Departments of 1 Cell Biology and 2 Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510
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ABSTRACT |
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Na-K-ATPase and H-K-ATPase are highly homologous ion pumps that
exhibit distinct plasma membrane distributions in epithelial cells. We
have studied the -subunits of these heterodimeric pumps to identify
the protein domains responsible for their polarized sorting. A chimeric
-subunit construct (N519H) was generated in which the first 519 amino acid residues correspond to the Na-K-ATPase sequence and the
remaining 500 amino acids are derived from the H-K-ATPase sequence. In
stably transfected LLC-PK1 cell
lines, we found that the N519H chimera is restricted to the basolateral surface under steady-state conditions, suggesting that residues within
the NH2-terminal 519 amino acids
of the Na-K-ATPase
-subunit contain a basolateral sorting signal.
H-K-ATPase
-subunit expressed alone in
LLC-PK1 cells accumulates at the
apical surface. When coexpressed with N519H, the H-K-ATPase
-subunit
assembles with this chimera and accompanies it to the basolateral
surface. Thus the NH2-terminal
basolateral signal in the Na-K-ATPase
-subunit masks or is dominant
over any apical sorting information present in the
-polypeptide. In
gastric parietal cells, the H-K-ATPase
-subunit targets the
H-K-ATPase to an intracellular vesicular compartment which fuses with
the plasma membrane in response to secretagogue stimulation. To test
whether the chimera-H-K-ATPase
-subunit complex is directed to a
similar compartment in LLC-PK1 cells, we treated transfected cells with drugs that raise intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels.
Elevation of cytosolic cAMP increased the surface expression of both
the N519H chimera and the H-K-ATPase
-subunit. This increase in
surface expression, however, appears to be the result of
transcriptional upregulation and not recruitment of chimera to the
surface from a cAMP-inducible compartment.
cell polarity; protein chimera; ion pump; LLC-PK1 cells
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INTRODUCTION |
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EPITHELIAL CELLS PERFORM a number of physiological tasks that require specific proteins to reside solely in the apical or basolateral domain of the plasma membrane. Achieving this polarized distribution requires that newly synthesized proteins be sorted to their appropriate cellular destinations and be retained there after delivery (5, 25). The signals specifying a protein's location are believed to be embedded within its primary amino acid sequence or tertiary structure (25).
We have sought to identify the sorting signals that determine the
apical or basolateral targeting of the highly homologous H-K-ATPase and
Na-K-ATPase. The H-K-ATPase is found in gastric parietal cells, where
it serves to secrete acid into the lumen of the stomach (16, 17). The
Na-K-ATPase is found in almost every animal cell (32). In epithelial
cells, it generates the cation gradients required for vectorial fluid
and solute transport. The Na-K-ATPase and H-K-ATPase are each composed
of a catalytic -subunit (62% identical between
H-K-ATPase and Na-K-ATPase), which is predicted to span the membrane 10 times, and a
-subunit (31% identity) which spans the membrane one
time in a type II orientation (23, 30). Both
-subunits must be
associated with their respective
-subunits to exit the endoplasmic
reticulum (ER) and be processed along the secretory pathway (12, 14). The
-subunits appear to associate with residues in COOH-terminal portions of the
-subunits, and there is little evidence for either
-subunit forming stable interactions with the
-subunit
of the other pump species when expressed in mammalian cells (14).
Despite the similar structure of the
- and
-subunits of these
proteins, it has been shown in gastric parietal cells and in
transfected LLC-PK1 epithelial
cell lines that the H-K-ATPase is a resident of the apical plasma
membrane, whereas the Na-K-ATPase is located at the basolateral plasma
membrane (5, 31). The large degree of amino acid identity and predicted
structural conservation shared by the Na-K-ATPase and H-K-ATPase invite
the hypothesis that the sorting signals that direct the Na-K-ATPase and
the H-K-ATPase to opposite surfaces reside within those protein domains
that are substantially divergent. The high degree of homology also allows for the construction of chimeras that retain the general structure of the native pumps (14). The sorting behavior of an
-subunit chimera consisting of the first 519 amino acids from the
H-K-ATPase and the remaining amino acids from the Na-K-ATPase (H519N)
has been studied in transfected
LLC-PK1 cells. These studies suggested the presence of an apical sorting signal within the NH2-terminal half of the
H-K-ATPase
-subunit (13). Additionally, it was noted that the
-subunit of H-K-ATPase contains apical sorting information distinct
from that embodied within the
NH2-terminal portion of the
H-K-ATPase
-subunit (13).
