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The Journal of Neuroscience, October 1, 2002, 22(19):8391-8401
ER-X: A Novel, Plasma Membrane-Associated, Putative Estrogen
Receptor That Is Regulated during Development and after Ischemic Brain
Injury
C. Dominique
Toran-Allerand1, 3, 5, 6,
Xiaoping
Guan2, 6,
Neil J.
MacLusky2, 6,
Tamas L.
Horvath7,
Sabrina
Diano7,
Meharvan
Singh2, 6,
E. Sander
Connolly Jr4,
Imam S.
Nethrapalli2, 6, *, and
Alexander A.
Tinnikov2, 6, *
Departments of 1 Anatomy and Cell Biology,
2 Obstetrics and Gynecology, 3 Neurology, and
4 Neurosurgery, and Centers for 5 Neurobiology
and Behavior and 6 Reproductive Sciences, Columbia
University College of Physicians and Surgeons, New York, New York
10032, and 7 Department of Obstetrics and Gynecology, Yale
University School of Medicine, New Haven, Connecticut 06510
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ABSTRACT |
We showed previously in neocortical explants, derived from
developing wild-type and estrogen receptor (ER)- gene-disrupted (ERKO) mice, that both 17 - and 17 -estradiol elicit the rapid and
sustained phosphorylation and activation of the mitogen-activated protein kinase (MAPK) isoforms, the extracellular signal-regulated kinases ERK1 and ERK2. We proposed that the ER mediating activation of
the MAPK cascade, a signaling pathway important for cell division, neuronal differentiation, and neuronal survival in the developing brain, is neither ER- nor ER- but a novel, plasma
membrane-associated, putative ER with unique properties. The data
presented here provide further evidence that points strongly to the
existence of a high-affinity, saturable, 3H-estradiol
binding site (Kd, ~1.6
nM) in the plasma membrane. Unlike neocortical ER- ,
which is intranuclear and developmentally regulated, and neocortical
ER- , which is intranuclear and expressed throughout life, this
functional, plasma membrane-associated ER, which we have designated
"ER-X," is enriched in caveolar-like microdomains (CLMs) of
postnatal, but not adult, wild-type and ERKO neocortical and uterine
plasma membranes. We show further that ER-X is functionally
distinct from ER- and ER- , and that, like ER- , it is
re-expressed in the adult brain, after ischemic stroke injury. We also
confirmed in a cell-free system that ER- is an inhibitory regulator
of ERK activation, as we showed previously in neocortical cultures.
Association with CLM complexes positions ER-X uniquely to interact
rapidly with kinases of the MAPK cascade and other signaling pathways,
providing a novel mechanism for mediation of the influences of
estrogen on neuronal differentiation, survival, and plasticity.
Key words:
caveolae/caveolar-like microdomains; 17 -estradiol; 17 -estradiol; ERK1/2; ERKO; brain; neocortex; uterus; development
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INTRODUCTION |
Two mammalian estrogen receptor (ER)
genes are now known, encoding, respectively, ER- (~67 kDa) (White
et al., 1987 ), which mediates many of the known transcriptional
actions of estrogen in the brain, and the more recently cloned ER-
(Kuiper et al., 1996 ) (60 kDa in mouse ovary) (Fitzpatrick et al.,
1999 ), whose neural role is less well defined. A third, more distantly
related member of the ER family, ER- , was recently cloned in
teleosts only (Hawkins et al., 2000 ). ER- and ER- appear
to be complementary but not redundant. Under steady-state conditions
they are predominantly intranuclear and differ with respect to the
homology of their functional domains, binding affinities, and ligand
specificities (Kuiper et al., 1997 ). ER- and ER- act as
ligand-inducible transcriptional enhancers (Landers and Spelsberg,
1992 ; Beato and Klug, 2000 ), binding to cognate estrogen response
elements (EREs) in DNA to regulate gene expression. Their
spatiotemporal expression and distribution differ with developmental
stage. Thus, although neocortical ER- is present throughout life
(Shughrue et al., 1997 ), neocortical ER- expression is
developmentally regulated and normally expressed at high levels only
during neocortical differentiation (Shughrue et al., 1990 , 1997 ),
suggesting a more restricted developmental role.
Some responses to estradiol cannot be attributed to ER- or ER-
(Singh et al., 1999 , 2000 ), such as the ability of estrogen to regulate
non-ERE-containing genes (Sukovich et al., 1994 ) and the very
rapid (seconds to minutes) effects of estrogen (Kelly et al.,
1978 ; Chiaia et al., 1983 ; Garcia-Segura et al., 1987 ; Migliaccio et al., 1993 ). Whereas such rapid responses appear inconsistent with direct transcriptional modulation via intranuclear receptors, they could be explained by the presence of plasma
membrane-associated ERs that may be coupled to signal transduction
pathways, typically associated with rapid activation by growth factors.
Neurotrophin activation of the mitogen-activated protein kinase (MAPK)
cascade is mediated by cognate transmembrane receptors associated with
caveolar-like microdomains (CLMs) of neuronal plasma membranes, the
neuronal homologs of caveolae found in most cell types other than
neurons (Huang et al., 1999 ). Caveolae/CLMs form important signaling
modules that compartmentalize, modulate, and integrate growth
factor-induced signaling events at the cell surface (Anderson, 1998 ;
Okamoto et al., 1998 ). Like the neurotrophins, estrogen is an important
neural trophic factor throughout life, with influences on neuronal
differentiation (Toran-Allerand, 1976 , 1980 ), survival (Green and
Simpkins, 2000 ; Garcia-Segura et al., 2001 ), and plasticity
(Matsumoto and Arai, 1981 ). 17 -estradiol activates many
signaling kinases, including protein kinase C (PKC) (G. Sétáló, Jr. and C. D. Toran-Allerand,
unpublished observations), c-src (Nethrapalli et al., 2001 ), and
members of the MAPK cascade (Watters et al., 1997 ; Singh et al., 1999 ,
2000 ). Rapid and sustained activation of cytoplasmic ERK1/2 is followed
by nuclear translocation of phosphorylated ERK
(Sétáló et al., 2001 ). We have proposed (Singh et
al., 1999 , 2000 ) that the ER mediating estrogen-induced activation of
ERK1/2 in the developing brain is neither ER- nor ER- and have
hypothesized that, like neurotrophin receptors, this ER might be
associated with CLMs (Toran-Allerand, 2000 ). Here we provide evidence
for the existence of a novel and unique, plasma membrane-associated
(CLM-associated) putative ER that we have designated "ER-X"
(Toran-Allerand, 2000 ).
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MATERIALS AND METHODS |
All animal experiments were conducted in a humane manner, and
animals were maintained according to protocols approved by the Institutional Animal Care and Use Committee at Columbia University. To
identify and characterize ER-X, we analyzed, by immunoprecipitation, Western blotting and both light and electron microscopy, cell lysates,
detergent-free, highly purified CLM preparations (Smart et al., 1995 ),
plasma membranes, postnuclear supernatants (PNS), and tissue sections
obtained from postnatal day 7 (P7) and adult wild-type and ER-
gene-disrupted (ERKO) neocortex and uterus.
Mice. Wild-type and ERKO mice were obtained from our
breeding colony from matings of C57BL/6J × 129 mice heterozygous
(+/ ) for the ER- gene disruption (Lubahn et al., 1993 ) and
identified by genotyping (Singh et al., 2000 ) as either wild-type (+/+)
or homozygous ( / ) for the disruption.
Genotyping. Tail snips were obtained from P3-P4 pups and
used for genotyping, as previously described (Singh et al., 2000 ). Briefly, tissues were digested with Proteinase K at 56°C for 90 min,
followed by a 99°C incubation for 10 min. The samples were then
vortexed vigorously, and insoluble material was pelleted in a
microfuge. Supernatants were used in a PCR reaction that used one
primer pair [primer 1: 5'-CGG TCT ACG GCC AGT CGG GCA TC-3'; primer
2: 5'-GTA GAA GGC GGG AGG GCC GGT GTC-3'] for the ER- gene product
(product size, 239 bp), and one primer pair [primer 2 from above with
NEO Primer: 5'-GCT GAC CGC TTC CTC GTG CTT TAC-3'] for the neomycin
insert-containing gene product (product size, 790 bp). The PCR program
was performed as follows: 1 cycle at 94°C for 3 min, 30 cycles of
94°C for 45 sec, 62°C for 1 min, and 72°C for 1 min 40 sec,
followed by a final extension cycle of 72°C for 7 min. Products were
analyzed by agarose gel electrophoresis. Wild-type animals revealed the
smaller 239 bp band, homozygous knock-outs (ERKO) showed the larger 790 bp band, and heterozygotes displayed both bands.
Neocortical cultures. Organotypic explant cultures, obtained
from 360 µm hemicoronal slices of the frontal and cingulate neocortex of P2 wild-type and ERKO mice (day of birth = P1), were explanted onto collagen-coated, poly-D-lysine precoated
coverslips and maintained in roller tube culture with gonadal
steroid-deficient (gelding serum) and phenol red-free nutrient medium,
as previously described (Singh et al., 1999 , 2000 ). The nutrient medium
was supplemented with 17 -estradiol (2 nM;
Sigma, St. Louis, MO) for 1 week, to optimize the development of CNS
cultures from estrogen target regions (C. D. Toran-Allerand,
unpublished observations).
Immunoprecipitation and Western blot analysis. Tissues were
harvested into protease and phosphatase inhibitor-containing lysis buffer (50 mM Tris-base, pH 7.4, 150 mM NaCl, 10% glycerol, 1 mM EGTA, 1 mM
Na3VO4, 5 µM ZnCl2, 100 mM NaF, 10 µg/ml aprotinin, 1 µg/ml
leupeptin, 1 mM PMSF, and 1% Triton X-100) and
prepared for immunoprecipitation and polyacrylamide gel
electrophoresis, as previously described (Singh et al., 1999 , 2000 ).
