| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Detected on the Plasma Membrane of Aldehyde-Fixed GH3/B6/F10 Rat Pituitary Tumor Cells by Enzyme-Linked Immunocytochemistry1
Department of Human Biological Chemistry and Genetics (A.M.N., C.S.W.), Department of Pharmacology and Toxicology (M.L.T.), University of Texas Medical Branch, Galveston, Texas 77555; and Department of Pediatrics (B.G.), Medical College of Wisconsin, Milwaukee, Wisconsin 53226
Address all correspondence and requests for reprints to: Cheryl S. Watson, Ph.D., Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555-0645. E-mail: cswatson{at}utmb.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(ER) proteins, located at the
plasma membrane, is postulated to mediate the rapid, nongenomic
responses of GH3/B6/F10 pituitary cells to estrogen. To
demonstrate the presence of ER at the plasma membrane and to
distinguish this receptor population from that in the nucleus,
GH3/B6/F10 cells were first prepared in 2%
paraformaldehyde/0.1% glutaraldehyde in PBS (P/G) without detergent,
then exposed to one of several antibodies (Abs) raised against nuclear
ER. Ab binding was visualized as a fluorescent/chromagenic reaction
product catalyzed by avidin-biotin-complexed alkaline
phosphatase. With P/G fixation, Abs could only access antigens at the
cell surface, as evidenced by the inability of 70K mol wt
dextrans to permeate cells and the absence of intracellular staining by
Abs to cytoplasmic or nuclear antigens. ER Abs generated membrane,
but not nuclear, staining in P/G-fixed cells; nuclear receptor labeling
could only be detected in detergent-treated cells. Specificity of
staining for ER was confirmed by three approaches: first, treatment
with an antisense oligodeoxynucleotide to nuclear ER mRNA reduced
immunolabeling of both membrane and nuclear ER; second, labeling by
two Abs raised against different ER oligopeptides was neutralized by
competing peptide; third, six Abs (ER21, H226, R4, H222, MC20, and
C542) that recognize unique epitopes on rodent ER produced
immunolabeling, but neither primate-specific ER Ab nor Ab to ERß
caused staining. In addition to demonstrating the plasma membrane ER
in GH3/B6/F10 cells, this method should be applicable to
other cell types that exhibit nongenomic responses to estrogen or other
steroid hormones. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(ER) functions
as a transcription factor, which localizes to the nucleus and, upon
activation by estrogen, assumes a conformation that binds with high
affinity to estrogen response elements on chromatin (1, 2). The
interaction of the liganded receptor with DNA is a well-characterized
mechanism by which estrogens regulate the transcription of target
genes. However, in addition to directing nuclear events, estrogens
rapidly activate ion channels and signaling cascades at the plasma
membrane (3, 4, 5). Reports of rapid, nongenomic responses to estrogen in
many systems have ignited interest in the putative receptor(s)
mediating these membrane-initiated effects. Radioligand binding
studies, using labeled 17ß-estradiol (E2), have revealed
specific, saturable binding sites in plasma membrane preparations from
different cell types (6, 7). In whole cells (8, 9, 10, 11, 12), estrogen binding
sites on the surface of cells have been observed using
17ß-E2 conjugated to fluorescently tagged proteins,
molecular complexes that are too large to diffuse across the plasma
membrane. Antibodies (Abs) raised against the nuclear ER have
provided another tool for the study of estrogen binding sites on the
cell surface: under experimental conditions in which the Abs do not
enter the cell, binding of ER Abs occurs at the plasma membrane (8, 11, 12, 13, 14, 15). These findings furnish evidence for the existence of a
population of ERs, structurally related to ER, that are located on
the cell membrane.
