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INTRODUCTION |
Na+/H+ exchangers
(NHEs)1 mediate the
electroneutral, amiloride-sensitive exchange of Na+ and
H+ across plasma membranes in most eukaryotic cells. In the
mammalian kidney this activity is particularly important as it
contributes to the maintenance of acid-base balance and NaCl
homeostasis. Although there are at least four NHE isoforms expressed in
the kidney (1-8), recent studies from this laboratory (7, 9, 10) and
from other (11) laboratories have shown that
Na+/H+ exchanger isoform NHE3 is present on the
apical (brush border) membrane of the proximal tubule and is the
isoform responsible for most, if not all, of the described
Na+/H+ exchange activity in this membrane
domain (12).
Transporters often exist as components of multimeric protein complexes
that stabilize their localization in specific membrane domains and/or
ensure proximity to signaling pathways (13-15). Recent studies have
shown that two homologous proteins known as NHE-RF
(Na+/H+ exchanger
regulatory factor) and E3KARP (NHE3
kinase A regulatory
protein) play critical roles in the cAMP-mediated
inhibition of NHE3 (16, 17). Both proteins have PDZ domains that may
interact directly with NHE3 or may be adapters that link NHE3 to the
cytoskeleton (18). Much of the data provided by these studies have been
derived from experiments in cell culture or from yeast two-hybrid analysis.
We therefore investigated whether NHE3 exists in assemblies with other
proteins in native kidney membranes. We report that the sedimentation
coefficient for NHE3 solubilized from renal brush border membranes is
greater than predicted for monomeric NHE3, indicating the presence of
multimeric complexes. Moreover, by use of a strategy involving the
generation of monoclonal antibodies to immunopurified NHE3 complexes,
we find that a significant pool of NHE3 exists in association with the
putative scavenger receptor megalin.
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MATERIALS AND METHODS |
Antibodies to NHE3--
In a previous paper we described in
detail the development and characterization of monoclonal antibodies to
a restricted region of the carboxyl terminus of NHE3 (9). mAbs 2B9,
4F5, and 19F5 were raised to a fusion protein (fpNHE3-702-832) that
reproduced the carboxyl-terminal 131 amino acids of the rabbit
Na+/H+ exchanger NHE3 (1). By study of
Na+/H+ exchanger-deficient LAP cells
transfected with each of NHE1-4, we demonstrated that these three mAbs
are specific for the 80-kDa NHE3 polypeptide (9). No cross-reactivity
was observed to other NHE isoforms or any other proteins. By
immunocytochemistry, all three mAbs stained the brush border of
proximal tubule cells (9). These mAbs were used as purified IgG from
hybridoma supernatants.
A polyclonal antibody, raised in guinea pigs to fpNHE3-702-832, was
also used in this study. This antibody is also specific for NHE3 (19)
and was used to immunoblot NHE3 in immune complexes precipitated with
the mAbs.
Additional Primary Antibodies--
A mAb raised to NHE1 (4E9)
has been described previously (19). This antibody was raised to a
fusion protein representing the carboxyl terminus (amino acids
514-818) of porcine NHE1 (4). A mAb (mouse IgG) to villin was
purchased from AMAC (Westbrook, ME). This mAb was raised to the
carboxyl-terminal headpiece region of purified pig villin (20). A
polyclonal antibody raised to the renal brush border Na-Pi
cotransporter, NaPi-2, was provided by Drs. Heini Murer and
Jurg Biber, Institute of Physiology, University of Zurich-Irchel,
Zurich, Switzerland (21). A mAb raised to megalin was provided by Drs.
Dontscho Kerjaschki and Markus Exner, University of Vienna, Austria
(35).
Antibody Conjugates--
Fluorescein isothiocyanate-conjugated
rabbit anti-mouse IgG (heavy and light chain), used at 10 µg/ml, was
purchased from Zymed Laboratories Inc. (San Francisco,
CA). Horseradish peroxidase-conjugated rabbit anti-guinea pig IgG
(heavy and light chain-specific), goat anti-mouse (
chain-specific),
or goat anti-rabbit (heavy and light chain-specific) were purchased
from Zymed Laboratories Inc. (San Francisco CA) and
used at 0.5 µg/ml.
Preparation of Rabbit Renal Membrane Fractions--
Adult male
New Zealand White rabbits (Gabrielle Farms, Woodstock, CT) were
sacrificed by intravenous injection of sodium pentobarbital (Butler
Co., Columbus, OH). Brush border membrane vesicles (BBMV) or microsomes
were prepared from renal cortex as described previously (7). Protease
inhibitors (Sigma) pepstatin A (0.7 µg/ml), leupeptin (0.5 µg/ml),
phenylmethylsulfonyl fluoride (40 µg/ml), and EDTA (1 mM)
each were included in the preparation. Protein concentrations were
determined by the method of Lowry et al. (22).
