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Endosomal Recycling of the Na+/H+Exchanger NHE3 Isoform Is Regulated by the Phosphatidylinositol 3-Kinase Pathway*

Open AccessPublished:August 14, 1998DOI:https://doi.org/10.1074/jbc.273.33.20828
      The NHE3 isoform of the Na+/H+ exchanger localizes to both the plasmalemmal and endosomal compartments in polarized epithelial and transfected Chinese hamster ovary (AP-1) cells. It is unclear how the distribution of NHE3 between these compartments is regulated. In this study, we examined the potential involvement of phosphatidylinositol 3′-kinase (PI3-K) in regulating the activity and distribution of NHE3, as this lipid kinase has been implicated in modulating vesicular traffic in the endosomal recycling pathway. Wortmannin and LY294002, both potent inhibitors of PI3-K, markedly inhibited NHE3-mediated H+ extrusion across the plasma membrane in a concentration- and time-dependent manner. The subcellular distribution of the antiporters was monitored by transfecting epitope-tagged NHE3 into AP-1 cells. In parallel with the inhibition of transport, PI3-K antagonists induced a pronounced loss of NHE3 from the cell surface and its accumulation in an intracellular compartment, as assessed by immunofluorescence microscopy and enzyme-linked immunosorbent assays. Further analysis using cells transfected with antiporters bearing an external epitope tag revealed that the redistribution reflected primarily a decrease in the rate of recycling of intracellular NHE3 to the cell surface. The wortmannin-induced inhibition and redistribution of NHE3 were prevented when cells were incubated at 4 °C, consistent with the known temperature dependence of the endocytic process. These observations demonstrate that NHE3 activity is controlled by dynamic endocytic and recycling events that are modulated by PI3-K.
      Cellular processes such as pH homeostasis, volume regulation, and transepithelial ion and water transport are mediated, in part, by the active extrusion of cytosolic H+ in exchange for extracellular Na+ (
      • Boron W.F.
      ,
      • Grinstein S.
      • Rotin D.
      • Mason M.J.
      ,
      • Knickelbein R.G.
      • Aronson P.S.
      • Dobbins J.W.
      ). This process is electroneutral and mediated by a family of integral membrane Na+/H+ antiporters or exchangers (NHE).
      The abbreviations used are: NHE
      Na+/H+ exchanger
      BCECF
      2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
      CHO
      Chinese hamster ovary
      ELISA
      enzyme-linked immunosorbent assay
      HA
      hemagglutinin
      αMEM
      α-minimal essential medium
      PBS
      phosphate-buffered saline
      pHi
      intracellular (cytosolic) pH
      PI3-K
      phosphatidylinositol 3'-kinase
      PAGE
      polyacrylamide gel electrophoresis.
      1The abbreviations used are: NHE
      Na+/H+ exchanger
      BCECF
      2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
      CHO
      Chinese hamster ovary
      ELISA
      enzyme-linked immunosorbent assay
      HA
      hemagglutinin
      αMEM
      α-minimal essential medium
      PBS
      phosphate-buffered saline
      pHi
      intracellular (cytosolic) pH
      PI3-K
      phosphatidylinositol 3'-kinase
      PAGE
      polyacrylamide gel electrophoresis.
      These transporters may also play a role in cell proliferation (
      • Grinstein S.
      • Rotin D.
      • Mason M.J.
      ) and adhesion (
      • Grinstein S.
      • Woodside M.
      • Waddell T.K.
      • Downey G.P.
      • Orlowski J.
      • Pouyssegur J.
      • Wong D.C.
      • Foskett J.K.
      ,
      • Schwartz M.A.
      • Ingber D.E.
      • Lawrence M.
      • Springer T.A.
      • Lechene C.
      ). In mammalian cells, six NHE isoforms (NHE1 to NHE6) have been identified to date (
      • Knickelbein R.G.
      • Aronson P.S.
      • Dobbins J.W.
      ,
      • Schwartz M.A.
      • Ingber D.E.
