for 0–24 h and either vehicle or 10 mM EDAC for 60 min at 22 C, and whole cell lysates or purified PM fractions were obtained. Immunoblot analysis was then performed for ER. Results shown are representative of three independent experiments.
NOS Activation
eNOS activation was assessed in intact COS-7 cells transfected with cDNAs for the enzyme and for ER constructs by measuring 3H-L-arginine conversion to 3H-L-citrulline, using previously reported methods (12). Adherent cells grown in six-well plates were placed in L-arginine-deficient, serum-free endothelial-SFM growth media (Life Technologies, Inc.) for 2 h, and then preincubated in PBS (pH 7.4) containing 120 mM NaCl, 4.2 mM KCl, 2.5 mM CaCl2, 1.3 mM MgSO4, 7.5 mM glucose, 10 mM HEPES, 1.2 mM Na2HPO4, and 0.37 mM KH2PO4 for 15 min at 37 C. The ensuing 15 min incubation for eNOS activity was initiated by replacing the preincubate with PBS containing 1.5 µCi/ml 3H-L-arginine. After 15 min, the reaction was stopped by adding 1 N trichloroacetic acid, the cells were freeze-fractured in liquid nitrogen and scraped with a rubber spatula, the contents of each well were ether extracted, and the 3H-L-citrulline generated was isolated using Dowex AG50WX-8 columns and quantified by liquid scintillation spectroscopy.
To evaluate eNOS activation by estrogen, 3H-L-arginine conversion to 3H-L-citrulline was measured in intact cells either under basal conditions or in the presence of 10–8 M E2 during 15-min incubations. In previous experiments, the effect of E2 was demonstrable within 5 min, the maximal response was obtained at 10–8 M, and the threshold concentration was 10–10 M (4). The role of estrogen receptors in eNOS activation was confirmed by the addition of ICI 182,780 (10–5 M) during the 15 min incubation for NOS activity. In control experiments, E2 did not stimulate eNOS in COS-7 cells expressing the enzyme alone. Both basal and stimulated NOS activity were completely inhibited by 2.0 mM nitro-L-arginine methyl ester. NOS activity was expressed relative to basal levels, which are designated as 100%, in the same six-well plate. All findings were confirmed in at least three independent studies.
In addition to being localized and modulated on the PM, there is evidence that eNOS can be phosphorylated and stimulated in the Golgi complex (25). Recent studies also suggest that estrogen can cause the activation of Akt kinase in the Golgi apparatus (44). To provide a focused evaluation of PM eNOS regulation by PM-associated ER, eNOS activation was assessed not only in whole cells but also in purified PMs reconstituted in 50 mM Tris HCl buffer (pH 7.4) with 0.1 mM EDTA, 10 µg/ml pepstatin A, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml N-p-tosyl-L-lysine chloromethyl ketone, 10 nM phenylmethylsulfonyl fluoride, 3 mM dithiothreitol, and 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (12). Membranes (10 µg) were incubated with 2.0 µCi/ml of 3H-L-arginine and 2 M cold L-arginine, and citrulline generation was evaluated over 60 min at 37 C. The assay was terminated by the addition of 400 µl of 40 mM HEPES buffer (pH 5.5), with 2 mM EDTA and 2 mM EGTA. 3H-L-citrulline generated was isolated and quantified as described for the intact cell experiments. The variability in basal activity in isolated membranes is typically ± 10%. All observations were confirmed in a minimum of three separate studies.
To determine the effect of estrogen on eNOS in the isolated membranes, incubations for activity were performed in the absence or presence of 10–8 M E2. In control experiments, E2 did not stimulate eNOS in membranes from COS-7 cells lacking ER. To confirm the role of membrane-associated ER in E2 responses, studies were done in the absence or presence of ICI 182,780 (10–5 M). NOS activity in all membrane samples was fully inhibited by 2 mM nitro-L-arginine methyl ester.
Coimmunoprecipitation
The immunoprecipitation of ER was performed by methods described previously (19). Briefly, COS-7 cells transfected with wild-type ER or mutant forms of the receptor 24 h earlier were placed in DMEM without serum for another 24 h. The cells were treated with vehicle or E2 (10–8 M) for 0–15 min and the postnuclear supernatants were isolated. The wild-type or mutant receptors were immunoprecipitated from 200 µg of postnuclear supernatant using monoclonal ER antibody (AER320; Labvision). Coimmunoprecipitated p85 subunit of PI3 kinase was detected using polyclonal anti-p85 antibody (Santa Cruz Biotechnology, Inc.).
Statistical Analysis
Differences in observations between treatment groups were evaluated by one-way ANOVA after establishing equivalence of variances and normal distribution of data. The level of significance of differences between mean values was assessed by the Student-Newman-Keuls method. Values shown are mean ± SEM, with n = 3 or more. Significance was accepted at the 0.05 level of probability.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Current address for L.S.: Department of Pediatrics, Yale University School of Medicine, P.O. Box 208064, 333 Cedar Street, New Haven, Connecticut 06520.
First Published Online October 14, 2004
1 K.L.C. and L.S. contributed equally to this work. 
Abbreviations: CM, Caveolae membrane; E2, 17ß-estradiol; EDAC, l-ethyl-3-[3-(dimethylamino) propyl] carbodiimide; eNOS, endothelial nitric oxide synthase; ER, estrogen receptor; ERE, estrogen response element; NLS, nuclear localization signal; NO, nitric oxide; PI3 kinase, phosphoinositide 3-kinase; PM, plasma membrane; RACK1, receptor for activated C-kinase.
Received for publication January 9, 2004. Accepted for publication October 5, 2004.
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, P < 0.05 vs. E2 alone. Similar findings were obtained in three independent experiments.
