Vol. 276, Issue 1, F10-F17, January 1999
Guanine nucleotide binding proteins in cultured renal
epithelia: studies with pertussis toxin and aldosterone
Sarah
Sariban-Sohraby1,
Michal
Svoboda2, and
Frédérique
Mies1
1 Laboratoire de Physiologie
and 2 Laboratoire de Chimie
Biologique, Université Libre de Bruxelles, 1070 Brussels,
Belgium
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ABSTRACT |
The GTP-binding proteins from cultured A6 epithelia were
examined in isolated membrane preparations. Binding of
[35S]GTP
S revealed
a class of binding sites with an apparent
Kd value of 100 nM and a Bmax of 220 pmol/mg
protein. Short-term aldosterone treatment of the cells did not modify
the binding kinetics, whereas pertussis toxin (PTX) decreased
Bmax by 50%. The mRNA levels for
G
i-3,
G0,
Gs, and
Gq were not increased after
aldosterone. The patterns of small
Mr G proteins and
of PTX-ribosylated proteins were identical in membranes of both control and aldosterone-treated cells. Cross-linking of
[-32P]GTP, in
control membranes, showed either no labeling or a faint band of
Mr 59.5 kDa. This
protein became prominent after aldosterone, and its labeling decreased
with spironolactone. Thus short-term aldosterone does not promote
increased expression of known heterotrimeric G proteins in epithelial
membranes but activates resident PTX-sensitive Gi proteins and stimulates the
expression of a specific GTP-binding protein of
Mr 59.5 kDa.
A6 cells; photoaffinity labeling; sodium transport; GTP hydrolysis
rate constant
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INTRODUCTION |
EPITHELIAL SODIUM CHANNELS are regulated by
aldosterone, although the molecular mechanisms are still largely
uncharacterized. The basal activity of these channels can be modulated
by GTP (9, 19) as well as by
Gi-3 (6). When purified from A6
renal epithelial cells or bovine renal papillae, these
Na+ channels comprise five to six
polypeptides (3, 15), one of which, namely, a 90-kDa protein, is
methylated after stimulating Na+
transport by short-term aldosterone (less than 4 h; Ref. 24). Methylation of this 90-kDa protein is stimulated in vitro by guanosine 5'-O-(3-thiotriphosphate) (GTPS) in control
membranes but not in membranes from cells exposed to aldosterone.
Furthermore, aldosterone treatment of A6 cells results in a doubling of
the rate of GTP hydrolysis by the isolated membranes (25). These
observations support the idea that activation of G proteins mediates
the early phase of aldosterone stimulation of apical
Na+ permeability, possibly via
methylation of the channels. Long-term exposure of A6 cells to
aldosterone (16 h or more) is associated with increased metabolic
labeling of the 41-kDa i-3 G
protein in the apical membrane and with a 1.6- to 2-fold increase in
the Gi-3 mRNA (22). The aim of
the present study was to identify the various GTP-binding proteins
associated with the isolated membranes and study the effect of
pertussis toxin (PTX) and short-term aldosterone on the expression of
these proteins with regard to increased GTPase activity. We detected
several GTP-binding proteins in A6 membranes and showed that their
levels of expression as well as mRNA were not modified by aldosterone.
This indicates that the hormonal stimulation of the GTP hydrolysis rate
is not linked to an increase in the membrane concentration of these G proteins and involves their activation through one or more additional regulatory steps. Interestingly, a novel 59.5-kDa GTP-binding protein
was specifically expressed in the membranes of cells exposed to
aldosterone. This protein may play a role in the reported G protein-mediated control of Na+
transport by aldosterone.
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MATERIALS AND METHODS |
Cell culture. A6 cells from
Xenopus laevis toad kidney (American
Type Culture Collection, Rockville, MD) were grown at 28°C in a
humidified incubator gassed with 1%
CO2 in air. Cultures were carried
on plastic dishes and on porous supports as described previously (24).
