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q/11-Subunit with Cytoskeleton in Adrenal Glomerulosa Cells: Role in Receptor-Effector Coupling1
Service of Endocrinology, Department of Medicine (M.C., N.G.-P.), and the Department of Physiology and Biophysics (M.D.P.), Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4; and INSERM U-401 (M.-N.D., G.G.), Montpellier, France
Address all correspondence and requests for reprints to: Dr. Nicole Gallo-Payet, Service of Endocrinology, Department of Medicine, Faculty of Medicine, University of Sherbrooke, 3001 12th Ave North, Sherbrooke, Quebec, Canada J1H 5N4. E-mail:n.gallo{at}courrier.usherb.ca
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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q/11-subunit of
the Gq/11 protein was associated with both structures. Ang
II stimulation induced a rapid translocation of
q/11, microfilaments, and microtubules to
the membrane and induced a time-dependent increase in the level of
q/11 associated with both microfilaments
and microtubules. Moreover, double immunofluorescence staining clearly
showed a colocalization of the
q/11-subunit of the Gq/11
coupling protein and microfilament distribution. These associations and
plasma membrane redistribution under Ang II stimulation indicate that
microfilaments and microtubules are both involved in phospholipase C
activation and inositol phosphate production. Moreover, our results
indicate that the q/11 protein is closely
associated with cytoskeletal elements and is found both at the plasma
membrane level as well as on intracellular stress fibers. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several studies have clearly shown that the cytoskeleton plays an
important role in the process of adrenal steroidogenesis (10). Indeed,
microfilaments are involved in the transport of cholesterol from lipid
droplets to endoplasmic reticulum and mitochondria (11, 12), whereas
microtubules are involved in the fusion of low density lipoprotein
vesicles with lysosomes (13, 14). Moreover, the cytoskeleton is
involved in the early steps of ACTH action. In a recent study, we
demonstrated that ACTH stimulation of cells induces a rapid
redistribution of microfilaments and microtubules at the membrane,
enhancing the amount of s associated with the membrane
(15); these changes are essential for adenylyl cyclase activation. As
for Ang II stimulation, Feuilloley et al. (16) have shown
that cytochalasin B inhibits Ang II-induced phosphoinositide breakdown
and steroid secretion in frog adrenocortical cells, whereas colchicine
is without effect, at least on aldosterone secretion (17). More recent
studies conducted on WRK1 cells, a tumoral cell line, have demonstrated
that q/11 protein is closely associated
with microfilaments, and that this association is necessary in inducing
PLC activation (18). In addition, studies conducted with purified
tubulin also indicate that q/11 protein
may be associated with microtubules (19).
There is no information regarding cellular distribution of
q/11 protein in rat adrenal glands or
concerning the early effects of Ang II on this protein or on the
cytoskeleton. The aim of the present study was to investigate 1) the
involvement of microfilaments and microtubules on inositol phosphate
(InsP) production in rat adrenal glomerulosa cells, 2) whether the
early events of Ang II action in adrenal glomerulosa cells are
accompanied by changes in the distribution of microfilaments and
microtubules, and 3) whether the
q/11-subunit of the
Gq protein is associated with either
microfilaments or microtubules. We show that Ang II induces a rapid
redistribution of actin filaments to the membrane and that localization
of the q/11-subunit of the heterotrimeric
G protein overlaps that of microfilament distribution. Studies on InsP
production indicate that both microfilaments and microtubules are
essential for the effects of Ang II on PLC activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anti-q/11 antibody was produced and
characterized in the laboratory (18). This polyclonal antibody is
directed against the common last 11 amino acids of the C-terminus of
the -subunits of Gq and G11 proteins and was
produced in a New Zealand White rabbit. Immunization was performed with
a conjugate of the synthetic peptide LQLNLKEYNLV and thyroglobulin
(THR). An additional cysteine was placed at the N-terminal sequence of
the peptide to facilitate its coupling to the protein. Activation of
the TRH by treatment with
sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
achieved the coupling. After elimination of the antibodies directed
against THR, the remaining antibodies directed against the synthetic
peptide were purified. This antibody did not recognize other C-terminal
synthetic peptides of the G protein family (18). Thus, the term
anti-q/11 will be used for this
antibody.