Immunoprecipitation was performed, using an indirect technique with
magnetic Dynabead separation (Dynal ASA, Oslo, Norway). All procedures
were performed at 4°C. In brief, P7 wild-type and ERKO cerebral
cortices were homogenized by passing the sample eight times through a
syringe fitted with a 20-gauge needle. The homogenate was centrifuged at 100,000 × g at 4°C for 15 min, and the protein
concentration of the supernatant was determined (Lowry's method;
Bio-Rad Detergent Compatible Protein Assay kit; Bio-Rad, Hercules, CA).
For coimmunoprecipitation experiments, detergent was omitted from the
lysis buffer. Depending on the species of the antibodies to be used,
the clarified lysates were precleared with either anti-mouse or anti
rabbit IgG-coated Dynabeads to reduce nonspecific antibody-antigen
binding. For immunoprecipitation of ER-X, the precleared lysates,
recovered from the supernatant, were then incubated at 4°C for 12-24
hr with gentle shaking on a Nutator with 6F11, a mouse monoclonal ER
antibody raised against the full-length mouse ER- molecule, which
has proven to be optimal for immunoprecipitation of ER-X (Novocastra;
Vector Laboratories, Burlingame, CA; 1:50-1:100). Primary antibody
incubation was followed by the addition of anti-mouse IgG-coated
Dynabeads for 3 hr to capture and precipitate the antibody-antigen complexes. The ER antibodies and coimmunoprecipitated proteins were
separated from the Dynabeads by the addition of 1× sample loading
buffer, containing 5% -mercaptoethanol, and boiling for 5 min. The
Dynabeads were removed from the supernatant, using Dynal Magnetic
Particle concentrators. The immunoprecipitated proteins were boiled at
95-100°C for 5 min, and 300-500 µg samples were loaded onto 10%
SDS-PAGE gels and separated based on molecular size. Prestained rainbow
markers (Bio-Rad) were used as molecular mass standards. The gels were
then electroblotted onto polyvinylidene difluoride (PVDF) membranes.
Immunodetection of the protein of interest was performed by first
blocking the membrane in 5% nonfat dry milk (Carnation; Nestle USA) in
TBS-Tween 20 (10 mM Tris-base, 150 mM
NaCl, and 0.2% Tween 20, pH 8.0), followed by addition of the primary
antibody. Wherever feasible, the PVDF membranes were probed with
antibodies different from those used for immunoprecipitation to
maximize the specificity of the immunoreactive product obtained. For
ER-X in particular, we used either of two antibodies highly specific for ER- : one specific for the ligand binding domain (LBD) of ER-
(MC20; 1:500; Santa Cruz Biotechnology, Santa Cruz, CA) and the other
raised against amino acids 586-600 of the C terminus of ER- (C1355;
1:2000; Friend et al., 1997 ; Upstate Biotechnology, Lake Placid, NY).
Both antibodies recognize ER-X on Western immunoblots and by
immunohistochemistry, but C1355 is not effective for
immunoprecipitation. ER- was identified with antibodies directed
against the LBD of ER- (1:250; Zymed, South San Francisco, CA).
Negative controls to test for the specificity of the interactions were
run in parallel and were performed by immunoprecipitation of the
precleared protein lysates with preimmune mouse IgG and subsequently
probed with the appropriate antibody. Additionally, a control peptide
or lysate (uterus, ovary) was always used as a positive
control to verify the identity of the band in the experimental
lanes. The specificity of the signal was determined by the
apparent molecular weight (MW) of the protein detected.
Antibody binding to protein was detected, using a secondary antibody
conjugated to horseradish peroxidase (1:40,000; Pierce, Rockford, IL),
and visualized autoradiographically on film, using enzyme-linked
chemiluminescence (ECL; Amersham Biosciences, Arlington Heights, IL),
as previously described (Singh et al., 1999 , 2000 ). All blots were
stripped and reprobed with the appropriate antibody to verify equal
loading of protein across lanes and were analyzed densitometrically.
For studies of ERK phosphorylation, the blots were first probed with
phosphospecific ERK antibodies to detect phospho-ERK1/2
[phospho-p44/42 MAP Kinase,
(Thr202/Tyr204) (1:1000; Cell Signaling, Beverly, MA)]. The same blot
was reprobed for total (nonphosphorylated) ERK protein to verify equal
loading [ERK-1 (C-16), or ERK-2 (C-14)] (1:1000; Santa Cruz
Biotechnology). All antibodies were diluted in the blocking solution.
Densitometric analyses. Densitometric analyses of ERK
protein levels were performed to ensure similar levels of protein
loaded across lanes. Autoradiograms were scanned in triplicate, using an HP Scanjet 6200C (Hewlett Packard Company, Greeley, CO) and analyzed
using Kodak 1D Image Analysis Software (Eastman Kodak, Rochester NY).
Net intensity values were calculated by subtracting the background
within the area measured for each band from the total intensity within
this same measured area to account for any variation in background
intensity across the film.
CLM preparation. Membrane fractions were prepared by
adapting the detergent-free method of Smart et al. (1995) . Briefly,
pools of 40-50 P7 ERKO neocortices were homogenized in 20 mM Tricine, pH 7.8, buffer, containing 1 mM EDTA, 0.25 M sucrose,
and 1 mM dithiothreitol (TESD buffer), then
centrifuged at 1000 × g at 4°C for 10 min. The
pellet was resuspended in TESD buffer, recentrifuged, and the
supernatants were pooled. The combined supernatants were subjected to
Percoll gradient fractionation in the same buffer to isolate the plasma
membrane fraction. In some binding experiments (indicated below)
Percoll-purified plasma membranes were used without further
fractionation. For preparation of CLMs, plasma membranes were sonicated
and further separated by centrifugation on a linear 20 to 10% OptiPrep
(iodixanol) gradient (Nycomed Pharma A. S, Oslo, Norway). Based on
their light-buoyant density, CLMs were separated and purified from
non-CLMs, using two OptiPrep density gradients. The purity of the CLM
preparation was verified immunologically by demonstrating the presence
of CLM-enriched proteins: flotillin (1:250), PKC- and PKC-
(1:1000) (Transduction Labs, Lexington, KY), and absence of the
non-CLM-associated cytoskeletal protein paxillin (1:10,000;
Transduction Labs). Electrophoretically separated CLMs on PVDF
membranes were probed with antibodies specific for ER- [C1355,
(Upstate Biotechnology); MC20 (Santa Cruz Biotechnology)]; ER-
(Zymed); flotillin (Transduction Labs) and other caveolar-resident proteins: e.g., PKC- and PKC- (Transduction Labs) and
noncaveolar-resident proteins: e.g., paxillin (Transduction
Labs)].
Phosphorylation of ERK1/2 in CLM and non-CLM preparations.
Phosphorylation of ERK1/2 in ERKO CLM and non-CLM preparations was
examined following the method of Liu et al. (1997) , except that basal
medium Eagle (BME) was used in the place of MEM. Nine parts of CLM or
non-CLM preparations were mixed with one part of 10× BME, pH 7.4, containing BSA 800 µg/ml, 10 mM NaF, 2 mM Na3VO4, leupeptin 100 µg/ml, soybean trypsin
inhibitor 100 µg/ml, 10 mM
MgCl2, and 1 mM ATP. For
MAP kinase kinase (MEK)1/2 inhibition of ERK activation, the
CLMs were pretreated with the MEK1/2 inhibitor, U0126 (10 µM; Cell Signaling, Beverly, MA) for 30 min
before pulsing them with the appropriate estradiol. Aliquots of ERKO
CLMs and non-CLMs were exposed for 30 min at 37°C to either
17 -estradiol (0.1 nM), 17 -estradiol (10 nM), U0126 (10 µM) or a
sham control and processed for ERK1/2 phosphorylation, using antibodies
to phosphorylated p44/42MAP Kinase
(ERK1/2) (Thr202/Tyr204) (Cell Signaling Technology), as previously described (Singh et al., 1999 , 2000 ).
Isolation of PNS. To increase the yield of ER-X and to test
in a cell-free system whether the presence of ER- is inhibitory for
ERK activation, as we had shown previously in neocortical cultures
(Singh et al., 2000 ), we also studied PNS, a cell-free system that
contains all the cell organelles except the nucleus. PNS was isolated
from P7 wild-type and ERKO neocortices, according to the method of
Smart et al. (1995) . Three or four P7 wild-type and ERKO neocortices
were homogenized, using a Teflon homogenizer in 1 ml of 20 mM Tricine, pH 7.8, buffer, containing 1 mM EDTA, 0.25 M sucrose, 10 µg/ml aprotinin, and 1 µg/ml leupeptin. The homogenate was
centrifuged at 1000 × g at 4°C for 10 min. The supernatant obtained is the PNS. The pellet was resuspended in 500 µl
of the homogenization buffer, recentrifuged, and the PNS obtained was
pooled with the first PNS. ERKO and wild-type PNS were mixed with 10×
phosphorylation buffer, and the MAPK assay was performed as described
above. PNS samples were exposed to 17 -estradiol (0.1 nM), 17 -estradiol (10 nM), the ER- -selective ligand propylpyrazole
triol (PPT) (100 nM) (Stauffer et al., 2000 ) (a
gift from J. A. Katzenellenbogen, University of Illinois,
Champaign/Urbana), the MEK inhibitor U0126 (10 µM), BDNF (100 ng/ml), ethanol (0.001%), DMSO
(0.001%), and a sham control; first, for 10 min at 4°C, followed by
10 min at 37°C.
