Divergent Pathways Regulate Ligand-Independent Activation of ER{alpha} in SK-N-BE Neuroblastoma and COS-1 Renal Carcinoma Cells

Cesare Patrone, Elisabetta Gianazza, Sabrina Santagati, Paola Agrati and Adriana Maggi

Centre Molecular Pharmacology Laboratory (C.P., S.S., P.A., A.M.) and Atherosclerosis Laboratory (E.G.) Institute of Pharmacological Sciences University of Milan Milan, Italy 1–20133


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The {alpha}-estrogen receptor (ER{alpha}) transcriptional activity can be regulated either by binding to the cognate ligand or by intracellular signaling pathways responsive to a variety of factors acting through cell membrane receptors. Studies carried out in HeLa and COS-1 cells demonstrated that the cross-coupling between estrogen and growth factor receptors is mediated by p21ras and requires phosphorylation of a specific serine residue (Ser 118 in the human ER{alpha} and Ser 122 in mouse ER{alpha}) located in the ER{alpha} N-terminal activation function 1 (AF-1). Likewise, in the SK-N-BE neuroblastoma cell line p21ras is involved in the cross-coupling between insulin and ER{alpha} receptors. However, in this cell line Ser 122 is not necessary for insulin-dependent activation of unliganded ER{alpha}. In addition, after insulin activation, the electrophoretic mobility associated to serine hyperphosphorylation of ER{alpha} in SK-N-BE and in COS-1 cells is different. Our study rules out the possibility of tyrosine phosporylation in unliganded ER{alpha} activation by means of transactivation studies of ER{alpha} tyrosine mutants and analysis of Tyr phosphorylation immunoreactivity. The two cofactors for steroid receptors RIP 140 and SRC-1 do not seem to be specifically involved in the insulin-induced ER{alpha} transactivation. The present study demonstrates the possibility of an alternative, cell-specific pathway of cross-coupling between intracellular and membrane receptors, which might be of importance for the understanding of the physiological significance of this mode of activation in the nervous system.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogens represent an important class of hormones for the maturation and functioning of several tissues including the nervous system (1, 2, 3). Estrogen action is mediated by intracellular receptors [named estrogen receptor (ER){alpha} and ERß], members of a large family of hormonally inducible transcription factors. After binding to the cognate ligand, the ERs modulate the transcription rate of target genes by means of protein-protein interactions affecting the stability of the preinitiation complex (4, 5, 6, 7). With regard to the ER{alpha}, it is known that these interactions are mediated by two transactivation regions: AF-1 located in the ER{alpha} N terminus and AF-2 located in the hormone-binding domain at the C terminus (8, 9). The two transactivating regions may function independently, or cooperate, depending on the target promoter and the presence of tissue-specific factors (10, 11, 12).

Phosphorylation plays an important role in the modulation of steroid hormone receptor functions, including receptor processing and shuttling, DNA binding, and transcriptional activation (13, 14, 15, 16). In general, this posttranslational modification occurs on serine residues (16, 17, 18, 19, 20, 21), but tyrosine phosphorylation has also been reported (22, 23). The finding that ER{alpha} can be phosphorylated by p21ras- or cAMP-dependent pathways indicates that this posttranslational modification may be involved in the cross-coupling between nuclear receptors and other signal transduction pathways. Indeed, several signaling pathways responsive to a variety of hormones, including dopamine (24), epidermal growth factor (25), insulin, and insulin growth factor-1 (IGF-I) (26, 27), have been shown to activate ER{alpha} in a ligand-independent fashion. The mutation of the major phosphorylation site (Ser 118 in human and Ser 122 in mouse ER{alpha}) proved that the p21ras-dependent pathway involves AF-1 in fibroblast-like COS-1 (28) and epithelial-like HeLa cells (29). Because of the distant embryological origin of COS-1 and HeLa cells, it has been assumed that p21ras-dependent activation occurs via an identical mechanism in all cell types. However, our previous studies in a cell line of neural origin (SK-N-BE neuroblastoma cells) proved AF-2 and not AF-1 as essential for p21ras-dependent ER{alpha} activation (30). This finding prompted us to further investigate the diversities of ER{alpha}-independent activation in our cell system.

