NHE2 contains subdomains in the COOH terminus for growth factor and protein kinase regulation

The cloned epithelial cell-specific Na+/H+ exchanger (NHE) isoform NHE2 is stimulated by fibroblast growth factor (FGF), phorbol 12-myristate 13-aceta te (PMA), okadaic acid (OA), and fetal bovine serum (FBS) through a change in maximal velocity of the transporter. In the present study, we used COOH- terminal truncation mutants to delineate specific domains in the COOH termi nus of NHE2 that are responsible for growth factor and/or protein kinase re gulation. Five truncation mutants (designated by the amino acid number at t he truncation site) were stably expressed in NHE-deficient PS120 fibroblast s. The effects of PMA, FGF, OA, FBS, and W-13 [a Ca2+/ calmodulin (CaM) inh ibitor] were studied. Truncation mutant E2/660, but not E2/573, was stimula ted by PMA. OA stimulated E2/573 but not E2/540. FGF stimulated E2/540 but not E2/499. The most truncated mutant, E2/499, wats stimulated by FBS. W-13 stimulated the basal activity of the wild-type NHE2. However, W-13 had no effect on E2/755. By monitoring the emission spectra of dansylated CaM fluo rescence, we showed that dansylated CaM bound directly to a purified fusion protein of glutathione S-transferase and the last 87 amino acids of NHE2 i n a Ca2+-dependent manner, with a stoichiometry of 1:1 and a dissociation c onstant of 300 nM. Our results showed that the COOH terminus of NHE2 is org anized into separate stimulatory and inhibitory growth factor/protein kinas e regulatory subdomains. This organization of growth factor/protein kinase regulatory subdomains is very similar to that of NHE3, suggesting that the tertiary structures of the putative COOH termini of NHE2 and NHE3 are very similar despite the minimal amino acid identity in this part of the two pro teins.