      • Lawrence M.
      • Springer T.A.
      • Lechene C.
      ,
      • Orlowski J.
      • Kandasamy R.A.
      • Shull G.E.
      ,
      • Orlowski J.
      ,
      • Field M.
      • Rao M.C.
      • Chang E.B.
      ,
      • Klanke C.A.
      • Su Y.R.
      • Callen D.F.
      • Wang Z.
      • Meneton P.
      • Baird N.
      • Kandasamy R.A.
      • Orlowski J.
      • Otterud B.E.
      • Leppert M.
      • Shul G.
      ). NHE1 and NHE6 are present in nearly all cells, whereas the other isoforms have a more restricted tissue distribution and fulfill more specialized functions. Among the latter, the best characterized is NHE3, which is confined to certain epithelial cells of the kidney, gastrointestinal tract, and gallbladder, where it participates actively in transepithelial Na+ and HCO3 absorption. The subcellular distribution of the isoforms also differs. NHE1, the “housekeeping” isoform, is present in the plasmalemma of most cells and accumulates in the basolateral membrane of epithelial cells. The other ubiquitous isoform, NHE6, is found in mitochondria (
      • Numata M.
      • Petrecca K.
      • Lake N.
      • Orlowski J.
      ). The epithelial isoform NHE3 is detectable in the apical (“brush border”) membrane, and also in subapical vesicles, possibly endosomes (
      • Biemesderfer D.
      • Rutherford P.A.
      • Nagy T.
      • Pizzonia J.H.
      • Abu-Alfa A.K.
      • Aronson P.S.
      ). Interestingly, this distribution is recapitulated when NHE3 is heterologously expressed in non-polarized cells, such as the antiport-deficient Chinese hamster ovary cell line AP-1 (
      • Rotin D.
      • Grinstein S.
      ). In this case, NHE3 is present and active on the plasma membrane, but is also abundant in a juxtanuclear vesicular compartment. Co-localization with transferrin receptors and with cellubrevin, and dispersal by colchicine indicated that this compartment overlaps with, or is identical to, the recycling endosomes (
      • D'Souza S.
      • Garcia-Cabado A.
      • Yu F.
      • Teter K.
      • Lukacs G.
      • Skorecki K.
      • Moore H.-P.
      • Orlowski J.
      • Grinstein S.
      ). These observations raised the possibility that, as in the case of other transporters, Na+/H+ exchange may be regulated in epithelia by recruitment of NHE3 to and from the apical membrane. Indeed, earlier fractionation experiments revealed that the density of the subcellular compartment expressing NHE3 shifted following treatment of renal cells with parathyroid hormone, or after induction of hypertension (
      • Hensley C.B.
      • Bradley M.E.
      • Mircheff A.K.
      ,
      • Zhang Y.
      • Mircheff A.K.
      • Hensley C.B.
      • Magyar C.E.
      • Warnock D.G.
      • Chambrey R.
      • Yip K.P.
      • Marsh D.J.
      • Holstein-Rathlou N.H.
      • McDonough A.A.
      ). These findings are consistent with the migration of NHE3 molecules from one intracellular compartment to another in response to physiological stimuli.
      Despite these suggestive observations, the mobilization of NHE3 has not been documented directly. Moreover, little is known about the molecular mechanisms regulating the putative translocation process. Some insight may be derived from recent findings made in Caco-2 cells expressing NHE3. These colon carcinoma cells responded to epidermal growth factor with an increase in the rate of Na+/H+exchange, which was eliminated by wortmannin, a potent inhibitor of phosphatidylinositol 3′-kinase (PI3-K). This enzyme catalyzes the phosphorylation of the 3′-position of the inositol ring of phosphoinositides, yielding polyphosphoinositides that seem to play active roles in signal transduction, including the recruitment and activation of several kinases and adaptors to the plasma membrane (see Ref.
      • Shepherd P.R.
      • Reaves B.J.