The present results show that not only is Ser 122 dispensable for p21ras-activation of ER{alpha} in neuroblastoma cells, but also the phosphorylation of ER{alpha} and its capability of interaction with coactivators can differ in this cell line. Our present study therefore suggests the existence of cell-specific events controlling unliganded ER{alpha} activity.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutation of Serine 122 Suppresses p21ras-Dependent Activation of ER{alpha} in COS-1 but not in SK-N-BE Cells
In human ER{alpha}, phosphorylation of Ser118 is important for the transcriptional activity of AF-1 (18). This site was recently shown to be a target for mitogen-activated protein kinases and essential for the cross-coupling between epidermal growth factor, IGF-I, and ERs (28, 29). These findings are in contrast with the observation that deletions encompassing the entire AF-1 domain of the mouse ER{alpha} did not affect the capability of insulin/IGF-I to activate unliganded ER{alpha} in neuroblastoma cells (30).

To examine the function of the Ser122 phosphorylation site (the mouse counterpart of the human Ser118) on p21ras-dependent activation of ER{alpha} in neuroblastoma, SK-N-BE cells were cotransfected with the dominant positive mutant of p21ras [ p21(Leu61)Hras] in the presence of the native (wtER) or Ser122-Ala mutated mouse ER{alpha}. The reporter plasmid used was pVEREtkCAT. Monitoring of the chloramphenicol acetyltransferase (CAT) immunoreactivity indicated that, in SK-N-BE cells, the p21ras dominant positive mutant increased the transcriptional activity of unliganded wt ER{alpha} about 4-fold above the control levels. In these cells, the activated p21ras could still increase ER{alpha} transcriptional activity despite the presence of the Ser122 to Ala mutation (Fig. 1Go, top). De facto, p21ras activated the mutated receptor to an even greater extent (~8-fold) than the wild type. Conversely, in COS-1 cells, the 2- to 3-fold p21ras-dependent activation of ER{alpha} was completely abolished by the presence of this amino acid substitution (Fig. 1Go, bottom). In both cells lines, the constitutive, but not the estrogen-dependent, transcriptional activity of the Ser122-Ala mutant was considerably lower than to wtER (-57% and -73% in COS-1 and SK-N-BE cells, respectively) proving that also in SK-N-BE cells the integrity of this phosphorylation site is of importance for ER{alpha} transactivation functions.



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Figure 1. p21 ras-Dependent Activation of the Mouse ER{alpha} Mutant Ser 122

SK-N-BE (top) and COS-1 (bottom) cells were cotransfected with 1.2 ng/µl of the reporter pVEREtkCAT, 0.6 ng/µl of the plasmids containing either the native or the Ser122 mutant of the ER{alpha}, and 0.2 ng/µl of pCMVßgal in the presence or absence of the p21ras dominant positive mutant (30 ng/µl). Where indicated, 1 nM 17ß-estradiol was added. The cells were harvested 24 h after the hormonal treatment. Data are expressed as fold induction with respect to the CAT content of cells transfected with the native ER{alpha} and the reporter gene. The bars represent the mean ± SD of four independent experiments each done in duplicate.

 
Western Blot Analysis of Native and Activated ER{alpha} in COS-1 and SK-N-BE Cells
ER{alpha} can be phosphorylated in several serine residues, and the accumulation of this phosphate groups changes its electrophoretic migration profile (19, 21). To determine whether insulin could influence the phosphorylation state of ER{alpha}, we studied its migration profile in COS-1 and SK-N-BE cells after 18 h treatment with 1 µM insulin. A band of 66- kDa molecular mass, corresponding to the uterine ER{alpha}, was revealed by the anti-ER{alpha} monoclonal antibody (Ab) H222 in untreated COS-1 and SK-N-BE cells stably transfected with ER{alpha} [SK-ER3 (31)] (Fig. 2Go, lanes 1 and 4). In some, but not all, of the experiments, bands at lower molecular mass could be detected by the H222 Ab. These bands could be due to the presence of degradation products. After insulin stimulation, ER{alpha} migrated differently in COS-1 and SK-N-BE cells. In COS-1 cells, ER{alpha} migrated as a triplet (the majority of the receptor still remained in the 66-kDa band; about 30% migrated in the two upper bands), while no changes in the banding pattern were observed in SK-N-BE cells (Fig. 2Go, lanes 2 and 5).



