s t e r o i d s 7 3 ( 2 0 0 8 ) 992–996

avai lab le at www.sc iencedi rec t .com

journa l homepage: www.e lsev ier .com/ locate /s tero ids

Modulation of A�-induced neurotoxicity by estrogenreceptor alpha and other associated proteins in lipid rafts

Raquel Marina,∗, Cristina Ramıreza, Araceli Moralesa, Miriam Gonzaleza,Rafael Alonsoa, Mario Dıazb

a Laboratory of Cellular Neurobiology, Department of Physiology & Institute of Biomedical Technologies,University of La Laguna, School of Medicine, Santa Cruz de Tenerife, Spainb Laboratory of Animal Physiology, Department of Animal Biology & Institute of Biomedical Technologies,Faculty of Biology, University of La Laguna, Santa Cruz de Tenerife, Spain

a r t i c l e i n f o

Published on line 23 December 2007

Keywords:

Estrogen receptors

a b s t r a c t

Some evidences have demonstrated the participation of estrogen receptors (ERs) in rapid,

non-genomic actions of estrogen to promote neuroprotection against different toxic agents.

However, there is still very little information about the structural nature of these receptors

and the manner these proteins may be integrated into the plasma membrane. One of the

Voltage-dependent anion channel

Lipid rafts

Caveolin-1

plausible possibilities is that they may be localized in lipid rafts microstructures where they

would be associated with other, still unknown, molecules which may modulate their physio-

logical activities related to cell survival. In this work, we have identified in caveolar fractions

of murine septal and hippocampal neurons a membrane-related ER shown to physically

interact with, both, a voltage-dependent anion channel and scaffold protein caveolin-1.

1. Homologous ER�-like located at theplasma membrane of neurons

Estrogens have the capacity to exert protective actions in avariety of neurotoxic circumstances such as excess of glu-tamate, serum-deprivation and amyloid-beta (A�) exposurethrough rapid, still unclear, mechanisms of action initiated atthe plasma membrane [1]. In particular, a variety of examplesin different neuronal types have documented that estro-gen can prevent the development of A� toxicity through avariety of pleiotropic actions exerted by, both, genomic andnon-genomic mechanisms. Rapid effects of estrogen againstdegenerative insults have been demonstrated to be activated

within minutes of hormone exposure, coupled to the activa-tion of different downstream intracellular signalling cascades,such as phosphoinositide 3-kinase (PI3K), mitogen-activated

∗ Corresponding author. Tel.: +34 922 319 411; fax: +34 922 319 397.E-mail address: rmarin@ull.es (R. Marin).

0039-128X/$ – see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.steroids.2007.12.007

© 2007 Elsevier Inc. All rights reserved.

protein kinase (MAPK) pathways, and cyclic-AMP-responsiveelement binding (CREB) [2]. These pathways observed in dif-ferent neuronal types have been associated with the putativeparticipation of membrane estrogen receptors (mERs) [3–5].Consistent with this view, we provided in a murine septalSN56 cell line the first evidence of the participation of a mERalpha coupled to downstream MAPK activation to promoteneuroprotection against A�-induced toxicity [6]. However, themolecular identity of these receptors that localize at theplasma membrane remains to be clarified, due in part tothe technical difficulties to identify such molecules. Someexamples in different neuronal types have demonstrated thatendogenous membrane receptors may share a common ori-

gin with nuclear receptors, based upon immunocytochemicalstudies [7]. In particular in SN56 cells and hippocampal-derived HT22 cells [8], a battery of antibodies directed tothe different domains of classical ER� were able to recognize

s t e r o i d s 7 3 ( 2 0 0 8 ) 992–996 993

Fig. 1 – A battery of antibodies directed to different regions of ER� immunoreact with homologous ERs in membranefractions of septal and hippocampal neurons. (A) Protein extracts from plasma membrane fractions (M) of SN56 and HT22cells were loaded on SDS-PAGE for Western blot analyses using antibodies against different domains of nuclear ER�. As acontrol of antibody immunoreactivity, whole cell extracts (T) were also loaded. (B) Protein extracts from microsomes (MC) ofseptal and hippocampal tissues were immunoblotted with the same battery of antibodies known to recognize nuclear ER�.T epitof

a(mrfftaapdacmErg(mams[

2n

Igw

he diagram on the top depicts the relative positions of therom Ref. [13]).

