C-terminal domains of Na(+)/H(+) exchanger isoform 3 are involved in the basal and serum-stimulated membrane trafficking of the exchanger.
When expressed either in polarized epithelial cells or in fibroblasts, two Na(+)/H(+) exchanger isoforms, NHE1 and NHE3, have different subcellular distributions. Using a quantitative cell surface biotinylation technique, we found PS120 cells target approximately 90% of mature NHE1 but only 14% of NHE3 to the cell surface, and this pattern occurs irrespective of NHE protein expression levels. In this study, we examined surface fractions of NHE3 C-terminal truncation mutants to identify domains involved in the targeting of NHE3. Removing the C-terminal 76 amino acids doubled surface fractions to 30% of total and doubled the V(max) from 1300 to 2432 microM H(+)/s. Removal of another 66 amino acids increased surface levels to 55% of total with an increase in the V(max) to 5794 microM H(+)/s. Surface fractions did not change with a further 105 amino acid truncation. We postulated that inhibition of the basal recycling of NHE3 could result in the surface accumulation of the NHE3 truncations. Accordingly, we found that, unlike wild-type NHE3, the truncations were shown to internalize poorly and were not affected by PI3 kinase inhibition. However, while the truncations demonstrated reduced basal recycling, they retained the same serum response as full-length NHE3, with a mobilization of approximately 10% of total NHE to the surface. We conclude that basal recycling of NHE3 is controlled by endocytic determinants contained within its C-terminal 142 amino acids and that serum-mediated exocytosis is independently regulated through a different part of the protein.
Akhter
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Division of Gastroenterology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Cavet
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Donowitz
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D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D019921:Biotinylation; D015153:Blotting, Western; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D015290:Second Messenger Systems; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D020033:Protein Isoforms; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1; D000077191:Wortmannin