©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Separate C-terminal Domains of the Epithelial Specific Brush Border Na/H Exchanger Isoform NHE3 Are Involved in Stimulation and Inhibition by Protein Kinases/Growth Factors (*)

Susan A. Levine , Samir K. Nath , C. H. Chris Yun , Jeannie W. Yip (§) , Marshall Montrose , Mark Donowitz (¶) , C. Ming Tse

From the (1) Departments of Medicine and Physiology, Gastrointestinal Unit, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

NHE3, a cloned intestinal and renal brush border Na/H exchanger, has previously been shown to be both stimulated and inhibited by different protein kinases/growth factors. For instance, NHE3 is stimulated by serum and fibroblast growth factor (FGF) and inhibited by protein kinase C. In the present study, we used a series of NHE3 C terminus truncation mutants to identify separate regions of the C-terminal cytoplasmic tail responsible for stimulation and inhibition by protein kinases/growth factors. Five NHE3 C terminus truncation mutant stable cell lines were generated by stably transfecting NHE3 deletion cDNAs into PS120 fibroblasts, which lack any endogenous Na/H exchanger. Using fluorometric techniques, the effects of the calcium/calmodulin (CaM) inhibitor W13, calcium/CaM kinase inhibitor KN-62, phorbol myristate acetate, okadaic acid, FGF, and fetal bovine serum on Na/H exchange were studied in these transfected cells. Inhibition of basal activity of full-length NHE3 is mediated by CaM at a site C-terminal to amino acid 756; this CaM effect occurs through both kinase dependent and independent mechanisms. There is another independent inhibitory domain for protein kinase C between amino acids 585 and 689. In addition, there are at least three stimulatory regions in the C-terminal domain of NHE3, corresponding to amino acids 509-543 for okadaic acid, 475-509 for FGF, and a region N-terminal to amino acid 475 for fetal bovine serum. We conclude that separate regions of the C terminus of NHE3 are involved with stimulation or inhibition of Na/H exchange activity, with both stimulatory and inhibitory domains having several discrete subdomains. A conservative model to explain the way these multiple domains in the C terminus of NHE3 regulate Na/H exchange is via an effect on associated regulatory proteins.


INTRODUCTION

A family of Na/H exchangers (NHE)() has been cloned from multiple mammalian species, and the structural and functional properties of three isoforms (NHE1, NHE2, and NHE3) have been characterized in detail (1-7). The Na/H exchangers cloned to date have certain common structural features, including an N-terminal transmembrane domain with a hydrophobicity profile which predicts 10-12 transmembrane segments, and a long C-terminal cytoplasmic domain, that contains multiple potential consensus sites for protein phosphorylation by a variety of protein kinases. Although the NHE isoforms have similarities in amino acid sequence and in structure as deduced from hydropathy plots, as well as similarities in some transport properties such as sodium kinetics and the allosteric nature of proton transport, they differ in second messenger regulation, amiloride sensitivity, tissue distribution, and presumably in physiological function. The first isoform cloned, designated NHE1, is activated by growth factors, phorbol esters, and cell shrinkage and is highly sensitive to inhibition by amiloride (8, 9) . NHE1 is present in almost all cell types, is located on the basolateral membrane in polarized cells, and is thought to function in maintenance of cell pH and cell volume, and perhaps in cell division. NHE2 is also activated by growth factors and phorbol esters, although the kinetics of this activation are different than for NHE1 (3, 6) . NHE2 message is found in rabbit kidney, intestine, and adrenal gland, and this isoform is less sensitive to inhibition by 5-amino substituted forms of amiloride compared with NHE1 (3) . The NHE3 isoform is activated by growth factors similarly to NHE2; however, it is inhibited by phorbol esters (2, 4, 5) . In addition, this amiloride-resistant exchanger has been found primarily on the apical membrane of intestinal and kidney proximal tubule epithelial cells and is thought to carry out transcellular sodium absorption (2, 5, 10) .

The isoforms NHE1, 2, and 3 have 40-60% overall amino acid identity, with the membrane spanning domains, which transport Na and H, being most highly conserved and the C-terminal cytoplasmic domains, which regulate rate of transport, having very little homology among the exchanger isoforms, especially concerning the many putative protein kinase consensus sequences found there. Understanding of how the C-terminal cytoplasmic domain regulates the rate of Na/H exchange is best understood for NHE1, in which all regulation is by an increase in affinity for intracellular [H]. It has been proposed that there are phosphorylation sites within the most C-terminal domain of NHE1 (amino acids 635-815), which are necessary for full activation by a variety of agonists, and a single region within the cytoplasmic tail (amino acids 567-635) which is required for the effect of all agonists, including those acting via different signal transduction pathways, activation via cell shrinkage, and growth factor regulation in the absence of changes in phosphorylation of NHE1 (9, 11) . The model described for NHE1 suggests that effects on Na and H transport are the consequences of this single regulatory region within the cytoplasmic tail causing an increase in proton affinity of the allosteric site in the N terminus, with a resultant decrease in K (H) and activation of transport (11, 12, 13) .

It seemed likely that NHE3 might be regulated in a manner different from NHE1 as the kinetics, second messenger regulation, and presumptive physiological function of NHE3 are different from those of NHE1. NHE3 is activated by serum and FGF via an increase in V, with no change in intracellular proton affinity, while phorbol esters cause a marked inhibition of exchanger activity, also with no change in proton affinity (4) . We hypothesized that it was likely that for NHE3 there was more than one region of the C-terminal cytoplasmic domain involved in the activation and inhibition by second messengers. To explore the relationship between the structure (amino acid sequence) and the regulation of the NHE3 isoform, we studied the second messenger regulation of mutant forms of the NHE3 protein which were truncated at various sites along the cytoplasmic tail.