Radioligand binding analyses of nuclear and plasma membrane
fractions have suggested that the number of estrogen binding sites on
the surface of cells is lower, by an order of magnitude, than the
number in the nucleus (6, 12). Hence, to study the membrane receptors,
conditions must be met that minimize contamination by the nuclear
signal and which afford enough sensitivity to detect the relatively low
levels of the membrane protein. In the present report, an
immunocytochemical system for detection of membrane ER was developed
in GH3/B6/F10 cells, a rat pituitary tumor line that
exhibits rapid responsiveness to estrogen and expresses high levels of
membrane ER (13). The cells were rendered impermeable to Abs by
aldehyde fixation, thereby eliminating interference from the
intracellular ER population (16). Additionally, by using fixed
cells, the avidin-biotinylated enzyme complex (ABC) method of
immunocytochemistry (17), in conjunction with a fluorescent/chromagenic
reaction product, could be employed to amplify the signal, thereby
enhancing the sensitivity of detection. This method provides a
convenient technique for sensitive and specific detection of plasma
membrane ER, and the technique has potential for application to
membrane steroid receptors in many other cell types.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Abs
ER Abs. Seven Abs that recognize different determinants
on the rat ER were employed in this study; Fig. 1
illustrates the approximate location of
the epitope for each Ab on the rat ER protein. One Ab, D75,
recognizes primate, but not rodent, ER protein and was used as an
experimental control. H151 (18) and C542 (19) are mouse monoclonal Abs
(mAbs) raised against oligopeptides derived from the human ER
sequence; these Abs, kindly provided by Drs. Nancy L. Weigel and Dean
P. Edwards, are now commercially available (StressGen Biotechnologies,
Victoria, Canada). H222, H226, ER21, and D75 are rat mAbs raised
against human ER (20); they were a generous gift from Dr. Geoffrey
Greene. Ab R4 is a peptide affinity-purified rabbit polyclonal Ab that
was raised in our laboratory against an oligopeptide from the rat ER
(13). Ab MC20, a peptide affinity-purified rabbit polyclonal Ab to an
oligopeptide based on the mouse ER, was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
|
Ab controls. Antiserum against rat PRL (AFP-131581570) was obtained through the National Hormone and Pituitary Program and the National Institute of Diabetes and Digestive and Kidney Disease. A mouse mAb that recognizes an epitope on all 5 histone proteins (Anti-Histon Pan) was purchased from Boehringer Mannheim (Indianapolis, IN). A rabbit polyclonal Ab (PA1–310) to a C-terminal peptide based on the rat ERß sequence was purchased from Affinity BioReagents, Inc. (Golden, CO). Tetramethylrhodamine-labeled dextran, of 70K mol wt, was purchased from Molecular Probes, Inc. (Eugene, OR).
Fluorescence immunocytochemistry
The ABC method (protocol I). GH3/B6/F10 cells
were washed three times in Dulbecco’s PBS (DPBS). The fixative and
fixation conditions were modified from Brink et al. (16), as
follows: For nonpermeabilized cells, the fixative contained 2.0%
paraformaldehyde (Sigma Chemical Co.) and 0.1%
glutaraldehyde (Electron Microscopy Sciences) in PBS, adjusted to pH
7.4; this fixative [2% paraformaldehyde/0.1% glutaraldehyde in PBS
(P/G)] was freshly prepared and applied to the cells for 30 min at 20
C. For permeabilized cells, 0.5% Nonidet P-40 (NP-40) and 0.15
M sucrose were included in the P/G solution; cells were
exposed to the detergent-containing fixative for 1 min at 20 C. 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, before a 1-h blocking step at 37
C with 10% BSA (ICC-grade, Sigma Chemical Co.) and 0.1%
gelatin (from cold water fish skin, Sigma Chemical Co.) in
DPBS. The primary Abs were diluted in 0.5% BSA, 0.1% gelatin in DPBS
(PBG) and were added to the cells for 2 h at 20 C; next, the cells
were washed six times over 30 min in PBG. For the ABC method (17), the
following steps were conducted at 20 C using reagents from a
Vectastain ABC-Alkaline Phosphatase kit in conjuction with
Vector Red substrate (Vector Laboratories, Inc.,
Burlingame, CA): Biotinylated universal antirabbit/antimouse IgG was
diluted to 4 µg/ml in PBG and added to the cells for 1 h; then,
the cells were washed six times over 30 min in PBG. The ABC-alkaline
phosphatase 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% ethanol, 95% ethanol, and xylene, then mounted using
Cytoseal 280 (Stephens Scientific, Riverdale, NJ).
