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Division of Molecular Parasitology and Centre of Biological-Medical Research, Heinrich-Heine-University, 40225 Duesseldorf, Germany, Centre National de la Recherche Scientifique, Unité Propre de Recherche 1524, Institute National de la Recherche Agronomique, 78352 Jouy-en-Josas, France
Address all correspondence and requests for reprints to: Prof. Dr. F. Wunderlich, Division of Molecular Parasitology, Heinrich-Heine-University, Universitaetsstrasse 1, 40225 Duesseldorf, Germany. E-mail: frank.wunderlich{at}uni-duesseldorf.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß. The aim of this study was to
investigate transcription-independent E2-signaling in mouse
IC-21 macrophages. E2 and E2-BSA induce a rapid
rise in the intracellular free Ca2+ concentration
([Ca2+]i) of Fura-2 loaded IC-21 cells as
examined by spectrofluorometry. These changes in
[Ca2+]i can be inhibited by pertussis toxin,
but not by the ER-blockers tamoxifen and raloxifene. The
E2-signaling initiated at the plasma membrane is mediated
through neither ER nor ERß, but rather through a novel G
protein-coupled membrane E2-receptor as revealed by RT-PCR,
flow cytometry, and confocal laser scanning microscopy. A special
feature of this E2-receptor is its sequestration upon
agonist stimulation. Sequestration depends on energy and temperature,
and it proceeds through a clathrin- and caveolin-independent pathway. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Recent findings, however, have revealed the existence of still
another intracellular receptor, the so-called iERß
(6, 7, 8, 9, 10).
There is also increasing evidence for transcription-independent actions
of estrogens, as for other steroid hormones too (11).
These actions manifest themselves as rapid responses of target cells in
the range of seconds to minutes. For instance, E2
can induce a fast rise in the intracellular free
Ca2+ concentration
([Ca2+]i) due to influx
of external Ca2+ and/or release of
Ca2+ from intracellular
Ca2+ stores (12, 13, 14, 15, 16, 17). Such
nongenomic actions are initiated at the plasma membrane and are
postulated to be mediated by plasma membrane-associated estrogen
receptors (mER). The current debate focuses on the nature and
properties of these mERs. There is some evidence that the mER is
identical with at least one form of the iER. For instance, Pappas
et al. have shown in pituitary cells that the mER is
very similar—if not identical—with iER, because mER cross-reacts with
iER-recognizing antibodies (18). In accordance, recent
transfection studies with iER and iERß complementary DNAs in CHO
cells have revealed about 3% of both iER and iERß in plasma
membrane enriched fractions (19). However, there are also
reports stating that the mER is different to iER and iERß
(13, 20, 21, 22). Here, we show the existence of mER in the
murine macrophage cell line IC-21 with totally different properties as
hitherto revealed: the mER is neither ER nor ERß, but is a G
protein-coupled receptor which mediates both Ca2+
mobilization and Ca2+ influx, and which is
sequestrable upon agonist stimulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemicals
17ß-estradiol (E2), 17ß-estradiol
6-(O-carboxymethyl)oxime/BSA (E2-BSA),
17-estradiol, tamoxifen, nifedipine, verapamil, and pertussis toxin
were from Sigma (St. Quentin, Fallavier, France), and
raloxifene from Eli Lilly & Co. (Saint-Cloud, France).
1-(6-((17ß-3-metoxyestra-1,3,5(10)-trien-17-yl)-amino)hexyl)-1H-pyrrole-2,5-dione
(U-73122) and
1-(6-((17ß-3metoxyestra-1,3,5(10)-trien-17-yl)-amino)-hexyl)-2,5-pyrrolidine-2,5-dione
(U-73343) were from BIOMOL Research Laboratory (Plymouth,
MA). Fura-2/AM was from Amersham Pharmacia Biotech (Les
Ulis, France). 17ß-estradiol 6-(O-carboxymethyl)oxime:
BSA-fluoresceine isothiocyanate conjugate
(E2-BSA-FITC) was from Sigma
(Deisenhofen, Germany) and Concanavalin A (Con A)-rhodamine from Vector
(Burlingame, CA). Vectashield was delivered from Vector (Burlingame,
CA) and 1,4-diazobicyclo-[2.2.2]octane (DABCO) from Merck & Co., Inc. (Darmstadt, Germany).
