Proc. Nati. Acad. Sci. USA Vol. 89, pp. 2424-2428, March 1992 Biochemistry
The Na+/H' antiporter cytoplasmic domain mediates growth factor signals and controls "H+-sensing" (Na-H exchanger/pH regulation/HW sensor/signal transduction/protein phosphorylation)
SHIGEO WAKABAYASHI, PIERRE FAFOURNOUX, CLAUDE SARDET, AND JACQUES POUYSS#GUR Centre de Biochimie, Centre National de la Recherche Scientifique, Parc Valrose, 06034 Nice, France
Communicated by Edward A. Adelberg, December 11, 1991
The amiloride-sensitive Na+/H' exchanger ABSTRACT (NHE1 human isoform) is activated in response to diverse mitogenic and oncogenic signals presumably through phosphoranism, a set of ylation. To get Insight into the activating deletion mutants within the C-terminal cytoplasmic domain of NHE1 has been generated. These mutant forms expressed in antiporter-deficient fibroblasts revealed that deletion of the complete cytoplasmic domain (i) preserves amiloride-sensitve Na+/H+ exchange and activation by intracellular H+, (is) reduces the affinity of the internal "H+-modifler site" in a manner mimicked by cellular ATP depletion, and (Us) alih growth factor-induced cytoplasmic alkanition. We conclude that NHEl can be separated into two distinct functional domains. One is an N-terminal transporter domain (T) that has all the features required to catalyze amiloride-sensitive Na+/H+ exchange with a built-in HW-modifier site. The other is a C-terminal cytoplasmic regulatory domain (R) that (i) determines the set point value of the exchanger and (a) mediates growth factor signals by interacting with the "H+-sensor" in a phosphorylation-dependent manner.
functional domains: the transporter with a built-in "H' sensor" and a cytoplasmic regulatory domain that controls the set point value and mediates extracellular signals.
MATERIALS AND METHODS Materials. 22NaCI (carrier free) was from Radiochemical
Centre (Amersham), and [7-14C]benzoic acid was from New England Nuclear. 5-(N-Methyl-N-propyl)amiloride (MPA) was a gift from E. Cragoe, Jr. (Merck Sharp & Dohme). All other chemicals were of the highest purity available. Cells and Culture Conditions. The Chinese hamster lung fibroblast line CCL39 (American Type Culture Collection), the Na+/H' antiporter-deficient derivative PS120, and the corresponding transfectants were maintained in Dulbecco's modified Eagle's medium (H21, GIBCO) containing 25 mM NaHCO3 and supplemented with 7.5% (vol/vol) fetal calf serum, penicillin (50 units/ml), and streptomycin (50 .g/ml). Cells were maintained at 370C in presence of 5% CO2. Construction of Na+/H+ Antiporter Deletion Mutants. c28 cDNA (cloned in pBluescript), coding for the human Na+/H+ antiporter (NHE1 isoform) (3, 15), was first inserted into the EcoRI site of the eukaryotic expression vector pECE (17) under the control of the early simian virus 40 (SV40) promoter (plasmid designated pEAP). For the construction of cDNA depleted of the 5' untranslated region (plasmid pEAPAS'), 355 base pairs (bp) of the 5' untranslated region was removed by using the restriction enzyme Nci I on pBluescript c28, and then the insert was cloned into Sal I/EcoRI sites of pECE. The construction of various 3'-end-deleted mutants was carried out by digestions of pEAP-A5' with EcoRI orXba I on the cloning site (3' end of the insert) and with the appropriate restriction enzymes on the insert, followed by blunt-ending with Klenow or T4 DNA polymerase and ligation with T4 ligase. Each deletion construct encodes an artificial stretch of one to three amino acids in the C-terminal end because of codons provided by a polylinker sequence prior to the universal termination codon of the pECE vector. The internal deletion mutant (pEA567-635), deleted of the cytoplasmic loop (residues 567-635), was constructed by using two different restriction enzymes, Eco 47III and Bsu 36I. Transient or Stable Expression of Mutant Antiporters in Hamster Fibroblasts. For transient expression, the antiporter-deficient mutant cell line PS120 (106 cells per 100-mm dish) was cotransfected with each plasmid construct (20 ,g) and pSVCAT (2 Ag) by using the calcium phosphate coprecipitation technique as described (18). After 2 days, transient expression of functional exchanger was evaluated by measuring amiloride analogue (MPA)-sensitive 22Na+ uptake. The 22Na' uptake activity was normalized by comparison to
