RAPID COMMUNICATION
Ezrin binding domain-deficient NHERF attenuates cAMP-mediated
inhibition of Na+/H+ exchange in OK
cells
Edward J.
Weinman1,2,3,
Deborah
Steplock1,
James
B.
Wade2, and
Shirish
Shenolikar4
1 Department of Medicine, 2 Department of Physiology,
University of Maryland School of Medicine, and 3 Medical
Service, Department of Veterans Affairs Medical Center, Baltimore,
Maryland 21201; and 4 Department of Pharmacology and Cancer
Biology, Duke University Medical Center, Durham, North Carolina
27710
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ABSTRACT |
Na+/H+ exchanger regulatory factor
(NHERF), an essential protein cofactor in cAMP-mediated inhibition of
Na+/H+ exchange transporter 3 (NHE3),
facilitates the formation of a signal complex of proteins that includes
NHE3, NHERF, and ezrin. This model for NHE3 regulation was developed in
fibroblasts and its applicability to epithelial cells remains to be
established. Opossum kidney (OK) cells were transfected with
either empty vector (control), full-length mouse (m) NHERF(1-355),
or a truncated mNHERF(1-325) that lacked ezrin binding and had
been demonstrated in fibroblasts to bind NHE3 but not mediate its
cAMP-associated inhibition. 8-Bromoadenosine 3',5'-cyclic
monophosphate (8-BrcAMP) at 10
4 M inhibited
Na+/H+ exchange activity in control and OK
cells expressing wild-type mNHERF(1-355) by >60% but by <10%
in cells expressing mNHERF(1-325). NHE3 coimmunoprecipitated with
mNHERF(1-325), but cAMP phosphorylation of NHE3 was impaired in
cells expressing mNHERF(1-325). The inhibitory effect of
hyperosmolality on NHE3 activity and the uptake of
3-O-methyl-D-glucose was the same in all three
cell lines. Cell surface expression of NHE3 was not changed by cAMP in
any of the cells lines. These data indicate that disruption of the
NHERF-ezrin signal complex attenuates the inhibitory effect of cAMP on
NHE3 activity in OK cells and provides evidence supporting the proposed
model of protein kinase A regulation of NHE3 in epithelial cells.
renal electrolyte transport; PSD-95/Dlg/ZO proteins; protein kinase
A; sodium/hydrogen exchange transporter 3; acid-base physiology; opossum kidney cells
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INTRODUCTION |
THE ACUTE REGULATION
OF RENAL apical membrane Na+/H+
exchange transporter 3 (NHE3) by agonists that increase intracellular
cAMP requires participation of a newly described protein called the Na+/H+ exchanger regulatory factor (NHERF)
(18, 17, 21). Studies in fibroblast cells have provided
evidence that NHERF functions to facilitate formation of a signal
complex of proteins including ezrin, NHE3, and cAMP-dependent protein
kinase A (PKA) (17, 22). This multiprotein complex
mediates the phosphorylation of NHE3, thereby inhibiting its activity
(17, 22). Despite extensive study of the interactions
among NHERF, ezrin, PKA, and NHE3 in fibroblast cells, a continuing
issue with the proposed model has been the lack of functional data to
indicate its applicability to epithelial cells.
In the course of study of the role of the ezrin binding domain of
NHERF, we expressed a 30-amino acid COOH-terminal truncated form of
mouse NHERF [mNHERF(1-325)] that lacked the
putative ezrin binding domain in PS-120 cell fibroblasts
(17). Compared with wild-type NHERF
[mNHERF(1-355)], the ezrin binding domain-deficient construct
did not bind to ezrin, did not phosphorylate NHE3, and did not support
cAMP inhibition of NHE3 activity. This construct, however, did bind to
NHE3 in vivo (17). We reasoned, therefore, that this
truncated form of NHERF might be a useful reagent to develop a
dominant-negative renal epithelial cell line to address the important
question of whether NHERF is required for cAMP-associated inhibition of
NHE3 in epithelial cells. In the present experiments, opossum kidney
(OK) cells, a proximal tubule cell line that expresses endogenous NHERF
and Na+/H+ exchanger activity, were transfected
with full-length mNHERF(1-355) or mNHERF(1-325), and the
effect of cAMP on the activity and cell surface expression of the
transporter was determined.
