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J. Biol. Chem., Vol. 275, Issue 24, 18219-18224, June 16, 2000

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The B1 Subunit of the H⁺ATPase Is a PDZ Domain-binding Protein

COLOCALIZATION WITH NHE-RF IN RENAL B-INTERCALATED CELLS^*

Sylvie Breton^§¶, Thorsten Wiederhold^**, Vladimir Marshansky, Ndona N. Nsumu, Vijaya Ramesh^§, and Dennis Brown

From the  Renal Unit and Program in Membrane Biology and the  Molecular Neurogenetics Unit, Massachusetts General Hospital East, Charlestown, Massachusetts 02129 and the Departments of  Pathology and ^§ Medicine, Harvard Medical School, Boston, Massachusetts 02114

Received for publication, December 13, 1999, and in revised form, March 2, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The 56-kDa B1 subunit of the vacuolar H⁺ATPase has a C-terminal DTAL amino acid motif typical of PDZ-binding proteins that associate with the PDZ protein, NHE-RF (Na⁺/H⁺ exchanger regulatory factor). This B1 isoform is amplified in renal intercalated cells, which play a role in distal urinary acid-base transport. In contrast, proximal tubules express the B2 isoform that lacks the C-terminal PDZ-binding motif. Both the B1 56-kDa subunit and the 31-kDa (E) subunit of the H⁺ATPase are pulled down by glutathione S-transferase NHE-RF bound to GSH-Sepharose beads. These subunits associate in vivo as part of the cytoplasmic V1 portion of the H⁺ATPase, and the E subunit was co-immunoprecipitated from rat kidney cytosol with NHE-RF antibodies. The interaction of H⁺ATPase subunits with NHE-RF was inhibited by a peptide derived from the C terminus of the B1 but not the B2 isoform. NHE-RF colocalized with H⁺ATPase in either the apical or the basolateral region of B-type intercalated cells, whereas NHE-RF staining was undetectable in A-intercalated cells. In proximal tubules, NHE-RF was located in the apical brush border. In contrast, H⁺ATPase was concentrated in a distinct membrane domain at the base of the brush border, from which NHE-RF was absent, consistent with the expression of the truncated B2 subunit isoform in this tubule segment. The colocalization of NHE-RF and H⁺ATPase in B- but not A-intercalated cells suggests a role in generating, maintaining, or modulating the variable H⁺ATPase polarity that characterizes the B-cell phenotype.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Membrane transport proteins are directed toward and inserted into specific cell surface domains by an elaborate series of sorting mechanisms (1). A relatively recent development in understanding how some of these proteins are concentrated into functionally differentiated regions of the plasma membrane has been the discovery of the so-called PDZ domain family of proteins and associated PDZ-binding proteins. Named after the initial three members of the family (PSD-95, Drosophila discs large protein, and ZO-1), PDZ proteins contain 80-100-amino acid stretches that allow them to interact with other proteins that have a four amino acid PDZ-binding cassette, usually at the extreme C terminus of the cytoplasmic domain (2). One such PDZ-binding cassette is the amino acid sequence D(S/T)XL. Thus, putative PDZ-binding proteins can be identified by screening their amino acid sequence for these concensus cassettes. Among the many proteins identified so far are the cystic fibrosis transmembrane conductance regulator (3), the ₂-adrenergic receptor (4), neuronal nitric-oxide synthase (5), GLUT1 (6), and some potassium channels (7-9). One PDZ protein to which the cystic fibrosis transmembrane conductance regulator (10), the ₂-adrenergic receptor, and P2Y1 receptor (4) bind is the Na⁺/H⁺ exchanger regulatory factor, NHE-RF, originally identified in rabbit kidney as a soluble factor that participates in the regulation of the NHE-3 Na⁺/H⁺ exchanger at the apical pole of proximal tubule epithelial cells (11, 12). NHE-RF also binds to the ERM family of actin-binding proteins via its C terminus outside the PDZ-binding motif (13, 14), thus potentially linking many ion channel and receptors to the actin cytoskeleton. Furthermore, NHE-RF has been reported to interact with the Na⁺/HCO₃ co-transporter, a basolateral protein in proximal tubules (15).