Sucrose Velocity Gradient Centrifugation--
Velocity gradient
sedimentation was carried out according to Copeland et al.
(23). Rabbit renal microsomes were solubilized in lysis buffer (pH 7.4)
containing 20 mM MES, 30 mM Tris, 100 mM NaCl, and 1% Triton X-100. Insoluble material was
removed by centrifugation at 200,000 × g in a Beckman
ultracentrifuge for 1 h at 4 °C. The supernatants were applied
to the top of 5-25% continuous sucrose gradients. Sucrose solutions
were prepared with lysis buffer containing 0.1% Triton X-100. After
centrifugation for 12 h at 4 °C at 40,000 rpm in an SW 41 rotor, the gradients were fractionated by hand from the top. Sucrose
concentrations of each fraction were calculated from the refractive
index. Sedimentation coefficients were determined by comparison to
standard proteins with known S values (aldolase,
s20,w = 7.3; catalase, s20,w = 11.3; horse spleen apoferritin,
s20,w = 16.5; and bovine thyroglobulin,
s20,w = 19.3), or by using the formula
s20,w =
I/
2t where I is the
time integral,
is rotor speed (radians/s), and t is
time, as described by Griffith (24). Buffers (Tris and MES), Triton
X-100, and apoferritin were purchased from Sigma. Aldolase, catalase,
and thyroglobulin were from Amersham Pharmacia Biotech.
Immunoaffinity Purification of Native NHE3--
An anti-NHE3
affinity matrix was prepared by cross-linking affinity purified mAb 2B9
to protein A-Sepharose CL-4B beads (Amersham Pharmacia Biotech) with
dimethylpimelimidate dihydrochloride (Sigma) according to the "two
layer" method described by Schneider et al. (25). Rabbit
anti-mouse IgG (Zymed Laboratories Inc.) was used as a
bridging antibody.
For purification of native NHE3 complexes, brush border membrane
vesicles were solubilized at 4 °C in 0.1% octyl glucoside (Roche
Molecular Biochemicals) in Tris-buffered saline (pH 7.4). Samples were
cleared by centrifugation at 15,000 × g for 10 min. The supernatants were applied to the affinity matrix and allowed to
incubated overnight in the cold (4 °C). The column was washed extensively with solubilization buffer and monitored for protein by
measuring the absorbance at 280 nm using a Gilford Spectrophotometer 260 (Gilford Instruments, Inc., Oberlin, OH). When the wash buffer was
determined to be free of unbound protein, bound NHE3 complexes were
eluted using glycine HCl (pH 2.5) and immediately neutralized to pH 7.4 using 1 M Tris base. When the efficiency of the column was
analyzed by immunoblotting samples of the unbound flow-through and of
the acid eluant, we found that most (>90%) of the total NHE3 had been
bound to the column (data not shown).
Immunization of Mice and Production of Hybridomas--
BALB/c
mice (Jackson Laboratories, Bar Harbor, ME) were immunized
intraperitoneally with affinity purified rabbit NHE3 complexes from 1 mg of BBMV using a pertussis/alum protocol (26). Animals were boosted
by injecting intraperitoneally the same amount of antigen in PBS. Mice
whose serum showed specific reactivity to renal brush borders by
immunocytochemistry were selected for fusion. Spleen cells were fused
with Ag8 cells and grown according to established procedures (9).
Selected hybridomas were cloned and subcloned by limiting dilution
using Cloning FactorTM (IGEN, Inc., Rockville, MD).
mAbs were purified from hybridoma supernatants by affinity
chromatography using protein G-Sepharose 4B (Amersham Pharmacia Biotech) according to manufacturer's protocols. Purified mAb (1-10 mg/ml) to which was added BSA at 1 mg/ml was stored in 50%
glycerol/PBS at
20 °C.
ELISA (Enzyme-linked Immunosorbent Assay)--
ELISAs were
performed as described previously (4). As antigen, BBMV (protein
concentration 30 mg/ml) were diluted 1:1000 in PBS, 100 µl applied to
each well of 96-well microtiter plates (Corning Costar, Kennebunk, ME),
and the plates allowed to incubate overnight at 4 °C. The following
day the plates were washed 4× in PBS (pH 7.4) with 0.1% Triton X-100
(PBS/Triton X-100) and once in PBS with 1% BSA and 0.1% Triton X-100
(PBS/BSA/Triton X-100) to block nonspecific antibody binding. After
blocking for 10 min, 25 µl of hybridoma supernatant was added to each
well and allowed to incubate for 1 h at room temperature. Plates
were washed 4× in PBS/Triton X-100 and once in PBS/BSA/Triton X-100. Bound antibody was detected by incubation for 1 h in horseradish peroxidase-conjugated goat anti-mouse IgG (
-chain specific;
Zymed Laboratories Inc., San Francisco, CA) diluted
1:2000 in PBS/BSA/Triton X-100. The plates were washed 5× in
PBS/Triton X-100 and once in distilled water, and bound horseradish
peroxidase was detected with o-phenylenediamine (Sigma). The
optical density was measured at 492 nm using a Titretek Multiskan
MCC/340 spectrophotometer.
