IDENTIFICATION OF 2 NEW HYDROPHOBIC RESIDUES ON BASIC FIBROBLAST-GROWTH-FACTOR IMPORTANT FOR FIBROBLAST-GROWTH-FACTOR RECEPTOR-BINDING

Basic fibroblast growth factor (bFGF) is implicated in the pathogenesi s of several types of vascular and connective diseases. A key step in the discovery of bFGF receptor antagonists to mitigate these actions i s to define the functional epitopes required for receptor binding of t he growth factor. Using structure-based site-directed mutagenesis, two critical areas on the bFGF surface for the high affinity receptor bin ding have already been identified [Springer,B.A., Pantoliano,M.W., Bar beral,F.A., Gunyuzlu,P.L., Thompson,L.D., Herblin,W.F., Rosenfeld,S.A. and Book,G.W. (1994) J. Biol. Chem., 269, 26879-26884; Zhu,H.Y., Ramn arayan,K., Anchin,J., Miao,Y., Sereno,A., Millman,L., Zheng,J., Balaji ,V.N. and Wolff,M.E. (1995) J. Biol. Chem., 270, 21869-21874; Zhu,H.Y. , Anchin,J., Ramnarayan,K., Zheng,J., Kawai,T., Mong,S. and Wolff,M.E. (1997) Protein Engng, 10, 417-421]. According to these studies, one r eceptor binding site includes two polar residues Glu96 and Asn104 on b FGF whereas the other includes four hydrophobic residues Tyr24, Tyr103 , Leu140 and Met142. Using a protein modelling technique, we report he re the identification of a new hydrophobic patch on bFGF which include s residues Tyr73, Val88 and Phe93. The role of this area on receptor b inding affinity was evaluated by mutating each of these residues indiv idually and determining the mutated protein's (mutein's) receptor bind ing affinity. In addition, we examined the role of two other hydrophob ic residues, Phe30 and Leu138, on bFGF for high-affinity receptor bind ing. These two residues are the neighbors of the hydrophobic residues Tyr24 and Tyr103, respectively. Replacement of Val88 and Phe93 with al anine reduced the receptor binding affinity about 10- and 80-fold, res pectively, compared with wild-type bFGF. In contrast, substitution of Phe30 and Leu138 with alanine has no effect on the receptor binding af finities. We conclude that the newly identified hydrophobic residues, Val88 and Phe93, are crucial for the receptor binding. The present dat a, together with the previous identification of four hydrophobic resid ues (Tyr24, Tyr103, Leu140 and Met142), suggests that there are two hy drophobic receptor binding sites on the bFGF surface. Our findings can be employed in the discovery and design of potent bFGF antagonists us ing computational methods.