Cholesterol depletion. To determine whether disruption of
CLMs impairs estrogen activation of the MAPK cascade, neocortical explants were pretreated on P9 with the sterol binding agent Nystatin (50 µg/ml) (Sigma), a compound used extensively to document the association of growth factor receptors with caveolae/CLMs (Huang et
al., 1999 ). This concentration of Nystatin has been shown to result in
a significant reduction of cellular cholesterol content without
appreciably affecting cell viability (Rothberg et al., 1990 ). P9
neocortical explants were exposed to Nystatin (50 µg/ml) (Sigma),
BDNF (100 ng/ml), or vehicle control (PBS) for 1 hr before pulsing with
10 nM 17 -estradiol for 30 min, in the
continued presence of inhibitor, BDNF, or vehicle. Explants were then
processed by Western immunoblot analysis for phosphoERK expression,
using antibodies to phosphorylated p44/42 MAP
Kinase (ERK1/2) (Thr202/Tyr204) (Cell Signaling
Technology), as previously described (Singh et al., 1999 , 2000 ).
In situ hybridization. Explants of the ERKO neocortex were
processed for in situ hybridization, after 7 d in
vitro, by a very sensitive, nonisotopic (digoxigenin) method,
using a 48 base oligodeoxyribonucleotide (oligonucleotide) to an
-specific sequence of the ER- LBD (BER2), as previously described
(Miranda and Toran-Allerand, 1992 ). Briefly, the probe was
3'-end-labeled with digoxigenin-labeled deoxyuridine triphosphate
(dUTP) by terminal deoxynucleotidyl transferase (TdT) (Invitrogen,
Grand Island, NY). After hybridization of the synthetic oligonucleotide
to the target cDNA, the hybrids were detected by enzyme-linked
immunohistochemistry, using anti-digoxigenin antibodies (Fab fragment),
conjugated to alkaline phosphatase (1:500; Boehringer Mannheim,
Indianapolis, IN), and an enzyme-catalyzed blue-color reaction
(5-bromo-4-chloro-3-indolyl phosphate and nitro-blue tetrazolium salt).
Immunocytochemistry. P7 ERKO and wild-type mice were
anesthetized by hypothermia and killed painlessly by transcardial
perfusion of saline, followed by 4% paraformaldehyde and 1%
glutaraldehyde fixation. The neocortex was processed by pre- and
post-embedding immunocytochemistry for ER- and flotillin,
respectively. Sections (50 µm) were incubated in anti-ER-
antibodies (C1355, 1:1000; or 6F11, 1:50), washed, and incubated in
biotinylated horse-anti-rabbit or anti-mouse IgG, 1:250 (Vector
Laboratories), incubated with avidin-biotin-peroxidase, 1:50 (Vector
Laboratories), and followed by diaminobenzidine (DAB) (brown reaction
product). Sections were then processed for electron microscopy,
dehydrated, and flat embedded in Durcupan (EM Science, Gibbstown, NJ).
Alternate ultrathin sections (Reichert-Jung Ultramicrotome) of the
neocortex, immunolabeled for ER- , were further labeled for flotillin
(1:50). Sections were washed and incubated in gold-conjugated (15 nm)
goat anti-rabbit IgG (1:20) (EM Science), then washed and contrasted
with saturated uranyl acetate. Ultrathin sections were examined using a
Philips CM-10 electron microscope.
Estrogen binding assay. Duplicate aliquots of 1 mg each of
protein lysate from ERKO P7 neocortex or wild-type adult uterus were
precleared for 30 min, using anti-rabbit IgG-coated magnetic beads
(Dynal AS). Precleared protein lysates were immunoprecipitated with
anti-ER- antibodies [6F11 (Novocastra) or MC20 (Santa Cruz Biotechnology)] at 4°C overnight. Immunoprecipitated samples, Percoll-purified plasma membrane fractions, and Optiprep-purified CLM
preparations (50 µg each) from P7 wild-type or ERKO neocortex were
incubated with 3H-estradiol (2, 4, 6, 7, 16, 17-3H-estradiol, 100 Ci/mmol; NEN Life
Sciences, Boston, MA) at 4°C for 18 hr. The incubation was terminated
by adsorption of the binding sites onto an equal volume of
hydroxylapatite (HAP) slurry in TESD buffer. HAP pellets were washed
four times with Tris-buffered saline containing 0.2% Tween 20 buffer
and extracted with 1 ml of absolute ethanol overnight at room
temperature. The ethanol supernatants were transferred to liquid
scintillation fluid (5 ml) and counted. Control tubes, used in
assessing HAP adsorption of free steroid, contained HAP and the same
buffer constituents, without addition of the membranes. Nonspecific
binding was assayed in the membranes using the same amount of
radioactive ligand plus 200-fold molar excess of unlabeled
diethylstilbestrol (DES) (Sigma). Specific binding was calculated by
subtracting nonspecific from total binding. The apparent affinity of
the membrane binding sites was determined by incubation with a range of
concentrations of 3H-estradiol (0.25-10
nM). The specificity of the binding sites was
studied by coincubation of purified membranes with 2 nM 3H-estradiol in
the presence of unlabeled progesterone, 17 -estradiol, or
17 -estradiol, added at either 25-fold or 500-fold molar excess.
Transient cerebral ischemia model. Details of the murine
model of focal cerebral ischemia, using an intraluminal suture, have been described previously (Huang et al., 2000 ). Briefly, mice were
anesthetized with 0.3 ml of intraperitoneal ketamine (10 mg/ml) and
xylazine (0.5 mg/ml) and positioned supine on a rectal temperature-controlled operating surface (Yellow Springs Instruments, Yellow Springs, OH). Animal core temperature was maintained at 37 ± 2°C during surgery and for 90 min after surgery. A midline neck
incision exposed the right carotid sheath under the operating microscope (Leica). The common carotid artery was isolated and the
occipital, pterygopalatine, and external carotid arteries were each
isolated, cauterized, and divided. Middle cerebral artery occlusion was
accomplished by advancing a 13 mm heat-blunted 6-0 nylon suture via an
arteriotomy made in the external carotid stump. After placement of the
occluding suture, the external carotid artery was cauterized to prevent
bleeding through the arteriotomy, and arterial flow was established.
After 45 min the occluding suture was removed, and electrocautery was
used to close the arteriotomy. The wound was closed with surgical
staples. After 24 hr, the mice were anesthetized, decapitated, and
brains were removed intact and placed in a mouse brain matrix
(Activational Systems Inc, Warren, MI) for 1 mm sectioning. Sections
were immersed in 2% triphenyltetrazolium chloride (Sigma) in 0.9%
saline and incubated for 12 min at 37°C. Infarcted brain was
identified as an area of unstained tissue. Slices containing tissue
from the region surrounding the infarct (penumbra) and from the
comparable region of the noninfarcted hemisphere were processed for
immunoprecipitation and Western analysis, using 6F11 and MC20
antibodies to ER- , respectively. A total of 8 wild-type mice were studied.
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RESULTS |
P7 neocortex contains an ~62-63 kDa protein that is neither
ER- nor ER- and is enriched in CLMs of the plasma membrane
Using antibodies directed against ER- and ER- , we found, by
immunoprecipitation and Western immunoblot analysis of wild-type and
ERKO P7 neocortical cell lysates, PNS, and CLM preparations, an
hitherto unidentified protein, immunoreactive for the LBD of ER- but
not ER- . We propose that this protein represents ER-X. Although
immunoreactive for ER- , this protein has an apparent MW of ~62-63
kDa that is clearly different from that of ovarian ER- (67 kDa) and
ER- (60 kDa) (Fitzpatrick et al., 1999 ) (Fig. 1a). Cell lysates and
detergent-free, highly purified, CLM preparations (Smart et al., 1995 )
of both P7 neocortical wild-type and ERKO plasma membranes expressed
this ~62-63 kDa protein (Fig. 1b). Although P7 wild-type
neocortex expressed both the 67 kDa ER- band and the ~62-63 kDa
ER-X band, P7 ERKO neocortex contained only the ~62-63 kDa band. P7
wild-type and ERKO neocortical CLM preparations were greatly enriched
with the ~62-63 kDa protein (Fig. 1b). A striking
reversal of the 67 kDa/~62-63 kDa ratio was seen in wild-type P7
neocortical CLM preparations, which, although highly enriched in the
~62-63 kDa form, were greatly diminished in the 67 kDa ER- band.
The specificity and significance of the association of the ~62-63
kDa protein with CLMs was emphasized by the failure to detect
immunoreactivity for other steroid receptors, such as ER- in CLM,
non-CLM, and plasma membrane preparations (Fig. 1c),
although its presence was clearly demonstrable in P7 neocortical cell
lysates and in the nuclear fraction and PNS (Fig. 1c).

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Figure 1.
ER-X is neither ER- nor ER- .
a, Western immunoblots of P7 wild-type and ERKO
neocortex and adult wild-type mouse ovary, using antibodies to the LBDs
of ER- (Santa Cruz Biotechnology; MC-20; ovary and neocortex) and
ER- (Zymed; ovary). The apparent MW of ER-X (~62-63 kDa) is
clearly different from the MW of the mouse ER- (67 kDa) and ER-
(60 kDa) ovarian controls. b, Whereas P7 wild-type
neocortex contained both the 67 kDa ER- and the ~62-63 kDa ER-X
bands, P7 ERKO tissues expressed only the ~62-63 kDa ER-X band. P7
wild-type and ERKO neocortical CLM preparations were greatly enriched
with the ~62-63 kDa protein. A striking reversal of the ER- /ER-X
ratio was seen in wild-type CLM preparations, in which the ~62-63
kDa form was highly enriched, whereas authentic 67 kDa ER- was
considerably diminished. c, Absence of ER- from the
plasma membrane, CLM, and non-CLM regions. Note the total absence of
ER- from the wild-type plasma membrane and the CLM and non-CLM
fractions. Note also the nuclear concentration of the 60 and 64 kDa
isoforms of ER- . PM, Plasma membrane;
non-CLM, non-caveolar-like membrane; CLM,
caveolar-like membrane.
