NHE2 contains subdomains in the COOH terminus for growth factor and protein kinase regulation

Samir K. Nath, Ravi Kambadur, C. H. Chris Yun, Mark Donowitz, and Chung-Ming Tse

Gastrointestinal Unit, Departments of Physiology and Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The cloned epithelial cell-specific Na+/H+ exchanger (NHE) isoform NHE2 is stimulated by fibroblast growth factor (FGF), phorbol 12-myristate 13-acetate (PMA), okadaic acid (OA), and fetal bovine serum (FBS) through a change in maximal velocity of the transporter. In the present study, we used COOH-terminal truncation mutants to delineate specific domains in the COOH terminus of NHE2 that are responsible for growth factor and/or protein kinase regulation. Five truncation mutants (designated by the amino acid number at the truncation site) were stably expressed in NHE-deficient PS120 fibroblasts. The effects of PMA, FGF, OA, FBS, and W-13 [a Ca2+/calmodulin (CaM) inhibitor] were studied. Truncation mutant E2/660, but not E2/573, was stimulated by PMA. OA stimulated E2/573 but not E2/540. FGF stimulated E2/540 but not E2/499. The most truncated mutant, E2/499, was stimulated by FBS. W-13 stimulated the basal activity of the wild-type NHE2. However, W-13 had no effect on E2/755. By monitoring the emission spectra of dansylated CaM fluorescence, we showed that dansylated CaM bound directly to a purified fusion protein of glutathione S-transferase and the last 87 amino acids of NHE2 in a Ca2+-dependent manner, with a stoichiometry of 1:1 and a dissociation constant of 300 nM. Our results showed that the COOH terminus of NHE2 is organized into separate stimulatory and inhibitory growth factor/protein kinase regulatory subdomains. This organization of growth factor/protein kinase regulatory subdomains is very similar to that of NHE3, suggesting that the tertiary structures of the putative COOH termini of NHE2 and NHE3 are very similar despite the minimal amino acid identity in this part of the two proteins.

sodium/hydrogen exchanger; calmodulin; fibroblast growth factor; phorbol 12-myristate 13-acetate; okadaic acid


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE THREE WELL-CHARACTERIZED cloned mammalian Na+/H+ exchanger (NHE) isoforms, NHE1, NHE2, and NHE3, have multiple structural and functional properties in common (for reviews, see Refs. 35, 41). At a structural level, they possess an NH2-terminal ion-transporting domain that consists of 10-12 transmembrane segments, as predicted by hydrophobicity analysis, and a long COOH-terminal cytoplasmic domain that contains multiple putative protein kinase consensus sequences (27, 35, 41). At a functional level, all NHEs are similar in terms of the stoichiometry of Na+ and H+ transport, the Na+ kinetics, and the allosteric nature of proton transport (15, 17, 22, 28, 35, 38, 41). Although NHEs have similar primary sequences in the NH2 termini, they have much less homology in their cytoplasmic COOH termini (27, 35, 41). NHEs also differ in second messenger regulation, amiloride sensitivity, tissue distribution, and specialized physiological functions (4, 14, 15, 17, 18, 21-23, 27, 28, 35, 38, 41).

There are major differences in the mechanisms by which NHEs are acutely regulated. The housekeeping isoform NHE1 is stimulated by growth factors and hyperosmolarity via an increase in the affinity of the transporter for intracellular H+ (K'[H+]i) (3, 25, 31, 32). In contrast, regulation of the epithelial isoforms NHE2 and NHE3 by growth factors and/or protein kinases and by hyperosmolarity is generally through a change in maximal velocity of the transporter (Vmax), with no change in K'[H+]i (15, 17, 21, 22, 38). The epithelial isoforms also differ in the nature of their regulation. For instance, the effects of phorbol 12-myristate 13-acetate (PMA) are opposite for NHE2 (stimulatory) and NHE3 (inhibitory) (14, 15, 17, 28). Hyperosmolarity stimulates NHE1 and inhibits NHE3 (3, 15, 21) but has different effects on NHE2 depending on the species and the cells in which NHE2 is expressed. Hyperosmolarity stimulates rat NHE2 expressed in the Chinese hamster ovary fibroblast-derived cell line AP-1 (15), whereas it inhibits rabbit NHE2 expressed in the Chinese hamster lung fibroblast-derived cell line PS120 (21).

The NHE COOH terminus is responsible for regulation of NHE activity. When NHE1 and NHE3 are truncated to contain only 19 amino acids of the putative COOH terminus, they become unresponsive to most growth factors and protein kinases (18, 32). However, these truncation mutants are still stimulated by serum, and the truncated NHE3 is still inhibited by hyperosmolarity (18). Deletion studies showed that, for NHE1, one crucial domain between amino acids 567 and 635 is required for all growth factor/protein kinase regulation (13, 32). On the other hand, the COOH-terminal cytoplasmic domain of NHE3 contains separate growth factor/protein kinase stimulatory and inhibitory subdomains (18).