Immunoprecipitation of NHE3--
Immunoprecipitation of soluble
renal proteins was performed essentially as described previously (27).
Renal membranes (BBMV or microsomes) were solubilized at 4 °C in TBS
(pH 7.4) containing 1% Triton X-100 and protease inhibitors
phenylmethylsulfonyl fluoride, leupeptin, pepstatin, and EDTA as
described above. The samples were subjected to either low speed
(15,000 × g for 10 min) or high speed (200,000 × g for 1 h) centrifugation using a table top
(HermleTM model Z230 M, National Labnet Co.,
Woodbridge, NJ) centrifuge or a Beckman ultracentrifuge, respectively.
To the above supernatants were added primary antibody, approximately 50 µg for mAbs or 10 µl of serum. Primary antibodies were allowed to
incubate at 4 °C for 1 h. Immune complexes were collected using
5 mg/sample of protein G-Sepharose 4B (Amersham Pharmacia Biotech). The
beads were washed 5× in solubilization buffer and then incubated in 50 µl of SDS-PAGE sample buffer for 5 min at 100 °C, and the samples were prepared for SDS-PAGE and immunoblotting. Although we have found
that heating NHE3 increases its tendency to form aggregates (see Fig.
5), heating is necessary in these experiments in order to completely
reduce IgG to monomeric heavy and light chains.
SDS-PAGE and Immunoblotting--
Protein samples were
solubilized in SDS-PAGE sample buffer and separated by SDS-PAGE using
7.5% polyacrylamide gels according to Laemmli (28). For
immunoblotting, proteins were transferred to PVDF (Millipore
Immobilon-P) at 500 mA for 6-10 h at 4 °C with a
TransphorTM transfer electrophoresis unit (Hoefer
Scientific Instruments, San Francisco) and stained with Ponceau S in
0.5% trichloroacetic acid. Immunoblotting was performed as follows.
Sheets of PVDF containing transferred protein from entire gels were
incubated first in Blotto (5% non-fat dry milk in PBS (pH 7.4)) for
1-3 h to block nonspecific binding of antibody, followed by overnight incubation in primary antibody. Primary antibodies, diluted in Blotto,
were used at dilutions ranging from 1:1000 to 1:5000. The sheets were
then washed in Blotto and incubated for 1 h with an appropriate
horseradish peroxidase-conjugated secondary antibody diluted 1:2000 in
Blotto. After washing 3× in Blotto, 1× in PBS (pH 7.4), and 1× in
distilled water, bound antibody was detected with the ECLTM
chemiluminescence system (Amersham Pharmacia Biotech) according to the
manufacturer's protocols. In some experiments (see Figs. 1, 2, and 5),
PVDF blots were reprobed with additional primary antibodies after
stripping away the first antibody. This was accomplished by incubating
the PVDF sheets in 2% SDS, 100 mM
-mercaptoethanol, 50 mM Tris (pH 6.9) for 60 min at 70 °C.
Tissue Preparation for Immunocytochemistry--
Rabbits were
anesthetized with sodium pentobarbital injected intravenously, and the
kidneys were perfusion-fixed with paraformaldehyde/lysine/periodate fixative (29) as described previously (30). Fixed tissue was processed
and stained using the immunoperoxidase method exactly as described
previously (30). When screening hybridomas, the culture supernatant or
hybridoma media, which served as controls, were used undiluted for
labeling. Thin sections of Epon-embedded tissue were examined using a
Zeiss 910 electron microscope.
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RESULTS |
Solubility Properties of Renal Na+/H+
Exchangers--
Our initial studies were designed to establish the
optimal conditions for solubilizing the apical
Na+/H+ exchanger NHE3 from rabbit renal
membranes. In these experiments, rabbit renal microsomes were
solubilized with several nonionic (Triton X-100, octyl glucoside, and
C12E8) or zwitterionic (CHAPS) detergents and
then subjected to differential centrifugation. The resulting fractions
were analyzed by immunoblotting. For comparison, we assessed the
solubility of both NHE3 and the basolateral
Na+/H+ exchanger, NHE1, which is also expressed
at high levels in rabbit renal cortex (8).