Preparation of glomerulosa cells
The zonae glomerulosa were obtained from adrenal glands of
female Long-Evans rats, weighing 200–250 g, and were isolated
according to a previously described method (7, 20). The successive
steps of zona glomerulosa isolation and cell dissociation were
performed in MEM Eagle’s medium (supplemented with 100 U/ml penicillin
and 100 µg/ml streptomycin). After a 20-min incubation at 37 C in
collagenase (2 mg/ml; four capsules per ml) and deoxyribonuclease (25
µg/ml), the cells were disrupted by gentle aspiration with a sterile
10-ml pipette, filtered, and centrifuged for 10 min at 100 x
g. They were then resuspended in Opti-MEM medium
supplemented with 2% FBS, 100 U/ml penicillin, and 100 µg/ml
streptomycin and plated in 35- or 60-mm tissue culture dishes (for
InsPs experiments) or 16-mm multiwell plates (for steroid measurements)
at a density of approximately 1 x 105 cells/multiwell
or 35-mm dish, respectively. The cells were cultured at 37 C in a
humidified atmosphere of 95% air-5% CO2. The culture
medium was changed every day, and the cells were used after 3 days of
culture. At this time, cell density was approximately 1–3.0 x
105 cells/dish or well in a multiwell plate.
Incubations for measurement of aldosterone secretion
Before each experiment, the medium of cultured cells was
removed, and the cells were washed twice with cold Hanks’ buffered
saline (HBS; 130 mM NaCl, 3.5 mM KCl, 1.8
mM CaCl2, 0.5 mM MgCl2,
2.5 mM NaHCO3, and 5 mM HEPES)
supplemented with 1 g/liter glucose and 0.5% BSA. The cells were
incubated in 1 ml medium consisting of 0.9 ml HBS-glucose supplemented
with 0.5% BSA, 0.1 mg/ml bacitracin, and 0.1 ml stimulus. After a 2-h
incubation at 37 C in an atmosphere of 95% air-5% CO2,
the incubation medium was removed by aspiration and stored at -20 C
until RIA determinations of aldosterone in the medium, using specific
antisera and tritiated steroid as tracer.
Measurement of InsP accumulation
Experiments were performed as described previously (21).
Briefly, cells were grown for 2 days in Opti-MEM culture medium
containing 2 µCi/ml myo-[3H]inositol. The radioactive
medium was then discarded, and the cells were incubated in isotope-free
culture medium. After 1 h, cells were washed and incubated for 15
min in HBS-glucose-LiCl (10 mM) medium. After a medium
change, cells were further incubated for 15 min at 37 C with fresh
HBS-glucose-LiCl medium containing the hormones and drugs to be tested.
Incubation was ended by aspiration of the medium and addition of 1 ml
5% (vol/vol) HClO4 and 200 µl BSA (20 mg/ml). InsPs were
separated by ion exchange chromatography on Dowex 1 x 8 columns.
The radioactivity found in the InsP fractions was determined by
scintillation counting in gel phase in a Beckman ß-counter (Palo
Alto, CA), with a counting efficiency of 18%. All results were
corrected for quenching and were expressed in disintegrations per
min.
Membrane preparation
After hormonal stimulation, 3-day cultured cells were washed
twice with HBS buffer, followed by 10 mM ice-cold Tris-HCl
buffer [containing 0.5 mM EDTA, 1 mM
MgCl2, 2.8 mM phenylmethylsulfonylfluoride
(PMSF), 0.04 TIU/ml aprotinin, and 1 mM benzamidine, pH
8.0]. The cells were then scraped from the substratum with a rubber
policeman and disrupted by sonication at 0 C. Cell extracts were
centrifuged at 700 x g for 10 min, and the resulting
supernatant was centrifuged at 30,000 x g for 30 min
to obtain the membrane fraction. The membrane fraction was resuspended
in 50 mM Tris-HCl buffer (containing 2 mM EDTA,
5 mM MgCl2, and 250 mMsucrose) and
stored at -20 C for subsequent Western blot assays.