The B1 (56-kDa) subunit of the vacuolar proton-pumping ATPase (H⁺ATPase) has a C-terminal DTAL motif, which suggests that it is a candidate PDZ-binding protein. This subunit of the H⁺ATPase is expressed in several tissues, but it is strongly amplified in specialized proton-translocating intercalated cells in the kidney (16), as well as similar cells in the epididymis (17). In contrast, a highly homologous 56-kDa isoform, the B2 isoform, is expressed in the kidney proximal tubule, but it has a C-terminal truncation and lacks the terminal PDZ-binding cassette (16). Most if not all of the PDZ-binding proteins so-far reported are transmembrane proteins, but the B1 H⁺ATPase subunit is part of the V1 portion of the holo-enzyme and has no membrane spanning domain. It is tethered to the membrane by interaction with other subunits of the enzyme, some of which span the lipid bilayer (18). Furthermore, multi-subunit complexes consisting of the cytoplasmic portion of the H⁺ATPase can exist as free, cytosolic entities (18).

In kidney cortex, intercalated cells show a complex regulation of H⁺ATPase expression at the cell surface (19), and subpopulations of these cells with apical, basolateral, diffuse, or even bipolar H⁺ATPase localization can be detected by immunocytochemistry (20). A-cells always have H⁺ATPase at their apical pole and the Cl/HCO₃ exchanger AE1 at their basolateral pole (21). These cells secrete protons into the tubule lumen. B-cells have no detectable AE1 in either plasma membrane domain, but the H⁺ATPase can be apical, basolateral, or bipolar in these AE1-negative cells. Cells with basolateral H⁺ATPase are bicarbonate-secreting cells. Although systemic acidosis results in more intercalated cells having apical H⁺ATPase, and alkalosis shifts more cells to a basolateral pattern of localization (22, 23), the cell biological mechanisms underlying these dramatic shifts in the polarized expression of a membrane protein remain unknown in situ. In this report, we show that the 56-kDa B1 subunit of the proton pump is a PDZ-binding protein that can associate with NHE-RF and that NHE-RF colocalizes with the H⁺ATPase in all B-intercalated cells, wherever the pump is located within any individual cell. We propose that the interaction of this subunit of the proton pump with NHE-RF could be responsible for the anchoring and/or targeting of membrane-associated H⁺ATPase molecules in this cell type. NHE-RF was barely detectable in A-type intercalated cells. In contrast, NHE-RF was abundant in the proximal tubule brush border, but its intracellular location was clearly distinct from that of the H⁺ATPase in these cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Animals-- Male Harlan Sprague-Dawley rats were anesthetized with sodium pentobarbital (Nembutal, 0.1 ml of a 50 mg/ml solution/100 g of body weight), and kidneys were fixed by perfusion through the abdominal aorta with a fixative containing 4% paraformaldehyde, 10 mM sodium periodate, 70 mM lysine (PLP), and 5% sucrose as described previously (24, 25). After 5 min of perfusion, kidneys were removed, sliced, and fixed by immersion for a further 6 h before rinsing and storage in PBS¹ (10 mM sodium phosphate buffer containing 0.9% NaCl, pH 7.4). For preparation of 4-µm sections, tissues were cryoprotected in 30% sucrose before sectioning with a Reichert Frigocut microtome using disposable knives.

Immunostaining-- Tissue sections picked up on Fisher Superfrost Plus slides (Fisher Scientific) were rinsed for 10 min in PBS and then treated with 1% SDS for 5 min. This step augments antigenicity of many proteins in frozen sections of PLP-fixed tissues, as described previously (26). After three more rinses (5 min each) in PBS to remove the SDS, sections were incubated for 20 min in PBS/1% bovine serum albumin to block nonspecific background staining. Primary anti-NHE-RF antibody (affinity purified rabbit polyclonal antibody IC270 raised against GST-NHE-RF fusion protein amino acids 270-358) was applied for 2 h at room temperature at a dilution of 1:4. This antibody has been characterized previously (27). After washing twice for 5 min in high salt PBS (PBS containing 2.7% NaCl) to reduce nonspecific staining and one further washing for 5 min in normal PBS, secondary anti-rabbit antibodies (diluted 1:800) coupled to CY3 (Jackson Immunologicals) were applied for 60 min. After further washing as above, sections were mounted in Vectashield anti-fading solution (Vector Labs., Burlinghame, CA), diluted 1:1 in 0.1 M Tris-HCl, pH 8.0.