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The purity of the CLM preparations was verified by demonstrating the
presence of the CLM integral protein flotillin (Bickel et al., 1997 )
(Fig. 2a) and such
CLM-enriched resident proteins as PKC- (Fig. 2b) and
PKC- (data not shown) (Smart et al., 1995 ), and by the absence of
the cytosolic protein paxillin (Fig. 2c), a cytoskeletal
component associated with non-CLM regions of plasma membranes (Smart et
al., 1995 ).

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Figure 2.
Characterization and purity of the CLM
preparations. a, Western immunoblots of CLMs show
enrichment in flotillin, the neuron-specific, integral CLM protein. The
purity of CLM preparations was verified by the presence of
caveolar-enriched resident proteins such as PKC-
(b), and by the absence of the cytosolic protein
paxillin, a cytoskeletal component associated with non-CLM regions
(c).
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ER-X has an entirely different steroid specificity than
either ER- or ER-
The steroid specificity for estrogen-induced
activation of ERK1/2 phosphorylation is radically different from that
of either ER- or ER- : ERK1/2 is not activated by either
ER- -selective ligands such as 16 -iodo-17 -estradiol (Singh et
al., 2000 ) and PPT (Stauffer et al., 2000 ) (100 nM) (see
Fig. 4b,c) or by ER- -selective ligands such as genistein
and coumestrol (Singh et al., 2000 ) but is activated equally well by
picomolar concentrations of 17 - and 17 -estradiol (Fig.
3a,b). In wild-type cultures
17 -estradiol, a natural stereoisomer of 17 -estradiol that is
generally considered to be transcriptionally inactive, elicited a
stronger, sustained activation of ERK1/2 at the 1-10
pM (10 12
M) range (Fig. 3b) than did
17 -estradiol (0.1-10 nM) (Fig.
3a). What makes this response so astonishing is that
17 -estradiol, which, like 17 -estradiol, is derived from
aromatization of androgens, but whose site of synthesis is unclear, has
a 100-fold lower affinity for ER- than 17 -estradiol (Hajek et
al., 1997 ). Significantly, higher levels of 17 -estradiol were
required for ERK activation in wild-type neocortical cultures (Fig.
3a), perhaps reflecting the need to overcome the inhibitory
effect of ER- on ERK1/2 phosphorylation (Singh et al., 2000 ) (Fig.
4), which, unlike 17 -estradiol,
17 -estradiol activates as well. That the inhibitory presence of
ER- influences dose responsiveness is suggested by observation that
in the ER- -deficient ERKO neocortical explants, 17 -estradiol,
like 17 -estradiol, is also able to elicit activation of ERK in the
1-10 pM range (data not shown).

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Figure 3.
ER-X is exquisitely sensitive to picomolar
concentrations of 17 - and 17 -estradiol. Western immunoblot of
ERK1/2 phosphorylation elicited in wild-type neocortical explants by
17 -estradiol (a) and 17 -estradiol
(b). Bottom blots, Reprobing with
antibodies to total nonphosphorylated ERK1/2 to verify equal loading of
ERK1/2 protein across lanes. pERK, phosphoERK.
Densitometry confirmed equal loading. Note that significantly higher
levels of 17 -estradiol were required for ERK activation, perhaps
reflecting the need in wild-type cultures to overcome the inhibitory
effect of ER- on ERK phosphorylation, which, unlike 17 -estradiol,
17 -estradiol activates as well.
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Figure 4.
Estrogen-induced activation of ERK1/2 in CLMs and
PNS. Western immunoblots: a, exposure of highly
purified, P7 ERKO neocortical CLMs to 17 -estradiol (0.1 nM) and 17 -estradiol (10 nM) for 30 min
elicited MEK-dependent (U0126) phosphorylation of ERK1 and ERK2.
Non-CLM regions were unresponsive. Densitometry confirmed equal loading
of protein. b, Exposure of P7 wild-type neocortical PNS
to 17 -estradiol (0.1 nM) and 17 -estradiol (10 nM) for 10 min, 37°C elicited MEK-dependent (U0126)
phosphorylation of ERK1 and ERK2. Note that, not only did the
ER- -selective ligand PPT reduce ERK phosphorylation levels below
baseline (0) very significantly, but that the
level of ERK1/2 phosphorylation, elicited by 17 -estradiol, was also
significantly lower than after exposure to 17 -estradiol. This
difference may be attributed to the fact that P7 wild-type neocortex is
also enriched in ER- which, because it is activated by 17 - (but
not 17- ) estradiol and exerts its inhibitory effect on ERK1/2, as
was also seen after exposure to PPT. Bottom blots,
Reprobing with antibodies to nonphosphorylated ERK1/2 to verify equal
loading of ERK protein across lanes. Densitometry confirmed equal
loading. c, Densitometric analysis of ERK activation in
wild-type PNS shown in b. These findings confirm that
ER- is a strong inhibitor of ERK activation, a measure of which is
shown by the ability of PPT to effectively prevent ERK activation even
in the face of the strong activation of ERK elicited by the PPT vehicle
ethanol.
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Estrogen elicits ERK1/2 activation in CLMs
To provide direct evidence that the CLM-associated ~62-63 kDa
ER-X protein is connected with estrogen-induced ERK1/2 activation, we
showed that exposure of highly purified, P7 ERKO neocortical CLMs to
17 -estradiol (10 nM) and 17 -estradiol (0.1 nM) for 30 min elicited phosphorylation of ERK1/2 (Fig.
4a). In both instances ERK activation was inhibited by the
MEK inhibitor U0126 (Fig. 4a). In contrast, non-CLM regions
of the plasma membrane, exposed similarly, did not respond (Fig.
4a).
ER- is an inhibitory regulator of ERK1/2 activation in PNS
We then investigated in wild-type PNS, a cell-free system, whether
ER- is an inhibitory regulator of estrogen-induced ERK1/2 activation, as we had shown previously in neocortical explants (Singh
et al., 2000 ). Using the ER- -selective ligand PPT (Stauffer et al.,
2000 ) (100 nM) in wild-type neocortical PNS, we found that
MEK-inducible ERK1/2 phosphorylation was dramatically reduced below
baseline (Fig. 4b). Of particular note, furthermore, were the findings that the levels of 17 -estradiol-induced ERK1/2
phosphorylation were significantly less than after exposure to
17 -estradiol, although both were inhibited by the MEK inhibitor
U0126. This difference in responsiveness may be attributed to the fact
that, at P7, wild-type neocortex is also enriched with maximal levels of ER- (Gerlach et al., 1983 ) which, when activated by 17 - (but not 17- ) estradiol, is able to exert its inhibitory effect on ERK1/2, as is also seen after exposure to PPT. These findings confirm
that ER- is a strong inhibitor of ERK1/2 activation, a measure of
which is given by the ability of PPT to effectively prevent activation
of ERK1/2 even in the face of strong ERK1/2 activation, elicited by the
PPT vehicle ethanol (Fig. 4b,c). These findings provide not
only additional proof that ER- does not mediate activation of the
MAPK cascade but also compelling evidence confirming the role of ER-
as an inhibitory modulator of ERK1/2 activation.
Cholesterol disruption in CLMs decreases estrogen activation
of ERK
CLMs, like caveolae, are highly enriched in cholesterol,
glycosphingolipids, sphingomyelin, and lipid-anchored membrane
proteins, which serve as multivalent scaffolding onto which many
signaling kinases assemble to generate preassembled signaling
complexes. Eighty to ninety percent of plasma membrane cholesterol is
concentrated within caveolae/CLMs, where it plays a critical role in
maintaining receptor protein association within the CLM domain
(Rothberg et al., 1990 ). The sterol-binding agent Nystatin has been
used extensively to document the association of growth factor receptors
with caveolae/CLMs (Huang et al., 1999 ). To determine whether selective
disruption of cholesterol in CLMs impairs the ability of estrogen to
elicit ERK1/2 phosphorylation, we exposed P9 neocortical explants to Nystatin (50 µg/ml) for 1 hr before pulsing with 17 -estradiol (10 nM), BDNF (100 ng/ml), or the vehicle control (PBS) for 30 min (Fig. 5) and then processing for
ERK1/2 phosphorylation by Western blot analysis. We found that
disruption of membrane cholesterol decreased the ability of both
estradiol and BDNF to elicit ERK1/2 phosphorylation, providing
additional evidence of the contributions of CLMs to estradiol-induced
ERK1/2 activation.

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Figure 5.
Disruption of cholesterol in CLMs impairs ERK
activation. Selective disruption of membrane cholesterol by Nystatin in
9-d-old wild-type neocortical explants decreased the ability of
estradiol and the BDNF control to elicit ERK phosphorylation.
Bottom blots, Reprobing with antibodies to
nonphosphorylated ERK1/2 to verify equal loading of ERK protein across
lanes. Densitometry confirmed equal loading.
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ER-X has homology with ER- LBD and is expressed in the
plasma membrane
Using an oligonucleotide probe directed against an -specific
region of the ER- LBD (BER2) (Miranda and Toran-Allerand, 1992 ), we
found widespread distribution of the blue ER- -like hybridization signal in neurons of cultured slices of the ER- -deficient P2 ERKO
neocortex, 17 d in vitro (Fig.
6). This pattern of hybridization in ERKO
neocortex suggests that, in view of the absence of ER- , the
oligonucleotide sequence used may share some homology with ER-X
mRNA.

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Figure 6.
ER-X has homology with the LBD of ER- .