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METHODS |
OK cells were maintained at 37°C in a humidified
atmosphere with 5% CO2 in DMEM/F-12 medium (1:1)
supplemented with 10% (vol/vol) FCS, penicillin (100 U/ml), and
streptomycin (100 µg/ml). Cells were grown to confluence in serum,
split, and regrown to sub-confluency in the presence of serum.
mNHERF(1-355) and mNHERF(1-325) cDNAs were cloned
into pcDNA3.1/Hygro+ vectors and transfected
into OK cells using Lipofectin (GIBCO-BRL) (17, 21, 22).
Cells transfected with the pcDNA3.1/Hygro+ vector alone
served as the control. Cells resistant to 600 U/ml hygromycin were
selected through eight passages before study. Transfected cells were
maintained at 37°C in a humidified atmosphere with 5%
CO2 in DMEM/F-12 medium (1:1) supplemented with 10%
(vol/vol) FCS, penicillin (100 U/ml), streptomycin (100 mg/ml), and 600 U/ml hygromycin.
Na+/H+ exchange activity was assayed in cells
grown on 40-mm glass coverslips using the pH-sensitive fluorescent
dye, the acetoxymethyl ester of
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM) (Molecular Probes) (17, 21, 22). Cells were serum deprived for 24-36 h and then loaded with 6.5 µM BCECF-AM in an assay
buffer of 20 HEPES, pH 7.4, containing (in mM) 40 NH4Cl, 90 NaCl, 5 KCl, 1 MgSO4, 2 CaCl2, 1 tetramethylammonium (TMA)-PO4 and 25 glucose for 20 min at
room temperature. Coverslips were transferred to an FSC2 chamber
(Bioptechs) and rapidly perfused at a constant flow rate with solutions
warmed to 37°C. Cells were acidified for 5 min (in mM) with 20 mM
HEPES, pH 7.5, 130 TMA-Cl, 5 KCl, 1 MgSO4, 2 CaCl2, 1 TMA-PO4, and 25 glucose.
Na+/H+ exchange transport was initiated by
substituting 130 mM NaCl for 130 mM TMA-Cl in the perfusion solution.
BCECF fluorescence was measured at excitation wavelengths of 500 and
440 nm and an emission wavelength of 530 nm. The NH4Cl
pulse was used to achieve an initial intracellular pH (pHi)
of 6.0, and only cells with initial pHi values between 6.0 and 6.2 were included for analyses. Na+/H+
exchange, expressed as change in pHi
(
pHi)/min, represented the initial slope of
transport activity measured between 5 and 10 s of sodium-dependent
pHi recovery. Over this brief time period, the relationship
between pHi and time was essentially linear. To analyze the
effects of cAMP on NHE3 activity, cells were pretreated with
10
4 M 8-bromoadeosine 3',5'-cyclic monophosphate
(8-BrcAMP) during the final 15 min of dye loading and continuously
throughout the perfusion process. At the end of each experiment, the
cells were equilibrated in pH-clamp media containing (in mM) 20 HEPES,
20 MES, 115 KCl, 14 NaCl, 1 MgSO4, 1 CaCl2, 1 TMA-PO4, 25 glucose, and 10 µM nigericin at pH 6.0 and
7.3. On any given coverslip, Na+/H+ exchange
was measured in several fields, and the determinations were averaged to
constitute a single observation. All measurements were made on cells at
the same passage on the same day. Where indicated, the cells were
studied as above except that the osmolality of the incubation media was
increased by the addition of 80 mM mannitol 60 min before study.
To determine the specificity of the observed response, the
sodium-dependent uptake of radiolabeled
3-O-methyl-D-glucose was measured in OK cells
grown in 9.6-cm2 wells. Before study, cells were grown in
serum-free media for 24 h and then thoroughly washed with
D-PBS.
