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modulate rapid prolactin release from rat pituitary tumor cells through plasma membrane estrogen receptors
* Department of Human Biological Chemistry and Genetics, and
Department of Pharmacology, University of Texas Medical Branch, Galveston, TX 77555, USA; and
Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 53226, USA
1Correspondence: Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77555-0645, USA. E-mail: cswatson{at}utmb.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(ER) were
used to investigate the role of ER proteins located at the plasma
membrane in mediating the rapid, estrogen-stimulated secretion of
prolactin (PRL) from rat pituitary GH3/B6/F10 cells.
Exposure of the cells to 1 nM 17ß-estradiol (E2)
significantly increased PRL release after 3 or 6 min. When ER Abs
that bind specifically to ER but are too large to diffuse into cells
were tested for activity at the cell membrane, Ab R4, targeted to an
ER hinge region sequence, increased PRL release in a time- and
concentration-dependent fashion. Ab H151, directed against a different
hinge region epitope, decreased PRL release and blocked the stimulatory
action of E2. Abs raised against the DNA binding domain
(H226) or the carboxyl terminus (C542) were not biologically active.
When each Ab was examined for recognition of ER on the cell surface
by immunocytochemistry, all except H151 generated immunostaining in
aldehyde-fixed cells. In live cells, however, Ab H151 but not Ab R4
blocked the membrane binding of fluorescently tagged
E2-BSA. Overall, the data indicate that plasma membrane
ER proteins mediate estrogen-stimulated PRL release from
GH3/B6/F10 cells. These results may also convey information
about conformationally sensitive areas of the membrane form of ER
involved in rapid, nongenomic responses to estrogens.—Norfleet,
A. M., Clarke, C. H., Gametchu, B., Watson, C. S.
Antibodies to the estrogen receptor- modulate rapid prolactin
release from rat pituitary tumor cells through plasma membrane estrogen
receptors.
Key Words: nongenomic effects • membrane steroid receptors • immunocytochemistry
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Since then, ‘rapid’ responses have been described
for all classes of steroid hormones (2
3
4)
. Such effects
occur within seconds or minutes of exposure to the hormone and often
involve second messenger systems or ion channels present in the plasma
membrane. The short latency of these responses is considered to
preclude the involvement of de novo mRNA or protein
synthesis, a consideration that has been corroborated in some
experiments using pharmacological inhibitors of transcription and
translation (5)
. From these observations, a model has
emerged in which steroid hormones interact with receptors to initiate
events at the plasma membrane within a very brief time frame, in
addition to interacting with nuclear receptors to regulate gene
expression over a longer period of time. Although these two
chronological phenomena have been investigated separately in the
laboratory, they most likely work in concert to promote the full
physiological spectrum of steroid hormone action within the cell
(3
, 4
, 6)
.
One controversial feature of this model of the dual nature of steroid
hormone action is the identity of the molecular entity that mediates
the rapid effects of steroids. Binding sites for steroid hormones have
been demonstrated on the plasma membranes of various cell types through
the use of steroids labeled with radioisotopes or fluorescent dyes
(7
8
9)
, steroids conjugated to large proteins that are
sterically impeded from entering the cell (8
, 10
11
12)
, and
antibodies directed against the nuclear form of the steroid receptor
(12
13
14
15
16)
. Although in most cases these plasma membrane
binding sites have not been isolated and characterized, four categories
of putative receptor proteins have been experimentally delineated. One
category is based on evidence that for a given steroid hormone, a
protein that structurally resembles its cognate nuclear receptor is
found at the plasma membrane (9
, 12
13
14)
. The second
category contains membrane proteins that are specific for a given
steroid, but differ significantly in size or sequence from the nuclear
form of the receptor (11
, 17)
. The third category has
evolved from reports that steroids ‘cross talk’ with surface
receptors whose cognate ligand is a different molecule, such as a
growth factor (18)
or neurotransmitter (19)
.