The fact that apical sorting information is present in the
NH2-terminal portion of the
H-K-ATPase -subunit suggested the possibility that the
NH2-terminal portion of the
Na-K-ATPase
-subunit may contain a signal directing the Na-K-ATPase
to the basolateral surface. To test this hypothesis, we expressed a
chimera designed to be the reciprocal of H519N. In this chimera,
referred to as N519H, the first 519 amino acids are derived from the
Na-K-ATPase and the remaining COOH-terminal residues are from the
H-K-ATPase sequence (14). We found that the
NH2-terminal half of the
Na-K-ATPase
-subunit appears to encode basolateral sorting
information, because the N519H
-subunit chimera accumulates at the
basolateral surface of LLC-PK1
cells. The H-K-ATPase
-subunit, which is apically sorted when
expressed alone, interacts with the N519H chimera and is brought to the
basolateral surface of cotransfected
LLC-PK1 cells via this
interaction.
As mentioned above, the H-K-ATPase is found at the apical surface of
gastric parietal cells stimulated to secrete acid into the stomach. In
unstimulated parietal cells, the H-K-ATPase resides in subapical
tubulovesicular structures which can fuse with the apical surface after
histamine stimulation (11, 36). Interestingly, the H-K-ATPase
-subunit is also found in a subapical vesicular population when
expressed alone in LLC-PK1 cells
(13). Because the N519H chimera associates with the H-K-ATPase
-subunit, we wondered whether this complex might be recruited from
an intracellular pool to the surface of epithelial cells in response to
stimulation by secretagogues. We found that elevating intracellular
adenosine 3',5'-cyclic monophosphate (cAMP) levels with
forskolin and 3-isobutyl-1-methylzanthine (IBMX) results in a marked
increase in the population of N519H at the cell surface. This effect,
however, seems to be due to an increase in the level of mRNA coding for
the chimeric protein and is not the result of recruitment of chimera to
the surface from a vesicular storage pool.
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MATERIALS AND METHODS |
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Plasmid construction.
Generation of the cDNA encoding N519H has been described (14). Briefly,
the full-length rat Na-K-ATPase -subunit cDNA (kindly provided by E. Benz, Johns Hopkins University) was cloned into the Hind III
site (all restriction enzymes are from New England Biolabs, Beverly,
MA, unless noted otherwise) of the Bluescript vector (Promega, Madison,
WI). The H-K-ATPase cDNA (kindly provided by G. Shull, University of
Cincinnati) was subcloned into the Cla
I and Sma I sites of Bluescript. The
N519H chimera was generated by subcloning the 1.7-kilobase fragment
generated by digestion of Na-K Bluescript with
Nar I and
Cla I into Bluescript cut with Nar I and
Cla I. The
Cla
I-Nar I fragment insert includes the sequence encoding amino acids 1-519 of the Na-K-ATPase
-subunit. The full-length N519H chimeric cDNA was excised from the
Bluescript vector with Cla I and
Xba I and then directly subcloned into
the same sites of the pCB6 vector (kindly provided by M. Roth, D. Russell, and C. Brewer, University of Texas, Southwestern). The generation of the H519N and H-K-ATPase
-constructs has been
previously described (14).
Cell culture and transfection.
LLC-PK1 cells were grown in
-minimal essential medium (GIBCO BRL, Gaithersburg, MD) supplemented
with 10% fetal calf serum (Sigma, St. Louis, MO) and 50 U/ml each of
penicillin and streptomycin (GIBCO BRL) in a humidified incubator at
5% CO2. Transfections were
performed by using CaPO4
precipitation with 40 µg of cDNA (20 µg each of both N519H and
H-K-ATPase
-subunit cDNA) followed by selection in Geneticin
(0.45-0.9 g/l) (GIBCO BRL). Resistant colonies were isolated
2-3 wk after transfection and screened for expression by Western
blotting.