Although NHE1 and NHE3 are phosphoproteins (20, 25, 31, 37), changes in the amount of phosphorylation of the exchangers do not account for all growth factor/protein kinase regulation (31, 37). This suggests that some regulation involves associated regulatory proteins. For NHE1, these include calmodulin (CaM), p24 (identified only by its size), and a calcineurin homologous protein (2, 10, 19, 30). Concerning NHE3, the identified associated regulatory proteins include CaM, NHE regulatory factor, and NHE3 kinase A regulatory protein (18, 36, 39).

In the present study, we determined by COOH-terminal truncation mutation studies whether these growth factors/protein kinases stimulate NHE2 by acting on a common region in the COOH terminus or separate subdomains. In addition, we studied the regulatory role of CaM. Intestinal brush-border Na+/H+ exchange is inhibited by CaM (6, 8), and NHE2, in addition to NHE3, is an intestinal brush-border isoform exchanger (12). Our surprising finding is not only that the NHE2 COOH terminus is organized into several subdomains that are necessary for the specific growth factor/protein kinase regulation of NHE2, but also that this organization is also very similar to that of NHE3.


    METHODS
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INTRODUCTION
METHODS
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Cell Culture

PS120 cells are derived from the Chinese hamster lung fibroblast cell line Dede (American Type Culture Collection CCL-39; Ref. 24) and are deficient in all NHEs. Both control and transfected PS120 cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS), 25 mM NaHCO3, 10 mM HEPES, 50 IU/ml penicillin, and 50 mg/ml streptomycin (pH 7.4) in a 5% CO2-95% air incubator at 37°C. Transfected cells were used up to 20 passages after transfection and were grown in the presence of G418 (400 µg/ml) for 24 h after plating. Subsequently, they were grown in medium without G418 and were further selected by function with the method of "acid killing" once a week as described previously (9, 17).

Generation of Truncation Mutants

NHE2 cDNA in pBluescript II KS was linearized by digestion with Apa I and Bgl II. The linearized cDNA was unidirectionally deleted from its 3'-end by exonuclease III/mung bean nuclease, followed by self-ligation. The extent of deletion was determined by sequencing. Five NHE2 COOH-terminal truncation mutant cDNAs were randomly selected and excised from pBluescript II by digestion with Sma I and Kpn I and subsequently subcloned into the mammalian expression vector pECE, which was digested with Hind III, blunt ended with Klenow, and digested with Kpn I. The five truncation mutants are E2/755, E2/660, E2/573, E2/540, and E2/499; the final number in the name of each mutant indicates the amino acid number at the truncation site. These pECE constructs bearing NHE2 truncations were stably transfected into PS120 cells by calcium phosphate precipitation.

Measurement of Na+/H+ Exchange Activity

Cells grown on glass coverslips were maintained in serum-free culture medium for 12-18 h before the experiment. Na+/H+ exchange rates were measured with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM as described previously (17). Solutions used included Na+ medium, containing (in mM) 130 NaCl, 5 KCl, 2 CaCl2, 1 MgSO4, 0.8 Na2HPO4, 0.2 NaH2PO4, 25 glucose, and 20 HEPES (pH 7.4). Tetramethylammonium (TMA) medium was identical to Na+ medium except that Na+ was replaced with equimolar TMA, and NH4Cl solution was identical to TMA medium except that it contained 40 mM NH4Cl and 90 mM TMA chloride instead of 130 mM TMA chloride.

In okadaic acid (OA) experiments, cells were preincubated with 1 µM OA in TMA medium for 15 min before addition of Na+ medium containing OA. In W-13 (50 µM), W-12 (50 µM), and PMA (1 µM) experiments, cells were preincubated with these agents in TMA medium for 15, 15, and 5 min, respectively, before Na+ was introduced. Fibroblast growth factor (FGF; 10 ng/ml) and dialyzed FBS (10%) were added simultaneously with the Na+ medium. Chosen concentrations of different substances were those that had been shown in earlier studies to elicit maximal or near maximal responses (17). The H+ efflux rate (in µM/s), equivalent to the rate of Na+/H+ exchange, was determined by multiplying the rate of change in intracellular pH (pHi) by the cellular buffering capacity at the corresponding pHi values. Intracellular buffering capacity for the transfected cells was determined using the method previously described (17, 28) and was quantitatively similar for the different transfected PS120 cell lines studied.