Steroid binding proteins present in serum form the fourth category,
since these proteins, upon binding to specific steroids, interact with
components of the plasma membrane to activate cell functions
(20)
. Hence, it appears that a ‘mixed binding system’
exists in the plasma membrane in which all four categories of steroid
hormone binding proteins may serve different physiological functions in
the cell (3)
. Linking a cellular function to a putative
receptor site is necessary, then, to confirm the relevance of the site
and elucidate its role in the overall response of the cell to the
steroid.
The present study was undertaken to investigate the role of membrane
receptors in mediating a rapid response to the steroid hormone,
estrogen. A plasma membrane population of the estrogen receptor-
(ER) has been demonstrated in the rat pituitary tumor cell line,
GH3/B6; furthermore, the F10 subclone derived
from them was selected for its expression of high levels of membrane
ER (21)
. GH3/B6 cells and the F10
subclone are known to both constitutively secrete prolactin (PRL) and
to respond to estrogens within minutes by releasing an additional bolus
of PRL (21
22
23)
. Therefore, experiments were conducted in
GH3/B6/F10 cells using antibodies (Abs) that
specifically recognize ER as experimental probes to identify the
membrane receptor by immunocytochemistry while analyzing the function
of the receptor in PRL release. The results showed that one of the
ER Abs tested was found to trigger PRL release whereas another
blocked E2-stimulated release, demonstrating a
functional correlation between the rapid, estrogen-stimulated release
of PRL and the plasma membrane population of ER.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
cells were used between passages 10 and 30. The cells were routinely
cultured in serum-supplemented medium (SSM), consisting of Ham’s F-10
medium (Life Technologies, Inc.-BRL, Grand Island, N.Y.), 12.5%
heat-inactivated horse serum, 2.5% heat-inactivated
defined-supplemented calf serum, and 1.5% heat-inactivated fetal calf
serum (all sera supplied by Hyclone, Logan, Utah). For experiments,
cells were plated at a density of 1.25 x
105 cells/cm2 and cultured
in SSM for 24 h. The culture medium was then changed to a
serum-free, phenol red-free defined medium (DIB), which contained DMEM
(Life Technologies, Inc.-BRL), 5 µg/ml insulin, 5 µg/ml
transferrin, 5 ng/ml selenium (Sigma, St. Louis, Mo.), and 0.1% bovine
serum albumin (BSA; Sigma). The cells were cultured for 72 h in
DIB prior to use in experiments.
Antibodies
Four antibodies (R4, H151, C542, H226) that recognize
determinants on the rat ER were used; one Ab (D75), which recognizes
primate but not rat protein, was used as an experimental control. Both
H151 and C542 are mouse monoclonal Abs (mAbs), which were kindly
provided by Drs. Nancy Weigel and Dean P. Edwards. These Abs are now
commercially available from StressGen Biotechnologies (Victoria, B.C.).
Ab H151 was raised against a synthetic oligopeptide (RAANLWPSPLMIKR)
derived from the hinge region of the human ER sequence (Dr. Weigel,
personal communication; ref 24
). Ab C542 is directed
against an oligopeptide (YYITGEAEGFPATV) corresponding to a
carboxyl-terminal sequence of human ER (Dr. Weigel, personal
communication; ref 25
). Abs H226 and D75 were generous
gifts from Dr. Geoffrey Greene. These are rat mAbs that were raised
against the human ER (26)
. The antigenic determinant
for Ab H226 is located in the DNA binding domain, whereas the epitope
for Ab D75 lies between the DNA binding region and the steroid binding
region of the receptor. Ab R4 is a rabbit polyclonal Ab raised in our
laboratory against a synthetic oligopeptide (LKHKRQRDDLEGRNE) from the
hinge region of the rat ER; the Ab, which was purified from immune
serum on a peptide affinity column, has previously been characterized
(21)
. See Fig. 9
for an illustration of these epitope
locations.
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For immunoprecipitation of ER from rat uterus and subsequent Western
analyses, the Ab used was MC-20, a peptide affinity-purified rabbit
polyclonal raised against a carboxyl-terminal sequence of the mouse
ER (Santa Cruz Biotechnology Inc, Santa Cruz, Calif.).
