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Short Report 2577 Identification of an estrogen receptor a non covalent ubiquitin-binding surface: role in 17b-estradiolinduced transcriptional activity Valeria Pesiri, Piergiorgio La Rosa, Pasquale Stano and Filippo Acconcia* Department of Science, Section Biomedical Science and Technologies, University Roma Tre, Viale Guglielmo Marconi, 446, 00146, Rome, Italy *Author for correspondence ([email protected]) Journal of Cell Science Accepted 11 March 2013 Journal of Cell Science 126, 2577–2582 ß 2013. Published by The Company of Biologists Ltd doi: 10.1242/jcs.123307 Summary Ubiquitin (Ub)-binding domains (UBDs) located in Ub receptors decode the ubiquitination signal by non-covalently engaging the Ub modification on their binding partners and transduce the Ub signalling through Ub-based molecular interactions. In this way, inducible protein ubiquitination regulates diverse biological processes. The estrogen receptor alpha (ERa) is a ligand-activated transcription factor that mediates the pleiotropic effects of the sex hormone 17b-estradiol (E2). Fine regulation of E2 pleiotropic actions depends on E2dependent ERa association with a plethora of binding partners and/or on the E2 modulation of receptor ubiquitination. Indeed, E2induced ERa polyubiquitination triggers receptor degradation and transcriptional activity, and E2-dependent reduction in ERa monoubiquitination is crucial for E2 signalling. Monoubiquitinated proteins often contain UBDs, but whether non-covalent Ub–ERa binding could occur and play a role in E2–ERa signalling is unknown. Here, we report an Ub-binding surface within the ERa ligand binding domain that directs in vitro the receptor interaction with both ubiquitinated proteins and recombinant Ub chains. Mutational analysis reveals that ERa residues leucine 429 and alanine 430 are involved in Ub binding. Moreover, impairment of ERa association to ubiquitinated species strongly affects E2-induced ERa transcriptional activity. Considering the importance of UBDs in the Ub-based signalling network and the central role of different ERa binding partners in the modulation of E2-dependent effects, our discoveries provide novel insights into ERa activity that could also be relevant for ERa-dependent diseases. Key words: 17b-estradiol, Estrogen receptor, Ubiquitin, Ubiquitin binding domains, Signal transduction Introduction Ubiquitination influences the functions of the target proteins by either affecting their stability (i.e. Ub proteolytic functions) or endowing them with additionally signalling properties as well as by creating new surfaces for intra- and intermolecular interactions (i.e. Ub non-proteolytic functions). Thus, Ub is an intracellular messenger, whose nature as a signal resides in the Ub modification. Single Ub moieties (i.e. monoubiquitination) or polyubiquitin chains (e.g. K63-linked chains) attached to the substrate create structural determinants that can be further exploited for molecular interactions. Consequently, cells have evolved Ub receptors that bind to the ubiquitinated protein by non-covalently contacting the Ub modification through specific UBDs and in this way decode, transduce and amplify the ubiquitination signal diversity into regulation of physiological functions (e.g. cell proliferation and migration) (Acconcia et al., 2009; Husnjak and Dikic, 2012; Woelk et al., 2007). The ERa is a ligand-activated transcription factor that mediates the cellular effects of the steroid hormone E2 through modifications in the pattern of gene expression. Regulation of ERa gene transcription requires the receptor recruitment to target gene promoters either directly through interaction with estrogen responsive elements (i.e. ERE sequences) or indirectly through ERa interaction with other transcriptional factors (e.g. AP-1; Sp1) (Ascenzi et al., 2006; Smith and O’Malley, 2004). Mounting evidence indicates that ERa activities are modulated by Ub (La Rosa and Acconcia, 2011). Polyubiquitination controls the E2dependent regulation of 26S-proteasome ERa degradation, which is interlaced with ERa transcriptional activity (La Rosa and Acconcia, 2011; La Rosa et al., 2012). Non-proteolytic Ub functions in E2:ERa signalling also occur and depend on ERa monoubiquitination, which is negatively modulated by E2 and controls E2-dependent cell