Some sections were double-stained with anti-H⁺ATPase antibodies to identify the cells that were positive for NHE-RF in the collecting duct. After application of the anti-NHE-RF antibody followed by secondary antibody coupled to CY3, an affinity purified chicken polyclonal antibody against the 31-kDa E subunit of the H⁺ATPase (a marker of A- and B-intercalated cells, diluted 1:40) was applied for 2 h, followed by a donkey anti-chicken IgG coupled to FITC, diluted 1:200 (Jackson Immunologicals). Sections were mounted in Vectashield diluted 1:1 in 0.1 M Tris-HCl, pH 8.0.

Some sections were double stained using a rabbit anti-AE1 Cl/HCO₃ exchanger antibody that has been previously characterized (28). Because this antibody is also raised in rabbit, an amplification procedure was used to allow staining of sections with two primary antibodies raised in the same species. Briefly, the first primary, anti-AE1, was applied at a dilution of 1:32,000, a concentration that is too low to be detected by conventional application of a fluorescent secondary antibody, as determined in preliminary experiments. The dilute AE1 antibody was detected using a tyramide amplification kit (NEN Life Science Products) with tyramide-CY3 as a fluorescent reagent, according to the manufacturer's instructions. The sections were then incubated conventionally with anti-NHE-RF and secondary goat anti-rabbit FITC as described above. No cross-reactivity between the two sets of reagents was detectable under these conditions. Sections were photographed in color on Kodak Ektachrome 400 Elite film exposed at 2500 ASA using a Nikon Eclipse 800 epifluorescence microscope equipped with specific CY3 and FITC filter combinations. Using the specific CY3 filter combination and analog photography, CY3 emission appears yellow (see Fig. 1, A and C). Some micrographs were prepared from digital images captured from the Nikon Eclipse 800 using a Hamamatsu Orca digital camera. Pseudocolored images were merged using IP Lab Spectrum software (Scanalytics Inc, Vianna). In these images, CY3 fluorescence appears red, and FITC is green (see Fig. 2). Control incubations were performed in which the primary NHE-RF antibody was incubated with the GST-NHE-RF fusion protein (at a final concentration of 0.2 mg/ml) for 1 h at room temperature prior to applying the antibody to the sections.

Preparation of Rat Kidney Inner Stripe Cytosol-- Kidneys of anesthetized rats were perfused with cold PBS for 1-2 min to remove blood, and the inner stripe was separated under a dissecting microscope. Pieces of inner stripe (0.4 g) were homogenized in 2 ml of homogenization buffer (0.25 M sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4, with Complete^TM, Roche Molecular Biochemicals, mixture of protease inhibitors) using a Wheaton glass potter fitted with a Teflon pestle (20 complete strokes). Rat kidney inner stripe cytosol was prepared by centrifugation of the homogenate for 1 h at 100,000 × g (41,000 rpm) using a Beckman, TL-100 Ultracentrifuge equipped with a TLA 55 rotor. Protein concentration of cytosol was measured with the Pierce BCA protein assay reagent using albumin as a standard.

Affinity Precipitation and Peptide Competition Assay-- Pull-down experiments were performed essentially as described previously (27) with minor modifications. Briefly, rat kidney inner stripe cytosol (0.4 mg of total protein) was incubated overnight at 4 °C with 600 pmol of GST-NHERF or GST alone as a control immobilized on GSH-Sepharose 4B beads. For the peptide competition assay, peptides B1 (PQDTEADTAL) and B2 (EFYPRDSAKH) were dissolved in distilled water, and 300 µg of peptide were preincubated with the GST-NHE-RF beads for 1 h at 4 °C. Cytosol containing 300 µg of peptide was then added to the beads, and the mixture was incubated overnight at 4 °C. The beads were then washed extensively with phosphate-buffered saline containing Pefabloc and resuspended in Laemmli sample buffer for SDS-PAGE.

Immunoprecipitation Assay-- Rat kidney cytosol was prepared as for the pull-down assay, except that cytosol from the entire cortex and outer medulla was used to ensure that adequate amounts of NHE-RF were present in the preparation. NHE-RF was immunoprecipitated from 500 µl (2.8 mg of total protein) of precleared (by preincubation with protein A-agarose beads alone) cytosol using 10 µl of anti-NHE-RF antiserum (IC270 serum or IC270 preimmune serum) and 50 µl of protein A-agarose beads (Roche Molecular Biochemicals). Immunoprecipitates were washed extensively with PBS and eluted by boiling in SDS sample buffer (as for the pull-down assay). The immunoprecipitates were run on SDS-PAGE and blotted using a monoclonal anti-E subunit antibody (E11) as described below.

