ABSTRACT
Four forms of basic fibroblast growth factor (bFGF or FGF-2) result from an alternative initiation of translation involving one AUG (155-amino acid form) and three CUGs (210-, 201- and 196-amino acid forms). These different forms of bFGF show different intracellular biological activities. To identify their intracellular targets, the 210- and 155-amino acid forms of bFGF were independently transfected into CHO cells and their correct subcellular localizations were verified, the 155-amino acid bFGF form being essentially cytoplasmic whereas the 210-amino acid protein was nuclear. The radiation fragmentation method was used to determine the target size of the different bFGF isoforms in the transfected CHO cells and to show that the 210- and 155-amino acids bFGF isoforms were included in protein complexes of 320 and 130 kDa respectively. Similar results were obtained using the SK-Hep1 cell line, which naturally expressed all forms of bFGF. Co-immunoprecipitation assays using different chimaeric bFGF-chloramphenicol acetyltransferase proteins showed that different cellular proteins are associated with different parts of the bFGF molecule. We conclude that bFGF isoforms are involved in different molecular complexes in the cytosol and nucleus, which would reflect different functions for these proteins.
Subject(s)
Fibroblast Growth Factor 2/chemistry , Fibroblast Growth Factor 2/physiology , Intracellular Fluid/chemistry , Intracellular Fluid/physiology , Animals , CHO Cells , COS Cells , Carcinoma, Hepatocellular , Cricetinae , Dose-Response Relationship, Radiation , Fibroblast Growth Factor 2/biosynthesis , Fibroblast Growth Factor 2/genetics , Gamma Rays , Genetic Vectors/metabolism , Humans , Intracellular Fluid/metabolism , Isomerism , Macromolecular Substances , Precipitin Tests , Subcellular Fractions/chemistry , Subcellular Fractions/metabolism , Subcellular Fractions/radiation effects , Transfection , Tumor Cells, CulturedSubject(s)
Diabetic Retinopathy/therapy , Endothelial Growth Factors/physiology , Fibroblast Growth Factors/physiology , Heparin/metabolism , Lymphokines/physiology , Animals , Endothelial Growth Factors/genetics , Fibroblast Growth Factors/genetics , Humans , Lymphokines/genetics , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/physiology , Receptors, Fibroblast Growth Factor/physiology , Receptors, Growth Factor/genetics , Receptors, Growth Factor/physiology , Receptors, Vascular Endothelial Growth Factor , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
Four forms of basic fibroblast-growth factor (bFGF or FGF-2) using one AUG (155 amino acids) and three upstream CUG (210, 201 and 196 amino acids) start codons, were synthesized through an alternative use of initiation codons. The 210-amino acid form of FGF-2 (210FGF-2) was expressed in a plasmid vector under the control of a bacteriophage T7 RNA polymerase promoter system in Escherichia coli. Characterization of the purified protein was performed by electrospray mass spectrometry and Edman degradation. The recombinant 210FGF-2 produced in E. coli had a mitogenic activity similar to the 146-amino acid form extracted from tissues.
Subject(s)
Fibroblast Growth Factor 2/genetics , Fibroblast Growth Factor 2/isolation & purification , Amino Acid Sequence , Base Sequence , Cell Division , Cells, Cultured , Escherichia coli/genetics , Fibroblast Growth Factor 2/pharmacology , Genetic Variation , Mass Spectrometry , Molecular Sequence Data , Recombinant Proteins/isolation & purification , Sequence AnalysisABSTRACT
The human basic Fibroblast Growth Factor (bFGF) gene was shown to encode four polypeptides by an alternative use of initiation codons (three CUG and one AUG). In this report, we present a comparative study of the fate and intracellular localization of individual bFGF isoforms. For this purpose, we have produced the various bFGF isoforms in E. coli and purified them to homogeneity: the 210 amino acid form initiated at CUG1 that contains a nuclear localization sequence (NLS), the 155 amino acid form (AUG-mediated initiation) and the 146 amino acid form (processed form extracted from tissues). While the different bFGFs were taken up by the cell with equal efficiency, more of the 210 amino acid form accumulated in the nucleus and represented 36% of the internalized bFGF compared with 15% in the others. A chimeric protein containing the minimal SV40 Large T NLS (SV40NLS) fused to the 155 amino acid bFGF form (SVbFGF) behaves like the native 155 amino acid form, indicating that nuclear accumulation of exogenous bFGF is not mediated by the NLS-associated function. These results suggest that the amino-terminal part of the 210 amino acid bFGF contains a sequence responsible for its nuclear retention. Bioactivities of the different forms were tested on adult bovine aortic endothelial (ABAE) cells. The bFGF degradation pathways, mitogenic activity and stimulation of rRNA synthesis appeared to be the same for all bFGFs but the stimulation of plasminogen activator was enhanced by the 210 amino acid form and correlated with nuclear accumulation.
