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-2) 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-2 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-2 and their only known molecular target: the alpha-subunit of Na+, K+-ATPase. Export of FGF-2 from COS-1 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.
Florkiewicz
R Z
RZ
PRIZM Pharmaceuticals, San Diego, California 92121, USA. rzflork@prizmpharm.com
Anchin
J
J
Baird
A
A
D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002298:Cardenolides / Q000494:pharmacology*; D002384:Catalysis; D016222:Fibroblast Growth Factor 2 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection
D002298:Cardenolides; D016222:Fibroblast Growth Factor 2; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium