Growth factor activation and "H(+)-sensing" of the Na+/H+ exchanger isoform 1 (NHE1). Evidence for an additional mechanism not requiring direct phosphorylation.
Growth factors stimulate the Na+/H+ exchange activity (NHE1 human isoform) and at the same time increase the phosphorylation state of the exchanger at serine residues. To determine the role of NHE1 phosphorylation, a set of deletion and point mutants has been generated. Functional characterization of deletion mutants expressed in fibroblastic cells revealed that the cytoplasmic region between amino acids 567 and 635 is required for both growth factor-induced cytoplasmic alkalinization and maintenance of high intracellular pH (pHi) sensitivity. In contrast to the loss of growth factor activation and pHi sensitivity caused by the deletion of amino acids 567-635, the same deletion had no apparent effect on the pattern of growth factor-induced phosphorylation. In addition, individual replacement of any of the serine residues between amino acids 567 and 635 with alanine also had no effect on growth factor activation of the exchange activity. Comparison of phosphopeptide maps for the wild type with those for the internal deletion mutant exchangers and the expressed cytoplasmic domain revealed that all major in vivo phosphorylation sites including growth factor-sensitive ones map to the cytoplasmic tail (amino acids 636-815). Deletion of these sites preserves high pHi sensitivity and reduces by only 50% growth factor-induced cytoplasmic alkalinization. Taken together, these data support the existence of a mechanism not requiring direct phosphorylation of NHE1, by which growth factor signals transmit to the "H(+)-sensor" and control the set point value of the exchanger. We propose that a regulatory factor(s) controls NHE1, presumably through its interaction with the critical cytoplasmic region prior to amino acid 635.
Wakabayashi
S
S
National Cardiovascular Center Research Institute, Osaka, Japan.
Bertrand
B
B
Shigekawa
M
M
Fafournoux
P
P
Pouysségur
J
J
D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary; D006133:Growth Substances / Q000494:pharmacology*; D006801:Humans; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D010449:Peptide Mapping; D010766:Phosphorylation; D017354:Point Mutation; D017384:Sequence Deletion; D017923:Sodium-Hydrogen Exchangers / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D006133:Growth Substances; D017923:Sodium-Hydrogen Exchangers; D006859:Hydrogen