Proline-rich Motifs of the Na+/H+
Exchanger 2 Isoform
BINDING OF Src HOMOLOGY DOMAIN 3 AND ROLE IN APICAL TARGETING IN
EPITHELIA*
Chung-Wai
Chow
§,
Michael
Woodside
,
Nicolas
Demaurex
,
Frank H.
Yu¶,
Pamela
Plant
,
Daniela
Rotin
,
Sergio
Grinstein
, and
John
Orlowski¶**
From the
Cell Biology Programme, Hospital for Sick
Children and the § Division of Respiratory Medicine,
Department of Medicine, University of Toronto, Ontario M5G 1X8 and
the ¶ Department of Physiology, McGill University, Montreal,
Quebec H3G 1Y6, Canada
 |
ABSTRACT |
The NHE2 isoform of the
Na+/H+ exchanger (NHE) displays two
proline-rich sequences in its C-terminal region that resemble SH3 (Src
homology 3)-binding domains. We investigated whether these regions
(743PPSVTPAP750, termed Pro-1, and
786VPPKPPP792, termed Pro-2) can bind to SH3
domains and whether they are essential for NHE2 function and targeting.
A fusion protein containing the Pro-1 region showed promiscuous binding
to SH3 domains of several proteins in vitro, whereas a
Pro-2 fusion bound preferentially to domains derived from kinases. In
contrast, cytoplasmic regions of NHE1, NHE3, or NHE4 failed to
interact. When expressed in antiporter-deficient cells, truncated NHE2
lacking both Pro-rich regions catalyzed Na+/H+
exchange, retained sensitivity to intracellular ATP, and was activated
by hyperosmolarity, resembling full-length NHE2. The role of the
Pro-rich regions in subcellular targeting was examined by transfection
of epitope-tagged forms of NHE2 in porcine renal epithelial
LLC-PK1 cells. Both full-length and Pro-2-truncated NHE2
localized almost exclusively to the apical membrane. By contrast, a
mutant devoid of both Pro-1 and Pro-2 was preferentially sorted to the
basolateral surface but also accumulated intracellularly. These
observations indicate that the region encompassing Pro-1 is essential
for appropriate subcellular targeting of NHE2.
 |
INTRODUCTION |
The Na+/H+ exchangers
(NHEs)1 are a family of
proteins found in virtually all mammalian cells, where they catalyze
the electroneutral exchange of intracellular H+ for
external Na+ with a 1:1 stoichiometry (see Refs. 1 and 2
for review). In most cells, NHE activity is important for intracellular
pH (pHi) homeostasis and also for maintenance of normal cellular volume (3). In addition, in epithelia of the kidney, gastrointestinal tract, and other organs, NHE plays a central role in
the absorption of NaCl, bicarbonate, and water. Six distinct NHE
isoforms have been identified to date in mammalian cells (1). Although
the primary sequence homology between the isoforms is limited
(20-60%), they all share the same predicted membrane topology, consisting of 12 membrane-spanning segments at the N terminus and a
hydrophilic C-terminal tail thought to extend into the cytoplasm (Ref.
1, but see Ref. 4 for alternative view). Functional characterization of
deletion mutants of NHE1 and NHE3 has localized the site of
Na+/H+ exchange to the N-terminal
(transmembrane) region of the protein (1). The C-terminal region is
believed to be responsible for modulation of transport by such diverse
agents as growth promoters, hormones, and changes in medium osmolarity
(1). The cytosolic domain is also thought to encompass the site that
confers ATP dependence to Na+/H+ exchange, a
hallmark of NHE that is common to all the isoforms studied to date (1,
2).
The pattern of expression of individual NHE isoforms varies among
tissues. NHE1 and NHE6 are expressed in virtually all cells, whereas
other isoforms have a more restricted tissue distribution (1). NHE2 is
preferentially expressed in the gastrointestinal tract and, to a lesser
extent, in the kidney, uterus, and brain (5, 6). Despite its discovery
several years ago, comparatively little is known about the physiology
of NHE2. Its selective expression pattern initially suggested a role
for this isoform in fluid and electrolyte balance, similar to the
function fulfilled by NHE3 in epithelial cells. However, creation of a
null mutation of NHE2 in mice caused no significant perturbations of
organismal acid-base or salt homeostasis (7). Instead, the only
apparent defect was a significant loss of net acid secretion in the
stomach due to a severe reduction in the viability of parietal cells of
the gastric mucosa. Although the underlying mechanism is unclear, it
was proposed that NHE2 is part of the basolateral transport system that
maintains parietal cell volume during high acid secretion at its apical
surface and that disruption of NHE2 function causes a chronic state of
volume depletion, leading to cellular necrosis (7).
Whereas an attractive hypothesis, the subcellular distribution of NHE2
in stomach has yet to be determined and, indeed, remains a
controversial issue in other tissues examined. On one hand, NHE2
activity was reported to be restricted to the basolateral membrane of
an inner medullary collecting duct cell line, mIMCD-3 (8). In contrast,
others detected NHE2 activity predominantly on the apical membranes of
ileal tissue (9) and in a cortical collecting duct cell line (10).
Similar observations were made when NHE2 was transiently expressed in a
colonic carcinoma cell line (11), and apical localization was also
documented immunologically in the medullary thick ascending limb (12).