      • Davidson H.W.
      for review). Importantly, multiple lines of evidence have implicated PI3-K(s) in vesicular trafficking. The p110 catalytic subunit of the mammalian PI3-K is 55% homologous to theSaccharomyces cerevisiae protein VPS-34 (
      • Hiles I.D.
      • Otsu M.
      • Volinia S.
      • Fry M.J.
      • Gout I.
      • Dhand R.
      • Panayotou G.
      • Ruiz-Larrea F.
      • Thompson A.
      • Totty N.F.
      • Hsuan J.J.
      • Courtneidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      ). Disruption of the VPS-34 gene abolishes PI3-K activity in yeast and causes missorting of vacuolar hydrolases to the secretory pathway (
      • Schu P.V.
      • Takegawa K.
      • Fry M.J.
      • Stack J.H.
      • Waterfield M.D.
      • Emr S.D.
      ). In mammalian systems, PI3-K inhibitors such as wortmannin and LY294002 interfere with membrane traffic in a variety of cell types. They obliterate histamine secretion in RBL-2H3 cells, exocytosis in neutrophils stimulated with chemotactic agents, and translocation of GLUT4 glucose transporters in insulin-stimulated adipocytes (
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ,
      • Yano H.
      • Nakanishi S.
      • Kimura K.
      • Hanai N.
      • Saitoh Y.
      • Fukui Y.
      • Nonomura Y.
      • Matsuda Y.
      ,
      • Thelen M.
      • Wymann M.P.
      • Langen H.
      ,
      • Arcaro A.
      • Wymann M.P.
      ,
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Yeh J.-I.
      • Gulve E.A.
      • Rameh L.
      • Birnbaum M.J.
      ,
      • Lee A.D.
      • Hansen P.A.
      • Holloszy J.O.
      ). Finally, mutagenesis of the two PI3-K binding sites of the platelet-derived growth factor receptor disrupts the post-endocytic sorting of this receptor (
      • Joly M.
      • Kazlauskas A.
      • Corvera S.
      ).
      In view of these precedents, we considered the possibility that traffic of NHE3 between the plasmalemmal and endosomal compartments could be similarly controlled by the activity of PI3-K. We therefore examined the effect of wortmannin and LY294002 on the activity and subcellular distribution of NHE3. In epithelial cells, NHE3 generally co-exists with other isoforms of the exchanger. To circumvent the complicating contribution of other exchangers to H+ flux determinations, NHE3 was heterologously expressed in AP-1 cells, which are antiport-deficient Chinese hamster ovary (CHO) cells isolated by the H+-suicide method of Pouyssegur et al. (
      • Pouyssegur J.
      • Sardet C.
      • Franchi A.
      • L'Allemain G.
      • Paris S.
      ). These cells are also advantageous for microscopic localization studies, as they are flatter than epithelial cells and markers for their intracellular compartments are well defined. Finally, identification of NHE3 was facilitated by attaching an epitope tag derived from the influenza virus hemagglutinin (HA). For some of the experiments, a triple HA tag was engineered in a putative extracellular loop of NHE3, enabling us to quantify the number of exchangers exposed to the extracellular surface and to compare their abundance with the functional activity recorded.

      REFERENCES

        • Boron W.F.
        Annu. Rev. Physiol. 1986; 8: 377-388
        • Grinstein S.
        • Rotin D.
        • Mason M.J.
        Biochim. Biophys. Acta. 1989; 988: 73-97
        • Knickelbein R.G.
        • Aronson P.S.
        • Dobbins J.W.
        J. Clin. Invest. 1988; 82: 2158-2163
        • Grinstein S.
        • Woodside M.
        • Waddell T.K.
        • Downey G.P.
        • Orlowski J.
        • Pouyssegur J.
        • Wong D.C.
        • Foskett J.K.
        EMBO J. 1993; 12: 5209-5218
        • Schwartz M.A.
        • Ingber D.E.
        • Lawrence M.
        • Springer T.A.
        • Lechene C.
        Exp. Cell Res. 1991; 195: 533-535
        • Orlowski J.