Whole-mount of a P2 ERKO neocortical explant, 17 d in
vitro. The culture was stained for ER- mRNA by in
situ hybridization with a 48 base oligonucleotide probe to an
-specific region of the ER- LBD (BER2; Miranda and
Toran-Allerand, 1992 ) and shows the ER- -like mRNA hybridization
signal in neocortical neurons. Residual, untranslated ER- mRNA? A
splice variant of ER- mRNA? Or the mRNA for a novel ER, ER-X?
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Direct evidence that ER-X may be a neuronal plasma membrane-associated
ER protein with some homology to ER- was also obtained in the ERKO
neocortex by means of light and electron microscopic immunohistochemistry (Fig.
7a-e). Using polyclonal
antibodies, generated against the final 14 C-terminal amino acids of
the rat ER and highly specific for ER- (C1355, Upstate
Biotechnology) (Schreihofer et al., 1999 ), large numbers of immature
ERKO neocortical neurons with unstained nuclei were seen (Fig.
7a,b). Immunoreactivity was clearly localized to the cell
membrane and cytoplasm and not in the nucleus. In Figure 7b,
a blood vessel (V) is in close proximity to a labeled
dendrite, an association which suggests a mechanism by which estrogen
could get even more efficiently onto ER-X. On the other hand, using
monoclonal antibodies generated against full-length mouse ER- (6F11;
Novocastra) (Fig. 7D,E) and said to recognize the 5' N
terminus region, the opposite result was obtained: nuclear labeling was
observed but no cytoplasmic or membrane labeling was seen. Because we
have found that 6F11 cross-reacts significantly with ER- by Western
blotting (data not shown), the nuclear labeling observed here most
likely reflects intranuclear ER- , which is normally expressed in
both wild-type and ERKO neocortex. Association of the ~62-63 kDa
protein with CLMs was further documented at the ultrastructural level
on ultrathin cryostat sections of P7 ERKO neocortex by demonstrating
immunoreactive flotillin, labeled by gold particles, colocalized with
horseradish peroxidase-labeled immunoreactivity for ER- on a
neocortical dendritic spine (Fig. 7C).

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Figure 7.
Direct evidence in ERKO that ER-X is a neuronal
plasma membrane-associated receptor with some homology to the ER-
LBD. A, Using antibodies highly specific for an
-specific region of the LBD of ER- (C1355), large numbers of
immature immunoreactive neocortical ERKO neurons with unstained nuclei
are seen. B, The immunoreactivity is clearly localized
to the cell membrane and cytoplasm and not in the nucleus.
D, E, Antibodies, raised against the
full-length ER- molecule, said to recognize epitopes in the 5',
N-terminal region (6F11), but which we have found also to cross-react
significantly with ER- , show widespread nuclear labeling with no
cytoplasmic or membrane labeling seen. The nuclear labeling observed
most likely reflects intranuclear ER- , which is normally expressed
in both wild-type and ERKO neocortical neurons. C, CLM
association of ER-X in ERKO neocortical neurons was further documented
at the ultrastructural level by demonstrating immunoreactive flotillin
(gold particles), colocalized with immunoreactivity for the ER- LBD
(horseradish peroxidase) on a mushroom-like neocortical
dendritic spine. Scale bars, 10 µm.
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ERKO neocortical plasma membranes contain an estrogen-binding
protein (ER-X)
We determined that neocortical plasma membranes contain a unique
estrogen-binding protein by scintillation counting of
3H-estradiol binding to the ~62-63 kDa
ER-X protein in highly purified P7 ERKO CLM preparations. In these
preparations, the only detectable ER-immunoreactive material present
was the ~62-63 kDa protein (Fig. 1). Binding of 10 nM
3H-estradiol to P7 ERKO CLMs appeared to
be specific and saturable, in that it was suppressed in the presence of
unlabeled diethylstilbestrol (DES). Neocortical CLM preparations from
P7 ERKO mice, shown to be highly enriched in ER-X, were similarly
highly enriched in DES-sensitive estrogen binding (282.12 fmol/mg CLM
protein), as compared with P7 ERKO neocortical lysates (9.94 fmol/mg
lysate protein) and wild-type adult uterine lysates (38.85 fmol/mg
lysate protein). Further characterization of the membrane binding sites was achieved using Percoll-fractionated plasma membranes, containing both CLM and non-CLM components, to increase the yield of total membrane sufficiently to allow construction of binding isotherms and
performance of specificity studies. In Percoll-purified membranes from
P7 ERKO neocortices, as in CLMs, the only detectable ER-immunoreactive protein present was the ~62-63 kDa band (data not shown). Membranes from both P7 ERKO and P7 wild type neocortex contained a high-affinity, saturable 3H-estradiol binding site
(Kd, ~1.6 nM)
(Fig. 8A).
Addition of 50 nM unlabelled 17 -estradiol or
17 -estradiol markedly inhibited binding of
3H-estradiol. In the presence of a 1 µM concentration of either estrogen, binding of
the tritiated ligand was reduced to the nonspecific levels observed in
the presence of excess DES (Fig. 8B). Unlabelled progesterone, by contrast, was less effective than either estrogen, progesterone only partially suppressing binding of
3H-estradiol when added in 500-fold molar
excess (Fig. 8B).

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Figure 8.
Binding of 3H-estradiol to
Percoll-purified plasma membranes from P7 ERKO and wild-type mouse
neocortex. A, Identical amounts of membrane protein (50 µg/tube) were incubated with varying concentrations of
3H-estradiol (0.3-8 nM) for 18 hr at 4C. The
reaction was terminated by addition of hydroxylapatite
(HAP). The membranes and HAP were sedimented by
centrifugation in a microfuge, and the pellet was washed four times to
remove free steroid. Radioactivity in the pellets was extracted with
ethanol and counted. Nonsaturable binding, assessed in the presence of
1 µM unlabeled DES, was subtracted from the total counts,
and the saturable binding was plotted as the ratio of bound-unbound
ligand versus the concentration of bound 3H-estradiol.
Similar concentrations of high-affinity binding (equilibrium
dissociation constant, Kd, ~1.6
nM) were observed in wild-type and ERKO membranes.
B, Specificity of the binding site in Percoll-purified
membranes from P7 ERKO mouse neocortex. Aliquots of plasma membrane
were incubated with 2 nM 3H-estradiol for 18 hr
at 4°C in the presence and absence of different concentrations (50 nM and 1 µM) of 17 -estradiol,
17 -estradiol, or progesterone. Bound 3H-estradiol was
separated by sedimentation with HAP and counted at an efficiency of
50%. Data represent the number of bound counts (after subtraction of
HAP-only blank control tubes, containing no membrane protein) expressed
as the means ± SD of triplicate determinations. The
horizontal dashed line indicates the level of
nonspecific binding observed in the presence of 1 µM
DES.
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ER-X is developmentally regulated in the brain and uterus
Expression of the ~62-63 kDa ER-X protein is developmentally
regulated and is maximally expressed ~P7-P10 in both the neocortex and uterus (Fig. 9a,b). During
the first postnatal month, wild-type and ERKO neocortical and uterine
levels of the ~62-63 kDa protein declined until P21 and became
dramatically reduced in the adult, which expressed little of this
protein.

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Figure 9.
ER-X is developmentally regulated. ER-X expression
is developmentally regulated and is maximally expressed at ~P7-P10
in the neocortex (a) and the uterus
(b). During the first postnatal month, wild-type
and ERKO neocortical ER-X levels decline dramatically and become barely
visible in the adult.
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ER-X is upregulated in a rodent model of brain injury
To test whether re-expression of the developmentally regulated
ER-X might return after brain injury in the adult, as has been reported
for the developmentally regulated ER- (Dubal et al., 2001 ), we
analyzed a mouse ischemic stroke model, elicited by transient
intraluminal middle cerebral artery occlusion (Huang et al., 2000 ).
Tissue from the region surrounding the infarct (the penumbra) was
compared with the comparable region of the noninfarcted neocortex of
the opposite side, 24 hr after occlusion. Using immunoprecipitation,
followed by Western blotting, we found upregulation of the ~62-63
kDa protein in the penumbra (Fig. 10) to levels comparable with those present during development, as compared with the noninfarcted side that remained unchanged. There
was also upregulation of ER- (Fig. 10), as has been shown previously
(Dubal et al., 2001 ).

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Figure 10.
ER-X is upregulated after ischemic brain injury
in the adult. Comparison of ER- and ER-X expression in the infarcted
and noninfarcted adult neocortex. After a large ischemic infarct in the
neocortex produced by middle cerebral artery occlusion, there was not
only upregulation of ER- expression in the penumbra of the ligated,
ischemic side but also upregulation of ER-X therein as well, suggesting
re-expression of a developmental mechanism normally latent in the
adult. Note the lack of significant ER-X expression on the noninfarcted
side. MCF-7 mammary tumor cells and adult uterus = ER-
controls; P7 neocortex = ER-X control.
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DISCUSSION |
These data point strongly to the existence of a novel, plasma
membrane-associated, putative estrogen receptor (ER-X). Although membrane ERs have been identified immunologically as ER- in several cell and tissue systems (Blaustein, 1992 ; Watson et al., 1999 ; Razandi
et al., 1999 ; Milner et al., 2001 ), our findings suggest that ER-X is a
unique, functionally distinct, and hitherto unidentified receptor,
based on its MW, ligand specificity, cellular localization, and
apparent response characteristics. Although ER-X reacts with antibodies
to the ER- LBD, ER-X is not membrane-associated ER- . Its apparent
MW of ~62-63 kDa is clearly different from that of both ER- (67 kDa) and ER- (60 kDa) (Fig. 1a). Although a functional isoform of ER- with an additional 18 amino acids inserted in the LBD
has been identified in rat and mouse tissues (ER- 2) (Petersen et
al., 1998 ), ER-X cannot represent ER- 2, because (1) antibodies directed against the ER- LBD cross-react with ER-X and do not recognize intranuclear ER- . (2) The anti-ER- antibody we used (Zymed) does not react with ER- but does cross-react on Western blots with the molecular isoforms of rat ER- observed in tissue lysates (N. J. MacLusky and I. S. Nethrapalli, unpublished
observations): no immunoreactivity was detected with this antibody in
blots from CLMs enriched in ER-X. (3) ERK1/2 is not activated by ER-
or ER- -selective agonists (Singh et al., 2000 ) (Fig. 4b).