Thus, NHE2 is mainly sorted to the apical surface of most polarized
epithelial cells examined, as reported for NHE3. By contrast, the
functional properties of NHE2 differ considerably from NHE3 and more
closely resemble those of NHE1 when assessed in transfected mammalian
cells: it is activated by agonists of the protein kinase A and C
pathways as well as by hyperosmotic-induced cell shrinkage, and its
pHi sensitivity is moderately reduced by cellular depletion of
ATP (3, 13-15).
The structural determinants that confer this unique behavior have not
been identified. Perusal of the primary structure of NHE2 revealed that
the C-terminal region, which is most divergent between isoforms,
contains two proline-rich motifs: residues 743-750 (PPSVTPAP), which
will be called hereafter Pro-1, and residues 786-792 (VPPKPPP),
designated Pro-2. The latter conforms to the consensus sequence of
proteins capable of binding Src homology 3 (SH3) domains,
PpXP, where
is a hydrophobic residue, X is any amino acid, and p tends to be (but is not always) Pro (16-18). Pro-1 approximates but does not match perfectly the SH3 consensus structure. These proline-rich regions are unique to NHE2 and may be
important in defining the distinctive behavior of this isoform.
SH3 domains are sequences of 50-75 amino acids found in diverse
proteins that include cytoskeletal components, such as spectrin (19),
adaptor and signaling molecules, such as Grb2 (20, 21) and Ras-GAP
(22), and protein and lipid kinases, including Src, Abl, and the p85
subunit of phosphatidylinositol 3'-kinase (23, 24). Coupling of
proteins via their SH3 domains has been implicated in a variety of
functions, including regulation of cell growth and proliferation (25,
26), endocytosis (27, 28), and activation of the respiratory burst (29,
30). The importance of SH3 domains in other systems prompted us to
analyze whether the Pro-rich motifs of NHE2 are capable of interacting
with SH3-containing proteins and to assess the functional role of this
interaction. To this end, we studied the ability of fusion proteins
encoding the Pro-1 and Pro-2 regions of NHE2 to bind SH3 domains
in vitro. In addition, we used transfection of
epitope-tagged constructs to compare the functional properties and
subcellular distribution of the full-length NHE2 with those of mutants
lacking either Pro-1 or Pro-2.
 |
EXPERIMENTAL PROCEDURES |
Materials and Media--
All polymerase chain reaction (PCR)
reagents and Escherichia coli DH5-
were purchased from
Life Technologies, Inc. pGEX-2T and E. coli HB101 were from
Amersham Pharmacia Biotech. Isopropylthio-
-D-galactoside was purchased from Calbiochem. Luria broth (LB) was obtained from the
Ontario Cancer Institute (Toronto, Ontario, Canada).
Fluorescein-labeled lectins, leupeptin, pepstatin, aprotinin,
phenylmethylsulfonyl fluoride, EDTA, antimycin A, reduced glutathione,
and glutathione-agarose beads were purchased from Sigma. The NHS-LC
biotinylation reagent was purchased from Pierce, and the enhanced
chemiluminescence (ECL) detection kit was from Amersham Pharmacia
Biotech. Nigericin and 2',7'bis-(2-carboxyethyl)-5(6)
carboxyfluorescein (BCECF) acetoxymethylester were purchased from
Molecular Probes, Inc.
Monoclonal antibodies to a peptide derived from the influenza virus
hemagglutinin (HA) were purchased from BAbCo. Goat anti-mouse Cy3-conjugated IgG and goat peroxidase-coupled anti-mouse secondary antibody were purchased from Jackson Immunoresearch Laboratories. Horseradish peroxidase coupled to avidin was purchased from Cappel.
Phosphate-buffered saline (PBS) contained 140 mM NaCl, 10 mM KCl, 8 mM sodium phosphate, 2 mM
potassium phosphate, pH 7.4. The sodium chloride solution contained 117 mM NaCl, 1.66 mM MgSO4, 1.36 mM CaCl2, 5.36 KCl mM, 25 mM Na-HEPES, 5.55 mM glucose, pH 7.4. Sodium-free solutions were made with equimolar substitution of
N-methyl-D-glucammonium solutions of the
appropriate salts. Unless otherwise indicated, all solutions were
nominally bicarbonate-free and were adjusted to 290 ± 10 mosM with the major salt. Hypertonic NaCl solution was
prepared by addition of sufficient NaCl to raise the osmolarity to
600 ± 10 mosM.
Preparation of GST Fusion Proteins--
GST fusion proteins of
the Pro-rich regions of NHE2 were prepared by PCR amplification of the
appropriate regions of rat NHE2 cDNA (31). The PCR product
encompassing the more N-terminal Pro-rich region, which contains amino
acids 743PPSVTPAP750 (Pro-1) plus 19 and 16 flanking residues at the 5'- and 3'-ends, respectively, was amplified
using the primers 5'-ccggatcctcaccagcctac-3' and
5'-ggaattcgggctgcctcag-3'. The primers used to amplify the more
C-terminal proline-rich region containing amino acids
786VPPKPPP792 (Pro-2) plus 12 and 21 flanking
residues at the 5'- and 3'-ends, respectively, were
5'-ccggatccggccggggcagg-3' and 5'-ggaattcaccacaagtctgc-3'. The PCR
products were purified following electrophoresis in 1.8% agarose
(Nu-Sieve Agarose, Biocan) using the Qiaex DNA extraction kit,
according to the manufacturer's directions. The Pro-1 and Pro-2
sequences were subsequently subcloned into the BamHI and EcoRI sites of pGEX-2T (Amersham Pharmacia Biotech). HB101
E. coli transformed with the resulting plasmid were grown at
37 °C in Luria broth supplemented with 50 µg/ml ampicillin to
log-phase, and then expression of the fusion protein was induced with 1 mM isopropyl-1-thio-
-D-galactopyranoside for
5 h at 37 °C. Bacteria were lysed in the presence of protease
inhibitors (10 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml
leupeptin, 1 µg/ml pepstatin, 1 µg/ml aprotinin, and 5 mM EDTA), and the fusion proteins were purified from the lysate using glutathione-agarose beads. GST fusion protein encompassing amino acids 560-690 of NHE3 and 667-717 of NHE4 were prepared similarly. A GST fusion protein containing the last 178 residues of the
cytosolic tail of rabbit NHE-1 was the kind gift of Dr. L. Fliegel
(University of Alberta, Edmonton, Alberta, Canada).