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Figure 2. Western Blot Analysis of Native and Activated ER{alpha} in COS-1 and SK-N-BE Cells

Mouse ER{alpha} transiently transfected COS-1 cells (COS-1) and stably transfected SK-N-BE cells (SK-ER3) were grown at semiconfluence and then treated for 18 h with 1 µM insulin or 1 nM 17ß-estradiol. Whole cell extract (100 µg/lane) was resolved by SDS-PAGE and transferred to nitrocellulose. ER{alpha} protein was detected by immunostaining using the H222 Ab. The experiment was repeated three times with superimposable results.

 
After treatment with estradiol, a triplet was recognized by the H222 Ab (Fig. 2Go, lane 6) in both cell lines (Fig. 2Go, lane 3 and 6). As shown by several authors, this effect is most likely due to the ER{alpha} hyperphosphorylation occurring after ligand activation (17, 21, 32). In both cell lines, estradiol treatment produced a 40–50% decrease of ER{alpha} immunoreactivity due to autologous down-regulation (33).

Since we have previously demonstrated that p21ras is essential in insulin-dependent activation of ER{alpha} in neuroblastoma cells, these data support the finding of the previous transient transfection experiments, suggesting that the molecular events induced by insulin differ in the two cell lines studied. Insulin-dependent hyperphosphorylation of ER{alpha} can be observed only in COS-1 cells. However, SK-N-BE cells seem to be supplied with the whole enzymatic apparatus required for ligand-induced ER{alpha} posttranslational modifications, as demonstrated by the fact that estrogen treatment induces changes of ER{alpha} electrophoretic mobility superimposable to those observed in COS-1 cells.

Tyrosine Phosphorylation Is Not Involved in Insulin-Dependent Activation of ER{alpha} in SK-N-BE Cells Stably Transfected with ER{alpha}
ER{alpha} has been reported to be phosphorylated also on tyrosine. The phosphorylated site in this case seems to be unique (23) and localized in a sequence that is phylogenetically very well conserved (Tyr 537 in human and Tyr 541 in mouse ER{alpha}).

To investigate whether the insulin-dependent activation of ER{alpha} involved this residue, we next compared the transcriptional activity of native ER{alpha} with mutants in which two different amino acids had been substituted for tyrosine 541 (Y541-A and Y541-F). The mutants were screened using a pVEREtkLUC reporter (Fig. 3Go). The substitution of tyrosine with phenylalanine in position 541 did not affect the transactivation induced by insulin (or ß-estradiol) suggesting that this residue is not a target for insulin activity. Alanine substitution in position 541 significantly increased (+100%) the ER{alpha} constitutive transcriptional activity, according to observations of other authors.



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Figure 3. Estrogen- and Insulin-Dependent Activation of ER{alpha} Tyrosine 541 Mutant in SK-N-BE

Cells were transfected with the expression plasmid carrying the wtER{alpha} or ER{alpha} mutants in which either phenylalanine (Y541-F) or alanine (Y541-A) substituted for tyrosine 541; the reporter gene was pVEREtkLUC. The extracts were prepared 24 h after treatment with 1 nM 17ß-estradiol or 1 µM insulin and assayed for ß-galactosidase and luciferase activity. Results are expressed as luciferase activity normalized for variations in transfection efficiency using the ß-galactosidase internal control. The bars represent the average ± SD of three separate experiments in which all the samples were assayed in triplicate.