3-O-methyl-D-glucose uptake was
initiated by the addition of D-PBS containing tracer amounts of
[14C]3-O-methyl-D-glucose (0.25 µCi/ml). Where studied, 10
4 M 8-BrcAMP was added 15 min
before the start of the uptake phase and was present for the duration
of the experiment. Uptake was terminated after 15 min by replacement of
the radioactive solution with ice-cold D-PBS. Cells were
washed several times with cold D-PBS. The monolayers were
then solubilized for 18 h using 1 ml 0.05% Triton X-100 and
counted for 14C content in a scintillation counter. The
time of uptake was in the linear range of
3-O-methyl-D-glucose as determined in
preliminary studies.
The phosphorylation of NHE3 in OK cells was determined by a
back-phosphorylation assay. Cells were washed with serum-free Dulbecco's modified media lacking any antibiotics, after which half of
the cells were used as control and the other half were treated with
10
4 M 8-BrcAMP for 15 min. Cells were scraped and
resuspended in inositol phosphate (IP) buffer consisting of 10 mM
NaPO4, pH 7.4, containing 100 mM NaCl, 5 mM EDTA, 1 mM
Na3VO4, 50 mM NaF, and a mixture of protease
inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 1 mM phenanthroline,
and 5 ug/ml each of aprotinin, leupeptin, pepstatin, and trypsin
inhibitor). Cells were sedimented by centrifugation for 10 min at
12,000 g in an Eppendorf centrifuge, resuspended in 1-ml IP
buffer containing 1% Triton X-100 (IPT buffer), lysed by drawing the
cells several times through a 27-gauge needle, agitated on a rotating
rocker at 4°C for 30 min, and subjected to centrifugation at 12,000 g for 30 min to remove cell debris. The resulting
supernatants were precleared with protein A-Sepharose CL 4B beads
washed with IPT buffer by rocking for 1-2 h. The beads were spun
down, and the supernatants were incubated overnight with 15 ml of
anti-NHE3 antibody. Protein A-Sepharose CL 4B beads previously washed
with IPT buffer were then added and slowly rocked for an
additional 2-4 h. The bound antibody-antigen complex was eluted from the beads with 100 ml of 30 mM glycine-HCl, pH 2.8, and immediately neutralized with the addition of 10 ml 1 M Tris, pH 11. Immunoprecipitated NHE3 was then back phosphorylated in vitro with PKA
in 21 mM glycine-100 mM Tris, pH 7.4, containing 50 mM ATP-Mg, 100 mM
MgCl2, 180 units of PKA catalytic subunit (Promega), and 50 mCi [
-32P]ATP. After 10 min at
30°C, the reaction was terminated (boiling in Laemmli buffer for 2 min). The phosphoproteins were subjected to 10% SDS-PAGE, transferred
to nitrocellulose, and visualized by autoradiography. After
autoradiography, the filter was reacted with anti-NHE3 antibody to
determine loading. Autoradiographs and Western immunoblots were
quantitated by laser densitometry.
Western immunoblots of OK cell lysates were performed using an antibody
to full-length rabbit NHERF that also recognizes OK cell NHERF
(16). Immunocytochemistry was performed as previously described from this laboratory with minor modifications
(15). OK cells grown on coverslips were fixed in 3.7%
paraformaldehyde for 30 min, followed by washing and quenching of
aldehyde groups with 50 mM NH4Cl. Cells were then
permeabilized by 0.1% Triton X-100 for 30 min and treated with 6 M
guanidine for 10 min to unmask antigenic sites. Slides were incubated
with a rabbit polyclonal antibody to NHE3 or ezrin (SC-6407, Santa Cruz
Biotechnology, Santa Cruz, CA) overnight at 4°C. After being washed,
they were incubated with secondary antibody for 2 h at 4°C. The
antibody was coupled to Alexa 488 or 568 dyes (Molecular Probes,
Eugene, OR) and diluted 1:100. Specimens were examined with a Zeiss
LSM410 confocal microscope. Protein concentrations were determined by the method of Lowry et al. (13). The results were analyzed
by ANOVA (9).