We assessed solubility based on the presence of protein in the
supernatant following centrifugation. In this study, low speed centrifugation is defined as 15,000 × g for 10 min,
and high speed centrifugation is carried out at 200,000 × g for 1 h. Therefore, the presence of a protein in the
high speed supernatant indicates complete solubilization. In Fig.
1, lane 1 represents proteins present in the low speed pellet, lane 2 the high speed
pellet, and lane 3 the high speed supernatant.

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Fig. 1.
Western blots showing the solubility of renal
Na/H exchangers. Rabbit renal cortical microsomes (200 µg) were
added to buffer (TBS, pH 7.4) containing either no detergent, 1%
Triton X-100, 4% CHAPS, 2% octyl glucoside, or 0.1%
C12E8. Incubation was performed at 4 °C for
30 min. Samples were centrifuged at 15,000 × g for 10 min, and the resulting low speed pellet is seen in panels A
and B, lane 1. The resulting supernatant was
centrifuged for 1 h at 200,000 × g. The high
speed pellet is seen in lane 2, and 1/10 of the high speed
supernatant is seen in lane 3. The blots were probed first
with a mAb to NHE1 (4E9) (panel A). The blot was then
stripped of bound antibody, and the blot was reprobed with a polyclonal
antibody to NHE3 (panel B).
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As indicated in Fig. 1, the solubility properties of NHE1 and NHE3 are
different. Although NHE1 can be detected in the high speed pellets
following CHAPS and octyl glucoside solubilization, most of this
isoform is soluble (present in the high speed supernatant) in Triton
X-100 and C12E8 (Fig. 1, panel A).
In contrast, NHE3 was completely insoluble (present in the pellets)
when we used CHAPS or octyl glucoside and was only partially soluble in
Triton X-100 and C12E8 (Fig. 1, panel
B). Such detergent insolubility of membrane proteins has
frequently been shown to result from specific protein-protein
(cytoskeletal) (14) or protein-lipid (31) interactions.
Sucrose Velocity Gradient Centrifugation--
Studies of
Na+/H+ exchangers expressed in cell culture
have shown them to form homomultimeric assemblies (32). Also, since the
data presented in Fig. 1 suggested that NHE3 may interact with other
brush border proteins, we next sought to estimate the size of the
native renal Na+/H+ exchangers. To this end, we
determined the sedimentation coefficients of NHE1 and NHE3 using
sucrose velocity gradient centrifugation (Fig.
2). In these studies, the high speed
Triton X-100 supernatant, prepared as described for Fig. 1, was applied
to the top of 5-25% sucrose gradients, and the samples were
centrifuged for 12 h as described under "Materials and
Methods." The distribution of the Na+/H+
exchangers (NHE1 and NHE3) in the gradient was determined by probing
immunoblots of the fractions with isoform-specific antibodies. Their
sedimentation coefficients were calculated and compared with standard
proteins with known S values (Fig. 2). NHE1 sedimented as a
single discrete peak with a sedimentation coefficient of approximately
s20,w = 6.5. In contrast, NHE3 had a
very broad distribution with a significantly higher sedimentation
coefficient. These data suggest that NHE3 exists in multimeric
assemblies under these conditions. Moreover, the broad distribution of
NHE3 in the gradient suggests that these assemblies are
heterogeneous.

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Fig. 2.
Sucrose velocity gradient centrifugation:
sedimentation coefficients of renal Na/H exchangers. Rabbit renal
cortical microsomes (7 mg) were solubilized in 10 ml of MES buffer
containing 1% Triton X-100. After centrifugation at 200,000 × g for 1 h, 1 ml of the supernatant was applied to the
top of a 5-25% sucrose gradient, and the gradient was centrifuged in
a Ti41 rotor at 40,000 for 12 h at 4 °C. 0.75-ml fractions were
collected, and 100 µl of each were prepared for immunoblotting. Blots
were probed first for NHE1 (upper panel) using mAb 4E9, then
stripped and probed with a polyclonal antibody for NHE3 (lower
panel). The locations of standard proteins (aldolase, catalase,
apoferritin, and thyroglobulin) are shown at the bottom of
the figure.