 
To rule out the implication of other tyrosine residues in insulin activation of ER{alpha}, SK-N-BE cells stably transfected with ER{alpha} (SK-ER3 cells) were treated for 18 h with insulin, 17ß-estradiol, or the appropriate solvent, and then harvested and lysed. Equal amounts of whole-cell extract proteins were resolved by denaturing gel electrophoresis. After blotting, the band corresponding to ER{alpha} was detected with the H222 anti-ER{alpha} monoclonal Ab (Fig. 4Go, right). The membrane was then stripped and reprobed with the selective 4G10 anti-phosphotyrosine monoclonal Ab (Fig. 4Go, left). As shown before, estrogen-dependent hyperphosphorylation produced several forms of ER{alpha} migrating at apparent 66–68 kDa. Insulin treatment neither changed the migration profile of the receptor nor influenced the staining intensity of the 66-kDa protein with the 4G10 Ab. The same experiment was repeated with immunoprecipitated ER{alpha}. Also in this case, in insulin-treated cells, the intensity of the 4G10 staining was not significantly different from control cells (Fig. 5Go). A further proof that the two antibodies, H222 and 4G10, were both recognizing the ER{alpha} is given by the experiment done in ER{alpha}-negative SK-N-BE cells in which both antibodies failed to recognize any protein of 66–68 kDa (Fig. 5Go).



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Figure 4. Constitutive or Insulin- and Estradiol-Induced Phosphorylation of ER{alpha} Stably Transfected in SK-N-BE Cells (SK-ER3)

Whole cell extracts (200 µg) were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was stained with the H222 Ab to label the ER{alpha} (right panel), stripped, and reprobed with the 4G10 antiphosphotyrosine Ab (left panel). See Materials and Methods for further experimental details. The autoradiograms represent one of three separate experiments, all providing superimposable results.

 


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Figure 5. Constitutive and Insulin-Induced Tyrosine Phosphorylation of Immunoprecipitated ER in SK-ER3 Cells

ER{alpha} was immunoprecipitated using the monoclonal Ab C542, resolved on SDS-PAGE, as described in Materials and Methods, and probed with the anti-ER{alpha} monoclonal Ab H222 (right panel). The membrane was then stripped and reprobed with the 4G10 antiphosphotyrosine Ab to visualize ER{alpha} tyrosine phosphorylation (left panel).

 
It is of interest to point out that, in spite of the down-regulation of ER{alpha}, due to the estradiol treatment, the intensity of the 4G10 staining in Fig. 4Go does not change. This suggests that, after estrogen activation, tyrosine residues of ER{alpha} are phosphorylated.

The observations that mutations of tyrosine 541 do not block insulin-dependent activation of ER{alpha} and that tyrosine phosphorylation of ER{alpha} is not modified by insulin support the conclusion that tyrosine phosphorylation of ER{alpha} is not relevant for insulin-dependent activation in SK-N-BE cells.

SRC-1 and RIP-140 Do Not Play a Significant Role in Insulin-Dependent Activation of ER{alpha} in SK-N-BE Cells
Cofactors and coactivators play an important role in the transcriptional activity of ER{alpha}. We therefore examined whether, in SK-N-BE cells, the ligand-independent activation of ER{alpha} was modulated by the presence of two factors, namely RIP 140 (34) and SRC-1 (35), known to operate through AF-2. Figure 6Go shows that RIP 140 at various concentrations did not exhibit any facilitator effect on insulin-dependent transcriptional efficiency of ER{alpha}. In agreement with reports from other cell lines, this factor facilitates estrogen-dependent activation of ER{alpha}, and its presence at high concentration influences the basal transcriptional activities of ER{alpha} (34). SRC-1 showed a concentration-dependent effect on the constitutive transcriptional activity of the unliganded ER{alpha}. The presence of insulin did not further augment ER{alpha} activity (Fig. 7Go). The effect of SRC-1 on ER{alpha} constitutive transcription makes it difficult to establish whether this coactivator displays any facilitator effect on insulin action.



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Figure 6. RIP 140 Cotransfection Does Not Affect Insulin-Dependent Activation of Unliganded ER{alpha}

SK-N-BE cells were transfected as described in Materials and Methods in the presence or absence of increasing concentrations of a vector expressing RIP 140 (0.03, 0.3, and 3 ng). The bars represent the average ± SD of three independent experiments, each done in triplicate.

