From the Department of Molecular Physiology, National Cardiovascular Center Research Institute, Fujishiro-dai 5-7-1, Suita, Osaka 565-8565, Japan
Received for publication, January 12, 2001, and in revised form, February 23, 2001
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ABSTRACT |
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The Na+/H+
exchangers (NHEs) comprise a family of transporters that
catalyze cell functions such as regulation of the pH and volume of a
cell and epithelial absorption of Na+ and bicarbonate.
Ubiquitous calcineurin B homologous protein (CHP or p22) is
co-localized and co-immunoprecipitated with expressed NHE1, NHE2, or
NHE3 independently of its myristoylation and Ca2+ binding,
and its binding site was identified as the juxtamembrane region within
the carboxyl-terminal cytoplasmic domain of exchangers. CHP
binding-defective mutations of NHE1-3 or CHP depletion by injection of
the competitive CHP-binding region of NHE1 into Xenopus oocytes resulted in a dramatic reduction (>90%) in the
Na+/H+ exchange activity. The data
suggest that CHP serves as an essential cofactor, which supports the
physiological activity of NHE family members.
The Na+/H+ exchanger
(NHE)1 is an electroneutral
plasma membrane transporter that catalyzes H+-extrusion
coupled to Na+-influx (1, 2). Six identified NHE isoforms
exhibit different tissue expression patterns (1-4): NHE1, in all
tissues; NHE2-4, mostly in epithelial cells; NHE5, in brain; and NHE6,
in mitochondria. These isoforms seem to have distinctive properties
despite their overall structural similarity. For example, NHE1 and
NHE3, which have been the most intensively studied isoforms, are
involved in regulation of intracellular pH and cell volume in all cell types and Na+ and bicarbonate absorption in the epithelial
cells, respectively. These two isoforms exhibit very different modes of
regulation by many physiological factors as well as a large difference
in the sensitivity to inhibitors such as amiloride derivatives (1, 2).
Furthermore, targeted gene disruption of NHE1, NHE2, or NHE3 produces
remarkably different phenotypes in mice; epilepsy and seizure for NHE1,
reduced acid secretion in stomach for NHE2, and reduced salt absorption
in kidney and low blood pressure for NHE3, respectively (5-7). The
functional diversity may suggest that NHE isoforms have fundamental
differences in the regulatory mechanism.
All NHE molecules comprise two major domains, amino-terminal
transmembrane (~500 amino acids) and carboxyl-terminal cytoplasmic domains (~300 amino acids). The latter has been suggested to function as a regulatory domain involving multiple accessory factors (1, 2). For
example, calmodulin (8, 9) and the NHE3 regulatory factor (10) have
been suggested to regulate NHE1 and NHE3 by interacting with their
cytoplasmic domains, respectively. Five years ago, Lin and Barber (11)
identified a novel Ca2+-binding protein CHP that interacts
with NHE1 and exerts regulatory influences on its activity. CHP is
ubiquitously expressed and homologous to the calcineurin B subunit
(11). This same protein has been identified independently as a factor
(called p22) required for the vesicular transport of proteins (12).
More recently, this protein has been reported to inhibit the
calcineurin phosphatase activity (13) or to associate with microtubules
(14). Lin and Barber (11) reported that overexpression of CHP with a
tag for detection in CCL39 fibroblastic cells inhibits the activation of NHE1 induced by serum or small G proteins and suggested that CHP may
be dissociated from the exchanger upon mitogenic stimulation (11).
However, the role of endogenous CHP in NHE1 has not yet been clarified.
Our preliminary experiment showed that CCL39 cells express a
significant amount of endogenous CHP that may be sufficient to form a
complex with endogenous NHE1 at a 1:1 molar ratio. Therefore, the
interpretation of the reported effect of CHP overexpression in NHE1
function (11) may not be straightforward. Clearly, to assign the
definitive role to CHP, it is necessary to compare the exchanger
functions in the presence or absence of bound CHP.
In this study, we have characterized the role of endogenous CHP in the
functions of NHE1 and other NHE isoforms not determined previously. We
show here that CHP is a key molecule that supports the activities of
multiple exchanger isoforms. This is the first study to show that
different exchangers require physical interaction with a common protein
to express physiological activity.