Ca2+ measurement
IC-21 cells were assayed for
[Ca2+]i as described
(23). In brief, cells were grown on
poly-L-lysine-coated glass coverslips until confluence and
then loaded with 1 µM Fura-2/AM for 30 min at room
temperature. The Ca2+ was measured in a
temperature-controlled (37 C) Hitachi F-2000
spectrofluorometer. Steroids and reagents were added directly to the
cuvette under continuous stirring (13, 15). Estrogens were
dissolved in ethanol; the final concentration of ethanol never exceeded
0.01%, and this concentration had no effect on
[Ca2+]i.
E2-BSA was treated with charcoal to remove any
free E2 or 17ß-estradiol
6-(O-carboxymethyl)oxime (24). Charcoal treatment had no
effect on the ability of E2-BSA to increase
[Ca2+]i
(13). The Fura-2 fluorescence response to
[Ca2+]i was calibrated
from the ratio of the 340/380 nm fluorescence values after subtraction
of the background fluorescence of the cells at 340 nm and 380 nm
(25). The dissociation constant of
Fura-2-Ca2+ complex was taken as 224
nM. The values for Rmax and
Rmin were calculated from measurements made using
25 µM digitonin, and 4 mM EGTA and enough
Tris base to raise the pH to 8.3 or higher. Each measurement on Fura-2
loaded cells was followed by a parallel experiment under identical
conditions with cells not loaded with Fura-2.
Labeling with E2-BSA-FITC
IC-21 cells were washed twice with phosphate-buffered salt
solution (PBS+; 140 mM NaCl, 2.7
mM KCl, 6.4 mM
Na2HPO4, 1.4 mM
KH2PO4, 0.5 mM
MgCl2, 0.9 mM
CaCl2, pH 7.2), and incubated at the indicated
temperatures for varying periods with 1.5 x
10-5 M
E2-BSA-FITC, or with BSA-FITC or BSA alone as
controls. For internalization experiments, intact IC-21 cells were
incubated at room temperature or 37 C for 15 min or 1 h with
E2-BSA-FITC, BSA-FITC or Con A-rhodamine (1:50)
or a rat antimouse F4/80 antibody (2 µg/ml; gift from H. Mossmann,
MPI for Immunobiology, Freiburg, Germany) and with Biotin-SP-conjugated
AffiniPure mouse antirat IgG (H+L) (1:500; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) as a secondary antibody and
streptavidin-fluoresceine (6 µg/107 cells;
Amersham Pharmacia Biotech, Braunschweig, Germany).
Colocalization was performed in intact cells using LysoTracker Red
DND-99 (10 µM; Molecular Probes, Inc.,
Göttingen, Germany) or transferrin conjugated with
tetramethylrhodamine (20 µg/ml; Molecular Probes, Inc.,
Göttingen, Germany). Then, the samples were postfixed with 1%
paraformaldehyde (PFA) (26). Cells prefixed with 0.5% PFA
were incubated with the anticlathrin antibody HC (N-19) (2 µg/ml;
Santa Cruz Biotechnology, Inc., Heidelberg, Germany) and
with a donkey antigoat-Cy3 antibody (1:200; gift from P. Traub, MPI for
Cell Biology, Ladenburg, Germany) as secondary antibody, or with the
anti-caveolin antibody caveolin-1 (N-20) (2 µg/ml; Santa Cruz Biotechnology, Inc.) using a TRITC-conjugated AffiniPure goat
antirabbit IgG (H+L) antibody (1:80; Jackson ImmunoResearch Laboratories, Inc.) as secondary antibody. The cells were
postfixed with 3% PFA (23).
Localization of ER
Intact IC-21 cells as well as cells prefixed with 0.5% PFA and
permeabilized with PBS+ containing 0.05%
Tween-20 and 0.5% BSA were labeled with the different ER-antibodies
ER (MC-20), ER (H-184), and ERß (Y-19) (all Santa Cruz Biotechnology, Inc.) in concentrations of 2 µg/ml for 1 h
at room temperature. Antirabbit IgG (whole molecule) FITC conjugate
(working dilution 1:320; Sigma, Deisenhofen, Germany) and
a donkey antigoat-FITC antibody (working dilution 1:100; gift from P.
Traub, MPI for Cell Biology, Ladenburg, Germany) were used as secondary
antibodies for 45 min. The cells were postfixed with 1% PFA (23, 26).