EXPERIMENTAL PROCEDURES

Cell Culture

Transfected PS120 fibroblasts were grown in Dulbecco's modified Eagle's medium supplemented with 25 mM NaHCO, 10 mM HEPES, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 10% fetal bovine serum in a 5% CO, 95% air incubator at 37 °C. Geneticin (400 µg/ml) was used to maintain selection pressure and was added immediately after each subculturing. In addition, cells were exposed to an acid load consisting of 50 mM NHCl/saline solution for 1 h, followed by 1 h in an isotonic 3 mM Na solution. Surviving cells were then placed in normal culture medium and allowed to reach 30-50% confluence. The acidification process was initially repeated every 2-3 days until >50% of cells survived and was then repeated every other week to maintain high Na/H exchange activity.

Construction and Expression of NHE3 Truncation Mutants

The amino acid sequence of the C-terminal cytoplasmic tail of NHE3 was mapped for putative protein kinase consensus sites using a computer program, PC/GENE (PROSITE) (14) . Identified putative protein kinase consensus sequences included: 10 protein kinase C, four calmodulin kinase II, four cAMP-dependent protein kinase, and two tyrosine kinase sites. This formed the strategy for studying which region(s) of the C terminus of the exchanger was involved in regulation by protein kinases and growth factors.

We previously cloned the NHE3 cDNA into the NheI and SaII sites of pMAMneo to create the NHE3/pMAMneo construct, which was stably transfected into PS120 cells (1) . To create NHE3 C terminus truncation mutants, the NHE3/pMAMneo construct was linearized with SalI, followed by a ``fill-in'' reaction with -phosphorothioate nucleotides and Klenow, and then digested with EcoRV. This EcoRV restriction site was introduced between the 3`-end of the NHE3 cDNA and the SalI restriction site during the construction of the NHE3/pMAMneo construct. The NHE3 cDNA was then digested from its 3`-end by exonuclease III/mung bean nuclease, followed by self-ligation and transformation into Escherichia coli to generate NHE3 cDNA deletion clones. These clones were then sequenced by the dideoxy method of Sanger to determine the extent of deletion from the 3`-end and to be sure that they were in frame with the TGA sequence (stop codon) 15 base pairs downstream of the SalI site. Four constructs of NHE3/pMAMneo were selected (the notation used was E3/X/pMAMneo, where X is the amino acid number at the truncation site): E3/509/pMAMneo, E3/585/pMAMneo, E3/686/pMAMneo, and E/756/pMAMneo. The construct E3/475/pMAMneo was obtained by cloning into the NheI and XhoI sites of pMAMneo the NHE3 cDNA fragment (nucleotides 24-1422) which was obtained by polymerase chain reaction using NHE3/pBluescript as the template and the primers (5`-primer: GCCGCTCTAGAACTAGTGG (flanking Bluescript); 3`-primer: GCAGCTTCTCGAGCAGCTTGGGCTCCC (flanking NHE3)). These five NHE3 deletion constructs were then stably transfected into PS120 fibroblasts to generate corresponding NHE3 truncation mutants.

Measurement of Na/H Exchange Activity

For fluorometry experiments, cells were seeded on glass coverslips, grown overnight in serum-free medium, and studied when they reached 50-70% confluence. The cells were loaded with the acetoxymethyl ester of 2`,7`-bis(carboxymethyl)5-6-carboxyl-fluorescein (BCECF-AM, 5 µM) in ``Na medium'' (containing 130 mM NaCl, 5 mM KCl, 2 mM CaCl, 1 mM MgSO, 1 mM NaHPO, 25 mM glucose, 20 mM HEPES, pH 7.4) for 60-90 min at 23 °C, then washed with ``TMA medium'' (containing 130 mM tetramethylammonium-Cl, 5 mM KCl, 2 mM CaCl, 1 mM MgSO, NaHPO, 25 mM glucose, 20 mM HEPES, pH 7.4) to remove the extracellular dye, and the coverslip was mounted at an angle of 60° in a 100-µl fluorometer cuvette designed for perfusion as described (15) and thermostatted at 37 °C. The cells were pulsed with 40 mM NHCl in TMA-Cl for 15-20 min; removal of NHCl and perfusion with TMA medium resulted in acidification of the cells. In the okadaic acid experiments, cells were incubated with okadaic acid (OA) (1 µM) in TMA medium for 15 min at 37 °C before addition of Na medium containing OA, and measurement of pH recovery. In other separate experiments, N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W13), N-(4-aminobutyl)-naphthalene-sulfonamide (W12), 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), and phorbol myristate acetate (PMA, 1 µM) were added 10, 10, 10, and 5 min, respectively, before Na was introduced, and fibroblast growth factor (FGF 10 ng/ml) and dialyzed fetal bovine serum (FBS, 10%) were added simultaneously with the Na medium. Concentrations of agonists were chosen as those which had been shown in earlier studies to elicit maximal or near maximal responses. Amiloride-sensitive Na-dependent pH recovery was measured in the presence and absence of the various agonists. Fluorescence measurements, determination of intracellular buffering capacity, and calibration of experiments were performed as previously reported (16) .