proliferation (Eakin et al., 2007; La Rosa et al., 2011a; La Rosa et al., 2011b; Ma et al., 2010). Monoubiquitinated proteins are often Ub receptors that possess at least one UBD (Woelk et al., 2006). Although many different UBDs exist, no specific conservation in terms of UBDs 3Dstructure has been recognized. In fact, UBDs appear to share a common ‘shape’: structural folds that have a regular secondary structure (e.g. a-helix and/or Zn2+-finger) are thought to be the only UBDs common features (Dikic et al., 2009; Husnjak and Dikic, 2012; Woelk et al., 2007). Interestingly, ERa biochemical anatomy consists in six modular domains (A to F). The A/B, D and F domains do not display a folded structure, the C domain (i.e. the DNA-binding domain, DBD) consists in two Zn2+-finger motifs and the E domain (i.e. the ligand binding domain, LBD) is composed of 12 a-helices (Ascenzi et al., 2006). On this basis, we speculated that an UBD could be present in ERa. Here, we report the identification of an ERa Ub-binding surface (ERa-UBS) that is required for E2-dependent ERa transcriptional activity. 2578 Journal of Cell Science 126 (12) Results and Discussion Journal of Cell Science Identification of an Ub-binding surface on ERa In vitro pull-down experiments were done to investigate the possibility that ERa binds Ub. We found that the A/B and the E domains but not the C domain were able to pull-down ubiquitinated species from total cellular lysates (Fig. 1A). Because the A/B domain is non-structured and displays weak association to ubiquitinated species when compared to the 12 ahelix-containing E domain, we focused on the E domain Ubbinding ability. Fig. 1B shows that this receptor portion is also able to pull-down recombinant K63-linked polyubiquitin chains, thus demonstrating that the ERa E domain non-covalently contacts Ub on the Ub-modified proteins. The E domain also bound recombinant K48-linked polyubiquitin chains but not purified mono-Ub (supplementary material Fig. S1A). Thus, as in the case of other UBDs (Woelk et al., 2007), the ERa Ub-binding surface (ERa-UBS) in the E domain displays preferential Ubbinding ability and could possess a low Ub-binding affinity. We next investigated which portion of the ERa E domain is necessary for Ub-binding. The N-terminal part of the E domain (i.e. amino acids 301–439) but not the E domain C-terminus (i.e. amino acids 439–547) was able to pull-down ubiquitinated species from total cellular lysates (Fig. 1C) and recombinant K63-linked polyubiquitin chains (Fig. 1D) although with different amount of bound materials with respect to the intact E domain (Fig. 1A,B). This evidence indicates that the region of the ERa E domain that non-covalently associates in vitro with Ub is located within the protein region encompassing the amino acids 301–439. Interestingly, our data indicate that disruption of the ERa E domain reduces the ERa Ub binding abilities. Unfortunately, we could not narrow down a smaller section of the E domain that Fig. 1. Ub-binding ability of ERa. (A,C) Schematic of ERa (A, top) and ERa E (C, left) domain deletions are depicted. Amino acid positions and domains are indicated. In vitro pull-down assay using the indicated GSTtagged ERa constructs. GST-fusion proteins were incubated with total cellular lysates extracted from growing HeLa cells and analyzed by immunoblot as indicated. (B,D) As for A and C except that fusion proteins were incubated with synthetic polyUb2-7 linked by Lys63. associated to ubiquitinated species by using rational deletions of the N-terminal protein portion (i.e. 301–439) (data not shown). Thus, it is most likely that the ERa-UBS requires an intact E domain 3D-folding. In turn, in silico molecular modelling experiments would help rationalize the biochemical structural requirements for ERa E domain:Ub interaction. For these reasons, we decided to introduce specific point mutations within the minimal Ub-binding region (i.e. 301–439) in the context of the full-length E domain in order to find the critical residues required for Ub-binding. The residues I358 and the Q375 are important for in vitro ERa monoubiquitination (Eakin et al., 2007), thus they may be part of the ERa-UBS (Woelk et al., 2006). Because the E domain double-mutated in I358 and Q375 to A (IQAA) is native-like (Eakin et al., 2007) but did not display any significant differences