SDS-PAGE and Western Blot Analysis-- Electrophoresis was performed using 12% SDS-Tris-glycine-PAGE gels. Proteins were transferred to polyvinylidene difluoride membranes and analyzed by Western blotting. The following antibodies were used as detailed in the figure legends: affinity-purified chicken polyclonal anti-E H⁺ATPase subunit (1:1,000) and affinity-purified rabbit polyclonal anti-B1 H⁺ATPase subunit (1:1,000), raised against the bovine subunits, and a monoclonal anti-E subunit antibody (E11) kindly provided by Dr. Steven Gluck (University of Florida, Gainesville). Images were scanned and analyzed with NIH Image (version 1.62) software.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Localization of NHE-RF in the Kidney-- NHE-RF was localized in the kidney on PLP-fixed cryostat sections that had been treated with SDS. As expected from the reported distribution of NHE-3 (29), by far the greatest amount of NHE-RF was found in proximal tubules. The entire brush border was strongly stained in all proximal tubule segments (Fig. 1, A and C). In addition, however, staining was seen in some cells of the cortical collecting ducts and connecting segments (Fig. 1, A and C). The extent and intracellular location of this staining was variable, with some cells showing distinct basolateral staining (Fig. 1A), others showing a more diffuse cytoplasmic staining (Fig. 1C), and yet others showing apical staining (Fig. 1C). These cells were identified as intercalated cells by double incubations using a chicken polyclonal antibody against the 31-kDa (E) subunit of the H⁺ATPase (Fig. 1, B and D). We have previously shown that the three major subunits of the cytoplasmic domain of this enzyme colocalize in intercalated cells (30). Thus, the E subunit is a reliable marker for the entire cytoplasmic domain of the H⁺ATPase, which also contains the 56 B kDa subunit. In these double-stained sections, the NHE-RF staining colocalized with the H⁺ATPase staining in intercalated cells, whereas connecting tubule cells that also contain the H⁺ATPase do not express detectable levels of NHE-RF (Fig. 1, C and D). However, in some intercalated cells, apical H⁺ATPase staining was seen with no detectable NHE-RF staining (Fig. 1A). Principal cells in some tubule segments showed a faint, finely granular staining in the cytoplasm that was much less intense than in the adjacent intercalated cells.

View larger version (47K): [in this window] [in a new window]   Fig. 1.   Sections of rat kidney cortex double stained for NHE-RF (A and C) and the 31-kDa subunit of the H+ATPase (B and D). The brush border is strongly stained in all proximal tubules. Some cells of the collecting ducts (CD) and connecting segments (CNT) are also stained. Different patterns of NHE-RF staining are detected, and the staining overlaps with that of the H+ATPase in intercalated cells. Some cells have basolateral staining (1), some have diffuse cytoplasmic staining (2), some have apical staining (3), and others have little or no NHE-RF staining but show apical H+ATPase staining (4). In connecting segments (CNT), all cells have strong H+ATPase staining, as described previously (30), but only a few of these are clearly NHE-RF-positive. The staining in A and C, which represent single exposures of the NHE-RF staining, appears yellow because of the use of a highly specific filter combination used for the CY3 fluorophore. Cy3 emission is yellow when the correct filter combination is used. Bar, 15 µm.

To distinguish between A- and B-intercalated cells, anti-AE1 antibodies, which label only A-cells (21), were used in conjunction with anti-NHE-RF antibodies. All cells with distinct apical, diffuse, or basolateral NHE-RF staining were negative for AE1 (Fig. 2). Furthermore, only A-intercalated cells are found in the inner stripe of the outer medulla, and these cells showed no detectable staining with the NHE-RF antibody (not shown). Thus, NHE-RF is most highly expressed in B-intercalated cells, and its variable intracellular localization pattern overlaps with that of the H⁺ATPase in each individual B-cell in the cortical collecting duct and connecting segment.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Double staining of rat kidney cortex collecting ducts with antibodies against NHE-RF (green) and AE1 (red). The red AE1 staining is basolateral in all A-intercalated cells. These cells do not stain for NHE-RF. In A, two cells with apical NHE-RF staining are found alongside an A-intercalated cell with basolateral AE1 staining. In B, two cells with basolateral NHE-RF staining are found in the same tubule as A-intercalated cells with basolateral AE1. Other cells are negative for both antigens. C shows a collecting duct with a mixture of AE1 positive/NHE-RF negative A-intercalated cells and NHE-RF-positive/AE1-negative B-intercalated cells. A small amount of punctate NHE-RF staining is seen in cells that are probably principal cells. In these merged digital images, CY3 staining is pseudocolored red to allow it to be more readily distinguished from the adjacent green FITC staining. Bar, 10 µm.