Total cell homogenate
After hormonal stimulation in 60-mm petri dishes, cells were
washed twice with HBS buffer containing aprotinin (0.04 TIU/ml), PMSF
(1 mM), and benzamidine (1 mM). The cells were
then scraped from the substratum with a rubber policeman, transferred
in a 15-ml conical tube, centrifuged at 100 x g for 10
min at room temperature, and solubilized in Tris buffer (100
mM; pH 6.8) containing 4% SDS, 20% glycerol, and 10%
2-mercaptoethanol. Cell extracts were heated at 100 C for 10 min. After
centrifugation at 10,000 x g for 5 min, the
supernatant was stored at -20 C for subsequent Western blot
analysis.
Preparation of microtubules
Preparations enriched in microtubules were extracted from cells
grown in 60-mm petri dishes as described by Solomon (22) with some
modifications. The cells were pretreated with 1 mM taxol
for 2 h before extraction of microtubules. At this concentration,
taxol stabilizes microtubules without promoting polymerization. The
culture medium was then aspirated and replaced with PM2G buffer (0.1
M PIPES, 2 M glycerol, 5 mM
MgCl2, 2 mM EGTA, 0.04 TIU/ml aprotinin, 2
mM PMSF, and 1 mM benzamidine, pH 6.9)
containing taxol (1 mM). Cells were scraped from the
substratum, centrifuged, and extracted with PM2G buffer containing 1%
Nonidet P-40 and 1 mM taxol. After a 15-min incubation at
37 C, the suspension was centrifuged at 1000 x g for 5
min at 37 C. The pellet containing the microtubules was then processed
as described previously for total extracts. Electron microscopy studies
indicate that the microtubule preparations were free of
microfilaments.
Extraction of microfilaments
Enriched microfilament preparations were extracted from cells
grown in 60-mm petri dishes as described by Phillips et al.
(23). Culture medium was aspirated and changed for HBS buffer. Cells
were scraped from the substratum with a rubber policeman and
transferred in a 15-ml conical tube. Cells were centrifuged at 100
x g for 5 min at room temperature. One hundred microliters
of Triton solution (1% Triton X-100, 10 mM EGTA, and 0.1
M Tris-HCl, pH 7.4) was added, and the solution was
transferred to 1.5-ml microcentrifuge tubes. After a 10-min incubation
at 0 C, the preparation was centrifuged at 8000 x g
for 4 min at room temperature. The Triton-soluble G-actin fraction was
contained in the supernatant. The pellet, which corresponds to the
Triton-insoluble fraction, was solubilized in 2% SDS-2%
2-mercaptoethanol (vol/vol). After a 10-min incubation at 100 C,
F-actin was solubilized. Both fractions were aliquoted and frozen for
subsequent Western blot analysis. Electron microscopy studies indicate
that the microfilament preparations were free of microtubules.
Western blotting
Samples from equivalent cell numbers were compared in each
experiment. Samples were separated on 4–15% SDS-polyacrylamide gels.
Proteins were transferred electrophoretically to polyvinylidene
difluoride membranes. Membranes were blocked with 1% gelatin-0.05%
Tween-20 in Tris-buffered saline (TBS; pH 7.5). After three washes with
TBS-Tween-20 (0.05%), membranes were incubated with anti-ß-tubulin
(dilution, 1:250), anti-actin (dilution, 1:100), or
anti-q/11 (dilution, 1:1000) for 3 h
at room temperature, followed by four washes with TBS-Tween-20.
Detection was accomplished using horseradish peroxidase-conjugated
antimouse antibody for actin and tubulin (Amersham) or antirabbit for
q and an enhanced chemiluminescence detection system
(Amersham, Oakville, Canada). The immunoreactive bands were scanned by
laser densitometry and expressed in arbitrary units. Note that both
isoforms of q/11 proteins were analyzed
together.
Immunoprecipitation
Glomerulosa cells grown in 60-mm petri dishes were washed once
and stimulated with Ang II (100 nM) at 37 C. The cells were
then washed twice with ice-cold HBS buffer and lysed in TSA buffer
[0.1 M Tris-HCl (pH 8.0), 0.14 M NaCl, 0.025%
NaN3, 1% Nonidet P-40, 1% BSA, 1 mM PMSF, 1
mM iodoacetamide, 0.2 TIU/ml aprotinin, and 1
mM benzamidine] for 60 min at 4 C. Lysates were clarified
with protein A-Sepharose for 2 h at 22 C, followed by
centrifugation at 200 x g for 1 min. For
immunoprecipitation of actin or tubulin, the lysates were incubated for
2 h with 2 mg/ml monoclonal antibodies at 22 C. Protein
A-Sepharose was added, and incubation was performed overnight at 4 C.