Data Analysis

Na+/H+ exchange rates (calculated as H+ efflux rate) were analyzed using a nonlinear regression analysis program (ENZFITTER, Biosoft) as described previously (17, 21, 28). In this analysis, the data were fitted to a general allosteric model described by the Hill equation V = Vmax · [S]napp/(K' + [S]napp), where V is velocity, [S] is H+ concentration, K' estimates were used to compare the relative affinities of the exchanger for intracellular H+ concentration, and the apparent Hill coefficient (napp) was calculated using data from the velocity range of approximately one-half of Vmax, as described by Segel (26). Each experiment was performed on at least three separate days, and at least 10 coverslips were used in each experiment. Statistical significance of an effect was determined by comparing means ± SD of the Vmax and K'[H+]i of Na+/H+ exchange rate, using two-tailed unpaired Student's t-tests and considering the number of coverslips studied. In these analyses, SD was provided from the nonlinear curve-fitting routine (17).

Quantitative Measurement of CaM Binding to COOH-Terminal 87 Amino Acids of NHE2

CaM was dansylated as described by Bertrand et al. (2). Briefly, purified bovine CaM (1 mg) in 0.5 ml was incubated with 1 µl of dansyl chloride (20 mg/ml) in 20 mM NH4HCO3 (pH 7.4) and 1 mM CaCl2 for 2 h at room temperature. The mixture was passed through Sephadex G25 to remove the nonreactive reagent from dansylated CaM, and the dansylated CaM was eluted with 1 ml of 20 mM NH4HCO3.

The fusion protein, glutathione S-transferase (GST), and the last COOH-terminal 87 amino acids of NHE2 (GST/E2/C87) were purified as previously described (29). The binding of GST/E2/C87 to dansylated CaM was measured by monitoring the fluorescence emission spectra (400-600 nm) of dansylated CaM (100 nM) with different concentrations of purified fusion protein (0-3 µM) in a solution containing 10 mM Tris-HEPES (pH 7.2), 30 mM NaCl, and 0.1 mM CaCl2 at 25°C, using a spectrofluorometer with an excitation wavelength of 340 nm.

Materials

PS120 fibroblasts used for transfection were provided by J. Pouyssegur (University of Nice, Nice, France). FBS was obtained from HyClone (Logan, UT), DMEM was from Life Technologies, and TMA chloride was from Fluka Chemical (Ronkonkoma, NY). BCECF-AM and nigericin were from Molecular Probes (Eugene, OR). OA (sodium salt) was from LC Laboratories (Woburn, MA). All other reagents were purchased from Sigma.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
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Construction of NHE2 COOH-Terminal Truncation Mutants

All cloned NHEs contain multiple putative protein kinase consensus sequences on their COOH termini. Although the functional significance of these protein kinase consensus sequences is not fully known, the COOH termini of NHE1 and NHE3 have been shown to be responsible for growth factor/protein kinase regulation of the exchangers (13, 18, 32, 37). To probe the organization of the growth factor/protein kinase-responsive subdomains on the COOH terminus of NHE2, five NHE2 COOH-terminal truncation mutants were constructed. Figure 1 depicts these NHE2 truncation mutants. Because NHE2 is stimulated by protein kinase C (PKC), potential PKC phosphorylation sites contained within the COOH terminus of NHE2 were predicted using the computer program PC/GENE. The wild-type NHE2 is predicted to have seven putative PKC phosphorylation sites in its COOH terminus, whereas the most truncated mutant, E2/499, contains one such site (Lys-Arg-Ser491-Asn-Lys-Lys).


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Fig. 1.   Wild-type (WT) and truncation mutants of Na+/H+ exchanger isoform 2 (NHE2) used in this study. Putative membrane-spanning domain of NHE2 (gray bars) is 480 amino acids long, and putative COOH-terminal cytoplasmic domain (open bars) is 329 amino acids long. Number of the amino acid at truncation site is indicated at end of each bar and in names of mutants. Protein kinase C (PKC) sites in NHE2 COOH terminus were predicted by PC/GENE software and number of such PKC (C-kinase) sites is shown for each truncation mutant.

Effect of PMA, FGF, FBS, and OA on NHE2 Truncation Mutants

We previously showed that serum, FGF, and PMA stimulated NHE2 by a mechanism involving a Vmax increase (17, 28). To map the serum-, FGF-, and PMA-sensitive subdomains on the COOH terminus of NHE2, we tested whether the truncation mutants responded to these agents.