Confocal laser scanning microscopy
IC-21 cells (2 x 106 cells/ml) were
allowed to adhere onto poly-L-lysine-coated glass
coverslips overnight, then labeled as described above, and embedded in
a 1:1 (vol/vol) mixture of glycerol and vecta-shield containing 2%
(wt/vol) DABCO (23). The confocal laser scanning
microscope (CLSM) Leica Corp. TCS NT version 1.5.451
(Leica Corp. Lasertechnik, Heidelberg, Germany) was used
for analysis of the specimens with FITC fluorescence excitation at 488
nm or Cy3 and TRITC fluorescence at 568 nm, respectively. Z-series
optical sections taken at 0.5 µm intervals were evaluated using Adobe
Photoshop 5.0 for Windows and Corel-Draw 8 for Windows (15, 27).
Flow cytometry
Aliquots of 150 µl IC-21 cells (107
cells/ml in PBS+) were centrifuged, and the cell
pellets were labeled as described above. Cells were analyzed in a
FACScan (Becton Dickinson and Co., Sunnyvale, CA) with a
sample size of 10,000 cells gated on the basis of forward and side
scatter. The data were stored and processed using the FACScan software
(26).
RNA isolation
RNA was isolated from IC-21 cells and ovaries removed from 8- to
10-week-old C57BL/10 mice according to the GTC/CsCl method
(28).
RT-PCR
The initial random-primed RT was performed with 1 µg of total
RNA, M-MLV Reverse Transcriptase (Promega Corp., Madison),
dNTPs (PCR Nucleotid Mix; Roche Molecular Biochemicals, Mannheim, Germany), and random primer
(Perkin-Elmer Corp., Weiterstadt, Germany) in a MJ
Minicycler (MJ Research, Inc., Biozym, Hess. Oldendorf,
Germany) for 10 min at 25 C, 1 h at 42 C and 5 min at 95 C.
Thereafter, the samples were purified with a QIAquick PCR Purification
Kit (QIAGEN, Hilden, Germany). For PCR, we used the
template complementary DNA, Taq DNA Polymerase
(Promega Corp., Madison, WI), dNTPs (PCR Nucleotid Mix;
Roche Molecular Biochemicals), and six different
oligonucleotide primer pairs. The carboxy terminus of the ER was
probed with two different primer pairs: (1) ERP2–1434
(5'-ACAGGAATCAAGGTAAATGTGTGG-3') and ERM1–1807
(5'-CTCCAGGAGCAGGTCATAGAGG-3'); as well as (2) ERP9–1350
(5'-GGCTGGAGATTCTG- ATGAFTGG-3') and ERM5–1935
(5'-GGGTATGTAGTAGGTTTGTA- AGG-3'). The primer pair (3)
ERP16–589 (5'-CTACTACCTGGAG-AACGAGCC-3') and ERM21–1029
(5'-GAAGCACCCATTTCATTTCGGC-3') was used for the DNA-binding domain of
ER. The DNA-binding domain of ERß was probed with the primer pair
(4) ERßP5–224 (5'-CTTGCCTGTAAACAGAGAGACC-3') and ERßM4–709
(5'-GACGGCTCACTAGCACATTGG-3'). The steroid binding domain of the ERß
was probed with the primer pairs (5) ERßP7–710
(5'-CAATGTGCTAGTGAGCCGTCC-3') and ERßM4–1209
(5'-CTGCTGCTGGGAAGAGATTCC-3') and (6) ERßP3–855
(5'-CAAGTCCGCCTCTTGGAAAGC-3') and ERßM1–1160
(5'-CATCTGTCACTGCGTTCAATAGG-3'). The amplification was performed with
36 cycles at 94 C for 1 min, at 56 C for 1 min, and at 72 C for 1 min
and at the end of the last cycle for 15 min at 72 C.
DNA sequencing
PCR fragments were separated in 2% Tris borate-EGTA gels,
eluted, and cloned into the vector pGEM-TEasy (Promega Corp., Madison). The clones were sequenced with Thermo Sequenase
fluorescent-labeled sequencing kit (Amersham Pharmacia Biotech), and analyzed with the LICOR sequencer (MWG
Biotech, Ebersberg, Germany).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
This Ca2+ rise dropped after 20–40 sec and,
then, turned into a sustained plateau. At 0.1 nM,
E2 caused a weaker Ca2+
spike by only about 50 nM Ca2+ (Fig. 1A). In contrast, 17-estradiol did not induce any significant
increase in [Ca2+]i (Fig. 1A). Moreover, a rise in
[Ca2+]i could also be
induced by 100 nM plasma membrane-impermeable
E2-BSA conjugate, whereas BSA alone did not
influence the [Ca2+]i
(Fig. 1B). It is not clear why 100 times higher
E2-BSA concentrations were required to elicit the
same response as free E2. One reason may be that,
because of steric hindrance, only one or two of the
E2 molecules bound to BSA are able to induce a
Ca2+ response. Another reason may be that
coupling of E2 to BSA via carboxymethyl oxime
(CMO) reduces the capacity of E2 to increase
[Ca2+]i
(13). In this context, it is also noteworthy that we
removed any E2 and E2-CMO
possibly released from E2-BSA with the
stripped-charcoal technique described by Lieberherr et al.