(4) Unlike ER- or ER- , ER-X is not stereospecific, responding
equally well to picomolar concentrations of 17 - and 17 -estradiol
(Fig. 3a,b), whereas ER- and ER- exhibit a markedly
higher affinity for 17 - than for 17 -estradiol (Kuiper et al.,
1997 ).
ER-X is part of a multimolecular CLM complex, comprising
immunoreactivity for ER- (but not ER- ) in association with hsp90, members of the MAPK cascade (Toran-Allerand et al., 1999 ; Singh et al.,
1999 ; Toran-Allerand, 2000 ) and flotillin, the multivalent, 48 kDa
scaffolding protein and neuronal homolog of the caveolar protein
caveolin (Bickel et al., 1997 ). Two recent studies (Levin, 2002 ;
Razandi et al., 2002 ), published after this paper was submitted, reported association of ER- immunoreactivity with caveolae in vascular and breast cancer (MCF-7) cells. Although caveolin-associated ER was identified by the authors as ER- (Razandi et al.,
2002 ), the MW of the immunoreactive band was stated to be 62 kDa, not 67 kDa, as would be expected for authentic full-length ER- .
Rather than demonstrating caveolar association of ER- , as Razandi et al. (2002) concluded, these data are consistent with the observations presented here. In vascular and MCF-7 cells, like neuronal CLMs, caveolar-associated ER- immunoreactivity represents primarily a
protein with an apparent MW ~5 kDa less than that of authentic ER- . In brain, both P7 wild-type and ERKO neocortical CLM
preparations were greatly enriched with the immunoreactive ~62-63
kDa ER-X protein (Fig. 1b) and depleted of ER- and ER-
(Fig. 1b,c), supporting the selectivity and specificity of
the ER-X association with CLMs.
Surprisingly, in both wild-type and ERKO neocortical explants and CLMs,
17 -estradiol, the natural stereoisomer of 17 -estradiol with
100-fold lower affinity for ER- , (Hajek et al., 1997 ) also elicited
sustained MEK-dependent activation of ERK1/2 in the picomolar range
(Figs. 3b, 4a,b). We showed earlier that ER- -
and ER- -selective ligands failed to elicit ERK1/2 activation in
wild-type neocortical explants (Singh et al., 2000 ) and suggested that
ER- may be an inhibitory regulator of ERK activation. This has been
confirmed in the PNS cell-free system (Fig. 4b,c). The
absence of an inhibitory response in ERKO PNS (data not shown) is
consistent with the absence of authentic 67 kDa ER- from ERKO brains.
Nystatin disrupts cholesterol in cell membranes (Iwabuchi et al., 2000 )
by forming globular deposits that alter the planar organization of the
membrane (McGookey et al., 1983 ), thereby selectively inhibiting
caveolar trafficking without altering other cell functions such
clathrin-mediated endocytosis (Ros-Baro et al., 2001 ) or intracellular
receptor trafficking back to the cell surface (Subtil et al., 1999 ).
Nystatin (50 µg/ml) has been shown to significantly reduce cellular
cholesterol content without appreciably affecting cell viability. This
concentration of Nystatin impaired estradiol induced ERK1/2 activation
(Fig. 5).
The existence of plasma membrane-associated ERs (Pietras and Szego,
1977 ) has been controversial because of previous failures to isolate
and characterize such a membrane-associated receptor. Hypothetical
mechanisms have included plasma membrane versions of classical
intranuclear ER- and ER- (Blaustein, 1992 ; Watson et al., 1999 ;
Razandi et al., 1999 ; Milner et al., 2001 ), novel members of the ER
family (Das et al., 1997 ; Gu et al., 1999 ; Nadal et al., 2000 ),
G-protein-coupled receptors (Kelly and Wagner, 1999 ; Filardo et al.,
2000 ; Wyckoff et al., 2001 ), or even growth factor-like receptor
tyrosine kinases (Anuradha et al., 1994 ).
That ER-X may have sequence homology with the ER- LBD was suggested
by (1) the strong hybridization signal obtained in ERKO neocortical
explants with an oligonucleotide probe specific for the ER- LBD
(Miranda and Toran-Allerand, 1992 ) (Fig. 6) and (2) ER- -like
immunoreactivity in ERKO neocortex, using antibodies to the ER- LBD
(Fig. 7A,B) but not with those recognizing the N-terminal
region (Fig. 7D,E). To generate ERKO, the ER- gene was
disrupted by insertion of a 1.8 kb PGK-Neomycin sequence in the region
of exon 2, ~280 bp downstream of the transcription start codon (N
terminus) (Lubahn et al., 1993 ), a region far upstream from the LBD
(exons 4-8). Therefore, ER- -like mRNA found in ERKO neocortex may
represent either (1) residual, untranslated ER- mRNA, (2) a splice
variant of ER- , or (3) ER-X mRNA itself. Residual, weak estrogen
binding not attributable to ER- has been reported in both ER-
(ERKO) and ER- /ER- - (double) knock-out adult mouse brains
(Shughrue et al., 2002 ). This binding was identified in ERKO only as a
splice variant of ER- at exon 2 that may regulate the progesterone
receptor. Nonetheless, there are compelling reasons to believe that
ER-X does not represent the protein product of such a splice variant. A
splice variant at exon 2 would contain exactly the same LBD sequence as
authentic ER- . However, the ligand specificity of ER-X is clearly
different from that of ER- in that ER-X responds equally well to
picomolar concentrations of 17 - and 17 -estradiol (Fig.
3a,b). Finally, ER-X simply cannot represent expression of a
protein derived from the targeted gene disruption, used to generate
ERKO mice, because ER-X is present at comparable levels in P7 wild-type
and ERKO neocortex (Fig. 1b). Earlier studies of cellular
variations in ER mRNA translation (Toran-Allerand et al., 1992 ) have
provided data consistent with the hypothesis that some of the
ER- -like mRNA detected by in situ hybridization may
actually represent ER-X mRNA. Although estrogen binding and ER mRNA
expression always colocalized, neurons expressing ER mRNA did not
always exhibit nuclear binding, and there was no clear-cut relationship
between the widespread hybridization signal (Miranda and
Toran-Allerand, 1992 ) and the limited extent of estrogen binding
(Gerlach et al., 1983 ); evidence of ER-X mRNA?
Our data do not prove that the ~62-63 kDa ER- -immunoreactive
protein binds estrogen. The SDS-PAGE conditions required to separate
the ~62-63 kDa protein are incompatible with retention of binding
site integrity. Nevertheless, circumstantial evidence suggests that the
~62-63 kDa protein binds estradiol and, moreover, that this binding
reaction may mediate activation of ERK1/2. The ~62-63 kDa band and
the estradiol binding site are both present in P7 ERKO neocortical
membranes that contain neither ER- nor ER- . In ERKO mouse
neocortex, 17 - and 17 -estradiol both activate ERK1/2: both also
compete strongly for membrane binding of
3H-estradiol (Fig. 8). Levels of membrane
binding are similar in ERKO and wild type neocortex, consistent with
the observation that similar concentrations of the ~62-63
kDa-immunoreactive band are present in membranes from ERKO and
wild-type P7 mice (Fig. 1). Finally, progesterone, which does not bind
intranuclear ER- or ER- but does activate ERK in developing brain
(Singh, 2001 ), is capable of competing with
3H-estradiol for the membrane binding
site, albeit less effectively than 17 - and 17 -estradiol.
ER-X expression is developmentally regulated in both neocortex and
uterus and is maximally expressed at ~P7-P10. Wild-type and ERKO
neocortical and uterine ER-X levels declined during the first postnatal
month and became dramatically reduced in the adult, which expressed
little ER-X (Fig. 9a,b). Transient, neocortical expression
of ER-X mimics the developmental pattern of estrogen binding (Gerlach
et al., 1983 ). That an ER other than ER- may have developmental
importance is even suggested by the original ERKO paper (Lubahn et al.,
1993 ). Because loss of functional ER- did not appear to influence
prenatal sexual development, the authors concluded that development of
the reproductive tract can occur in the absence of ER-mediated
responsiveness. An alternate explanation is that early development may
depend on another ER, such as ER-X.
Developmentally regulated estrogen receptors may be upregulated and
re-expressed in the adult brain. Previous studies have demonstrated
that 17 - and 17 -estradiol protect against ischemic CNS injury, as
well as neuronal cell death induced by exposure to peroxides or
-amyloid (for review, see Green and Simpkins, 2000 ). The
neuroprotective efficacy of 17 -estradiol has been interpreted as
evidence of a direct antioxidant, as opposed to an ER-dependent,
mechanism (Behl et al., 1997 ; Green et al., 1997 ). Our results suggest
an alternative explanation: responses to 17 -estradiol may, in part,
reflect activation of membrane ER-X response pathways. Developmentally
regulated ERs, such as neocortical ER- and ER-X, latent in the brain
since development, may be re-expressed in the adult after injury caused
by ischemia, loss of trophic support, or steroid deprivation. ER-X and
its signaling pathways could therefore underlie not only the
differentiative effects of estrogen in the developing brain but some of
its neuroprotective actions in the adult (Simpkins et al., 1997 ; Dubal
et al., 1998 ; Green and Simpkins, 2000 ).
