Engineering of fibroblast growth factor: alteration of receptor binding specificity.
A five-residue loop structure in basic fibroblast growth factor (FGF-2) which extends from amino acid residue 118 to residue 122 was replaced, by cassette mutagenesis, with the corresponding seven-residue loop structure from the structural homologue acidic fibroblast growth factor (FGF-1) or the corresponding five-residue loop from interleukin-1 beta to give FGF-2LA and FGF-2LI, respectively. The mutants were expressed in Escherichia coli and purified to homogeneity, and their heparin and receptor binding and biological properties were examined. The ability of FGF-2LA to induce endothelial cell proliferation was the same as that of FGF-2. Affinities of the mutants to heparin and to cells that express FGF receptor-1 (FGFR-1) were identical to those of the wild-type protein. The role of the loop structure in FGF-1 and FGF-2 was elucidated by using soluble FGF receptor systems, which display distinct ligand binding specificities. Thus, FGF-2LA bound, with the same affinity as FGF-1 and FGF-2, to FGFR-1 and FGFR-2, whereas only FGF-1 and the FGF-1 loop-containing mutant, FGF-2LA, bound to the keratinocyte growth factor receptor. A change in receptor binding specificity was not observed with the FGF-2LI engineered mutant. That the binding specificity of FGF-2 was dramatically altered by transfer of a loop structure from FGF-1 to resemble the binding profile of the donor protein provides strong evidence that this motif is a receptor binding specificity determinant of fibroblast growth factors.
Seddon
A P
AP
Department of Protein Chemistry, American Cyanamid Company, Pearl River, New York 10965.
Aviezer
D
D
Li
L Y
LY
Böhlen
P
P
Yayon
A
A
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D003432:Cross-Linking Reagents; D017931:DNA Primers / Q000737:chemistry; D005346:Fibroblast Growth Factors / Q000737:chemistry* / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D003432:Cross-Linking Reagents; D017931:DNA Primers; D017468:Receptors, Fibroblast Growth Factor; D005346:Fibroblast Growth Factors; D006493:Heparin