PMA effect. It was found that the truncation mutants E2/755 and E2/660 were both stimulated by PMA, whereas E2/573, E2/540, and E2/499 were unresponsive. Figure 2 shows the effect of PMA on E2/660 and E2/573. PMA (1 µM, 5-min preincubation) increased the Vmax (PMA, 1,772 ± 240 µM/s; control, 1,230 ± 234 µM/s; P < 0.001) of E2/660 without affecting the K'[H+]i (PMA, 0.26 ± 0.09 µM; control, 0.27 ± 0.11 µM) (Fig. 2A). In contrast, PMA did not affect the kinetic parameters of E2/573 (Fig. 2B; Vmax, 480 ± 23 µM/s for PMA vs. 498 ± 39 µM/s for control; K'[H+]i, 0.06 ± 0.02 µM for PMA vs. 0.12 ± 0.04 µM for control). Thus the PMA-sensitive stimulatory domain of NHE2 is between amino acids 573 and 660. 


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Fig. 2.   Stimulation of NHE2 by phorbol 12-myristate 13-acetate (PMA) requires COOH-terminal amino acids between 573 and 660. A: PMA stimulates E2/660. In cells pretreated with PMA (1 µM, 5 min) and recovered in Na+ medium containing 1 µM PMA (black-triangle), maximal velocity (Vmax) of exchanger was 44% higher than in control untreated cells (open circle ), with no change in either affinity of transporter for intracellular H+ (K'[H+]i) or apparent Hill coefficient (napp). B: PMA does not affect kinetic parameters of E2/573. Experiment was performed as described for A. Kinetic parameters of Na+-dependent intracellular pH recovery rates of PMA-pretreated cells (black-triangle, dotted line) and untreated control cells (open circle , solid line) were not significantly different. Results are from identical experiments performed on 3 different days.

FGF effect. All truncation mutants studied were stimulated by FGF except E2/499. As shown in Fig. 3A, FGF (10 ng/ml) stimulated the Vmax of E2/540 from 1,054 ± 105 to 1,429 ± 128 µM/s (P < 0.001) without affecting the K'[H+]i (0.31 ± 0.09 vs. 0.32 ± 0.09 µM in control and FGF, respectively). Unlike other truncation mutants, the basal activity of E2/499 was too low for quantitative analysis of the effect of FGF by initial rate studies. Qualitative assessment of the effect of FGF on E2/499 was performed by adding FGF to acidified E2/499 cells. As demonstrated in Fig. 3B, FGF did not alter the rate of pHi recovery in E2/499 cells, suggesting that FGF has no effect on this truncation mutant. As a positive control, serum stimulates E2/499 (see below). This indicates that the regulatory site of FGF is situated between amino acids 499 and 540. 


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Fig. 3.   Stimulation of NHE2 by fibroblast growth factor (FGF) requires COOH-terminal amino acids between 499 and 540. A: FGF stimulates E2/540. Cells pretreated with FGF (10 ng/ml; black-triangle, dotted line) stimulated Vmax of exchanger by 36% compared with control untreated cells (open circle , solid line), with no change in K'[H+]i. Results are representative of 4 similar experiments. B: FGF does not stimulate E2/499. Spectrofluorometric trace representative of 5 identical experiments shows that addition of FGF (10 ng/ml) did not affect activity of E2/499.

FBS effect. All truncation mutants were stimulated by 10% FBS. As shown in Fig. 4, when 10% dialyzed FBS was added to acidified E2/499 cells, FBS stimulated the rate of the pHi recovery. This stimulation was blunted by 1 mM amiloride, confirming that the pHi change represented Na+/H+ exchange. E2/499 contains only 19 amino acids remaining in the putative cytoplasmic tail of NHE2, which indicates that the site of regulation by FBS is located in the putative membrane-spanning portion of the exchanger or in the 19 amino acids remaining in the cytoplasmic tail of E2/499.


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Fig. 4.   Fetal bovine serum (FBS) stimulates NHE2 truncated at amino acid 499. This representative spectrofluorometric trace from 4 independent experiments shows that FBS (10%, dialyzed) stimulated Na+/H+ exchange activity of E2/499. FBS was added during perfusion with Na+ medium to cells previously acidified with NH4Cl. Amiloride (1 mM) was then added as indicated to confirm that response to FBS represented amiloride-sensitive Na+-dependent intracellular alkalinization.