The plasma membrane and amiloride-sensitive Na+/H' exchangers are electroneutral transporters that regulate intracellular pH (pH1) in virtually all eukaryotic cells (1, 2). The first Na+/H' exchanger isoform cloned (3), referred to as NHE1, is ubiquitously expressed, localized in the basal membrane of intestinal epithelial cells (4), and is activated by oncogenic transformation and in response to widespread external signals (5-8). Activation results from an increased affinity of the antiporter for the intracellular H' at an allosteric "modifier" site(s) (2, 9-11) that is thought to be distinct from the Na+/H+ transport sites (9). This activation leads to a cytoplasmic alkalinization, most prominent in the absence of bicarbonate (12, 13), that persists as long as the extracellular signal is present (14). Our cloning and immunological studies established that the human NHE1 isoform is a phosphoglycoprotein of 815 amino acids with a N-terminal domain (500 amino acids) that contains 10 to 12 putative transmembrane spanning segments followed by a large C-terminal hydrophilic cytoplasmic domain (3, 15). Stimulation by growth-promoting agents (epidermal growth factor, a-thrombin, phorbol esters, okadaic acid) increases the phosphorylation of the antiporter exclusively on serine residues and on apparently identical phosphopeptides, suggesting early integration of distinct transmembrane signals (15, 16). To study the structure-function of the Na+/H+ antiporter and get insight into the molecular mechanism of growth factor activation, we have generated a set of cDNA deletion mutants within the cytoplasmic domain and expressed them in the antiporter-deficient mutant cell line PS120. Here we show that the Na+/H+ antiporter can be separated into two distinct
Abbreviations: NHE1, Na+/H+ exchanger isoform 1; pH1, intracellular pH; ChoCi, choline chloride; SV40, simian virus 40; MPA, 5-(N-methyl-N-propyl)amiloride; CAT, chloramphenicol acetyltransferase.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
2424
Biochemistry: Wakabayashi et al. the chloramphenicol acetyltransferase (CAT) activity. For stable expression, PS120 cells (2 x 105 cells per 60-mm dish) were transfected the same way but only with 10 ug of each plasmid. Three to 4 days after transfection, the cells were submitted to the "HW-killing" selection test that eliminates recipient cells that do not express a functional Na+/H' antiporter. The test was performed as follows: cells were loaded with NH4' for 1 hr at 37TC in Hepes buffered saline (HBS) containing 50 mM NH4CI and 70 mM choline chloride (ChoCl) (pH 7.5), then rapidly washed twice with 120 mM ChoCl, pH 7.0, and further incubated for 1 hr in HBS containing 120 mM NaCI (pH 7.5). This treatment, which results in an acute and lethal acid load, was repeated five times over a period of 3 weeks, and the resulting stable transfectant cells were used for the experiment and stocked in liquid N2Immunoblot Analysis of the Exchanger Protein Expressed in the Membrane. PS120 cells (-4 x 107 cells) stably transfected with plasmid constructs were washed twice with cold water and placed for 20 min at 00C in hypotonic solution containing 3 mM KCl, 5 mM EDTA, 10 mM Tris (pH 7.4), and protease inhibitors (phenylmethylsulfonyl fluoride, o-phenanthrolin, and iodoacetamide, 1 mM each), and then harvested cells were homogenized with a Potter-type homogenizer (30 strokes). The cell homogenate was centrifuged for 10 min at 500 x g, and the supernatant was further