in Ub-binding when compared with the wt Fig. 2. Characterization of the ERa-UBS. (A,C,D) Top: schematic of ERa E domain. Amino acid positions are indicated. Bottom: In vitro pull-down assay using the indicated GST-tagged ERa constructs. GST-fusion proteins were incubated with total cellular lysates extracted from growing HeLa cells (A and C) or with synthetic polyUb2-7 linked by Lys63 (D) and analyzed by immunoblot as indicated. * indicates significant differences with respect to the relative wild-type sample. (B) Top: ClustalW (http://www.ebi.ac.uk/ Tools/msa/clustalw2/) alignment of ERa E domain with RNF168 UMI domain (Pinato et al., 2011). Middle: Projected helices using the Helical Wheel Projections software tool (http://rzlab.ucr.edu/scripts/wheel/wheel. cgi). Bottom: Three-dimensional structure of the human ERa LBD-E2 complex (1ERE) (Brzozowski et al., 1997); E2 has been removed. The structure is drawn in green; conserved amino acids between the ERa E domain and RNF168 UMI domain are space-filled and stained as indicated. Journal of Cell Science ERa ubiquitin-binding surface E domain (Fig. 2A), I358 and Q375 are not necessary for ERaUBS. Interestingly, bioinformatic analysis identified L428, L429 and A430 in the E domain to have a spatial distribution reminiscent of the UBD called UMI (Pinato et al., 2011), with L428 buried in the E domain core protein matrix and L429 and A430 at least partially exposed to solvent (Fig. 2B). Consistently, while single substitution of L429 or A430 barely affected the Ub-binding abilities of the E domain (Fig. 2C), double mutation of L429 and A430 (L429A,A430G; LAAG) strongly prevented the E domain Ub-binding (Fig. 2C,D). Notably, introduction of the LAAG mutation in ERa did not significantly affect proper receptor folding, as indicated by circular dichroism (CD) on the E-domain GST-fusion proteins (supplementary material Fig. S1B). Based on this evidence, we report that two Ub-binding surfaces could exist within ERa and that the residues L429 and A430 within the E domain are necessary for Ub-binding. Interestingly, as the A/B and the E domains (Ascenzi et al., 2006) are located at the N-terminus and at the C-terminus of the ERa monoubiquitination sites (K302 and K303) (Eakin et al., 2007) respectively, it is tempting to speculate that avidity-based Ubbinding interactions could direct intra-molecular communications between distant ERa domains. In this respect, structural crosstalk between distant ERa A/B and E domains has been reported to be required for the modulation of ERa activities (Métivier et al., 2002). Role of ERa-UBS in E2-induced ERa gene transcription Next, we sought to determine the role of ERa non-covalent Ubbinding on receptor transcriptional activity. Thus, the LAAG 2579 mutation was generated in the context of the full length receptor (wt) (Figs 3, 4) and transiently transfected in HeLa or HEK293 cells. Initially, we verified if the LAAG mutation in the context of the full length ERa reduces Ub-binding also in cells by evaluating if wt ERa was able to associate to ubiquitinated proteins in cells. The anti-Ub blot performed after wt ERa immunoprecipitation resulted in a clear smear only in HEK293 transfected with wt ERa but not in non-transfected HEK293 cells (Fig. 3A). Because ubiquitinated ERa interactors can in principle associate to the receptor both directly through the Ubbased modification and through a different protein surface (Woelk et al., 2006) and the anti-Ub antibody cannot distinguish between these two possibilities, we also performed wt ERa immunoprecipitation by treating the lysates with SDS in order to reduce the amount of receptor interactors (Woelk et al., 2006). Under these conditions, wt ERa was still able to associate with ubiquitinated proteins although less efficiently than in the non-treated lysates (Fig. 3A), thus demonstrating that ERa binds ubiquitinated proteins in cells. Prompted by these results, we next tested the ability of the LAAG mutant receptor to associate with ubiquitinated proteins in cells under the above-mentioned stringent conditions. Immunoprecipitation analysis revealed that introduction of the double mutation L429A,A430G in ERa strongly prevents the receptor ability to associate with ubiquitinated proteins (Fig. 3B). These data demonstrate that the ERa-UBS directs Ub-binding also in cells. Next, we determined