The rate of Na-dependent alkalinization was obtained by calculating the first order derivative of the Na-dependent pH recovery curve. Data points were recorded from every 3- to 12-s interval during the rapid phase of pH recovery, with longer (30 s) intervals between data points as the rate of alkalinization slowed. To minimize variability, a similar number of data points were collected in control and test cells in each experiment. Hydrogen ion efflux rates (µM H/s), equivalent to rate of Na/H exchange, were then determined by multiplying the rate of change in intracellular pH by the cellular buffering capacity at the corresponding pH values. Scatter plots of H efflux rate versus intracellular [H] were then constructed. Control cells (no agonist) were studied at the same time in parallel with treated cells to control for variability in basal exchange rate among cells from different cell passages and acid selection. All quantitative comparisons presented are based on analysis of equal number of control/test cells studied at the same time.

Data Analysis

Kinetic analyses did not include data from cells with intracellular pH greater than 7.1, as there is an endogenous acidification process in PS120 cells only when the intracellular pH is over 7.1, a phenomenon previously described by Tse et al. (5). At pH values <7.1, the pH recovery was entirely Na dependent and amiloride-sensitive. Na/H exchange rate data were analyzed using a nonlinear regression data analysis program (ENZFITTER, Biosoft Corp.) (17) which allowed fitting of data to a general allosteric model described by the Hill equation (v = V * [S] /K`+[S] ) with estimates for V and K`, as well as fitting to a hyperbolic curve, such as expected with Michaelis-Menten kinetics. Standard errors generated were used as an indication of the accuracy of the parameter estimates, as suggested in the program manual. In this analysis, K` estimates were used to compare the relative affinities of the exchangers for intracellular [H]. Apparent Hill coefficients (n) were calculated using data from the velocity range around 1/2 V, as described by Segel (18) .

The computer program used to analyze the data establishes a line which best fits the data based on the kinetic model. Estimates for K` and V with standard errors represent the 95% confidence interval for the fitted line. Lack of overlap of the 95% confidence intervals of the lines indicates significant differences exist between groups being compared (treatment and control). This can be assessed visually from the figures where there is minimal or no overlap of data points at V and from the kinetic parameter estimates (± S.E.) reported.

Materials

PS120 fibroblasts used for transfection were originally provided by J. Pouyssegur. Fetal bovine serum was obtained from Hyclone Corp (Logan UT) and was dialyzed to remove constitutents M < 60,000; Dulbecco's modified Eagle's medium was from Life Technologies, Inc., and TMA was from Fluka Chemical Corp (Ronkonkoma, NY). BCECF-AM and nigericin were from Molecular Probes (Eugene, OR), okadaic acid (sodium salt) was from LC Laboratories (Woburn, MA), FGF (basic) was from Boehringer Mannheim, W13, W12, and KN-04 from Seikagaku America, Inc. (Rockville, MD), and KN-62 was from Calbiochem (La Jolla, CA), while other reagents were purchased from Sigma.


RESULTS

A series of PS120/NHE3 truncation mutants with portions of the C-terminal cytoplasmic domain removed was generated; these mutants and the number of putative consensus sites for protein kinase C in each truncation are shown diagrammatically in Fig. 1.


Figure 1: Wild type and truncation mutant forms of NHE3. The membrane spanning portion of the NHE3 protein is shown as a hatched bar, while the portion of the C-terminal cytoplasmic portion remaining is shown as an open bar, with the amino acid position at the truncation site indicated for each mutant. The number of putative protein kinase C sites predicted to be present in the cytoplasmic tail of each truncation mutant is shown.



Second Messenger Regulation of Truncation Mutants: Inhibitory Domain

Second messenger regulation of the truncation mutants stably expressed in PS120 cells was investigated, and from these studies functionally distinct inhibitory and stimulatory regions of the C-terminal cytoplasmic domain of NHE3 were identified.

Effect of W13, W12, and KN-62

Previous studies have demonstrated regulation of ileal brush border Na/H exchange under basal conditions by calmodulin (CaM) (19, 20) . Furthermore, a putative CaM-binding site was identified by protein sequence analysis at the distal C-terminal end of NHE3 (see ``Discussion''). Thus the effects of inhibitors of CaM were studied on NHE3. Pretreatment of PS120/NHE3 cells with the CaM inhibitor W13 (50 µM, 10 min) caused a 71% increase in Na/H exchange activity compared with untreated controls (V 918 ± 81 µM/s versus 537 ± 55 µM/s for W13-treated and control cells, respectively), with no change in K` (0.14 ± 0.06 µMversus 0.15 ± 0.07 µM for W13 and control cells, respectively) or n (2.0 and 1.8 for W13 and control cells, respectively) (Fig. 2A). In a separate series of experiments (), the W13 effect was shown to be concentration dependent: 10 µM W13 did not affect NHE3 (V 1048 ± 69 µM/s versus 1023 ± 46 µM/s) for W13-treated and control cells, respectively; 25 µM W13 increased the V by 76% (2156 ± 164 µM/s) compared to untreated controls (1225 ± 51 µM/s). The magnitude of the stimulation of V was similar when 45 µM W13 was studied (75% increase) (2395 ± 531 µM/s versus 1369 ± 143 µM/s for W13-treated and control cells, respectively). Preincubation of the cells for 10 min with the W13 hydrophobic control, W12 (45 µM), did not affect NHE3 activity (1771 ± 229 µM/s versus 1704 ± 151 µM/s for W12-treated and control cells, respectively) ().