PRL release experiments
GH3/B6/F10 cells, seeded in SSM and
cultured in 24-well plates for 72 h in DIB, were washed once and
preincubated for 15 min in DMEM containing 20 mM HEPES and 0.1% BSA
(DHB). The cells were maintained at 37°C for the preincubation and
test incubations. The preincubation medium was removed and replaced at
time zero with test medium (DHB ± 0.001% EtOH ± test
agents). The test agents included 17ß-estradiol
(E2) and the ER Abs. A stock solution of
E2 (Sigma) in 95% EtOH was diluted such that the
final EtOH concentration was 0.001%, which was used in all control and
test conditions. After 3 or 6 min, test medium was collected into
prechilled tubes, immediately microfuged at 4°C to pellet any
dislodged cells, and the supernatants were stored at -20°C. PRL
concentrations in the test media were determined by radioimmunoassay
using antiserum (AFP-131581570) provided by the National Institute of
Diabetes and Digestive and Kidney Disease and the National Hormone and
Pituitary Program. The intra-assay variability did not exceed 7%. PRL
release observed in the vehicle control group was considered to be
100%. Data were normalized by dividing sample values by the mean of
the vehicle control group, multiplying by 100%, and subtracting 100%
to yield ‘percent change from control’. A one-way analysis of
variation comparing treatment groups to the control was performed using
SigmaStat version 2.0 (Jandel, San Rafael, Calif.); post hoc group
differences were analyzed using Dunnett’s test. Statistical
significance was accepted at P < 0.05.
Immunocytochemistry
GH3/B6/F10 cells were seeded in SSM onto
poly-D-lysine-coated glass coverslips for attachment, then cultured as
described above in DIB for 72 h. The cells were washed three times
in Dulbecco’s phosphate-buffered saline (DPBS). Fixation conditions
were modified from Brink et al. (27)
, as follows. For
nonpermeabilized cells, the fixative contained 2.0% paraformaldehyde
(Sigma) and 0.1% glutaraldehyde (Electron Microscopy Sciences, Fort
Washington, Pa.) in phosphate-buffered saline (PBS), adjusted to pH
7.4; this fixative (P/G) was freshly prepared and applied to the cells
for 30 min at 20°C. For pemeabilized cells, 0.5% Nonidet-40 and 0.15
mM sucrose were added to the P/G fixative, which was applied to the
cells for 1 min. At the end of the fixation period, the cells were
washed three times with DPBS, and free aldehyde groups were reduced
with a 15 min incubation in 100 mM NH4Cl, prior
to a 1 h blocking step at 37°C with 10% BSA (ICC-grade, Sigma)
and 0.1% gelatin (from cold water fish skin, Sigma) in DPBS. The
primary Abs were diluted in 0.5% BSA, 0.1% gelatin in DPBS (PBG) and
added to the cells for 2 h at 20°C; next, the cells were washed
six times over 30 min in PBG. The following steps were conducted at
20°C using reagents from a Vectastain ABC-alkaline phosphatase (AP)
kit in conjuction with Vector red substrate (Vector Labs, Burlingame,
Calif.). Biotinylated universal anti-rabbit/anti-mouse immunoglobulin G
(IgG) was diluted to 4 µg/ml in PBG and added to the cells for 1 h; the cells were then washed six times over 30 min in PBG. The ABC-AP
reagent was diluted in DPBS and added to cells for 60 min, followed by
six washes over 30 min in DPBS. Vector red substrate was prepared
according to the manufacturer’s instructions and added to the cells
for 20 min; the reaction was stopped by rinsing the wells with water.
The cells were dehydrated and cleared by successive treatments with
70% EtOH, 95% EtOH, and xylene, then mounted using Cytoseal 280
(Stephens Scientific, Riverdale, N.J.).