OA effect. NHE1 and NHE3 are stimulated by OA, a serine/threonine phosphatase 1 and 2A inhibitor. This is consistent with the observations that NHE1 and NHE3 are phosphoproteins and that phosphoserine is the only phosphoamino acid in NHE3 (37). NHE2 is also a phosphoprotein (Tse, unpublished results). Therefore, we tested whether OA regulates NHE2 and mapped the OA regulatory domain by studying the effect of OA on NHE2 truncation mutants. Preincubation with OA (1 µM, 15 min) increased the Vmax of full-length NHE2 by 37% (572 ± 82 vs. 417 ± 53 µM/s; P < 0.001) without affecting the K'[H+]i (0.22 ± 0.1 vs. 0.21 ± 0.09 µM in control and OA, respectively) (Fig. 5A). OA also stimulated E2/755, E2/660, and E2/573 but not E2/540 or E2/499. As shown in Fig. 5B, OA stimulated E2/573 by 29% (646 ± 16 vs. 500 ± 37 µM/s; P < 0.0001) without affecting the K'[H+]i (0.12 ± 0.03 vs. 0.11 ± 0.01 µM in control and OA, respectively). OA did not affect the kinetic parameters of truncation mutant E2/540 (Fig. 5C; Vmax, 1,042 ± 96 µM/s for control vs. 1,002 ± 105 µM/s for OA; K'[H+]i, 0.24 ± 0.04 µM for control vs. 0.18 ± 0.07 µM for OA).


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Fig. 5.   Okadaic acid (OA) stimulates wild-type NHE2 but not its truncation mutant E2/540. A: preincubation of PS120/NHE2 cells with OA (1 µM, 15 min; black-triangle) increased Vmax of full-length NHE2 by 37% relative to control (open circle ) without affecting K'[H+]i. B: OA (black-triangle, dotted line) stimulated E2/573 by 29% relative to control (open circle , solid line) without affecting K'[H+]i. C: OA did not affect kinetic parameters of truncation mutant E2/540. Vmax and K'[H+]i of control (open circle , solid line) and OA-treated (black-triangle, dotted line) cells are not statistically different. Results are from identical experiments performed on 3 different days.

Regulation of NHE2 by CaM

Because NHE2 is a brush-border protein and brush-border NHE is inhibited by CaM at basal levels of intracellular Ca2+ concentration ([Ca2+]i) (6, 8), we tested whether NHE2 in PS120 cells is regulated by CaM, using the CaM inhibitor W-13. As shown in Fig. 6A, pretreatment of NHE2-containing cells with W-13 (50 µM, 15 min) caused an 89% increase in the Vmax (644 ± 40 µM/s) compared with untreated controls (340 ± 25 µM/s; P < 0.0001) with no change in K'[H+]i (0.14 ± 0.02 and 0.11 ± 0.04 µM for control and W-13-pretreated cells, respectively). As a control, W-12, the inactive analog of W-13, did not affect the kinetic parameters of NHE2 activity (data not shown).


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Fig. 6.   Effect of calmodulin (CaM) inhibitor W-13 on NHE2 and E2/755. A: CaM antagonist W-13 stimulates basal NHE2 activity. Cells were grown on glass coverslips and were acidified by NH4Cl prepulse. One set of cells (black-triangle) was pretreated with 50 µM W-13 for 15 min in tetramethylammonium solution, while control cells (open circle ) remained untreated. Cells were then allowed to recover to a steady-state intracellular pH in Na+ solution, and H+ efflux rates were measured as detailed in METHODS. In W-13-pretreated cells, Vmax of exchanger was 89% higher than control, with no change in K'[H+]i. B: W-13 did not affect kinetic parameters of E2/755. W-13 pretreatment (50 µM, 15 min) (black-triangle, dotted line) did not affect kinetic parameters of E2/755 compared with controls (open circle , solid line). Results are representative of 4 similar experiments.

W-13 had no effect on any of the NHE2 truncation mutants studied. As an example, the effect of W-13 on E2/755 is shown in Fig. 6B. Pretreatment of E2/755 cells with W-13 (50 µM, 15 min) did not affect the kinetic parameters of E2/755 activity (Vmax, 486 ± 57 µM/s for control vs. 468 ± 44 µM/s for W-13; K'[H+]i, 0.18 ± 0.07 µM for control vs. 0.17 ± 0.06 µM for W-13). Thus these results suggest that CaM inhibits NHE2 under basal [Ca2+]i conditions and that this inhibition requires the last COOH-terminal 54 amino acids of NHE2, i.e., the domain between amino acids 756 and 809.