Figure 2: Effects of calmodulin inhibitors W13 and KN-62 on NHE3. A, the calmodulin antagonist W13 stimulates Na/H exchange in PS120/NHE3 cells. Cells were acidified by incubation with NHCl, followed by perfusion with either TMA solution for control cells (), or with TMA solution containing 50 µM W13 () for 10 min. Cells were then allowed to recover to steady state pH in Na medium. In this figure, H efflux rates, equivalent to Na/H exchange, are plotted against intracellular [H]. Na/H efflux rates were calculated at various pH, and lines were fit to the data using an allosteric model, and kinetic parameters (V, K`(H), and n) were estimated. W13-treated cells had a 71% higher V (918 ± 81 µM/s) compared with untreated control cells (V 537 ± 55 µM/s), with no change in K` or n. These data were obtained from 15 similar experiments. B, effects of W13 (50 µM) were determined in E3/756 cells studied under the same conditions described in Fig. 2A. W13 had a minimal effect on E3/756 compared with its effect on wild type NHE3. Results are from six similar experiments. C, effects of KN-62 (50 µM) under same conditions used to study W13 in Fig. 2A on PS120/NHE3. KN-62-treated cells had a 51% higher V (2135 ± 138 µM/s versus 1418 ± 114 µM/s in KN-62-treated and control, respectively) with no change in K` or n. These data were obtained from four similar experiments. D, effects of KN-62 (50 µM) under same conditions used to study W13 in Fig. 2A on E3/756. KN-62 did not affect E3/756. Results are from four similar experiments.



In order to investigate whether the Ca/CaM-induced inhibition of NHE3 was mediated through Ca-CaM-dependent protein kinase II, we studied the effect of the Ca-CaM-dependent protein kinase II inhibitor KN-62 (). Pretreatment of the cells with KN-62 (5 µM, 10 min) stimulated the NHE3 V by 36% (1771 ± 230 µM/s) versus control (1301 ± 104 µM/s). Similar preincubation with 25 µM KN-62 stimulated V by 51% (2135 ± 138 µM/s) compared to controls (1418 ± 114 µM/s) (no change occurred in K` or nK`: 0.10 ± 0.03 µMversus 0.16 ± 0.05 µM in KN-62-treated and control cells, respectively; n: 2.1 ± 0.2 versus 2.1 ± 0.2 in KN-62-treated and control cells, respectively) (Fig. 2C), and the magnitude of stimulation of V was not further affected by increasing the concentration of KN-62 to 50 µM (2013 ± 42 µM/s versus 1369 ± 143 µM/s for KN-62 and untreated control, respectively; 47% increase). Pretreatment of the PS120/NHE3 cells with KN-04 (50 µM, 10 min), the negative control substance for KN-62 did not affect the kinetic parameters of NHE3 (data not shown).

To determine whether the effects of W13 and KN-62 were additive, the effects of a maximum concentration of W13 (45 µM), were determined in cells pretreated for 10 min with a concentration of KN-62 which maximally stimulated NHE3 (50 µM). In these studies, cells exposed to KN-62 for 10 min had initial Na/H exchange rates determined with or without W13 (45 µM). W13 caused an increase in Na/H exchange rate of 26% in KN-62 exposed cells (2521 ± 316 µM/s versus 2006 ± 380 µM/s in W13 plus KN-62 treated cells versus KN-62 treated alone; studied at the same time, Na/H exchange rate in W13-treated and control cells was 2553 ± 127 and 1403 ± 30 µM/s, respectively).

Truncation mutant E3/756 was studied to determine if amino acids 757-832 were required for the W13 and KN-62 stimulation of NHE3. Pretreatment with W13 (50 µM, 10 min) (Fig. 2B) and KN-62 (50 µM, 10 min) (Fig. 2D) failed to cause significant stimulation of E3/756. The presence of an inhibitory domain between amino acids 756 and the C terminus is indicated by the much faster Na/H exchange rate for E3/756, although the amount of NHE3 protein has not been considered.

Effect of PMA

A second inhibitory site within the C-terminal cytoplasmic domain of NHE3 was identified in experiments involving pretreatment of the cells with phorbol ester to activate protein kinase C. As we have previously reported (4) , full-length NHE3 was inhibited by 5 min of preincubation with PMA (1 µM) with a 64% decrease in V (890 ± 81 µM/s for control and 318 ± 42 µM/s for PMA-treated cells) but no change in K` (0.19 ± 0.07 µM for control and 0.16 ± 0.12 µM for PMA-treated cells) or n (2.0 for control and 1.9 for PMA) (Fig. 3A). E3/756, with two putative protein kinase C sites removed and eight remaining, was similarly inhibited by PMA (data not shown), as were cells transfected with truncation 689, with four protein kinase C sites removed (six remaining) (Fig. 3B). The inhibition of E3/689 by PMA was reflected by a 39% decrease in V from 1525 ± 96 µM/s for untreated to 924 ± 56 µM/s for PMA-treated cells, with no change in K` (0.09 ± 0.04 µM for untreated and 0.07 ± 0.03 µM for PMA treated cells) or in the apparent Hill coefficient (1.8 untreated, 1.9 PMA treated). In contrast, E3/585 cells showed no inhibition (or stimulation) of Na/H exchange by PMA, with V estimates of 1233 ± 94 µM/s for control and 1293 ± 73 µM/s for PMA-treated cells (Fig. 3C) and no change in K` (0.14 ± 0.06 and 0.18 ± 0.06 µM for control and PMA-treated cells, respectively) or in n (1.7 for both groups). These results indicate that the regulatory site(s) for PMA were located between amino acid 585 and amino acid 689. There are five putative PKC consensus sites within this region.