These supports were homemade
102-cm2 filter-bottomed cups that
allowed the collection of large amounts of cells. The growth medium was
Dulbecco's modified Eagle's medium (GIBCO) containing 75 mM NaCl and
8 mM NaHCO3 and supplemented with
5% fetal bovine serum (Hyclone, Logan, UT). When appropriate, cultures
on porous supports were exposed to 100 nM aldosterone placed in the
basolateral growth medium. Transepithelial measurements of voltage and
resistance were performed on cells grown on
0.33-cm2 structures (Costar) using
an EVOM voltohmmeter (World Precision Instruments) as described
previously (25). The corresponding sodium current was calculated from
these values obtained in the presence or absence of amiloride.
Membrane preparation. Cells grown to
confluence on 102-cm2 porous
supports were washed three times with ice-cold homogenization medium
(HM) composed of 30 mM mannitol, 10 mM Tris-HEPES, and 10 mM
MgCl2, pH 7.4, scraped in HM
supplemented with phenylmethylsulfonyl fluoride (175 µg/ml), and
homogenized with a Potter homogenizer (20 strokes). The homogenate was
spun 15 min at 5,500 g, and the pellets were discarded. The supernatants were centrifuged 20 min at
43,000 g. The resulting pellets were
resuspended in 100 mM mannitol and 10 mM Tris-HEPES, pH 7.4, and
centrifuged once more at 43,000 g. The
final pellets, enriched 10-fold in apical membranes (23), were
resuspended and kept on ice or frozen at 
80°C after snap-freezing in a dry-ice/ethanol slush.
Binding of
[35S]GTPS
to A6 membranes.
Binding of
[35S]GTPS to A6
membranes was assessed with the rapid-filtration technique described by
Northup et al. (18). Ten micrograms of membrane protein were diluted in
140 µl of Tris · HCl, pH 8.0, 1 mM dithiothreitol
(DTT), 100 mM NaCl, and 30 mM
MgCl2 (buffer
A) containing 0.025 µM
[35S]GTPS (1.5 × 105 cpm). Nonspecific
binding was determined in the presence of 0.1 mM unlabeled GTPS.
Incubations were carried at 28°C for either 5 or 30 min, and the
samples were then applied to 25-mm nitrocellulose filters (Millipore,
HAWP 0.45) presoaked in buffer A. The
filters were rapidly washed (under suction) with four successive 2-ml volumes of buffer A, oven-dried,
dissolved, and counted in scintillation fluid (Insta-gel Plus,
Packard). Specific binding was calculated as the difference in bound
radioactivity in the absence or presence of 0.1 mM unlabeled GTPS.
Blank values were 211 ± 31 cpm (n = 28), i.e.,
0.14% of the applied radioactivity. This filtration method showed a
linear increase in binding with increasing protein concentration in the
range tested (5-41 µg/sample). Binding was linear with time up
to 10 min. Initial rates and equilibrium binding were thus measured,
respectively, after 5 and 30 min of incubation with the radioactive ligand.
RT-PCR. Total RNA was extracted from
control and aldosterone-treated A6 cells using RNA Now (Biogentex). The
RNA samples were treated with DNase I and reverse transcribed with
Superscript II (GIBCO-BRL) using random primers at 15 µg/ml
(GIBCO-BRL). cDNAs were amplified using primers designed to enhance
published sequences of frog
Gi-1 (forward 658-678;
reverse 894-874; GenBank accession no. X56089),
Gi-3 (forward 447-467;
reverse 683-663; no. X56090), G0 (forward 606-627;
reverse 837-816; no. X14636), and
Gq (forward 528-549;
reverse 764-743; no. U10502). Glucose-6-phosphate dehydrogenase
(G3PDH) was used as control
(forward 564-584; reverse 1015-996; no. U41753). As a
positive control of aldosterone effect on mRNA levels, we used primers
designed from ASUR 1, a cloned DNA sequence from A6 cells kindly
provided by F. Verrey. This mRNA was shown to increase 400-500%
after short-term aldosterone treatment (27). Primers were as follows:
ASUR 1 forward, GTA CCC AGG TCA AGG GTC AA; and ASUR 1 reverse, ACT GGC
TGC TTT TAT TCA TTC C. PCR reactions were performed in a total volume
of 20 µl containing 1.5 mM
MgCl2, 50 ng each of forward and
reverse primers, and 0.25 U of Gold Star polymerase (Eurogentec). The PCR amplification was performed in a Crocodile II DNA thermal cycler
(Appligene) with the following protocol: 94°C for 3 min (1 cycle),
then 94°C for 30 s, 58°C for 1 min, and 72°C for 1 min (30 cycles), with a 10-min extension at 72°C at the end of the cycling.