Immunocomplexes were washed five times before electrophoresis on
4–15% SDS-polyacrylamide gels and analyzed by immunoblotting.
Immunofluorescence
For immunofluorescence studies, cells were plated on plastic
coverslips (Starsted, St. Laurent, Canada), grown for 3 days, and
treated with the appropriate stimuli. For visualization of
microfilaments, cells were fixed for 1 min with 3% (vol/vol)
formaldehyde in PBS buffer, permeabilized for 10 min in PBS-0.1%
Triton X-100, and incubated for 20 min at room temperature with 1 U
rhodamine/phalloidin solution. For microtubules and
q/11 detection, cells were fixed for 1
min with 3% (vol/vol) formaldehyde in 80 mM PIPES (pH
6.5), 5 mM EDTA, and 2 mM MgCl2 and
fixed for an additional 8 min with 3% (vol/vol) formaldehyde in 100
mM sodium borate (pH 11) (18). Cells were then incubated
for 30 min in PBS-0.1% (vol/wt) sodium borohydride, permeabilized by
incubation in PBS-0.2% Triton X-100, and incubated overnight at 4 C
with anti-ß-tubulin (1:50) or anti-q/11
(1:50). After washing, cells were further incubated for 60 min at 37 C
with a secondary conjugated anti-IgG antibody coupled with fluorescein
isothiocyanate (FITC). For double immunofluorescence studies, cells
were fixed and permeabilized as described for microtubule and G protein
detection and processed successively with anti-q and
anti-ß-tubulin or rhodamine/phalloidin as described above. After
washings, cells were postfixed for 20 min with 3% formaldehyde-PBS and
incubated in the presence of 50 mM NH4Cl for 10
min. The coverslips were then mounted in Vectashield mounting medium
and examined on a Nikon DM 400 microscope equipped for epifluorescence
using B-1E FITC and G-2A rhodamine filters (Nikon, Melville, NY).
Data analysis
The data are presented as the mean ± SE.
Statistical analyses of the data were performed using the one-way ANOVA
test. Homogeneity of variance was assessed by Bartlett’s test, and
P values were obtained from Dunnett’s tables. n indicates
the number of experiments, each performed in triplicate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 100 nM Ang II induced a 15-fold
increase in InsPs production in rat adrenal glomerulosa cells. A 30-min
preincubation with 10 µM colchicine (a
microtubule-disrupting agent) or 10 µM cytochalasin B (a
microfilament-disrupting agent) decreased basal InsPs accumulation by
25%, and both induced a 51 ± 3% inhibition of the Ang II effect
(P < 0.01; n = 3). Fluoroaluminate
(AlF-4; a nonspecific activator of all
heterotrimeric G proteins (24) increased the stimulation of InsP
production 10-fold. The results shown in Fig. 1A indicate that both
agents decreased AlF-4-induced InsP production
by 41 ± 3% and 57 ± 4%, respectively.
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show that 30-min preincubation of glomerulosa
cells with 10 µM colchicine or 10 µM
cytochalasin B decreased by 30% the basal output of aldosterone
secretion, whereas stimulation induced by 100 nM Ang II was
reduced by 57 ± 4% and 44 ± 2% (n = 3;
P < 0.01). Fluorescence studies show that these
experimental conditions (10 µM; 30 min) were sufficient
to achieve a complete disruption of the microtubular and
microfilamentous network (data not shown).
