A basolateral sorting signal is encoded in the alpha -subunit of Na-K-ATPase

T. R. Muth1, C. J. Gottardi1, D. L. Roush1, and M. J. Caplan2

Departments of 1 Cell Biology and 2 Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -subunits of these heterodimeric pumps to identify the protein domains responsible for their polarized sorting. A chimeric alpha -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 alpha -subunit contain a basolateral sorting signal. H-K-ATPase beta -subunit expressed alone in LLC-PK1 cells accumulates at the apical surface. When coexpressed with N519H, the H-K-ATPase beta -subunit assembles with this chimera and accompanies it to the basolateral surface. Thus the NH2-terminal basolateral signal in the Na-K-ATPase alpha -subunit masks or is dominant over any apical sorting information present in the beta -polypeptide. In gastric parietal cells, the H-K-ATPase beta -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 beta -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 beta -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

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -subunit (62% identical between H-K-ATPase and Na-K-ATPase), which is predicted to span the membrane 10 times, and a beta -subunit (31% identity) which spans the membrane one time in a type II orientation (23, 30). Both alpha -subunits must be associated with their respective beta -subunits to exit the endoplasmic reticulum (ER) and be processed along the secretory pathway (12, 14). The beta -subunits appear to associate with residues in COOH-terminal portions of the alpha -subunits, and there is little evidence for either alpha -subunit forming stable interactions with the beta -subunit of the other pump species when expressed in mammalian cells (14). Despite the similar structure of the alpha - and beta -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 alpha -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 alpha -subunit (13). Additionally, it was noted that the beta -subunit of H-K-ATPase contains apical sorting information distinct from that embodied within the NH2-terminal portion of the H-K-ATPase alpha -subunit (13).

The fact that apical sorting information is present in the NH2-terminal portion of the H-K-ATPase alpha -subunit suggested the possibility that the NH2-terminal portion of the Na-K-ATPase alpha -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 alpha -subunit appears to encode basolateral sorting information, because the N519H alpha -subunit chimera accumulates at the basolateral surface of LLC-PK1 cells. The H-K-ATPase beta -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 beta -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 beta -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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Plasmid construction. Generation of the cDNA encoding N519H has been described (14). Briefly, the full-length rat Na-K-ATPase alpha -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 alpha -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 beta -constructs has been previously described (14).

Cell culture and transfection. LLC-PK1 cells were grown in alpha -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 beta -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.

To raise intracellular cAMP levels, cells were incubated with 10 µM forskolin (Sigma) and 0.2 mM IBMX (Sigma) diluted from 1,000× stocks in dimethyl sulfoxide (DMSO). Cells incubated for 0 h with forskolin and IBMX were treated with the same concentration of the DMSO vehicle for 18 h.

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.

For surface immunofluorescence with the H-K-ATPase beta -subunit antibody (gift of D. Chow and J. Forte, University of California, Berkeley), cells were washed three times in PBS-Ca-Mg and then incubated with the antibody diluted 1:100 in PBS-Ca-Mg for 4-6 h at 4°C. The cells were then fixed in 4.0% paraformaldehyde. Permeabilization, blocking in GSDB, and incubation with secondary antibody were carried out as described above.

All images were generated with a Zeiss LSM 410 laser scanning confocal microscope. Contrast and brightness settings were chosen to ensure that all pixels were within the linear range. Images are the product of eightfold line averaging. The xz cross sections were produced using a 0.2-µm motor step.

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 alpha -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 alpha -subunit mRNA, we generated an Xba I-Bgl II fragment from the Na-K-ATPase alpha -subunit cloned into the Bluescript vector. This fragment is derived from the 3'-end of the Na-K-ATPase alpha -subunit, which is not present in the N519H chimeric construct. For detection of the H-K-ATPase beta -subunit mRNA, we generated a Mlu I-Xba I fragment from the H-K-ATPase beta -construct cloned into the pCB6 vector. This DNA fragment has little homology with the Na-K-ATPase beta -subunit and does not hybridize with endogenous Na-K-ATPase beta -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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -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 alpha -subunit and the complementary COOH-terminal sequences of the H-K-ATPase alpha -subunit. The H519N chimera derives its first 519 amino acids from the H-K-ATPase alpha -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 beta -subunit were generated using a standard calcium phosphate precipitate transfection protocol (29). Cotransfection with the H-K-ATPase beta -subunit was required, because we have previously shown that this alpha -subunit chimera must assemble with the H-K-ATPase beta -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 alpha -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 beta -subunit is detected at both surfaces (Fig. 2B). We have previously found that the H-K-ATPase beta -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 alpha -chimera, the H-K-ATPase beta -subunit appears to be present at both surfaces. These data suggest that any apical sorting information present in the H-K-ATPase beta -subunit is either masked or overridden by a basolateral signal in the N519H chimera. According to this interpretation, those H-K-ATPase beta -subunit proteins not associated with the alpha -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 beta -subunit.


