C-terminal domains of Na+/H+ exchanger isoform 3 are involved in the basaland 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 distri butions. Using a quantitative cell surface biotinylation technique, we foun d PS120 cells target similar to 90% of mature NHE1 but only 14% of NHE3 to the cell surface, and this pattern occurs irrespective of NHE protein expre ssion levels. In this study, we examined surface fractions of NHE3 C-termin al truncation mutants to identify domains involved in the targeting of NHE3 . Removing the C-terminal 76 amino acids doubled surface fractions to 30% o f total and doubled the V-max from 1300 to 2432 mu M H+/s. Removal of anoth er 66 amino acids increased surface levels to 55% of total with an increase in the V-max to 5794 mu M H+/s. Surface fractions did not change with a fu rther 105 amino acid truncation. We postulated that inhibition of the basal recycling of NHE3 could result in the surface accumulation of the NHE3 tru ncations. Accordingly, we found that, unlike wild-type NHE3, the truncation s were shown to internalize poorly and were not affected by PI3 kinase inhi bition. However, while the truncations demonstrated reduced basal recycling , they retained the same serum response as full-length NHE3, with a mobiliz ation of similar to 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 indep endently regulated through a different part of the protein.