Figure 3: Inhibition of NHE3 by phorbol ester occurs at sites between amino acids 585 and 689. A, phorbol ester inhibits Na/H exchange in PS120/E3 cells. Cells were acidified with NHCl followed by TMA medium, then perfused with either Na medium (), or with Na medium containing phorbol myristate acetate (-PMA, 1 µM, added 5 min before perfusion with sodium medium) and their Na-dependent pH recovery in HCO-free medium measured. PMA-treated cells had a 64% lower V (318 ± 42 µM/s) compared with untreated control cells (V 890 ± 81 µM/s) with similar K`(H) and Hill coefficients. B, phorbol ester also inhibits Na/H exchange in E3/689 cells. In a similar experiment as described in A above, acidified cells pretreated with PMA (PMA (), 1 µM) showed a slower rate of pH recovery than did untreated control cells (). PMA-treated cells had a 39% lower V (924 ± 56 µM/s) than did the controls (1525 ± 96 µM/s), while K`(H) and n estimates were not different. C, phorbol ester does not alter Na/H exchange in PS120 E3/585 cells. Control () and PMA-treated cells () were acidified and their Na-dependent pH recovery measured. There was no difference in Na/H exchange rates for untreated or treated cells, with similar V, K`(H), and n estimates for control and PMA-treated cells. These data are from at least 12 experiments with each cell line. D, the inhibitory effect of PMA on PS120/NHE3 cells was independent of the effect of W13. Cells were incubated with W13 (50 µM) for 10 min () as described in Fig. 2, or with W13 for 10 min and PMA (1 µM) for the last 5 min (), then allowed to recover to steady state pH in Na medium. PMA following incubation with W13 caused a reduction in V by 47% (from 1110 ± 37 to 585 ± 48 µM/s) compared with cells incubated with W13 alone, with no effect on K` or n. These data represent six separate experiments with W13- and W13/PMA-treated cells.



When PS120/NHE3 cells were pretreated with 50 µM W13, the degree of inhibition by PMA was similar to that seen in cells not pretreated with W13 (Fig. 3D), with a decrease in V from 1110 ± 37 to 585 ± 48 µM/s in cells only treated with W13 compared to cells treated with W13 followed by 1 µM PMA, respectively. This result suggests that regulation by PMA is independent of the CaM effect.

Second Messenger Regulation of Truncation Mutants: Stimulatory Domain

In addition to the inhibitory regions, several distinct but clustered stimulatory regions of the C-terminal cytoplasmic tail of NHE3 were identified through experiments using OA, FGF, and FBS. Okadaic acid is a serine/threonine phosphatase 1 and 2a inhibitor that was used to examine the role of basal phosphorylation in the activity of the exchanger. Preincubation with OA (1 µM) for 15 min, stimulated Na/H exchanger activity in PS120/NHE3 cells, with a 26% increase in V from 1550 ± 172 µM/s for control to 1948 ± 62 µM/s for OA-treated cells (Fig. 4A), with no change in K` (0.19 ± 0.08 and 0.16 ± 0.06 µM for control and OA-treated cells, respectively). In E3/585 cells, in which the two identified inhibitory domains of NHE3 were removed, a much greater percent stimulation by OA was seen. There was a 68% increase in V from 557 ± 69 µM/s in untreated controls to 936 ± 104 µM/s in OA-treated cells (Fig. 4B), and no change in K` (0.15 ± 0.06 and 0.21 ± 0.08 µM for control and OA-treated cells, respectively). In contrast, there was minimal, if any, stimulation by OA in E3/509 cells (Fig. 4C), with little difference in V or K` estimates for control (V 87 ± 13 µM/s, K` 0.24 ± 0.10 µM) and for OA-treated cells (V 98 ± 16 µM/s, K` 0.18 ± 0.10 µM). This suggested that a response element(s) for okadaic acid was located between amino acids 509 and 585.


Figure 4: Stimulation of NHE3 by okadaic acid occurs between amino acids 509 and 585. A, OA stimulates Na/H exchange in PS120/NHE3 cells. Experiments were performed as described above, with cells treated with OA (, 15 min preincubation, 1 µM) compared to untreated cells (). OA stimulated the V by 26% (1948 ± 62 versus 1550 ± 172 µM/s) without affecting the K`(H) or n. At 3 µM, OA caused similar stimulation of PS120/NHE3 and also did not affect the K(H) or n of the exchanger (data not shown). B, OA stimulates Na/H exchange in PS120 E3/585 cells. Experiments were performed as described above, with cells treated with OA ((), 15 min preincubation, 1 µM) compared with untreated cells (). OA-treated cells had a 68% higher Na/H exchange rate that did controls, with V 936 ± 104 and 557 ± 69 µM/s, respectively, with no change in K`(H). C, OA does not significantly stimulate Na/H exchange in PS120 E3/509 cells. In similar experiments as with E3/585 cells, E3/509 cells showed no difference in Na/H exchange or in V estimates compared with control cells (98 ± 16 and 87 ± 13 µM/s, respectively). These results represent data from six separate experiments for each cell line.



Fibroblast growth factor caused a 51% stimulation of exchanger activity in PS120/NHE3 cells, with a V 404 ± 36 µM/s for FGF- (10 ng/ml) treated cells compared with untreated control cells (V 267 ± 28 µM/s) with no change in K` (0.10 ± 0.07 and 0.09 ± 0.06 µM for FGF-treated and control cells, respectively) or n (FGF-treated 1.9 versus 2.0 for control) (Fig. 5A). E3/509 cells (Fig. 5B) also responded to FGF with a 76% increase in V (153 ± 9 µM/s versus 87 ± 13 µM/s FGF-treated and control cells, respectively), also without a change in K` (0.16 ± 0.03 µM for FGF-treated and 0.24 ± 0.10 µM for control). In contrast, there was no longer a response to FGF in E3/475 cells as shown in the representative fluorometer trace in Fig. 5C. The basal rate of E3/475 was so low that it was not possible to perform quantitative analysis of the initial rate, so FGF was added during pH recovery for qualitative assessment of the effect. There was no change in the rate of pH recovery after FGF was added to E3/475 cells. This indicates that the regulatory site for FGF is in the region between amino acids 475 and 509.