Cell Culture and cDNA Transfection--
The
exchanger-deficient PS120 cells (15) were cultured in Dulbecco's
modified Eagle's medium (Life Technologies, Inc.) supplemented with
7.5% fetal calf serum at 37 °C in an atmosphere of 95% air and 5%
CO2. Plasmids were transfected into PS120 cells (5 × 105 cells/10-cm dish) by means of the calcium-phosphate
co-precipitation technique. Cell populations that stably express NHE
variants were selected by means of the repetitive
H+-killing selection procedure (16). For stable expression
of CHP·GFP fusion protein variants, single clones expressing proteins were isolated using GFP fluorescence as a marker after selection with G418.
Construction of Expression Vectors for NHE and CHP
Variants--
Plasmids carrying cDNAs for human NHE1, rat NHE2,
and rat NHE3, and their mutants were all cloned into the mammalian
expression vector pECE. All these constructs were produced by means of
the polymerase chain reaction (PCR)-based strategy (16). The cDNA for CHP was obtained from human blood by reverse transcriptase-PCR based on the reported sequence (11). CHP·GFP fusion protein plasmids
were constructed using vector pEGFP-N1 (Clontech) by means of the
PCR-based method. Inserted DNA fragments containing mutations were
confirmed by sequencing plasmids with a DNA sequencer model 377 (ABI)
to ensure the fidelity of construction.
Expression and Purification of NHE and CHP Fusion
Proteins--
MBP or GST fusion protein plasmids were constructed
using vector pMAL-c (New England Biolabs) or pGEX-2TK (Amersham
Pharmacia Biotech) by means of the PCR-based method. Proteins were
purified according to the manufacturer's protocol. For production of
recombinant CHP, the full CHP sequence with 6 His residues was cloned
into bacterial expression vector pET11 (Stratagene). This plasmid was incorporated into Escherichia coli cells (BL21) either alone
or together with pBB131 carrying cDNA for yeast
N-myristoyltransferase (kindly provided by Dr. Gordon,
Washington University). After protein induction, the transformed
bacteria were sonicated and centrifuged. The pellet was suspended in
the lysis buffer containing 6 M guanidine hydrochloride.
CHP proteins were purified on a ProBondTM resin column (Invitrogen),
according to the manufacturer's protocol. Proteins were renatured by
dialysis overnight against 150 mM NaCl and 10 mM Hepes, Tris, pH 7.4. His-tagged NHE1 proteins were also purified from E. coli in a similar way.
Immunoprecipitation, Immunoblotting, and Surface Labeling with
NHS-Biotin--
Polyclonal anti-NHE1 was previously described (8).
Polyclonal anti-NHE3 or anti-CHP antibodies were produced by immunizing rabbits with the MBP·NHE3-(470-831) or GST·CHP-(1-195) fusion proteins. Immunoprecipitation and immunoblotting were performed essentially as described previously (8, 16). Briefly, cells were
solubilized with 1% Triton X-100 in 20 mM Hepes, Tris, pH 7.4, 150 mM NaCl, and protease inhibitors, and the cell
lysate was incubated with the antibodies indicated and protein
A-Sepharose. Precipitated materials were separated on 7.5 or 12%
polyacrylamide gels and transferred to Immobilon membranes (Millipore
Japan). After blocking, incubation with antibodies, and washing,
protein signals were visualized using enhanced chemiluminescence
(Amersham Pharmacia Biotech).
For surface labeling of NHE1 or NHE3, cells were incubated with 1 mM NHS-biotin for 30 min at room temperature, solubilized with the lysis buffer, and then incubated for 1 h with
streptavidin-agarose beads as described (17). After washing, the
proteins were released from the beads by boiling in 3% SDS buffer and
then subjected to immunoblot analysis with the antibodies indicated.
Far-Western Analysis and Pull-down Assay--
For far-Western
analysis, blots for various MBP·NHE1 fusion proteins were incubated
for 1 h with 100 µg/ml GST·CHP proteins, and then bound
GST·CHP fusion proteins were visualized by immunoblotting with
anti-GST antibody (Amersham Pharmacia Biotech). For the pull-down assay
for CHP proteins, non-myristoylated or myristoylated CHP (150 µg
each) was incubated for 30 min at 4 °C with 30 µl of amylose resin
pretreated with the MBP·NHE1-(503-815) fusion protein (200 µg) in
buffer (in mM: NaCl, 150; Hepes/Tris, pH 7.4, 10; and
CaCl2, 1; or EDTA, 1). After washing, the proteins were
eluted from resin with 50 mM maltose, electrophoresed, and
then visualized by Coomassie Brilliant Blue staining.