E2-BSA binding
GH3/B6/F10 cells were cultured in plastic
35 mm dishes for 72 h in DIB, then washed once in DHB and chilled
to 4°C over 15 min. The medium was replaced with ice-cold DHB ±
ER Ab and the cells were incubated for 5 min at 4°C. This medium
was replaced with ice-cold DHB containing fluorescein-isothiocyanate
(FITC) -labeled 17ß-E2-BSA conjugate (Sigma)
for 20 min in the dark at 4°C. In parallel, control samples were
incubated with FITC-BSA (without E2) to assess
nonspecific binding. The cells were washed three times with ice-cold
DPBS, then fixed with 4% paraformaldehyde for 15 min. Coverslips were
mounted with Fluoromont-G (Electron Microscope Sciences) and the
samples were immediately examined for fluorescence.
Photomicrography
Photomicrographs were taken with Kodak Tmax 3200 black and white
negative film or Ektapress 1600 Plus color negative film using an
Olympus AHBT microscope equipped with a fluorescence attachment (Model
AH2-RFL) and camera (Model C-35AD-4).
Immunoprecipitation
GH3/B6/F10 cells cultured in SSM or uterus
tissue from an 18 day pregnant rat were prepared for
immunoprecipitation using reagents contained in a protein G
immunoprecipitation kit (Boehringer-Mannheim, Indianapolis, Ind.). The
cell lysate from GH3/B6/F10 cells was incubated
overnight with Ab H151 (10 µg/ml); the tissue lysate was incubated
overnight with Ab MC-20 (5 µg/ml). The immunoprecipitated material
was analyzed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) on a 4–20% acrylamide gradient gel (Novex,
San Diego, Calif.) and transferred onto a 0.45 µM nitrocellulose
membrane (Novex). Nonspecific binding sites were blocked with PBS
containing 1% goat serum and 0.5% Tween-20. Individual lanes were
probed with or without Ab MC-20 (1 µg/ml). After incubation with a
goat anti-rabbit IgG alkaline phosphatase conjugate (Sigma), the pH was
raised by two successive washes in assay buffer (Ambion, Austin, Tex.)
prior to a 5 min incubation with the chemiluminescent AP substrate,
CDP-Star (Ambion). Bands were visualized by exposure of the immunoblot
to X-ray film.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). When ER Abs were added to the test incubation medium instead of
E2, two Abs were found to affect PRL release,
whereas others were inactive. Ab R4 stimulated PRL release in a time-
and concentration-dependent fashion (Fig. 2
). Significant PRL release above basal levels occurred only after a 6
min incubation with the Ab, compared to a 3 min incubation with
E2 (see Fig. 1
). Added together with 1 nM
E2, Ab R4 (1:100) did not stimulate PRL to a
greater extent than 1 nM E2 alone (data not
shown). In contrast to Ab R4, Ab H151 inhibited basal PRL release
(Fig. 3
). The response to Ab H151 was time and concentration dependent, with
statistically significant responses appearing only at the 6 min time
point, although the inhibitory trend was evident at all concentrations
and times. When Ab H151 and E2 were added to the
cells simultaneously, the Ab blocked stimulation of PRL release when
tested at the 3 min time point (Fig. 4
).
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Unlike these two functionally active Abs, two other Abs that recognize
rodent ER had no significant effect on PRL release at the times and
concentrations examined (Fig. 5
, top and middle). Ab D75 (Fig. 5
, bottom), which did not affect
activity, was included as a negative control since it only recognizes
antigen derived from primate sources. Each of these three Abs was
tested at a low concentration of 1 µg/ml and a high concentration of
at least 10 µg/ml, but no changes in basal PRL secretion were
observed (data not shown).
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Ab recognition of plasma membrane ER
Due to the differential abilities of the Abs to modulate PRL
secretion, immunocytochemical analyses were conducted to determine
whether the differences in bioactivity were associated with any
differences in recognition of the membrane ER by the Abs. We
previously characterized an immunocytochemical system for specifically
and sensitively detecting ER on the plasma membrane of cells fixed
with paraformaldehye and glutaraldehyde to prevent the Abs from
entering the cells (28)
. Figure 6
depicts the typical patterns of fluorescence immunolabeling associated
with each of the five Abs that were tested in the PRL release assay.