centrifuged for 30 min at 100,000 x g. The resulting pellet (crude cell membrane fraction) was solubilized with 0.3 ml of 1% Nikkol in the above hypotonic solution without KCl and centrifuged for 30 min at 100,000 x g. Solubilized proteins in the resulting supernatant were separated on 7.5% acrylamide gel and electrophoretically transferred to Hybond C extra-supported membranes (Amersham) in 25 mM Tris/0.19 M glycine. Membranes were blocked in TN (50 mM Tris-HCl, pH 8.0/150 mM NaCl) containing 5% nonfat dry milk. The blots were then incubated with the antibody RP1-c28 (15) (1:100) in blocking solution overnight at 4°C, washed in TN, and incubated with horseradish peroxidase-conjugated goat antirabbit IgG (1:1000, Sigma) in TN for 1 hr. The blots were developed by enhanced chemiluminescent (ECL) detection system (Amersham). Measurement of 22Na+ Uptake Activity. Amiloride analogue (MPA)-sensitive 22Na' uptake was measured by either the Li+- or NH4+-loading methods as described (19-21). For Li'-loading, cells were incubated for 2 hr at 37°C in HBS containing 120 mM LiCl (pH 7.4) (20) and then washed twice with Li+/K+-free HBS containing 120 mM ChoCl (pH 7.4). The cells then received the same ChoCl medium containing 1,uCi (1 Ci = 37 GBq) of carrier-free 22Na' per ml and 1 mM ouabain with and without 0.1 mM MPA. The radioactivity taken up was measured after 15 min. Intracellular pHdependence of amiloride-sensitive 22Na' uptake was carried out as described (11) but with several modifications and with two stable transfectants (pEAP-A5' and pEA515). A change in pHi was produced by loading the cells with different concentrations of NH4' for 30 min at 37°C in a NH4+-loading medium [HBS containing 0-50 mM NH4Cl (the osmolarity was adjusted with ChoCl) and 5 mM glucose (pH 7.0 or 7.4)] and then by washing the cells rapidly with NH4+-free HBS containing 120 mM ChoCl. Then, the radioactivity of 22Na' taken up was measured after 1 min. In ATP-depletion experiments, 5 mM glucose was replaced by 5 mM 2-deoxyglucose and 2 ,g of oligomycin per ml for preincubation in the NH4+-loading medium. Thirty minutes after the addition of metabolic inhibitors, the cellular ATP level was reduced to
The Na+/H' antiporter cytoplasmic domain mediates growth factor signals and controls "H+-sensing" (Na-H exchanger/pH regulation/HW sensor/signal transduction/protein phosphorylation)
SHIGEO WAKABAYASHI, PIERRE FAFOURNOUX, CLAUDE SARDET, AND JACQUES POUYSS#GUR Centre de Biochimie, Centre National de la Recherche Scientifique, Parc Valrose, 06034 Nice, France
Communicated by Edward A. Adelberg, December 11, 1991
The amiloride-sensitive Na+/H' exchanger ABSTRACT (NHE1 human isoform) is activated in response to diverse mitogenic and oncogenic signals presumably through phosphoranism, a set of ylation. To get Insight into the activating deletion mutants within the C-terminal cytoplasmic domain of NHE1 has been generated. These mutant forms expressed in antiporter-deficient fibroblasts revealed that deletion of the complete cytoplasmic domain (i) preserves amiloride-sensitve Na+/H+ exchange and activation by intracellular H+, (is) reduces the affinity of the internal "H+-modifler site" in a manner mimicked by cellular ATP depletion, and (Us) alih growth factor-induced cytoplasmic alkanition. We conclude that NHEl can be separated into two distinct functional domains. One is an N-terminal transporter domain (T) that has all the features required to catalyze amiloride-sensitive Na+/H+ exchange with a built-in HW-modifier site. The other is a C-terminal cytoplasmic regulatory domain (R) that (i) determines the set point value of the exchanger and (a) mediates growth factor signals by interacting with the "H+-sensor" in a phosphorylation-dependent manner.
functional domains: the transporter with a built-in "H' sensor" and a cytoplasmic regulatory domain that controls the set point value and mediates extracellular signals.