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Affinity Isolation of the Native NHE3 Transporter Complex and
Preparation of Monoclonal Antibodies--
In order to evaluate further
the possibility that NHE3 exists in assemblies with other proteins, we
generated mAbs to the native NHE3 complex as a strategy to identify
proteins that specifically associate with the
Na+/H+ exchanger. The NHE3 transporter complex,
solubilized with C12E8, was isolated using an
affinity column prepared with mAb 2B9 raised to the carboxyl terminus
of rabbit NHE3 (9). Mice were immunized and boosted with the column
eluate purified from approximately 1 mg of rabbit brush border membrane
vesicles. Hybridomas were prepared using standard protocols, and their
supernatants were screened using successive assays that included ELISA,
immunoblotting, immunocytochemistry, and immunoprecipitation. Our goal
was to first identify mAbs that were capable of immunoprecipitating
native NHE3. Once these were identified, each antibody was
characterized in order to determine if it was specific for NHE3 or an
associated protein. Of the approximately 180 hybridomas that were
positive by ELISA to rabbit brush border membranes, we identified 10 mAbs that stained the brush border of the proximal tubule by
immunocytochemistry and that immunoprecipitated NHE3 from Triton X-100
or C12E8-solubilized brush border membrane vesicles.
In order to identify mAbs that were directed to NHE3, we performed
immunoblotting experiments using microsomes prepared from LAP cells
that had been transfected with NHE3 or other isoforms of NHE as
controls (6, 7). Only one of the 10 mAbs (3G11) labeled NHE3 by
immunoblotting (data not shown). This mAb also cross-reacted with NHE1,
indicating that its epitope is shared between the two isoforms. Of the
remaining 9 mAbs, one mAb (10A3) has been characterized in detail, and
these studies are presented below.
Characterization of mAb 10A3--
Although mAb 10A3
immunoprecipitated NHE3 (see Fig. 5), when used for immunoblotting it
labeled a very large molecular weight protein that migrated by SDS-PAGE
above the 200-kDa standard near the top of the 7.5% gel (Fig.
3, panel A). When
used for immunocytochemistry, this mAb also exhibited a unique staining
pattern. As seen by immunoelectron microscopy in Fig. 3,
panel B, although some staining was detected on
microvilli, most of the staining was restricted to the coated pit
region of the brush border of proximal tubules. No staining was
observed in the controls (data not shown). These observations raised
the possibility that the 10A3 antigen may be the putative scavenger
receptor megalin, which is located most abundantly in the coated pit
region of the renal brush border (33) and which has a predicted
molecular mass of over 500 kDa (34).

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Fig. 3.
Characterization of mAb 10A3. mAb 10A3
was used for immunoblotting (panel A) and
immunocytochemistry (panel B) in rabbit kidney. Rabbit renal
microsomes (100 µg) were separated by SDS-PAGE and prepared for
immunoblotting (panel A). mAb 10A3 stained primarily a high
molecular mass (>200 kDa) protein. Panel B shows the 10A3
antigen localized at the level of the electron microscope using the
immunoperoxidase method. Shown here is the apical region of two
proximal tubule cells. The electron dense reaction product is localized
to the microvilli, the coated pit region of the brush border, and
within the endosome-like compartments ( ). Magnification × 16,000.
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In order to test this hypothesis, we evaluated whether mAb 10A3 and a
well characterized anti-megalin mAb (DC6) (35) would immunoprecipitate
the same protein from solubilized renal microsomes. Indeed, as
indicated by the solid arrow in Fig.
4, both 10A3 and DC6 immunoprecipitated
the same protein that was visualized on the immunoblot by staining with
both antibodies. This large (>200 kDa) protein was not precipitated by
a control mAb (anti-villin). The smaller protein bands observed in the
lower part of the blot (open arrow in Fig. 4) were due to
staining by the anti-mouse IgG secondary antibody of the heavy chains
of the primary antibodies (including the control) used for
precipitation. The data in Fig. 4 confirm that mAbs 10A3 and DC6 are
both directed at the same renal protein, namely megalin.

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Fig. 4.
The 10A3 antigen is megalin. Rabbit
renal microsomes (100 µg) were solubilized in TBS containing 1%
Triton X-100. After clearing by centrifugation at 15,000 × g, the supernatants were subjected to immunoprecipitation
using either mAb 10A3, DC6, or anti-villin (negative control). The
immune complexes were separated by SDS-PAGE and prepared for
immunoblotting. The blots were stained with either mAb 10A3 or
DC6.
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Specificity of NHE3-Megalin Association--
During hybridoma
screening, mAb 10A3 had been selected based on its ability to
co-precipitate NHE3 from the low speed (15,000 × g for
10 min) supernatants of Triton X-100-solubilized renal microsomes. But,
as demonstrated earlier (Fig. 1), a significant amount of NHE3 could be
pelleted from detergent-solubilized membranes when centrifuged at high
speed for a longer time (200,000 × g for 1 h),
indicating that some of the transporter is found in large, insoluble
aggregates. Co-localization of NHE3 and megalin in such insoluble
aggregates might account for their apparent co-precipitation from the
low speed supernatants of detergent-solubilized renal membranes.