The fluorescent secondary Ab method (protocol II). The cells were treated, as described above, up to the addition of the biotinylated secondary Ab incubation. Instead of the biotinylated Ab, a tetramethylrhodamine isothiocyanate (TRITC)-labeled antirabbit IgG (Sigma Chemical Co.) was added to the cells in the dark for 1 h at 20 C. The cells were washed six times over 30 min in DPBS, then mounted with Fluoromount G (Electron Microscopy Sciences), and stored in the dark at 4 C until examined for fluorescence (Eastman Kodak Co., Rochester, NY).
Photomicrography. Photomicrographs were taken with Kodak Pro400MC or Kodak Ektapress 1600 Plus color negative film using an Olympus Corp. (Melville, NY). AHBT microscope equipped with a fluorescence attachment (Model AH2-RFL) and camera (Model C-35AD-4).
Antisense experiments
A 15-mer antisense oligonucleotide (5'GGGTCATGGTCATGG3') and a
scrambled control (5'GTGGTGGATCGTGAC3') were synthesized from
unmodified bases by Genosys (Life Technologies, Inc.,
Rockville, MD). The ability of the antisense oligo to reduce ER
levels in vitro has been previously reported (21).
After 3 days of culture in SSM, as described above, GH3/B6/F10 cells were rinsed two times with a serum-free, defined medium composed of phenol red-free DMEM, 0.1% BSA, 5 µg/ml insulin, 5 ng/ml selenium, and 5 µg/ml transferrin. The cells were then cultured for 24 h in this medium in the absence or presence of oligonucleotides. In the untreated control condition, cells were cultured without any oligonucleotide. For oligonucleotide controls, the scrambled oligo was present at a concentration of 0.5 or 1.0 µM, as stated. Antisense-treated cells were cultured with 0.05, 0.5, or 1.0 µM of the antisense oligo, as stated.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Ab C542. Figure 2A
represents the typical pattern of fluorescence that was generated using
ABC-alkaline phosphatase in conjunction with Vector Red substrate; the
corresponding brightfield image is presented in Fig. 2B. For
comparison, Fig. 2C depicts the labeling observed using indirect
immunofluorescence with a TRITC-labeled antimouse IgG; Fig. 2D is the
corresponding brightfield image. Only low levels of background
fluorescence were detected when no ER Ab was added to incubations in
either protocol I (Fig. 2, E and F) or protocol II (Fig. 2, G and
H). Although no staining of nuclear ER was detectable by protocols I
and II when the cells were prepared in the P/G fixative with no added
detergent, cells permeabilized by the addition of NP-40 to the P/G
fixative exhibited distinct nuclear staining in both
immunocytochemistry protocols (for results using protocol 1, see Fig. 3). Results similar to those presented in
Fig. 2 were obtained using ER Abs R4 and MC-20 (data not shown).
|
|
,
subsequent experiments focused on characterization of this system.