Effect of Ang II on the distribution of microfilaments,
microtubules, and
q/11
Immunofluorescence studies using rhodamine/phalloidin indicate
that in control rat glomerulosa cells, actin filaments consisted of
thin and discrete clusters of parallel stress fibers crisscrossing the
entire surface of the cell and forming a thin cortical ring (Fig. 2A). Ang II induced rapid and reversible changes in the
organization of the microfilamentous network. After only 1 min, Ang II
had increased actin labeling intensity at the cell periphery (Fig. 2, B
vs. A). Between 5 and 30 min, labeling also increased in the
cytoplasm (Fig. 2, C–E), but returned to basal levels near the plasma
membrane (Fig. 2F). It should be noted that the nucleus was always
evident during the first 15 min of Ang II application (compare Fig. 2, C–E with A and E). In contrast, microtubules appeared as long and thin
filaments, loosely distributed throughout the cell (Fig. 3A). Apart from a small visible increase at the
perinuclear region, Ang II treatment did not significantly modify
microtubular distribution inside the cell (Fig. 3B). Immunofluorescence
experiments using q/11 antibody indicate
that the pattern of q/11 localization in
control (Fig. 4A) or Ang II-stimulated cells is similar
to that observed with actin. Figure 4, B and C, clearly indicates that
q/11 labeling increased at the membrane
level after 1- or 5-min incubation with Ang II compared with that after
either a control (Fig. 4A) or 2-h incubation with Ang II (Fig. 4D).
Double immunofluorescence experiments clearly confirm that
q/11 labeling overlapped actin labeling
in both control (Fig. 4, E and F) and Ang II-stimulated cells (data not
shown). No labeling was observed when
anti-q/11 was inactivated by heating at
100 C or when secondary IgG was used alone (data not shown).
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q/11 protein
under Ang II stimulation , left panels)
or anti-ß-tubulin antibodies (Fig. 5, right panels). As
shown in Fig. 5A, a 1-min incubation with Ang II increased
membrane-associated actin (1.71 ± 0.02-fold increase; n = 3;
P < 0.001 compared with control; Fig. 5A, lane 2
vs. lane 1). This membrane association decreased after 15
min and remained slightly higher (20% increase) than control values
after a 2-h incubation with Ang II (1.31 ± 0.02- and 1.19 ±
0.03-fold increase after 15 min and 2 h, respectively; n = 3;
Fig. 5A, lanes 3 and 4). The effect of Ang II also stimulated actin
polymerization, as actin levels increased in the Triton X-100
preparation after a 1-min stimulation (3.73 ± 0.03-fold increase;
n = 3; P < 0.001 compared with the control; Fig. 5B, lane 2 vs. lane 1) and remained slightly above basal
values after 15 min and 2 h of incubation (Fig. 5B, lanes 3 and 4
vs. lane 1). This increase is due to actin polymerization
and not to actin synthesis, as actin levels did not increase in total
cell homogenates (Fig. 5C). Figure 5D indicates that Ang II application
also induced a rapid and transient increase in the level of
microtubules associated with the membrane (4.3 ± 0.2-, 2.4
± 0.2-, and 2.4 ± 0.3-fold increase; n = 3) after 1 min, 15
min, and 2 h, respectively (lanes 2, 3, and 4). All of these
values were significantly increased compared with the control value
(P < 0.001). In contrast to actin, the amount of
polymerized tubulin decreased over the 2-h incubation (Fig. 5E),
although the total amount of tubulin in the cell did not change (Fig. 5F).
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q/11 antibody revealed two bands of
42 and 43 kDa. The total content of q/11
in the homogenate did not change significantly during the 2-h
incubation with Ang II (Fig. 6A; 1.12 ± 0.14-,
0.98 ± 0.11-, and 1.01 ± 0.07-fold increase; n = 3)
after 1 min, 15 min, and 2 h, respectively (lanes 2, 3, and 4). In
contrast, the amount of q/11 specifically
associated with the membrane increased after a 1-min incubation (Fig. 6B) and over the entire period of incubation in the anti-actin
immunoprecipitate samples (Fig. 6C). The association of
anti-q/11 with the antitubulin
immmunoprecipitates transiently increased between 1 and 15 min (Fig. 6D). Densitometric analysis indicated that the level of
q associated with microfilaments (Fig. 6C) and
microtubules (Fig. 6D) increased by 3.73 ± 0.03- and 2.31 ±
0.08-fold, respectively, after 1 min (lanes 2 vs. lane 1;
n = 3; P < 0.001 compared with control).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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q/11-subunit of the
Gq protein is highly associated with cytoplasmic stress
fibers, but also with microtubules. These associations are essential
for PLC activation and InsP production. Until now, much of the data regarding the role of the cytoskeleton in adrenal steroidogenesis have been obtained from experiments in which the cytoskeleton was disrupted by drug treatment (10). Large variations in dosage (0.1 µM to 10 mM) and duration (10 min to 24 h) may explain the opposite stimulatory or inhibitory effects described in the literature. Discrepancies may be also due to differences in animal species. For example, Feuilloley et al. (16, 17) were unable to find any effect of 10 µM colchicine on the Ang II response in frog adrenal cells, whereas in the present study, the same concentration decreased Ang II-induced InsPs production and aldosterone secretion by 50%. Moreover, we found that cytochalasin decreased, whereas in frog cells, cytochalasin blocked InsPs production induced by Ang II.