The PCR reaction products were resolved on 1.5% agarose gels in 0.5 TBE buffer (1× TBE: 45 mM Tris, 45 mM boric acid, and 1 mM EDTA)
containing 0.5 µg/ml ethidium bromide. DNA fragments were visualized
on a ultraviolet (UV) transluminator (UVP). The fluorescence intensity
was quantified with a BIO-1D video-image analysis system
(Viber-Loumat).
RNase protection assay. PCR products
of Gi-3,
G3PDH, and ASUR 1 described above
were subcloned in pCR2.1 plasmids. Recombinant plasmids were
characterized by restriction enzyme analysis and sequencing. Plasmids
with antisense inserts were cleaved by
BamH I. One microgram of cleaved
plasmid was transcribed with T7 RNA polymerase in the presence of
[-32P]UTP, using
the Maxiscript SP6/T7 in vitro transcription kit (Ambion). The labeled
antisense RNAs were purified on acrylamide gels, eluted, and hybridized
with total RNA from control and aldosterone-treated A6 cells using the
RPA II kit from Ambion. Hybridized material was digested and separated
on 6% acrylamide gels. The gels were dried, autoradiographed, and
relative absorptions were quantified with a BIO-1D video-image analysis
system (Viber Loumat).
PTX activation. For in vitro
ADP-ribosylation, PTX was activated by incubation with 10 mM DTT and 1 mM NAD in 100 mM NaCl and 20 mM Na2HPO4,
pH 7.0, for 20 min at 30°C.
ADP-ribosylation of A6 membranes. The
protocol was adapted from Ribeiro-Neto et al. (21). Membranes
resuspended in ribosylation buffer (1 mM ADP-ribose, 1 mM DTT, 10 mM
thymidine, 1 mM NAD, 10 mM phosphocreatine, 2 U/10 mg protein of CPK,
and 10 mM Tris · HCl, pH 7.5) were incubated for 30 min at 28°C in the presence of
[32P]NAD (10 µM; SA:
8 Ci/mmol) with or without activated PTX (1 µg/ml). The reaction was
stopped by the addition of an excess cold phosphate-buffered solution,
and the samples were ultracentrifuged (100,000 g for 1 h). The pellets were
resuspended in sample buffer and separated by SDS-PAGE (8%
acrylamide). Bound radioactivity was detected by autoradiography.
Films were developed by autoprocessing and scanned using a laser
densitometer with built-in integrator (Ultroscan XL, LKB).
Binding of
[-32P]GTP
on Western blots.
Membrane proteins separated by SDS-PAGE (12% polyacrylamide gels) were
transferred to nitrocellulose paper in transfer buffer (25 mM Tris, 192 mM glycine, and 20% methanol) using the Trans-Blot apparatus from
Bio-Rad (30 V overnight). Blots were incubated for 90 min at room
temperature with
[-32P]GTP (1 µCi/ml, 0.1 ml/cm2) in binding
buffer (50 mM Tris · HCl, pH 7.5, 0.3% Tween 20, 5 mM MgCl2, and 1 mM EGTA) with or
without 1 µM unlabeled GTP. Blots were then rinsed with seven to
eight changes of binding solution over 1 h and air dried. Bound
radioactivity was detected by autoradiography.
Photoaffinity labeling of membrane
proteins. The UV-mediated cross-linking of
[-32P]GTP to
membrane proteins was adapted from Basu and Modak (2). The incubation
mixture, in a total volume of 55 µl, contained 20 mM
Tris · HCl, pH 7.4, 1 mM ATP, 1 mM DTT, 100 mM NaCl,
30 mM MgCl2, 0.1%
Lubrol, 0.25 µM
[-32P]GTP (800 Ci/mmol), and 100 µg protein. After a 10-min incubation on ice, the
samples were exposed to broad-spectrum UV light for 7 min at a distance
of 15 cm and at 4°C. The reaction was stopped by the addition of 20 µl of 2 mM GTP. Samples were resuspended in sample buffer (70 mM
Tris · HCl, pH 6.8, 2% SDS, 12.5% glycerol, 0.02%
bromophenol blue, and 5% 
-mercaptoethanol) and separated on 12%
polyacrylamide gels by SDS-PAGE in a Bio-Rad Protean II gel apparatus. Bound radioactivity was detected by autoradiography.
