Subcellular localization and biological activity of mr 18,000 basic fibroblast growth factor: Site-directed mutagenesis of a putative nuclear translocation sequence

Marco Presta, Anna Gualandris, Chiara Urbinati, Marco Rusnati, Daniela Coltrini, Antonella Isacchi, Paolo Caccia, Laura Bergonzoni

Research output: Contribution to journalArticlepeer-review

Abstract

Residues 27-31 (Lys-Asp-Pro-Lys-Arg) of the 155-amino acid form of basic fibroblast growth factor (bFGF) are in good agreement with a consensus sequence for nuclear translocation. To evaluate the role of this sequence in mediating the intracellular localization and biological activity of bFGF, basic residues Lys-27, Lys-30, and Arg-31 were changed to neutral glutamine residues by site-directed mutagenesis of the human bFGF cDNA. The bFGF mutant (MIQ-bFGF) was expressed in eukaryotic cells and in prokaryotic cells, from which it was purified to homogeneity. Transient expression of bFGF cDNA and of MIQ-bFGF cDNA in simian COS-1 cells followed by immunolocalization and by subcellular fractionation indicated that both molecules localize in the nucleus, as well as in the cytoplasm of transfected cells, and interact with nuclear chromatin and with eukaryote DNA in a similar manner. Prokaryotic expression of MIQ-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 capacity to induce the production of urokinase-type plasminogen activator (uPA) in endothelial cells. The uPA-inducing activity of M1Q-bFGF was fully 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 structure of the growth factor, thus affecting some of its biological properties.

Original languageEnglish
Pages (from-to)269-278
Number of pages10
JournalGrowth Factors
Volume9
Issue number4
DOIs
Publication statusPublished - 1993

Keywords

  • Fibroblast growth factor
  • Nucleus
  • Plasminogen activator

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Endocrinology
  • Cell Biology

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