In proximal tubules, NHE-RF was present in the brush border (Fig. 3), as predicted from previous studies that showed abundant NHE-3 in this location (11, 29). However, the H⁺ATPase staining was, as we have previously described (30), the most concentrated in a tight subapical band at the base of the brush border. This subapical domain, showing intense H⁺ATPase staining in the S3 segment, did not contain detectable levels of NHE-RF (Fig. 3). This is consistent with the known expression of the C-terminally truncated B2 isoform of the 56-kDa subunit in these cells, which lacks the DTAL PDZ-binding motif (16). NHE-RF was not detected in the basolateral domain of proximal tubules.

View larger version (62K): [in this window] [in a new window]   Fig. 3.   Proximal tubule from rat kidney cortex showing the distribution of NHE-RF (red) on the brush border microvilli, and the H+ATPase (31-kDa subunit; green) on submicrovillar vesicles and invaginations. The two proteins are not co-localized in this S3 tubule segment. A differential interference contrast image is shown in the lower panel for orientation purposes. Bar, 10 µm.

To determine the specificity of labeling with anti-NHE-RF antibodies in kidney cortex, parallel incubations were performed with normal anti-NHE-RF antibody and with antibody that had been preincubated with 0.2 mg/ml of the GST-NHE-RF fusion protein that was used as an immunogen. The results show that the proximal tubule staining, as well as the apical, basolateral, and bipolar intercalated cell staining were completely abolished by preincubation with the immunizing fusion protein (Fig. 4).

View larger version (115K): [in this window] [in a new window]   Fig. 4.   Control for the specificity of NHE-RF staining in rat renal cortex. Under normal incubation conditions (A), NHE-RF stains the brush border of proximal tubules, as well as intercalated cells in variable patterns (apical, basolateral, and bipolar). This staining is completely inhibited by preincubation of the NHE-RF antibody with the immunizing GST-NHE-RF fusion protein (B). Bar, 10 µm.

Binding of Soluble H⁺ATPase Complexes to a GST-NHE-RF Affinity Matrix-- Sepharose beads to which a GST-NHE-RF fusion protein was bound were used as an affinity matrix to extract potential binding proteins from renal medullary cytosol. A cytosolic preparation from the inner medulla was used for these experiments because (a) kidney cytosol contains large amounts of free cytosolic H⁺ATPase subunits (Fig. 5A, Cytosol), (b) this kidney region contains high levels of the B1-H⁺ATPase isoform, located in A-type intercalated cells, and (c) this region contains no proximal tubules, which express abundant endogenous NHE-RF that could potentially compete with protein binding to the affinity matrix. Under these conditions, Western blots of the affinity-purified material bound to the beads showed that both the B1 and the E subunits of the H⁺ATPase were present (Fig. 5A). Control experiments using the matrix with GST alone showed little or no H⁺ATPase subunit antigenicity associated with the beads in the absence of NHE-RF, indicating the specificity of the association. The binding of H⁺ATPase to the NHE-RF-GST affinity matrix was completely inhibited by incubation of the beads with an 11-amino acid C-terminal peptide derived from the B1 H⁺ATPase subunit, which contains the C-terminal motif DTAL (PQDTEADTAL). However, binding was not inhibited by preincubation with a peptide containing the 11 C-terminal amino acids of the B2 56-kDa subunit isoform (EFYPRDSAKH), which does not contain the PDZ-binding motif (Fig. 5B).

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Results of the GST-NHE-RF pull down assay and co-immunoprecipitation. A shows that both the B1 and the E H+ATPase subunits are pulled down by GST-NHERF beads, but not by beads coupled to GST alone. B shows that the ability of the GST-NHERF beads to pull down the H+ATPase (in this case the E subunit) is completely inhibited by a peptide derived from the B1 H+ATPase subunit (which contains the DTAL motif) but is not competed away by the B2 subunit peptide. C shows that the E subunit of the H+ATPase is co-immunoprecipitated by anti-NHE-RF antibodies but not by preimmune serum. In all of these blots, the E subunit runs slightly higher than the predicted 31-kDa molecular mass for this subunit. IP, immunoprecipitate.

