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• W2065220616 abstract "The NHE2 isoform of the Na+/H+ exchanger (NHE) displays two proline-rich sequences in its C-terminal region that resemble SH3 (Src homology 3)-binding domains. We investigated whether these regions (743PPSVTPAP750, termed Pro-1, and786VPPKPPP792, termed Pro-2) can bind to SH3 domains and whether they are essential for NHE2 function and targeting. A fusion protein containing the Pro-1 region showed promiscuous binding to SH3 domains of several proteins in vitro, whereas a Pro-2 fusion bound preferentially to domains derived from kinases. In contrast, cytoplasmic regions of NHE1, NHE3, or NHE4 failed to interact. When expressed in antiporter-deficient cells, truncated NHE2 lacking both Pro-rich regions catalyzed Na+/H+exchange, retained sensitivity to intracellular ATP, and was activated by hyperosmolarity, resembling full-length NHE2. The role of the Pro-rich regions in subcellular targeting was examined by transfection of epitope-tagged forms of NHE2 in porcine renal epithelial LLC-PK1 cells. Both full-length and Pro-2-truncated NHE2 localized almost exclusively to the apical membrane. By contrast, a mutant devoid of both Pro-1 and Pro-2 was preferentially sorted to the basolateral surface but also accumulated intracellularly. These observations indicate that the region encompassing Pro-1 is essential for appropriate subcellular targeting of NHE2. The NHE2 isoform of the Na+/H+ exchanger (NHE) displays two proline-rich sequences in its C-terminal region that resemble SH3 (Src homology 3)-binding domains. We investigated whether these regions (743PPSVTPAP750, termed Pro-1, and786VPPKPPP792, termed Pro-2) can bind to SH3 domains and whether they are essential for NHE2 function and targeting. A fusion protein containing the Pro-1 region showed promiscuous binding to SH3 domains of several proteins in vitro, whereas a Pro-2 fusion bound preferentially to domains derived from kinases. In contrast, cytoplasmic regions of NHE1, NHE3, or NHE4 failed to interact. When expressed in antiporter-deficient cells, truncated NHE2 lacking both Pro-rich regions catalyzed Na+/H+exchange, retained sensitivity to intracellular ATP, and was activated by hyperosmolarity, resembling full-length NHE2. The role of the Pro-rich regions in subcellular targeting was examined by transfection of epitope-tagged forms of NHE2 in porcine renal epithelial LLC-PK1 cells. Both full-length and Pro-2-truncated NHE2 localized almost exclusively to the apical membrane. By contrast, a mutant devoid of both Pro-1 and Pro-2 was preferentially sorted to the basolateral surface but also accumulated intracellularly. These observations indicate that the region encompassing Pro-1 is essential for appropriate subcellular targeting of NHE2. 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein Chinese hamster ovary glutathione S-transferase hemagglutinin Na+/H+ exchanger phosphate-buffered saline intracellular (cytosolic) pH polyacrylamide gel electrophoresis Src homology domain 3 human embryonic kidney epithelial Na+channel The Na+/H+ exchangers (NHEs)1 are a family of proteins found in virtually all mammalian cells, where they catalyze the electroneutral exchange of intracellular H+ for external Na+ with a 1:1 stoichiometry (see Refs. 1Orlowski J. Grinstein S. J. Biol. Chem. 1997; 272: 22373-22376Crossref PubMed Scopus (520) Google Scholar and 2Wakabayashi S. Shigekawa M. Pouyssegur J. Physiol. Rev. 1997; 77: 51-74Crossref PubMed Scopus (562) Google Scholarfor review). In most cells, NHE activity is important for intracellular pH (pHi) homeostasis and also for maintenance of normal cellular volume (3Kapus A. Grinstein S. Wasan S. Kandasamy R. Orlowski