Isotopes were obtained from New England Nuclear, and PTX
was from Sigma. Gel electrophoresis reagents and standards were from Bio-Rad. Proteins were measured using the BCA Protein Assay kit from
Pierce (Rockford, IL). Antibodies against the -subunits of
Gi,
G0,
Gs, and
Gq proteins were from Calbiochem
(San Diego, CA).
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RESULTS |
In previous studies, we observed an increase of both the sodium
transport rate and GTPase activity in membranes isolated from A6 cells
exposed to aldosterone (25). It was possible that the increase in
activity was due simply to an increase in the number of hydrolysis
sites. We tested this directly in the present studies. Experiments were
carried out in vitro on membrane preparations which are enriched
10-fold in apical markers (23).
Equilibrium binding of
[35S]GTPS
to A6 membranes.
Binding of
[35S]GTPS was
measured in membranes prepared from control cells and from cells
exposed to 100 nM aldosterone for 4 h. In the presence of
MgCl2, binding was linear up to 10 min and reached equilibrium after 30 min at 28°C. Binding was a
saturable function of GTPS concentration with similar
characteristics in membranes from control and aldosterone-treated
tissues (Fig.
1A). The membranes were enriched 7- to 10-fold in binding sites compared with their respective cell homogenates
(n = 3). Linearization of the binding
data according to Scatchard is shown in Fig. 1B. The values
of Kd and Bmax, summarized in Table
1, are not significantly different for
control and aldosterone-treated tissues (unpaired t-test). Likewise, the
initial rate of binding (1.24 ± 0.08 pmol · min
1 · mg
protein1) was unmodified
after aldosterone (1.20 ± 0.05 pmol · min1 · mg
protein1). Both GTP and
GDP behaved as competing nucleotides for the binding of GTPS with an
EC50 value of 1 µM for GTP and 5 µM for GDP
(n = 2).
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Fig. 1.
[35S]GTPS
equilibrium binding to A6 membranes.
A: equilibrium binding vs. GTPS
concentration. Membranes (10 µg protein) were incubated for 30 min at
28°C with the indicated concentrations of nucleotide. Specific
binding was calculated by subtracting the counts obtained in the
presence of 100 µM GTPS and by correcting for specific activity of
[35S]GTPS at each
nucleotide concentration. Assays were performed with membranes prepared
from control ( ) or aldosterone-treated ( ) cells;
n = 6. B: linearization of binding data
according to Scatchard. PTX, pertussis toxin; Aldo, aldosterone. Values
of Kd and
Bmax are given in Table 1.
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Table 1.
Maximum rates of binding and half-saturation constants obtained after
linearization of the data as presented in Fig. 1B
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Clearly, the total number of GTPS binding sites was not changed by
aldosterone. These studies were pursued on tissues treated with PTX.
This toxin specifically ADP-ribosylates
Gi proteins and was found to
inhibit both the aldosterone-stimulated GTPase activity and sodium
transport (25).
After incubating the membranes with activated toxin, equilibrium
binding was inhibited by 50.4 ± 4%
(n = 12) in control membranes and 53 ± 5% (n = 12) in membranes from
aldosterone-treated cells (Fig. 2). In both
conditions, the inhibition by PTX was related to a decrease in
Bmax with little change in
affinity (Fig. 1B; Table 1). From the
binding data and the values of GTPase activity, we calculated rate
constants for GTP hydrolysis,
kcatGTP (moles of
GTP hydrolyzed/number of binding sites) after aldosterone
and/or PTX (Table
2).1
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Fig. 2.
Effect of pertussis toxin (PTX) on
[35S]GTPS binding.
