The inhibition of fibroblast growth factor-2 export by cardenolides implies a novel function for the catalytic subunit of Na+,K+-ATPase

Basic fibroblast growth factor (FGF-S) is one of a select group of proteins that can exit cells through an alternate, endoplasmic reticulum/Golgi apparatus independent exocytic pathway, This alternate pathway has been termed protein export, In an attempt to better understand this process, we have identified a family of related compounds, "cardenolides," that inhibit FGF-2 export, The cardenolides inhibit FGF-2 export in a time and concentration dependent fashion, Inhibition of FGF-S export is specific in that the cardenolides have no effect on conventional protein secretion as measured by their inability to block release of the secreted protein human chorionic gonadotropin-alpha. Because cardenolides are known to inhibit ion transport activity mediated by Na+,K+-ATPase, we investigated whether there are functional interactions between FGF-S and their only known molecular target: the alpha-subunit of Na+,K+-ATPase. Export of FGF-2 from COS-l cells is selectively inhibited when co-transfected with expression vectors encoding the alpha-subunit and FGF-2. Moreover, antibodies to the alpha-subunit specifically co-immunoprecipitate FGF-2 along with the alpha-subunit while conversely, antibodies to FGF-2 specifically co-immunoprecipitate the alpha-subunit along with FGF-2. Finally, the ion transporting activities of the Na+,K+-ATPase can be uncoupled from protein export, Varying the external concentration of K+ has little effect on export of FGF-2. Taken together, these data: 1) identify a novel activity for cardenolides; 2) suggest a previously unknown role for the alpha-subunit of Na+, K+-ATPase in FGF-2 export; and 3) raise the possibility that the alpha-subunit itself may be an integral component of this alternate exocytic pathway mediating translocation of cytosolic FGF-2 to the cell surface.