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RESULTS |
OK cells express NHERF with a lower apparent molecular size on
SDS-PAGE than mNHERF, as seen in Western immunoblot of cell lysates
(Fig. 1). The abundance of OK NHERF was
not altered by the overexpression of mNHERF(1-355) or
mNHERF(1-325). As seen in lanes 2 and 3, the
NHERF antibody recognized an additional band not present in lane
1 representing mNHERF(1-355) and mNHERF(1-325). Immunoprecipitates of anti-OK cell NHE3 were probed with anti-NHERF antibody and, as seen in Fig. 2, only the
smaller OK cell NHERF was recovered from control cells. By contrast,
anti-NHE3 immunoprecipitates from cells expressing
mNHERF(1-355) showed a broader band that reflected both
mNHERF and OK NHERF. Surprisingly, the larger mNHERF was the
predominant species recovered with the anti-NHE3 immunoprecipitates from the mNHERF(1-325)-expressing cells. Figure
3 shows representative tracings of the
initial sodium-dependent pHi recovery in the absence and
presence of cAMP in the three cell lines. Table
1 summarizes the measurements of the rate
of Na+/H+ exchange and the effect of cAMP.
Control OK cells transfected with the pcDNA vector alone had a rate of
sodium-dependent pHi recovery (
pHi/min) from
an acidifying stimulus of 0.035 ± 0.004 in the absence of cAMP
and 0.010 ± 0.002 in the presence of 10
4 M 8-BrcAMP
(P < 0.01). OK cells expressing
mNHERF(1-355) had a rate of 0.040 ± 0.006
pHi/min in the absence of cAMP and 0.010 ± 0.002 in the presence of cAMP (P < 0.01). OK cells
expressing mNHERF(1-325) had a rate of 0.045 ± 0.003
pHi/min in the absence of cAMP and 0.043 ± 0.002 in the presence of cAMP [P = not significant (NS)].

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Fig. 1.
Western immunoblots of opossum kidney (OK) cell lysates
using an anti-Na+/H+ exchanger regulatory
factor (NHERF) antibody that recognizes OK cell and mouse (m) NHERF.
Lane 1, OK cells transfected with the pcDNA vector alone
(control); lane 2, OK cells transfected with full-length
mNHERF(1-355); and lane 3, OK cells transfected
with NHERF(1-325).
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Fig. 2.
Na+/H+ exchange transporter 3 (NHE3) was immunoprecipitated from OK cell lysates using an antibody
against OK cell NHE3. The presence of NHERF in the immunoprecipitates
was analyzed by immunoblotting with an anti-NHERF antibody. Lane
1, control cells; lane 2, OK cells transfected with
mNHERF(1-355); lane 3, from OK cells transfected
with mNHERF(1-325).
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Fig. 3.
Representative tracings of the sodium-dependent
intracellular pH (pHi) recovery from an acid load in the
absence (solid lines) or presence (dashed lines) of 10 4 M
8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) for 15 min in OK
cells transfected with the pcDNA vector alone (control), full-length
mNHERF(1-355), or NHERF(1-325). Sodium was
introduced at time 0.
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Table 1.
The effect of cAMP on
Na+/H+
exchange in OK cells expressing mouse wild-type NHERF or an ezrin
binding domain-deficient NHERF
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We next analyzed the covalent modification of NHE3 in OK cells using an
in vitro back-phosphorylation assay of immunoprecipitated NHE3. In
control cells treated with cAMP, the amount of 32P
incorporated in vitro determined by autoradiography and normalized for
the amount of NHE3 by Western immunoblot was decreased by 37.1 ± 7.1% (n = 3, P < 0.05) compared with
cells not treated with cAMP (Fig. 4).
This suggested the prior phosphorylation of this fraction of NHE3 in OK
cells in response to cAMP. In cells expressing mNHERF(1-355), cAMP
treatment decreased back phosphorylation of NHE3 by 44.6 ± 4.6%
(n = 3, P < 0.05). By contrast, in the mNHERF(1-325)-expressing cells, the amount of 32P
incorporated in vitro into the immunoprecipitated NHE3 was essentially the same whether the cells were previously treated with cAMP or not
(%change = +9.4 ± 3.7%).

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Fig. 4.
