Characterization of the interaction between α2-macroglobulin and fibroblast growth factor-2:: the role of hydrophobic interactions

Basic fibroblast growth factor (FGF-2) is important in development, wound healing and angiogenesis. The human plasma proteinase inhibitor alpha(2)-macroglobulin (alpha(2)M) binds to and regulates the biological activity of various growth factors, including FGF-2. FGF-2 binds specifically and saturably to native alpha(2)M and conformationally modified alpha(2)M (alpha(2)M*); however, the K-D for FGF-2 binding to alpha(2)M* is 10-fold lower. This study investigates the biochemical nature of the interaction between FGF-2 and a(2)M* and localizes a possible FGF-2 binding site in the alpha(2)M subunit. FGF-2 binding to alpha2M* was not affected by shifts in pH between 6.5 and 10: however, increasing temperature decreased the K-D for this interaction. The binding affinity of FGF-2 for alpha(2)M* also increased with increasing ionic strength. These results are consistent with the hypothesis that hydrophobic interactions predominate in promoting FGF-2 association with alpha2M*. Consistent with this hypothesis, FGF-2 bound to a glutathione S-transferase fusion protein containing amino acids 591-774 of the alpha(2)M subunit (FP3) and to a hydrophobic 16-amino-acid peptide (amino acids 718-733) within FP3. Specific binding of FGF-2 to the 16-amino-acid peptide was inhibited by excess transforming growth factor-beta1. When the 16-amino-acid peptide was chemically modified to neutralize the only two charged amino acids, FGF-2-binding activity was unaffected, supporting the predominant role of hydrophobic interactions. FGF-2 presentation to signalling receptors is influenced by growth factor binding to heparan sulphate proteoglycans (HSPGs), which is electrostatic in nature. Our results demonstrate that the interactions of FGF-2 with alpha(2)M* and HSPGs are biochemically distinct, suggesting that different FGF-2 sequences are involved.