J. J. Biol. Chem. 1994; 269: 23544-23552Abstract Full Text PDF PubMed Google Scholar). In addition, in epithelia of the kidney, gastrointestinal tract, and other organs, NHE plays a central role in the absorption of NaCl, bicarbonate, and water. Six distinct NHE isoforms have been identified to date in mammalian cells (1Orlowski J. Grinstein S. J. Biol. Chem. 1997; 272: 22373-22376Crossref PubMed Scopus (520) Google Scholar). Although the primary sequence homology between the isoforms is limited (20–60%), they all share the same predicted membrane topology, consisting of 12 membrane-spanning segments at the N terminus and a hydrophilic C-terminal tail thought to extend into the cytoplasm (Ref.1Orlowski J. Grinstein S. J. Biol. Chem. 1997; 272: 22373-22376Crossref PubMed Scopus (520) Google Scholar, but see Ref. 4Biemesderfer D. DeGray B. Aronson P.S. J. Biol. Chem. 1998; 273: 12391-12396Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar for alternative view). Functional characterization of deletion mutants of NHE1 and NHE3 has localized the site of Na+/H+ exchange to the N-terminal (transmembrane) region of the protein (1Orlowski J. Grinstein S. J. Biol. Chem. 1997; 272: 22373-22376Crossref PubMed Scopus (520) Google Scholar). The C-terminal region is believed to be responsible for modulation of transport by such diverse agents as growth promoters, hormones, and changes in medium osmolarity (1Orlowski J. Grinstein S. J. Biol. Chem. 1997; 272: 22373-22376Crossref PubMed Scopus (520) Google Scholar). The cytosolic domain is also thought to encompass the site that confers ATP dependence to Na+/H+ exchange, a hallmark of NHE that is common to all the isoforms studied to date (1Orlowski J. Grinstein S. J. Biol. Chem. 1997; 272: 22373-22376Crossref PubMed Scopus (520) Google Scholar,2Wakabayashi S. Shigekawa M. Pouyssegur J. Physiol. Rev. 1997; 77: 51-74Crossref PubMed Scopus (562) Google Scholar). The pattern of expression of individual NHE isoforms varies among tissues. NHE1 and NHE6 are expressed in virtually all cells, whereas other isoforms have a more restricted tissue distribution (1Orlowski J. Grinstein S. J. Biol. Chem. 1997; 272: 22373-22376Crossref PubMed Scopus (520) Google Scholar). NHE2 is preferentially expressed in the gastrointestinal tract and, to a lesser extent, in the kidney, uterus, and brain (5Collins J.F. Honda T. Knobel S. Bulus N.M. Conary J. Dubois R. Ghishan F.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3938-3942Crossref PubMed Scopus (130) Google Scholar, 6Wang Z. Orlowski J. Shull G.E. J. Biol. Chem. 1993; 268: 11925-11928Abstract Full Text PDF PubMed Google Scholar). Despite its discovery several years ago, comparatively little is known about the physiology of NHE2. Its selective expression pattern initially suggested a role for this isoform in fluid and electrolyte balance, similar to the function fulfilled by NHE3 in epithelial cells. However, creation of a null mutation of NHE2 in mice caused no significant perturbations of organismal acid-base or salt homeostasis (7Schultheis P.J. Clarke L.L. Meneton P. Harline M. Boivin G.P. Stemmermann G. Duffy J.J. Doetschman T. Miller M.L. Shull G.E. J. Clin. Invest. 1998; 101: 1243-1253Crossref PubMed Scopus (222) Google Scholar). Instead, the only apparent defect was a significant loss of net acid secretion in the stomach due to a severe reduction in the viability of parietal cells of the gastric mucosa. Although the underlying mechanism is unclear, it was proposed that NHE2 is part of the basolateral transport system that maintains parietal cell volume during high acid secretion at its apical surface and that disruption of NHE2 function causes a chronic state of volume depletion, leading to cellular necrosis (7Schultheis P.J. Clarke L.L. Meneton P. Harline M. Boivin G.P. Stemmermann G. Duffy J.J. Doetschman T. Miller M.L. Shull G.E. J. Clin. Invest. 