Immunofluorescence and microscopy.
LLC-PK1 cells were grown to
confluence on 24-mm Transwell filter supports (Corning-Costar,
Cambridge, MA). Cell monolayers were washed two times in
phosphate-buffered saline (PBS) with calcium (0.1 mM
CaCl2) and magnesium (1.0 mM
MgCl2) (PBS-Ca-Mg) and then
fixed in 20°C methanol for 9 min or in 4.0%
paraformaldehyde at room temperature for 30 min. Cells were rinsed one
time in PBS-Ca-Mg and then incubated for 15 min in cell
permeabilization buffer (PBS-Ca-Mg, 0.3% Triton X-100, and 0.1%
bovine serum albumin). The monolayers were then incubated in 1×
goat serum dilution buffer (GSDB; 16% filtered goat serum, 0.3%
Triton X-100, 20 mM sodium phosphate, pH 7.4, and 150 mM NaCl) for 30 min at room temperature to block nonspecific immunoglobulin G (IgG)
binding sites (4). Primary antibody solution was prepared by diluting
monoclonal antibody (MAb) 6H (1:100) in 1× GSDB. Filters were
excised from the filter support with a razor blade and placed on a
100-µl droplet of primary antibody solution resting on a piece of
Parafilm (American National Can, Neenah, WI). Incubations were
performed in a humidified chamber for either 1.5 h at room temperature
or overnight at 4°C. The filters were then washed three times for 5 min each in permeabilization buffer to remove unbound primary
antibodies and then incubated with fluorescein isothiocyanate
(FITC)-conjugated anti-mouse secondary antibody (Sigma) at a 1:100
dilution in 1× GSDB. Secondary incubations were for 1 h at room
temperature or overnight at 4°C. Filters were then washed three
times for 5 min each in permeabilization buffer and one time for 10 min
in 10 mM sodium phosphate, pH 7.5. Filters were mounted in freshly
prepared mounting solution (75% glycerol-PBS with 0.1%
p-phenylenediamine) or Vectashield
(Vector Laboratories, Burlingame, CA). A glass coverslip was placed
over the filter and sealed in place with nail polish.
Cell surface biotinylation and Western blot analysis. Biotinylation experiments were performed as described previously (15). Briefly, LLC-PK1 cells were grown to confluence on 24-mm Transwell filter supports as described for the immunofluorescence experiments. Cell monolayers were placed on ice and washed three times with PBS-Ca-Mg prechilled to 0°C. Either the apical or the basolateral surface of the monolayers was then incubated with N-hydroxysuccinimide-SS-biotin (Pierce, Rockford, IL) (1.5 mg/ml) in biotinylation buffer (10 mM triethanolamine, 150 mM NaCl, 2 mM CaCl2, pH to 9.0 with 1.0 N HCl) two times for 25 min each on ice continuously mixed with a gentle horizontal motion. The cell surface not exposed to biotin was bathed in biotinylation buffer. Unreacted biotin was then quenched by incubating monolayers with PBS-Ca-Mg containing 100 mM glycine for 20 min at 4°C. Filters were excised from the filter supports, and the cells were lysed in 1.0 ml of lysis buffer [1.0% Triton X-100, 150 mM NaCl, 5.0 mM EDTA, 50 mM tris(hydroxymethyl)aminomethane (Tris), pH 7.5] for 1 h on ice. Solubilized cells were scraped from the filters with a rubber policeman and centrifuged at 14,000 g for 10 min at 4°C. The supernatant was then transferred to a new microcentrifuge tube and incubated overnight at 4°C with 100 µl of washed ImmunoPure immobilized streptavidin-agarose beads (Pierce). The beads were washed three times with lysis buffer, two times with high-salt wash buffer (0.1% Triton X-100, 500 mM NaCl, 5.0 mM EDTA, 50 mM Tris, pH 7.5), and one time with no-salt wash buffer (10 mM Tris, pH 7.5). Proteins were then removed from the agarose beads by incubation in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer (80 mM dithiothreitol, 5.6% SDS, 0.008% bromphenol blue, 0.24 M Tris · HCl, pH 8.9, and 16% glycerol).