homologous 67-kDa band at plasma membrane fractionsFig. 1A), leading us to the conclusion that mER conserves

ost epitopes of ER�. More interestingly, these results wereeproduced in mouse septal and hippocampal microsomalractions (Fig. 1B), indicating that these may be a widespreadeature. Furthermore, other ERs with different molecular fea-ures have also been identified, such as ER-X [9] and GPR30,seven-transmembrane G-protein-coupled receptor localizedt the endoplasmic reticulum [10]. In addition, some exam-les, including our own, in different neuronal types haveetected multiple bands of higher Mw than classical ER� thatre also recognized by anti-ER� antibodies [11,12]. Empiri-ally, these bands may be explained by post-transcriptionalodifications, such as glycosylation, which may facilitate

R insertion at the plasma membrane, although the 80-kDaeceptor observed in septal and hippocampal cells is neither alycoprotein [8] nor the product of ubiquitin- or SUMO-bindingunpublished data). The most plausible explanation is that it

ay be the result of an alternative splicing variant of ER gene,s previously observed in MCF-7 cells [13], that may be furtherodified by lipids (i.e., palmitoylation) as a requirement for

table localization and functionality at the plasma membrane14].

. Integration of ER� in lipid rafts ofeurons

mportant questions remain with respect to the precise inte-ration of hydrophilic ERs lacking transmembrane domainsithin the hydrophobic structure of the lipid bilayer. One

pes recognized by the different antibodies. (Figure adapted

of the possibilities is that lipid rafts may be the integrat-ing elements of ERs into the plasma membrane. Thesecaveolar microstructures are invaginations of the plasmamembrane recovered by caveolin scaffolded protein andhighly enriched in cholesterol, glycosphingolipids, sphin-gomyelin and lipid-anchored membrane proteins. Theycompartmentalize signal transduction molecules and inte-grate growth factor-induced signalling events at the cellsurface [15,16]. In this regard, caveolin co-purifies with avariety of lipid-modified signaling molecules, including G-proteins, Src family tyrosine kinases, Ras, and eNOS [17,18].Other proteins associated with caveolae are APP (amyloid pre-cursor protein), PI3K and some components of MAPK pathway[19].

Previous studies in endothelial and smooth muscle cells[20,21] have shown a subpopulation of ERs located in caveolarfractions, suggesting that lipid rafts may serve as functionalsignalling domains to promote intracellular signalling andlipid/protein trafficking. Furthermore, in neurons, plasmamembrane receptors have been reported to localize mainly atdiscrete caveolar-like microdomains (CLMs) [22], the neuron-specific caveolae. The presence of ER in these neuronalmicrodomains has been reported in neocortical explants,derived from developing wild-type and ER-� gene-disrupted(ERKO) mice [9], where the association with CLM complexessettles the ER-X to interact rapidly with the MAPK cascadeand other signalling pathways. In agreement with this pos-

sibility, we have provided in septal (SN56) and hippocampal(HT22) cells the first evidence of a raft-located ER� in neu-rons [23]. A very interesting finding in these microdomains,as evidenced by immunoprecipitation and immunocytochem-

994 s t e r o i d s 7 3 ( 2 0 0 8 ) 992–996

Fig. 2 – Association of mER� with VDAC and caveolin-1 at the plasma membrane of septal and hippocampal neurons. (A)Immunoprecipitation assays were performed on plasma membrane fractions from SN56 cultured cells (IPM) or murineseptal (S) and hippocampal (H) microsomes (IPMC) using a specific anti-ER� antibody. The resultant precipitated proteinswere immunoblotting with the corresponding antibodies directed to VDAC or caveolin-1. As a control ofimmunoprecipitation efficiency, total protein extracts were also used to purify ER� protein (IPT). (B) Co-localization of mERwith VDAC and caveolin-1 at the cell surface of SN56 neurons. Cells were fixed under detergent-free non-permeabilizedconditions, and incubated with anti-ER�, anti-VDAC or anti-caveolin-1 antibodies. After washing, cultures were exposed tocorresponding secondary biotinylated antibodies, followed by incubation with cyanine 2 (green) or cyanine 3 (red)dye-conjugated streptavidin. Panel on the right illustrates overlapped pixels (indicated by black spots) of the digital imagedcomposed by fluorescent signals containing both green and red color distributions. (Figure adapted from [23]). (For