Shown in Fig. 6
, panels A, B, respectively, are the low
levels of background fluorescence observed when no ER Ab was added
and when Ab D75 specific for primate ER was added. When the
biologically inactive Abs were tested, C542 displayed intense membrane
staining (Figs. 6C
) and H226 yielded consistently low, but
detectable, levels of staining (Fig. 6D
), suggesting that
these Abs bound the
membraneER without activating the receptor in PRL release experiments. The
stimulatory Ab R4 produced strong labeling (Fig. 6E
), but no
signal above background was detected with the inhibitory Ab H151 (Fig. 6F
). No immunofluorescence was visualized using Ab H151 at
concentrations ranging from 1 to 20 µg/ml in fixed, nonpermeabilized
GH3/B6/F10 cells (data not shown). Moreover, when
tested for recognition of nuclear ER in permeabilized cells fixed in
the presence of detergent, no labeling was observed with Ab H151,
whereas dense nuclear staining was produced by Abs R4, C542, and H226
(data not shown). [Although we previously reported immunostaining of
GH3/B6/F10 cells with Ab H151 using a 10-fold
higher concentration of glutaraldehyde (29)
, the earlier
fixation method resulted in higher levels of nonspecific labeling and
has now been replaced by the current staining protocols.]
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To verify that H151 recognized ER in the
GH3/B6/F10 cell line, immunoprecipitation
experiments were undertaken with the Ab. As illustrated in Fig. 7
, a protein of the appropriate size for ER
(Mr 68,000) was precipitated by the
Ab. These results suggested that the epitope recognized by Ab H151 was
altered by the aldehyde fixation procedure used in the
immunocytochemical method and that the antigenic determinant was
thereby rendered nonimmunogenic. Hence, live cells were used to examine
the interaction of Ab H151 with membrane ER, using
FITC-E2-BSA binding.
|
Binding of FITC-E2-BSA to the cells was not
affected by preincubation with Ab R4 (Fig. 8A
) compared to cells preincubated with no ER Ab (data not
shown). In contrast, as presented in Fig. 8B
, preincubating
the cells with Ab H151 reduced binding of the
FITC-E2-BSA conjugate to the background levels
seen in controls incubated with FITC-BSA with no
E2 (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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confirm the
presence of a population of ER proteins on the extracellular face of
the plasma membrane and verify a functional role for these receptors in
mediating a rapid, nongenomic response to estrogen in
GH3/B6/F10 rat pituitary cells. Because the Abs
specifically recognize ER but are too large to diffuse into the
cell, they represent ideal tools for probing the function of ER
residing at the cell surface. Even though two of the Abs tested had no
effect on the secretion of PRL, the ability of one ER Ab to elicit
and one to block E2-stimulated PRL release
provides compelling evidence that the surface molecule recognized by
the Abs is involved in the nongenomic, estrogen-mediated regulation of
PRL secretion in these cells. Furthermore, since the epitopes for both
bioactive Abs lie in the hinge region of the ER protein, the results
may reveal information about conformationally sensitive areas of the
membrane receptor involved in its function. A summary of these findings
in relationship to the epitope map of ER34 is presented in Fig. 9
.
Abs targeted to the ligand binding domain of ER have been used to
study the functional role of membrane receptors for estrogen. Morey et
al. (30)
found that Ab H222 reversed the rapid, inhibitory
effects of E2 on mitogen-stimulated MAP kinase
activity in vascular smooth muscle cells. Using ID5, an anti-idiotypic
Ab that interferes with estrogen binding to the ER, Somjen et al.