Extracellular vs. intracellular immunostaining
The next set of experiments was designed to determine whether the
observed immunostaining of ER occurred on the extracellular surface
of the cells, i.e. whether the fixation conditions were
sufficient to prevent Abs from penetrating the cell membrane. Cells
fixed in P/G without detergent displayed membrane labeling (but not
nuclear labeling) after incubation with ER Ab C542 (Fig. 3A
). No
nuclear staining by an antihistone Ab was detected when the cells were
fixed with P/G in the absence of detergent (Fig. 3B). Staining of the
plasma membrane by antihistone Ab was not observed, as would be
predicted, given the nuclear location of histone proteins. The lack of
membrane labeling by the antihistone Ab is of further significance
because the Ab is a mouse monoclonal of the same IgG1
subtype as the ER mAb C542 and was used at a 2.5-fold higher
concentration than mAb C542 in this experiment. Besides providing
evidence that the Abs did not enter the nonpermeabilized cells and bind
nuclear antigens, these data verify that the observed immunostaining by
mAb C542 did not result from nonspecific binding of the Fc region of
the IgG1 molecule. In contrast to nonpermeabilized cells,
cells permeabilized by the addition of NP-40 to the P/G fixative
exhibited intense nuclear fluorescence when exposed to either the ER
Ab (Fig. 3C) or the antihistone Ab (Fig. 3D). Nuclear staining was
evident at even lower Ab concentrations than employed here (0.5
µg/ml; data not shown). In another set of experiments, consistent
results were obtained using rabbit antiserum to rat PRL.
GH3/B6 cells (22) and the F10 cell line derived from them
(13) constitutively synthesize and secrete PRL. Yet, no intracellular
labeling of the cells was evident unless the cells were fixed in the
presence of detergent (data not shown). Taken together, these data from
permeabilized and nonpermeabilized cells demonstrate that the Abs are
able to recognize their respective antigens if the appropriate cellular
compartment is accessible.
Dextran-tetramethylrhodamine (dextran-TMR) was also used to label
nonpermeabilized and detergent-treated, permeabilized cells. The
dextran-TMR preparation contains molecules distributed in size around
70K mol wt; hence, these molecules are significantly smaller than the
IgG Abs (approximately 150K mol wt). After a 2-h incubation with
dextran-TMR at 20 C (the same conditions used for Abs), no labeling
could be visualized inside the P/G-fixed cells. The data presented in
Fig. 4A illustrate that the fluorescent
dextran molecules were present in the solution surrounding the cells
but that even these relatively small molecules were excluded from cells
under the fixation and incubation conditions used in these experiments.
However, when cells were permeabilized, dextran-TMR diffused throughout
the interior of the cells and was readily viewed inside the cell after
the solution containing the labeled dextran was rinsed off the cells
(Fig. 4B). To depict the margins of the cells in Fig. 4. A and B, the
corresponding brightfield images are shown in Fig. 4, C and D,
respectively.
|
,
expression of the protein was knocked out by treating the cells with a
nuclear ER antisense oligonucleotide for 24 h before
immunocytochemical analysis with ER Abs. These experiments were
conducted in a serum-free, defined medium to minimize degradation of
the oligonucleotides by serum components. Removal of serum caused the
GH3/B6/F10 cells to assume a more spindle-shaped
morphology, yet the cells continued to express membrane ER. In the
control condition shown in Fig. 5A, cells
had been incubated with a scrambled oligonucleotide; this treatment did
not affect the level of immunostaining, compared with cells that were
not exposed to any oligonucleotide (data not shown). In contrast, the
degree of immunolabeling decreased in a concentration-dependent fashion
in cells that had been treated with antisense ER oligonucleotide at
0.05 and 0.5 µM (Fig. 5, B and C, respectively). Fig. 5, D, E, and F, the brightfield images corresponding to Fig. 5, A, B, and
C, respectively, illustrate that the density of cells in the three
fields is equivalent. Note that red staining is visible in the
brightfield views because the Vector Red reaction product is a
chromagen, as well as a fluorophore. Although the extent to which the
fluorescent signal was reduced by antisense treatment varied within and
between experiments, from partial to complete elimination of the
immunostaining, the decrease was also detected with ER Abs directed
against other epitopes on the receptor protein, including Ab R4 (Fig. 6) and Ab ER21 (data not shown). In
separate experiments, 24-h treatment with 0.5 µM ER
antisense caused partial reduction of Ab C542 labeling of the nucleus
in permeabilized cells (data not shown).