, the

interpretation of the references to color in this figure legend

ical assays, was the physical interaction of mER with, both,a voltage-dependent anion channel (VDAC), and scaffoldprotein caveolin-1 (Fig. 2). Furthermore, the interaction ofthese three proteins was corroborated in microsomal frac-tions of septal and hippocampal areas from mouse, therefore,indicating that this association may be a widespread phe-nomenon, at least in neurons. We believe that this firstevidence of the association of ER� with VDAC at the plasmamembrane level may provide novel insights in the factorsinvolved in the alternative mechanisms of estrogen neuro-protection mediated by ER. First, VDAC may participate inestrogen neuroprotective actions. This is supported by ourprevious demonstration of the participation of this porin inthe mechanism of A�-induced toxicity [23], which is in linewith previous data claiming a role of VDAC as an apoptotic

modulator [24]. Second, some data from neuroblastoma cellshave suggested a direct modulation of estrogens to controlVDAC activation via post-translational modifications of thephosphorylation status [25]. Thus, VDAC in a complex with

reader is referred to the web version of the article.)

caveolin-1 may be a candidate to modulate mER functionalityat the plasma membrane level related to cell preservation andintegrity.

Together with VDAC, the participation of caveolin-1, actingas an anchoring protein in this complex with mER, may pro-vide additional stability to the hydrophilic receptor moleculeto be integrated into the lipid raft. We have searched in thedatabase for amino acid sequences present in mouse ER� pri-mary structure susceptible of binding to caveolar scaffoldingdomain (CSD) of caveolin-1, known to interact with differ-ent signalling proteins [18], detecting a consensus sequence�X�XXXXHy, (�, aromatic amino acid; Hy, bulky hydropho-bic amino acid) at 463–470 of ER ligand binding domain (LBD)(sequence Y463TFLSSTL470). Interestingly, LBD has been previ-ously demonstrated to be crucial for the receptor recruitment

in a palmitoylation-dependent manner [26]. Moreover, weanalyzed the predicted secondary structure of mouse (Musmusculus) VDAC (showing a 99.6 percentage similarity withhuman VDAC) using different bioinformatic tools [27,28]. Pre-

s t e r o i d s 7 3 ( 2 0 0 8 ) 992–996 995

Fig. 3 – Hypothetical model of the association between mER, caveolin-1 and VDAC in caveolar microdomains. Caveolin-1may constitute the pivotal element of binding between mER and VDAC. Caveolin-1 monomers may separately bind in CSDt nd “

dmtosishotbttbreiYattlAfpiiaprob

r

o sequences “YTFLSST” of the ER ligand binding domain, a

ictions highlighted that, both, murine and human VDACay be formed by thirteen �-sheets and a potential N-

erminal �-helix (data not shown). Similar to ER�, analysisf VDAC primary structure also revealed a motif �X�XX�XHy

usceptible of CSD binding, located at 62–76 amino acidsn the intracellular loop between the third and fourth �-trand (sequence Y62RWTEYGL76). Overall, these analysesave demonstrated in, both, mER� and VDAC, the existencef consensus regions that may allow the physical interac-ion with caveolin-1 CSD. Inspired by these data, we haveeen tempted to elaborate a hypothetical model to illustratehe interaction of these three proteins (Fig. 3). According tohis paradigm, caveolin-1 may constitute the main linkeretween mER and VDAC, forming homo-oligomers within lipidafts. On the one hand, we postulate that mER may be ori-nted to the intracellular side of the phospholipid bilayer,nteracting with CSD at 463–470 residues of LBD (sequence