Therefore, it was important to verify that NHE3 and megalin could also
be co-precipitated from the truly soluble supernatant after high speed
centrifugation. In addition, because megalin is thought to be a
multi-ligand receptor (36-41), we considered the possibility that
solubilized megalin might associate with multiple renal membrane
proteins nonspecifically. Accordingly, it was important to test whether
other brush border proteins could be co-precipitated with megalin.
To address these issues, we performed immunoprecipitation experiments
(shown in Fig. 5) using both the low
speed and high speed supernatants of Triton X-100-solubilized renal
microsomes. In addition to antibodies to NHE3 (mAb 2B9) and megalin
(mAb 10A3), we used antibodies to the microvillar core protein villin
and the brush border Na-Pi cotransporter NaPi-2
(42) as controls. Immune complexes formed with the precipitating
antibodies (indicated along the bottom of Fig. 5) were then
analyzed by immunoblotting for NHE3, megalin, NaPi-2, or
villin (indicated at the left of Fig. 5).

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Fig. 5.
Anti-megalin mAb 10A3 specifically
immunoprecipitates NHE3. Immunoprecipitations were carried out
using the following soluble fractions (supernatants) of rabbit renal
cortical microsomes. Low speed (LS) supernatants were
prepared by solubilizing 200 µg of microsomes in 1 ml of 1% Triton
X-100 in TBS (pH 7.4) and centrifuging for 10 min at 15,000 × g. High speed (HS) supernatants were prepared by
solubilizing 2 mg of microsomes in 10 ml of 1% Triton X-100 in TBS (pH
7.4) and centrifuging for 1 h at 200,000 × g.
Both low speed and high speed supernatants were subjected to
immunoprecipitation using either a mAb to NHE3 (2B9), a mAb to megalin
(10A3), a polyclonal antibody to the brush border Na+,
Pi cotransporter NaPi-2, or a mAb to the
microvillar core protein villin. Immune complexes, as well as a sample
(100 µg) of rabbit renal cortical microsomes (lane M),
were prepared for immunoblotting, and the blot was probed successively
with a polyclonal antibody to NHE3 (top panel),
anti-NaPi-2, anti-villin, or the mAb 10A3. Monomeric NHE3
(80 kDa) is marked by an arrow and an aggregate of NHE3 by
an asterisk. Molecular weights, expressed as
10 3 × Mr, are presented on the
right.
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In Fig. 5, lanes 1 and 2, immune complexes formed
with the anti-NHE3 mAb 2B9 were analyzed. Only NHE3 was detected in
these complexes. We have frequently observed that after exposure to nonionic detergents NHE3 migrates by SDS-PAGE as both a monomer (solid arrow) and as a higher molecular weight aggregate
(asterisk). The formation of the high molecular weight
aggregate of NHE3 is highly variable and is not consistently found to
be dependent on interaction with megalin. Surprisingly, the anti-NHE3
antibody 2B9 failed to co-precipitate megalin, an issue addressed in
experiments described below. In lanes 3 and 4,
immune complexes formed with the anti-megalin mAb 10A3 were analyzed.
In addition to the >500-kDa megalin seen in the bottom
panel, significant amounts of NHE3 were also co-precipitated from
both the low (LS) and high speed (HS)
supernatants. Note that neither villin nor NaPi-2
co-precipitated with megalin. Finally, as seen in lanes
5-8, when antibodies to either NaPi-2 or villin were
used to immunoprecipitate, neither NHE3 nor megalin were detected in
the immune complexes.
The results in Fig. 5 confirm the specificity of the NHE3-megalin
interaction. The fact that NHE3 co-precipitated with megalin even from
the high speed, well solubilized supernatant indicates that the
association between the two proteins does not result from their
co-localization in large insoluble membrane aggregates. Moreover, the
observation that neither villin nor NaPi-2
co-precipitated with megalin indicates that the NHE3-megalin
association does not reflect promiscuous binding of solubilized megalin
to brush border proteins nonspecifically.
NHE3 Exists in Megalin-bound or Megalin-free States--
Although
Fig. 5 shows specific NHE3-megalin interaction, it also demonstrates
that when our anti-NHE3 mAbs directed to the carboxyl-terminal 131 amino acids are used to immunoprecipitate NHE3, we cannot
co-immunoprecipitate megalin. One explanation for these data is that
renal brush border NHE3 exists in two forms that are structurally
different. One form is not associated with megalin and is available for
binding (immunoprecipitation) with our anti-NHE3 mAbs. A second form is
associated with megalin in such a way that the carboxyl-terminal
epitopes of the Na+/H+ exchanger are blocked
from antibody binding.
In order to test directly this hypothesis (Fig.