Proteins were separated by electrophoresis on a 10% polyacrylamide gel. Before loading, protein samples in sample buffer were heated to 80°C for 5 min. The gels were transferred to nitrocellulose, and the resultant blots were blocked in blocking buffer [5.0% powdered milk in Tris-buffered saline-Tween (20 mM Tris · HCl, 150 mM NaCl, pH 7.5, and 0.1% Tween-20)] for 1 h. Primary antibody incubations were carried out for 1.5 h in the blocking buffer with the appropriate antibody: MAb 6H (1:500), rabbit polyclonal HK2 (1:500), or rabbit polyclonal HK9 (1:250). Blots were washed three times for 15 min each in blocking buffer to remove unbound primary antibodies before addition of horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (1:1,000 in blocking buffer) for a 1-h incubation. Blots were then washed three times for 15 min each in wash solution (20 mM Tris · HCl, 150 mM NaCl, pH 7.5, and 0.1% Tween-20) before enhanced chemiluminescence (ECL) detection (Amersham) was performed. Total cell lysates were prepared from the cell monolayers washed three times in PBS-Ca-Mg. Excised filters were incubated with 1.0 ml of SDS-PAGE sample buffer. Lysates were scraped from the filters, and insoluble material was pelleted as described above. Lysates were heated to 80°C for 5 min before they were loaded on a 10% polyacrylamide gel.Northern blot analysis.
The RNA used in these experiments was from
LLC-PK1 cells maintained under
normal growth conditions with or without drug treatment. We used TRIzol
reagent (GIBCO BRL) to isolate RNA from the cells (after the
manufacturer's protocol). The RNA was then separated by mobility in an
agarose gel and transferred to nitrocellulose using a standard Northern
blot protocol. A Mlu
I-Xba I fragment was
excised from the 3'-end of the H-K-ATPase -subunit cDNA. This
fragment was used to generate a
[32P]dATP-labeled
oligonucleotide probe with the Ready-to-Go DNA labeling kit, according
to the manufacturer's protocol (Pharmacia, Uppsala, Sweden). This
probe was used to specifically detect mRNA encoding the N519H chimera,
which contains the 3'-half of the H-K-ATPase coding sequence. For
detection of the endogenous Na-K-ATPase
-subunit mRNA, we generated
an Xba
I-Bgl II fragment from the Na-K-ATPase
-subunit cloned into the Bluescript vector. This fragment is derived
from the 3'-end of the Na-K-ATPase
-subunit, which is not
present in the N519H chimeric construct. For detection of the
H-K-ATPase
-subunit mRNA, we generated a
Mlu
I-Xba I fragment from the H-K-ATPase
-construct cloned into the pCB6 vector. This DNA fragment has little
homology with the Na-K-ATPase
-subunit and does not hybridize with
endogenous Na-K-ATPase
-mRNA under these conditions. The actin probe
was kindly provided by K. Gillen and L. Roman (Yale University). The
mRNA blots were blocked for 1 h at 42°C in prehybridization
solution [6× saline sodium citrate (SSC),
0.5% SDS, 100 µg/ml salmon sperm DNA, 50% formamide, and 5×
Denhardt's solution in diethyl pyrocarbonate (DEPC)-treated distilled
deionized water]. After blocking, labeled probe was added to
hybridization solution (6× SSC, 0.5% SDS, 100 µg/ml salmon sperm DNA, 50% formamide in DEPC-treated distilled deionized water), and the blot was then incubated overnight at 42°C. The blot was washed for 15 min at room temperature in 1× SSC and 0.1% SDS for 15 min at 37°C in 1× SSC and 0.1% SDS for 15 min at room
temperature in 0.2× SSC and 0.1% SDS and finally for 15 min at
37°C in 0.2× SSC and 0.1% SDS. The blot was then wrapped in
cellophane and exposed to Hyperfilm-MP (Amersham, Arlington Heights,
IL) for 28 h.