|
|
Ab binding to the surface
of GH3/B6/F10 cells was derived from peptide competition
experiments in which a given Ab was preabsorbed to the oligopeptide
against which it was raised. Figure 7, A
and B are photomicrographs of P/G-fixed cells that were incubated with
ER Ab C542 in the absence or presence, respectively, of competing
peptide. An unrelated, control peptide had no effect on Ab C542 binding
(data not shown). Cells that were permeabilized by the addition of
detergent to the fixative displayed a distinct pattern of nuclear
staining (Fig. 7C), which was also reduced by competing peptide (Fig. 7D). Immunostaining by Ab R4 of the membrane in nonpermeabilized cells
and of the nucleus in permeabilized cells was also competitively
inhibited by preabsorption of the Ab with its cognate peptide (data not
shown).
|
(see Fig. 1) was examined in this assay system. Ab MC20, whose epitope
overlaps that of C542 near the C-terminus of the receptor, produced
consistently intense staining reactions, similar to C542, at
concentrations as low as 2 µg/ml. Intermediate levels of labeling
were generated by Ab ER21 (10 µg/ml), whose epitope lies at the
N-terminus, and by Ab R4 (1:200), a peptide affinity-purified rabbit
polyclonal whose epitope is found in the hinge region. Interestingly,
Ab H151, a mouse monoclonal whose epitope is adjacent to that of R4,
yielded no staining above background at concentrations ranging from
1–20 µg/ml. Ab H222 (directed at the ligand binding domain of the
receptor) and Ab H226 (directed against the DNA binding domain) both
generated weak, but detectable, signals in this system at 10 µg/ml.
As expected, Ab D75, a mAb only recognizing ER from primate species,
did not label the rat protein at concentrations ranging from 1–10
µg/ml. Also, no membrane labeling of GH3/B6/F10 cells was
detected using a rabbit polyclonal Ab to the C-terminus of rat ERß
(1–10 µg/ml). In this regard, it is noteworthy that the ER
peptides against which C542, MC-20, or R4 were raised share no homology
with sequence reported for rat ERß (23). |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
at the plasma membrane and clearly
distinguishes this population from ER located in the nucleus. By
first fixing the cells in paraformaldehyde and glutaraldehyde in the
absence of detergent, the ABC-alkaline phosphatase method could be
employed to enhance detection of the membrane receptor, which is
present in low abundance, relative to the nuclear receptor. This
technique may have applicability to other steroid hormone membrane
receptors and other cell types, provided it can be determined, in a
given system, that fixation conditions prevent entry of the Abs into
the cell and that the immunolabeling is specific for the receptor under
consideration. In validating the methodology for the membrane ER in
GH3/B6/F10 cells, several approaches to each of these
technical issues were employed.
To exclude Abs from the intracellular compartment, glutaraldehyde was
combined with paraformaldehyde in the fixative solution, as previously
described by Brink et al. (16). Under these fixation
conditions, the pattern of binding exhibited by ER Abs is clearly
extranuclear. The lack of staining by Abs to histone proteins or to PRL
[which is found in the cytoplasm of these cells (13, 22)], provides
additional evidence that large IgG molecules are unable to penetrate
the cell when fixed with P/G. Brink et al. reported that no
staining of the nuclear glucocorticoid receptor could be visualized
under similar P/G fixation conditions. Moreover, these investigators
found it was necessary to add the detergent, NP-40, together with
(rather than after) the aldehyde mixture, to permeabilize the cell
membrane and allow access of Abs to intracellular compartments. In our
experiments, permeabilization of GH3/B6/F10 cells with
NP-40 permitted nuclear labeling by ER Abs, in striking contrast to
the pattern of surface staining obtained when no detergent is added.
