The contribution of individual basic amino acids within three putative "consensus sequences" for heparin binding of fibroblast growth factor-1 have been examined by site-directed mutagenesis. The results indicate that a significant reduction in the apparent affinity of fibroblast growth factor-1 for heparin is only observed when basic residues in one of the three regions are mutated. Mutation in the other regions are without affect on heparin binding. The heparin binding properties of synthetic peptides based on the three "consensus sequences" paralleled the mutagenesis results. That is, synthetic peptides corresponding to regions of the protein that were affected by mutagenesis with respect to heparin binding exhibited a relatively high affinity for immobilized heparin, whereas those corresponding to regions of similar charge density that were unaffected by mutagenesis did not. In addition, amino acid substitution of a nonbasic residue in the heparin-binding peptide could abolish its heparin binding capacity. The heparin-binding peptide could antagonize the mitogenic activity of FGF-1, probably because of the heparin dependence of this activity. Together these data demonstrate that the heparin binding properties of fibroblast growth factor-1 are dictated by structural features more complex than clusters of basic amino acids. The results of these and other studies indicate that consensus motifs for heparin-binding require further definition. More importantly, the results provide a basis for the design of peptide-based inhibitors of FGF-1.The fibroblast growth factor (FGF) 1 family consists of at least nine structurally related proteins (1-3). Two of these proteins, FGF-1 and FGF-2, have been characterized under many different names, most often as acidic FGF and basic FGF, respectively. Although there is a large amount of overlap in the spectrum of biological activities and receptor-binding properties of the FGFs, the only known function shared by all members of the family is a relatively high affinity for heparin or heparan sulfate proteoglycans (HSPGs). It has been established that heparin can potentiate the mitogenic activity of FGF-1 (4 -6) and protect both FGF-1 and FGF-2 from proteolytic and heat inactivation (7-9). In addition, heparin increases the apparent affinity of FGF-1 for high affinity FGF receptors (10, 11). Recently, an obligatory role for heparin or HSPGs in mediating the binding of FGF-1 or FGF-2 to the high affinity, tyrosine kinase receptors has been suggested (12-17). It has also been reported that cell surface HSPGs are capable of binding and internalizing FGF-2 (18). A direct role of HSPGbound FGF in mediating the various functions of this growth factor family has not been established.We reported previously that a change of lysine 132 in FGF-1 to a glutamic acid (K132E) by site-directed mutagenesis reduced the apparent affinity of the recombinant protein for heparin (4). The K132E mutant is fully capable of binding to and activating the high-affinity tyrosine kinase FGF receptors and can i...