Proof that ER-X is a novel ER must await sequencing of the protein,
currently in progress. The data presented here, however, suggest that
this protein is functional and is associated with estradiol-induced
activation of the MAPK cascade. Our data further suggest that responses
to estrogen during development and after injury are not necessarily
mediated via either ER- or ER- . Association with CLMs positions
ER-X uniquely to interact with colocalized signaling kinases, providing
a novel mechanism for mediation of the influences of estrogen on
neuronal differentiation (Toran-Allerand, 1976 ), survival
(Garcia-Segura et al., 2001 ), and plasticity (Matsumoto and Arai,
1981 ).
 |
FOOTNOTES |
Received Nov. 21, 2002; revised July 16, 2002; accepted July 16, 2002.
*
I.S.N. and A.A.T. contributed equally to this work.
This work was funded in part by National Institutes of Health (National
Institute on Aging), National Institute of Mental Health, National
Science Foundation, the Alzheimer's Association (Burks B. Lapham grant
and T.L.L. Temple Foundation Discovery Award), and an Alcohol, Drug
Abuse, and Mental Health Administration Research Scientist Award
(all to C. D. T-A). We thank R. G. Anderson (University of
Texas Southwestern) for helpful suggestions, T. M. Jessell
(Columbia University) for constructive comments on previous versions of
this manuscript, D. B. Lubahn (University of Missouri-Columbia)
for the generous gift of heterozygote breeding pairs to found the ERKO
mouse breeding colony, C. A Mason and H. H. Melikyan (Columbia
University) for the gift of adult and P7 wild-type C57BL/6J mice,
R. B. Hochberg (Yale University) and J. Katzenellenbogen
(University of Illinois, Champaign/Urbana) for the gifts of 16 -iodo
17 -estradiol and propylpyrazole triol (PPT), respectively, I. S. Edelman (Columbia University) for providing valuable discussions,
and N. Nair and I. Laufer (Columbia University) for providing infarcted
and noninfarcted neocortex.
Correspondence should be addressed to Dr. C. Dominique Toran-Allerand,
Department of Anatomy and Cell Biology, 650 West 168th Street, Room
BB1615, Columbia University College of Physicians and Surgeons, New
York, NY 10032. E-mail: cdt2{at}columbia.edu.
 |
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28(4):
387 - 439.
[Abstract]
[Full Text]
[PDF]
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A. Zheng, A. Kallio, and P. Harkonen
Tamoxifen-Induced Rapid Death of MCF-7 Breast Cancer Cells Is Mediated via Extracellularly Signal-Regulated Kinase Signaling and Can Be Abrogated by Estrogen
Endocrinology,
June 1, 2007;
148(6):
2764 - 2777.
[Abstract]
[Full Text]
[PDF]
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Y.-C. Hsieh, H.-P. Yu, M. Frink, T. Suzuki, M. A. Choudhry, M. G. Schwacha, and I. H. Chaudry
G Protein-Coupled Receptor 30-Dependent Protein Kinase A Pathway Is Critical in Nongenomic Effects of Estrogen in Attenuating Liver Injury after Trauma-Hemorrhage
Am. J. Pathol.,
April 1, 2007;
170(4):
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[Abstract]
[Full Text]
[PDF]
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G. Coiret, A.-S. Borowiec, P. Mariot, H. Ouadid-Ahidouch, and F. Matifat
The Antiestrogen Tamoxifen Activates BK Channels and Stimulates Proliferation of MCF-7 Breast Cancer Cells
Mol. Pharmacol.,
March 1, 2007;
71(3):
843 - 851.
[Abstract]
[Full Text]
[PDF]
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N. Vasudevan and D. W. Pfaff
Membrane-Initiated Actions of Estrogens in Neuroendocrinology: Emerging Principles
Endocr. Rev.,
February 1, 2007;
28(1):
1 - 19.
[Abstract]
[Full Text]
[PDF]
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R. X-D Song, P. Fan, W. Yue, Y. Chen, and R. J Santen
Role of receptor complexes in the extranuclear actions of estrogen receptor {alpha} in breast cancer
Endocr. Relat. Cancer,
December 1, 2006;
13(Supplement_1):
S3 - S13.
[Abstract]
[Full Text]
[PDF]
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P. Mannella and R. D. Brinton
Estrogen receptor protein interaction with phosphatidylinositol 3-kinase leads to activation of phosphorylated akt and extracellular signal-regulated kinase 1/2 in the same population of cortical neurons: a unified mechanism of estrogen action.
J. Neurosci.,
September 13, 2006;
26(37):
9439 - 9447.
[Abstract]
[Full Text]
[PDF]
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C. L. Bevan, D. M. Porter, C. R. Schumann, E. Y. Bryleva, T. J. Hendershot, H. Liu, M. J. Howard, and L. P. Henderson
The Endocrine-Disrupting Compound, Nonylphenol, Inhibits Neurotrophin-Dependent Neurite Outgrowth
Endocrinology,
September 1, 2006;
147(9):
4192 - 4204.
[Abstract]
[Full Text]
[PDF]
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R. X.-D. Song and R. J. Santen
Membrane Initiated Estrogen Signaling in Breast Cancer
Biol Reprod,
July 1, 2006;
75(1):
9 - 16.
[Abstract]
[Full Text]
[PDF]
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Z. Wang, X. Zhang, P. Shen, B. W. Loggie, Y. Chang, and T. F. Deuel
A variant of estrogen receptor-{alpha}, hER-{alpha}36: Transduction of estrogen- and antiestrogen-dependent membrane-initiated mitogenic signaling
PNAS,
June 13, 2006;
103(24):
9063 - 9068.
[Abstract]
[Full Text]
[PDF]
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J. Qiu, M. A. Bosch, S. C. Tobias, A. Krust, S. M. Graham, S. J. Murphy, K. S. Korach, P. Chambon, T. S. Scanlan, O. K. Ronnekleiv, et al.
A G-Protein-Coupled Estrogen Receptor Is Involved in Hypothalamic Control of Energy Homeostasis
J. Neurosci.,
May 24, 2006;
26(21):
5649 - 5655.
[Abstract]
[Full Text]
[PDF]
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M. Singh, J. A. Dykens, and J. W. Simpkins
Novel Mechanisms for Estrogen-Induced Neuroprotection.
Experimental Biology and Medicine,
May 1, 2006;
231(5):
514 - 521.
[Abstract]
[Full Text]
[PDF]
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J. W. Gatson, P. Kaur, and M. Singh
Dihydrotestosterone Differentially Modulates the Mitogen-Activated Protein Kinase and the Phosphoinositide 3-Kinase/Akt Pathways through the Nuclear and Novel Membrane Androgen Receptor in C6 Cells
Endocrinology,
April 1, 2006;
147(4):
2028 - 2034.
[Abstract]
[Full Text]
[PDF]
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T. R. Pak, W. C. J. Chung, J. L. Roberts, and R. J. Handa
Ligand-Independent Effects of Estrogen Receptor {beta} on Mouse Gonadotropin-Releasing Hormone Promoter Activity
Endocrinology,
April 1, 2006;
147(4):
1924 - 1931.
[Abstract]
[Full Text]
[PDF]
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A. J. Mhyre, R. A. Shapiro, and D. M. Dorsa
Estradiol Reduces Nonclassical Transcription at Cyclic Adenosine 3',5'-Monophosphate Response Elements in Glioma Cells Expressing Estrogen Receptor Alpha
Endocrinology,
April 1, 2006;
147(4):
1796 - 1804.
[Abstract]
[Full Text]
[PDF]
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C. D. Toran-Allerand, A. A. Tinnikov, R. J. Singh, and I. S. Nethrapalli
17{alpha}-Estradiol: A Brain-Active Estrogen?
Endocrinology,
September 1, 2005;
146(9):
3843 - 3850.
[Abstract]
[Full Text]
[PDF]
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N. R. Miller, T. Jover, H. W. Cohen, R. S. Zukin, and A. M. Etgen
Estrogen Can Act via Estrogen Receptor {alpha} and {beta} to Protect Hippocampal Neurons against Global Ischemia-Induced Cell Death
Endocrinology,
July 1, 2005;
146(7):
3070 - 3079.
[Abstract]
[Full Text]
[PDF]
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J. A. Arreguin-Arevalo and T. M. Nett
A Nongenomic Action of 17{beta}-Estradiol as the Mechanism Underlying the Acute Suppression of Secretion of Luteinizing Hormone
Biol Reprod,
July 1, 2005;
73(1):
115 - 122.
[Abstract]
[Full Text]
[PDF]
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A. Hasbi, B. F. O'Dowd, and S. R. George
A G Protein-Coupled Receptor For Estrogen: The End Of The Search?
Mol. Interv.,
June 1, 2005;
5(3):
158 - 161.
[Abstract]
[Full Text]
[PDF]
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M. I. Boulware, J. P. Weick, B. R. Becklund, S. P. Kuo, R. D. Groth, and P. G. Mermelstein
Estradiol Activates Group I and II Metabotropic Glutamate Receptor Signaling, Leading to Opposing Influences on cAMP Response Element-Binding Protein
J. Neurosci.,
May 18, 2005;
25(20):
5066 - 5078.
[Abstract]
[Full Text]
[PDF]
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B. Liu, J. C. Eisenach, and C. Tong
Chronic Estrogen Sensitizes a Subset of Mechanosensitive Afferents Innervating the Uterine Cervix
J Neurophysiol,
April 1, 2005;
93(4):
2167 - 2173.
[Abstract]
[Full Text]
[PDF]
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L. Bjornstrom and M. Sjoberg
Mechanisms of Estrogen Receptor Signaling: Convergence of Genomic and Nongenomic Actions on Target Genes
Mol. Endocrinol.,
April 1, 2005;
19(4):
833 - 842.