(15)
observed increased levels of intracellular calcium
within 5 s of Ab addition to cultured osteoblasts. The biological
effects of Abs H222 and ID5 have been interpreted as resulting from
direct interaction of the Abs at the estrogen binding site of the
membrane receptor. In our experiments, Abs whose epitopes lie outside
of the ligand binding domain were found to be biologically active. Ab
R4, which mimicked the stimulatory activity of E2
upon rapid PRL release, was raised in a rabbit immunized with a
synthetic oligopeptide corresponding to amino acids 270 through 284 of
the rat ER sequence (21
, 31)
. Ab R4 did not increase
PRL release as rapidly or to the same degree as
E2, indicating that the steroid hormone is more
efficacious than the Ab in activating the response and suggesting that
the effect of the Ab cannot be attributed to residual serum estrogens
contaminating the Ab preparation. In this regard, the Ab was purified
from the antiserum on a peptide affinity column, a process of dilution,
washing, elution, and dialysis involving a total volume of buffers
equivalent to 10,000-fold the volume of antiserum. The
affinity-purified Ab preparation was further diluted 50- to 200-fold
for use in the PRL release assay. It seems unlikely, then, that
serum-derived estrogen contamination accounts for the effects of the Ab
preparation. Ab H151, which inhibited basal PRL release and blocked
E2-stimulated release, was raised in a mouse
against a human ER hinge region sequence that aligns with amino
acids 292 through 305 of the rat sequence (31)
. Although
this Ab did not detect membrane or nuclear ER by immunocytochemical
analysis in aldehyde-fixed GH3/B6/F10 cells, the
Ab precipitated a single protein of the appropriate size for ER from
the cells using Western analysis and blocked the binding of
FITC-E2-BSA binding to the surface of live
(unfixed) cells presenting native ER structure. It is noteworthy
that Ab H151 and the biologically inactive Ab C542 are of the same
mouse IgG1 subtype, providing evidence that the
effect of H151 on PRL secretion does not result from a nonspecific
interaction of the Fc region of the molecule with the cell.
The antigenic determinants for the biologically active Abs R4 and H151
are found between amino acids 270 and 305 of the receptor protein; Abs
directed at epitopes outside this region (to the carboxyl terminus and
DNA binding domain) were inactive. Therefore, it appears that this 36
amino acid stretch of the membrane ER may be conformationally
sensitive and that the stimulatory Ab R4 and the inhibitory Ab H151 may
induce distinct conformational changes on binding to this portion of
the receptor. Consistent with this idea, Ab H151 was found to block the
binding of FITC-E2-BSA to the receptor, whereas
Ab R4 had no effect, suggesting that attachment of the Ab H151
structurally constrains the receptor in a manner that prevents estrogen
from binding. It should be noted that structural changes induced by
hormonal analogues are associated with functional consequences for the
nuclear receptor as well (32)
. Another possible
explanation for these data is that Ab H151 sterically inhibits the
macromolecular E2-BSA conjugate from attaching to
the receptor, although this possibility is less likely because the Ab
does not target the ligand binding domain, and Ab R4, whose epitope is
nearby, had no effect on E2-BSA binding.
Besides conformational changes induced in the receptor structure by Ab
binding, aggregation of some receptor molecules after Ab binding is
another mechanism known to trigger receptor activation. Patching and
capping of receptors on the surface of cells after exposure to
anti-receptor Abs have been described for peptide hormone receptors
(33
, 34)
, as well as for steroid hormone receptors. Sabeur
et al. (16)
reported that an anti-progesterone receptor Ab
was observed to localize in a distinct band on the head of sperm cells.
The Ab also inhibited the rapid, progesterone-stimulated acrosomal
response in sperm cells, possibly by blocking the binding of the
steroid to the receptor. Using a monoclonal Ab directed against the
progesterone molecule, another group demonstrated rapid stimulation of
calcium influx and acrosomal exocytosis in sperm cells in the presence
of the anti-progesterone Ab and subthreshold doses of progesterone
(17)
. Presumably the bivalent Ab acted by enhancing
aggregation of receptors since monovalent fragments of the Ab were
inactive. In our own laboratories, we have observed that glucocorticoid
receptor Abs caused patching and capping of receptors on the surface of
human leukemic cells (35)
and that ER Abs induced
clustering of surface receptors in GH3/B6 cells
(21
, 23)
. We and others (8)
have also
reported that receptor aggregation can be elicited by estradiol
conjugated to BSA, which is also multivalent, having multiple steroids
molecules conjugated to a single protein. However, it is not known
whether the process of receptor aggregation in these systems performs a
biological function related to receptor activation or simply represents
clustering of receptors due to the binding of large multivalent
macromolecules to the cell.