whether the reduction in ERa Ub-binding could affect basal ERa transcriptional activity. As shown in Fig. 3C, equal expression of either wt ERa or of ERa LAAG Fig. 3. Characterization of the full length ERa-UBS mutant. (A,B) Ubiquitin and ERa western blot analysis of ERa immunoprecipitation in non-transfected (Nt) and pcDNA FLAG wild-type ERa (Wt) HEK293 cells (A) and in pcDNA FLAG-ERa L429A, A430G (LAAG) mutant transfected HEK293 cells (B). SDS (0.05%) was added to the lysates in A where indicated (+) and in B before immunoprecipitation and the immunoprecipitated complexes were washed in RIPA buffer containing 0.1% SDS. (C) Luciferase assay on the HeLa cells transiently transfected with pcDNA FLAG, pcDNA FLAG-ERa (Wt) or the pcDNA FLAG-ERa L429A, A430G (LAAG) mutant together with the 36ERE TATA reporter plasmid. Insets show Wt and mutant FLAG-ERa expression normalized on vinculin. (D) ERa dimerization analysis in growing HEK293 cells transiently co-transfected with pcDNA FLAG-ERa (Wt) and pEGFP-ERa (Wt), and with pcDNA FLAG-ERa (LAAG) and pEGFP-ERa (LAAG) mutants, as indicated. After transfection, cell lysates were immunoprecipitated with anti-FLAG antibody, followed by immunoblot with anti-ERa antibody (rabbit). (E) Confocal miscrocopy analysis of the HeLa cells transiently transfected with pEGFP-ERa (Wt) or the pEGFP-ERa (LAAG) mutant kept in growing conditions. 2580 Journal of Cell Science 126 (12) Journal of Cell Science Fig. 4. Effect of ERa Ub-binding ability on E2-induced receptor transcriptional activity. (A,C,D) Luciferase assay on the HeLa cells transiently transfected with pcDNA FLAG-ERa (wt) or the pcDNA FLAG-ERa (LAAG) mutant together with either the 36ERE TATA reporter plasmid (A), with pXP2-D1-2966 cyclin D1 reporter plasmid (pD1) (C) or with the protein complement 3 (pC3) reporter plasmid (D) and then treated for 24 hours with different doses of E2 (A) or with 10-9 M E2 (C,D). (B) Western blot analysis of the Bcl-2 protein levels in the HeLa cells transiently transfected with pcDNA FLAG-ERa (wt) or the pcDNA FLAG-ERa (LAAG) mutant and then treated for 24 hours with different doses of E2. Loading control was done by evaluating vinculin expression in the same filter. * indicates significant differences with respect to the relative C sample; ˚ indicates significant differences with respect to the relative wt ERa E2-treated sample. mutant triggered the activation of the artificial ERE-containing reporter constructs (i.e. 3xERE-TATA, pERE) (La Rosa et al., 2012) to comparable levels. ERa binds DNA to ERE sequences as a homodimer (Ascenzi et al., 2006) and co-immunoprecipitation analysis revealed that both wt and mutant receptors were equally able to dimerize (Fig. 3D), thus further suggesting that receptor dimerization is not affected by the LAAG mutation. Moreover, ERa-based signalling is a function of receptor intracellular localization (i.e. nuclear and extra-nuclear) (Ascenzi et al., 2006; La Rosa et al., 2012). Thus, we also tested the effect of LAAG mutation on ERa sub-cellular distribution. Immunofluorescence analysis showed that both GFP-ERa and GFP-ERa LAAG mutant have the same nuclear, cytoplasmic and membranetethered localization (Fig. 3E). Thus, we conclude that mutation of L429 and A430 residues does not influence basal ERa transcriptional activity (e.g. ERE binding), receptor dimerization and sub-cellular localization. Analysis of the ability of E2 to trigger ERa transcriptional activity revealed that the introduction of the LAAG mutation in ERa strongly reduced, with respect to the wt ERa, the receptor ability to activate the 3xERE-TATA reporter constructs as a dose-dependent function of E2 (Fig. 4A) without affecting E2 binding affinity (Table 1). This effect was still evident when the E2-induced ERa LAAG mutant transcriptional activity was tested in the presence of a reporter construct containing the ERE sequence in the context of the protein complement 3 (pC3) natural promoter (La Rosa et al., 2011b) (Fig. 4B). Besides the direct association of ERa to ERE located in the target gene promoters (Reid et al., 2003), the E2-activated receptor can regulate the transcription of non-ERE containing genes (e.g. cyclin D1) through indirect association with other transcription factors (e.g. AP-1, Sp-1) (Ascenzi et al., 2006; Marino et al., 2002; Marino et al., 2003). Even in this