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RESULTS |
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The N519H and H519N chimeras were generated to identify
the location of critical sorting information in the sequences of the H-K-ATPase and Na-K-ATPase -subunits (14). These two chimeras are
structurally reciprocal versions of one another. The N519H construct is
composed of the first 519 amino acid residues of the Na-K-ATPase
-subunit and the complementary COOH-terminal sequences of the
H-K-ATPase
-subunit. The H519N chimera derives its first 519 amino acids from the H-K-ATPase
-subunit, whereas the remainder are
contributed by the Na-K-ATPase (Fig. 1).
The fusion point resides within the putative ATP binding site, which is
highly conserved between the two pumps.
LLC-PK1 cells stably expressing
both the N519H chimera and the H-K-ATPase
-subunit were generated
using a standard calcium phosphate precipitate transfection protocol
(29). Cotransfection with the H-K-ATPase
-subunit was required,
because we have previously shown that this
-subunit chimera must
assemble with the H-K-ATPase
-subunit to exit the ER and reach the
cell surface (14). The distribution of the N519H chimera was initially
examined by cell surface biotinylation (Fig.
2). The
LLC-PK1 cells were grown to
confluence on Transwell polycarbonate filter supports and were
biotinylated according to methods described in detail elsewhere (15,
31). The cells were then lysed in 1.0% Triton X-100, and the
biotinylated cell surface proteins were isolated with streptavidin
beads, separated by SDS-PAGE, and transferred electrophoretically to
nitrocellulose for Western blotting. The distribution of N519H chimera,
detected using the HK2 antibody (14) which is specific for an epitope in the COOH-terminal half of the H-K-ATPase
-subunit (amino acid residues 565-585), is shown in Fig. 2. The HK2 antibody only
recognizes the N519H chimera and does not cross-react with the
endogenous Na-K-ATPase. As can be seen in Fig. 2, the chimera is
biotinylated predominantly at the basolateral surface, with only a
small fraction detectable at the apical surface (Fig.
2A). The H-K-ATPase
-subunit is
detected at both surfaces (Fig. 2B).
We have previously found that the H-K-ATPase
-subunit stably
expressed by itself in LLC-PK1 is
predominantly directed to the apical surface (13). In contrast, when it
is associated with the N519H
-chimera, the H-K-ATPase
-subunit
appears to be present at both surfaces. These data suggest that any
apical sorting information present in the H-K-ATPase
-subunit is
either masked or overridden by a basolateral signal in the N519H
chimera. According to this interpretation, those H-K-ATPase
-subunit
proteins not associated with the
-chimera are delivered to the
apical surface. The small quantity of N519H detected at the apical
surface by biotinylation is also likely to be associated with the
H-K-ATPase
-subunit.
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We have previously shown that quantitative biotinylation of proteins at the apical surface domain is difficult to achieve in LLC-PK1 cells (15). Although we used conditions that we have previously found to maximize the availability of apical proteins in LLC-PK1 to surface biotinylation (pH 9.0 and salt concentrations of 250 mM), the possibility that an apical pool of N519H is underrepresented in our biotinylation experiments cannot be excluded. Indirect immunofluorescence was used as an additional method to assess the distribution of the N519H chimera in LLC-PK1.
Cells expressing both N519H and the H-K-ATPase -subunit were grown
as described for the biotinylation experiments. Unfortunately, the HK2
antibody, which is specific for the N519H chimera, does not effectively
label the chimera under conditions used for immunofluorescence and
therefore could not be used in these studies. Instead, we used the 6H
antibody, which recognizes an epitope at the extreme NH2 terminus of the Na-K-ATPase
-subunit and which is thus able to detect both the N519H chimera and
endogenous Na-K-ATPase
-subunits (14). Because the endogenous
Na-K-ATPase is strictly basolateral in
LLC-PK1 cells, any apical
reactivity detected with 6H would indicate the presence of the N519H
chimera at the apical surface. Filters were prepared for
immunofluorescence as described in MATERIALS AND
METHODS. As expected, the endogenous Na-K-ATPase in
untransfected LLC-PK1 is present
exclusively at the basolateral membrane (Fig. 3A). The
same distribution of 6H reactivity is found in
LLC-PK1 cells stably cotransfected
with N519H and H-K-ATPase
-subunit (Fig.
3B). These cells manifest little or
no 6H staining at their apical surfaces or in intracellular
compartments, demonstrating that the chimera must be predominantly
restricted to the basolateral surface.