6), we subjected an aliquot of
solubilized BBMV to repeated precipitation with anti-NHE3 mAb 2B9
(1st three lanes). After three precipitations, all of the NHE3 available for binding to this mAb was removed. Then, in
order to determine if there was any remaining NHE3 that was bound to
megalin and that mAb 2B9 could not detect, an additional immunoprecipitation was performed from the same sample using mAb 10A3.
As seen in the 4th lane of this figure, a
significant amount of NHE3 (that could not be precipitated by anti-NHE3
mAb 2B9) was co-precipitated with megalin.

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Fig. 6.
Megalin binding blocks the carboxyl terminus
of NHE3. Rabbit brush border membrane vesicles (50 µg) were
solubilized in 1% Triton X-100 in TBS. Repeated (three times)
immunoprecipitations were performed with anti-NHE3 mAb 2B9. A fourth
immunoprecipitation was performed from the same sample using the
anti-megalin mAb 10A3. The immune complexes were washed, prepared for
immunoblotting, and the blots probed for NHE3 using an anti-NHE3
polyclonal antibody. NHE3 (arrow) appears as an 80-kDa
band.
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We sought to examine further these pools of NHE3 by estimating their
sedimentation coefficients using sucrose velocity gradient centrifugation. We predict that NHE3 that is assembled with megalin will have a greater sedimentation coefficient than "free" NHE3. Therefore, we subjected the high speed supernatant of Triton
X-100-solubilized microsomes to sedimentation through 5-25% sucrose
gradients. Fractions were collected across the gradients, and one-half
of each fraction was subjected to immunoprecipitation with either mAb
2B9 or mAb 10A3. The presence of NHE3 in the resulting immune complexes
was analyzed by immunoblotting, as shown in Fig.
7. The peak of NHE3 precipitated with
anti-carboxyl-terminal NHE3 mAb 2B9 (top panel, arrow) had a
sedimentation coefficient of s20,w = 9.6. In contrast, the anti-megalin mAb 10A3 co-immunoprecipitated NHE3 (bottom panel, asterisk) from sucrose fractions having a
sedimentation coefficient of s20,w = 21. Importantly, mAb 2B9 did not immunoprecipitate any NHE3 from the high
density fractions that were associated with megalin. The experiments
presented in Figs. 6 and 7 confirm that renal NHE3 exists in both
megalin-free and megalin-bound forms, the latter incapable of binding
mAb 2B9.

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Fig. 7.
Sucrose velocity gradient centrifugation
identifies two pools of NHE3. Rabbit renal cortical microsomes
were solubilized in 1% Triton X-100 in MES buffer (pH 7.4). A
200,000 × g supernatant was applied to the top of
5-25% sucrose gradients that were then centrifuged at 40,000 rpm for
12 h in a Ti41 rotor. 1-ml fractions were collected across the
gradient. One-half of each gradient was subjected to
immunoprecipitation with either anti-NHE3 mAb 2B9 (top
panel) or the anti-megalin mAb 10A3 (bottom panel).
Immune complexes were prepared for immunoblotting, and the blots were
probed using an anti-NHE3 polyclonal antibody. The location of protein
standards is shown at the bottom.
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The NHE3 Megalin Association Is Not
Ca2+-dependent--
Megalin has been shown to
be an important Ca2+-binding protein in the kidney (43). In
fact, binding of most of the reported ligands for megalin, including
that of the receptor-associated protein (44), has been shown to be
Ca2+-dependent. Therefore, we sought to
determine if the binding of NHE3 to megalin required Ca2+
or if this interaction represented a different class of association. As
shown in Fig. 8, co-precipitation of NHE3
by anti-megalin mAb 10A3 was unaffected by the presence or absence of
Ca2+ in the solubilization buffer. These data show that the
interaction of NHE3 with megalin is not
Ca2+-dependent.

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Fig. 8.
Association of NHE3 and megalin is not
Ca2+-dependent. Rabbit renal microsomes
(200 µg) were solubilized in TBS and 1% Triton X-100 containing
either 5 mM EDTA (1st lane) or 2 mM Ca2+ (2nd lane).
Immunoprecipitations were performed with mAb 10A3 using either the EDTA
or Ca2+ buffers throughout the procedure. Immune complexes
were collected, prepared for immunoblotting, and probed with goat
anti-NHE3. Monomeric NHE3 is seen as an 80-kDa band (arrow),
and an aggregate of NHE3 is seen near the top of the gel
(asterisk). Molecular weights, expressed as
10 3 × Mr, are presented on the
left.