In addition to verifying the functional significance of membrane ER,
the results of this study confirm the structural relationship between
the plasma membrane and nuclear population of receptors. The data
reported here extend observations made by us and others that Abs whose
epitopes span the length of the ER protein recognize both membrane
and nuclear receptors. This structural relationship has been further
substantiated by recent experiments in which cells that do not express
estrogen receptors have been transfected with ER cDNA. The
transfected cells express ER on the plasma membrane, as well as in
the nucleus (12)
, and they exhibit rapid responsiveness to
estrogen (12
, 36)
. Little is known, however, about the
structural properties of the receptor that are causes or consequences
of its residence at the membrane, and our results represent some of the
first data to address functionally important regions of the membrane
ER protein.
In summary, we have applied an immunocytochemical method and
E2-BSA binding to verify the presence of ER on
the GH3/B6 rat pituitary cell subclone, F10. The
use of several Abs recognizing different ER epitopes substantiates
the identification of this membrane receptor as well as its full
exposure on the outside surface of the cell. Moreover, the ability of
two of these Abs to modulate a biological response through their
binding to mER reveals a functional role for these receptors and
suggests that alteration of receptor conformation or of receptor
aggregation is involved in the rapid, membrane-initiated estrogenic
response. Therefore, specific Abs directed against steroid receptors
continue to be powerful tools for probing the identity and function of
membrane-associated steroid receptors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ERß expressed in Chinese hamster ovary cells. Mol. Endocrinol. 13,307-319. Steroids 64,5-13[Medline]
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J. G. Tasker, S. Di, and R. Malcher-Lopes Rapid Glucocorticoid Signaling via Membrane-Associated Receptors Endocrinology, December 1, 2006; 147(12): 5549 - 5556. [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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N. N. Bulayeva, A. L. Wozniak, L. L. Lash, and C. S. Watson Mechanisms of membrane estrogen receptor-{alpha}-mediated rapid stimulation of Ca2+ levels and prolactin release in a pituitary cell line Am J Physiol Endocrinol Metab, February 1, 2005; 288(2): E388 - E397. [Abstract] [Full Text] [PDF]
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B. Du, M. Ohmichi, K. Takahashi, J. Kawagoe, C. Ohshima, H. Igarashi, A. Mori-Abe, M. Saitoh, T. Ohta, A. Ohishi, et al. Both estrogen and raloxifene protect against {beta}-amyloid-induced neurotoxicity in estrogen receptor {alpha}-transfected PC12 cells by activation of telomerase activity via Akt cascade J. Endocrinol., December 1, 2004; 183(3): 605 - 615. [Abstract] [Full Text] [PDF]
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J. Q. Chen, M. Delannoy, C. Cooke, and J. D. Yager Mitochondrial localization of ER{alpha} and ER{beta} in human MCF7 cells Am J Physiol Endocrinol Metab, June 1, 2004; 286(6): E1011 - E1022. [Abstract] [Full Text] [PDF]
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S. Mabuchi, M. Ohmichi, A. Kimura, Y. Nishio, E. Arimoto-Ishida, N. Yada-Hashimoto, K. Tasaka, and Y. Murata Estrogen Inhibits Paclitaxel-Induced Apoptosis via the Phosphorylation of Apoptosis Signal-Regulating Kinase 1 in Human Ovarian Cancer Cell Lines Endocrinology, January 1, 2004; 145(1): 49 - 58. [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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A. M. Braun and P. Thomas Androgens Inhibit Estradiol-17{beta} Synthesis in Atlantic Croaker (Micropogonias undulatus) Ovaries by a Nongenomic Mechanism Initiated at the Cell Surface Biol Reprod, November 1, 2003; 69(5): 1642 - 1650. [Abstract] [Full Text] [PDF]