(13). Repeated additions of E2 or
E2-BSA leads to repeated
Ca2+ spikes (23). Even a
pretreatment of IC-21 cells with 1 or 10 nM
E2 for 4 h did not reduce the
Ca2+ response to E2 (data
not shown). Pretreatment of cells with tamoxifen or raloxifene, which
are blockers of classical iERs, prevented neither the
E2-BSA-induced nor the
E2-induced increase in
[Ca2+]i (Fig. 1C).
However, pertussis toxin totally inhibited the
E2- induced Ca2+ spike
(Fig. 1D).
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). When intracellular
Ca2+ mobilization was inhibited by the direct
phospholipase C inhibitor U-73122, there was still an
E2-induced increase in
[Ca2+]i due to influx of
extracellular Ca2+ (Fig. 1F). Partial inhibition
of Ca2+ increase was achieved with
Ni2+ concentrations known to inhibit the release
of Ca2+ from intracellular stores (Fig. 1G, 0.5
mM), whereas the influx was completely inhibited by higher
Ni2+ concentrations that also block plasma
membrane Ca2+ channels (Fig. 1G, 5
mM). Also, the E2-induced
Ca2+ influx could be reduced with the two
blockers of voltage-gated Ca2+ channels
nifedipine (L-type channels) and verapamil (Fig. 1H).
Sequestrable surface E2-binding sites
To test the presence of putative surface
E2-receptors, the IC-21 cells were incubated with
the ligand E2-BSA-FITC conjugate. After labeling
for 5 sec, flow cytometry detected a significant increase in
fluorescence intensity compared with unlabeled control cells (Fig. 2A). However, the fluorescence intensity
increased gradually with progressing labeling periods reaching a
maximum after about 1 h (Fig. 2A). In parallel, the cells were
investigated by confocal laser scanning microspopy (CLSM). After
labeling for 5 sec and 1 min, the fluorescence was exclusively
localized on the cell surface. After 5 min, however, weak punctate
fluorescence emerged inside of the cells at their periphery, besides
surface fluorescence. This punctate fluorescence increased in intensity
after labeling for 1 h and was distributed throughout the whole
cytoplasm (Fig. 2B).
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).
The latter were stained with LysoTracker Red
DND-99 and did not colocalize with the green punctate fluorescence of
E2-BSA-FITC (Fig. 3A). Also, the sequestrated
E2-BSA-FITC colocalized neither with clathrin as
detected by anti-clathrin antibodies (Fig. 3B) and by
transferrin-tetramethylrhodamin, which is an indicator of sequestration
through clathrin-coated vesicles (Fig. 3C), nor with caveolin as
monitored by anticaveolin antibodies (Fig. 3D). The internalization
process could be inhibited by treatment of cells with hypertonic medium
(Fig. 3E) but not by the tubulin-blocker nocodazole and the
microfilament-blocker cytochalasin B (data not shown).
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, A and B). Moreover,
internalization occurred neither with surface-bound ConA-rhodamine nor
with the macrophage specific surface marker F4/80 identified by a rat
monoclonal antibody against F4/80, even if the surface labelings were
performed in the presence of E2 (Fig. 4B).
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-estradiol (Fig. 5A). By
contrast, pretreatment of cells with 17ß-E2 for
2 h before incubation with E2-BSA-FITC alone
did not prevent sequestration (Fig. 3F). Neither testosterone nor
testosterone-BSA nor 1-dehydrotestosterone were able to competitively
reduce the sequestration of E2-BSA-FITC (Fig. 5A). Pretreatment of IC-21 macrophages with pertussis toxin resulted in
a decrease of internalized fluorescence intensity (Fig. 5A). However,
the phospholipase C inhibitor U-73122 as well as its inactive control
compound U-73343 did not block the internalization of surface-bound
E2-BSA-FITC (data not shown). Furthermore, the
internalization of membrane E2-receptors depended
on energy. Depletion of ATP by sodium azide resulted in a decrease of
the fluorescence intensity by about 30% (Fig. 5A), which was localized
almost exclusively on the surface of IC-21 cells. Finally, the
sequestration of membrane E2-receptors was also
dependent on temperature. Temperatures at 16 C and below largely
inhibited the sequestration of surface-bound
E2-BSA-FITC. By contrast, the binding of
E2-BSA-FITC to the cell surface was not affected
by temperature (Fig. 5B).
