The Release of Fibroblast Growth Factor-1 from NIH 3T3 Cells in Response to Temperature Involves the Function of Cysteine Residues

摘要: Fibroblast growth factor (FGF)-1 is released from NIH 3T3 cells in response to heat shock as a biologically inactive protein that unable bind heparin and requires activation by (NH4)2SO4 generate active extracellular heparin-binding (Jackson, A., Friedman, S., Zhan, X., Engleka, K. Forough, R., Maciag, T.(1992) Proc. Natl. Acad. Sci. USA 89, 10691-10695). To further study the mechanism of FGF-1 release (42°C), we examined kinetics FGF-1-transfected observed require at least 1 h exposure conditions for FGF-1. Interestingly, agents interfere with function endoplasmic reticulum-Golgi apparatus, exocytosis, multidrug resistance pathway (brefelden A, methylamine, verapamil, respectively) do not inhibit temperature; rather, they exaggerate Because immunoblot analysis conditioned medium heat-shocked revealed presence minor band an apparent molecular weight homodimer because have previously shown FGF-1, but FGF-2, able form chemical oxidation CuCl2 (Engleka, J. Biol. Chem. 267, 11307-11315), whether reducing would substitute activate Indeed, dithiothreitol reduced glutathione are individually monomer cell transfectants. confirm cysteine residues involved temperature, used mutagenesis prepare human Cys-free mutant which Cys30, Cys97, Cys131 were converted serine. Analysis transfected demonstrated into temperature. transfectants brefelden A followed also absence mutant. Finally, ion-exchange reverse-phase chromatographies analyzed resolve under nonreducing conditions. These data demonstrate utilizes important component its vitro temperature exits affinity binding biological activities.