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Fig. 1.   Wild-type and chimeric constructs. N519H and H519N chimeras were created by exchanging the NH2-terminal halves of the H-K-ATPase and Na-K-ATPase alpha -subunits. Fusion point resides within putative ATP binding site. This diagram shows predicted 10 membrane-spanning domains of both native alpha -subunits and predicted structure of chimeras created from these proteins. Segments shown in gray represent H-K-ATPase, and segments shown in black represent Na-K-ATPase.


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Fig. 2.   N519H and H-K-ATPase beta -subunits can be biotinylated at basolateral (Bl) membranes of polarized LLC-PK1 cells. LLC-PK1 cells stably expressing both N519H chimera and H-K-ATPase beta -subunit were seeded at confluent density and maintained until day of experiment. Two filter monolayers were biotinylated, 1 apically and 1 basolaterally, according to method of Lisanti et al. (23a) with minor modifications. Cells were then solubilized in 1.0% Triton X-100, and biotinylated proteins were precipitated with streptavidin-agarose and subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and subsequent Western blot analysis. A: blot probed with antibody (HK2) directed against H-K-ATPase. B: same blot as in A, reprobed with anti-H-K-ATPase beta -subunit antibody. Both N519H chimera and H-K-ATPase beta -subunit are located predominantly at Bl surface. H-K-ATPase beta -subunit protein, presumably unassociated with N519H chimera, is also detected at apical (Ap) surface.

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 beta -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 alpha -subunit and which is thus able to detect both the N519H chimera and endogenous Na-K-ATPase alpha -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 beta -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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Fig. 3.   N519H is present at Bl surface in stably transfected LLC-PK1 cells. Untransfected LLC-PK1 cells or LLC-PK1 cells stably transfected with N519H chimera and H-K-ATPase beta -subunit (HKbeta ) were grown to confluence on Costar Transwell filter supports and then fixed and probed with 6H antibody, which recognizes both the N519H chimera and endogenous Na-K-ATPase. Confocal xz cross sections of untransfected cell monolayers reveal that endogenous Na-K-ATPase is detected at Bl surface (A). Absence of any significant staining from Ap surface of cells expressing N519H suggests that this chimera is restricted to Bl surface as well (B). Ap and Bl surfaces are indicated with arrows.

Because the N519H chimera at the cell surface is complexed with the H-K-ATPase beta -subunit, we used an antibody directed against the beta -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 beta -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 beta -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 beta -subunit (Fig. 4, C and D). This observation is consistent with the interpretation that H-K-ATPase beta -subunit unassociated with N519H is directed to the apical surface, whereas the H-K-ATPase beta -subunit associated with the N519H alpha -chimera is found at the basolateral membrane.


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Fig. 4.   H-K-ATPase beta -subunit staining in LLC-PK1 cells stably transfected with H-K-ATPase beta -subunit and N519H chimera. Cells were grown as described for Fig. 3 and then incubated with antibody directed against H-K-ATPase beta -subunit in both Ap and Bl media compartments for 4 h at 4°C. Cells were then fixed, and bound immunoglobin was detected with fluorescein isothiocyanate-conjugated secondary antibody. When viewed both en face (A and C) and in xz cross section (B and D), it is apparent that distribution of H-K-ATPase beta -subunit is exclusively Ap in singly transfected cells (C and D), whereas it is Ap and Bl in cells that coexpress N519H chimera (A and B). Arrows denote position of Ap and Bl surfaces.

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 beta -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 beta -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 beta -subunit. Time course experiments demonstrate that an increase in the population of H-K-ATPase beta -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 beta -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 alpha -chimera (data not shown).