We then tested whether CaM directly binds to NHE2, using a purified fusion protein of GST and the last COOH-terminal 87 amino acids of NHE2 (GST/E2/C87) and dansylated CaM and monitoring the emission spectra of dansylated CaM fluorescence. As shown in Fig. 7A, binding of GST/E2/C87 to CaM is indicated by an increase in fluorescence intensity and a shift in peak emission wavelength (from 520 to 480 nm) of dansylated CaM on addition of GST/E2/C87 in the presence of 0.1 mM Ca2+. This increase in the fluorescence intensity of dansylated CaM occurs in a fusion protein concentration-dependent manner (0-3 µM) and is blunted in the presence of 1 mM EGTA (data not shown). Control experiments using GST had no effect on the fluorescence intensity of dansylated CaM, in either the presence or the absence of 0.1 mM Ca2+ (data not shown). We further determined the affinity of the Ca2+/CaM binding sites to GST/E2/C87 by dansylated CaM fluorescence as described by Bertrand et al. (2) and Ehlers et al. (7). As shown in Fig. 7B, Scatchard analysis shows that Ca2+/CaM binds to a single binding site of the COOH-terminal fusion protein of NHE2 with an intermediate affinity [dissociation constant (Kd) = 300 nM]. The maximum binding (Bmax; indicated by the x-intercept) is 105 nM and is almost the same as the CaM concentration (100 nM) used in the study, suggesting that CaM binds to GST/E2/C87 with a stoichiometry of 1:1. Thus these results suggest that CaM inhibits NHE2 in PS120/NHE2 cells under basal [Ca2+]i conditions (transport studies) and binds to the last 87 amino acids of NHE2 (dansylated CaM binding studies).


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Fig. 7.   Direct binding of CaM to purified NHE2 fusion protein GST/E2/C87. A: fluorescence emission spectra (400-600 nm) of dansylated CaM with or without GST/E2/C87. Fluorescence was monitored with a spectrofluorometer at 25°C in a mixture containing 10 mM Tris (pH 7.2), 30 mM NaCl, 0.1 mM CaCl2, 100 nM dansylated CaM, and various concentrations of GST/E2/C87 (0-3 µM). Excitation wavelength was 340 nm. Peak fluorescence of dansylated CaM alone (0 µM fusion protein) was at 520 nm. On binding of fusion protein, peak fluorescence of dansylated CaM shifted to a new peak at 480 nm. Binding of GST/E2/C87 to CaM increased fluorescence intensity of dansylated CaM in a fusion protein concentration-dependent manner. B: Scatchard analysis. Fraction of bound dansylated CaM (Fb) was calculated from fractional fluorescence increase at 480 nm using the relationship Fb = (f - fo)/(falpha  - fo), where fo is fluorescence when all dansylated CaM is free, falpha is fluorescence when all dansylated CaM is bound, and f is fluorescence for any mixture of GST/E2/C87 and CaM. From these values, concentrations of bound dansylated CaM, bound fusion protein, and free fusion protein were determined and used to construct Scatchard plots as previously described (2, 7). Line was fitted to data using the single-site Scatchard relationship B/F = -B/Kd + Bmax/Kd, where B is amount of fusion protein bound, Bmax is maximum binding, F is amount of free fusion protein, and Kd is dissociation constant. Kd was determined as negative reciprocal of slope of line fitted to data by least squares method (Kd = 300 nM).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

NHE2 and NHE3 are epithelial cell specific and are colocalized to the brush border of intestinal epithelial cells (12). This study shows that the COOH terminus of the cloned NHE2 is involved in its regulation by growth factors/protein kinases. Like NHE1, NHE2 is stimulated by serum, FGF, OA, and PMA (17). NHE1 is regulated by OA, FGF, and phorbol esters, and this regulation requires a single domain in the COOH terminus, amino acids 537-635, although 50% of the regulation involves the amino acids COOH-terminal to amino acid 635 (13, 32). In contrast, we have identified multiple separate COOH-terminal regulatory subdomains sensitive to these growth factors/protein kinases in NHE2. In the present study, we find that the organization of the growth factor/protein kinase-responsive subdomains of NHE2 is very similar to that of NHE3, which is stimulated by serum, FGF, and OA but is inhibited by PMA (Fig. 8).


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Fig. 8.   Comparison of stimulatory and inhibitory regions of NHE2 COOH terminus with those of NHE3. In both isoforms, there are inhibitory and stimulatory domains. Inhibitory domain is located at distal part of COOH terminus, whereas stimulatory domain is nearer NH2 terminus. Inhibitory domain of NHE3 has 2 subdomains, 1 for PMA and 1 for CaM, whereas inhibitory domain of NHE2 consists of CaM-responsive area alone. In NHE2, PMA regulatory domain is a part of stimulatory domain, and domain between amino acids 660 and 755 (denoted by ?) has no identified regulatory role (it is not known whether it is stimulatory or inhibitory). Organization of sites responsible for OA, FGF, and FBS for NHE2 and NHE3 is similar. For both isoforms, the shortest truncations studied, which contained only 20 amino acids of putative COOH terminus for NHE3 and 19 for NHE2, are regulated by serum and hyperosmolarity. Osm, hyperosmolarity; Inhibit., inhibitory.