Top: representative autoradiographs of the
back phosphorylation of NHE3 immunoprecipitated from control OK cells,
OK cells transfected with mNHERF(1-355), or cells
transfected with mNHERF(1-355). Studies were performed
in the absence ( ) or presence (+) of in vivo treatment of the cells
with 10 4 M 8-BrcAMP for 15 min. Bottom:
Western immunoblots to assess loading of the individual lanes.
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In separate experiments, we examined the physiological effect of
acutely increasing the osmolality of the incubating media, a non-cAMP
stimulus previously shown to inhibit NHE3 activity (1). As
summarized in Table 2, control OK cells
had a rate of sodium-dependent pHi recovery of 0.051 ± 0.009 pHi/min and 0.034 ± 0.006 in control media
and in media containing 80 mM of added mannitol, respectively
(n = 4, P < 0.05). OK cells expressing NHERF(1-355) had a rate of 0.035 ± 0.003
pHi/min and 0.022 ± 0.002 in control media and in
media containing 80 mM of added mannitol, respectively
(n = 5, P < 0.05). OK cells expressing mNHERF(1-325) had a rate of 0.047 ± 0.005 in control
media and 0.027 ± 0.004
pHi/min in media
containing 80 mM of added mannitol (n = 4, P < 0.05). To demonstrate the specificity of the
effect observed in the mNHERF(1-325) cells, a non-cAMP
regulated transporter was examined. As summarized in Table
3, the sodium-dependent uptake of the
nonmetabolized sugar 3-O-methyl-D-glucose at 15 min was 710.0 ± 17.3 counts · min
1
(cpm) · µg protein
1 in control cells, 703.3 ± 76.0 cpm/µg protein in cells expressing mNHERF(1-355),
and 678.9 ± 110.8 cpm/µg protein in cells expressing mNHERF (1). cAMP did not affect the uptake of
the sugar in any of the cell lines.
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Table 2.
The effect of hyperosmolality on
Na+/H+
exchange in OK cells expressing mouse wild-type NHERF or an ezrin
binding domain-deficient NHERF
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Table 3.
The effect of cAMP on sodium-dependent
3-O-methyl-D-glucose uptake in OK cells expressing mouse
wild-type NHERF or an ezrin binding domain-deficient NHERF
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A physiological role for NHERF in membrane trafficking of some
transporters, receptors, and signaling proteins has been suggested by
recent studies (8, 14, 18). To determine if NHERF affected the cellular trafficking of NHE3, the three cell lines were examined by
immunocytochemistry and confocal microscopy for NHE3 and ezrin. The
pattern of distribution of ezrin was not different between the cell
lines and did not change in response to cAMP (data not shown). Using
ezrin as a marker, the distribution of cell surface NHE3 was
determined. As shown in Fig. 5, there was
no clear difference in cell surface expression of NHE3 among the three
cell lines in response to treatment with 10
4 M 8-BrcAMP
for 15 min or in the mNHERF(1-355)- and
mNHERF(1-325)-expressing cells after 60 min of treatment with
cAMP.

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Fig. 5.
Cell surface expression of NHE3 in OK cells transfected with
mNHERF(1-355) (A-C),
mNHERF- (1) (D-F), or
with an empty pcDNA vector (G and H) in the
absence ( cAMP, A, D, G) or presence
(+cAMP, B, C, E, F,
H) of 10 4 M 8-BrcAMP for 15 min or 60 min.
Cells were stained with a polyclonal antibody against rabbit NHE3. Bar,
10 µm.
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DISCUSSION |
Recent experiments have indicated that NHERF is a necessary
cofactor in cAMP-mediated inhibition of NHE3 (16-20).
