The release of fibroblast growth factor-1 from NIH 3T3 cells in response to temperature involves the function of cysteine residues.
Fibroblast growth factor (FGF)-1 is released from NIH 3T3 cells in response to heat shock as a biologically inactive protein that is unable to bind heparin and requires activation by (NH4)2SO4 to generate a biologically active extracellular heparin-binding growth factor (Jackson, A., Friedman, S., Zhan, X., Engleka, K. A., Forough, R., and Maciag, T. (1992) Proc. Natl. Acad. Sci. USA 89, 10691-10695). To further study the mechanism of FGF-1 release in response to heat shock (42 degrees C), we examined the kinetics of FGF-1 release from FGF-1-transfected NIH 3T3 cells and observed that the cells require at least 1 h of exposure to heat shock conditions for the release of FGF-1. Interestingly, agents that interfere with the function of the endoplasmic reticulum-Golgi apparatus, exocytosis, and the multidrug resistance pathway (brefelden A, methylamine, and verapamil, respectively) do not inhibit the release of FGF-1 in response to temperature; rather, they exaggerate the release of FGF-1. Because immunoblot analysis of FGF-1 in the conditioned medium of heat-shocked NIH 3T3 cells revealed the presence of a minor band with an apparent molecular weight of a FGF-1 homodimer and because we have previously shown that FGF-1, but not FGF-2, is able to form a homodimer in response to chemical oxidation by CuCl2 (Engleka, K. A., and Maciag, T. (1992) J. Biol. Chem. 267, 11307-11315), we examined whether reducing agents would substitute for (NH4)2SO4 and activate extracellular FGF-1. Indeed, dithiothreitol and reduced glutathione are able to individually generate a FGF-1 monomer as a heparin-binding protein from the conditioned medium of heat-shocked NIH 3T3 cell transfectants. To confirm that cysteine residues are involved in the release of FGF-1 in response to temperature, we used mutagenesis to prepare a human FGF-1 Cys-free mutant in which Cys30, Cys97, and Cys131 were converted to serine. Analysis of the release of the FGF-1 Cys-free mutant in NIH 3T3 cells transfected with the FGF-1 Cys-free mutant demonstrated that the FGF-1 Cys-free mutant is not released into the conditioned medium in response to temperature. Interestingly, exposure of the NIH 3T3 cell FGF-1 Cys-free transfectants to brefelden A followed by heat shock also demonstrated the absence of the extracellular FGF-1 Cys-free mutant.(ABSTRACT TRUNCATED AT 400 WORDS)
Jackson
A
A
Department of Molecular Biology, Holland Laboratory, American Red Cross, Rockville, MD 20855.
Tarantini
F
F
Gamble
S
S
Friedman
S
S
Maciag
T
T
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D003545:Cysteine / Q000378:metabolism*; D017931:DNA Primers; D016220:Fibroblast Growth Factor 1 / Q000737:chemistry / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data
D017931:DNA Primers; D016220:Fibroblast Growth Factor 1; D003545:Cysteine