1998; 101: 1243-1253Crossref PubMed Scopus (222) Google Scholar). Whereas an attractive hypothesis, the subcellular distribution of NHE2 in stomach has yet to be determined and, indeed, remains a controversial issue in other tissues examined. On one hand, NHE2 activity was reported to be restricted to the basolateral membrane of an inner medullary collecting duct cell line, mIMCD-3 (8Soleimani M. Singh G. Bizal G.L. Gullans S.R. McAteer J.A. J. Biol. Chem. 1994; 269: 27973-27978Abstract Full Text PDF PubMed Google Scholar). In contrast, others detected NHE2 activity predominantly on the apical membranes of ileal tissue (9Wormmeester L. Sanchez de Medina F. Kokke F. Tse C.M. Khurana S. Bowser J.M. Cohen M.E. Donowitz M. Am. J. Physiol. 1998; 274: C1261-C1272Crossref PubMed Google Scholar) and in a cortical collecting duct cell line (10Guerra L. Di Sole F. Valenti G. Ronco P.M. Perlino E. Casavola V. Reshkin S.J. Kidney Int. 1998; 53: 1269-1277Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Similar observations were made when NHE2 was transiently expressed in a colonic carcinoma cell line (11Tse C.M. Levine S.A. Yun C. Montrose M.H. Little P.J. Pouyssegur J. Donowitz M. J. Biol. Chem. 1993; 268: 11917-11924Abstract Full Text PDF PubMed Google Scholar), and apical localization was also documented immunologically in the medullary thick ascending limb (12Sun A.M. Liu Y. Dworkin L.D. Tse C.M. Donowitz M. Yip K.P. J. Membr. Biol. 1997; 160: 85-90Crossref PubMed Scopus (60) Google Scholar). Thus, NHE2 is mainly sorted to the apical surface of most polarized epithelial cells examined, as reported for NHE3. By contrast, the functional properties of NHE2 differ considerably from NHE3 and more closely resemble those of NHE1 when assessed in transfected mammalian cells: it is activated by agonists of the protein kinase A and C pathways as well as by hyperosmotic-induced cell shrinkage, and its pHi sensitivity is moderately reduced by cellular depletion of ATP (3Kapus A. Grinstein S. Wasan S. Kandasamy R. Orlowski J. J. Biol. Chem. 1994; 269: 23544-23552Abstract Full Text PDF PubMed Google Scholar, 13Tse C.M. Levine S. Yun C. Brant S. Counillon L.T. Pouyssegur J. Donowitz M. J. Membr. Biol. 1993; 135: 93-108Crossref PubMed Scopus (126) Google Scholar, 14Kandasamy R.A. Yu F.H. Harris R. Boucher A. Hanrahan J.W. Orlowski J. J. Biol. Chem. 1995; 270: 29209-29216Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 15Singh G. Orlowski J. Soleimani M. J. Membr. Biol. 1996; 151: 261-268Crossref PubMed Scopus (14) Google Scholar). The structural determinants that confer this unique behavior have not been identified. Perusal of the primary structure of NHE2 revealed that the C-terminal region, which is most divergent between isoforms, contains two proline-rich motifs: residues 743–750 (PPSVTPAP), which will be called hereafter Pro-1, and residues 786–792 (VPPKPPP), designated Pro-2. The latter conforms to the consensus sequence of proteins capable of binding Src homology 3 (SH3) domains, φPpXP, where φ is a hydrophobic residue, X is any amino acid, and p tends to be (but is not always) Pro (16Ren R. Mayer B.J. Cicchetti P. Baltimore D. Science. 1993; 259: 1157-1161Crossref PubMed Scopus (1022) Google Scholar, 17Viguera A.R. Arrondo J.L. Musacchio A. Saraste M. Serrano L. Biochemistry. 