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【关键词】 antibodies
Departments of Pediatrics, Internal Medicine, and Cellular and Molecular Physiology at Yale University School of Medicine, New Haven, Connecticut
Department of Physiology, McGill University, Montreal, Quebec, Canada
ABSTRACT
Transfection studies using mutant constructs have implicated one or both protein kinase A (PKA) consensus phosphorylation sites [serines 552 and 605 in rat Na+/H+ exchanger type 3 (NHE3)] as critical for mediating inhibition of NHE3 in response to several stimuli including dopamine. However, whether one or both of these sites is actually phosphorylated in endogenous NHE3 in proximal tubule cells is unknown. The purpose of this study was to generate phosphospecific antibodies so that the state of phosphorylation of these serine residues in endogenous NHE3 could be assessed in vitro and in vivo. To this end, polyclonal and monoclonal phosphospecific peptide antibodies were generated against each PKA consensus site. Phosphospecificity was established by ELISA and Western blot assays. We then used these antibodies in vitro to evaluate the effect of dopamine on phosphorylation of the corresponding PKA sites (serines 560 and 613) in NHE3 endogenously expressed in opossum kidney cells. Baseline phosphorylation of both sites was detected that was significantly increased by dopamine. Next, we determined the baseline phosphorylation state of each serine in rat kidney NHE3 in vivo. We found that serine 552 of NHE3 is phosphorylated to a much greater extent than serine 605 at baseline in vivo. Moreover, we detected a distinct subcellular localization for NHE3 phosphorylated at serine 552 compared with total NHE3. Specifically, NHE3 phosphorylated at serine 552 localized to the coated pit region of the brush-border membrane, where NHE3 is inactive, while total NHE3 was found throughout the brush-border membrane. These findings strongly suggest that phosphorylation of NHE3 plays a role in its subcellular trafficking in vivo. In conclusion, we successfully generated phosphospecific antibodies that should be useful to assess the phosphorylation of endogenous NHE3 at its two PKA consensus sites under a variety of physiological conditions in vitro and in vivo.
dopamine; proximal tubule; microvilli; coated pits; protein kinase A
SODIUM/HYDROGEN EXCHANGER TYPE 3 (NHE3), a member of the Na+/H+ exchanger family, plays a crucial role in acid-base and volume homeostasis by mediating the majority of sodium and bicarbonate reabsorption in the proximal tubule of the kidney (17). Although Na+/H+ exchangers are similar in structure and function, each exchanger has a unique profile in terms of location and regulation. In this regard, NHE3 functions as a sodium/hydrogen exchanger on the apical membrane of the proximal tubule and thick ascending limb of the kidney. The predicted structure of NHE3 involves 1012 transmembrane domains at the NH2 terminus, which are responsible for mediating ion exchange, and a long COOH terminus, which is the regulatory portion of the protein (17). The activity of NHE3 is tightly controlled by physiological and hormonal factors.
Multiple molecular mechanisms have been identified as important in the acute regulation of NHE3, including direct phosphorylation (17, 18, 25, 29), protein trafficking (6, 7, 10, 14), and accessory regulatory proteins (22, 23). However, the specific roles and interplay of each of these mechanisms remain to be elucidated.
There is abundant evidence that phosphorylation of NHE3 plays an important role in its regulation. There are multiple protein kinase A (PKA) consensus sites on the COOH terminus of NHE3, and mutation of some of these sites leads to aberrant NHE3 regulation (15, 29). Furthermore, certain hormones, such as parathyroid hormone (PTH) and dopamine, inhibit NHE3 activity via a PKA-dependent pathway (2, 3, 12, 24, 28). In fact, recent studies show that dopamine-induced inhibition of NHE3 is associated with an increase in total NHE3 phosphorylation along with an increase in endocytosis of NHE3 (4, 12).
Previous investigations of the specific sites of NHE3 phosphorylation have been carried out exclusively in cells transfected with NHE3 (15, 29). These transfection studies using mutant constructs have implicated the importance of serine 605 and possibly serine 552 in phosphorylatory regulation of rat NHE3 (corresponding to residues 613 and 560 of opossum NHE3). Although phosphorylation of endogenous NHE3 in opossum kidney (OKP) cells in response to agonists such as dopamine has been demonstrated by use of 32P labeling and autoradiography (29), the specific phosphorylated residues were not identified. Thus it remains unknown whether one or both residues implicated in the regulation of NHE3 on the basis of transfection studies are actually phosphorylated in NHE3 endogenously expressed in proximal tubule cells.