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and iERß in IC-21 cells was first
examined by RT-PCR. Using different primers of the carboxy terminus and
the DNA-binding domain of ER, RT-PCR revealed the expected bands of
ER in IC-21 cells and mouse uterus, which was used as a positive
control (Fig. 6A). DNA sequencing
confirmed that the PCR fragments derived from uterus and IC-21 cells
contained the predicted regions of the ER. However, RT-PCR did not
detect any ERß in IC-21 cells, whereas the uterus was ERß positive
(Fig. 6A). In accordance, incubation of IC-21 cells with the anti-ERß
antibody ERß (Y-19) directed against an epitope corresponding to the
amino terminus of the ERß did not result in any significant labeling
of intact or permeabilized IC-21 cells as detected by flow cytometry
and CLSM (data not shown).
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was predominantly localized in the cytoplasm and to a lesser
extent in the nucleus as detected in permeabilized cells by CLSM using
the anti-ER antibody ER (MC-20) directed against an epitope in
the carboxy terminus of the ER as well as the anti-ER antibody
ER (H-184) directed against an epitope in the amino terminus of the
ER (Fig. 6C). Both antibodies specifically reacted with
permeabilized cells, because the antibody reaction could be
competitively displaced by specific blocking peptides as revealed by
flow cytometry (Fig. 6B). However, ER was not accessible on the
outer surface of intact cells as probed by flow cytometry (Fig. 6B) and
CLSM (data not shown) using the two different ER antibodies
described above. Furthermore, no sequestration of peripheral ER was
found. After 1 h labeling with the anti-ER antibodies in the
presence of E2, there was no increase in
fluorescence intensity of intact cells. Moreover, when intact IC-21
cells were incubated with the ER (MC-20) antibody and, in parallel,
with E2-BSA-FITC for 1 h, CLSM did reveal
only internalized E2-BSA-FITC, but not any
internalized ER (data not shown; see Fig. 2B). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The E2-induced increase in
[Ca2+]i of IC-21 cells is
initiated on the cell surface via specific
E2-receptors. This view is supported by our
findings that also the plasma membrane-impermeable ligand
E2-BSA induces a rise in
[Ca2+]i. In addition, the
fluorescent conjugate E2-BSA-FITC specifically
binds to the surface of intact IC-21 cells as detected by flow
cytometry and CLSM. Moreover, our data show that the rise in
[Ca2+]i can be blocked by
pertussis toxin, and the phospholipase C inhibitor U-73122 inhibits the
release of intracellular Ca2+. Obviously, the
surface E2-receptors belong to that class of
membrane receptors which are coupled to phospholipase C via a pertussis
toxin-sensitive G protein. In accordance, recent studies show that
E2 activates ß
subunits of a pertussis
toxin-sensitive G protein coupled to a PLC-ß2 in osteoblasts
(21, 30).
The plasma membrane G protein-coupled receptors for
E2 (E2-GPCR) in IC-21 cells
exhibit properties that are typically found for other G protein-coupled
receptors (GPCR). For instance, a wide variety of GPCRs, as
e.g. the prototypic ß2-adrenergic
receptor and the angiotensin II type 1A receptor, become sequestrated
after ligand binding (31, 32, 33). Also, the
E2-GPCR become sequestrated a few minutes after
binding of E2 as visualized by labeling with
E2-BSA-FITC. This internalization process is
ligand-specific, i.e. internalization of
E2-BSA-FITC is competitively inhibited by
17ß-E2 and 17ß-E2-BSA,
but not by 17-estradiol and internalization depends on temperature
and energy. Moreover, pertussis toxin reduces sequestration, indicating
that E2-GPCR internalization is dependent on
Ca2+. In addition, E2-GPCR
internalization is selective, i.e. only distinct plasma
membrane domains are internalized, which exclude, for example,
macrophage-specific surface molecules such as F4/80. In general, the
sequestration of GPCR occurs via the clathrin-coated vesicle-mediated
endocytotic pathway (34, 35, 36) or via caveolae (37, 38). The clathrin pathway can be prevented by hypertonic media
(39, 40, 41). Also, we can find in IC-21 cells that the
E2-GPCR internalization is inhibited by
hypertonic sucrose. Nevertheless, the E2-GPCR
sequestration is mediated by a clathrin- and caveolin-independent
pathway because there is no colocalization of vesicles containing
E2-BSA-FITC with caveolin, clathrin, and
transferrin. In accordance, there is some information available that
internalization of GPCRs does not necessarily occur through clathrin-
or caveolin-dependent pathways (35, 42). In general, GPCR
sequestration is considered to be important for regulation of
signaling, recycling, down-regulation and responsiveness or essential
for the activation of specific signal transduction factors (33, 43, 44, 45). Though the reason for E2-induced
E2-GPCR internalization is still unknown, a
possible down-regulation does not seem very likely because pretreatment
of cells with E2 for 2 h did not prevent
sequestration of E2-GPCRs and pretreatment for
4 h did not reduce the Ca2+ response to
E2. Thus, it seems more plausible that
internalization of E2-GPCR may be involved in the
activation of specific signaling pathways.
