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A six-amino acid deletion in basic fibroblast growth factor dissociates its mitogenic activity from its plasminogen
Abstract:
A recombinant deletion mutant of the 155-amino acid form of human basic fibroblast growth factor (bFGF), lacking amino acid residues 27-32 (Lys-Asp-Pro-Lys-Arg-Leu), was expressed in Escherichia coli and purified to homogeneity by heparin-Sepharose affinity chromatography. When maintained in the presence of an equimolar concentration of soluble heparin, the bFGF mutant (M1-bFGF) is as potent as bFGF in stimulating cell proliferation in normal and transformed fetal bovine aortic endothelial cells, in adult bovine aortic endothelial cells, and in NIH 3T3 fibroblasts. However, under the same experimental conditions, M1-bFGF is at least 100 times less efficient than bFGF in stimulating plasminogen activator (PA) production in endothelial cells, as assayed by chromogenic PA assay, SDS/PAGE zymography, and Northern blot analysis of urokinase-type PA mRNA. In the presence of heparin, M1-bFGF binds to bFGF plasma membrane receptors present on endothelial cells in a manner undistinguishable from bFGF. It also induces the same tyrosine phosphorylation pattern when added to NIH 3T3 cells. The data suggest that the PA-inducing activity of bFGF may depend upon a functional domain that differs from those involved in the mitogenic activity of the growth factor and that the binding of bFGF to its plasma membrane receptor may not be sufficient to induce urokinase-type PA production in endothelial cells.
Insights
A modified basic fibroblast growth factor (bFGF) mutant shows reduced plasminogen activator production while retaining mitogenic activity, suggesting distinct functional domains for these cellular responses.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Basic fibroblast growth factor (bFGF) is a potent mitogen involved in cell proliferation and differentiation.
- bFGF signaling is mediated through binding to cell surface receptors and requires heparin for full activity.
- The precise functional domains responsible for bFGF's diverse cellular effects are not fully elucidated.
Purpose of the Study:
- To investigate the role of specific amino acid residues in bFGF's mitogenic and plasminogen activator (PA) inducing activities.
- To determine if the mitogenic activity of bFGF is separable from its ability to induce PA production.
- To explore the relationship between bFGF receptor binding and downstream signaling events.
Main Methods:
- Expression and purification of a recombinant bFGF deletion mutant (M1-bFGF) lacking residues 27-32.
- Assays for cell proliferation using various endothelial cells and NIH 3T3 fibroblasts.
- Measurement of plasminogen activator production via chromogenic assay, SDS/PAGE zymography, and Northern blot analysis.
Main Results:
- The M1-bFGF mutant exhibited mitogenic potency comparable to wild-type bFGF in the presence of heparin.
- M1-bFGF was at least 100-fold less efficient than bFGF in stimulating plasminogen activator production.
- M1-bFGF bound to bFGF receptors and induced similar tyrosine phosphorylation patterns as bFGF.
Conclusions:
- The PA-inducing activity of bFGF may involve a distinct functional domain separate from its mitogenic domain.
- Heparin-dependent bFGF receptor binding alone is insufficient to trigger urokinase-type PA production in endothelial cells.
- These findings provide insights into the complex structure-function relationships of bFGF.