Immunofluorescence and Western blots analyses indicate that a 1-min
incubation with Ang II induced the formation of an intense cortical
ring of actin (Fig. 2, B vs. A), which corresponded to an
increase in the amount of F-actin associated with the membrane (Fig. 5A). Ang II also increased the number of actin fibers at least during
the first 15 min of stimulation (Fig. 5B), a result in agreement with
the numerous studies describing the essential role of microfilaments in
the process of steroidogenesis (10). In several models, cell activation
is associated with a rapid and transient increase in F-actin content.
Jennings et al. (25) demonstrated that a 15-sec stimulation
of platelets with thrombin increased the amount of F-actin by 65% and
increased the organization of actin filaments with other cytoskeletal
proteins; identical results were found in neutrophils (26). Receptors
have been also shown to be associated with actin filaments (27, 28, 29).
Such observations suggest that the cytoskeleton could operate as a
matrix, improving the efficiency of the signal transduction
cascade.
Changes in microtubule distribution under hormonal stimulation is less documented. Similar to bovine adrenocortical cells (30), our immunofluorescence studies show that Ang II did not significantly affect microtubular arrangement. However, Western blot experiments clearly indicate that Ang II stimulation was accompanied by an increase in membrane-associated microtubules. The absence of effect in immunofluorescence studies may be due to alteration of the membrane during the process of permeabilization needed to introduce the anti-tubulin antibody into the cell.
One of the most important observations in this study is that the
distribution of the q/11-subunit of
Gq protein is exactly the same as that of microfilaments
(Fig. 4). Moreover, the activation of
q/11, which is responsible for PLC
activation, relies on cytoskeleton integrity. Western blot results
indicate that Ang II stimulation induces a rapid increase in the
association of q/11 with both
microtubules and microfilaments, all of which are translocated to the
membrane. The colocalization of
q/11 protein with
microfilaments has also been recently described in WRK1
cells, where the same q/11
antibody was used and characterized (18). This close functional
association between G proteins and the cytoskeleton has been
extensively described during the last 5 yr (19, 31, 32, 33, 34). In addition,
evidence for direct control of microfilament polymerization by G
proteins is increasing. For example, in electropermeabilized
neutrophils, fluoroaluminate and guanosine
5'-3-O-(thio)triphosphate (GTP
S) were found to induce an
increase in F-actin content, even while PLC activity was inhibited
(35). Exposure of mast cells to GTPS induced disassembly of cortical
F-actin at the cortex, but induced an increase in F-actin in the cell
interior (36). However, the exact contribution or role of each
structure (microtubules or/and microfilaments) in receptor-effector
coupling is not yet clearly established. Several data indicate that
microfilaments interact directly or indirectly, via actin-binding
proteins, with the plasma membrane. Very little is known about how G
proteins are transported and retained before activation. Several
possibilities can be considered. A G protein may either be transported
as a heterotrimer, or the - and ß-subunits may be localized
independently of each other. For the Go protein,
localization of the -subunit in the membrane depends on the subunit
itself and not on the ß-subunits or the receptors (37). Although
the -subunit of a G protein is known to interact with the
ß-subunits to activate receptor and the downstream effector,
Nübe and Neer (37) have shown that o localization
in the membrane is independent of receptor interaction and does not
require formation of ß complexes, suggesting that direct
interaction with actin or tubulin may be implicated in the targeting of
o in the membrane. Several data support the observation
that microfilaments control the dissociation of GTP-binding protein
associated with PLC (27). Ibarrondo et al. have shown that
association of Gq/11 with microfilaments is essential to
promote PLC activation (18). Ozawa et al. (38) and Suzuki
et al. (39) have also demonstrated that integrity of the
heterotrimer q/ß and its association with the
membrane are necessary for PLC activation. On the other hand, Ravindra
et al. (19) demonstrated that Gq is involved in
the polymerization of tubulin. Our results are in agreement with both
conclusions, as we found that
q/11 is associated with both
structures and that these associations are hormone regulated.