Fusion proteins of the SH3 domain of c-Abl, p85, Ras-GAP, N-Src, Src,
and
-spectrin were obtained and generated as described previously
(32). GST-SH3 domain fusions were subsequently biotinylated using
NHS-LC biotin (Pierce), as described (19). In Vitro Far Western Binding Assays
Fusion proteins (8 µg) of the indicated regions of the exchangers were separated by SDS-PAGE (12% acrylamide), transferred to nitrocellulose, and incubated overnight in blocking buffer (0.25% gelatin, 10% ethanolamine, 0.1 M Tris, pH
9.0) supplemented with 5% powdered milk. The blots were next incubated
with 2 µg of the appropriate biotinylated SH3 fusion protein in 10 ml
of blocking buffer for 2 h. The blots were then washed and
incubated for 2 h at room temperature with streptavidin-peroxidase
(12.5 µg/ml) in a buffer containing 0.25% gelatin, 0.05% Nonidet
P-40, 0.15 M NaCl, 5 mM EDTA, 50 mM
Tris, pH 7.5, followed by washing and detection using ECL.
cDNA Constructs--
The rat NHE2 cDNA was engineered to
contain a series of unique restriction endonuclease sites that did not
alter the primary structure, but created convenient DNA cassettes for
mutagenesis. The modified NHE2 cDNA was inserted into the pCMV
mammalian expression vector (plasmid renamed pNHE2) as described
previously (6, 33). To allow for immunological detection of the
protein, the influenza virus HA epitope YPYDVPDYAS, preceded by a
single G amino acid linker (inserted to create peptide flexibility),
was inserted at the very C terminus of NHE2 (after amino acid 813) using PCR amplification of a C-terminal cDNA fragment. Similarly, the NHE2
777HA and NHE2
731HA deletion
mutants were engineered by inserting the HA epitope plus a stop codon
immediately following positions 777 and 731 of the wild type
transporter. The PCR fragments were sequenced prior to substitution
into NHE2 to confirm the presence of the mutations and to ensure that
other random mutations were not introduced.
Cells--
AP1 is a cell line devoid of endogenous
Na+/H+ exchange activity that was derived from
wild type Chinese hamster ovary (CHO) cells by the
"H+-suicide" technique (34). These cells and the
transfectants derived thereof were grown in
-minimal essential
medium (Ontario Cancer Institute, Toronto, Ontario, Canada). HEK-T is a
subclone of the transformed human embryonic kidney (HEK) cell line
containing the large T antigen. LLC-PK1 are epithelial
cells derived from porcine kidney proximal tubule. HEK-T and
LLC-PK1 cells were grown in Dulbecco's minimal essential
medium (Ontario Cancer Institute, Toronto, ON). Both
-minimal
essential medium and Dulbecco's minimal essential medium contained 25 mM NaHCO3 and were supplemented with 10% fetal
calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (Life
Technologies, Inc.). Cells were incubated in a humidified environment
containing 95% air and 5% CO2 at 37 °C. Cultures were
re-established from frozen stocks regularly, and cells from passages
3-20 were used for the experiments.
Where indicated, intracellular ATP was depleted by incubating the cells
for 10 min in glucose-free medium with 5 mM
2-deoxy-D-glucose and 1 µg/ml antimycin A, to inhibit
both glycolysis and oxidative phosphorylation. This protocol has
previously been shown to deplete >90% of the ATP in CHO cells within
10 min (35). Subsequent fluorescence measurements were performed in
glucose-free medium containing 5.5 mM
2-deoxy-D-glucose.
Transfection and Selection--
AP-1, LLC-PK1, and
HEK-T cells were transfected with plasmids containing the
epitope-tagged wild type or truncated NHE2 constructs by the calcium
phosphate-DNA co-precipitation technique of Chen and Okayama (36). For
selection of stable lines, the AP-1 cells were selected for survival by
repeated acute acid loads (5-6 times over a 2-week period), in order
to discriminate between Na+/H+
exchanger-positive and negative-transfectants, starting 48 h after
transfection (33, 34). For stable transfection of LLC-PK1 cells, which express endogenous NHE, we subcloned NHE2HA,
NHE2
777HA, and NHE2
731HA into the pBK
vector (Stratagene), which contains the neomycin resistance gene.
Stably expressing cells were selected by incubation with 500 µg/ml
G418 and then screened by immunofluorescence for expression of the wild
type or mutant NHEs.
