Participation of Na,K-ATPase in FGF-2 secretion: rescue of ouabain-inhibitable FGF-2 secretion by ouabain-resistant Na,K-ATPase alpha subunits.
We have examined the relationship between Na,K-ATPase and FGF-2 secretion in transfected primate cells. FGF-2 lacks a classic hydrophobic export signal, and the mechanisms mediating its secretion are unknown. To monitor secretion, a FLAG epitope tag was inserted into the carboxyl terminus of the 18 kDa form of human FGF-2, and the construct was transfected into either human HEK 293 or monkey CV-1 cells. Exported FGF-2 was detected in the culture medium using the FLAG-specific monoclonal antibody M2. FGF-2 secretion from HEK 293 or CV-1 cells was linear over time and sensitive to inhibition by the cardiac glycoside ouabain, a specific inhibitor of the Na,K-ATPase. In contrast, the secretion of FGF-8 (an FGF family member that contains a hydrophobic secretory signal) was not inhibited by treatment of HEK 293 or CV-1 cells with ouabain. FGF-2 secretion was also assayed in CV-1 cells expressing the naturally ouabain-resistant rodent Na,K-ATPase alpha1 subunit. In cells expressing the rodent alpha1 subunit, FGF-2 secretion was unaffected by high levels of ouabain, indicating that the rodent alpha1 subunit was capable of rescuing ouabain-inhibitable FGF-2 export. Expression of ouabain-resistant mutants of the rodent alpha2 and alpha3 subunits, or the naturally ouabain-resistant rodent alpha4 subunit, also supported FGF-2 secretion in ouabain-treated cells. Taken together, our studies are consistent with the idea that the Na,K-ATPase plays a prominent role in regulating FGF-2 secretion, although none of the alpha subunit isoforms exhibited specificity with regard to FGF-2 export.
Dahl
J P
JP
Department of Pharmacology and IBIOS Graduate Program in Molecular Medicine, Penn State College of Medicine, Hershey, Pennsylvania 17033, USA.
Binda
A
A
Canfield
V A
VA
Levenson
R
R
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D020126:Brefeldin A / Q000494:pharmacology; D002478:Cells, Cultured; D004351:Drug Resistance; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D051524:Fibroblast Growth Factor 8; D005346:Fibroblast Growth Factors / Q000378:metabolism; D000882:Haplorhini; D006801:Humans; D009842:Oligopeptides; D010042:Ouabain / Q000494:pharmacology*; D010455:Peptides; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
C496456:FGF8 protein, human; D009842:Oligopeptides; D010455:Peptides; D021122:Protein Subunits; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D051524:Fibroblast Growth Factor 8; D020126:Brefeldin A; D010042:Ouabain; D005346:Fibroblast Growth Factors; C085706:FLAG peptide; D000254:Sodium-Potassium-Exchanging ATPase