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DISCUSSION |
In this study we have shown that the renal brush border
Na+/H+ exchanger (NHE3) exists as part of least
two distinct oligomeric units. In one form the
Na+/H+ exchanger has a sedimentation
coefficient of 9.6 S and can be immunoprecipitated by specific mAbs to
carboxyl-terminal epitopes (amino acids 702-832) within the
transporter. A second form of NHE3 has a sedimentation coefficient of
21 S and cannot be immunoprecipitated by the carboxyl-terminal mAbs.
This second form of the Na+/H+ exchanger is
part of a molecular complex which includes the brush border protein megalin.
Megalin, also called gp330 and Heymann nephritis antigen, was first
identified by Kerjaschki and Farquhar as the autoantigen for Heymann
nephritis in 1982 (45). These early studies, showing megalin to be
concentrated in the clathrin-coated pits of the renal brush border,
suggested that it might function as a receptor in the proximal tubule
(Ref. 46; for reviews see Refs. 36 and 47). This notion has been
supported by molecular cloning studies that show megalin to be a member
of the low density lipoprotein receptor gene family (34, 48). However,
although in vitro studies have shown megalin to be capable
of binding various ligands including plasminogen (49), albumin (38),
vitamin B12 complexes (40), and calcium (43), a complete understanding
of the renal function of this very large (>500 kDa) glycoprotein is
still incomplete.
Because our finding of an association between NHE3 and megalin was
unexpected, we sought to exclude carefully the possibility that our
observation was a result of experimental artifact. Specifically, we
wanted to rule out the possibilities that NHE3 and megalin were
co-precipitated because of indirect interactions with other poorly
soluble cellular elements such as the cytoskeleton and/or resulted from
promiscuous nonspecific binding of megalin to solubilized brush border
proteins due its proposed protein scavenger function (38). To address
these concerns, we demonstrated (see Figs. 5 and 7) that NHE3 could be
co-precipitated with megalin even from the high speed supernatant of
Triton X-100-solubilized renal microsomes. These are conditions that
should pellet any insoluble cytoskeletal elements. The interaction of
NHE3 and megalin did not appear to result from a general protein
scavenger function of megalin since we were unable to detect its
association with other cytoskeletal (villin) or membrane
(NaPi-2) proteins of the brush border.
The interaction of NHE3 with megalin may represent a novel class of
binding to megalin. The extracellular domain of megalin contains four
putative ligand-binding domains, each of which consists of
cysteine-rich repeats that are characteristic of the low density lipoprotein receptor gene family (34). To date, the best characterized binding partner of megalin is receptor-associated protein, which binds
with high affinity in a Ca2+-dependent manner
and competes with all previously known ligands for megalin (35). Recent
studies by Orlando and co-workers (35) have identified the second
cluster of ligand-binding repeats (specifically in the region of amino
acids 1111-1210) as the probable binding domain for
receptor-associated protein and many, if not most, of the known
ligands. The fact that in our study the interaction of NHE3 with
megalin is not Ca2+-dependent suggests that
this interaction may be mediated through a different binding domain
within megalin. The fact that epitopes within the carboxyl-terminal
hydrophilic domain of NHE3 (amino acids 702-832) are blocked in this
complex raises the possibility that this region of NHE3 represents part
of the domain mediating association with megalin itself or linking protein(s).
Although both NHE-RF and E3KARP are thought to interact with NHE3, the
direct relationship of these proteins with either NHE3 or megalin in
kidney is not known. Lamprecht and co-workers (18) have suggested that
these proteins function as adapters that link NHE3 to cytoskeletal
elements such as ezrin. Since ezrin binds protein kinase A type II and
since NHE3 is phosphorylated by protein kinase A, these authors propose
that such linkage is a mechanism whereby protein kinase A is brought
into close proximity to NHE3. In future studies, it will be important
to determine if NHE-RF and/or E3KARP are part of either the 9.6 S or
21 S pools of NHE3.
The fact that there is a significant pool of NHE3 in the proximal
tubule that is inaccessible to antibodies raised to regions of the
carboxyl terminus of the transporter raises questions regarding previously published studies that immunolocalized NHE3 in the kidney
(7, 9, 11). Since all of these studies utilized antibodies raised to
the carboxyl terminus of NHE3, there is a distinct possibility that
these studies did not detect all of the NHE3 present in the proximal
tubule. In particular, the inability of available anti-NHE3 antibodies
to detect NHE3 associated with megalin may explain the absence of
detectable staining for NHE3 in coated pits and coated vesicles in
previous studies (9). A complete description of the localization of
NHE3 in the proximal tubule will require immunocytochemical studies
using antibodies that are capable of binding both free and
megalin-bound forms of the brush border Na+/H+
exchanger. Such future studies will be important for evaluating the
possibility that association with megalin is involved in the described
regulation of NHE3 activity by endocytosis in proximal tubule cells
(50, 51).