Immunoblotting--
For immunoblot analysis, the cells were
grown to confluence on 10-cm2 plastic dishes. The cells
were washed three times in PBS, lysed in a hypotonic buffer (10 mM Hepes, 18 mM potassium acetate, 1 mM EDTA, pH 7.2, 50 mosM) and solubilized at
4 °C in radioimmune precipitation buffer (150 mM NaCl,
20 mM Tris HCl, 0.1% SDS, 0.5% deoxycholate, 1% Triton
X-100, pH 8.0) containing 1 mM iodoacetamide, 1 mM pepstatin, and 1 mM phenylmethylsulfonyl
fluoride. The soluble fraction was subjected to SDS-PAGE (10%
acrylamide) and transferred to nitrocellulose. The blot was incubated
overnight in blocking buffer (see above) and then incubated with
anti-HA antibody (1:5000) for 1 h in 0.25% gelatin, 0.05%
Nonidet P-40, 0.15 M NaCl, 5 mM EDTA, 50 mM Tris, pH 7.5, supplemented with 5% milk, prior to labeling with peroxidase-coupled anti-mouse secondary antibody (1:5000). Chemiluminescence was then detected using the Amersham Pharmacia Biotech ECL detection kit.
Immunofluorescence--
Cells were grown to subconfluence on
sterile 18 mm glass coverslips (Thomas Scientific, Swedesboro, NJ),
washed with PBS, and fixed for 20 min with 4% paraformaldehyde, and
the excess formaldehyde was quenched with 100 mM glycine
for 15 min. Where indicated, the fixed cells were stained with a
mixture of fluorescein isothiocyanate-labeled lectins (peanut, orange,
wheat germ, and pea, 2 µg/ml each) for 45 min at 4 °C. The cells
were then permeabilized with 0.1% Triton X-100 supplemented with 5%
bovine serum albumin and incubated with anti-HA antibody (1:1000) at
room temperature for 45 min. The cells were washed again with PBS and
incubated with Cy3-conjugated anti-mouse antibody (1:5000) for 45 min.
The coverslips were mounted using Dako mounting reagent and visualized using a Leica fluorescence microscope. Images were obtained using the
Winview program and processed using Adobe Photoshop.
Measurements of Cytosolic pH--
The pHi of small
groups of cells was determined by microphotometry of the fluorescence
emission of BCECF using dual wavelength excitation. Cells grown to
confluence on 25-mm glass coverslips (Thomas Scientific, Swedesboro,
NJ) were loaded with BCECF by incubation with 2 µg/ml of the
precursor acetoxy-methylester form for 10 min at 37 °C. The
coverslips were then mounted in a Leiden coverslip dish (Medical System
Corp., Greenvale, NY) and placed into a thermostatted holding chamber
heated to 37 °C (open perfusion microincubator, Medical Systems
Corp., Greenvale, NY) attached to the stage of a Nikon Diaphot TMD
inverted microscope (Nikon Canada, Toronto, Ontario, Canada). Cells
were visualized using a Nikon Fluor × 40/1.3 numerical aperture
oil immersion objective and a Hoffman modulation contrast video system
with an angled condenser (Modulation Optics) through a CCD-72 video camera and control unit (Dage-MTI, Michigan City, IN) connected to a
Panasonic monitor. Clusters of 6-12 cells from the confluent culture
were selected for analysis with an adjustable diaphragm.
The chamber was continuously perfused at ~ 0.5 ml/min to allow
for complete exchange of the bath solution once every minute using a
gravity-driven system and a Leiden aspirator. When rapid solution
changes were required, three aliquots of 1 ml of the new medium were
quickly pipetted (<15 s) into the chamber and perfusion was continued
using the new medium (37). Fluorescence measurements were made using an
M Series dual wavelength illumination system from Photon Technologies,
Inc. (South Brunswick, NJ) in a dual excitation/single emission
configuration. Excitation light provided by a Xenon lamp was
alternately selected using 495 ± 10 and 445 ± 10 nm filters
(Omega Optical, Brattleboro, VT) at a rate of 50 Hz and then reflected
to the cells by a 510 nm dichroic mirror. Emitted light was first
selected by a 520 nm long-pass filter and then separated from the red
light used for Hoffman imaging by a 550 nm dichroic mirror and directed
to the photometer through a 530 ± 30 nm band-pass filter. This
optical system allowed for continuous visualization of cells without
interfering with fluorescence measurements. Photometric data was
acquired at 10 Hz using a 12 bit A/D board (Labmaster, National
Instruments, Austin, TX) interfaced to a Dell 486 computer and analyzed
with the Felix software (Photon Technologies Inc., South Brunswick, NJ). Calibration of the fluorescence intensity to pHi was
performed in the presence of 5 mM nigericin in high
potassium medium (140 mM KCl, 20 mM HEPES, 1 mM MgCl2, and 5 mM glucose) as
detailed previously (38). Each coverslip was calibrated at the end of
the experiment using at least three pH values. Quantification of
cell-associated fluorescence was performed using the Felix software
package (Photon Technologies, Inc., South Brunswick, NJ). The rate of
pHi change was derived by linear regression of the pHi
versus time curve over 4 s intervals using the Origin
software (MicroCal Software Inc., Northampton, MA).
 |
RESULTS |
Interaction of Proline-rich Regions with SH3 Domains in
Vitro--
To assess whether SH3 domains are capable of binding
specifically to the Pro-rich motifs of the cytosolic domain of NHE2 we initially tested the ability of the Src-SH3 domain to bind to this
region in an in vitro overlay assay. A GST fusion protein encompassing residues 724-766 and including the Pro-1 region
(743PPSVTPAP750) of NHE2 was subjected to
SDS-PAGE, transferred to nitrocellulose, and overlaid with the
biotinylated SH3 domain of Src, and binding was detected with
streptavidin-horseradish peroxidase and ECL. In order to assess the
specificity of the interaction, GST alone and GST fusion proteins
prepared from the cytoplasmic segments of other NHE isoforms, which
have no Pro-rich domains, were analyzed simultaneously. As illustrated
in Fig. 1, the Src SH3 domain bound to
the GST-NHE2-Pro-1 fusion protein but failed to interact with either
GST alone or with the cytoplasmic regions of NHE1, NHE3, or NHE4. This
observation suggests that SH3 domains bind to the cytosolic segment of
the exchangers only when Pro-rich sequences are present.