NHE2 activity is stimulated by PMA, and the COOH-terminal amino acids 573-660 of NHE2 are required for PMA stimulation of NHE2 activity. In this region, there are three putative PKC consensus sequences (Leu-Thr-Ala-Asp-Thr-Ser629-Glu-Arg-Gln-Ala-Lys, Ile-Arg-Arg-Arg-His-Ser643-Ile-Arg-Glu-Ser647-Ile-Arg-Lys-Asp-Asn). Although preliminary studies showed that NHE2 is a phosphoprotein, it is not known whether PMA acting through PKC stimulates NHE2 by changing the total amount of its phosphorylation or whether PMA activates another regulatory protein(s), which in turn regulates NHE2.

OA stimulates basal NHE2 activity, and the OA-sensitive domain is mapped to a region between amino acids 540 and 573. This region does not overlap with the PMA-sensitive domain, suggesting that OA is not potentiating the effect of PKC by inhibiting serine/threonine phosphatase(s). This is supported by the similar percentage of NHE2 stimulation by OA in full-length NHE2 and E2/573 (Fig. 5, B and C). There are three serine residues (Ser550, Ser551, and Ser554) and one threonine (Thr570) residue in the OA-responsive region, but none of these is predicted to be a PKC consensus sequence by the PC/GENE program. It is not known whether a kinase regulates NHE2 by phosphorylating NHE2 in this domain, with the phosphorylation status and the activity of NHE2 being balanced by serine/threonine phosphatase activity, or whether a serine/threonine-phosphorylated protein that binds to this domain is involved in regulating NHE2 under basal conditions.

For both NHE1 and NHE3, both phosphorylation-dependent and phosphorylation-independent regulation occurs (11, 20, 31, 34, 37). It has been demonstrated that, although NHE3 is a phosphoprotein, PMA inhibits NHE3 and FGF stimulates NHE3 without changing the total amount of phosphorylation of NHE3 in PS120 cells (37). However, cAMP inhibits NHE3 activity and increases the amount of NHE3 phosphorylation (20). Recently, Kurashima and colleagues (16) further showed that Ser605 and Ser634 are responsible for cAMP inhibition of rat NHE3 expressed in AP-1 cells. There was a threefold increase in Ser605 phosphorylation in response to cAMP inhibition. On the other hand, Ser634 was not phosphorylated on cAMP inhibition but was required for the effect of cAMP. For NHE1, 50% of the growth factor/protein kinase regulation, including PMA stimulation, is phosphorylation independent and involves accessory regulatory proteins (31).

We have identified separate domains of NHE2 that respond to different agonists. It is not known whether these domains indicate areas of NHE2 that are phosphorylated and/or bind to regulatory proteins. The fact that some of the domains do not contain appropriate kinase consensus sequences suggests but does not prove the involvement of associated regulatory proteins. It is possible that each NHE2 agonist affects a different accessory regulatory protein(s), which in turn interacts with the corresponding agonist-sensitive subdomain. In this regard, although the site of action of FGF on the NHE2 COOH terminus is located between amino acids 499 and 540, there is no tyrosine kinase consensus sequence in this region. This indicates that the receptor tyrosine kinase does not directly phosphorylate NHE2 in the FGF-sensitive domain. Preliminary studies with Western blotting of NHE2 immunoprecipitated with anti-phosphotyrosine antibody also suggest that NHE2 is not tyrosine phosphorylated (Tse, unpublished result). Similarly, serum stimulates NHE2. The truncation mutant E2/499, which contains only 19 amino acids of the putative COOH terminus, is unresponsive to FGF and PKC but is still stimulated by serum. Thus at least the effects of serum and FGF on NHE2 likely occur via intermediates. Whether these intermediates are protein kinases or accessory regulatory proteins that bind to NHE2 at the corresponding FGF- or serum-sensitive domains is not known. The very low Na+/H+ exchange rate of E2/499 is not totally understood. One possibility is that E2/499 expresses a different percentage of the total cell NHE on the plasma membrane than does wild-type NHE2. We predict that a low percentage of E2/499 is present on the plasma membranes under basal conditions and that this is one explanation for its failure to function in the basal state, although it retains the ability to be stimulated by serum. It has been reported that wild-type NHE3 and its truncation mutants express different percentages of total cell NHE on the plasma membrane and that serum increases the amount of plasma membrane NHE3 (1).

Although the domains involved in regulation by PMA of NHE2 and NHE3 are located in similar parts of the COOH termini, PMA inhibits NHE3 but stimulates NHE2. Whether PMA directly acts on the COOH terminus or regulates transport indirectly by acting on accessory proteins remains to be investigated. In the latter case, the nature of the accessory proteins must have an opposite effect for NHE2 (stimulatory) and NHE3 (inhibitory).

