The Na+/H+ exchanger isoform 1 (NHE1) is a novel member of the calmodulin-binding proteins. Identification and characterization of calmodulin-binding sites.
The Na+/H+ exchange activity (NHE1 human isoform) is rapidly activated in response to growth factors and hyperosmotic stress. To get insight into the mechanism of NHE1 activation, we studied the direct interaction of a ubiquitous Ca(2+)-dependent regulatory factor, calmodulin (CaM) with NHE1. Binding experiments with CaM-Sepharose, as well as fluorescence measurements with dansylated CaM, revealed that the NHE1 cytoplasmic domain strongly binds CaM in a Ca(2+)-dependent manner. Fusion protein analysis with deletion mutants provided evidence for high (Kd approximately 20 nM) and intermediate (Kd approximately 350 nm) affinity CaM-binding sites located in neighboring regions of NHE1 (amino acids 636-656 and 657-700). To assess a regulatory role of CaM-binding sites, several cDNAs having deletion and point mutations in the high affinity site were generated and expressed in the exchanger-deficient fibroblast cell line PS120. Deletion and point mutations of positively charged residues of the high affinity CaM-binding site resulted in up to 50 and 80% reductions of cytoplasmic alkalinization caused by growth factors (alpha-thrombin, etc.) and 100 mM sucrose, respectively. In these mutants, the reduction in alkalinization was apparently in proportion to that of the CaM-binding ability. These results suggest that binding of Ca2+/CaM to the high affinity site is involved at least partly in the activation of NHE1 in response to different extracellular signals.
Bertrand
B
B
Department of Molecular Physiology, National Cardiovascular Center Research Institute, Osaka, Japan.
Wakabayashi
S
S
Ikeda
T
T
Pouysségur
J
J
Shigekawa
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D017923:Sodium-Hydrogen Exchangers / Q000302:isolation & purification* / Q000378:metabolism
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D017923:Sodium-Hydrogen Exchangers; D002118:Calcium