Immunostaining--
Cells were fixed and permeabilized with cold
methanol and incubated with anti-NHE1 antibody, followed by incubation
with rhodamine-labeled second antibody as described (18). For
observation of GFP fluorescence, cells stably expressing CHP·GFP
proteins were placed in serum-free Dulbecco's modified Eagle's medium
without phenol red. Images were taken under a fluorescent microscope
equipped with a CoolSNAP imaging system (RS Photometrics).
Crude Membrane Preparation from Xenopus Oocytes--
Xenopus oocytes were stripped and defolliculated
enzymatically with 1 mg/ml collagenase in a Ca2+-free ND96
solution (in mM: NaCl, 96; KCl, 2; MgCl2, 1;
and Hepes/NaOH, pH 7.5, 5) for 30 min at room temperature. For membrane
preparation, 100 oocytes injected with each protein were homogenized
for 60 s with Physcotron (Nition Co.) in a medium (in
mM: NaCl, 20; Mg-acetate, 10; phenylmethylsulfonyl
fluoride, 1; and Tris-HCl, pH 7.6, 20), and then subjected to
discontinuous sucrose gradient centrifugation. After centrifugation for
30 min at 15,000 g, the 20-50% interface was collected as the crude
membrane fraction. Membranes were solubilized with lysis buffer (in
mM: KCl, 100; MgCl2, 5; CaCl2, 1;
phenylmethylsulfonyl fluoride, 1; and Tris-HCl, pH 8.2, 100) containing
1% Triton X-100, 0.5% SDS, and 1% sodium deoxycholate and was then
subjected to immunoprecipitation.
Measurement of 22Na+ Uptake--
The
rates of ethylisopropyl-amiloride (EIPA)-sensitive
22Na+ uptake by PS120 cells expressing NHE
variants were measured using cells pHi-clamped at 5.6 by
the K+/nigericin method (19). For measurement of
22Na+ uptake by oocytes, cells were
preincubated for 1 h in NH4Cl medium (in
mM: NH4Cl, 80; CaCl2,1;
MgCl2, 1; and Hepes/Tris, pH 7.4, 10), washed twice with
choline-Cl medium (in mM: choline-Cl, 80; CaCl2, 1; MgCl2, 1; and Hepes/Tris, pH 7.4, 10)
and then incubated for 15 min in the same medium additionally
containing 1 mM 22NaCl (370 kBq/ml), 1 mM ouabain, and 0 or 0.1 mM EIPA. Oocytes were
washed four times with ice-cold, nonradioactive choline-Cl medium, and
then 22Na radioactivity was counted.
Identification and Characterization of the CHP-binding Domain in
NHEs--
CHP is a myristoylated protein with four
Ca2+-binding motifs (EF-hands) (Refs. 11, 12; Fig.
1a). CHP is expressed in
virtually all cells (11) including the exchanger-deficient PS120 cells used in our expression study (Fig. 1b).
Co-immunoprecipitation studies involving PS120 cells stably expressing
NHE1 revealed that CHP is tightly associated with NHE1 (Fig.
1b). To identify the CHP-binding domain in NHE1, we produced
a series of MBP fusion proteins containing various regions of the
carboxyl-terminal cytoplasmic domain (amino acids 503-815) of NHE1
(see Fig. 1a) and examined their interaction with GST·CHP
fusion proteins by far-Western analysis (Fig. 1, c and
d). The analysis revealed that amino acids 515-530 of NHE1
are required for CHP-binding (Fig. 1, c and d), which was further confirmed by co-immunoprecipitation experiments involving cells expressing several deletion mutants of NHE1 (Fig. 1e). The identified CHP-binding domain is different from the
previously reported one (amino acids 567-635; Ref. 11). The sequence
of the CHP-binding domain was found to be well conserved among
mammalian NHE isoforms NHE1-5 (see Fig.
2a), suggesting that all these
isoforms may interact with CHP. In fact, CHP bound to NHE3 (see Fig.
