SUBCELLULAR-LOCALIZATION AND BIOLOGICAL-ACTIVITY OF MR-18,000 BASIC FIBROBLAST GROWTH-FACTOR - SITE-DIRECTED MUTAGENESIS OF A PUTATIVE NUCLEAR TRANSLOCATION SEQUENCE

Residues 27-31 (Lys-Asp-Pro-Lys-Arg) of the 155-amino acid form of bas ic fibroblast growth factor (bGF) are in good agreement with a consens us sequence for nuclear translocation. To evaluate the role of this se quence in mediating the intracellular localization and biological acti vity of bFGF, basic residues Lys-27, Lys-30, and Arg-31 were changed t o neutral glutamine residues by site-directed mutagenesis of the human bFGF cDNA. The bFGF mutant (M1Q-bFGF) was expressed in eukaryotic cel ls and in prokaryotic cells, from which it was purified to homogeneity . Transient expression of bFGF cDNA and of M1Q-bFGF cDNA in simian COS -1 cells followed by immunolocalization and by subcellular fractionati on indicated that both molecules localize in the nucleus, as well as i n the cytoplasm of transfected cells, and interact with nuclear chroma tin and with eukaryote DNA in a similar manner. Prokaryotic expression of M1Q-bFGF cDNA yields a polypeptide endowed with a receptor-binding capacity and mitogenic activity similar to that exerted by wild-type bFGF. However, recombinant M1Q-bFGF showed a drastically reduced capac ity to induce the production of urokinase-type plasminogen activator ( uPA) in endothelial cells. The uPA-inducing activity of M1Q-bFGF was f ully restored by the presence of soluble heparin in the culture medium . In conclusion, the sequence bFGF(27-31) does not appear to represent a nuclear translocation and/or retention sequence for bFGF. However, neutralization of its basic residues seems to modify the tertiary stru cture of the growth factor, thus affecting some of its biological prop erties.