Co-immunoprecipitation of NHE-RF and the H⁺ATPase-- to strengthen the evidence for an in vivo interaction between NHE-RF and the H⁺ATPase, evidence for co-immunoprecipitation of the two proteins from rat kidney cytosol was sought. As shown in Fig. 5C, the E subunit of the H⁺ATPase was co-immunoprecipitated from kidney cytosol by the anti-NHE-RF antibody but not by preimmune serum.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The present data show that the 56-kDa B1 subunit isoform of the H⁺ATPase is a PDZ-binding protein that allows association of the cytosolic (V1) portion of the H⁺ATPase with NHE-RF, a PDZ protein that is expressed in the kidney. Immunolocalization indicates that in the collecting duct and connecting segment, this association occurs in a specialized subtype of intercalated cell, the B-cell, which has a highly variable pattern of intracellular localization of the H⁺ATPase (19-21, 23). Other collecting duct cell types, including principal cells and A-intercalated cells, showed a very low level of staining. The greatest amount of NHE-RF staining was observed, as expected in proximal tubules, but some thin limbs of Henle in the medulla were also strongly stained (not shown).

Especially intriguing is the failure to detect NHE-RF in A-type intercalated cells, which also contain high levels of the B1-H⁺ATPase subunit but which always have an apical staining for this protein, either on the plasma membrane or on numerous subapical vesicles. Thus, NHE-RF expression is amplified in the AE1-negative B-cell population, in which the pattern of H⁺ATPase localization is widely variable. This result suggests that the B-cell represents a distinct cell type with respect to NHE-RF expression and that NHE-RF could be involved in generating and/or maintaining apical and basolateral H⁺ATPase polarity in B-cells. Because A-cells, which insert H⁺ATPase uniquely into the apical domain, contain little or no detectable NHE-RF, we conclude that NHE-RF is in some way involved in the ability of B-cells to display plasticity of membrane H⁺ATPase insertion. This could occur via the association of NHE-RF with merlin and/or ezrin, members of the ERM (ezrin, radixin, moesin) family of actin binding proteins, which bind to the C-terminal of NHE-RF (27, 31). Other factors, including an extracellular matrix protein named hensin, have also been implicated in generating the plasticity of the intercalated cell phenotype at least in vitro (19, 32). An additional interesting finding is that both the B1 and B2 subunits of the H⁺ATPase are capable of binding actin directly via their N-terminal domains (33). Thus, it is possible that the H⁺ATPase interacts with the actin cytoskeleton both directly and indirectly (via NHE-RF). The respective roles and the regulation of these two mechanisms of interaction now need to be evaluated in different cell types. Indeed, phosphorylation of NHE-RF has been shown to disrupt the indirect interaction of the ₂-adrenegric receptor with the actin cytoskeleton and to affect the endocytic sorting of this receptor (34).

Although antibodies against all of the H⁺ATPase subunits were not utilized in this study, at least two major cytosolic subunits associated with the NHE-RF-GST beads in an affinity binding assay, and the E (31 kDa) subunit of the H⁺ATPase was co-immunoprecipitated from rat kidney cytosol by anti-NHE-RF antibodies. Because of interference from the rabbit-derived polyclonal NHE-RF IgG used for immunoprecipitation, we were unable to determine whether the 56-kDa H⁺ATPase subunit was also co-immunoprecipitated from these samples. In addition, our data show that a peptide derived from the C terminus of the B1 56-kDa subunit isoform, but not from the B2 isoform, inhibits interaction of the H⁺ATPase with NHE-RF. Coupled with previous data showing that preassembled H⁺ATPase cytoplasmic domains exist in the cytosol (18), our data suggest that the cytosolic portion of the H⁺ATPase can bind NHE-RF via a specific interaction with the C-terminal DTAL motif that is unique to the B1 subunit. Furthermore, our data provide some evidence that the interaction occurs via the C-terminal domain of the 56-kDa subunit, because the rest of the protein sequence of these two isoforms is similar except for a short N-terminal sequence difference (16, 35). It is likely that when this 56-kDa subunit is assembled in a membrane together with the transmembrane portion of the H⁺ATPase (the Vo sector), the entire H⁺ATPase assembly could thereby be coupled to NHE-RF and thus be anchored into a selected membrane domain. However, it is interesting that this domain can be either the apical or the basolateral domain in the B-intercalated cell. It has been stated recently that NHE-RF expression is restricted to the apical domain of epithelial cells (36), but this is clearly not the case in B-intercalated cells. Thus, NHE-RF may not determine the polarity of H⁺ATPase expression per se, but NHE-RF might stabilize the complex once it has reached its target membrane. In other cell types, NHE-RF appears to stabilize the cystic fibrosis transmembrane conductance regulator in the apical plasma membrane (3), whereas in Caenorhabditis elegans, PDZ proteins have been proposed to be involved in basolateral anchoring and/or targeting of a TGF- homolog (37). Recently, a Drosophila PDZ protein, discs lost, was also reported to have a dual role in maintaining apical and basolateral epithelial cell polarity (38). However, the diffuse, intracellular pattern of NHE-RF and H⁺ATPase localization seen in some B-intercalated cells raises the possibility that NHE-RF might participate in the trafficking or targeting of the H⁺ATPase. Whether PDZ proteins are involved in anchoring, targeting, or both processes remains to be determined, but our results clearly indicate that this process is not unidirectional (i.e. exclusively apical) in every cell type.