2d). Furthermore, we produced two chimeric exchangers,
NHE1(N2) and NHE1(N4), in which amino acids 503-600 of NHE1 were
replaced by the corresponding region of NHE2 or NHE4, respectively
(Fig. 1c). CHP also bound to these exchangers (Fig.
1f). Thus, NHE1-4 all contain CHP-binding sites in the
juxtamembrane region of the carboxyl-terminal cytoplasmic domain.
We have produced His-tagged recombinant CHP proteins with and without a
myristoyl moiety using an E. coli expression system. Both
CHP proteins bound to NHE1 in the absence or presence of Ca2+, as revealed in the pull-down assay with MBP·NHE1
fusion proteins (Fig. 1g). Thus, myristoylation and
Ca2+ binding in CHP are not required for the interaction of
CHP with NHE1. It is noteworthy that the Ca2+-bound form of
myristoyl-CHP migrated faster (Fig. 1g). Thus, the
Ca2+-induced conformational change in CHP seems to require
its myristoylation as in the case of other Ca2+-binding
myristoylated proteins (20).
Effects of Mutation within CHP-binding Domains of NHEs on Surface
Expression, CHP-binding, and the Exchange Activity--
The
CHP-binding domain of NHE1 is predicted to form a conserved
Intriguingly, these mutations 4Q, 4R, 6Q, and 2Q/4R dramatically
reduced the exchange activity, as measured as EIPA-sensitive 22Na+ uptake at an acidic intracellular pH of
5.6, to <10% of the level in the wild-type (Fig. 2g). A
similar reduction in activity also occurred for deletion mutants
Injection of CHP-binding Region of NHE1 Dramatically Reduces the
Activity of Xenopus Oocyte Exchanger--
The above data strongly
suggest that CHP binding is required for the exchange activity of
NHE1-3. To examine this further, we used a Xenopus oocytes
expression system. Oocytes are known to have an endogenous NHE, whose
amino acid sequence is similar to that of human NHE1 (overall homology,
78%; Refs. 24, 25). CHP-binding domains were well conserved in the two
species (see Fig. 2a). Oocytes also have an endogenous CHP
detectable on immunoblotting with anti-human CHP antibody (Fig.
3a). We produced His-tagged human NHE1 proteins (amino acids 503-600) containing the wild-type sequence or CHP binding-defective mutations (Fig. 3b).
Injection of the His-tagged wild-type protein into oocytes, but not
that of mutant proteins, resulted in the disappearance of
Xenopus NHE1 from the CHP immunoprecipitates (Fig.
3c, middle). Furthermore, anti-CHP antibody
co-precipitated the His-tagged wild-type protein with endogenous CHP,
but not mutant proteins (Fig. 3c, bottom). These
data indicate that the injected human CHP-binding protein depletes
bound CHP from Xenopus NHE. Importantly, we found that injection of the His-tagged wild-type protein abolished the endogenous exchange activity in oocytes almost completely (Fig. 3d). A
relatively long time ( Subcellular Localization of GFP-tagged CHP Proteins--
CHP
fusion protein conjugated with green fluorescent protein (CHP·GFP)
was expressed uniformly in the cytosol of PS120 cells (Fig.
4c). This fusion protein
became partly localized in the cell surface when exogenous NHE1 was
co-expressed (Fig. 4d). However, CHP·GFP was not localized
in the surface membrane when NHE1 mutant 4Q was expressed in the
membrane (Fig. 4, b and e). Therefore, NHE1 seems
to be the principal target for CHP in the membrane. We stably
overexpressed two GFP-conjugated CHP mutant proteins (CHP2A·GFP and
CHPEF3·GFP) that lack the ability to be myristoylated or to bind
Ca2+ (12). Consistent with the in vitro binding
data (see Fig. 1g), these proteins were partly localized in
the surface membrane together with the NHE1 protein (Fig. 4,
f and g), suggesting that they might have
replaced endogenous CHP. Because expression of these proteins did not
inhibit 22Na+ uptake activity (Fig.
4l), it appears that neither myristoylation nor
Ca2+ binding is essential for exchange activity. CHP·GFP
was also partly localized in the surface membrane when it was
co-expressed with NHE2 or NHE3, although a significant amount of
CHP·GFP still remained in the cytosol or an intracellular compartment
(Fig. 4, h and j). However, such surface
expression was abolished by mutations (NHE2-3R and NHE3-4R) introduced
into these NHE isoforms (Fig. 4, i and k),
indicating that CHP binding was lost in these mutants.