1994; 33: 10925-10933Crossref PubMed Scopus (129) Google Scholar, 18Yu H. Chen J.K. Feng S. Dalgarno D.C. Brauer A.W. Schreiber S.L. Cell. 1994; 76: 933-945Abstract Full Text PDF PubMed Scopus (875) Google Scholar). Pro-1 approximates but does not match perfectly the SH3 consensus structure. These proline-rich regions are unique to NHE2 and may be important in defining the distinctive behavior of this isoform. SH3 domains are sequences of 50–75 amino acids found in diverse proteins that include cytoskeletal components, such as spectrin (19Rotin D. Bar-Sagi D. O'Brodovich H. Merilainen J. Lehto V.P. Canessa C.M. Rossier B.C. Downey G.P. EMBO J. 1994; 13: 4440-4450Crossref PubMed Scopus (218) Google Scholar), adaptor and signaling molecules, such as Grb2 (20Cussac D. Frech M. Chardin P. EMBO J. 1994; 13: 4011-4021Crossref PubMed Scopus (100) Google Scholar, 21Wong L. Johnson G.R. J. Biol. Chem. 1996; 271: 20981-20984Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) and Ras-GAP (22Briggs S.D. Bryant S.S. Jove R. Sanderson S.D. Smithgall T.E. J. Biol. Chem. 1995; 270: 14718-14724Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), and protein and lipid kinases, including Src, Abl, and the p85 subunit of phosphatidylinositol 3′-kinase (23Pisabarro M.T. Serrano L. Biochemistry. 1996; 35: 10634-10640Crossref PubMed Scopus (111) Google Scholar, 24Cicchetti P. Mayer B.J. Thiel G. Baltimore D. Science. 1992; 257: 803-806Crossref PubMed Scopus (420) Google Scholar). Coupling of proteins via their SH3 domains has been implicated in a variety of functions, including regulation of cell growth and proliferation (25Erpel T. Alonso G. Roche S. Courtneidge S.A. J. Biol. Chem. 1996; 271: 16807-16812Crossref PubMed Scopus (56) Google Scholar,26Broome M.A. Hunter T. J. Biol. Chem. 1996; 271: 16798-16806Crossref PubMed Scopus (117) Google Scholar), endocytosis (27McMahon H.T. Wigge P. Smith C. FEBS Lett. 1997; 413: 319-322Crossref PubMed Scopus (84) Google Scholar, 28Wigge P. Vallis Y. McMahon H.T. Curr. Biol. 1997; 7: 554-560Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), and activation of the respiratory burst (29de Mendez I. Adams A.G. Sokolic R.A. Malech H.L. Leto T.L. EMBO J. 1996; 15: 1211-1220Crossref PubMed Google Scholar,30Nakamura R. Sumimoto H. Mizuki K. Hata K. Ago T. Kitajima S. Takeshige K. Sakaki Y. Ito T. Eur. J. Biochem. 1998; 251: 583-589Crossref PubMed Scopus (72) Google Scholar). The importance of SH3 domains in other systems prompted us to analyze whether the Pro-rich motifs of NHE2 are capable of interacting with SH3-containing proteins and to assess the functional role of this interaction. To this end, we studied the ability of fusion proteins encoding the Pro-1 and Pro-2 regions of NHE2 to bind SH3 domainsin vitro. In addition, we used transfection of epitope-tagged constructs to compare the functional properties and subcellular distribution of the full-length NHE2 with those of mutants lacking either Pro-1 or Pro-2. All polymerase chain reaction (PCR) reagents and Escherichia coli DH5-α were purchased from Life Technologies, Inc. pGEX-2T and E. coli HB101 were from Amersham Pharmacia Biotech. Isopropylthio-β-d-galactoside was purchased from Calbiochem. Luria broth (LB) was obtained from the Ontario Cancer Institute (Toronto, Ontario, Canada). Fluorescein-labeled lectins, leupeptin, pepstatin, aprotinin, phenylmethylsulfonyl fluoride, EDTA, antimycin A, reduced glutathione, and glutathione-agarose beads were purchased from Sigma. The NHS-LC biotinylation reagent was purchased from Pierce, and the enhanced chemiluminescence (ECL) detection kit was from Amersham Pharmacia Biotech. Nigericin and 2′,7′bis-(2-carboxyethyl)-5(6) carboxyfluorescein (BCECF) acetoxymethylester were purchased from Molecular Probes, Inc. Monoclonal antibodies to a peptide derived from the influenza virus hemagglutinin (HA) were purchased from BAbCo. Goat anti-mouse Cy3-conjugated IgG and goat peroxidase-coupled anti-mouse secondary antibody were