In the proximal tubule, NHE-RF is abundant and is colocalized with NHE-3 in the apical brush border (data not shown). This scaffolding interaction is probably responsible for maintaining NHE-3 at a high concentration in the apical membrane (29). The B subunit of the H⁺ATPase that is expressed in this tubule segment lacks the C-terminal DTAL and should be incapable of interacting with PDZ domain proteins (16). Immunofluorescence shows that the H⁺ATPase is not colocalized with NHE-RF at the apical pole of proximal tubules. Because H⁺ATPase is involved in the extensive apical membrane endocytosis and recycling that occurs in proximal tubules (1), it may be advantageous for this cell type to express an isoform of the H⁺ATPase B subunit that cannot interact with NHE-RF at the apical membrane. Such an interaction would anchor the H⁺ATPase in the plasma membrane by cross-linking to the NHE-3/NHE-RF complex and might hinder the endocytotic recycling of this protein, leading to failure of the endosomal acidification process and defective recycling of apical membrane proteins.

We have previously reported that the 56-kDa B1 subunit of the H⁺ATPase is present on endosomes in the other collecting duct cell type, the principal cell (39). These endosomes are involved in recycling the water channel AQP2, they do not acidify their lumen, and they lack other subunits of the H⁺ATPase (39). We postulated that the 56-kDa subunit might be a promiscuous subunit that could associate with the membranes of these endosomes via another, as yet unidentified mechanism. This mechanism can now be envisaged to occur via the PDZ-binding domain, although principal cells were only weakly stained with the anti-NHE-RF antibody. Thus, it is possible that in principal cells, a different PDZ protein such as the NHE-RF related NHE-RF2 (3) might be associated with the 56-kDa H⁺ATPase B1 subunit.

In summary, we have shown that the B1 subunit isoform of the H⁺ATPase is a PDZ-binding protein that associates with NHE-RF and may therefore be responsible for linking the H⁺ATPase to the cytoskeleton in these cells. The two proteins are colocalized in B-intercalated cells but not in A-cells, suggesting a role in generating, maintaining, or modulating the B-cell phenotype. NHE-RF and H⁺ATPase are not colocalized in proximal tubules, which express a truncated B2 subunit isoform of the H⁺ATPase that lacks the PDZ-binding domain.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants DK38452 (to S. B.), DK42956 (to D. B.), and NS24279 (to V. R.) and by a U.S. Army grant (to V. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported by a Massachusetts General Hospital Claflin Distinguished Fellowship. To whom correspondence should be addressed: Renal Unit and Program in Membrane Biology, Massachusetts General Hospital East, 149 13^th St., Charlestown, MA 02129. Tel.: 617-726-5785; Fax: 617-726-5669; E-mail: sbreton@receptor.mgh.harvard.edu.

^** Supported by a Gottlieb Daimler and Karl Benzs Predoctoral Fellowship.

Published, JBC Papers in Press, March 30, 2000, DOI 10.1074/jbc.M909857199

	ABBREVIATIONS

The abbreviations used are: PBS, phosphate-buffered saline; GST, glutathione S-transferase; FITC, fluorescein isothiocyanate; PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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