Biochemical and cell studies have advanced evidence that NHERF binds to
both NHE3 and ezrin and is required for cAMP to phosphorylate NHE3 (17, 22). In this model, ezrin is considered to be the PKA anchor protein. NHERF is envisioned to function as an adaptor that
facilitates formation of a multiprotein complex that mediates the rapid
phosphorylation of NHE3 by bringing the PKA-ezrin complex into
proximity with NHE3 (18). The subsequent phosphorylation of NHE3 results in the acute downregulation of its activity. This model
of the signal complex regulation of NHE3 activity was developed exclusively from studies performed in PS-120 cell fibroblasts. An
important shortcoming of the proposed model, however, has been the
lack of its validation in an epithelial cell. The present studies
derive from recent experiments from our laboratory indicating that a
construct of NHERF that lacked the putative ezrin binding domain
[mNHERF(1-325)] did not support PKA-mediated phosphorylation of
NHE3 or inhibition of transport activity, but did bind to NHE3 in vivo
(17). Accordingly, we considered that if the ezrin binding domain-deficient NHERF could be expressed in an epithelial cell line,
it might displace native NHERF and, by virtue of its inability to bind
ezrin, disrupt cAMP regulation of NHE3 activity.
OK cells are a proximal tubule cell line that expresses endogenous NHE3
and NHERF. OK cell NHERF has a lower apparent molecular mass on
SDS-PAGE than mNHERF, and this size differential permitted identification of both the native OK cell NHERF and expressed mNHERF
using the same antibody. Stable expression of mNHERF(1-355) or
mNHERF(1-325) did not affect the abundance of OK cell NHERF. The
association of NHE3 with the specific NHERF proteins was examined by
immunoprecipitation of NHE3. In control cells, only the smaller OK cell
NHERF was recovered in association with NHE3. Anti-NHE3 immunoprecipitates from cells expressing mNHERF(1-355) showed a
broader band that reflected both mNHERF and OK NHERF. Surprisingly, the
larger mNHERF was the predominant form recovered with the anti-NHE3
immunoprecipitates from the mNHERF(1-325)-expressing cells. As the
expression of NHERF in both cell lines was equivalent, this suggests
that mNHERF(1-325) may have a higher affinity for OK NHE3 than
mNHERF(1-355). It is also possible that the association of
NHERF(1-355) with ezrin reduces its affinity for NHE3. Whatever the correct explanation, the data indicate an in vivo association between both the wild type and truncated form of NHERF with NHE3.
In control OK cells transfected with the pcDNA vector alone and in
cells transfected with full-length mNHERF(1-355), 15-min exposure to cAMP inhibited Na+/H+ exchange
activity by >60%. By contrast, the mNHERF(1-325) cells behaved
as NHERF dominant-negative cells, and the effect of cAMP to acutely
downregulate Na+/H+ exchange activity was
markedly attenuated. In PS-120 cells, NHERF is required for
cAMP-mediated phosphorylation of NHE3 (22). The
relationship between NHE3 phosphorylation and NHERF also appears to be
present in OK cells. In control cells and in cells expressing mNHERF(1-355), cAMP resulted in the phosphorylation of NHE3. On the other hand, in cells expressing mNHERF(1-325), the amount of
32P incorporated in vitro into the immunoprecipitated NHE3
was essentially the same whether the cells were previously treated with
cAMP. This indicates that mNHERF(1-325) disrupted the cellular
mechanism that promotes NHE3 phosphorylation in vivo in response to
cAMP and provides a biochemical explanation for the failure of cAMP to
inhibit NHE3 activity in these cells. The specificity of the effect
observed in the mNHERF(1-325) cells was examined in two additional
sets of experiments. Acute increases in the osmolality of the
incubation media have previously been demonstrated to inhibit NHE3
activity in OK cells by a mechanism presumed to be independent of cAMP
(1). In the present studies, incubation of the three cell
lines in media containing 80 mM of added mannitol for 60 min resulted
in an acute decrease in the rate of Na+/H+
exchange transport compared with cells studied in the normal media. We
also examined the sodium-dependent uptake of the nonmetabolized sugar
3-O-methyl-D-glucose to establish the integrity
of the sodium gradient across the cell and to rule out nonspecific
effects of the transfections on cell function. The sodium-dependent
uptake of the sugar was the same in all three cell lines, and cAMP
treatment had no effect on this transport process.