 


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Figure 7. SRC-1 Effect on ER{alpha} Transcriptional Activity in SK-N-BE Cells

SK-N-BE were transfected as in Fig. 5Go. The concentrations of the SRC-1-expressing plasmid were: 100, 200, and 600 ng. Bars represent a single experiment done in triplicate. The experiment was repeated five times even with a wider range of SRC-1 concentrations with superimposable results.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The main conclusion of the present study is that, in SK-N-BE neuroblastoma cells, insulin-dependent ER{alpha} activation occurs through a diverse mechanism from the cell lines so far studied. In fact, while insulin/IGF-I receptors and ER{alpha} are cross-coupled via the p21ras pathway in several cells lines, SK-N-BE included, the mechanism of unliganded ER{alpha} activation in neuroblastoma cells seems to differ downstream of p21ras. We provide two lines of evidence to support this conclusion: 1) insulin activates transcriptionally the ER{alpha} mutant S122A in SK-N-BE, but not in COS-1 cells (Fig. 1Go); and 2) insulin gives rise to two different ER{alpha} electrophoretic migration profiles in COS-1 and SK-N-BE cells (Fig. 2Go). These results support our previous studies in SK-N-BE, demonstrating that only the ER{alpha} C terminus AF-2 is crucial for unliganded ER{alpha} activation (30), and are in agreement with others (28, 29) proving the importance of serine phosphorylation in AF-1 in COS-1 cells.

Our study rules out Ser122 and ER{alpha} N terminus as a target for insulin activity; however, since other serines in the hinge region and in the C terminus were described as susceptible to phosphorylation, at present we can not exclude that, in SK-N-BE cells, insulin activates ER{alpha} via serine phosphorylation. On the other hand, an involvement of tyrosine phosphorylation appears as unlikely on the basis of our study with the mutants of Tyr541 (the well recognized tyrosine phosphorylation site in AF-2) (23) (Fig. 3Go) and of the immunoenzymatic labeling of ER{alpha} using the antiphosphotyrosine Ab 4G10 (Figs. 4Go and 5Go).

Transcription activation of ER{alpha}, as well as of other intracellular receptors, requires a series of events initiated by the dissociation of the receptor from the inhibitory proteins [heat shock protein (HSP), immunophilins, etc.]. The question arising is how insulin can activate the receptor. Considering that the receptor might not be entirely assembled with HSP and that, as recently suggested by Smith et al. (39), the ER{alpha}-HSP complexes might continuously dissociate and reassociate, we propose that the quota of free receptor could be the target for insulin activation. Hence, insulin might induce posttranslational modifications enabling ER{alpha} to better interact with transcription coactivators or might induce the synthesis of specific coactivators capable of modulating the unliganded receptor activity. Of interest is our observation that, in SK-N-BE cells, SRC-1 activates transcriptionally unliganded ER{alpha}. Because in other cell lines SRC-1 does not interact with unliganded ER{alpha}, our findings might suggest that in SK-N-BE the receptor conformation, possibly due to the presence of specific factors, is more ready to recognize SRC-1 or the transcriptional machinery. However, at this stage, we cannot point to any SK-N-BE-specific molecule determining this effect.

The peculiarity of the cell line here studied seems to be restricted to insulin-dependent activation of ER{alpha}. In fact, in the case of estrogen activation, the SK-N-BE ER{alpha} undergoes changes in the electrophoretic mobility (Fig. 2Go) similar to those described in other cell systems by Pateranne and co-workers and Le Goff et al. (19, 32). Furthermore, the results of the studies carried out with the Tyr 541 mutants (Fig. 3Go) are in complete agreement with those of White and associates (36) and Weis et al. (37). However it is noteworthy that, by the use of antiphosphotyrosine antibodies, we prove that estrogen-dependent activation of ER{alpha} causes an increased immunoreactivity (Fig. 4Go), suggesting the possibility of phosphorylation in tyrosines other than Tyr 541.