In the present study, we identified CaM as an NHE2-associated regulatory protein and demonstrated that CaM regulates NHE2 basal activity, since the CaM blocker W-13 stimulates NHE2 under basal conditions. By truncation mutant studies, we localized the CaM-sensitive domain to the last 54 amino acids of NHE2, since the truncation mutant E2/755 does not respond to W-13. We further demonstrated that dansylated CaM binds to the NHE2 fusion protein GST/E2/C87, which contains the last COOH-terminal 87 amino acids of NHE2, with a Kd of 300 nM and a stoichiometry of 1:1. This binding of CaM to GST/E2/C87 is Ca2+ dependent (it does not occur in the absence of Ca2+). Thus CaM inhibits NHE2 under basal conditions, and CaM binds the NHE2 COOH terminus. Moreover, there appears to be overlap between the domain of NHE2 that binds CaM and the one that is necessary for CaM inhibition. More detailed determination of the domains involved in CaM binding and regulation of NHE2 will be necessary to understand how CaM regulates NHE2.

Recently, Wakabayashi et al. (33) reported that elevating [Ca2+]i with ionomycin stimulated rat NHE2 expressed in PS120 fibroblasts by a mechanism that appeared to involve stimulation of both Vmax and K'(H+)i. In addition, they showed that rat NHE2 amino acids 607-627 appeared to act as an autoinhibitory domain when substituted in NHE1 for the NHE1 "autoinhibitory domain" (amino acids 637-656) and acted to increase Na+/H+ exchange when Ca2+ was elevated (eliminating the autoinhibition). They suggested that this domain of NHE2, which binds CaM with high affinity (130 nM) in the presence of Ca2+, was involved in the elevated Ca2+ stimulation of NHE2. Our studies do not directly address whether amino acids 607-627 of NHE2 constitute an autoinhibitory domain in NHE2, although it is possible that the NHE2 COOH terminus has multiple CaM binding domains.

This study and our previous studies show that the organization of the regulatory domains in the COOH termini of NHE2 and NHE3 are very similar (Fig. 8). This suggests that the tertiary structures of the putative COOH termini of NHE2 and NHE3 are very similar even though they do not have homology in their primary sequences (25% amino acid identity). Because separate growth factor/protein kinase domains have been identified on the putative COOH termini of NHE2 and NHE3, we hypothesize that COOH termini of NHE2 and NHE3 bind to multiple accessory regulatory proteins and act as a scaffold to bring the regulatory domain or regulatory proteins in contact with the NH2 terminus transport site directly or indirectly. Some of the defined areas in the COOH terminus could mark the site of interaction of a regulatory protein or be required for a bound regulatory protein to interact with the NH2-terminal transport domain. To this end, NHE2 and NHE3 are regulated by growth factors/protein kinases by a mechanism of Vmax change (17, 28). Chimera studies of the membrane-spanning domain (M domain) of one NHE with the COOH-terminal cytoplasmic domain (C domain) of other NHEs show that only the chimeras with NHE2 and NHE3 on both domains could be regulated by FGF and PMA, whereas chimeras between an epithelial isoform (NHE2 or NHE3) and the housekeeping isoform, NHE1, are insensitive to PMA and FGF (40). This further supports the concept of precise interactions of the regulatory domains and/or accessory proteins with the NH2 termini of NHE2 and NHE3. Thus not only are the COOH termini of NHE2 and NHE3 similar at the tertiary structure level as opposed to NHE1, but also the NH2 termini of NHE2 and NHE3 must also be structurally related, increasing the differences from NHE1. This probably explains why most chimeras of the transport domains made between NHE1 and NHE3 fail to function (5, 23).

In summary, this study shows that the organization of the growth factor/protein kinase subdomains of NHE2 and NHE3 is very similar. This organizational similarity most likely contributes to the previous observation that only M domain-C domain chimeras between epithelial isoforms NHE2 and NHE3 are responsive to FGF and PMA regulation, and it suggests that the tertiary structures of M and C domains of NHE2 and NHE3 are very similar and that there is precise communication between the C domains of epithelial isoform exchangers and their M domains. Furthermore, we identified CaM as an accessory regulatory protein that inhibits NHE2 at basal Ca2+ levels and directly binds to its last COOH-terminal 87 amino acids. Thus CaM is the first identified accessory regulatory protein that regulates all three well-characterized NHEs, NHE1, NHE2, and NHE3.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-26523, P01-DK-44484, R29-DK-43778, R01-DK-51116, and F32-DK-08672 and Training Grant T32-DK-07632, by the Meyerhoff Digestive Diseases Center, and by the Hopkins Center for Epithelial Disorders.


    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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: C.-M. Tse, Dept. of Medicine, GI Division, 918 Ross Research Bldg., Johns Hopkins University School of Medicine, 720 Rutland Ave., Baltimore, MD 21205 (E-mail: mtse{at}welchlink.welch.jhu.edu).

Received 23 July 1998; accepted in final form 13 January 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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