Surface estrogen receptors, identical or structurally related to at
least one form of the iER, have been recently localized in various
cells such as GH3/B6 rat pituitary tumor cells
(18, 46), human monocytes (17), rabbit uterus
cells (47), and transfected hamster ovary cells
(19). By contrast, the surface
E2-GPCR of IC-21 cells are neither ER nor
ERß. The latter is not expressed in IC-21 cells at all, and ER is
not accessible on the outer surface of intact cells, but can only be
detected intracellularly, i.e. in the cytoplasm and to a
lesser extent in the nucleus of permeabilized IC-21 cells. In
accordance, other studies have also revealed that classical ERs can be
localized in both the cytoplasm and the nucleus (48, 49, 50, 51).
On the basis of the cytoplasmic localization of ER, it could be
argued that a possible tight association of ER with the cytoplasmic
surface of the plasma membrane could lead to an activation of this
ER by E2-BSA. However, this can be excluded
because E2-BSA conjugates bind to neither ER
nor ERß as recently shown using several different assays
(52). Moreover, ER reveals properties which are clearly
distinct from those of the E2-GPCR on the surface
of IC-21 macrophages. For instance, ER cannot be induced to be
internalized by E2 or
E2-BSA-FITC, in contrast to the
E2-GPCR, though Kim et al. have
demonstrated the occurrence of ER in plasmalemmal caveolae
(53). Moreover, the iER-blockers tamoxifen and raloxifene
have no inhibitory effect on the rapid rise in
[Ca2+]i of IC-21 cells
induced by both E2 and
E2-BSA.
Recently, IC-21 cells have been also shown to contain sequestrable surface GPCRs for testosterone (T-GPCR) (23). However, T-GPCR exhibit properties different to those of E2-GPCR. First, the E2-GPCR mediates the E2-induced increase in [Ca2+]i via both Ca2+ release from intracellular stores and influx of extracellular Ca2+, whereas testosterone induces, via T-GPCR, only a mobilization of Ca2+ from intracellular stores. The phospholipase C inhibitor U-73122 completely blocks the testosterone-induced raise in [Ca2+]i, whereas the E2-induced raise is only reduced by approximately one half. Verapamil and nifedipine reduces the increase in [Ca2+]i after E2-treatment, whereas the testosterone-induced increase in [Ca2+]i is unaffected by these drugs. Moreover, testosterone and testosterone-BSA are not able to compete with E2 for the internalization of E2-GPCR. Sequestration of T-GPCR, but not that of E2-GPCR, is inhibited by the direct phospholipase C inhibitor U-73122 as well as by nocodazole and cytochalasin B. It remains to be seen as to whether the E2-GPCR and the T-GPCR are two different receptors or there is only one receptor with different binding sites for E2 and testosterone coupled to different signaling pathways.
Collectively, our data unequivocally show the presence of functional novel E2-GPCR in plasma membranes of IC-21 cells that do not mediate the classical genomic ER-response, but rather initiate a transcription-independent E2-signaling pathway involving Ca2+ as one of several other possible intracellular mediators.
Received March 2, 2000.