[Abstract]
[Full Text]
[PDF]
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N. M. Flake, D. B. Bonebreak, and M. S. Gold
Estrogen and Inflammation Increase the Excitability of Rat Temporomandibular Joint Afferent Neurons
J Neurophysiol,
March 1, 2005;
93(3):
1585 - 1597.
[Abstract]
[Full Text]
[PDF]
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J. L. Temple and S. Wray
Bovine Serum Albumin-Estrogen Compounds Differentially Alter Gonadotropin-Releasing Hormone-1 Neuronal Activity
Endocrinology,
February 1, 2005;
146(2):
558 - 563.
[Abstract]
[Full Text]
[PDF]
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I. S. Nethrapalli, A. A. Tinnikov, V. Krishnan, C. D. Lei, and C. D. Toran-Allerand
Estrogen Activates Mitogen-Activated Protein Kinase in Native, Nontransfected CHO-K1, COS-7, and RAT2 Fibroblast Cell Lines
Endocrinology,
January 1, 2005;
146(1):
56 - 63.
[Abstract]
[Full Text]
[PDF]
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N. J. MacLusky, V. N. Luine, T. Hajszan, and C. Leranth
The 17{alpha} and 17{beta} Isomers of Estradiol Both Induce Rapid Spine Synapse Formation in the CA1 Hippocampal Subfield of Ovariectomized Female Rats
Endocrinology,
January 1, 2005;
146(1):
287 - 293.
[Abstract]
[Full Text]
[PDF]
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F. Acconcia, P. Ascenzi, A. Bocedi, E. Spisni, V. Tomasi, A. Trentalance, P. Visca, and M. Marino
Palmitoylation-dependent Estrogen Receptor {alpha} Membrane Localization: Regulation by 17{beta}-Estradiol
Mol. Biol. Cell,
January 1, 2005;
16(1):
231 - 237.
[Abstract]
[Full Text]
[PDF]
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T. R. Chakraborty and A. C. Gore
Aging-Related Changes in Ovarian Hormones, Their Receptors, and Neuroendocrine Function
Experimental Biology and Medicine,
November 1, 2004;
229(10):
977 - 987.
[Abstract]
[Full Text]
[PDF]
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C.N. Mowa and R.E. Papka
The Role of Sensory Neurons in Cervical Ripening: Effects of Estrogen and Neuropeptides
J. Histochem. Cytochem.,
October 1, 2004;
52(10):
1249 - 1258.
[Abstract]
[Full Text]
[PDF]
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H. C. Evrard and J. Balthazart
Rapid Regulation of Pain by Estrogens Synthesized in Spinal Dorsal Horn Neurons
J. Neurosci.,
August 18, 2004;
24(33):
7225 - 7229.
[Abstract]
[Full Text]
[PDF]
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V. V. Chaban, A. J. Lakhter, and P. Micevych
A Membrane Estrogen Receptor Mediates Intracellular Calcium Release in Astrocytes
Endocrinology,
August 1, 2004;
145(8):
3788 - 3795.
[Abstract]
[Full Text]
[PDF]
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A. K. Murashov, R. R. Islamov, R. J. McMurray, E. S. Pak, and D. A. Weidner
Estrogen increases retrograde labeling of motoneurons: evidence of a nongenomic mechanism
Am J Physiol Cell Physiol,
August 1, 2004;
287(2):
C320 - C326.
[Abstract]
[Full Text]
[PDF]
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J. L. Temple, E. Laing, A. Sunder, and S. Wray
Direct Action of Estradiol on Gonadotropin-Releasing Hormone-1 Neuronal Activity via a Transcription-Dependent Mechanism
J. Neurosci.,
July 14, 2004;
24(28):
6326 - 6333.
[Abstract]
[Full Text]
[PDF]
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M. M. McCarthy
Out with the New, in with the Old: Classical Estrogen Receptors Mediate Novel Estradiol Actions in Brain
Endocrinology,
July 1, 2004;
145(7):
3053 - 3054.
[Full Text]
[PDF]
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N. J. MacLusky
Estrogen and Alzheimer's Disease: The Apolipoprotein Connection
Endocrinology,
July 1, 2004;
145(7):
3062 - 3064.
[Full Text]
[PDF]
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I. M. Abraham, M. G. Todman, K. S. Korach, and A. E. Herbison
Critical in Vivo Roles for Classical Estrogen Receptors in Rapid Estrogen Actions on Intracellular Signaling in Mouse Brain
Endocrinology,
July 1, 2004;
145(7):
3055 - 3061.
[Abstract]
[Full Text]
[PDF]
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O. Kretz, L. Fester, U. Wehrenberg, L. Zhou, S. Brauckmann, S. Zhao, J. Prange-Kiel, T. Naumann, H. Jarry, M. Frotscher, et al.
Hippocampal Synapses Depend on Hippocampal Estrogen Synthesis
J. Neurosci.,
June 30, 2004;
24(26):
5913 - 5921.
[Abstract]
[Full Text]
[PDF]
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V. M. Miller, D. J. Tindall, and P. Y. Liu
Of Mice, Men, and Hormones
Arterioscler Thromb Vasc Biol,
June 1, 2004;
24(6):
995 - 997.
[Full Text]
[PDF]
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S.-H. Yang, R. Liu, E. J. Perez, Y. Wen, S. M. Stevens Jr., T. Valencia, A.-M. Brun-Zinkernagel, L. Prokai, Y. Will, J. Dykens, et al.
Mitochondrial localization of estrogen receptor {beta}
PNAS,
March 23, 2004;
101(12):
4130 - 4135.
[Abstract]
[Full Text]
[PDF]
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C. D. Toran-Allerand
Minireview: A Plethora of Estrogen Receptors in the Brain: Where Will It End?
Endocrinology,
March 1, 2004;
145(3):
1069 - 1074.
[Abstract]
[Full Text]
[PDF]
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V. P. Korovkina, A. M. Brainard, P. Ismail, T. J. Schmidt, and S. K. England
Estradiol Binding to Maxi-K Channels Induces Their Down-regulation via Proteasomal Degradation
J. Biol. Chem.,
January 9, 2004;
279(2):
1217 - 1223.
[Abstract]
[Full Text]
[PDF]
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Y. Xu, R. J. Traystman, P. D. Hurn, and M. M. Wang
Membrane Restraint of Estrogen Receptor {alpha} Enhances Estrogen-Dependent Nuclear Localization and Genomic Function
Mol. Endocrinol.,
January 1, 2004;
18(1):
86 - 96.
[Abstract]
[Full Text]
[PDF]
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H.-b. Liu, K. K. Loo, K. Palaszynski, J. Ashouri, D. B. Lubahn, and R. R. Voskuhl
Estrogen Receptor {alpha} Mediates Estrogen's Immune Protection in Autoimmune Disease
J. Immunol.,
December 15, 2003;
171(12):
6936 - 6940.
[Abstract]
[Full Text]
[PDF]
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C. S. Watson and B. Gametchu
Proteins of Multiple Classes May Participate in Nongenomic Steroid Actions
Experimental Biology and Medicine,
December 1, 2003;
228(11):
1272 - 1281.
[Abstract]
[Full Text]
[PDF]
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M. C. Farach-Carson and P. J. Davis
Steroid Hormone Interactions with Target Cells: Cross Talk between Membrane and Nuclear Pathways
J. Pharmacol. Exp. Ther.,
December 1, 2003;
307(3):
839 - 845.
[Abstract]
[Full Text]
[PDF]
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J. Qiu, M. A. Bosch, S. C. Tobias, D. K. Grandy, T. S. Scanlan, O. K. Ronnekleiv, and M. J. Kelly
Rapid Signaling of Estrogen in Hypothalamic Neurons Involves a Novel G-Protein-Coupled Estrogen Receptor that Activates Protein Kinase C
J. Neurosci.,
October 22, 2003;
23(29):
9529 - 9540.
[Abstract]
[Full Text]
[PDF]
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S. D. Sullivan and S. M. Moenter
Neurosteroids Alter {gamma}-Aminobutyric Acid Postsynaptic Currents in Gonadotropin-Releasing Hormone Neurons: A Possible Mechanism for Direct Steroidal Control
Endocrinology,
October 1, 2003;
144(10):
4366 - 4375.
[Abstract]
[Full Text]
[PDF]
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L. L. DonCarlos, D. Garcia-Ovejero, S. Sarkey, L. M. Garcia-Segura, and I. Azcoitia
Androgen Receptor Immunoreactivity in Forebrain Axons and Dendrites in the Rat
Endocrinology,
August 1, 2003;
144(8):
3632 - 3638.
[Abstract]
[Full Text]
[PDF]
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V. N. Luine, L. F. Jacome, and N. J. MacLusky
Rapid Enhancement of Visual and Place Memory by Estrogens in Rats
Endocrinology,
July 1, 2003;
144(7):
2836 - 2844.
[Abstract]
[Full Text]
[PDF]
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D. Yokomaku, T. Numakawa, Y. Numakawa, S. Suzuki, T. Matsumoto, N. Adachi, C. Nishio, T. Taguchi, and H. Hatanaka
Estrogen Enhances Depolarization-Induced Glutamate Release through Activation of Phosphatidylinositol 3-Kinase and Mitogen-Activated Protein Kinase in Cultured Hippocampal Neurons
Mol. Endocrinol.,
May 1, 2003;
17(5):
831 - 844.
[Abstract]
[Full Text]
[PDF]
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C. B. Wade and D. M. Dorsa
Estrogen Activation of Cyclic Adenosine 5'-Monophosphate Response Element-Mediated Transcription Requires the Extracellularly Regulated Kinase/Mitogen-Activated Protein Kinase Pathway
Endocrinology,
March 1, 2003;
144(3):
832 - 838.
[Abstract]
[Full Text]
[PDF]
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