Activated PTX (1 µg/ml) was added to membranes for 30 min at
28°C. Equilibrium binding of
[35S]GTPS was
measured in presence or absence of 100 µM unlabeled GTPS. Control
membranes were treated identically, except without PTX. C, control
membranes; A, membranes from aldosterone-treated cells; C/PTX, toxin on
control membranes; A/PTX, toxin on membranes from aldosterone-treated
cells. Equilibrium binding was also measured at different GTPS
concentrations, as described in Fig.
1A, and Scatchard plots of the data
are shown in Fig. 1B.
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Table 2.
Rate constants of GTP hydrolysis calculated from GTPase and binding
activities in absence and presence of PTX
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Clearly, the PTX-sensitive component of
kcatGTP after
aldosterone was increased 13-fold over control. Based on the assumption that PTX-sensitive binding reflects the membrane concentration of
Gi proteins, this supports the
idea that aldosterone stimulates the activity and not the abundance of
this important G protein.
It is possible that the observed similarity of the proportional
decrease in binding sites after PTX was due to the instability (i.e.,
rapid turnover) of sites in the membrane. This was tested directly with
cycloheximide, which inhibits Na+
transport but not GTP hydrolysis (8). As shown in Fig.
3, in tissues exposed to cycloheximide,
inhibition of transepithelial Na+
current was measurable after 2 h and aldosterone stimulation was
completely prevented, but no change in initial rates of
binding was observed up to 24 h in either control or
aldosterone-treated cells. Thus we cannot attribute the decrease in
binding after PTX to a nonspecific, time-dependent loss of sites.
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Fig. 3.
Effect of cycloheximide on transepithelial
Na+ current and on
[35S]GTPS binding.
A6 cells grown to confluence on porous supports were exposed to 100 nM
aldosterone (), 1 µg/ml cycloheximide ( ), or 1 µg/ml
cycloheximide plus 100 nM aldosterone ( ) for the times indicated.
Na+ currents of control tissues
were 12.3 ± 0.16 µA/cm2 at
time 0 and stayed stable over 24 h.
Calculated amiloride-sensitive Na+
currents are shown. Initial rates of
[35S]GTPS binding,
in
pmol · min1 · mg
protein1, are also shown
(). Values of binding obtained in control and aldosterone-treated
tissues were indistinguishable (n = 6).
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Because binding studies measure total membrane concentration of sites,
they do not exclude the existence of a minor population of G proteins
specifically induced after aldosterone. Therefore, we verified by
RT-PCR and by the more sensitive RNase protection assay
(RPA) method the level of mRNA for the -subunits of
various heterotrimeric G proteins.
RT-PCR and RPA. mRNA abundance was
estimated by quantitative RT-PCR in identical amounts of total RNA from
cells grown in the absence or presence of 100 nM aldosterone.
The results of the amplification of
Gi-3 are shown in Fig.
4. We did not observe any increase in
Gi-3 mRNA with aldosterone up
to 2 h (Fig. 4A) or 24 h (data not
shown). This is in contrast to the increase in the positive control
ASUR 1 (27), which doubled after 30 min (Fig.
4B). Likewise,
Gi-1,
G0,
Gq, and
Gs mRNAs remained unchanged
(data not shown). The RT-PCR results were confirmed by RPA as shown in
Fig. 5. Aldosterone did not modify the
signal for Gi-3 compared with
ASUR 1 (positive control) and
G3PDH (negative control). These
data rule out the existence of even a small increase in mRNA which
would have been unnoticed by PCR.
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Fig. 4.
RT-PCR amplification (30 cycles) of
Gi-3
(A) and ASUR 1 (B). Total RNA was extracted from A6
cells grown in absence or in presence of 100 nM aldosterone. Results
are shown for incubation times with aldosterone up to 120 min.
Fluorescent amplified DNA fragments appear at the
top of each graph. These are
representative of 3 experiments. AU, absorbance units.
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Fig. 5.
RNase protection assays (RPA). RPAs of glucose-6-phosphate
dehydrogenase (G3PDH)
(group 1), ASUR 1 (group
2), and Gi-3
mRNA (group 3) were performed using total RNA
from control A6 cells (solid bars) or from cells treated with
aldosterone for 2.5 h (hatched bars) or 24 h (open bars).