Abs to other antigens that are present in intracellular compartments
(histone proteins in the nucleus and PRL in the cytoplasm) only
generated signal in the presence of detergent. Even a fluorescently
labeled dextran with a mol wt of 70K did not diffuse into the cells
unless detergent was included in the fixative. Hence, we conclude that
any immunostaining in cells prepared with P/G, in the absence of
detergent, could be attributed to Abs bound to the surface of the
cells.
The results presented here, using P/G-fixed cells, confirm and
extend observations reported from our laboratory using live, unfixed
cells. In live GH3/B6 cells, the indirect
immunofluorescence signal associated with ER Ab binding was
localized to the cell perimeter by confocal scanning laser microscopy
(8, 14). Additionally, in double-labeling experiments (8), an
E2-BSA conjugate (tagged with fluorescein) and rabbit R3
anti-ER Ab/goat antirabbit Ab (tagged with Cy3) were shown to
colocalize on the surface of the cells. In those studies, live cells
were incubated for 10 min with Abs at 4 C to prevent uptake of the Abs
before fixation of the cells with paraformaldehyde; this methodology
resulted in a punctate staining pattern on the periphery of cells, with
no observable nuclear staining. By increasing incubation times and
temperatures, aggregation of the signal was accentuated, consistent
with the phenomenon of Ab-induced patching and capping of antigens in a
fluid membrane. In the experiments reported here, by first fixing the
cells with P/G, it was possible to lengthen Ab incubations and apply
the ABC-alkaline phosphatase method to improve detection sensitivity
through enzymatic amplification of the signal (17). As expected, the
ABC technology generated a more intense signal, and a greater
percentage of cells were labeled using the amplification methodology,
compared with indirect immunofluorescence of live cells. Although it is
significant that the presence of surface labeling and the absence of
intracellular staining are similar in both live and P/G-fixed cells,
the punctate pattern of membrane signal seen with the ABC method arises
from accumulation of reaction product and is not related to Ab patching
and capping, because the cells were fixed before exposure to Ab.
To determine whether the membrane immunolabeling observed using fixed
cells was specific for ER, three experimental approaches were taken:
First, experiments were conducted with an antisense
oligodeoxynucleotide targeted to the region of the translation start
codon of all nuclear ER mRNAs (24, 25). This antisense sequence was
previously shown to reduce [3H]-17ß-E2
binding in a mouse colon cancer cell line in a dose- and time-dependent
fashion (21). In the present study, application of the antisense oligo
to the cells for 24 h resulted in a concentration-dependent
decline in immunostaining by ER Ab C542, whose epitope lies at the
C-terminus of the nuclear ER protein. Furthermore, after treatment
with nuclear ER antisense, we observed reduction of membrane
labeling by Abs to the hinge region (R4) and the N-terminus (ER21) of
the nuclear protein. Antisense treatment also decreased immunostaining
of nuclear ER, examined in permeabilized cells. No effect on
immunolabeling of membrane or nuclear receptors by any of these Abs was
noted after exposure of cells to a control, scrambled oligo, which
consisted of the same 15 nucleotides arranged in a sequence with little
or no homology to sequences in GenBank. Though these results confirm
the specificity of Ab staining for the ER protein in this system,
they also support the working hypothesis that the nuclear and plasma
membrane populations of ER are structurally linked. In this regard,
when Migliaccio et al. (26) transfected Cos-7 cells (which
do not express ER) with a plasmid containing the human nuclear ER
coding sequence, the cells became responsive to 17ß-E2,
in terms of rapid activation of the MAP kinase, ERK. More recently,
Razandi et al. (12) introduced a complementary DNA for ER
into Chinese hamster ovary cells (which also do not express ER) and
discovered that populations of membrane and nuclear receptors arose
from the same transcript. They further demonstrated, in the transfected
cells, that 17ß-E2 stimulated ERK through
membrane-associated ER; and they concluded that a population of
ER, residing at the plasma membrane, functions in the rapid,
nongenomic response to estrogen. In the same study, transfection of
Chinese hamster ovary (CHO) cells with an ERß complementary DNA also
yielded functional membrane and nuclear receptors. In our experiments
with GH3/B6/F10 cells, no membrane signal was detected using an Ab to
ERß, although it is not known whether ERß is expressed in our
cells.