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Fig. 5.   Effect of forskolin and 3-isobutyl-1-methylzanthine (IBMX) on surface expression of N519H chimera. Cells were grown to confluence on Costar 24-mm Transwell filters. Forskolin (10 µM) and IBMX (0.2 mM) were added 0, 2, or 18 h before biotinylation. Cells were biotinylated from Ap or Bl surface as described in MATERIALS AND METHODS. Biotinylated proteins were recovered on streptavidin-agarose beads, separated by electrophoresis on a 10% SDS-polyacrylamide gel, and then transferred to nitrocellulose. N519H chimera was detected with HK2 antibody via enhanced chemiluminescence technique. Lanes are labeled according to incubation time, with forskolin and IBMX (0, 2, or 18) and surface which was biotinylated. Arrow marks position of 100-kDa N519H chimera.

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 beta -protein but has no effect on the expression of the H519N chimera in cells transfected with this construct (Fig. 6).


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Fig. 6.   Expression of N519H in cells treated with forskolin and IBMX. Cells were grown on 24-mm Costar Transwell filters as described in Fig. 5. After cells reached confluence, cell lysates were prepared, separated on an SDS-polyacrylamide gel, and transferred to nitrocellulose. Blots were probed with antibodies directed against 2 alpha -subunit chimeras and H-K-ATPase beta -subunit. Length of time cells were incubated with forskolin and IBMX is indicated above each lane.

To test the possibility that forskolin and IBMX lead to increased accumulation of the mRNAs encoding the chimera and H-K-ATPase beta -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 beta -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 alpha -subunit polypeptide (Fig. 7). Taken together, these results suggest that forskolin and IBMX stimulate the expression of the H-K-ATPase beta -subunit and N519H but not that of the Na-K-ATPase alpha -subunit or the H519N chimera.


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Fig. 7.   Effect of forskolin (F) and IBMX (I) on expression of mRNA encoding N519H. mRNA was isolated from forskolin and IBMX-treated or untreated LLC-PK1 cells stably expressing N519H chimera and H-K-ATPase beta -subunit. RNA was separated by electrophoresis, transferred to nitrocellulose, and incubated with probes derived from cDNAs encoding N519H, H-K-ATPase beta -subunit, Na-K-ATPase alpha -subunit (NaKalpha ), and actin. Same blot was utilized with each of these probes, except for endogenous Na-K-ATPase alpha -subunit, for which a blot of total RNA was probed. In each case, right lane shows RNA from cells treated with forskolin and IBMX, and left lane shows RNA from untreated cells.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have previously shown that apical sorting information resides in the NH2-terminal half of the H-K-ATPase alpha -subunit (13). The H519N chimera used in that study was composed of the first 519 amino acids of H-K-ATPase alpha -subunit fused to the COOH-terminal 500 amino acids of Na-K-ATPase alpha -subunit. This protein associates with the endogenous Na-K-ATPase beta -subunit and brings it to the apical surface of LLC-PK1 cells. We also found that the H-K-ATPase beta -subunit is capable of exiting the ER without forming a heterodimeric complex with an alpha -subunit (14). In LLC-PK1 cells, the H-K-ATPase beta -subunit expressed alone is present primarily at the apical surface, indicating the possible presence of apical sorting information within the beta -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 alpha -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 beta -protein to indirectly assess the chimera distribution. We find that there is H-K-ATPase beta -subunit staining at the basolateral surface of LLC-PK1 cells stably coexpressing N519H and H-K-ATPase beta -subunit (Fig. 4, A and B). The basolateral staining is most likely due to H-K-ATPase beta -subunit associated with N519H chimera, because H-K-ATPase beta -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 beta -subunit seen in doubly transfected cells is thus likely to represent H-K-ATPase beta -protein which reaches the cell surface without associating with an alpha -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 beta -subunit. The presence of information in the NH2-terminal half of the alpha -subunit that is capable of redirecting a subordinate beta -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 alpha -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 alpha -subunit and associates with H-K-ATPase beta -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 beta -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 beta -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 beta -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 beta -subunit. Finally, no stimulation of actin or Na-K-ATPase alpha -mRNA expression was observed (Fig. 7). Our observations suggest the possibility that sequences associated with the H-K-ATPase beta -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 beta -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 alpha -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 beta -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 beta -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 beta -subunit and N519H expression reflects mechanisms that may be of physiological relevance.

    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.

    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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Results
Discussion
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