Autoradiograms were analyzed with a BIO-1D video-image analysis system
as described in MATERIALS AND METHODS.
Relative absorption (AU, absorbance units) of protected fragments is
shown, representative of 3 experiments.
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We also visualized various GTP-binding proteins by biochemical
approaches that classically target either heterotrimeric or small
Mr G proteins.
Binding of
[-32P]GTP
on Western blots.
Low-molecular-weight G proteins uniquely renature upon
Western blotting and bind GTP specifically. A6 membrane proteins were separated by SDS-PAGE and transferred to nitrocellulose. Exposure of
these blots to
[-32P]GTP resulted
in the specific labeling of a group of proteins in the
Mr range of
26-29 kDa as well as a 21-kDa polypeptide (Fig. 6). The same pattern was seen in membranes
from control (lane 1) or
aldosterone-treated cells (lane 2).
When [35S]GTPS was
used instead of
[-32P]GTP, we
observed specific labeling of a 26-kDa polypeptide only (data not
shown). Because aldosterone did not alter the expression or the pattern
of these small G proteins, they were not characterized further (see
DISCUSSION).
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Fig. 6.
Labeling of low-molecular-weight G proteins in A6 membranes.
Autoradiogram of a Western blot of membranes from control
(lane 1) and aldosterone-treated
cells (lane 2) separated on 12%
polyacrylamide gels. Each lane was loaded with 80 µg protein. Blots
were exposed to 12.5 nM
[-32P]GTP for 90 min, and nonspecific binding was examined in presence of 1 µM cold
GTP (lanes 3 and
4). Films were exposed for 2 wk at
80°C. Background shown is the best we could obtain even
after extensive rinsing of the blots. Molecular masses of the standards
are indicated on left.
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ADP-ribosylation of A6 membranes.
Since PTX-sensitive sites are clearly important in the response of
cells to aldosterone (see above), it was of interest to identify the G
proteins targeted by the toxin. Exposure of A6 membranes to
[32P]NAD in the
presence of PTX, followed by gel electrophoresis resulted in the
labeling of a single band of
Mr 41 kDa in
control membranes (Fig. 7,
lane 1). After aldosterone, the
amount of ADP-ribosylated protein remained unchanged
(lane 3). This 41-kDa polypeptide
was recognized by anti-Gi-3
antibodies on Western blots (data not shown; see Ref. 25). Unlike a
previous report by Ausiello et al. (1), we did not observe
ADP-ribosylation of other proteins associated with the
Na+ channels such as the 90- to
95-kDa polypeptide (1).
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Fig. 7.
ADP-ribosylation of A6 membrane proteins. Autoradiogram of A6 membrane
proteins (40 µg/lane) separated on 8% polyacrylamide gels after a
30-min exposure to
[-32P]NAD and PTX
(lanes 1 and
3 for control membranes and membranes
of cells treated with aldosterone for 4 h, respectively). In absence of
toxin, label incorporation was not observed (lane
2). Molecular masses of the standards are indicated
on left. Films were exposed for 2 days
at 80°C.
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Photoaffinity labeling of membrane
proteins. The GTP binding sites may be modified by SDS
solubilization. Therefore, we used photoaffinity covalent labeling to
examine the binding sites in their native form. The results of
photoincorporation of high specific activity
[-32P]GTP (0.25 µM) into membrane proteins are shown in Fig.
8. In the absence of UV irradiation, there
was no covalent binding of label. After UV irradiation of control
membranes (Fig. 8A,
lane 1), little or no incorporation
was observed. After aldosterone, however, intense labeling of a
59.5-kDa band was observed (Fig. 8A,
lane 2). Photoincorporation of the
label was completely blocked with 100 µM GTP (Fig.
8A, lane
3) but decreased only 16% with 100 µM ATP (Fig.
8B,
left lane) as measured by densitometry
scanning of the autoradiograms (n = 2). This 59.5-kDa protein was probed on Western blots by commercial
antibodies against either Gi-3, Go,
Gs, or
Gq, but no labeling was
observed (data not shown). Furthermore,
[32P]NAD ribosylation
of this protein in the presence of PTX did not occur (Fig.