        • Kandasamy R.A.
        • Shull G.E.
        J. Biol. Chem. 1992; 267: 9331-9339
        • Orlowski J.
        J. Biol. Chem. 1993; 268: 16369-16377
        • Field M.
        • Rao M.C.
        • Chang E.B.
        New Engl. J. Med. 1989; 321: 879-883
        • Klanke C.A.
        • Su Y.R.
        • Callen D.F.
        • Wang Z.
        • Meneton P.
        • Baird N.
        • Kandasamy R.A.
        • Orlowski J.
        • Otterud B.E.
        • Leppert M.
        • Shul G.
        Genomics. 1995; 25: 615-622
        • Numata M.
        • Petrecca K.
        • Lake N.
        • Orlowski J.
        J. Biol. Chem. 1998; 273: 6951-6959
        • Biemesderfer D.
        • Rutherford P.A.
        • Nagy T.
        • Pizzonia J.H.
        • Abu-Alfa A.K.
        • Aronson P.S.
        Am. J. Physiol. 1997; 273: F289-F299
        • Rotin D.
        • Grinstein S.
        Am. J. Physiol. 1989; 257: C1158-C1165
        • D'Souza S.
        • Garcia-Cabado A.
        • Yu F.
        • Teter K.
        • Lukacs G.
        • Skorecki K.
        • Moore H.-P.
        • Orlowski J.
        • Grinstein S.
        J. Biol. Chem. 1998; 273: 2035-2043
        • Hensley C.B.
        • Bradley M.E.
        • Mircheff A.K.
        Am. J. Physiol. 1989; 257: C637-C645
        • Zhang Y.
        • Mircheff A.K.
        • Hensley C.B.
        • Magyar C.E.
        • Warnock D.G.
        • Chambrey R.
        • Yip K.P.
        • Marsh D.J.
        • Holstein-Rathlou N.H.
        • McDonough A.A.
        Am. J. Physiol. 1996; 270: F1004-F1014
        • Shepherd P.R.
        • Reaves B.J.
        • Davidson H.W.
        Trends Cell Biol. 1996; 6: 92-97
        • Hiles I.D.
        • Otsu M.
        • Volinia S.
        • Fry M.J.
        • Gout I.
        • Dhand R.
        • Panayotou G.
        • Ruiz-Larrea F.
        • Thompson A.
        • Totty N.F.
        • Hsuan J.J.
        • Courtneidge S.A.
        • Parker P.J.
        • Waterfield M.D.
        Cell. 1992; 70: 419-429
        • Schu P.V.
        • Takegawa K.
        • Fry M.J.
        • Stack J.H.
        • Waterfield M.D.
        • Emr S.D.
        Science. 1993; 260: 88-91
        • Clarke J.F.
        • Young P.W.
        • Yonezawa K.
        • Kasuga M.
        • Holman G.D.
        Biochem. J. 1994; 300: 631-635
        • Yano H.
        • Nakanishi S.
        • Kimura K.
        • Hanai N.
        • Saitoh Y.
        • Fukui Y.
        • Nonomura Y.
        • Matsuda Y.
        J. Biol. Chem. 1993; 268: 25846-25856
        • Thelen M.
        • Wymann M.P.
        • Langen H.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4960-4964
        • Arcaro A.
        • Wymann M.P.
        Biochem. J. 1993; 296: 297-301
        • Cheatham B.
        • Vlahos C.J.
        • Cheatham L.
        • Wang L.
        • Blenis J.
        • Kahn C.R.
        Mol. Cell. Biol. 1994; 14: 4902-4911
        • Yeh J.-I.
        • Gulve E.A.
        • Rameh L.
        • Birnbaum M.J.
        J. Biol. Chem. 1995; 270: 2107-2111
        • Lee A.D.
        • Hansen P.A.
        • Holloszy J.O.
        FEBS Lett. 1995; 361: 51-54
        • Joly M.
        • Kazlauskas A.
        • Corvera S.