We have recently reported that NHERF, NHE3, and ezrin colocalize in the
apical membrane of the rat proximal tubule and that NHERF and ezrin
coimmunoprecipitate from OK cells (12, 15). Although
consistent with the presence of an association among the proposed
components of the signal complex, the above studies did not establish a
physiological role for the multiprotein complex in control of
Na+/H+ exchange transport activity in renal
tissue or kidney-derived cell lines. The demonstration herein that the
effect of cAMP on Na+/H+ exchange transport in
the mNHERF(1-325)-expressing cells is blunted, however, provides the required functional and biochemical data to
support the above studies (12, 15). When considered
together, the results indicate that PKA regulation of NHE3 activity in
an epithelial cell is mediated by a signal complex of proteins that includes NHERF.
A growing literature indicates that NHERF is involved in the
trafficking and recycling of transport proteins and receptors (3,
8, 14). Prior studies in renal tissue suggested that parathyroid
hormone caused a redistribution of Na+/H+
exchange activity and NHE3 protein from the cell surface to internal membrane pools (5, 11). Studies of Fan et al.
(5) in the rat proximal tubules showed that the acute
inhibitory effect of parathyroid hormone (PTH) on NHE3 activity
resulted from phosphorylation of the antiporter, whereas over a longer
time course of ~3 h, PTH facilitated the endocytic removal of NHE3
from the plasma membrane (5). In more recent studies, they
established a similar dual mode of regulation of NHE3 in OK cells in
response to PTH, with a significant decrease in cell surface NHE3 being
evident after only 30 min of hormone treatment (4).
Optical sectioning of the OK cells used in the present experiments
indicated that the distribution of ezrin, concentrated at the apical
surface, was similar in all cell lines and did not change in response
to cAMP. In comparable optical sections, there was no difference in
cell surface expression of NHE3 between the three cell lines either
before or after 15-min exposure to cAMP. Cell surface expression of
NHE3 was also not different after 60 min of treatment with cAMP in the
mNHERF(1-355)- and mNHERF(1-325)-expressing cells. In OK
cells, PTH has been demonstrated to use multiple second messenger
pathways (10). Thus the present findings, when considered with the results of Fan et al. (5), suggest that the
effect of PTH on the cellular redistribution of NHE3 is transduced by a
non-cAMP-mediated mechanism. We would conclude that the acute regulation of NHE3 by cAMP analyzed in the current experiments most
likely represents PKA phosphorylation of COOH-terminal residues in NHE3
mediated by the NHERF-ezrin signal complex (17, 22).
In summary, the present studies indicate that a truncated form of NHERF
deficient in ezrin binding functioned as a dominant-negative reagent
when expressed in OK cells. Although mNHERF(1-325) had little
effect on basal Na+/H+ exchange activity, it
markedly blunted the acute inhibitory response to cAMP. These studies,
then, provide the first functional validation for the signal-complex
model of NHE3 regulation in renal epithelial cells. Recent experiments
have established an association between NHERF and two other
cAMP-regulated transporters in the renal proximal tubule. The sodium
bicarbonate cotransporter (NBC) is inhibited by cAMP by a process that
requires NHERF (2). Gisler et al. (7)
recently showed that the COOH-terminus of the type IIa sodium phosphate
cotransporter associated with NHERF, although the physiological
relevance of this association was not established. The present
experiments suggest that the expression of the dominant-negative NHERF(1-325) in appropriate epithelial cells may help elucidate the role of the NHERF-ezrin complex in the hormonal control of NBC and
other renal transporters.
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ACKNOWLEDGEMENTS |
Dr. Mark Knepper kindly provided a polyclonal antibody to rat NHE3
(L546) (6), and Dr. Orson Moe provided a polyclonal antibody to OK cell NHE3. The authors acknowledge the technical support
of Christine Evangelista, Min-Zhi Liu, and Jie Liu.
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FOOTNOTES |
These studies were supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-55881 (E. J. Weinman and
S. Shenolikar) and DK-32839 (J. B. Wade) and a grant from the
Research Service, Department of Veterans Affairs (E. J. Weinman).
Address for reprint requests and other correspondence: E. J. Weinman, Div. of Nephrology, Rm. N3W143, UMH, Univ. of Maryland Hospital, 22 S. Greene Street, Baltimore, MD 21201 (E-Mail:
eweinman{at}medicine.umaryland.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
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in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 14 September 2000; accepted in final form 16 March 2001.
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