It has generally been considered that a single polypeptide for each NHE
isoform is sufficient for normal exchange activity. However, the
present data provide evidence that NHE1 requires an extra cofactor
protein, CHP, for it to express physiological activity. Our data also
suggest the same role of CHP for NHE2-4. A potential CHP-binding motif
also exists in NHE5 (Ref. 3; see Fig. 2a), but not in
mitochondrial NHE6 (4). Based on these findings, we propose that CHP
functions as an integral cofactor common to plasma membrane-type
Na+/H+ exchangers.
Na+/H+ exchange presumably occurs through the
transmembrane domain of an exchanger. Cells expressing a NHE1 mutant
(
Subdomain I contains the CHP-binding region (amino acids 515-530) as
well as two potential phosphatidylinositol 4,5-bisphosphate (PIP2) binding motifs (amino acids 509-516 and 552-560
for the human NHE1 sequence) identified by Aharonovitz et
al. (26) who provided evidence that a reduction of cell
PIP2 causes marked inhibition of the exchange activity of
NHE1. Their study, however, provided little information regarding
whether or not the effect of PIP2 is mediated by the above
binding motifs in situ. In this study, we found that the
injected His-tagged wild-type NHE1 protein (amino acids 503-600)
almost completely abolished the exchange activity in oocytes, whereas
mutant protein 4R lacking the ability to bind CHP did not (Fig.
3d). This finding seems to argue against the view that the
function of CHP is directly related to PIP2, because the
injected wild-type and mutant 4R proteins would not be much different
in their PIP2 binding (see Fig. 2a).
We observed that the surface localization of CHP·GFP in NHE1
transfectants was not affected significantly by ionomycin, ATP depletion, phorbol ester, serum, or other growth factors (not shown),
which is inconsistent with the previous proposal (11) that CHP may be
dissociated from NHE1 upon growth factor activation. Whether or not the
bound CHP is involved in acute regulation of the exchanger is not clear
at present. Although CHP interacts with multiple NHE isoforms, it is
still possible that the activity of the exchanger is regulated through
post-translational modifications of the associated CHP in response to
extracellular stimuli. Indeed, myristoylated CHP alters its
conformation in a Ca2+-dependent manner (see
Fig. 1g), similar to some other Ca2+-myristoyl
switch proteins (20). It is possible that such a conformational change
of bound CHP alters the structure of subdomain I and regulates the
transport activity.
In conclusion, the present study revealed that multiple exchangers
require physical interaction with a common protein, CHP for exchange
activity. This function does not appear to require Ca2+
binding and myristoylation in CHP. To our knowledge, this is the first
finding to show that a Ca2+-binding protein supports the
activity as an integral component of the secondary active transporter.
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INTRODUCTION
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REFERENCES
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REFERENCES
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Fig. 1.
Interaction of NHE with CHP.
a, models of the structures of NHE1 and CHP. NHE1 comprises
two major domains, amino-terminal transmembrane (amino acids 1-500)
and carboxyl-terminal cytoplasmic domains (amino acids 500-815). In
this figure, the secondary structure model of NHE1 is shown as reported
previously (17). b, co-immunoprecipitation of CHP and NHE1
proteins performed with specific antibodies. Lysates of
exchanger-deficient PS120 cells or NHE1 transfectants were subjected to
immunoprecipitation (IP) followed by immunoblot analysis
(IB) with the antibodies indicated in the figure as
described under "Materials and Methods." c, constructs
for MBP fusion proteins containing various regions of the NHE1
cytoplasmic domain and their CHP binding ability determined as in
b by far-Western assaying with GST·CHP fusion protein
(+++, strong; +, weak; , no binding). The last two constructs
represent chimeric exchangers in which amino acids 503-815 of NHE1
were replaced by corresponding regions of NHE2 or NHE4. d,
far-Western analysis was carried out as described under "Materials
and Methods." The left panel shows protein inputs (10 µg). e and f, cells expressing various NHE
variants were subjected to co-immunoprecipitation experiments with the
antibodies indicated in the figure. g, pull-down assay for
non-myristoylated or myristoylated CHP. Proteins were eluted through
amylose resin immobilized with MBP·NHE1 and visualized by Coomassie
Brilliant Blue staining as described under "Materials and Methods."