purchased from Jackson Immunoresearch Laboratories. Horseradish peroxidase coupled to avidin was purchased from Cappel. Phosphate-buffered saline (PBS) contained 140 mm NaCl, 10 mm KCl, 8 mm sodium phosphate, 2 mmpotassium phosphate, pH 7.4. The sodium chloride solution contained 117 mm NaCl, 1.66 mm MgSO4, 1.36 mm CaCl2, 5.36 KCl mm, 25 mm Na-HEPES, 5.55 mm glucose, pH 7.4. Sodium-free solutions were made with equimolar substitution ofN-methyl-d-glucammonium solutions of the appropriate salts. Unless otherwise indicated, all solutions were nominally bicarbonate-free and were adjusted to 290 ± 10 mosm with the major salt. Hypertonic NaCl solution was prepared by addition of sufficient NaCl to raise the osmolarity to 600 ± 10 mosm. GST fusion proteins of the Pro-rich regions of NHE2 were prepared by PCR amplification of the appropriate regions of rat NHE2 cDNA (31Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4243) Google Scholar). The PCR product encompassing the more N-terminal Pro-rich region, which contains amino acids 743PPSVTPAP750 (Pro-1) plus 19 and 16 flanking residues at the 5′- and 3′-ends, respectively, was amplified using the primers 5′-ccggatcctcaccagcctac-3′ and 5′-ggaattcgggctgcctcag-3′. The primers used to amplify the more C-terminal proline-rich region containing amino acids786VPPKPPP792 (Pro-2) plus 12 and 21 flanking residues at the 5′- and 3′-ends, respectively, were 5′-ccggatccggccggggcagg-3′ and 5′-ggaattcaccacaagtctgc-3′. The PCR products were purified following electrophoresis in 1.8% agarose (Nu-Sieve Agarose, Biocan) using the Qiaex DNA extraction kit, according to the manufacturer's directions. The Pro-1 and Pro-2 sequences were subsequently subcloned into the BamHI andEcoRI sites of pGEX-2T (Amersham Pharmacia Biotech). HB101E. coli transformed with the resulting plasmid were grown at 37 °C in Luria broth supplemented with 50 μg/ml ampicillin to log-phase, and then expression of the fusion protein was induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside for 5 h at 37 °C. Bacteria were lysed in the presence of protease inhibitors (10 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 μg/ml aprotinin, and 5 mm EDTA), and the fusion proteins were purified from the lysate using glutathione-agarose beads. GST fusion protein encompassing amino acids 560–690 of NHE3 and 667–717 of NHE4 were prepared similarly. A GST fusion protein containing the last 178 residues of the cytosolic tail of rabbit NHE-1 was the kind gift of Dr. L. Fliegel (University of Alberta, Edmonton, Alberta, Canada). Fusion proteins of the SH3 domain of c-Abl, p85, Ras-GAP, N-Src, Src, and α-spectrin were obtained and generated as described previously (32Staub O. Dho S. Henry P. Correa J. Ishikawa T. McGlade J. Rotin D. EMBO J. 1996; 15: 2371-2380Crossref PubMed Scopus (741) Google Scholar). GST-SH3 domain fusions were subsequently biotinylated using NHS-LC biotin (Pierce), as described (19Rotin D. Bar-Sagi D. O'Brodovich H. Merilainen J. Lehto V.P. Canessa C.M. Rossier B.C. Downey G.P. EMBO J. 1994; 13: 4440-4450Crossref PubMed Scopus (218) Google Scholar). In Vitro Far Western Binding Assays—Fusion proteins (8 μg) of the indicated regions of the exchangers were separated by SDS-PAGE (12% acrylamide), transferred to nitrocellulose, and incubated overnight in blocking buffer (0.25% gelatin, 10% ethanolamine, 0.1 m Tris, pH 9.0) supplemented with 5% powdered milk. The blots were next incubated with 2 μg of the appropriate biotinylated SH3 fusion protein in 10 ml of blocking buffer for 2 h. The blots were then washed and incubated for 2 h at room temperature with streptavidin-peroxidase (12.5 μg/ml) in a buffer containing 0.25% gelatin, 0.05% Nonidet P-40, 0.15 m NaCl, 5 mm EDTA, 