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Fig. 1.
Interaction between NHE fusion proteins and
the SH3 domain of src. GST fusion proteins of
regions of the C-terminal domain of NHE1, NHE2, NHE3, and NHE4, as well
as unconjugated GST (8 µg each), were separated by SDS-PAGE (12%
acrylamide) and blotted onto nitrocellulose. The blot was initially
stained with Ponceau S to reveal the proteins (top panel)
and then overlaid with biotinylated GST-Src-SH3 (bottom
panel). After extensive washing, bound GST-Src-SH3 was
detected using streptavidin-peroxidase and ECL. Double bands in some of
the Ponceau-stained lanes reflect proteolysis of the fusion proteins.
Shown is a representative of four similar experiments.
|
|
We next compared the ability of the Pro-1 and Pro-2 regions of NHE2 to
associate with a variety of SH3 domains, i.e. those of Abl,
Ras-GAP, p85 of phosphatidylinositol 3'-kinase,
-spectrin, Src, or
the neuronal (N-Src). As before, GST alone was included as a measure of
the specificity of the interaction. Our results show that none of the
indicated SH3 domains interacted measurably with GST (Fig.
2). By contrast, all of these bound to
the Pro-1 fusion protein, with Ras-GAP interacting most strongly,
whereas N-Src bound only marginally. The Pro-2 domain was less
promiscuous, interacting strongly with the SH3 domains of p85 and Src,
only moderately with N-Src and Abl, but not measurably with either Ras-GAP or
-spectrin. It therefore appears that both Pro-rich motifs
of NHE2 can bind SH3 domains in vitro, albeit with different specificity.

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Fig. 2.
Binding of various SH3 domains to the
Pro-rich regions of NHE2. GST fusion proteins of the Pro-1 and
Pro-2 regions of NHE2 (encompassing residues 724-766 and 774-813,
respectively), as well as unconjugated GST (8 µg each) were separated
by SDS-PAGE (12% acrylamide) and blotted onto nitrocellulose. The blot
was initially stained with Ponceau S to reveal the proteins (top
panel) and then overlaid with biotinylated GST fusion proteins
obtained from Abl, Ras-GAP, the p85 subunit of phosphatidylinositol
3'-kinase, -spectrin, Src, or neuronal Src (N-Src). After
extensive washing, bound GST-SH3 domains were detected using
streptavidin-peroxidase and ECL, as in Fig. 1. Shown is a
representative of five similar experiments.
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Functional Role of Pro-rich Regions of NHE2--
To assess the
functional role of the Pro-rich regions of NHE2, we compared the
behavior of the full-length protein with that of truncated mutants
lacking one or both Pro-rich regions. To this end, vectors encoding
epitope-tagged intact or truncated constructs were transfected into
mammalian cells and their expression and transport properties were
evaluated. As a preliminary experiment to evaluate the
immunodetectability of the epitope-tagged constructs, the cDNAs
were transiently transfected into HEK-T cells. As illustrated in Fig.
3, transfection of the full-length NHE2
(NHE2HA) construct resulted in expression of a 95-105-kDa
polypeptide that was recognized by an antibody directed to the HA
epitope, which was attached at the C terminus of the protein.
Transfection of constructs truncated at position 777 (NHE2
777HA), lacking the C-terminal Pro-2, or at
position 731 (NHE2
731HA), lacking both Pro-1 and Pro-2,
induced the expression of polypeptides of ~90-95 and ~85-92 kDa.
The levels of expression of the full-length and truncated molecules
were comparable and the observed differences in molecular mass are consistent with the deletion of 36 and 82 residues, respectively.

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Fig. 3.
Expression of full-length and truncated forms
of NHE2. HEK-T cells were transfected with either
NHE2HA, NHE2 777HA, NHE2 731HA
or with the empty pCMV vector alone (Sham). After 48 h,
the cells were lysed hypotonically and dissolved in Laemmli sample
buffer for SDS-PAGE analysis. Following electrophoresis, the samples
were transferred to nitrocellulose and blotted with monoclonal anti-HA
antibody (1:5,000), followed by goat peroxidase-coupled
anti-mouse (1:5,000) and detection by ECL. Identical amounts of
protein were loaded on each lane. Shown is a representative of at
least three experiments.
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The functional properties of the wild type and mutant NHE2s were
studied next. To quantify the ion exchange mediated by the intact and
truncated forms of NHE2, it was imperative to dissociate their activity
from that of endogenous Na+/H+ exchangers
present in the cells used for transfection. This was accomplished by
stably expressing the cDNAs in AP-1 cells, a mutant line of CHO
cells devoid of NHE activity (34). Transport activity was estimated as
the Na+-dependent changes in pHi,
measured fluorometrically using BCECF. In all experiments, pHi
was measured after overnight serum depletion to minimize confounding
effects of growth factors in the regulation of NHE2 activity.