In addition to the specific sites of phosphorylation, the ultimate mechanisms by which protein phosphorylation results in inhibition of endogenously expressed NHE3 in vivo remain to be determined. A theory with mounting supportive evidence proposes that NHE3 phosphorylation leads to changes in NHE3 intracellular trafficking. In support of this hypothesis, PTH and dopamine induce a phosphorylation-dependent endocytosis of NHE3 in cell culture models (8, 12). In vivo, changes in NHE3 localization in response to stimuli such as PTH and acute hypertension have been described (26, 27). However, the relationship between NHE3 phosphorylation and its subcellular localization in vivo is unknown.
To investigate the specific sites of endogenous NHE3 phosphorylation in vitro and in vivo, we generated multiple phosphospecific polyclonal and monoclonal antibodies. These antibodies target the two different PKA consensus sites on the COOH terminus of rat NHE3, serine 552, and serine 605, which were implicated in transfection studies. We characterized these antibodies and confirmed their phosphospecificity.
To demonstrate the utility of these antibodies in evaluating changes in endogenous NHE3 phosphorylation in vitro, we assayed changes in phosphorylation at the corresponding serine residues in OKP cells in response to dopamine. We report that dopamine induces phosphorylation of both residues in concert with inhibition of NHE3 transport activity.
In rat kidney in vivo, we determined the baseline phosphorylation status of NHE3 at serines 552 and 605 and studied the subcellular localization of NHE3 phosphorylated at these residues. We find that under baseline in vivo conditions, there is much greater phosphorylation of serine 552 than serine 605. Moreover, we demonstrate that the phosphorylated pool of NHE3 is preferentially localized to the coated pit region of the brush-border membrane.
MATERIALS AND METHODS
Materials
We purchased Imject Maleimide Activated Conjugation Kit, SulfoLink Coupling Gels, glycine-HCl elution buffer, and o-phenylenediamine dihydrochloride from Pierce; BALB/c mice from Charles River; Cloning Factor from IGEN; goat anti-rabbit horseradish peroxidase (HRP)-conjugated and goat anti-mouse HRP-conjugated secondary antibodies from Zymed; Lipofectamine 2000 from Invitrogen; DMEM, fetal calf serum, penicillin-streptomycin, and Na-pyruvate from GIBCO; polyvinylidene fluoride (PVDF) microporous membrane and Centricon 30 filters from Millipore; enhanced chemiluminescence system and Protein G Sepharose 4B from Amersham Pharmacia; 96-well vinyl assay plates from Costar; goat anti-mouse and goat anti-rabbit Alexa Fluor secondary antibodies from Molecular Probes; VectaShield from Vector Laboratories; Sprague-Dawley rats from Charles River Laboratories; Opti-Prep from Nycomed Pharma; and anti-gamma-glutamyltranspeptidase (GGT) was a kind gift from Dr. D. Castle (University of Virginia). All other reagents and chemicals were obtained from Sigma. National Institutes of Health densitometry program (Scion Image) was provided by Scion. All animal protocols were approved by the Institutional Animal Care and Use Committee of Yale University.
Methods
Polyclonal antibody production. Two 22-amino acid peptides with NH2 terminal cysteines were synthesized by the W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University. The peptides were selected from the rat NHE3 sequence and contained one of the two phosphoserines of interest. For serine 552, the peptide corresponded to amino acids 542563 (CSYVAEGERRGpSLAFIRSTPSTD), and for serine 605, the peptide corresponded to amino acids 594615 (CDMQSLEQRRRpSIRDTEDMVTH). These peptides are called PS552 and PS605, respectively. Peptides were conjugated to carrier proteins [keyhole-limpet hemocyanin (KLH) or ovalbumin (OVA)] using the Imject Maleimide Activated Immunogen Conjugation Kit from Pierce according to the manufacturer’s protocol.
Identical peptides with a nonphosphorylated central serine were also synthesized by the W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University and used for screening and purification as detailed below. The nonphosphorylated peptides are called NPS552 and NPS605.
Rabbits were immunized with the KLH- and OVA-conjugated peptides according to a standard protocol at Pocono Rabbit Farm and Laboratory (Canadensis, PA). Briefly, rabbits were immunized intradermally with 210 μg of KLH-conjugated PS552 or PS605 in Complete Freund’s Adjuvant, followed by subcutaneous booster injections at days 14 and 28 with 110 μg of KLH-conjugated peptides in Incomplete Freund’s Adjuvant. Subsequent booster injections were given subcutaneously with OVA-conjugated PS552 or PS605 in Incomplete Freund’s Adjuvant every 4 wk. The first dose of OVA-conjugated peptides was 200 μg and then all other doses were 50 μg. Sera were obtained from the animals before immunization and then every 4 wk starting on day 42 after the initial immunization.