7). However, labeling of the 59.5-kDa protein was decreased in
membranes from cells exposed to both aldosterone and spironolactone, a
competitive antagonist of the hormone, suggesting that this membrane
protein is specifically induced by aldosterone (Fig.
8C).
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Fig. 8.
Photoaffinity labeling of A6 membrane proteins.
A: membranes (100 µg per sample)
were incubated with 0.25 µM
[-32P]GTP in
presence and absence of an excess unlabeled GTP and exposed to
broad-spectrum UV light as described in MATERIALS AND
METHODS. Membrane proteins were separated on 12%
polyacrylamide gels and the gels were exposed to Hyperfilm-MP
(Amersham, Belgium) for 1 wk at 80°C. Molecular mass marker
positions are as follows, from top to
bottom: phosphorylase b (97 kDa),
bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase
(31 kDa), soybean trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa). Lane 1, control membranes;
lane 2, membranes from cells treated
with aldosterone for 4 h; lane 3, + 100 µM GTP. Photograph is representative of 3 experiments.
B: photoaffinity labeling of membranes
from cells treated with aldosterone
(right lane) and exposed to 100 µM
ATP during the irradiation period
(left lane).
C: photoaffinity labeling of membranes
from cells treated with aldosterone
(left lane) or with aldosterone and 10 µM spironolactone for 4 h (right
lane). For B and
C, molecular mass marker positions are
the same as in A (first 4 markers,
from 97 to 31 kDa). Exactly 100 µg of protein were loaded in each
lane (n = 2).
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DISCUSSION |
Evidence has accumulated that G proteins play significant roles in
controlling the activity of amiloride-inhibitable
Na+ channels at the apical
membrane of epithelia (see Ref. 26 for review) as well as in
lymphocytes, which also possess these channels (5). However, the G
protein-mediated pathway(s) activated by aldosterone in the control of
Na+ channels is clearly different
from the ones involved with membrane-bound G protein-coupled receptors
and diffusible second messengers (see Ref. 28 for a review).
Activation of Na+ channels located
in the apical membrane of responsive epithelia by aldosterone causes an
increase in the Na+ transport
rate. This results from an increase of the channel's open probability,
the number of functional channels, or both (11, 14). In
sodium-reabsorbing epithelia, there are no cell-surface receptors for
aldosterone. This delays the effect of the hormone on the
Na+ transport rate, which probably
involves the synthesis of a number of proteins, many of which, still
have not been identified. GTP-dependent methylation of the apical
Na+ channels appears to mediate
the short-term effects of aldosterone on apical
Na+ permeability (13, 24, 26).
Because aldosterone activates a specific membrane GTPase, we examined
the G proteins that reside in the cell membrane before and after
hormone treatment. Our membrane preparations are enriched 10-fold in
apical markers as well as in GTPS binding sites, but we cannot
exclude the presence of basolateral membranes.
Using techniques that target different G proteins, we identified the
following three groups of specific resident GTP-binding proteins:
1) several small G proteins of
molecular mass 21 and 26-29 kDa (the pattern as well as the level
of expression of these proteins did not change after
aldosterone); 2) a
41-kDa protein that was ADP-ribosylated by PTX, recognized by an
anti-Gi-3 antibody, and also
not modified by aldosterone; and 3)
a 59.5-kDa protein labeled by photoaffinity whose expression at the
cell membrane was triggered by treating cells with aldosterone. This
protein has not been described previously. Its labeling was blocked by an excess of unlabeled GTP but not by ATP. The protein was not recognized by antibodies directed against the -subunits of
Gi-3, Gs,
Go, or
Gq or by ADP ribosylation with
PTX. Spironolactone strongly diminished the protein labeling.
We also observed that the binding of
[35S]GTPS to apical
membrane preparations was not altered after 4 h of aldosterone in terms
of the kinetics (initial rate and equilibrium binding), the affinity
for the ligand, or the number of binding sites. Likewise, using two
independent approaches, we did not observe any increase in mRNA levels
for the -subunits of Gi-3,
Gs,
Go, or
Gq proteins in A6 cells exposed to
aldosterone for either 4 or 24 h. Rokaw et al. (22), using the Northern
blot technique, have reported an increase in the level of
Gi-3 mRNA in A6 cells after 16 h of aldosterone. However, this increase was relatively small (only 1.6- to 2-fold) and may not be physiologically relevant to the early,
4-h phase of the aldosterone response.