Figure 5: Stimulation of NHE3 by fibroblast growth factor occurs between amino acids 475 and 509. A. FGF stimulates Na/H exchange in PS120/E3 cells. Cells were acidified with NHCl followed by TMA solution and their rate of pH recovery measured in the presence () or absence () of FGF (10 ng/ml). FGF-treated cells showed a 51% increase in V compared with control (404 ± 36 versus 267 ± 28 µM/s for FGF-treated and control cells, respectively) and no change in K`(H) or n. B, FGF also stimulates E3/509 cells. Cells treated with FGF () showed a 76% increase in Na/H exchange rate compared with untreated control cells () (153 ± 9 versus 87 ± 13 µM/s for FGF and control cells, respectively), also with no significant change in K`(H). C, FGF has no effect on E3/475 cells. This figure shows a representative fluorometer trace with FGF (10 ng/ml) added to E3/475 cells during pH recovery following acidification, as the exchange rate was too slow to allow kinetic analysis. There was no change in the rate of pH recovery after addition of FGF. The kinetic plots for PS120/NHE3 and E3/509 represent at least six separate experiments, while the E3/475 experiment was repeated five times.



In separate studies, the interaction of two stimulatory domains was studied. In PS120/NHE3 cells which were exposed to OA (1 µM) for 15 min, subsequent addition of FGF at the steady state caused a further increase in exchanger activity as indicated by further alkalinization (data not shown).

All the truncated NHE3 isoforms studied were stimulated by 10% FBS, with an increase in Na/H exchange rate. E3/475 cells, with only 19 amino acids remaining of the putative cytoplasmic tail, showed an increase in pH recovery when 10% dialyzed FBS was added during pH recovery. The stimulation by FBS was inhibited by 3 mM amiloride, indicating that the response was indeed due to an increase in Na/H exchanger activity (Fig. 6). This suggests that the regulatory site for FBS may be located in the putative membrane spanning portion of the protein or in the small portion of cytoplasmic tail remaining in E3/475.


Figure 6: Fetal bovine serum stimulates the NHE3 exchanger truncated at amino acid 475. This representative fluorometer trace shows the response of acidified E3/475 cells to FBS (10%, dialyzed) added during perfusion with Na medium. Amiloride (3 mM) was then added at the time indicated to confirm that the response was amiloride sensitive Na-dependent alkalinization. These tracings are representative of five similar experiments.




DISCUSSION

In this study, we show that the C-terminal domain of the cloned brush border Na/H exchanger isoform NHE3 is involved in its regulation by protein kinases and growth factors. This is similar to what has been found for the housekeeping isoform NHE1. In contrast to NHE1, in which all regulation is stimulatory, we have identified distinct stimulatory and inhibitory regions in the C terminus of NHE3. Moreover, both these domains contain multiple subdomains with separate sites within the stimulatory region for the effects of OA, FGF, and FBS and within the inhibitory region for the effects of CaM and PMA (Fig. 7).


Figure 7: The stimulatory and inhibitory regions of the NHE3 cytoplasmic tail are indicated in this model of the NHE3 Na/H exchanger. The cytoplasmic tail contains separate sites for stimulation by FGF and OA and for inhibition by PMA and calmodulin, while the effect of FBS may be mediated either directly through the most proximal part of the cytoplasmic tail or the membrane spanning portion of the exchanger or possibly through an intermediate or accessory factor which then interacts with the exchanger. The mechanism by which the cytoplasmic tail interacts with the membrane spanning portion to cause the changes in Na/H exchange rate (V) is not known. This interaction may be due to conformational changes within the NHE3 molecule, perhaps in response to phosphorylation of amino acid residues in the cytoplasmic tail, or may involve intermediates, shown here as postulated accessory proteins R and R, which might mediate the stimulatory and inhibitory effects of the second messengers.



The stimulatory and inhibitory regions regulate NHE activity separately, and the exchanger can be both up- and down-regulated simultaneously. This is best shown by the response of NHE3 to FBS which contains both growth factors to stimulate the exchanger and protein kinase C activators to inhibit the exchanger. We previously reported PS120/NHE3 cells pretreated with the protein kinase C inhibitor H7 showed a greater stimulation by FBS compared with cells treated only with FBS (4) . This indicates that while the overall effect of FBS on NHE3 is stimulatory, this effect is modulated by a concurrent inhibition mediated by protein kinase C.

Phosphorylation of the exchanger or of intermediate factors appears to play a role in establishing basal Na/H exchanger activity. Evidence for this includes the effect of OA and KN-62 to increase exchanger activity. Okadaic acid, a phosphatase 1 and 2A inhibitor would only be expected to increase phosphorylation and Na/H if there were basal kinase activity. KN-62 is a CaM kinase inhibitor, originally described as specifically inhibiting CaM kinase II, and subsequently as also inhibiting CaM kinase III and V (21, 22, 23) . KN-62 would only be expected to alter basal Na/H exchange if CaM kinase were inhibiting basal Na/H exchange.

CaM inhibits NHE3 under basal conditions, 1) acting by both CaM kinase-dependent and -independent mechanisms and 2) with both effects requiring amino acids 757-832 in the C terminus of NHE3. Both W13 and KN-62 caused concentration-dependent stimulation of NHE3, while W12, the hydrophobic control for W13 did not affect Na/H exchange. The W13 concentration-dependent stimulation was in the concentration range demonstrated for other CaM-dependent processes (24) . That W13 stimulated NHE3 above that stimulated by a maximum concentration of KN-62 demonstrates that W13 is causing an effect in addition to that of KN-62; and thus we suggest that CaM acts by both CaM kinase-dependent and -independent mechanisms. Both forms of CaM regulation occur at basal [Ca]. This is not surprising for the kinase-independent regulation, since elevating Ca acts by increasing the affinity for CaM, and CaM effects at basal [Ca] are known to occur. However, CaM kinase II is normally activated by elevated Ca, and further studies are required to understand whether and how CaM kinase(s) regulates NHE3.

