Regulation of the epithelial brush border Na+/H+ exchanger isoform 3 stably expressed in fibroblasts by fibroblast growth factor and phorbol esters is not through changes in phosphorylation of the exchanger.
The epithelial brush border Na+/H+ exchanger isoform 3 (NHE3) is regulated by growth factors and protein kinases. When stably expressed in PS120 fibroblasts, NHE3 is stimulated by serum and fibroblast growth factor (FGF) and inhibited by phorbol esters. To examine the role of phosphorylation of NHE3 in growth factor/protein kinase regulation, NHE3 was C-terminally tagged with an 11-amino acid epitope of the vesicular stomatitis virus glycoprotein (VSVG) and stably expressed in Na+/H+ exchanger null PS120 fibroblasts (PS120/NHE3V). NHE3V was regulated by serum, FGF, and phorbol ester in a manner identical to wild type non-VSVG-tagged NHE3. Phosphorylation of NHE3V was evaluated via immunoprecipitation with anti-VSVG antibody after in vivo labeling of PS120/NHE3V cells with [32P]orthophosphate. NHE3V was phosphorylated under basal conditions. However, FGF and PMA, under conditions in which these agonists regulate NHE3V, altered neither the amount of phosphorylation of NHE3V as analyzed by one-dimensional SDS-polyacrylamide gel electrophoresis and autoradiography nor two-dimensional phosphopeptide maps of tryptic digests of NHE3V. In contrast, while changes in NHE3V phosphorylation were not observed with serum exposure by one-dimensional SDS-polyacrylamide gel electrophoresis, two-dimensional studies showed increases in two phosphopeptides. Under all these conditions, phosphoamino acid analysis showed that NHE3V was phosphorylated only on serine residues. By cell surface protein biotinylation studies under basal conditions, at least 27% of the NHE3V was expressed on the cell surface. To further analyze the phosphorylation status of the surface and intracellular forms of NHE3V under basal conditions and determine whether the amount of phosphorylation of the surface form changes upon serum, FGF, and PMA regulation, the surface form of NHE3V was separated from intracellular form by biotinylation/avidin-agarose precipitation. Under basal conditions, both intracellular and surface forms of NHE3V were phosphorylated. However, the amount of phosphorylation of the surface form of NHE3V did not change upon stimulation by serum and FGF and inhibition by PMA based on one-dimensional SDS-polyacrylamide gel electrophoresis and autoradiography. Thus, we conclude that when expressed in PS120 cells, while NHE3 is a phosphoprotein under basal conditions, its regulation by FGF and PMA is not by changes in the phosphorylation of NHE3, while regulation by serum may involve changes in its phosphorylation. Regulation of NHE3 probably involves intermediate associated regulatory proteins. The function of basal phosphorylation of NHE3 is not known.
Yip
J W
JW
Department of Medicine, GI Unit, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Ko
W H
WH
Viberti
G
G
Huganir
R L
RL
Donowitz
M
M
Tse
C M
CM
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media; D004848:Epithelium / Q000378:metabolism; D005346:Fibroblast Growth Factors / Q000494:pharmacology*; D005347:Fibroblasts / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008168:Lung; D008871:Microvilli / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism; D017384:Sequence Deletion; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection
D003470:Culture Media; D010446:Peptide Fragments; D010748:Phosphopeptides; D011994:Recombinant Proteins; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D005346:Fibroblast Growth Factors; D013755:Tetradecanoylphorbol Acetate