In conclusion, in all the cell types tested so far, the cross-coupling between ER{alpha} and other intracellular signaling pathways was demonstrated to occur via the AF-1 site. The neuroblastoma cell line SK-N-BE represents an exception to this rule. At present it is difficult to demonstrate that the peculiarity of the neuroblastoma cell line reflects a physiological requirement for ER{alpha} action in neural cells. However, in view of the multiple roles of ER{alpha} in the nervous system, both during brain development and later in the mature brain, we propose that neural cells may utilize insulin activation of AF-2 as a default pathway to ensure the transcription of AF-2-dependent target genes even in the presence of low blood level of estrogens. For instance, it could prove beneficial that neural growth in the developing brain, or survival and plasticity in both developing and mature brain, was under the control of a multiplicity of hormonal signals funnelled through the ER. Supporting this view is the observation that insulin and its receptor are present at high concentration in the same brain areas in which ER{alpha} appears to be localized (40).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reagents
Chemicals were purchased from Bracco (Milan, Italy) if not otherwise specified. Plasticware was from Corning (distributed by Disa, Milan, Italy). Tissue culture media from Sigma Chemical Corp. (Milan, Italy) and GIBCO (Paisley, U.K.). FBS (from Poxoid, Milan, Italy; distributed by Unipath, Milan, Italy). Insulin and 17ß-estradiol purchased from Sigma were resuspended and stored as previously described (30). The murine ER expression plasmids pMT2MOR, pMT2MOR S122, Y541-A, and Y541-F (37) and pBRIP 140 (34), were from M. G. Parker (London, U.K.); pVEREtkLUC, pVEREtkCAT, pCMVßgal, and pBK-CMVSRC-1 (35) were provided by M. J. Tsai (Houston, TX); the dominant positive mutant p21(Leu-61)H-ras (41) was supplied by E. Martegani (Milan, Italy).

Cell Culture and Transient Transfection
SK-N-BE and COS-1 cells were grown in RPMI 1640, without phenol red and supplemented with 10% charcoal-stripped serum, and transfected in 1% serum by the calcium phosphate coprecipitation method, as previously described (30). Generally, 50,000 cells were plated in 24-multiwell plates and grown for 24 h in 200 µl of medium. For the transfection, growth medium was replaced with 400 µl of DMEM to which 50 µl of the calcium phosphate/DNA suspension were added. In the 50-µl suspension the DNA concentration was as follows: 0.2 ng/µl of the internal control pCMVßgal, 0.6 ng/µl of plasmids pMT2MOR or MOR mutants, 1.2 ng/µl of the reporter plasmids and the carrier pGEM3Z to a final DNA concentration of 25 ng/µl. In some experiments, the above plasmids were cotransfected with expression plasmids for RIP 140, SRC-1, and p21(Leu-61)H-ras or the corresponding empty vectors. Details in the amounts used are reported in the figure legends. The reporters were either pVEREtkCAT or pVEREtkLUC. In transient transfections the hormonal treatments were done by addition of the specified hormones in the medium after removal of the cotransfection mixture. We used 1 nM 17ß-estradiol and 1 µM insulin; the treatments were carried out for 24 h, after which the cells were harvested and extracts were prepared for ß-gal, CAT, or luciferase assays as previously specified (30). Luciferase activity was measured by integrating the luminescence signal for 15 sec; experimental values are expressed as arbitrary luminescence units. CAT content was measured by an immunoenzymatic method (CAT ELISA kit, Boehringer Mannheim, Milan, Italy), and luciferase experimental values were normalized on the protein concentration of the extract (42) and the ß-galactosidase activity.