Calmodulin acting by kinase-independent mechanisms in regulation of transport proteins is well characterized, including regulation of Ca-ATPase (25, 26) and the recent demonstration of Ca/CaM inhibition of the olfactory cyclic-nucleotide-gated cation channel, in which Ca/CaM inhibits cyclic-nucleotide activation by an ATP-independent mechanism (27, 28) . Of note, this Ca/CaM inhibition involved a decrease in response to cAMP and not an effect on basal activity (27) . In a recent report, calmodulin and/or CaM kinase II was shown to inhibit Na/H exchange in LLC-PK1 cells, a piglet renal epithelial cell line, although in this study the inhibition followed stimulation by calcitonin, and there was no effect on basal activity (29) . The inhibition of Na/H exchange activity by calmodulin reported here differs from the effect of calmodulin on the plasma membrane Ca-ATPase where calmodulin is thought to interact with the CaM-binding domain to free the active site of the pump and allow access to the substrates, thereby activating transport (26) . Thus our study demonstrates a newly recognized function for CaM, that of causing basal inhibition of a transport protein.

Not known is how CaM affects NHE3. Amino acids 757-832 (the very C terminus of NHE3) of NHE3 are required for both the CaM kinase-dependent and -independent effects, but this area of NHE3 does not contain a CaM kinase consensus sequence. This suggests either involvement of at least one accessory protein or that CaM binds to this area of NHE3 and activates a CaM-dependent kinase which then phosphorylates a CaM kinase phosphorylation consensus sequence either in another part of the C terminus of NHE3 or in a regulatory protein. Of note, while NHE3 is a phosphoprotein under basal conditions,() it is not known whether there are changes in its phosphorylation as part of CaM regulation.

Direct binding of CaM to NHE3, especially between amino acids 757-832 has not been demonstrated. However, by visual inspection, there is a region between amino acid 777 and 791 of NHE3 which has some characteristics in common with several documented calmodulin-binding sites in several other CaM-binding proteins (30, 31) . This region of NHE3 contains hydrophobic residues and also several Arg (charged) residues but is atypical in that it also contains a Glu and several Pro residues (2) . Demonstration of direct CaM binding to NHE3 and determination of CaM effects on NHE3 phosphorylation are required to further understand the mechanism of CaM regulation of NHE3.

These effects of CaM demonstrated here with NHE3 in PS120 fibroblasts may explain the previously reported finding that under basal conditions CaM inhibits ileal NaCl absorption (32) and that in brush border membrane vesicles made from rabbit ileum, W13 (19) , and a specific Ca/CaM kinase II inhibitory peptide (20) both caused an increase in basal Na/H exchange activity. In those studies, CaM kinase II activity was implicated in the inhibition of basal exchanger activity because the peptide inhibitor was designed to inhibit kinase activity by binding to the CaM kinase II autoinhibitory domain (20) . An additional, direct role of calmodulin was not tested. The fact that at least partially similar regulation of NHE3 by CaM occurs in a fibroblast and in the highly specialized brush border domain of an intestinal epithelial cell suggests that this aspect of regulation is specific to the Na/H exchanger isoform rather than to the cell in which the Na/H exchange occurs.

Phosphorylation also appears involved in growth factor/kinase regulation of NHE3 since NHE3 is stimulated by FBS and FGF and inhibited by phorbol esters. It is likely that the kinase consensus sites identified in the different regions of the cytoplasmic tail are important for regulation of the exchanger; however, it is unclear which are involved in mediating the effects of the agonists. The effect of PKC is mediated by a region in which there are five putative protein kinase C consensus sequences, and future studies will be necessary to determine if one or more of these sites are phosphorylated with PMA addition.

Within the stimulatory domain of NHE3, there are several distinct regions which activate the exchanger with an increase in V, although it is not known if these sites are phosphorylated. The region between amino acids 509-585 appears to be activated under basal conditions as seen from the effect of OA, and the site between amino acids 475 and 509 appears to mediate the effect of FGF. Although the latter region does not contain a ``classical'' tyrosine kinase consensus sequence, there is a tyrosine as well as 2 serines present in the region. It is less clear which region of the protein N-terminal to amino acid 475 mediates the response to FBS, as there is only a single serine remaining within the cytoplasmic tail, and no sites which fit a computer algorithm (PCGENE) for a kinase consensus site motif. Serum may regulate NHE3 through an intermediate regulatory protein, but it is also possible that the N-terminal domain alone mediates activation by FBS. It is interesting that the exchanger is activated by FBS via a different site than that for FGF and suggests that either the different growth factors have separate binding sites, or that there is another factor in serum which is responsible for the activation. Further evidence that prediction of the effect of various second messengers on NHE activity based entirely on the putative phosphorylation sites is not possible comes from the observation that although there are four cAMP-dependent kinase consensus sequences within the cytoplasmic domain of NHE3, cAMP has no effect on the activity of the exchanger when transfected into either PS120 fibroblasts or Caco-2 intestinal epithelial cells (4 and data not shown).