MATERIALS AND METHODS Materials. 22NaCI (carrier free) was from Radiochemical
Centre (Amersham), and [7-14C]benzoic acid was from New England Nuclear. 5-(N-Methyl-N-propyl)amiloride (MPA) was a gift from E. Cragoe, Jr. (Merck Sharp & Dohme). All other chemicals were of the highest purity available. Cells and Culture Conditions. The Chinese hamster lung fibroblast line CCL39 (American Type Culture Collection), the Na+/H' antiporter-deficient derivative PS120, and the corresponding transfectants were maintained in Dulbecco's modified Eagle's medium (H21, GIBCO) containing 25 mM NaHCO3 and supplemented with 7.5% (vol/vol) fetal calf serum, penicillin (50 units/ml), and streptomycin (50 .g/ml). Cells were maintained at 370C in presence of 5% CO2. Construction of Na+/H+ Antiporter Deletion Mutants. c28 cDNA (cloned in pBluescript), coding for the human Na+/H+ antiporter (NHE1 isoform) (3, 15), was first inserted into the EcoRI site of the eukaryotic expression vector pECE (17) under the control of the early simian virus 40 (SV40) promoter (plasmid designated pEAP). For the construction of cDNA depleted of the 5' untranslated region (plasmid pEAPAS'), 355 base pairs (bp) of the 5' untranslated region was removed by using the restriction enzyme Nci I on pBluescript c28, and then the insert was cloned into Sal I/EcoRI sites of pECE. The construction of various 3'-end-deleted mutants was carried out by digestions of pEAP-A5' with EcoRI orXba I on the cloning site (3' end of the insert) and with the appropriate restriction enzymes on the insert, followed by blunt-ending with Klenow or T4 DNA polymerase and ligation with T4 ligase. Each deletion construct encodes an artificial stretch of one to three amino acids in the C-terminal end because of codons provided by a polylinker sequence prior to the universal termination codon of the pECE vector. The internal deletion mutant (pEA567-635), deleted of the cytoplasmic loop (residues 567-635), was constructed by using two different restriction enzymes, Eco 47III and Bsu 36I. Transient or Stable Expression of Mutant Antiporters in Hamster Fibroblasts. For transient expression, the antiporter-deficient mutant cell line PS120 (106 cells per 100-mm dish) was cotransfected with each plasmid construct (20 ,g) and pSVCAT (2 Ag) by using the calcium phosphate coprecipitation technique as described (18). After 2 days, transient expression of functional exchanger was evaluated by measuring amiloride analogue (MPA)-sensitive 22Na+ uptake. The 22Na' uptake activity was normalized by comparison to
The plasma membrane and amiloride-sensitive Na+/H' exchangers are electroneutral transporters that regulate intracellular pH (pH1) in virtually all eukaryotic cells (1, 2). The first Na+/H' exchanger isoform cloned (3), referred to as NHE1, is ubiquitously expressed, localized in the basal membrane of intestinal epithelial cells (4), and is activated by oncogenic transformation and in response to widespread external signals (5-8). Activation results from an increased affinity of the antiporter for the intracellular H' at an allosteric "modifier" site(s) (2, 9-11) that is thought to be distinct from the Na+/H+ transport sites (9). This activation leads to a cytoplasmic alkalinization, most prominent in the absence of bicarbonate (12, 13), that persists as long as the extracellular signal is present (14). Our cloning and immunological studies established that the human NHE1 isoform is a phosphoglycoprotein of 815 amino acids with a N-terminal domain (500 amino acids) that contains 10 to 12 putative transmembrane spanning segments followed by a large C-terminal hydrophilic cytoplasmic domain (3, 15). Stimulation by growth-promoting agents (epidermal growth factor, a-thrombin, phorbol esters, okadaic acid) increases the phosphorylation of the antiporter exclusively on serine residues and on apparently identical phosphopeptides, suggesting early integration of distinct transmembrane signals (15, 16). To study the structure-function of the Na+/H+ antiporter and get insight into the molecular mechanism of growth factor activation, we have generated a set of cDNA deletion mutants within the cytoplasmic domain and expressed them in the antiporter-deficient mutant cell line PS120. Here we show that the Na+/H+ antiporter can be separated into two distinct