Western Analysis of ER{alpha} Protein
ER{alpha}-containing extracts were prepared in which the cells were resuspended (~8 x 106) in 200 µl of lysis buffer [50 mM Tris-HCl, pH 8, at 20 C, 150 mM NaCl, 10% (vol/vol) glycerol, 1 mM EGTA, 1 mM sodium-orthovanadate, 5 µM ZnCl2, 100 mM NaF, 1 mM phenylmethylsulfonylfluoride, 1.3% (wt/vol) sodium-deoxycholate, 1% (vol/vol) Triton-X100]. The cell lysate was frozen and thawed twice in liquid nitrogen for 1 min/cycle and centrifuged at 100,000 x g for 15 min. The supernatants were collected and the protein content was measured by Bradford’s method (42). Equal amounts of protein extract (100–500 µg) from COS-1 and SK-N-BE cells were either immunoprecipitated with the C542 monoclonal anti-ER{alpha} Ab (Stressgene Biotechnology Corp., Victoria, British Columbia, Canada) or directly loaded (100–200 µg) on SDS-PAGE 4–12% T polyacrylamide gradients cast with discontinuous Laemli buffer and resolved using a Protean II apparatus at 500 V and 70 mA. ER{alpha} was immunoprecipitated from the supernatant of the whole-cell extracts above described. Five hundred micrograms of each sample were incubated overnight at 4 C with 5 µg of the ER{alpha} monoclonal Ab C542. Protein G Sepharose (10 mg) (Pharmacia Biotech, Uppsala, Sweden), prewashed in buffer A (50 mM Tris-HCl, pH 8, 5% albumin) and resuspended in 125 µl of lysis buffer, was added to the samples and incubated for 3 h at 4 C with rotation. The pellets, obtained after centrifugation for 15 sec at 4 C at 12,000 x g, were washed four times with 500 µl of lysis buffer and once with distilled water. Before loading on the gel, the immunoprecipitated proteins of each sample were eluted from Protein G Sepharose by boiling the pellet for 10 min in 30 µl electrophoresis buffer containing 10 µl of Laemmli buffer 3x (200 mM Tris-HCl, pH 8, 30% glycerol, 6% SDS, 15% ß-mercaptoethanol, and 0.075 bromophenol blue) and 20 µl of lysis buffer. Color markers (6.5–205 kDa, Sigma) were used as migration reference. The run was continued for 45 min after the exit of the tracking dye off the anodic edge of the slab. Electroblotting was at 400 mA for 9 h at 4 C in a Bio-Rad tank (Protean 228 II). After protein staining with Red Ponceau, 50-mm strips, centered on the albumin standard, were cut from nitrocellulose filters. The membrane was saturated with 5% (wt/vol) milk proteins in Tris borate buffer containing 0.1% Tween 20. Using the anti-ER rat monoclonal H222 (Abbott Laboratories, Rome, Italy) as primary Ab, the secondary Ab was a rabbit antirat-IgG Ab coupled with horseradish peroxidase, HRP (Vector Laboratories, Burlingame, Ca). Both antibodies were used at 1:10,000 dilution. When specified, the membrane was stripped free of the immunoreagent, tested for successful stripping, and reprobed with an antiserum raised against phosphotyrosine (4G10, UBI, distributed by DISA, Milan, Italy) at 1:8,000 dilution. In this case, the secondary Ab was an antimouse IgG HRP-coupled Ab (Vector Laboratories, Burlingame, CA). The zymogram for peroxidase was developed with the ECL reagent (Amersham, Milan, Italy). The Hyperfilm-MP (Amersham, Milan, Italy) was exposed to chemoluminescent radiation for 5 min to reveal ER bands and 20 min when the 4G10 Ab was used.


    ACKNOWLEDGMENTS
 
We are grateful to Raffaella Barbieri and Monica Rebecchi for their excellent secretarial and technical assistance, to Elisabetta Vegeto and Paolo Ciana for helpful comments, and to E. Martegani, M. Parker, M. J. Tsai, and Abbott Laboratories for providing us with reagents critical for the present study.


    FOOTNOTES
 
Address requests for reprints to: Adriana Maggi, Via Balzaretti 9, Milan Italy.

This work was supported by the European Economic Community (BIOMED Programme Pl962286), the Italian Association for Cancer Research (AIRC), Telethon Italy (E600), and the Istituto Superiore di Sanità (93/J/T61) and National Research Council (Target Project Biotechnologies).

Received for publication November 17, 1997. Revision received January 27, 1998. Accepted for publication February 11, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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