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References |
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 K. S. Monkkonen, R. Aflatoonian, K.-F. Lee, W. S.B. Yeung, S.-W. Tsao, J. T. Laitinen, and A. Fazeli Hormonal regulation of G{alpha}i2 and mPR{alpha} in immortalized human oviductal cell line OE-E6/E7 Mol. Hum. Reprod., December 1, 2007; 13(12): 845 - 851. [Abstract] [Full Text] [PDF]
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E. Filardo, J. Quinn, Y. Pang, C. Graeber, S. Shaw, J. Dong, and P. Thomas Activation of the Novel Estrogen Receptor G Protein-Coupled Receptor 30 (GPR30) at the Plasma Membrane Endocrinology, July 1, 2007; 148(7): 3236 - 3245. [Abstract] [Full Text] [PDF]
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 K. S. Monkkonen, R. Aflatoonian, K.-F. Lee, W. S.B. Yeung, S.-W. Tsao, J. T. Laitinen, E. M. Tuckerman, T.C. Li, and A. Fazeli Localization and variable expression of G{alpha}i2 in human endometrium and Fallopian tubes Hum. Reprod., May 1, 2007; 22(5): 1224 - 1230. [Abstract] [Full Text] [PDF]
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L. Canesi, C. Ciacci, L. C. Lorusso, M. Betti, T. Guarnieri, S. Tavolari, and G. Gallo Immunomodulation by 17beta-estradiol in bivalve hemocytes Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2006; 291(3): R664 - R673. [Abstract] [Full Text] [PDF]
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 M. Farooqui, Z. H. Geng, E. J. Stephenson, N. Zaveri, D. Yee, and K. Gupta Naloxone acts as an antagonist of estrogen receptor activity in MCF-7 cells. Mol. Cancer Ther., March 1, 2006; 5(3): 611 - 620. [Abstract] [Full Text] [PDF]
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X. Li, L. Strauss, A. Kaatrasalo, A. Mayerhofer, I. Huhtaniemi, R. Santti, S. Makela, and M. Poutanen Transgenic Mice Expressing P450 Aromatase as a Model for Male Infertility Associated with Chronic Inflammation in the Testis Endocrinology, March 1, 2006; 147(3): 1271 - 1277. [Abstract] [Full Text] [PDF]
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 J. A. Arreguin-Arevalo and Terry. M. Nett A Nongenomic Action of Estradiol as the Mechanism Underlying the Acute Suppression of Secretion of Luteinizing Hormone in Ovariectomized Ewes Biol Reprod, January 1, 2006; 74(1): 202 - 208. [Abstract] [Full Text] [PDF]
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 E. R. Levin Integration of the Extranuclear and Nuclear Actions of Estrogen Mol. Endocrinol., August 1, 2005; 19(8): 1951 - 1959. [Abstract] [Full Text] [PDF]
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 X. Guo, M. Razandi, A. Pedram, G. Kassab, and E. R. Levin Estrogen Induces Vascular Wall Dilation: MEDIATION THROUGH KINASE SIGNALING TO NITRIC OXIDE AND ESTROGEN RECEPTORS {alpha} AND {beta} J. Biol. Chem., May 20, 2005; 280(20): 19704 - 19710. [Abstract] [Full Text] [PDF]
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 N. Kanda and S. Watanabe 17{beta}-Estradiol enhances heparin-binding epidermal growth factor-like growth factor production in human keratinocytes Am J Physiol Cell Physiol, April 1, 2005; 288(4): C813 - C823. [Abstract] [Full Text] [PDF]
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P. Thomas, Y. Pang, E. J. Filardo, and J. Dong Identity of an Estrogen Membrane Receptor Coupled to a G Protein in Human Breast Cancer Cells Endocrinology, February 1, 2005; 146(2): 624 - 632. [Abstract] [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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 K. C. Lambert, E. M. Curran, B. M. Judy, D. B. Lubahn, and D. M. Estes Estrogen receptor-{alpha} deficiency promotes increased TNF-{alpha} secretion and bacterial killing by murine macrophages in response to microbial stimuli in vitro J. Leukoc. Biol., June 1, 2004; 75(6): 1166 - 1172. [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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 B. BARTHOLOME, C. M. SPIES, T. GABER, S. SCHUCHMANN, T. BERKI, D. KUNKEL, M. BIENERT, A. RADBRUCH, G.-R. BURMESTER, R. LAUSTER, et al. Membrane glucocorticoid receptors (mGCR) are expressed in normal human peripheral blood mononuclear cells and up-regulated after in vitro stimulation and in patients with rheumatoid arthritis FASEB J, January 1, 2004; 18(1): 70 - 80. [Abstract] [Full Text] [PDF]
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C. E. Navarro, S. A. Saeed, C. Murdock, A. J. Martinez-Fuentes, K. K. Arora, L. Z. Krsmanovic, and K. J. Catt Erratum Mol. Endocrinol., December 1, 2003; 17(12): A - 2658. [Abstract] [Full Text] [PDF]