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Because the N519H chimera at the cell surface is complexed with the
H-K-ATPase -subunit, we used an antibody directed against the
-subunit (HKB) as an additional tool with which to probe the
localization of the chimera (14). The HKB antibody reacts with an
ectodomain epitope of H-K-ATPase
-subunit, and thus this antibody
could be used for surface immunofluorescence on intact cells. For this
experiment, filter-grown cells were first washed in PBS-Ca-Mg and then
incubated with the HKB antibody at 4°C for 4-6 h before
fixation with 4% paraformaldehyde. The incubation was performed at
4°C to prevent the redistribution of antibody-bound surface
proteins. H-K-ATPase
-subunit staining can be detected at both the
apical and basolateral surfaces in chimera-expressing cells (Fig.
4, A and
B). In contrast, only apical labeling is found in cells
that express exclusively H-K-ATPase
-subunit (Fig. 4,
C and
D). This observation is consistent
with the interpretation that H-K-ATPase
-subunit unassociated with
N519H is directed to the apical surface, whereas the H-K-ATPase
-subunit associated with the N519H
-chimera is found at the
basolateral membrane.
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In the resting state, the H-K-ATPase of gastric parietal cells is
located in a subapical vesicular population, from which it can be
recruited to the apical surface under conditions that lead to a rise in
the intracellular cAMP levels (7, 10). We have recently shown that the
sorting information required to return the H-K-ATPase pump to the
intracellular storage compartment is embodied within a tyrosine-based
signal present in the cytoplasmic tail of the H-K-ATPase -protein
(8). Like the H-K-ATPase, the aquaporin 2 water channel of renal
principal cells is stored in an intracellular compartment which fuses
with the apical surface in response to stimulation of the ADH receptor
(3, 19). When this protein is expressed in
LLC-PK1 cells, it is retained in an intracellular storage compartment and delivered to the cell surface
in response to elevations in cytosolic cAMP, similar to its behavior in
its native principal cells (19). We wondered whether the surface
delivery of N519H/H-K-ATPase
-complexes could similarly be
stimulated by elevating cytosolic cAMP. To examine this possibility, we
used forskolin and IBMX to raise the levels of cAMP in
LLC-PK1 cells coexpressing N519H
and H-K-ATPase
-subunit. Time course experiments demonstrate that an
increase in the population of H-K-ATPase
-subunit and N519H
available to surface biotinylation can be seen after 2 h of exposure to
forskolin and IBMX (Fig. 5). The signal
continues to increase with time, reaching a maximum level after
16-18 h. It is important to note that the increased expression of
N519H does not affect its ability to accumulate predominantly at the
basolateral cell surface (Fig. 5). Similar results are also seen when
cells expressing N519H and H-K-ATPase
-subunit are treated with the
membrane-permeant cAMP analog dibutyryl cAMP, supporting the hypothesis
that the effect is due to an increased level of cAMP (data not shown).
Treatment with forskolin and IBMX had no apparent effect on the surface
expression of endogenous Na-K-ATPase or the H519N
-chimera (data not
shown).
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The biotinylation experiment does not differentiate between the
delivery of a pool of previously synthesized N519H to the basolateral
cell surface or an increase in the overall level of N519H within the
cells. The 2-h lag before any increase at the cell surface is seen
could reflect upregulation of N519H transcription, followed by its
synthesis and transport through the secretory pathway to the cell
surface. To differentiate between these two possibilities, we examined
whether there was an increase in the total amount of N519H protein
produced in response to forskolin and IBMX. The total amount of
cell-associated N519H was measured by probing Western blots of whole
cell lysates using the HK2 antibody. As can be seen in Fig.
6, forskolin and IBMX exposure induces a
time-dependent increase in the level of N519H (Fig. 6). This observation argues against the idea that N519H in vesicular elements is
recruited to the cell surface upon raising the levels of cAMP within
the cells. The forskolin and IBMX treatment also increases the pool of
cell-associated H-K-ATPase -protein but has no effect on the
expression of the H519N chimera in cells transfected with this
construct (Fig. 6).