463TFLSSTL470). Lipid modification at different residues, suchs palmitoylation at Cys447 [26] may further stabilize this pro-ein at this structure. On the other hand, VDAC may bindo caveolin-1 monomers through sequence Y62RWTEYGL76

ocated in the second intracellular loop of the N-terminal.lthough not explored here, other molecules may be also

orming part of this complex, therefore, adding more com-lexity to this hypothetical paradigm. In this order of ideas,

n the nervous system, intracellular ER� has been shown tonteract with insulin growth factor-1 (IGF-I) receptor prob-bly through an adapter protein, and with p85 subunit of

hosphatidil-inositol-3-kinase (PI3K) in the promotion of neu-onal survival [29]. Altogether, these evidences are indicativef the plethora of factors modulating and being modulatedy ERs located at the plasma membrane that may partici-

YRWTEYGL” of VDAC second loop.

pate in neuronal integrity and preservation. Future studieswill bring some additional clues to elucidate this complexpuzzle.

Acknowledgements

This work was supported by grants PI84/04, SAF2004-08316 C02-01, ISCIII/FISS PI04042460, SAF2007-66148-C02-01;SAF2007-66148-C02-02. RM is a fellow of the “Ramon y Cajal”Programme.

e f e r e n c e s

[1] Marin R, Guerra B, Alonso R, Ramırez CM, Dıaz M. Estrogenactivates classical and alternative mechanisms toorchestrate neuroprotection. Curr Neurovasc Res2005;4:287–301.

[2] Rønnekleiv OK, Malyala A, Kelly MJ. Membrane-initiatedsignalling of estrogen in the brain. Semin Reprod Med2007;25:165–77.

[3] Marin R, Guerra B, Morales A, Dıaz M, Alonso R. Anoestrogen membrane receptor participates in estradiolactions for the prevention of amyloid-� peptide1-40-inducedtoxicity in septal-derived cholinergic SN56 cells. JNeurochem 2003;85:1180–9.

[4] Toran-Allerand CD. Estrogen and the brain: beyondER-alpha, ER-beta, and 17beta-estradiol. Ann NY Acad Sci2005;1052:136–44.

[5] Wu TW, Wang JM, Chen S, Brinton RD. 17Beta-estradiolinduced Ca2+ influx via L-type calcium channels activatesthe Src/ERK/cyclic-AMP response element binding proteinsignal pathway and BCL-2 expression in rat hippocampalneurons: a potential initiation mechanism for

( 2 0

2004;1:32–5.

996 s t e r o i d s 7 3

estrogen-induced neuroprotection. Neuroscience2005;135:59–72.

[6] Guerra B, Dıaz M, Alonso R, Marin R. Plasma membraneoestrogen receptor mediates neuroprotection against�-amyloid toxicity through activation of Raf-1/MEK/ERKcascade in septal-derived cholinergic SN56 cells. JNeurochem 2004;91:99–109.

[7] Arvanitis DN, Wang H, Bagshaw RD, Callahan JW, Boggs JM.Membrane-associated estrogen receptor and caveolin-1 arepresent in central nervous system myelin andoligodendrocyte plasma membranes. J Neurosci Res2004;75:603–13.

[8] Marin R, Ramirez CM, Gonzalez M, Alonso R, Diaz M.Alternative estrogen receptors homologous to classicalreceptor alpha in murine neural tissues. Neurosci Lett2006;395:7–11.

[9] Toran-Allerand CD, Guan X, MacLusky NJ, Horvath TL, DianoS, Singh M, et al. ER-X: a novel, plasmamembrane-associated, putative estrogen receptor that isregulated during development and after ischemic braininjury. J Neurosci 2002;22:8391–401.

[10] Revankar CM, Cimino DF, Sklar LA, Arterburn JB, ProssnitzER. A transmembrane intracellular estrogen receptormediates rapid cell signaling. Science 2005;307:1625–30.

[11] Asaithambi A, Mukherjee S, Thakur MK. Expression of112-kDa estrogen receptor in mouse brain cortex and itsregulation with age. Biochem Biophys Res Commun1997;231:683–5.

[12] Rao BR. Isolation and characterization of an estrogenbinding protein which may integrate the plethora ofestrogenic actions in non-reproductive organs. J SteroidBiochem Mol Biol 1998;65:3–41.

