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.
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.
Levine
S A
SA
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Nath
S K
SK
Yun
C H
CH
Yip
J W
JW
Montrose
M
M
Donowitz
M
M
Tse
C M
CM
D001483:Base Sequence; D002147:Calmodulin / Q000502:physiology; D004848:Epithelium / Q000378:metabolism; D005346:Fibroblast Growth Factors / Q000494:pharmacology; D006133:Growth Substances / Q000502:physiology*; D008871:Microvilli / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011494:Protein Kinases / Q000502:physiology*; D015290:Second Messenger Systems / Q000502:physiology; D017923:Sodium-Hydrogen Exchangers / Q000502:physiology*; D013329:Structure-Activity Relationship; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002147:Calmodulin; D006133:Growth Substances; D017923:Sodium-Hydrogen Exchangers; D005346:Fibroblast Growth Factors; D011494:Protein Kinases; D013755:Tetradecanoylphorbol Acetate