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To test the possibility that forskolin and IBMX lead to increased
accumulation of the mRNAs encoding the chimera and H-K-ATPase -subunit, we performed Northern blot analysis. The experiments reveal a marked elevation of the levels of mRNA coding for the N519H
chimera and H-K-ATPase
-subunit in cells treated with forskolin and
IBMX (Fig. 7). Levels of actin mRNA did not
show any marked increase in the treated cells, nor was there an effect
on the level of mRNA encoding the endogenous Na-K-ATPase
-subunit
polypeptide (Fig. 7). Taken together, these results suggest that
forskolin and IBMX stimulate the expression of the H-K-ATPase
-subunit and N519H but not that of the Na-K-ATPase
-subunit or
the H519N chimera.
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DISCUSSION |
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We have previously shown that apical sorting information resides in the
NH2-terminal half of the
H-K-ATPase -subunit (13). The H519N chimera used in that study was
composed of the first 519 amino acids of H-K-ATPase
-subunit fused
to the COOH-terminal 500 amino acids of Na-K-ATPase
-subunit. This
protein associates with the endogenous Na-K-ATPase
-subunit and
brings it to the apical surface of
LLC-PK1 cells. We also found that
the H-K-ATPase
-subunit is capable of exiting the ER without forming
a heterodimeric complex with an
-subunit (14). In
LLC-PK1 cells, the H-K-ATPase
-subunit expressed alone is present primarily at the apical surface, indicating the possible presence of apical sorting information within
the
-subunit protein (13). In the current study, we have extended
these initial findings by demonstrating that there is basolateral
sorting information encoded within the
NH2-terminal half of Na-K-ATPase
-subunit.
Using cell surface biotinylation, we were able to detect the N519H
chimera predominantly at the basolateral surface of
LLC-PK1 (Fig. 2). Because the
biotinylation of apical proteins may not be efficient in
LLC-PK1 cells, we carried out
indirect immunofluorescence experiments, which also indicated that the
majority of the N519H chimera is present at the basolateral surface.
The 6H antibody, which recognizes the N519H chimera, showed an
exclusively basolateral staining pattern in transfected cells (Fig. 3).
Finally, we examined the distribution of the H-K-ATPase -protein to
indirectly assess the chimera distribution. We find that there is
H-K-ATPase
-subunit staining at the basolateral surface of
LLC-PK1 cells stably coexpressing N519H and H-K-ATPase
-subunit (Fig. 4,
A and
B). The basolateral staining is most
likely due to H-K-ATPase
-subunit associated with N519H chimera,
because H-K-ATPase
-subunit expressed alone is located at the apical
surface of LLC-PK1 cells (Fig. 4,
C and D). The apical pool of H-K-ATPase
-subunit seen in doubly transfected cells is thus likely to
represent H-K-ATPase
-protein which reaches the cell surface without
associating with an
-subunit. Taken together, these results strongly
suggest that N519H manifests basolateral sorting information.
Furthermore, this basolateral sorting signal is able to overcome or
mask whatever signals participate in the apical localization of the
singly transfected H-K-ATPase
-subunit. The presence of information
in the NH2-terminal half of the
-subunit that is capable of redirecting a subordinate
-subunit is
entirely in keeping with the previous studies of the H519N chimera. It
would appear therefore that both the Na-K-ATPase and the H-K-ATPase are
actively sorted to their respective destinations by virtue of sorting
signals that reside within the ~500
NH2-terminal amino acids of their
-subunits.
The H-K-ATPase holoenzyme is found in subapical tubulovesicular
compartments in unstimulated gastric parietal cells (33). When these
cells are stimulated with histamine, the tubulovesicular compartments
fuse with the apical surface, allowing the H-K-ATPase to pump
H+ ions into the lumen of the
stomach (11). We wondered whether the N519H chimera, which possesses
the COOH-terminal half of the H-K-ATPase -subunit and associates
with H-K-ATPase
-subunit, could also be recruited to the basolateral
cell surface from vesicular stores within the cell. This behavior has
been previously noted for aquaporin 2 expressed by transfection in
LLC-PK1 cells (20). To test this
possibility, we raised the intracellular levels of cAMP with forskolin
and IBMX, because cAMP is the same second messenger induced by
histamine stimulation of parietal cells (16). These agents do, in fact,
cause an increase in N519H detectable at the basolateral surface (Fig.