As a second approach, in addition to the antisense experiments,
the specificity of ER immunolabeling was substantiated by peptide
competition studies. Immunostaining by Abs C542 and R4 of membrane
ER in nonpermeabilized cells and of nuclear ER in permeabilized
cells was eliminated by preincubation of each Ab with its respective
cognate peptide. Peptide competible binding of an Ab in
immunocytochemical analyses provides evidence for specific recognition
of a given epitope by the Ab. This important control does not
guarantee, however, that a given epitope is not shared by more than one
protein in the cell. In the present study, the fact that immunolabeling
by two Abs raised against different ER peptides could be neutralized
by peptide competition, combined with the fact that both nuclear and
membrane staining by both Abs could be inhibited by peptide
competition, strengthens the argument for the specificity of the
observed staining for ER. Moreover, these data confirm the
structural relationship between the membrane and nuclear receptor
populations.
A third approach to validating the specificity of ER Ab binding to
the membrane was to test Abs whose antigenic determinants lie at
different regions of the protein. Abs, directed against different
epitopes, produced varying degrees of staining. Because the
antigenicity of an epitope can be altered during the protein
cross-linking associated with aldehyde fixatives, the observed
variation in immunolabeling may be related to the nature and extent of
cross-linking associated with the P/G fixation. Alternatively, the
variation in staining intensity by different Abs may relay information
about the conformation of the receptor on the membrane and the
accessibility of a particular epitope to its Ab, or about some
difference in amino acid composition.
Finally, it is important to note some differences between the two
fluorescence immunocytochemistry protocols applied to fixed cells in
this report: one based on the ABC method, in conjunction with a
fluorescent reaction product; and the other using a TRITC-labeled
secondary Ab. Both methods yielded similar results, in terms of the
nonnuclear, surface staining exhibited by nonpermeabilized cells
contrasted with the nuclear labeling found in permeabilized cells. We
focused on the ABC method because of the enhanced sensitivity stemming
from the enzyme amplification step and because the Vector Red
fluorophore is resistant to photobleaching (the Vector Red reaction
product can also be viewed as a red chromagen in brightfield
microscopy, further enhancing the flexibility of the method). At the
same time, it is important to note possible artifacts related to the
ABC methodology as employed here. Most obvious of these is the punctate
staining pattern associated with the ABC method. As substrate
accumulates, it forms large macromolecular complexes that appear as
aggregates on the cell surface; the use of a TRITC-labeled secondary Ab
revealed a more even, diffuse pattern of staining. Also, in the ABC
protocol, the cells were dehydrated before mounting, whereas they were
not dehydrated in the labeled secondary Ab method used in these
experiments. Dehydration was associated with some alteration of cell
morphology and reduction of cell size, which could have contributed to
the enlarged appearance of the aggregates of Vector Red substrate.
Therefore, conclusions about the physical arrangement of ER on the
plasma membrane will require further study.
