Differential localization of Na+/H+ exchanger isoforms (NHE1 and NHE3) in polarized epithelial cell lines

Journal of Cell Science, May 1996

J. Noel, D. Roux, J. Pouyssegur

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Differential localization of Na+/H+ exchanger isoforms (NHE1 and NHE3) in polarized epithelial cell lines

Josette Noel 0 Danile Roux 0 Jacques Pouyssgur 0 0 Centre de Biochimie-CNRS, Universite de Nice , Parc Valrose, 06108, Nice , France *These authors have contributed equally to this work Author for correspondence (e-mail: ) - Na+/H+ exchangers (NHEs) are transporters that exchange sodium and proton ions across the plasma membrane at the expense of their chemical gradient. In higher eukaryotes these transporters exist as multiple specialized isoforms. For example, NHE1, the ubiquitously expressed form is a major pH-regulating system whereas the epithelial NHE3 isoform is specialized in transepithelial Na+ transport. NHE1 and NHE3 can be very well distinguished pharmacologically with the HOE694 specific inhibitor and immunologically with specific polyclonal and monoclonal antibodies. With these molecular tools we investigated the specific steady state expression of the two NHE isoforms in polarized epithelial cells in culture. Endogenous NHE3 in OK cells or NHE3-VSVG transfected in either OK or MDCK cells showed an exclusive expression of the transporter at the apical membrane. Overexpression of NHE3 did not result in any spill over on the basal lateral side. These results obtained by functional measurement of NHE3 activity were fully consistent with its detection only at the apical side by immunofluorescence The Na+/H+ exchanger (NHE) is a plasma membrane protein that functions in higher eukaryotic cells primarily for the maintenance of intracellular pH but also contributes to the transepithelial transport of Na+ (for reviews see Grinstein et al., 1989; Tse et al., 1993a; Nol and Pouyssgur, 1995). H+ extrusion is ensured by the extreme sensitivity of NHE to intracellular H+ and by the inwarded-directed Na+ gradient established by the Na+, K+ ATPase. NHE is therefore powerful for restoring intracellular pH towards neutrality in response to acid-load insults (Pouyssgur et al., 1984). In intestine and renal epithelial cells, NHE, expressed at the luminal side contributes primarily in transepithelial transport of Na+, the plurality of functions being dictated by the location and the kinetic parameters of the NHE molecules. The first NHE to be cloned and referred to as NHE1 is a glycoprotein of 110 kDa (Sardet et al., 1989, 1990) that exists in the membrane as a homodimer (Fafournoux et al., 1994). The N-terminal half of the molecule possesses multiple transmembrane segments (10 to 12) and constitutes the transporter unit. and confocal microscopy. By contrast, using the same cells, the same culture conditions and the same detection methods, we clearly detected NHE1 at both specialized membranes of four different polarized epithelial cell lines. Furthermore, biotinylation of cell surface proteins of MDCK, OK and HT-29 cells followed by immunoprecipitation of NHE1 revealed expression of the transporter at both sides of the polarized epithelial cells. Interestingly, the cell surface expression correlated well with the corresponding NHE1 activities. In addition, immunodetection by fluorescence microscopy was found to be qualitatively consistent with the above-reported results. We therefore conclude that the epithelial and more specialized NHE3 isoform is exclusively restricted to the apical side of epithelial cells. In marked contrast, both endogenous or ectopically expressed NHE1 isoform, have the capacity to be expressed in both the apical and basal lateral membranes of polarized cells in cultures. The C-terminal cytoplasmic domain of about 300 amino acids, although dispensable for Na+/H+ exchange activity, controls the affinity for intracellular H+ and represents a regulatory domain referred to as the transducer unit. Indeed, partial or complete removal of the cytoplasmic domain reduces or abolishes NHE activation in response to various extracellular stimuli that include growth factors, hormones, fibronectin, hyperosmotic shock (Wakabayashi et al., 1992, 1994; Bianchini et al., 1995; C. Lechene and J. Pouyssgur, unpublished results). NHE, like essential proteins, and as previously predicted (Clark and Limbird, 1991), exists as a multigene family. Five isoforms encoded by distinct genes have so far been identified (Sardet et al., 1989; Orlowski et al., 1992; Tse et al., 1992, 1993b; Klanke et al., 1995). NHE1-4 possess the generic NHE1 hydropathy profile, and share between them about 40% identity at the amino acid level, with a stronger conservation in the transporter unit (Tse et al., 1993a; Nol and Pouyssgur, 1995). Whereas NHE1 is virtually expressed in all tissues, NHE 2, 3 and 4 have restricted expression to epithelial tissues and restricted expression within polarized cells. For instance, the location of NHE2 and NHE3 at the apical surface of epithelial cells confers a specialized function to these isoforms, pointing out the importance of the mechanisms involved in membrane protein sorting. A large effort has been devoted to this question over the last decade and although considerable progress and unders (...truncated)


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J. Noel, D. Roux, J. Pouyssegur. Differential localization of Na+/H+ exchanger isoforms (NHE1 and NHE3) in polarized epithelial cell lines, Journal of Cell Science, 1996, pp. 929-939, 109/5,