50 mmTris, pH 7.5, followed by washing and detection using ECL. The rat NHE2 cDNA was engineered to contain a series of unique restriction endonuclease sites that did not alter the primary structure, but created convenient DNA cassettes for mutagenesis. The modified NHE2 cDNA was inserted into the pCMV mammalian expression vector (plasmid renamed pNHE2) as described previously (6Wang Z. Orlowski J. Shull G.E. J. Biol. Chem. 1993; 268: 11925-11928Abstract Full Text PDF PubMed Google Scholar, 33Orlowski J. J. Biol. Chem. 1993; 268: 16369-16377Abstract Full Text PDF PubMed Google Scholar). To allow for immunological detection of the protein, the influenza virus HA epitope YPYDVPDYAS, preceded by a single G amino acid linker (inserted to create peptide flexibility), was inserted at the very C terminus of NHE2 (after amino acid 813) using PCR amplification of a C-terminal cDNA fragment. Similarly, the NHE2Δ777HA and NHE2Δ731HA deletion mutants were engineered by inserting the HA epitope plus a stop codon immediately following positions 777 and 731 of the wild type transporter. The PCR fragments were sequenced prior to substitution into NHE2 to confirm the presence of the mutations and to ensure that other random mutations were not introduced. AP1 is a cell line devoid of endogenous Na+/H+ exchange activity that was derived from wild type Chinese hamster ovary (CHO) cells by the “H+-suicide” technique (34Rotin D. Grinstein S. Am. J. Physiol. 1989; 267: C1158-C1165Crossref Google Scholar). These cells and the transfectants derived thereof were grown in α-minimal essential medium (Ontario Cancer Institute, Toronto, Ontario, Canada). HEK-T is a subclone of the transformed human embryonic kidney (HEK) cell line containing the large T antigen. LLC-PK1 are epithelial cells derived from porcine kidney proximal tubule. HEK-T and LLC-PK1 cells were grown in Dulbecco's minimal essential medium (Ontario Cancer Institute, Toronto, ON). Both α-minimal essential medium and Dulbecco's minimal essential medium contained 25 mm NaHCO3 and were supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (Life Technologies, Inc.). Cells were incubated in a humidified environment containing 95% air and 5% CO2 at 37 °C. Cultures were re-established from frozen stocks regularly, and cells from passages 3–20 were used for the experiments. Where indicated, intracellular ATP was depleted by incubating the cells for 10 min in glucose-free medium with 5 mm2-deoxy-d-glucose and 1 μg/ml antimycin A, to inhibit both glycolysis and oxidative phosphorylation. This protocol has previously been shown to deplete >90% of the ATP in CHO cells within 10 min (35Goss G.G. Woodside M. Wakabayashi S. Pouyssegur J. Waddell T. Downey G.P. Grinstein S. J. Biol. Chem. 1994; 269: 8741-8748Abstract Full Text PDF PubMed Google Scholar). Subsequent fluorescence measurements were performed in glucose-free medium containing 5.5 mm2-deoxy-d-glucose. AP-1, LLC-PK1, and HEK-T cells were transfected with plasmids containing the epitope-tagged wild type or truncated NHE2 constructs by the calcium phosphate-DNA co-precipitation technique of Chen and Okayama (36Chen C.A. Okayama H. BioTechniques. 1988; 6: 632-638Crossref PubMed Scopus (28) Google Scholar). For selection of stable lines, the AP-1 cells were selected for survival by repeated acute acid loads (5–6 times over a 2-week period), in order to discriminate between Na+/H+exchanger-positive and negative-transfectants, starting 48 h after transfection (33Orlowski J. J. Biol. Chem. 1993; 268: 16369-16377Abstract Full Text PDF PubMed Google Scholar, 34Rotin D. Grinstein S. Am. J. Physiol. 