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O. A. Sukocheva, L. Wang, N. Albanese, S. M. Pitson, M. A. Vadas, and P. Xia Sphingosine Kinase Transmits Estrogen Signaling in Human Breast Cancer Cells Mol. Endocrinol., October 1, 2003; 17(10): 2002 - 2012. [Abstract] [Full Text] [PDF]
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C. E. Navarro, S. Abdul Saeed, C. Murdock, A. J. Martinez-Fuentes, K. K. Arora, L. Z. Krsmanovic, and K. J. Catt Regulation of Cyclic Adenosine 3',5'-Monophosphate Signaling and Pulsatile Neurosecretion by Gi-coupled Plasma Membrane Estrogen Receptors in Immortalized Gonadotropin-Releasing Hormone Neurons Mol. Endocrinol., September 1, 2003; 17(9): 1792 - 1804. [Abstract] [Full Text] [PDF]
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S. Fiorini, M. E. Ferretti, C. Biondi, B. Pavan, L. Lunghi, G. Paganetto, and L. Abelli 17{beta}-Estradiol Stimulates Arachidonate Release from Human Amnion-Like WISH Cells through a Rapid Mechanism Involving a Membrane Receptor Endocrinology, August 1, 2003; 144(8): 3359 - 3367. [Abstract] [Full Text] [PDF]
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I. M. Abraham, S.-K. Han, M. G. Todman, K. S. Korach, and A. E. Herbison Estrogen Receptor {beta} Mediates Rapid Estrogen Actions on Gonadotropin-Releasing Hormone Neurons In Vivo J. Neurosci., July 2, 2003; 23(13): 5771 - 5777. [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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J. M. Hall and K. S. Korach Stromal Cell-Derived Factor 1, a Novel Target of Estrogen Receptor Action, Mediates the Mitogenic Effects of Estradiol in Ovarian and Breast Cancer Cells Mol. Endocrinol., May 1, 2003; 17(5): 792 - 803. [Abstract] [Full Text] [PDF]
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 Y. Zhu, C. D. Rice, Y. Pang, M. Pace, and P. Thomas From the Cover: Cloning, expression, and characterization of a membrane progestin receptor and evidence it is an intermediary in meiotic maturation of fish oocytes PNAS, March 4, 2003; 100(5): 2231 - 2236. [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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K. L. Chambliss and P. W. Shaul Estrogen Modulation of Endothelial Nitric Oxide Synthase Endocr. Rev., October 1, 2002; 23(5): 665 - 686. [Abstract] [Full Text] [PDF]
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Z. Guo, W. P. M. Benten, J. Krucken, and F. Wunderlich Nongenomic Testosterone Calcium Signaling. GENOTROPIC ACTIONS IN ANDROGEN RECEPTOR-FREE MACROPHAGES J. Biol. Chem., August 9, 2002; 277(33): 29600 - 29607. [Abstract] [Full Text] [PDF]
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 K. J. Ho and J. K. Liao Non-nuclear Actions of Estrogen: New Targets for Prevention and Treatment of Cardiovascular Disease Mol. Interv., July 1, 2002; 2(4): 219 - 228. [Abstract] [Full Text] [PDF]
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A. B. Ropero, B. Soria, and A. Nadal A Nonclassical Estrogen Membrane Receptor Triggers Rapid Differential Actions in the Endocrine Pancreas Mol. Endocrinol., March 1, 2002; 16(3): 497 - 505. [Abstract] [Full Text] [PDF]
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Z. Guo, J. Krucken, W. P. M. Benten, and F. Wunderlich Estradiol-induced Nongenomic Calcium Signaling Regulates Genotropic Signaling in Macrophages J. Biol. Chem., February 22, 2002; 277(9): 7044 - 7050. [Abstract] [Full Text] [PDF]
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E. J. Filardo, J. A. Quinn, A. R. Frackelton Jr., and K. I. Bland Estrogen Action Via the G Protein-Coupled Receptor, GPR30: Stimulation of Adenylyl Cyclase and cAMP-Mediated Attenuation of the Epidermal Growth Factor Receptor-to-MAPK Signaling Axis Mol. Endocrinol., January 1, 2002; 16(1): 70 - 84. [Abstract] [Full Text] [PDF]
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J. M. Hall, J. F. Couse, and K. S. Korach The Multifaceted Mechanisms of Estradiol and Estrogen Receptor Signaling J. Biol. Chem., September 28, 2001; 276(40): 36869 - 36872. [Full Text] [PDF]
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M. W. Knoferl, M. G. Schwacha, D. Jarrar, M. K. Angele, K. Fragoza, K. I. Bland, and I. H. Chaudry Estrogen pretreatment protects males against hypoxia-induced immune depression Am J Physiol Cell Physiol, May 1, 2002; 282(5): C1087 - C1092. [Abstract] [Full Text] [PDF]
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