Abbreviations: NHE1, Na+/H+ exchanger isoform 1; pH1, intracellular pH; ChoCi, choline chloride; SV40, simian virus 40; MPA, 5-(N-methyl-N-propyl)amiloride; CAT, chloramphenicol acetyltransferase.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
2424
Biochemistry: Wakabayashi et al. the chloramphenicol acetyltransferase (CAT) activity. For stable expression, PS120 cells (2 x 105 cells per 60-mm dish) were transfected the same way but only with 10 ug of each plasmid. Three to 4 days after transfection, the cells were submitted to the "HW-killing" selection test that eliminates recipient cells that do not express a functional Na+/H' antiporter. The test was performed as follows: cells were loaded with NH4' for 1 hr at 37TC in Hepes buffered saline (HBS) containing 50 mM NH4CI and 70 mM choline chloride (ChoCl) (pH 7.5), then rapidly washed twice with 120 mM ChoCl, pH 7.0, and further incubated for 1 hr in HBS containing 120 mM NaCI (pH 7.5). This treatment, which results in an acute and lethal acid load, was repeated five times over a period of 3 weeks, and the resulting stable transfectant cells were used for the experiment and stocked in liquid N2Immunoblot Analysis of the Exchanger Protein Expressed in the Membrane. PS120 cells (-4 x 107 cells) stably transfected with plasmid constructs were washed twice with cold water and placed for 20 min at 00C in hypotonic solution containing 3 mM KCl, 5 mM EDTA, 10 mM Tris (pH 7.4), and protease inhibitors (phenylmethylsulfonyl fluoride, o-phenanthrolin, and iodoacetamide, 1 mM each), and then harvested cells were homogenized with a Potter-type homogenizer (30 strokes). The cell homogenate was centrifuged for 10 min at 500 x g, and the supernatant was further centrifuged for 30 min at 100,000 x g. The resulting pellet (crude cell membrane fraction) was solubilized with 0.3 ml of 1% Nikkol in the above hypotonic solution without KCl and centrifuged for 30 min at 100,000 x g. Solubilized proteins in the resulting supernatant were separated on 7.5% acrylamide gel and electrophoretically transferred to Hybond C extra-supported membranes (Amersham) in 25 mM Tris/0.19 M glycine. Membranes were blocked in TN (50 mM Tris-HCl, pH 8.0/150 mM NaCl) containing 5% nonfat dry milk. The blots were then incubated with the antibody RP1-c28 (15) (1:100) in blocking solution overnight at 4°C, washed in TN, and incubated with horseradish peroxidase-conjugated goat antirabbit IgG (1:1000, Sigma) in TN for 1 hr. The blots were developed by enhanced chemiluminescent (ECL) detection system (Amersham). Measurement of 22Na+ Uptake Activity. Amiloride analogue (MPA)-sensitive 22Na' uptake was measured by either the Li+- or NH4+-loading methods as described (19-21). For Li'-loading, cells were incubated for 2 hr at 37°C in HBS containing 120 mM LiCl (pH 7.4) (20) and then washed twice with Li+/K+-free HBS containing 120 mM ChoCl (pH 7.4). The cells then received the same ChoCl medium containing 1,uCi (1 Ci = 37 GBq) of carrier-free 22Na' per ml and 1 mM ouabain with and without 0.1 mM MPA. The radioactivity taken up was measured after 15 min. Intracellular pHdependence of amiloride-sensitive 22Na' uptake was carried out as described (11) but with several modifications and with two stable transfectants (pEAP-A5' and pEA515). A change in pHi was produced by loading the cells with different concentrations of NH4' for 30 min at 37°C in a NH4+-loading medium [HBS containing 0-50 mM NH4Cl (the osmolarity was adjusted with ChoCl) and 5 mM glucose (pH 7.0 or 7.4)] and then by washing the cells rapidly with NH4+-free HBS containing 120 mM ChoCl. Then, the radioactivity of 22Na' taken up was measured after 1 min. In ATP-depletion experiments, 5 mM glucose was replaced by 5 mM 2-deoxyglucose and 2 ,g of oligomycin per ml for preincubation in the NH4+-loading medium. Thirty minutes after the addition of metabolic inhibitors, the cellular ATP level was reduced to