Control, no pretreatment with the MBP·NHE1 protein.
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Fig. 2.
Properties of NHE mutants. a,
amino acid sequence alignment of the CHP-binding domain of NHEs and the
mutant constructs. The CHP binding domains of mammalian NHE1-5,
Xenopus NHE, and the mutant constructs were aligned.
Hydrophobic amino acids are underlined. The helical wheel
diagram of the CHP-binding domain is shown for amino acids 518-535 of
NHE1. b, expression level of various NHE variants. Lysates
(20 µg) of cells expressing the mutant exchangers shown in
a were analyzed by immunoblotting with anti-NHE1
(left) or anti-NHE3 (right) antibody.
c, surface labeling of the NHE1 or NHE3 protein was carried
out using NHS-biotin as described under "Materials and Methods."
d, lysates of cells expressing various NHE variants were
subjected to co-immunoprecipitation experiments with anti-CHP antibody
followed by immunoblotting with ant-NHE1 or anti-NHE3 antibody.
e and f, the extents of surface labeling of NHE1
variant proteins with NHS-biotin as in c and the amounts of
them recovered on immunoprecipitation with anti-CHP antibody as in
d, normalized as to the expression level of the high
molecular weight NHE1 protein. The data represent means ± S.D.
(n = 3), relative to the values for wild-type NHE1
(WT). g, the rates of EIPA-sensitive
22Na+ uptake by NHE1 variants were measured
using cells pHi-clamped at 5.6. Inset, data for
NHE2 and NHE3 and their mutants. Data are means ± S.D.
(n = 3).
-helix
like the calcineurin B-binding domain within the calcineurin A subunit
(Refs. 21, 22; Fig. 2a). A helical wheel diagram for amino
acids 518-535 of NHE1 revealed a cluster of hydrophobic residues (Fig.
2a). Based on the previous finding that a hydrophobic interaction is important for the interaction between two calcineurin subunits (21, 22), we produced several mutant plasmids carrying amino
acid substitutions of hydrophobic to hydrophilic residues (Gln or Arg)
as well as other substitutions (to Ala) (see Fig. 2a for the
naming of mutants). When transfected into cells, these NHE1 variants
were expressed in high and low molecular weight forms that correspond
to glycosylated mature and immature NHE1 proteins, respectively (Ref.
23; Fig. 2b, not shown for some mutants). The mature
proteins from all these constructs were labeled efficiently with
membrane-impermeable NHS-biotin applied from the outside (Fig.
2c), indicating the surface expression of these proteins.
NHE1 mutants 4Q, 4R, 6Q, and 2Q/4R were not co-immunoprecipitated with
CHP (Fig. 2d). These findings suggest that CHP is not
required for the surface expression of NHE1.
510-530 and
515-530 (not shown). As summarized in Fig. 2,
f and g, the exchange activity changed in
parallel with the CHP-binding ability for various NHE1 mutants, despite
the fact that nearly the same amounts of proteins were expressed in the
surface (Fig. 2e). These data suggest that CHP binding is
required for optimal activity of NHE1. We could not further analyze
regulatory or kinetic properties of CHP binding-defective mutant
exchangers (4Q, 4R, 6Q, and 2Q/4R) because of their low activity
(Vmax). However, cells expressing a mutant
(518Q/522Q) with low affinity for CHP but with modest activity show
similar properties to the wild-type NHE1, i.e. an
intracellular pH dependence of 22Na+ uptake
with a pK of ~6.6 and cytoplasmic alkalinization by 0.1 ~ 0.2 pH unit in response to
-thrombin, platelet-derived growth factor,
phorbol ester, and hyperosmotic stress (data not shown). These
properties probably reflect those of the CHP-bound but not CHP-unbound
mutant exchanger because only the former exhibits measurable high
activity as shown in oocyte experiments described below. Similar to
NHE1 mutants, NHE2-3R or NHE3-4R mutants (see Fig. 2a) did
not interact with CHP (see Figs. 2d and 4, h-k). In addition, these CHP binding-defective mutants of NHE2 and NHE3 exhibited markedly reduced exchange activity (Fig. 2g,
inset).