Polyclonal antibody purification. Sera from each rabbit were purified by negative affinity purification followed by positive affinity purification as done previously by other investigators (16, 19). Negative and positive affinity purification was carried out using SulfoLink Coupling Gels from Pierce according to manufacturer’s protocol. For negative affinity purification, sera were incubated on a column in which the corresponding nonphosphorylated peptide (NPS552 or NPS605) was covalently bound. All flow-through fractions containing protein were collected and concentrated back to the original volume using Centricon 30 filters. For subsequent positive purification, this concentrated flow-through was then added to a second column in which the appropriate phosphorylated peptide (PS552 or PS605) was covalently bound. The second column was eluted with a glycine-HCl buffer from Pierce. Protein-containing fractions were concentrated to the original volume using Centricon 30 filters and then dialyzed into PBS with glycerol and sodium azide (50% PBS, 50% glycerol, 0.04% sodium azide). Purified antibodies were stored at 20°C.
Monoclonal antibody production. Monoclonal antibodies (mAbs) were also generated against the PS552 and PS605 peptides conjugated to KLH. BALB/c mice were immunized intraperitoneally with 100 μg of KLH-conjugated peptide using a pertussis/alum protocol (13). Monthly booster injections were given with 100 μg of the respective peptides in PBS. Sera were tested by ELISA and Western blotting against transfected COS-7 cells to assess reactivity to phosphorylated NHE3 (as will be discussed below). Spleen cells from mice with optimal reactivity against the respective phosphorylated peptide as well as phosphorylated NHE3 were then fused with AG8 cells and grown according to standard procedures (20). Hybridomas were then selected based on reactivity to the phosphorylated peptide by ELISA and phosphorylated NHE3 by Western blotting of transfected COS-7 cells. Selected hybridomas were cloned and subcloned by limiting dilution using Cloning Factor from IGEN. Hybridoma supernatants were purified by affinity chromatography using protein G-Sepharose 4B according to manufacturer’s protocols. Purified antibody was dialyzed into a PBS solution with 50% glycerol and 0.04% sodium azide and stored at 20°C.
ELISA. PS552, NPS552, PS605, and NPS605 peptides were solubilized in PBS at a concentration of 1 μg/ml. Solubilized peptides were used to coat 96-well vinyl plates by overnight incubation at 4°C. After overnight incubation, plates were washed with PBS/0.1% Triton and then blocked at room temperature for 10 min with PBS/0.1% Triton/1% BSA. Primary antibody was then added at various dilutions and allowed to incubate for 1 h at 4°C. Plates were again washed and blocked and then incubated with secondary antibody (HRP-conjugated goat anti-rabbit and anti-mouse from Zymed) at a dilution of 1:2,000 for 30 min at room temperature. After being washed thoroughly with PBS/Triton 0.1%, HRP substrate was added to each well (0.05 M citric acid, 0.1 M Na2HPO4, 0.4 mg/ml o-phenelyenediamine, and 0.12% H2O2) and incubated for 30 min at room temperature. The reaction was terminated with 6 N HCl and the OD read at 490 nm.
Transient transfection of NHE3 in COS-7 cells. COS-7 cells were transiently transfected with the rat wild-type NHE3 cDNA and used for testing antibody specificity. In addition to wild-type rat NHE3, cells were also transfected with mutant NHE3 in which Ser552 was replaced by alanine (S552A), or Ser605 was replaced by glycine (S605G) (15). COS-7 cells were grown in DMEM with 10% fetal calf serum, 50 U/ml penicillin, 50 mg/ml streptomycin, and 1 mM Na-pyruvate at 37°C in 5% CO2-95% air. Transfection was performed using the Lipofectamine 2000 system according to the manufacturer’s protocol. Cells were assayed 24 h after transfection.
OKP cell culture. OKP cells were grown in DMEM with 10% fetal calf serum, 50 U/ml penicillin, 50 mg/ml streptomycin, and 1 mM Na-pyruvate at 37°C in 5% CO2-95% air. Cells were transferred to 24-well culture plates, serum starved for 24 h, and then used at 9095% confluency for Western blot analysis or transport assays.