[13] Pink JJ, Wu SQ, Wolf DM, Bilimoria MM, Jordan VC. A novel80-kDa human estrogen receptor containing a duplication ofexons 6 and 7. Nucleic Acids Res 1996;24:404–10.

[14] Marino M, Ascenzi P, Acconcia F. S-palmitoylationmodulates estrogen receptor alpha localization andfunctions. Steroids 2006;71:298–303.

[15] Anderson RG. The caveolae membrane system. Annu RevBiochem 1998;67:199–225.

[16] Okamoto T, Schlegel A, Scherer PE, Lisanti MP. Caveolins, afamily of scaffolding proteins for organizing “pre-assembledsignaling complexes” at the plasma membrane. J Biol Chem1998;273:5419–22.

[17] Lisanti MP, Scherer P, Tang Z-L, Sargiacomo M. Caveolae,caveolin and caveolin-rich membrane domains: a signallinghypothesis. Trends Cell Biol 1994;4:231–5.

[18] Couet J, Li S, Okamoto T, Ikezu T, Lisanti MP. Identification ofpeptide and protein ligands for the caveolin-scaffolding

0 8 ) 992–996

domain. Implications for the interaction of caveolin withcaveolae-associated proteins. J Biol Chem 1997;272:6525–33.

[19] Arcaro A, Aubert M, Espinosa del Hierro ME, Khanzada UK,Angelidou S, Tetley TD, et al. Critical role for lipidraft-associated Src kinases in activation of PI3K-Aktsignaling. Cell Signal 2007;19:1081–92.

[20] Chambliss KL, Yuhanna IS, Mineo C, Liu P, German Z,Sherman TS, et al. Estrogen receptor alpha and endothelialnitric oxide synthase are organized into a functionalsignalling module in caveolae. Circ Res 2000;87:E44–52.

[21] Razandi M, Oh P, Pedram A, Schnitzer J, Levin ER. ERsassociate with and regulate the production of caveolin:implications for signaling and cellular actions. MolEndocrinol 2002;16:100–15.

[22] Huang CS, Zhou J, Feng AK, Lynch C, Klumperman J,DeArmond SJ, et al. Nerve growth factor signaling incaveolae-like domains at the plasma membrane. J BiolChem 1999;274:36707–14.

[23] Marin R, Ramirez CM, Gonzalez M, Gonzalez-Munoz E,Zorzano A, Camps M, et al. Voltage-dependent anionchannel (VDAC) participates in amyloid beta-inducedtoxicity and interacts with plasma membrane estrogenreceptor alpha in septal and hippocampal neurons. MolMemb Biol 2007;24:148–60.

[24] Elinder F, Akanda N, Tofighi R, Shimizu S, Tsujimoto Y,Orrenius S, et al. Opening of plasma membranevoltage-dependent anion channels (VDAC) precedes caspaseactivation in neuronal apoptosis induced by toxic stimuli.Cell Death Differ 2005;12:1134–40.

[25] Dıaz M, Bahamonde MI, Lock H, Munoz FJ, Hardy SP, Posas F,et al. Okadaic acid-sensitive activation of Maxi Cl− channelsby triphenylethylene antioestrogens in C1300 mouseneuroblastoma cells. J Physiol 2001;536:79–88.

[26] Acconcia F, Ascenzi P, Bocedi A, Spisni E, Tomasi V,Trentalance A, et al. Palmitoylation-dependent estrogenreceptor alpha membrane localization: regulation by17beta-estradiol. Mol Biol Cell 2005;16:231–7.

[27] Gromiha MM, Ahmad S, Suwa M. Neural network-basedprediction of transmembrane beta-strand segments in outermembrane proteins. J Comput Chem 2004;25:762–7.

[28] Bagos GP, Liakopoulos DT, Spyropoulos IC, Hamodrakas SJ.PRED-TMBB: a web server for predicting the topology ofb-barrel outer membrane proteins. Nucleic Acids Res

[29] Mendez P, Azcoitia I, Garcia-Segura LM. Interdependence ofoestrogen and insulin-like growth factor-I in the brain:potential for analysing neuroprotective mechanisms. JEndocrinol 2005;185:11–7.