case, mutation of the L429 and A430 residues within ERa prevented the E2-induced wt ERa mediated cyclin D1 promoter activation (pD1) (Fig. 4C). Therefore, mutation of the ERa-UBS prevents the E2-dependent activation of direct and indirect ERa transcriptional activity. Finally, we investigated the physiological expression of the endogenous E2-responsive Bcl-2 gene (Acconcia et al., 2005b). Dose-response analysis revealed that the LAAG mutation reduced the ability of the E2:ERa complex to increase the Bcl2 cellular levels as compared with the wt ERa (Fig. 4D). Taken together, these data demonstrate that the reduction in ERa non-covalent Ub-binding results in an overall lower ERa transcriptional activity. In conclusion, we identify here the structural determinants required for ERa to non-covalently associate to Ub and further demonstrate the involvement of the ERa-UBS in the E2induced ERa transcriptional activity. E2-dependent productive gene transcription involves the sequential formation of macromolecular complexes centred on ERa (Manavathi et al., 2012; Tarallo et al., 2011). Many of the ERa binding partners are either ubiquitinated proteins or enzymes of the ubiquitination cascades (i.e. Ub-ligases) (La Rosa and Acconcia, 2011; Métivier Table 1. E2 binding to wild-type and mutant ERa IC50 (M) Kit E2 1.0610 ERa Rec 28 ERa wt 28 3.060.24610 ERa LAAG 210 7.060.91610 2.660.40610210 In vitro ERa affinity (as indicated by the IC50 value) was determined using the HitHunter EFC assay (Discoverx) according to manufacturer instructions for E2 towards either recombinant ERa protein (ERa Rec), wild-type or LAAG mutant receptors (ERa wt or ERa LAAG, respectively) extracted from transfected HEK293 cells. Kit indicates the value of E2 affinity to ERa as reported in the manufacturer’s specification, and has been introduced as a positive control reference; the value is similar to that measured with ERa Rec (Panvera). Results show that the E2 affinity to ERa wt is the same as that for the mutant ERa LAAG. Overall affinity of ERa wt and ERa LAAG with respect to ERa Rec can be ascribed to the fact that ERa wt and ERa LAAG E2 binding abilities were measured in equivalent amounts of total cellular lysates. ERa ubiquitin-binding surface et al., 2003; Reid et al., 2003). Thus, our discoveries open the possibility that the assembly of E2-dependent ERa-based macromolecular complexes could be due to the presence of ERa UBS (present results), ERa monoubiquitination (Eakin et al., 2007; La Rosa et al., 2011a) or both. Remarkably, the ERa extranuclear signalling also integrates and controls receptor transcriptional activity in the modulation of the E2-dependent cellular processes (e.g. proliferation) (Ascenzi et al., 2006; La Rosa et al., 2012). Thus, ERa Ub-binding could play critical roles in the modulation of ERa nuclear and extra-nuclear activities and of E2-regulated cellular processes. Materials and Methods Cell culture and reagents HeLa and HEK293 cells were grown as previously described (La Rosa et al., 2011b). Specific antibodies against FLAG epitope (M2) and vinculin (Sigma, St. Louis, MO, USA); anti-ERa (HC-20) anti-Bcl-2 (C-2) and anti-ubiquitin (P4D1) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used. All other antibodies were purchased by Cell Signalling Technology Inc. (Beverly, MA, USA). All the products were from Sigma-Aldrich (St. Louis, MO, USA). Analytical or reagent grade products, without further purification, were used. 2581 proteins were measured at the concentration of 0.075 mg/ml. For each sample, nine sequential spectra were recorded and averaged. Data are shown, without smoothing, in terms of mean molar ellipticity per residue. Cellular and biochemical assays Protein extraction, immunoprecipitation assays and western blots as well as HeLa and HEK293 cells transfection, luciferase assays and confocal microscopy analysis were performed as previously described (La Rosa et al., 2011b). Statistical analysis Statistical analysis was performed using the ANOVA test with the InStat.3 software system (GraphPad Software Inc., San Diego, CA). In all analyses, P,0.01 was considered significant, but for densitometric analysis P,0.05 was considered significant. Data are means6s.d. of three independent experiments. Author contributions V.P. performed pull-down, transfection, western blot analysis, confocal