SDS-PAGE and immunoblotting. COS-7 or OKP cells were solubilized in sample buffer (10% SDS, 20% glycerol, 2% -mercaptoethanol, 2.9 mM Tris, pH 6.8) and then subjected to SDS-PAGE using 7.5% polyacrylamide gels. Proteins were then transferred from the polyacrylamide gel to a PVDF microporous membrane that was used for immunoblotting. The PVDF membrane was incubated with Blotto (5% nonfat dry milk and 0.1% Tween 20 in PBS, pH 7.4) for 1 h at room temperature to block nonspecific binding. The PVDF membrane was subsequently incubated overnight at 4°C with the primary antibody in Blotto at the following concentrations: anti-PS552 at 1:1,000, anti-PS605 at 1:500, mAb 10A8 at 1:1,000, mAb 22D5 at 1:500, mAb 14D5 at 1:2,000, mAb 1A4 at 1:500, or anti-NHE3 mAb 3H3 at 1:1,000. After the membrane was washed with Blotto, appropriate secondary antibody was added at a concentration of 1:2,000 and allowed to incubate for 1 h at room temperature. Goat anti-rabbit HRP-conjugated secondary antibody from Zymed was used for blots probed with polyclonal anti-PS552 and anti-PS605. Goat anti-mouse HRP-conjugated secondary antibody was used for blots probed with mAbs 10A8, 22D5, 14D5, 1A4, and 3H3. Membranes were again washed with Blotto and then rinsed with PBS. Antibody was visualized using an enhanced chemiluminescence system.
Radioactive sodium uptake. OKP cells were grown as described above. For 22Na uptake assay, cells were transferred to a 24-well plate and used at 90100% confluency. NHE3 activity was measured after acid-loading by the NH4Cl prepulse technique. After aspiration of the culture medium, the cells were incubated in an isotonic NH4Cl solution (30 mM NH4Cl, 90 mM choline chloride, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose, 1 mM ouabain, and 20 mM HEPES-Tris, pH 7.4) at room temperature for 20 min. This solution was then aspirated and a solution with 1 μCi/ml of 22Na was added for 5 min. The 22Na solution contained 1 mM NaCl, 120 mM choline chloride, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose, 1 mM ouabain, and 20 mM HEPES-Tris, pH 7.4. The influx of radioactive sodium was terminated after 5 min by three rapid washes of the cell monolayers with ice-cold choline chloride solution. Cells were then solubilized using 0.2 M NaOH and then neutralized with addition of an equal amount of 0.2 N HCl. The solubilized cells were then transferred to vials and the radioactivity was measured using a liquid scintillation counter.
Dopamine treatment of OKP cells. During the 30-min preincubation with the NH4Cl solution as above, cells were treated with H89 (104 M), forskolin (104 M) + IBMX (103 M), or dopamine (10 μM or 1 mM). Cells were then used for transport assays or immediately solubilized in sample buffer and subjected to SDS-PAGE and immunoblotting.
Preparation of rat kidney membranes. Adult male Sprague-Dawley rats were anesthetized with intraperitoneal pentobarbital sodium. The kidneys were removed and placed in a sucrose buffer (250 mM sucrose, 10 mM HEPES, 5 mM CaCl2, pH 7.0) with protease inhibitors (40 μg/ml PMSF, 0.5 μg/ml leupeptin, and 0.7 μg/ml pepstatin) and phosphatase inhibitors (50 mM NaF and 15 mM sodium pyrophosphate) for homogenization. The homogenate was subjected to a low-speed centrifugation (2,400 g for 10 min at 4°C) to remove any particulate material, nuclei, and mitochondria from the supernatant. The supernatant was then subjected to a high-speed centrifugation (47,000 g for 45 min at 4°C) creating a membrane pellet. The pellet was resuspended in the sucrose buffer and the protein concentration was determined by the method of Lowry.
Tissue preparation for immunohistochemistry. Adult male Sprague-Dawley rats were anesthetized with pentobarbital sodium. The kidneys were cleared with PBS and then fixed by perfusion of a modified high-osmolar PLP fixative (2% paraformaldehyde, 75 mM lysine, 10 mM sodium periodate, 750 mM sucrose in phosphate buffer, pH 7.4) through the distal aorta. The kidneys were removed, submerged in PLP fixative for 4 h, infiltrated with 30% sucrose overnight, and then cut into 4-μm cryosections for use by indirect immunofluorescence.