The activity of the full-length and truncated forms of NHE2 was
initially compared in cells that were acutely acid-loaded by
preincubation with 25 mM NH4Cl for 10 min,
followed by rapid removal of the weak base. When bathed in
Na+-free,
N-methyl-D-glucammonium+-rich
medium, the cells remained acidic, indicating that
Na+-independent systems contribute negligibly to
pHi restoration in the time frame studied. As reported earlier,
re-addition of Na+ elicited a rapid cytosolic
alkalinization in cells expressing the original unmodified full-length
NHE2, and a comparable pH change was obtained when NHE2 was
epitope-tagged, indicating that addition of the C-terminal HA
nonapeptide did not interfere with the basic function of the antiporter
(Fig. 4). Similar recoveries from the
acid load were obtained in cells transfected with
NHE2
777HA or with NHE2
731HA. Comparison
of the relative rates of recovery from the acid load indicates that the
pHi dependence of the exchange process was not markedly
affected by truncation of either one or both Pro-rich domains. The
full-length NHE2, as well as the truncated versions, became minimally
active between pHi 7.0 and 7.5, and their activity increased
steeply at more acidic pHi (Figs. 4 and
5). NHE2
777HA appeared to
become quiescent at a somewhat more acidic pH than the other constructs. We did not quantify the absolute rates of transport because
this parameter is not informative when comparing expression levels in
different stable transfectants.

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Fig. 4.
Na+-induced acid extrusion from
cells transfected with full-length and truncated NHE2 . Antiport-deficient Chinese hamster ovary cells (AP-1) were stably
transfected with full-length NHE2, with or without a C-terminal HA tag
(NHE2HA and NHE2, respectively), or with epitope-tagged
truncated forms lacking either Pro-2 only ( 777HA)
or both Pro-1 and Pro-2 ( 731HA). Cells were grown to
subconfluence on glass coverslips and loaded with BCECF as described
under "Experimental Procedures." The cells were acid-loaded by
prepulsing with 25 mM NH4Cl, and pHi
determination was initiated upon perfusion with Na+-free
solution. Where indicated, the solution was replaced by
Na+-rich medium. pHi was determined by ratio
microfluorometry and calibrated using nigericin/K+. Samples
that were acid-loaded to slightly different levels are illustrated, to
offset the traces and avoid overlap. Shown are representatives of at
least four determinations of each type.
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Fig. 5.
ATP dependence of the activity of full-length
and truncated NHE2 . pHi was measured fluorometrically in
AP-1 cells transfected with either full-length or truncated forms of
epitope-tagged NHE2, as in Fig. 4. Where indicated, the cells were
depleted of ATP by incubation for 10 min in glucose-free medium with 5 mM 2-deoxy-D-glucose and 1 µg/ml antimycin A. Fluorescence measurements in depleted cells were performed in
glucose-free medium with 5.5 mM
2-deoxy-D-glucose. A, representative recovery
from an acid load in control and ATP-depleted full-length NHE2
transfectants. B-D, plots of pHi versus
activity of control (solid squares) and ATP-depleted
(open squares) cells. Cells were transfected with
NHE2HA (B), NHE2 777HA
(C), and NHE2 731HA (D). For
comparison, the activity was normalized to the rate measured at pH 6.5. Data shown are from at least four experiments of each type.
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Although the basal operation of NHE2 is not grossly affected by
deletion of the Pro-rich regions, it is possible that its regulation
may be altered. Intracellular ATP is an important regulator of NHE2
activity (39). Although the precise mechanism of action of ATP is not
well defined, depletion of the nucleotide is known to depress the
activity of NHE2, shifting its activation threshold to more acidic pH
values. We analyzed whether the Pro-rich motifs influenced this
regulation. As illustrated in Fig. 5A, insertion of the HA
epitope at the C terminus of the full-length NHE2 did not modify the
responsiveness of the exchanger to metabolic depletion: the rate of
recovery from an acid load was greatly reduced by preincubation for 10 min in glucose-free medium with 5 mM
2-deoxy-D-glucose and 1 µg/ml antimycin A, conditions
shown earlier to deplete ATP in CHO cells by >90% within 10 min (35).
A comparable inhibition of exchange was noted in
NHE2
777HA and in NHE2
731HA transfected cells (Fig. 5, C and D). Therefore, the presence
of the Pro-rich sequences is not essential for regulation of NHE2 by
ATP. This result is not entirely unexpected because other NHE isoforms
are also regulated by ATP, albeit to differing extents, but lack
obvious SH3-binding domains.
Osmotic Activation of NHE2--
In addition to its important role
in maintaining pHi homeostasis, the NHE family of proteins
plays an important role in volume regulation and H2O
transport. In response to hypertonic stress, NHE2 is activated in
transfected CHO and porcine proximal tubule LLC-PK1 cells
(Refs. 3 and 15, but see Ref. 40 for a differing view). The mechanism
by which hypertonicity modulates antiport activity is not known, but
cytoskeletal involvement is suspected. Because several cytoskeletal
components contain SH3 domains, we considered the possibility that the
cognate Pro-rich motifs may play a role in volume regulation. For these
experiments, cells incubated in isotonic medium were challenged with a
hypertonic (600 ± 20 mosM) Na+-rich
solution. As illustrated in the representative traces shown in Fig.