1989; 267: C1158-C1165Crossref Google Scholar). For stable transfection of LLC-PK1cells, which express endogenous NHE, we subcloned NHE2HA, NHE2Δ777HA, and NHE2Δ731HA into the pBK vector (Stratagene), which contains the neomycin resistance gene. Stably expressing cells were selected by incubation with 500 μg/ml G418 and then screened by immunofluorescence for expression of the wild type or mutant NHEs. For immunoblot analysis, the cells were grown to confluence on 10-cm2 plastic dishes. The cells were washed three times in PBS, lysed in a hypotonic buffer (10 mm Hepes, 18 mm potassium acetate, 1 mm EDTA, pH 7.2, 50 mosm) and solubilized at 4 °C in radioimmune precipitation buffer (150 mm NaCl, 20 mm Tris HCl, 0.1% SDS, 0.5% deoxycholate, 1% Triton X-100, pH 8.0) containing 1 mm iodoacetamide, 1 mm pepstatin, and 1 mm phenylmethylsulfonyl fluoride. The soluble fraction was subjected to SDS-PAGE (10% acrylamide) and transferred to nitrocellulose. The blot was incubated overnight in blocking buffer (see above) and then incubated with anti-HA antibody (1:5000) for 1 h in 0.25% gelatin, 0.05% Nonidet P-40, 0.15 m NaCl, 5 mm EDTA, 50 mm Tris, pH 7.5, supplemented with 5% milk, prior to labeling with peroxidase-coupled anti-mouse secondary antibody (1:5000). Chemiluminescence was then detected using the Amersham Pharmacia Biotech ECL detection kit. Cells were grown to subconfluence on sterile 18 mm glass coverslips (Thomas Scientific, Swedesboro, NJ), washed with PBS, and fixed for 20 min with 4% paraformaldehyde, and the excess formaldehyde was quenched with 100 mm glycine for 15 min. Where indicated, the fixed cells were stained with a mixture of fluorescein isothiocyanate-labeled lectins (peanut, orange, wheat germ, and pea, 2 μg/ml each) for 45 min at 4 °C. The cells were then permeabilized with 0.1% Triton X-100 supplemented with 5% bovine serum albumin and incubated with anti-HA antibody (1:1000) at room temperature for 45 min. The cells were washed again with PBS and incubated with Cy3-conjugated anti-mouse antibody (1:5000) for 45 min. The coverslips were mounted using Dako mounting reagent and visualized using a Leica fluorescence microscope. Images were obtained using the Winview program and processed using Adobe Photoshop. The pHi of small groups of cells was determined by microphotometry of the fluorescence emission of BCECF using dual wavelength excitation. Cells grown to confluence on 25-mm glass coverslips (Thomas Scientific, Swedesboro, NJ) were loaded with BCECF by incubation with 2 μg/ml of the precursor acetoxy-methylester form for 10 min at 37 °C. The coverslips were then mounted in a Leiden coverslip dish (Medical System Corp., Greenvale, NY) and placed into a thermostatted holding chamber heated to 37 °C (open perfusion microincubator, Medical Systems Corp., Greenvale, NY) attached to the stage of a Nikon Diaphot TMD inverted microscope (Nikon Canada, Toronto, Ontario, Canada). Cells were visualized using a Nikon Fluor × 40/1.3 numerical aperture oil immersion objective and a Hoffman modulation contrast video system with an angled condenser (Modulation Optics) through a CCD-72 video camera and control unit (Dage-MTI, Michigan City, IN) connected to a Panasonic monitor. Clusters of 6–12 cells from the confluent culture were selected for analysis with an adjustable diaphragm. The chamber was continuously perfused at ∼ 0.5 ml/min to allow for complete exchange of the bath solution once every minute using a gravity-driven system and a Leiden aspirator. When rapid solution changes were required, three aliquots of 1 ml of the new medium were quickly pipetted (<15 s) into the chamber and perfusion was continued using the new medium (37Chow C.W. Kapus A. Romanek R. Grinstein S. J. Gen. Physiol. 