The focus of this report was the demonstration of ER proteins on the
plasma membrane of GH3/B6/F10 cells using aldehyde-fixed
cells in conjunction with the ABC method of immunocytochemistry. This
provides a convenient system for sensitively and specifically detecting
plasma membrane ER and for distinguishing the membrane population
from the nuclear population of receptors. This technique now provides
us with the opportunity to address such issues as: how membrane
localization of ER is regulated; and what functional interactions
exist between nuclear and membrane ER. In addition, this method
should have broader applicability, including other cell systems and
other membrane steroid receptors.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received February 12, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and ERß expressed in chinese hamster
ovary cells. Mol Endocrinol 13:307–319This article has been cited by other articles:
 |
|
 |
|
  Y. Pang, J. Dong, and P. Thomas Estrogen Signaling Characteristics of Atlantic Croaker G Protein-Coupled Receptor 30 (GPR30) and Evidence It Is Involved in Maintenance of Oocyte Meiotic Arrest Endocrinology, July 1, 2008; 149(7): 3410 - 3426. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. R. Hammes and E. R. Levin Extranuclear Steroid Receptors: Nature and Actions Endocr. Rev., December 1, 2007; 28(7): 726 - 741. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. J. Pietras and D. C. Marquez-Garban Membrane-Associated Estrogen Receptor Signaling Pathways in Human Cancers Clin. Cancer Res., August 15, 2007; 13(16): 4672 - 4676. [Full Text] [PDF]
|
|
|
|
 |
|
 W. Yin, J. M. Mendenhall, S. B. Bratton, T. Oung, W. G. M. Janssen, J. H. Morrison, and A. C. Gore Novel Localization of NMDA Receptors Within Neuroendocrine Gonadotropin-Releasing Hormone Terminals Experimental Biology and Medicine, May 1, 2007; 232(5): 662 - 673. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 A. Pedram, M. Razandi, and E. R. Levin Nature of Functional Estrogen Receptors at the Plasma Membrane Mol. Endocrinol., September 1, 2006; 20(9): 1996 - 2009. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 A. M. Braun and P. Thomas Biochemical Characterization of a Membrane Androgen Receptor in the Ovary of the Atlantic Croaker (Micropogonias undulatus) Biol Reprod, July 1, 2004; 71(1): 146 - 155. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
D. P. McDonnell Mining the Complexities of the Estrogen Signaling Pathways for Novel Therapeutics Endocrinology, October 1, 2003; 144(10): 4237 - 4240. [Full Text] [PDF]
|
|
|
|
 |
|
 J. K. Wong, H. H. Le, A. Zsarnovszky, and S. M. Belcher Estrogens and ICI182,780 (Faslodex) Modulate Mitosis and Cell Death in Immature Cerebellar Neurons via Rapid Activation of p44/p42 Mitogen-Activated Protein Kinase J. Neurosci., June 15, 2003; 23(12): 4984 - 4995. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Marino, F. Acconcia, and A. Trentalance Biphasic Estradiol-induced AKT Phosphorylation Is Modulated by PTEN via MAP Kinase in HepG2 Cells Mol. Biol. Cell, June 1, 2003; 14(6): 2583 - 2591. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. A. Orihuela, A. Parada-Bustamante, P. P. Cortes, C. Gatica, and H. B. Croxatto Estrogen Receptor, Cyclic Adenosine Monophosphate, and Protein Kinase A Are Involved in the Nongenomic Pathway by Which Estradiol Accelerates Oviductal Oocyte Transport in Cyclic Rats Biol Reprod, April 1, 2003; 68(4): 1225 - 1231. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Razandi, G. Alton, A. Pedram, S. Ghonshani, P. Webb, and E. R. Levin Identification of a Structural Determinant Necessary for the Localization and Function of Estrogen Receptor {alpha} at the Plasma Membrane Mol. Cell. Biol., March 1, 2003; 23(5): 1633 - 1646. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. R. Levin Bidirectional Signaling between the Estrogen Receptor and the Epidermal Growth Factor Receptor Mol. Endocrinol., March 1, 2003; 17(3): 309 - 317. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Razandi, A. Pedram, S. T. Park, and E. R. Levin Proximal Events in Signaling by Plasma Membrane Estrogen Receptors J. Biol. Chem., January 17, 2003; 278(4): 2701 - 2712. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. W. Singleton, Y. Feng, C. J. Burd, and S. A. Khan Nongenomic Activity and Subsequent c-fos Induction by Estrogen Receptor Ligands Are Not Sufficient to Promote Deoxyribonucleic Acid Synthesis in Human Endometrial Adenocarcinoma Ce |