The action of PTX in A6 cells appears complex. Several reports have
indicated that the sensitivity of
Na+ channels to PTX is modulated
by the biochemical state in which they reside. For example, their level
of phosphorylation/dephosphorylation may influence the channel's
response to GTPS (5, 7, 12, 19). In the present study, equilibrium
binding of GTPS to both control and aldosterone-treated tissues was
inhibited by 50% in the presence of PTX. Previously, we observed
similar decreases in the rates of
Na+ transport and GTP hydrolysis
in aldosterone-treated tissues exposed to PTX (25). Since this toxin is
a specific marker of Gi, this suggests that half of the GTP binding sites are involved in the toxin-sensitive stimulation of GTP hydrolysis following aldosterone. In
control membranes, we also observed a 50% decrease in GTPS binding
after PTX treatment. However, the toxin has only small effects
(<15%) on basal Na+ transport
and GTP hydrolysis (25). This indicates that under basal conditions,
Gi is present in the membrane
but contributes little to the control of steady-state
Na+ transport. In support of this
idea, we observed a dissociation of GTP binding from
Na+ transport after cycloheximide
treatment. This also indicates that the GTP binding sites
and the sodium channels have different residence times in the cell
membrane and suggests that their expression is independently regulated.
After aldosterone, the increase in the rate constant of GTP hydrolysis
results essentially from the activation of PTX-sensitive G proteins.
The kcatGTP
values obtained in this study are lower than the values found in
nonepithelial tissues equipped with soluble signaling proteins (17).
Values of kcatGTP
in epithelia are not available in the literature for comparison. The
large increase (over 13-fold) in the rate of PTX-sensitive GTPase
activity after aldosterone associated with a constant membrane
concentration of Gi-3 proteins
points to the activation of these proteins by an additional regulatory
component such as a member of the RGS ("regulators of G protein
signaling") or the GAP ("GTPase activating proteins") families
(4, 7, 16, 17). The basal, unstimulated PTX-insensitive GTPase activity
could be related to the presence of the low-molecular-weight G
proteins. In this regard, subcellular localization of G
proteins to the apical membrane of epithelia has been reported for
low-molecular-weight G proteins (20). One of these proteins, a 29-kDa
polypeptide located in the apical membrane of collecting duct cells of
mammalian kidney, was identified as
ral (11), but no specific function has
been assigned to it or to any other small G proteins in epithelia.
Recently, Spindler et al. (27) identified in A6 cells an early
adrenal-steroid-upregulated RNA (ASUR 5) as the A transcript of
Xenopus
K-ras2. Its role in the stimulation of
sodium transport by aldosterone has not been reported.
In summary, we found that short-term aldosterone does not promote the
expression of classic -subunits of heterotrimeric G proteins in A6
cells but stimulates the GTP hydrolysis rate by activating resident
PTX-sensitive G proteins. Aldosterone also increases specifically the
expression of a novel 59.5-kDa GTP-binding protein, the role of which,
as a regulatory component in the complex cellular response to
aldosterone, remains to be established.
| |
ACKNOWLEDGEMENTS |
We thank Dr. M. Lambert and D. Bui for advice and technical help
with the initial cross-linking and ADP-ribosylation experiments, C. Vanhoutte for help with the
[35S]GTPS binding
assay, and Dr. F. Verrey for the kind gift of ASUR 1.
| |
FOOTNOTES |
This work was funded by the Fonds National de la Recherche
Scientifique, Belgium, Grants 1.5.023.96 and 3.4591.97.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
1
Values of GTPase activities are taken from Ref.
25 and have been verified in this study.
Address for reprint requests: S. Sariban-Sohraby, Laboratoire de
Physiologie, Université Libre de Bruxelles, Bat. E.2.4.107./CP
604, 808, route de Lennik, 1070 Brussels, Belgium.
Received 4 May 1998; accepted in final form 10 September 1998.
| |
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