Both NHE1 and NHE3 are activated by FGF, OA, and FBS (4) ; however, we reported previously that there are differences in the mechanism of activation of the two isoforms as reflected by changes in different kinetic parameters. NHE1 is activated via an increase in affinity for H as a consequence of treatment with the agonists, whereas NHE3 responds with an increase in V with no change in H affinity. Both NHE1 and NHE3 require ATP for regulation, which is consistent with the exchangers or an intermediate being phosphorylated during regulation, although ATP-dependent non-phosphorylation-dependent control of transport processes are well recognized. Pouyssegur et al.(11) have studied the regulation of NHE1 in detail using a similar strategy to that presented here with deletion of various portions of the C-terminal cytoplasmic domain. NHE1 is stimulated by growth factors, thrombin, and phorbol esters. Although these agonists act via different second messenger pathways (receptor tyrosine kinase for EGF and a PKC-dependent pathway for thrombin and phorbol esters), they all stimulate the same increases in phosphorylation of NHE1 based on phosphopeptide maps. This implies that the second messenger pathways converge on a common activating kinase, which then phosphorylates the exchanger; this phosphorylation has been shown to be only on the portion of NHE1 C-terminal to amino acid 635. In addition, Wakabayashi et al.(11) identified a critical region in NHE1 between amino acids 567-635, without which no regulation of NHE1 occurs. In the absence of the portion of NHE1 which is phosphorylated, this region is sufficient to mediate part of the growth factor response and to preserve high pH sensitivity, leading to speculation that there must be additional regulatory factors which activate NHE1 through interaction with the cytoplasmic domain N-terminal to amino acid 635.

This model of a single regulatory region for NHE1 has not been confirmed in all reports. A study by Winkel et al.(33) using microinjection of an antibody directed at the 658-815 region of NHE1 found that this antibody altered regulation by endothelin-1 and -thrombin, but that PKC and osmotic induced activation, as well as pH sensing, were mediated via a separate, more proximal region of the cytoplasmic tail. In addition we found the NHE1/519, a truncated form of NHE1 having only 19 amino acids at the end of the N-terminal portion could be stimulated by serum.() These results suggest that sites in addition to the crucial regulatory region (567-635) are involved in hormonal regulation of NHE1.

In addition to insights concerning NHE3 regulation, these truncation studies also give insight concerning the location of the allosteric site involved in Na/H exchange. Removal of all but 19 amino acids in the cytoplasmic tail (truncation at amino acid 475) resulted in greatly reduced Na/H exchange activity, as seen by the extremely slow rate of recovery shown by E3/475 cells in Fig. 5C. It is not known if this reflects less plasma membrane location of the exchanger or less function of a normal amount of E3/475 in the plasma membrane. Although the basal rate of this truncation mutant was too slow for kinetic analysis, when the E3/475 was activated by FBS, the kinetic plot of Na/H exchange had a sigmoidal shape (data not shown) similar to that of the full-length NHE3 (Fig. 2A, 3A, and 4A). This suggested that the allosteric site(s) for H binding was preserved, and therefore most likely resides within the membrane spanning region. This is further supported by the finding that the other truncation mutants showed the same degree of allosteric interaction, with a Hill coefficient, n 2 for all the truncated exchangers, as well as for the full-length NHE3.

We propose a structure-function model for NHE3 which is shown in Fig. 7 . In this model there are distinct stimulatory and inhibitory regions within the cytoplasmic tail of the exchanger, and there are separate sites for regulation by the different agonists and inhibitors studied here. The nature of the interaction of the cytoplasmic tail of NHE3 with the membrane spanning region is unknown. However, since K`(H) is not altered during regulation, the tail probably does not affect affinity of the proton binding site(s). We postulate that the regulatory regions of the cytoplasmic tail of NHE3 interact either directly or indirectly with effector sites on the membrane-spanning domain to increase or decrease activity by changing exchanger activity or turnover number. As shown in Fig. 7 there may be intermediate ``regulators'' which interact with the regulatory areas in the C terminus identified in this study and/or with the effector site(s) on the N-terminal part of NHE3. We suggest the involvement of a stimulatory and an inhibitory protein or a single protein with stimulatory and inhibitory domains. The nature of these regulatory proteins is unknown, but analogy to the subunits of the class of heterotrimeric guanine nucleotide-binding proteins, which have isoforms which both stimulate and inhibit adenylate cyclase, is one possible model. We favor the existence and involvement of the intermediate protein(s) in regulation of NHE3 since we think it would be difficult for at least five separate regulatory parts of the C terminus of NHE3 to interact directly with the transport domain of NHE3.

  
Table: Effect of W13, W12, and KN-62 on PS120/NHE3

n, represents number of separate coverslips studied for control and inhibitor.



FOOTNOTES

*
This work was supported by National Institutes of Health Grants RO1DK26523, PO1DK44484, F32DK08820, F32DK08950, R29DK43778, Training Grant T32DK07632, and the Meyerhoff Digestive Diseases Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported in part by a Medical Research Council Travelling Fellowship, United Kingdom.

To whom correspondence should be addressed: GI Division, Dept. of Medicine, Johns Hopkins University School of Medicine, 925 Ross Research Bldg., 720 Rutland Ave., Baltimore, MD 21205. Tel.: 410-955-9675; Fax: 410-955-9677.

The abbreviations used are: NHE, Na/H exchanger; FGF, fibroblast growth factor; FBS, fetal bovine serum; BCECF-AM, 2`7`-bis(carboxyethyl)-5(6)-carboxyfluoroscein (acetoxymethyl ester); PMA, phorbol 12`-myristate 13-acetate; OA, okadaic acid; CaM, calmodulin.

C. M. Tse, J. W. Yip, and M. Donowitz, unpublished observations.

C. H. C. Yun, S. A. Levine, S. K. Nath, C. M. Tse, and M. Donowitz, unpublished observations.


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