5). However, because there was an increase in the size of the total
pool of N519H associated with the cells, the increase at the surface
appeared not to reflect a recruitment of preexisting chimera to the
surface from a vesicular population, but rather an overall increase in
expression (Fig. 6). Consistent with this interpretation, we detected
increased levels of N519H and H-K-ATPase
-mRNA in a Northern blot
analysis (Fig. 7).
In light of the evidence presented here, it seems likely that the
cAMP-induced increases in H-K-ATPase -subunit and N519H expression
are due to increased transcription of the mRNAs encoding these
proteins. Although cAMP increases the expression of transfected N519H
and H-K-ATPase
-subunit, no such stimulation (as judged by total
cellular protein levels) is observed with the chimera H519N (Fig. 6).
Expression of the H519N is mediated by the same cytomegalovirus
(CMV)-based vector as is utilized with N519H and the H-K-ATPase
-subunit. Finally, no stimulation of actin or Na-K-ATPase
-mRNA
expression was observed (Fig. 7). Our observations suggest the
possibility that sequences associated with the H-K-ATPase
-subunit
and N519H cDNAs themselves are responsible for the cAMP-induced upregulation.
Histamine stimulation, acting through elevations in cytosolic Ca and
cAMP, increases expression of the H-K-ATPase subunits in gastric
parietal cells (1, 24, 28, 34). Changes in cAMP are thought to exert
effects on gene expression by affecting the function of specific
transcription factors. Many genes that manifest cAMP-regulated
expression possess canonical sequences that appear to act as cAMP
response elements (CREs) (22). Transcription factors sensitive to cAMP
bind to CREs and modulate the expression of their associated genes.
Consensus CREs have been found in genes encoding the H-K-ATPase and
Na-K-ATPase. In both cases, the CREs are present in sequences from the
5'-end to the transcription start site and thus are not included
in our cDNA constructs. The 3'-untranslated region of the
H-K-ATPase -subunit cDNA does encode a consensus CRE. Furthermore, a
7/8 match for a consensus CRE is present in the coding sequence of the
Na-K-ATPase
-subunit. This sequence is present in the cDNA encoding
N519H and is absent in the complementary chimera H519N. Evidence for
functional cis-acting transcriptional
regulatory elements embedded within transcribed sequences has been
found in a number of systems (2, 9, 18, 21, 26, 27, 35). If these
putative CREs are functional in the context of our expression vector,
it might explain why cAMP elevates the expression of H-K-ATPase
-subunit and N519H but not of H519N. Whether these putative CREs
play any role in regulating pump subunit expression in vivo remains to
be established.
It should be noted that the CMV promoter present in our expression
vector includes three consensus CRE sequences (6). Because all three
constructs used in this study utilize this promoter, it would be
difficult to explain the differential effect of cAMP on H519N versus
N519H and H-K-ATPase -subunit if the cAMP stimulation were entirely
referenceable to the intrapromoter CREs. Further experiments will be
required to determine whether the cAMP-induced elevation of H-K-ATPase
-subunit and N519H expression reflects mechanisms that may be of
physiological relevance.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Gary Shull, Dar Chow, John Forte, Michael Reuben, George Sachs, Ed Benz, Michael Roth, and Laura Roman for generously providing reagents. We also thank the members of the Caplan lab for their support and indispensable input during the writing of the manuscript.
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FOOTNOTES |
---|
This work was supported by National Institutes of Health Grant GM-42136 awarded to M. J. Caplan and predoctoral National Research Service Awards to T. R. Muth, C. J. Gottardi, and D. L. Roush.
Present address of C. J. Gottardi: Department of Cellular Biochemistry and Biophysics, Sloan-Kettering Institute, 1275 York Ave., Box 564, New York, NY 10021.
Address for reprint requests: T. R. Muth, Dept. of Cell Biology, Yale Univ. School of Medicine, 333 Cedar St., Sterling Hall of Medicine, BE-26, New Haven, CT 06510.
Received 10 March 1997; accepted in final form 25 November 1997.
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