6, the hypertonic response described
earlier for the full-length NHE2 persists in the epitope-tagged
construct. A clear osmotic stimulation was also noted in cells
transfected with NHE2
731HA. By comparison, the response
recorded in cells expressing NHE2
777HA was considerably
smaller. This depressed response is not likely due to a reduced number
of copies of the exchanger, because the rate of recovery from an acid
load was comparable to that of the other forms. Instead, it may be a
consequence of the acidic shift in the pHi dependence of this
truncated form. Alternatively, removal of Pro-2 may have allowed
interaction of an inhibitory SH3 domain with Pro-1. The inhibition
caused by this interaction would be relieved upon truncation of Pro-1.
In any event, because the hypertonic response persists in
NHE2
731HA cells, which lack both Pro-1 and Pro-2, it is
clear that the SH3-binding domains are not essential for the osmotic
activation of NHE2.

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Fig. 6.
Osmotic responsiveness of full-length and
truncated NHE2. pHi was measured fluorometrically in AP-1
cells transfected with either full-length or truncated forms of
epitope-tagged NHE2, as in Fig. 4. The cells were initially suspended
in isotonic, Na+-rich medium (Iso). Where
specified, the osmolarity of the medium was increased to 600 ± 10 mosM (Hyper). Traces are representative of at
least four experiments.
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Localization of Full-length NHE2 and Its Truncation Mutants in
LLC-PK1 Cells--
Because the Pro-rich regions of NHE2
were seemingly not required for either basal
Na+/H+ exchange activity or for its regulation
by ATP or cell volume, we considered the possibility that they may be
involved in subcellular targeting. As mentioned in the introduction,
NHE2 is abundant in epithelial cells of the kidney and gastrointestinal
tract. In epithelial cells, where proper targeting of transporters is needed for vectorial movement of electrolytes and fluid (41, 42), the
distribution of individual NHE isoforms is polarized. NHE3 is
exclusively apical, whereas NHE1 is almost entirely basolateral (43-46). More uncertainty exists concerning NHE2, which has been reported by some to concentrate apically (9-11), whereas others have
found that it functions at the basolateral membrane (8).
To re-assess the localization of full-length NHE2 and to define the
role of the Pro-rich motifs in the establishment of polarity, we
transfected HA-tagged constructs into LLC-PK1 cells. Stably transfected cells were obtained by subcloning the constructs into the
pBK plasmid that encodes for neomycin resistance, and selected using
G418. Stable transfectants were grown to confluence and analyzed by
immunofluorescence and confocal laser scanning microscopy using anti-HA
antibodies followed by Cy3-conjugated secondary antibodies. Prior to
fixation, the apical membrane was counter-stained by overlaying the
cells with a mixture of fluoresceinated lectins (see under
"Experimental Procedures"). As illustrated in Fig. 7, both the full-length
(NHE2HA) and the NHE2
777HA, which lacks the
C-terminal Pro-rich domain (Pro-2), displayed a punctate appearance that followed the contour of the apical membrane. Accordingly, the
distribution of these proteins matched closely the distribution of
apically bound lectins (cf. Fig. 7, B and
C). There was little evidence of intracellular retention of
either the full-length NHE2HA or NHE2
777HA,
despite wide variation in the level of expression among individual
clones.

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Fig. 7.
Localization of full-length and truncated
NHE2 in LLC-PK1 cells. LLC-PK1 cells were
stably transfected with NHE2HA (A),
NHE2 777HA (B and C), or
NHE2 731HA (D-F) using the calcium phosphate
method and selected with G418 as described under "Experimental
Procedures." The cells were grown to confluence on coverslips, washed
with PBS prior to fixation with 4% paraformaldehyde, and subsequently
labeled with fluorescein isothiocyanate-labeled lectins. After
extensive washing, the cells were permeabilized with 0.1% Triton X-100
in 5% bovine serum albumin and labeled with 1:1000 anti-HA antibody,
followed by secondary labeling was with 1:1000 Cy3 labeled anti-mouse
IgG (A, C, D, and F).
Fluorescence microscopy was used to detect the distribution of the
lectin (B and E) or of the HA epitope, indicative
of NHE2. Images are representative of at least five experiments.
Bar in D is 10 µm, and all panels are the same
magnification.
|
|
By contrast, a large fraction of the cells expressing
NHE2
731HA appeared to retain the construct in the
endoplasmic reticulum and/or Golgi complex (Fig. 7D). This
was not due to clonal variation or differences in the level of
expression, because similar results were obtained in multiple clones
with varying degrees of expression. The fraction of cells that
successfully exported NHE2
731HA to the plasma membrane
displayed a distinct distribution pattern. When viewed en
face, the truncated exchangers appeared to delineate the cell
borders, a pattern characteristic of basolateral proteins. Accordingly,
this distribution differed from that of the apical lectins
(cf. Fig. 7, E and F) and was instead
similar to that of ZO-1, a tight-junctional protein (not illustrated).
Only a small fraction of NHE2
731HA appeared to be
present at the apical membrane.
A quantitative summary of these observations, obtained from 10 separate
observations made in polyclonal populations, is presented in Fig.
8. The data confirm that whereas
NHE2HA and NHE2
777HA are predominantly
apical, a large fraction of NHE2
731HA is retained intracellularly, and those truncated exchangers that reach the membrane
localize predominantly to the basolateral side of the cell. We conclude
that the Pro-1 motif is important for effective processing and
targeting of NHE2 to the apical membrane of epithelial cells.

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Fig. 8.
Quantitative estimation of the subcellular
distribution of full-length and truncated NHE2. LLC-PK1 cells
transfected with full-length or truncated forms of NHE2 were
stained and analyzed by epifluorescence microscopy using a × 100 objective as in Fig. 7. The subcellular distribution of each one of the
tagged exchangers was assessed in 10 random samples. Data summarize the
aggregate observations.