Indirect immunofluorescence microscopy. Cryosections were washed with TBS, then 500 mM ammonium chloride, and subsequently treated with 1% SDS in TBS. After being washed again with TBS, the sections were incubated with a blocking solution (TBS/0.1% BSA/10% goat serum) for 15 min. Sections were incubated for 1 h with the primary antibody diluted in blocking solution at the following concentrations: anti-NHE3 [4F5 (6)] at 1:100, anti-GGT at 1:5,000, anti-PS552 mAb (14D5) at 1:2, and anti-megalin [anti-MC-220 (30)] at 1:500. After thorough washing with a high-salt solution (TBS containing 2.5% NaCl instead of the typical 0.9% NaCl/0.1% BSA), the sections were incubated with the appropriate secondary antibody for 1 h. Following additional high-salt washes and a final TBS wash, sections were mounted in VectaShield and then visualized with a Zeiss Axiophot microscope.
Preparation of dense membranes. Dense membranes (DMs) were prepared as previously described (5). Briefly, adult male Sprague-Dawley rats were anesthetized with an intraperitoneal injection of pentobarbital sodium. After removal of the kidneys, the cortex was isolated and placed in an ice-cold buffer for homogenization (20 mM Tricine, pH 7.8, 8% sucrose) with protease inhibitors and phosphatase inhibitors (as detailed above under Preparation of rat kidney membranes). The homogenate was then subjected to a low-speed centrifugation (1,900 g) for 15 min, followed by a subsequent high-speed centrifugation of the supernatant (21,000 g) for 20 min. The supernatant and the upper, light portion of the pellet were saved and centrifuged at 48,000 g for 30 min. The resultant pellet (called postmitochondrial membranes) was then resuspended in the homogenization buffer and then separated by isopycnic centrifugation using OptiPrep density gradients. Fractions from this density gradient were collected and assayed for the presence of NHE3, villin, and megalin by Western blot analysis. Dense fractions enriched in NHE3 and megalin, but not villin, were pooled and then pelleted at 100,000 g. This pellet was resuspended in a K-HEPES buffer (200 mM mannitol, 80 mM HEPES, 41 mM KOH, pH 7.5), and protein concentration was determined by the method of Lowry.
Microvillar membrane vesicle preparation. Rat kidney cortex was isolated and homogenized in K-HEPES buffer (200 mM mannitol, 80 mM HEPES, 41 mM KOH, pH 7.5). The technique of differential centrifugation and magnesium precipitation as previously described by our lab was then used to isolate microvillar membrane vesicles (MMVs) (1). Protein concentration was determined by the method of Lowry, and samples were stored at 80°C. For experiments comparing DMs and MMVs, equal amounts of protein from each membrane preparation were loaded onto a gel, separated by SDS-PAGE, and then subjected to Western blot analysis with various antibodies.
RESULTS
Production of Phosphospecific Antibodies
Two polyclonal phosphospecific antibodies were generated, anti-PS552 and anti-PS605, by immunizing rabbits with the respective phosphorylated peptides. Each of these polyclonal antibodies is directed to a PKA consensus site on the COOH terminus of rat NHE3. As shown in Fig. 1, the unpurified anti-sera reacted strongly by ELISA against the respective phosphorylated peptides. However, there was also significant reactivity against the nonphosphorylated peptides. This is not surprising as there may be epitopes on the immunizing peptides that are distinct from the phosphorylated residues. In addition, dephosphorylation of the immunizing peptides may have occurred.
To selectively purify antibodies with phosphospecificity from the anti-sera, we used a two-stage affinity purification. First, each anti-serum was negatively purified by exposure to an affinity column containing the nonphosphorylated peptide. Second, the flow-through from the negative purification was positively purified on a column containing the phosphorylated peptide. As also illustrated in Fig. 1, the purification process successfully produced antibodies with preserved reactivity to the phosphorylated peptides and virtually no reactivity with the nonphosphorylated peptides.
Next, we sought to confirm the phosphospecificity of purified anti-PS552 and anti-PS605 within the context of the entire NHE3 protein. We used Western blot analysis in COS-7 cells transfected with rat NHE3 cDNA. To phosphorylate NHE3 in this system, we exposed the cells to forskolin and IBMX, both of which lead to PKA activation. As shown in Fig. 2, labeling with a general anti-NHE3 antibody (mAb 3H3) demonstrated expression of NHE3 in transfected cells, which was unaffected by PKA activation. In contrast, both anti-PS552 and anti-PS605 strongly labeled NHE3 when PKA was activated by forskolin and IBMX but had minimal reactivity with NHE3 in the absence of PKA activation. These findings verify that both anti-PS552 and anti-PS605 selectively label NHE3 that has been phosphorylated by PKA.