microscopy, binding and circular dicroism experiments; P.L.R. performed the dimerization assay; P.S. performed circular dicroism and analyzed the relative data; F.A. conceived the idea and planned the research, prepared the plamids, performed the transfection and binding experiments, analyzed the data and wrote the paper. Journal of Cell Science Plasmids and constructs The reporter plasmid 3xERE TATA, protein complement 3 (pC3) and the pXP2D1-2966 plasmid (pD1) and the pcDNA FLAG-ERa have been described (La Rosa et al., 2011b). The pEGFP-ERa was obtained by subcloning the ERa ORF from the pSG5-HE0 (Acconcia et al., 2005a) into the pEGFP-C2 obtained by Dr Simona Polo, IFOM, Milan. Site-directed mutagenesis of the ERa L429 and A430 residues were performed by using the QuikChange kit (Stratagene, La Jolla, CA) and the following oligonucleotides: 59-GAGATCTTCGACATGCTGGCGGCTACATCATCTCGGTTC-39 (for single L429A mutation) on the wt ERa ORF used as template; 59ATCTTCGACATGCTGCTGGGTACATCATCTCGGTTCCGC-39 (for single A430G mutation) on the wt ERa ORF used as template; 59-ATCTTCGACATGCTGGCGGGTACATCATCTCGGTTCCGC-39 (for double L429A, A430G mutation) on the ERa ORF L429A mutant used as template (bold underlined nucleotides differ from the wt ERa ORF). Plasmids were then sequenced to verify the introduction of the desired mutations. pGEX-6P-3 ERa E domain I358A, Q375A was subcloned from pET151D-ERa LBD I358A, Q375A generously provided by Dr Rachel Klevit (Eakin et al., 2007). To engineer constructs harboring ERa deletions domains, PCR amplification was performed with the oligonucleotides in supplementary material Table S1. PCR products were then cloned into pGEX-6P-3 (obtained by Dr Simona Polo, IFOM, Milan) using BamHI/XhoI sites. All constructs were sequence verified. GST pull-down assays and circular dichroism GST fusion proteins were expressed, purified and as described in (Belenghi et al., 2003) except that all ERa-E domain encoding constructs were prepared in the presence of 20 mM E2. Pull-down experiments were performed as previously reported (La Rosa et al., 2011b; Penengo et al., 2006) by incubating 30 mg of GSTfusion proteins with either 300 mg of growing HeLa cells total lysate for 3 hours at 4 ˚C or with 0.5 mg of polyUb2-7 linked by Lys63 (Boston Biochem) for 1 hour at 4 ˚C in the presence of 20 mM E2. All experiments were normalized by running 1/ 10 of the pull-down on an SDS-PAGE gel. Proteins were detected by Comassie Brilliant Blue staining (Com.). For circular dichroism analysis GST-fusion proteins were eluted and recovered from the beads by treatment with 20 mM free glutathione in 50 mM Tris HCl (pH 9) buffer. The elution buffer, which is unsuitable for spectral measurement in the far UV region, was exchanged by dialyzing the proteins (two times) against 100 volumes of 25 mM sodium phosphate (pH 7.5), 150 mM NaF at 4 ˚C, for 10 hours. UV absorption spectroscopy The molar extinction coefficients at 280 nm (e280) of GST-WT and GST-LAAG fusion proteins (476 residues, 54.8 kDa), calculated by the method of Gill and von Hipple (Gill and von Hippel, 1989), resulted to be 65,110 cm21 M21. The proteins were centrifuged (16 ˚C, 10 minutes, 15,000 rpm) to remove traces of scattering particles, and their absorption spectra were recorded on an Agilent HP8453 diode array spectrophotometer, using 1 cm quartz cell. Samples were perfectly transparent at all wavelengths greater than 300 nm. The protein concentrations were measured by reading the absorption at 280 nm. Circular dichroism measurements CD spectra in the far-UV region were recorded at 16 ˚C using a Jasco J-600 spectropolarimeter and 0.05 cm quartz cells. GST-WT and GST-LAAG fusion Funding This study was supported by grants from Associazione Italiana Ricerca sul Cancro (AIRC) [grant number MFAG12756] and Ateneo Roma Tre to F.A. Supplementary material available online at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.123307/-/DC1 References Acconcia, F., Ascenzi, P., Bocedi, A., Spisni, E., Tomasi, V., Trentalance, A., Visca, P. and Marino, M. (2005a). Palmitoylation-dependent estrogen receptor alpha membrane localization: regulation by 17beta-estradiol. Mol. Biol. Cell 16, 231237. Acconcia, F., Totta, P., Ogawa, S., Cardillo, I., Inoue, S., Leone, S., Trentalance, A., Muramatsu, M. and Marino, M. (2005b). 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