Microfilaments could store and transport
q/11 to the membrane, while
binding of tubulin to q/11 may be
required for anchoring these protein subunits in the plasma
membrane.
In addition, many recent studies indicate that several intracellular
targets of second messengers are associated with microtubules and
microfilaments, such as phosphatase (40), phosphoinositides,
diacylglycerol (41, 42), PLC (43), and mitogen-activated protein
kinase (or microtubule-associated protein kinase) (44). This is
particularly true for Ang II action in vascular smooth muscle,
fibroblasts, or cardiac cells (45, 46, 47, 48). Some studies now indicate that
Ang II stimulates not only PLCß, but also PLC (48), and indicate
that Ang II activates several effectors usually stimulated by the
growth factor family of receptors (49). This association with the
membrane may be direct or mediated by some of the numerous
actin-binding proteins, such as the focal adhesion kinase protein (45, 47) or paxillin (46). These interactions may be ensured by the
plecsktrin homology domain found in all of these structures
(50, 51, 52, 53).
In summary, both microtubules and microfilaments are involved in the
production of InsPs induced by Ang II in rat glomerulosa cells.
Moreover, our results indicate that distribution of the
q/11-subunit of the Gq/11
protein is linked to both microtubules and microfilaments and that
these associations are essential for PLC activation.
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Acknowledgments |
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Footnotes |
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2 Recipient of a scholarship from Les Fonds de La Recherche en
Santé du Québec.
Received January 15, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunits of Gq and G11 G proteins with actin
filaments in WRK1 cells: relation to G protein-mediated
phospholipase C activation. Proc Natl Acad Sci USA 92:8413–8417 on
soluble and polymerized tubulin pools in GH3 and AtT-20
cells. J Cell Biochem 61:392–401[CrossRef][Medline]
-phosphate
of GTP in its binding site. FEBS Lett 191:181–185[CrossRef][Medline]
and Gi1.
J Biol Chem 265:1239–1242o to growth cones in PC12 cells: role of
Go association with receptors and Gß. J Cell Sci 109:221–228[Abstract]
but not by soluble Gq. FEBS Lett 377:333–337[CrossRef][Medline]
1 to
the cytoskeleton in rat hepatocytes. J Biol Chem 269:7156–71621 in vascular smooth muscle cells. J Biol Chem 269:10935–10939 interactions with PH domains and ras-MAPK signaling pathways.
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M. Bensimon, A. I. Chang, M. L. K. de Bold, A. Ponce, D. Carreras, and A. J. De Bold Participation of G Proteins in Natriuretic Peptide Hormone Secretion from Heart Atria Endocrinology, November 1, 2004; 145(11): 5313 - 5321. [Abstract] [Full Text] [PDF]
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S. Campbell, M. Otis, M. Cote, N. Gallo-Payet, and M. D. Payet Connection between Integrins and Cell Activation in Rat Adrenal Glomerulosa Cells: A Role for Arg-Gly-Asp Peptide in the Activation of the p42/p44mapk Pathway and Intracellular Calcium Endocrinology, April 1, 2003; 144(4): 1486 - 1495. [Abstract] [Full Text] [PDF]
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) or 10 µM cytochalasin B
(
) and were further incubated for 15 min at 37 C in HBS medium in
the absence (C) or presence of 100 nM Ang II or 30
mM fluoroaluminate (AlF-4). Total
accumulated InsPs were separated on Dowex columns as described in
Materials and Methods. Results represent the mean
± SE of three experiments, each performed in triplicate.
When no error bars are shown, they are contained within the symbols. B,
After preincubation for 30 min at 37 C in HBS buffer in the absence (C)
or presence of 10 µM colchicine or 10 µM
cytochalasin B, as described in A, Ang II was added, and the cells were
incubated for an additional 2 h in a 95% O2-5%
CO2 atmosphere in HBS medium. Accumulation of aldosterone
was measured by specific RIA. Results are the mean ±
SE of three experiments, each performed in triplicate. *,
P < 0.01; +, P < 0.05
(difference compared with hormone-stimulated cells).