        J. Biol. Chem. 1995; 270: 13225-13230
        • Pouyssegur J.
        • Sardet C.
        • Franchi A.
        • L'Allemain G.
        • Paris S.
        Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4833-4837
        • Chen C.
        • Okayama H.
        Mol. Cell. Biol. 1987; 7: 2745-2752
        • Franchi A.
        • Perucca-Lostanlen D.
        • Pouyssegur J.
        Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9388-9392
        • Thomas J.A.
        • Buchsbaum R.N.
        • Zimniak A.
        • Racker E.
        Biochemistry. 1979; 18: 2210-2218
        • Vlahos C.J.
        • Matter W.F.
        • Hui K.Y.
        • Brown R.F.
        J. Biol. Chem. 1994; 269: 5241-5248
        • Spiro D.J.
        • Boll W.
        • Kirchhausen T.
        • Wessling-Resnick M.
        Mol. Biol. Cell. 1996; 7: 355-367
        • Shpetner H.
        • Joly M.
        • Hartley D.
        • Corvera S.
        J. Cell Biol. 1996; 132: 595-605
        • Okada T.
        • Kawano Y.
        • Sakakibara T.
        • Hazeki O.
        • Ui M.
        J. Biol. Chem. 1994; 269: 3568-3573
        • Kurashima K.
        • Yu F.H.
        • Cabado A.G.
        • Szabo E.Z.
        • Grinstein S.
        • Orlowski J.
        J. Biol. Chem. 1997; 272: 28672-28679
        • Nakanishi S.
        • Kakita S.
        • Takahashi I.
        • Kawahara K.
        • Tsukuda E.
        • Sano T.
        • Yamada K.
        • Yoshida M.
        • Kase H.
        • Matsuda Y.
        J. Biol. Chem. 1992; 267: 2157-2163
        • Cross D.A.
        • Alessi D.R.
        • Vandenheede J.R.
        • McDowell H.E.
        • Hundal H.S.
        • Cohen P.
        Biochem. J. 1994; 303: 21-26
        • Nakanishi S.
        • Catt K.J.
        • Balla T.
        Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5317-5321
        • Martys J.L.
        • Wjasow C.
        • Gangi D.M.
        • Kielian M.C.
        • McGraw T.E.
        • Backer J.M.
        J. Biol. Chem. 1996; 271: 10953-10962
        • Li G.
        • D'Souza-Schorey C.
        • Barbieri M.A.
        • Roberts R.L.
        • Klippel A.
        • Williams L.T.
        • Stahl P.D.
        Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10207-10211
        • Lin H.C.
        • Gilman A.G.
        J. Biol. Chem. 1996; 271: 27979-27982
        • Tsakiridis T.
        • McDowell H.E.
        • Walker T.
        • Downes C.P.
        • Hundal H.S.
        • Vranic M.
        • Klip A.
        Endocrinology. 1995; 136: 4315-4322
        • Sue-A-Quan A.K.
        • Fialkow L.
        • Vlahos C.J.
        • Schelm J.A.
        • Grinstein S.
        • Butler J.
        • Downey G.P.
        J. Cell. Physiol. 1997; 172: 94-108
        • Shepherd P.R.
        • Soos M.A.
        • Siddle K.
        Biochem. Biophys. Res. Comm. 1995; 211: 535-539
        • Jones A.T.
        • Clague M.J.
        Biochem. J. 1995; 311: 31-34
        • Khurana S.
        • Nath S.K.
        • Levine S.A.
        • Bowser J.M.
        • Tse C.-M.
        • Cohen M.E.
        • Donowitz M.
        J. Biol. Chem. 1996; 271: 9919-9927
        • Ma Y.H.
        • Reusch H.P.
        • Wilson E.
        • Escobedo J.A.
        • Fantl W.J.
        • Williams L.T.
        • Ives H.E.
        J. Biol. Chem. 1994; 269: 30734-30739
        • Orlowski J.
        • Kandasamy R.A.
        J. Biol. Chem. 1996; 271: 19922-19927