Identification of the fibroblast growth factor (FGF)-interacting domain in a secreted FGF-binding protein by phage display.
Fibroblast growth factor-binding proteins (FGF-BP) are secreted carrier proteins that release fibroblast growth factors (FGFs) from the extracellular matrix storage and thus enhance FGF activity. Here we have mapped the interaction domain between human FGF-BP1 and FGF-2. For this, we generated T7 phage display libraries of N-terminally and C-terminally truncated FGF-BP1 fragments that were then panned against immobilized FGF-2. From this panning, a C-terminal fragment of FGF-BP1 (amino acids 193-234) was identified as the minimum binding domain for FGF. As a recombinant protein, this C-terminal fragment binds to FGF-2 and enhances FGF-2-induced signaling in NIH-3T3 fibroblasts and GM7373 endothelial cells, as well as mitogenesis and chemotaxis of NIH-3T3 cells. The FGF interaction domain in FGF-BP1 is distinct from the heparin-binding domain (amino acids 110-143), and homology modeling supports the notion of a distinct domain in the C terminus that is conserved across different species. This domain also contains conserved positioning of cysteine residues with the Cys-214/Cys-222 positions in the human protein predicted to participate in disulfide bridge formation. Phage display of a C214A mutation of FGF-BP1 reduced binding to FGF-2, indicating the functional significance of this disulfide bond. We concluded that the FGF interaction domain is contained in the C terminus of FGF-BP1.
Xie
Bin
B
Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057, USA.
Tassi
Elena
E
Swift
Matthew R
MR
McDonnell
Kevin
K
Bowden
Emma T
ET
Wang
Shaomeng
S
Ueda
Yumi
Y
Tomita
York
Y
Riegel
Anna T
AT
Wellstein
Anton
A
D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000378:metabolism; D001667:Binding, Competitive; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry*; D002417:Cattle; D049109:Cell Proliferation; D002478:Cells, Cultured; D002633:Chemotaxis; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005346:Fibroblast Growth Factors / Q000737:chemistry*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D016366:Open Reading Frames; D019151:Peptide Library; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D017468:Receptors, Fibroblast Growth Factor / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D020349:Surface Plasmon Resonance; D013997:Time Factors
D002352:Carrier Proteins; D018076:DNA, Complementary; D004220:Disulfides; C489453:Fgfbp1 protein, mouse; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; D019151:Peptide Library; D017468:Receptors, Fibroblast Growth Factor; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C070699:FGFBP1 protein, human; D005346:Fibroblast Growth Factors; D003545:Cysteine