1997; 110: 185-200Crossref PubMed Scopus (21) Google Scholar). Fluorescence measurements were made using an M Series dual wavelength illumination system from Photon Technologies, Inc. (South Brunswick, NJ) in a dual excitation/single emission configuration. Excitation light provided by a Xenon lamp was alternately selected using 495 ± 10 and 445 ± 10 nm filters (Omega Optical, Brattleboro, VT) at a rate of 50 Hz and then reflected to the cells by a 510 nm dichroic mirror. Emitted light was first selected by a 520 nm long-pass filter and then separated from the red light used for Hoffman imaging by a 550 nm dichroic mirror and directed to the photometer through a 530 ± 30 nm band-pass filter. This optical system allowed for continuous visualization of cells without interfering with fluorescence measurements. Photometric data was acquired at 10 Hz using a 12 bit A/D board (Labmaster, National Instruments, Austin, TX) interfaced to a Dell 486 computer and analyzed with the Felix software (Photon Technologies Inc., South Brunswick, NJ). Calibration of the fluorescence intensity to pHi was performed in the presence of 5 mm nigericin in high potassium medium (140 mm KCl, 20 mm HEPES, 1 mm MgCl2, and 5 mm glucose) as detailed previously (38Thomas J.A. Buchsbaum R.N. Zimniak A. Racker E. Biochemistry. 1979; 18: 2210-2218Crossref PubMed Scopus (1767) Google Scholar). Each coverslip was calibrated at the end of the experiment using at least three pH values. Quantification of cell-associated fluorescence was performed using the Felix software package (Photon Technologies, Inc., South Brunswick, NJ). The rate of pHi change was derived by linear regression of the pHi versus time curve over 4 s intervals using the Origin software (MicroCal Software Inc., Northampton, MA). To assess whether SH3 domains are capable of binding specifically to the Pro-rich motifs of the cytosolic domain of NHE2 we initially tested the ability of the Src-SH3 domain to bind to this region in an in vitro overlay assay. A GST fusion protein encompassing residues 724–766 and including the Pro-1 region (743PPSVTPAP750) of NHE2 was subjected to SDS-PAGE, transferred to nitrocellulose, and overlaid with the biotinylated SH3 domain of Src, and binding was detected with streptavidin-horseradish peroxidase and ECL. In order to assess the specificity of the interaction, GST alone and GST fusion proteins prepared from the cytoplasmic segments of other NHE isoforms, which have no Pro-rich domains, were analyzed simultaneously. As illustrated in Fig. 1, the Src SH3 domain bound to the GST-NHE2-Pro-1 fusion protein but failed to interact with either GST alone or with the cytoplasmic regions of NHE1, NHE3, or NHE4. This observation suggests that SH3 domains bind to the cytosolic segment of the exchangers only when Pro-rich sequences are present. We next compared the ability of the Pro-1 and Pro-2 regions of NHE2 to associate with a variety of SH3 domains, i.e. those of Abl, Ras-GAP, p85 of phosphatidylinositol 3′-kinase, α-spectrin, Src, or the neuronal (N-Src). As before, GST alone was included as a measure of the specificity of the interaction. Our results show that none of the indicated SH3 domains interacted measurably with GST (Fig.2). By contrast, all of these bound to the Pro-1 fusion protein, with Ras-GAP interacting most strongly, whereas N-Src bound only marginally. The Pro-2 domain was less promiscuous, interacting strongly with the SH3 domains of p85 and Src, only moderately with N-Src and Abl, but not measurably with either Ras-GAP or α-spectrin. It therefore appears that both Pro-rich motifs of NHE2 can bind SH3 domains in vitro, albeit with different specificity. To assess the functional role of the Pro-rich regions of NHE2, we compared the" @default.
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