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 regul ated by growth factors and protein kinases. When stably expressed in P S120 fibro blasts, NHE3 is stimulated by serum and fibroblast growth f actor (FGF) and inhibited by phorbol esters. To examine the role of ph osphorylation of NHE3 in growth factor/protein kinase regulation, NHE3 was C-terminally tagged with an 11-amino acid epitope of the vesicula r stomatitis virus glycoprotein (VSVG) and stably expressed in Na+/Hexchanger 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 immunopre cipitation with anti-VSVG antibody after in vivo labeling of PS120/NHE 3V cells with [P-32]orthophosphate. NHE3V was phosphorylated under bas al conditions. However, FGF and PMA, under conditions in which these a gonists regulate NHE3V, altered neither the amount of phosphorylation of NHE3V as analyzed by one dimensional SDS-polyacrylamide gel electro phoresis and autoradiography nor two-dimensional phosphopeptide maps o f tryptic digests of NHE3V. In contrast, while changes in NHE3V phosph orylation were not observed with serum exposure by one dimensional SDS -polyacrylamide gel electrophoresis, two-dimensional studies showed in creases in two phosphopeptides. Under all these conditions, phosphoami no acid analysis showed that NHE3V was phosphorylated only on serine r esidues. By cell surface protein biotinylation studies under basal con ditions, at least 27% of the NHE3V was expressed on the cell surface. To further analyze the phosphorylation status of the surface and intra cellular forms of NHE3V under basal conditions and determine whether t he amount of phosphorylation of the surface form changes upon serum, F GF, and PMA regulation, the surface form of NHE3V was separated from i ntracellular 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 inh ibition by PMA based on one-dimensional SDS-polyacrylamide gel electro phoresis and autoradiography. Thus, we conclude that when expressed in PS120 cells, while NHE3 is a phosphoprotein under basal conditions, i ts regulation by FGF and PMA is not by changes in the phosphorylation of NHE3, while regulation by serum may involve changes in its phosphor ylation. Regulation of NHE3 probably involves intermediate associated regulatory proteins. The function of basal phosphorylation of NHE3 is not known.