Separate C-terminal domains of the epithelial specific brush border Na+/H+ exchanger isoform NHE3 are involved in stimulation and inhibition by protein kinases/growth factors

S. A. Levine, S. K. Nath, C. H.C. Yun, J. W. Yip, M. Montrose, M. Donowitz, C. M. Tse

Research output: Contribution to journalArticlepeer-review

Abstract

NHE3, a cloned intestinal and renal brush border Na+/H+ exchanger, has previously been shown to be both stimulated and inhibited by different protein kinases/growth factors. For instance, NHE3 is stimulated by serum and fibroblast growth factor (FGF) and inhibited by protein kinase C. In the present study, we used a series of NHE3 C terminus truncation mutants to identify separate regions of the C-terminal cytoplasmic tail responsible for stimulation and inhibition by protein kinases/growth factors. Five NHE3 C terminus truncation mutant stable cell lines were generated by stably transfecting NHE3 deletion cDNAs into PS120 fibroblasts, which lack any endogenous Na+/H+ exchanger. Using fluorometric techniques, the effects of the calcium/calmodulin (CAM) inhibitor W13, calcium/CaM kinase inhibitor KN- 62, phorbol myristate acetate, okadaic acid, FGF, and fetal bovine serum on Na+/H+ exchange were studied in these transfected cells. Inhibition of basal activity of full-length NHE3 is mediated by CaM at a site C-terminal to amino acid 756; this CaM effect occurs through both kinase dependent and independent mechanisms. There is another independent inhibitory domain for protein kinase C between amino acids 585 and 689. In addition, there are at least three stimulatory regions in the C-terminal domain of NHE3, corresponding to amino acids 509-543 for okadaic acid, 475-509 for FGF, and a region N-terminal to amino acid 475 for fetal bovine serum. We conclude that separate regions of the C terminus of NHE3 are involved with stimulation or inhibition of Na+/H+ exchange activity, with both stimulatory and inhibitory domains having several discrete subdomains. A conservative model to explain the way these multiple domains in the C terminus of NHE3 regulate Na+/H+ exchange is via an effect on associated regulatory proteins.

Original languageEnglish (US)
Pages (from-to)13716-13725
Number of pages10
JournalJournal of Biological Chemistry
Volume270
Issue number23
DOIs
StatePublished - 1995

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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