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 R. M. LOSEL, E. FALKENSTEIN, M. FEURING, A. SCHULTZ, H.-C. TILLMANN, K. ROSSOL-HASEROTH, and M. WEHLING Nongenomic Steroid Action: Controversies, Questions, and Answers Physiol Rev, July 1, 2003; 83(3): 965 - 1016. [Abstract] [Full Text] [PDF]
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B. D. Boyan, V. L. Sylvia, T. Frambach, C. H. Lohmann, J. Dietl, D. D. Dean, and Z. Schwartz Estrogen-Dependent Rapid Activation of Protein Kinase C in Estrogen Receptor-Positive MCF-7 Breast Cancer Cells and Estrogen Receptor-Negative HCC38 Cells Is Membrane-Mediated and Inhibited by Tamoxifen Endocrinology, May 1, 2003; 144(5): 1812 - 1824. [Abstract] [Full Text] [PDF]
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C. H. CAMPBELL, N. BULAYEVA, D. B. BROWN, B. GAMETCHU, and C. S. WATSON Regulation of the membrane estrogen receptor-{alpha}: role of cell density, serum, cell passage number, and estradiol FASEB J, December 1, 2002; 16(14): 1917 - 1927. [Abstract] [Full Text] [PDF]
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 K. J. Ho and J. K. Liao Nonnuclear Actions of Estrogen Arterioscler. Thromb. Vasc. Biol., December 1, 2002; 22(12): 1952 - 1961. [Abstract] [Full Text] [PDF]
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 M. Marino, F. Acconcia, F. Bresciani, A. Weisz, and A. Trentalance Distinct Nongenomic Signal Transduction Pathways Controlled by 17beta -Estradiol Regulate DNA Synthesis and Cyclin D1 Gene Transcription in HepG2 Cells Mol. Biol. Cell, October 1, 2002; 13(10): 3720 - 3729. [Abstract] [Full Text] [PDF]
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 R. G. Erben, D. W. Soegiarto, K. Weber, U. Zeitz, M. Lieberherr, R. Gniadecki, G. Moller, J. Adamski, and R. Balling Deletion of Deoxyribonucleic Acid Binding Domain of the Vitamin D Receptor Abrogates Genomic and Nongenomic Functions of Vitamin D Mol. Endocrinol., July 1, 2002; 16(7): 1524 - 1537. [Abstract] [Full Text] [PDF]
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C. Tiffoche, C. Vaillant, D. Schausi, and M.-L. Thieulant Novel Intronic Promoter in the Rat ER{alpha} Gene Responsible for the Transient Transcription of a Variant Receptor Endocrinology, September 1, 2001; 142(9): 4106 - 4119. [Abstract] [Full Text] [PDF]
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 E. Vegeto, C. Bonincontro, G. Pollio, A. Sala, S. Viappiani, F. Nardi, A. Brusadelli, B. Viviani, P. Ciana, and A. Maggi Estrogen Prevents the Lipopolysaccharide-Induced Inflammatory Response in Microglia J. Neurosci., March 15, 2001; 21(6): 1809 - 1818. [Abstract] [Full Text] [PDF]
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 E. Falkenstein, H.-C. Tillmann, M. Christ, M. Feuring, and M. Wehling Multiple Actions of Steroid Hormones---A Focus on Rapid, Nongenomic Effects Pharmacol. Rev., December 1, 2000; 52(4): 513 - 556. [Abstract] [Full Text] [PDF]
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 A. Nadal, A. B. Ropero, O. Laribi, M. Maillet, E. Fuentes, and B. Soria Nongenomic actions of estrogens and xenoestrogens by binding at a plasma membrane receptor unrelated to estrogen receptor alpha and estrogen receptor beta PNAS, October 10, 2000; 97(21): 11603 - 11608. [Abstract] [Full Text] [PDF]
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 A. K. Singh, B. D. Schultz, J. A. Katzenellenbogen, E. M. Price, R. J. Bridges, and N. A. Bradbury Estrogen Inhibition of Cystic Fibrosis Transmembrane Conductance Regulator-Mediated Chloride Secretion J. Pharmacol. Exp. Ther., October 1, 2000; 295(1): 195 - 204. [Abstract] [Full Text]
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