The antibodies were further characterized using mutant NHE3 constructs to confirm that each antibody is specific for its particular PKA consensus site. For this evaluation, we used mutant NHE3 constructs in which the serine of interest (552 or 605) was mutated to either alanine or glycine, respectively, and therefore could not be phosphorylated. Figure 3 displays Western blots prepared from COS-7 cells transfected with either wild-type NHE3, S552A mutant NHE3 (552 serine mutated to alanine), or S605G mutant NHE3 (605 serine mutated to glycine) and then probed with either anti-PS552 (Fig. 3A), anti-PS605 (Fig. 3B), or anti-NHE3 (Fig. 3C). Again, to activate PKA, cells were exposed to forskolin and IBMX in the lanes indicated. Anti-PS552 is unable to recognize NHE3 when serine 552 is mutated, but its reactivity to NHE3 is preserved when serine 605 is mutated. Likewise, anti-PS605 is unable to recognize NHE3 when serine 605 is mutated, but its reactivity to NHE3 is preserved when serine 552 is mutated. Anti-NHE3 antibody demonstrates good expression of NHE3 in all lanes with transfected cells. These results prove that each antibody is specific for its PKA consensus site and does not cross-react with the other PKA site. These findings also provide additional evidence for antibody phosphospecificity as no signal was seen in the presence of point mutations that prevent NHE3 phosphorylation.
As seen in Fig. 3 and to a far lesser extent in Fig. 2, both anti-PS552 and anti-PS605 can detectably label NHE3 in transfected COS-7 cells in the absence of forskolin/IBMX treatment, raising the possibility that the antibodies are not completely phosphospecific. However, it is quite possible that there is a low level of NHE3 phosphorylation at baseline in the absence of pharmacological activation of PKA. In fact, we noted large variability in labeling of NHE3 with our phosphospecific antibodies in the absence of forskolin/IBMX treatment, consistent with varying levels of baseline NHE3 phosphorylation. Moreover, as demonstrated in Fig. 3, mutation of the phosphorylation sites completely abolishes this baseline reactivity of anti-PS552 and anti-PS605 with NHE3. Taken together, these findings strongly suggest that baseline reactivity of anti-PS552 and anti-PS605 with NHE3 is due to variable phosphorylation of NHE3 rather than cross-reactivity of these antibodies with nonphosphorylated NHE3.
Having demonstrated the feasibility of producing phosphospecific anti-NHE3 antibodies, we next pursued generation of monoclonal antibodies that might offer the advantages of unlimited renewable quantities, lack of need for purification, and batch to batch consistency. Mice were immunized with the aforementioned phosphopeptides, and hybridoma supernatants were screened for selective reactivity with the phosphorylated vs. the nonphosphorylated peptides by ELISA. We successfully identified and cloned two hybridomas producing antibody specific for PS552 (14D5 and 1A4) and two hybridomas specific for PS605 (10A8 and 22D5).
As presented in Fig. 4, anti-PS552 mAb 14D5 and anti-PS605 mAb 10A8 were completely phosphospecific by ELISA, as no reactivity could be detected with the nonphosphorylated peptides. Western blot analysis in COS-7 cells transfected with rat NHE3 demonstrated strong reactivity in the presence of forskolin and IBMX for both anti-PS552 mAb 14D5 and anti-PS605 mAb 10A8 (Fig. 5). These findings confirm the abilities of the mAbs to recognize phosphorylated NHE3. Of note, a small amount of reactivity of the mAbs with NHE3 could be detected even without exposure to forskolin and IBMX, as seen earlier with the polyclonal antibodies anti-PS552 and anti-PS605. In view of the absolute phosphospecificity of the mAbs demonstrated by ELISA (Fig. 4), these findings provide further evidence that baseline levels of NHE3 phosphorylation are present in the absence of forskolin and IBMX. Results with 1A4 (mAb anti-PS552) and 22D5 (mAb anti-PS605) were similar to those shown for 14D5 and 10A8 in Figs. 4 and 5.