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|
 |
DISCUSSION |
The observations presented in this report provide evidence that
the cytoplasmically oriented Pro-rich regions of NHE2 can bind to SH3
domains. Pro-2 bound preferentially to SH3 domains derived from
tyrosine kinases and from the regulatory domain of phosphatidylinositol
3'-kinase, whereas Pro-1 was more promiscuous, interacting with a wider
variety of SH3 domains. Although promiscuous, this interaction was
nevertheless specific to SH3 domains, inasmuch as it was not observed
for GST alone, nor for fusion proteins derived from the cytosolic
domains of other NHE isoforms, which lack Pro-rich sequences.
The functional significance of these interactions was evaluated by
comparing the behavior of intact and truncated forms of NHE2, using
heterologous expression systems. Truncation of the C-terminal Pro-2
region (NHE2
777HA) had subtle yet reproducible functional consequences. The pHi dependence of the rate of
exchange was modestly shifted to more acidic values, and the osmotic
activation was depressed. However, an even more profound truncation
(NHE2
731HA) that eliminated both Pro-1 and Pro-2
restored both the normal pHi dependence and osmotic
responsiveness. This biphasic behavior is not unprecedented. Moderate
truncation of the C-terminal domain of NHE1 can produce an alkaline
shift in its pHi dependence, with loss of responsiveness to a
variety of stimuli (47, 49). The shift and loss of responsiveness are
reversed by more profound truncations, which can in fact induce an
acidic shift in the pHi versus activity relationship (50, 51). Thus, the exchangers can seemingly exist in several conformations that are exquisitely dependent on subdomains of the
cytosolic tail.
Despite the subtle effects noted upon deletion of Pro-2, it is clear
that neither Pro-1 nor Pro-2 is absolutely essential for the ability of
NHE2 to exchange Na+ for H+, nor for its
dependence on ATP or sensitivity to osmotic activation. This conclusion
stems from the observations made using the NHE2
731HA construct, which displayed near-normal behavior under these conditions. We therefore considered the possible role of the Pro-rich regions in
protein processing and subcellular targeting. This initially required
confirmation of the subcellular distribution of NHE2 in epithelial
cells, which has been the source of controversy. By transfecting the
full-length, epitope-tagged NHE2 into LLC-PK1 cells, we
found that this isoform accumulates in the apical membrane, as reported
by the majority of studies (9-12). The source of the discrepancies
with the results of Soleimani et al. (8), who detected
basolateral NHE2, remains unclear. Neither tissue nor species
differences appear to account for the inconsistency, because both
apical and basolateral locations have been reported for NHE2 in renal
medullary cells of rodents (8, 12).
Despite these uncertainties, comparison of the full-length and
truncated constructs in a single expression system provided useful
information concerning the role of the Pro-rich regions in targeting.
Briefly, we found that whereas deletion of Pro-2 was ineffectual, the
additional removal of Pro-1 greatly impaired the ability of NHE2 to
reach the apical membrane. A sizable fraction of the truncated
exchanger was retained in the secretory pathway, and the exchanger that
was exported to the plasmalemma was primarily found in the basolateral
membrane. It is tempting to speculate that the existence of splice
variants or posttranslational truncation of NHE2 may account for the
reports in which this isoform was detected basolaterally.
These observations suggest that the cytosolic region between residues
732 and 777, encompassing Pro-1, is necessary for appropriate targeting
of NHE2 to the apical surface. In this context, Pro-1 was found to bind
to the SH3 domain of
-spectrin, a protein that has previously been
reported to participate in the apical targeting of epithelial
Na+ channels (ENaCs) (19). Like NHE2, ENaC interacts with
the SH3 domain of
-spectrin in vitro. Moreover,
microinjection of a fusion protein containing the Pro-rich region of
ENaC revealed targeting to the apical membrane of epithelial cells
(19). Based on these observations, it was suggested that
-spectrin
may function to retain ENaC apically, a mechanism that may also apply
in the case of NHE2. Alternatively, interaction with spectrin present
in the Golgi (48) may be essential for export of the exchangers and channels to the plasmalemma. Clearly, other SH3-containing proteins may
also function in apical delivery and retention of NHE2. A complete
understanding of the role of Pro-rich domains awaits identification of
all the proteins that interact with the exchanger in
vivo.
 |
FOOTNOTES |
*
This work was supported by the Medical Research Council of
Canada and the Kidney Foundation of Canada.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.
International Scholar of the Howard Hughes Medical Institute
and the current holder of the Pitblado Chair in Cell Biology. Cross-appointed to the Department of Biochemistry of the University of Toronto.
**
Supported by a Scientist award from the Medical Research Council of
Canada. To whom correspondence should be addressed: Dept. of
Physiology, McGill University, McIntyre Medical Science Bldg., 3655 Drummond St., Montreal, Quebec H3G 1Y6, Canada. Tel.: 514-398-8335; Fax: 514-398-7452; E-mail: orlowski{at}physio.mcgill.ca.
 |
ABBREVIATIONS |
The abbreviations used are:
BCECF, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein;
CHO, Chinese hamster ovary;
GST, glutathione S-transferase;
HA, hemagglutinin;
NHE, Na+/H+ exchanger;
PBS, phosphate-buffered saline;
pHi, intracellular (cytosolic) pH;
PAGE, polyacrylamide gel electrophoresis;
SH3, Src homology domain 3;
HEK, human embryonic kidney;
ENaC, epithelial Na+
channel.
 |
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