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Subcellular localization and biological activity of M(r) 18,000 basic fibroblast growth factor: site-directed
M Presta1, A Gualandris, C Urbinati
1Department of Biomedical Sciences and Biotechnology, School of Medicine, University of Brescia, Italy.
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 (M1Q-bFGF) was expressed in eukaryotic cells 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 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 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 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.
Insights
The basic fibroblast growth factor (bFGF) nuclear translocation sequence (27-31) does not mediate nuclear localization. Mutating this sequence affects bFGF
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Basic fibroblast growth factor (bFGF) is a key regulator of cellular processes.
- A specific sequence (residues 27-31) in bFGF resembles known nuclear translocation signals.
- The role of this sequence in bFGF's intracellular localization and function requires investigation.
Purpose of the Study:
- To determine if the bFGF(27-31) sequence mediates nuclear translocation.
- To assess the impact of mutating basic residues in this sequence on bFGF localization and biological activity.
Main Methods:
- Site-directed mutagenesis was used to create a bFGF mutant (M1Q-bFGF) with altered residues 27-31.
- The mutant and wild-type bFGF were expressed in eukaryotic and prokaryotic cells.
- Immunolocalization, subcellular fractionation, receptor-binding assays, and mitogenic activity assays were performed.
Main Results:
- Both wild-type and mutant bFGF localized to the nucleus and cytoplasm and interacted with DNA similarly.
- M1Q-bFGF retained receptor-binding capacity and mitogenic activity comparable to wild-type bFGF.
- M1Q-bFGF exhibited reduced urokinase-type plasminogen activator (uPA) induction, which was restored by heparin.
Conclusions:
- The bFGF(27-31) sequence is not essential for nuclear translocation or retention.
- Mutation of basic residues in this sequence alters bFGF's tertiary structure, impacting specific biological activities like uPA induction.
- Heparin can modulate the activity of the altered bFGF, suggesting structural changes affect its interaction with cofactors.