3 h) was required for inhibition of the
exchange activity after injection of proteins (Fig. 3e),
suggesting that CHP is slowly released from Xenopus NHE. On
the other hand, the exchange activity of human NHE1 expressed in
oocytes was significantly increased when human CHP was co-expressed
(Fig. 3f). These data also strongly suggest that CHP is
required for high exchange activity.
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Fig. 3.
Analysis of NHE function in
Xenopus oocytes. a, immunoblot for
oocyte proteins (50 µg) with anti-CHP antibody. b,
purified recombinant His-tagged proteins (10 µg) containing the
wild-type sequence (amino acids 503-600 of NHE1) or CHP
binding-defective mutations were visualized by Coomassie Brilliant Blue
staining. c, His-tagged proteins (20 ng/50 nl) were injected
into oocytes, from which crude membranes were prepared 8 h later.
Using solubilized crude membranes, co-immunoprecipitation experiments
were performed with the antibodies indicated (anti-His antibody from
Roche Molecular Biochemicals. d, eight hours after the
injection of His-tagged proteins, 22Na+ uptake
into oocytes containing endogenous NHE was measured in the presence or
absence of 0.1 mM EIPA as described under "Materials and
Methods." The 22Na+ uptake was 40-50
pmol/oocyte/15 min in the presence of EIPA. e, time course
of EIPA-sensitive 22Na+ uptake after injection
of the His-tagged wild-type protein. f, three days after
injection of cRNAs (50 ng) into oocytes, EIPA-sensitive
22Na+ uptake was measured. Data are means ± S.D. of the values for at least 10 oocytes.
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Fig. 4.
Subcellular localization of CHP·GFP
proteins and exchange activity of various stable transfectants.
PS120 cells expressing various NHE variants and/or CHP·GFP proteins
were seeded onto 60-mm dishes. a and b,
immunostaining with anti-NHE1 antibody. c-k,
fluorescent images of cells stably expressing CHP·GFP proteins.
Gly-2 of CHP was replaced by Ala in CHP2A·GFP, whereas
Asp-133/Glu-134 in the third EF-hand motif were replaced by Ala/Met,
respectively, in CHPEF3·GFP. Scale bar, 20 µm.
l, EIPA-sensitive 22Na+ uptake
activity was measured using cells stably expressing NHE1 and various
CHP·GFP fusion proteins. Data are means ± S.D.
(n = 3).
515) with the complete carboxyl-terminal cytoplasmic domain (amino
acids 516-815) deleted still retain low exchange activity that can be detected by expressing a high copy number of a mutant molecule (16).
This is consistent with the present finding that mutants lacking the
CHP-binding site are able to exhibit low exchange activity (5-10% of
the wild-type level). We previously showed that the exchange activity
at physiological pHi is markedly decreased by deletion of
different regions in the amino terminus (subdomain I, amino acids
515-595) of the NHE1 cytoplasmic domain (19). These data suggest that
subdomain I is essential for normal exchange activity. This domain with
bound CHP would therefore function as a key structure that interacts
with the transmembrane domain (see Fig. 1a), thereby
permitting the ion translocation pathway to maintain its
physiologically relevant active conformation. More detailed structural
information including the crystal structure of the CHP·NHE complex is
necessary to reveal how CHP is involved in this important task of
subdomain I.
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ACKNOWLEDGEMENT |
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We thank Dr. T. Y. Nakamura for technical assistance in the Xenopus oocyte experiments.
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FOOTNOTES |
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* This work was supported by Grants-in-aid for Scientific Research, 09680642 and 10470013, from the Ministry of Education, Science, and Culture of Japan, and the Uehara Memorial Foundation.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. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 81-6-6833-5012;
Fax: 81-6-6872-7485; E-mail: wak@ri.ncvc.go.jp.
Published, JBC Papers in Press, February 28, 2001, DOI 10.1074/jbc.M100296200
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ABBREVIATIONS |
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The abbreviations used are: NHE, Na+/H+ exchanger; CHP, calcineurin B homologous protein; GFP, green fluorescent protein; MBP, maltose-binding protein; GST, glutathione S-transferase; PCR, polymerase chain reaction; NHS-biotin, N-hydroxysuccinimido-biotin; EIPA, ethylisopropyl-amiloride.
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