Dopamine Inhibition and Phosphorylation of NHE3
We used polyclonal antibodies anti-PS552 and anti-PS605 to evaluate whether the corresponding PKA consensus sites (Ser560 and Ser634) in endogenous opossum kidney cell NHE3 are phosphorylated in response to dopamine. Western blots were prepared from OKP cells incubated for 30 min with vehicle (Ctrl), a PKA inhibitor (H89), forskolin and IBMX, dopamine at 10 μM (Dopa 10 μM), or dopamine at 1 mM (Dopa 1 mM) and then probed with anti-PS552, anti-PS605, or anti-NHE3. As demonstrated in the representative experiment in Fig. 6, dopamine treatment increased NHE3 phosphorylation at both serine residues. However, even at 1 mM dopamine, phosphorylation was not as great as induced by forskolin/IBMX. This experiment was performed five times and then analyzed by densitometry. These results, depicted in Fig. 7, further verify that dopamine increases NHE3 phosphorylation at both sites.
To correlate changes in NHE3 phosphorylation with changes in NHE3 activity, we measured 22Na uptake under the same conditions. As indicated in Fig. 8, dopamine at the tested doses did inhibit NHE3 activity, although this inhibition was less than that caused by forskolin/IBMX. Comparison of Figs. 7 and 8 suggests a correlation between the degree of inhibition of NHE3 activity and the extent of NHE3 phosphorylation at both PKA consensus sites.
In Vivo Basal NHE3 Phosphorylation
We next used the phosphospecific monoclonal antibodies to evaluate the phosphorylation of NHE3 in vivo. To evaluate the extent of in vivo NHE3 phosphorylation at serines 552 and 605 under baseline conditions, rat kidney membranes were prepared from three different animals (rats 1-3) and probed with monoclonal anti-PS552, anti-PS605, and anti-NHE3 (Fig. 9). In the same experiment, we also probed COS-7 cells transfected with rat NHE3 and exposed to forskolin/IBMX to compare maximum in vitro phosphorylation to basal in vivo phosphorylation of NHE3 at serines 552 and 605. As seen in Fig. 9, approximately equal amounts of NHE3 are present in all four lanes, transfected cells, rat 1, rat 2, and rat 3. Likewise, NHE3 phosphorylated at both serines is also present in all four lanes; however, the extent of NHE3 phosphorylation in PKA-stimulated, transfected cells is much greater than that seen at baseline in vivo. Although basal NHE3 phosphorylation exists at both sites in vivo, serine 552 is phosphorylated to a much greater extent than serine 605. In fact, baseline in vivo NHE3 phosphorylation at serine 552 is 40% of the maximal in vitro phosphorylation at that site, whereas in vivo baseline phosphorylation at serine 605 is only 3% of its maximal in vitro phosphorylation.
All monoclonal antibodies were tested for immunofluorescence in COS-7 cells that were transfected with rat NHE3 and then stimulated with forskolin and IBMX. Reasonably strong signals were achieved with all antibodies (results not shown). When tested against PLP-fixed rat kidney cortex, monoclonal anti-PS552 and anti-PS605 both gave detectable signals, but the anti-PS605 signal was very weak compared with anti-PS552, despite relatively equal signals in transfected cells. These findings corroborate the Western blot data and suggest minimal in vivo baseline phosphorylation of serine 605. Further studies on immunolocalization of phosphorylated NHE3 were conducted only with the anti-PS552 antibody because immunofluorescence signals with anti-PS605 were barely detectable above background.
In Vivo Subcellular Localization of Phosphorylated NHE3
The phosphospecific monoclonal antibodies were used to determine whether phosphorylated NHE3 has a distinct subcellular localization. Figure 10A shows that total NHE3 completely colocalized with GGT, confirming its presence throughout the brush border. In contrast, Fig. 10B illustrates that NHE3 phosphorylated at serine 552 colocalized with anti-GGT primarily at the base of the brush border (yellow) but was relatively excluded from the microvillar domain (green).
The differential localization of total and phosphorylated NHE3 was further evaluated by colocalization with an anti-megalin antibody that labels a pool of megalin in the intermicrovillar coated pits and subapical dense tubules but does not label microvillar megalin (30). As shown in Fig. 10C, total NHE3 colocalized with megalin in the coated pit region (yellow) but was also present in the microvillar domain where megalin staining was absent (red). In contrast, indicated in Fig. 10D, NHE3 phosphorylated at serine 552 almost completely colocalized with megalin staining, confirming its restricted localization to the coated pit region of the brush-border membrane.
To further substantiate localization of phosphorylated NHE3 to the coated pit region of the brush border, biochemical analysis of phosphorylated NHE3 distribution was undertaken. For this purpose, two different types of rat kidney membranes were prepared: DMs enriched for coated pit markers (5) and purified MMVs (1). Previous studies from our group demonstrated that although the relative abundance of NHE3 in these two compartments is similar, NHE3 in microvillar membranes is active whereas that in the DMs is inactive (5). We compared the amount of NHE3 phosphorylated at either serine 552 or 605 to the total amount of NHE3 in each of these membrane fractions by Western blot analysis.
The representative experiment in Fig. 11A shows that the abundance of NHE3 phosphorylated at serine 552 was greater in the DMs (coated pits) than in the microvillar membranes, whereas the abundance of total NHE3 was similar in the two compartments. Densitometric analysis of three independent experiments revealed a 3.2-fold increase in the abundance of NHE3 phosphorylated at serine 552 in the DMs relative to the microvillar membranes. Analysis of densitometric data for anti-PS605 suggested that NHE3 phosphorylated at serine 605 also localizes to the coated pit region, but the differences were not statistically significant. The very low level of basal NHE3 phosphorylation at serine 605 makes it difficult to assay its distribution with certainty. Taken together, immunofluorescence microscopy and Western blot analysis of membrane fractions indicate that NHE3 phosphorylated at serine 552 has a subcellular localization distinct from total NHE3. Specifically, phosphorylated NHE3 preferentially localizes to the coated pit region of the brush-border membrane.
DISCUSSION
We developed the first phosphospecific antibodies to NHE3. Our antibodies are directed to two different PKA consensus sites of rat NHE3 (serine 552 and serine 605), mutation of which has been shown to affect NHE3 regulation (15, 29). We generated both polyclonal and monoclonal antibodies directed against each of these sites and established antibody phosphospecificity by ELISA against the phosphorylated and nonphosphorylated peptides and by Western blot analysis of transfected cells activated by PKA. These antibodies enable us to directly assess the phosphorylation state of these two PKA consensus sites in endogenous NHE3 under basal conditions and with different pharmacological and physiological manipulations.
To this end, we used our new phosphospecific antibodies to evaluate the changes in phosphorylation of these specific serine residues in endogenous NHE3 in response to dopamine. It had already been shown that mutation of these sites in NHE3 transfected into OKP cells blocks dopamine-induced endocytosis of the transporter (12). Although phosphorylation of endogenous NHE3 in OKP cells in response to dopamine was demonstrated (24), the specific phosphorylated residues were not identified. Our results definitively prove that dopamine increases phosphorylation of both PKA consensus sites (serines 560 and 613) in endogenous NHE3 expressed in OKP cells. Our results, when considered in the context of previous studies, provide strong evidence that dopamine-induced inhibition of NHE3 is mediated by phosphorylation of both serines 560 and 613 in OKP cells.
We also established the utility of these phosphospecific antibodies for the study of NHE3 phosphorylation in rat kidney in vivo. We established that serine 552 of NHE3 is phosphorylated to a much greater extent than serine 605 at baseline in vivo. Moreover, our newly developed phosphospecific antibodies have enabled us to detect a distinct subcellular localization for NHE3 phosphorylated at serine 552 compared with total NHE3. Specifically, NHE3 phosphorylated at serine 552 localizes to the coated pit region of the brush-border membrane, where NHE3 is inactive (5), while total NHE3 is found throughout the brush-border membrane. These findings strongly suggest that phosphorylation of NHE3 plays a role in its subcellular trafficking in vivo. For example, stimuli such as acute hypertension and PTH that have been observed to redistribute NHE3 from the microvillar domain to the base of the brush border (or coated pit region) (26, 27) may do so by inducing changes in NHE3 phosphorylation at serines 552 and/or 605.
Site-specific phosphospecific antibodies have been used successfully to study serine, threonine, and tyrosine phosphorylation of a wide array of proteins including glial fibrillary acidic protein, Bcr, Na-K-Cl cotransporter (NKCC1), and calponin (9, 21). In these cases, phosphospecific antibodies allowed for rapid and effective determination of phosphorylation at specific sites in the endogenously expressed proteins both in vitro and in vivo. For example, Gimenez and Forbush (11) recently used a phosphospecific antibody to show that vasopressin stimulation of the Na-K-Cl cotransporter NKCC2 in native kidney in vivo is associated with phosphorylation of regulatory threonine residues. We anticipate that the antibodies we generated will be similarly useful to understand better the mechanistic contributions of phosphorylation at discrete sites in the regulation of NHE3.
GRANTS
This work was supported by National Institutes of Health Grants DK-17433 and DK-33793 (to P. S. Aronson) and a Postdoctoral Research Fellowship from the National Kidney Foundation (to H. S. Kocinsky).
FOOTNOTES
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.
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