MULTIVALENT LIGAND-RECEPTOR BINDING INTERACTIONS IN THE FIBROBLAST GROWTH-FACTOR SYSTEM PRODUCE A COOPERATIVE GROWTH-FACTOR AND HEPARIN MECHANISM FOR RECEPTOR DIMERIZATION

The binding interactions for the three primary reactants of the fibrob last growth factor (FGF) system, basic FGF (bFGF), an FGF receptor, FG FR1, and the cofactor heparin/heparan sulfate (HS), were explored by i sothermal titrating calorimetry, ultracentrifugation, and molecular mo deling. The binding reactions were first dissected into three binary r eactions: (1) FGFR1 + bFGF <-> FGFR1/bFGF, K-1 = 41 (+/-12) nM; (2) FG FR1 + HS <-> FGFR1/HS, K-2 = 104 (+/-17) mu M; and (3) bFGF + HS <-> b FGF/HS, K-3 = 470 (+/-20) nM, where HS = low MW heparin, similar to 3 kDa. The first, binding of bFGF to FGFR1 in the absence of HS, was fou nd to be a simple binary binding reaction that is enthalpy dominated a nd characterized by a single equilibrium constant, K-1. The conditiona l reactions of bFGF and FGFR1 in the presence of heparin were then exa mined under conditions that saturate only the bFGF heparin site (1.5 e quiv of HS/bFGF) or saturate the HS binding sites of both bFGF and FGF R1 (1.0 mM HS). Both 3-and 5-kDa low MW heparins increased the affinit y for FGFR1 binding to bFGF by similar to 10-fold (K-d = 4.9 +/- 2.0 n M), relative to the reaction with no HS. In addition, HS, at a minimum of 1.5 equiv/bFGF, induced a second FGFR1 molecule to bind to another lower affinity secondary site on bFGF (K-4 = 1.9 +/- 0.7 mu M) in an entropy-dominated reaction to yield a quaternary complex containing tw o FGFR1, one bFGF, and at least one HS. Molecular weight estimates by analytical ultracentrifugation of such fully bound complexes were cons istent with this proposed composition. To understand these binding rea ctions in terms of structural components of FGFR1, a three-dimensional model of FGFR1 was constructed using segment match modeling. Electros tatic potential calculations confirmed that an elongated cluster, simi lar to 15 X 35 Angstrom, of nine cationic residues focused positive po tential (+2k(B)T) to the solvent-exposed beta-sheet A, B, E, C' surfac e of the D(II) domain model, strongly implicating this locus as the HS binding region of FGFR1. Structural models for HS binding to FGFR1, a nd HS binding to bFGF, were built individually and then assembled to j uxtapose adjacent binding sites for receptor and HS on bFGF, against m atching proposed growth factor and HS binding sites on FGFR1. The calo rimetric binding results and the molecular modeling exercises suggest that bFGF and HS participate in a concerted bridge mechanism for the d imerization of FGFR1 in vitro and presumably for mitogenic signal tran sduction in vivo. The thermodynamic driving force for receptor dimeriz ation can be explained in terms of allosteric multivalent binding reac tions that allow for the cooperative energetic coupling of heparin bin ding reactions on FGFR1 and bFGF, reactions 2 and 3, with growth facto r/receptor binding events, reactions 1 and 4. Finally, the observation that pentosan polysulfate binds to FGFR1 with a 10-fold higher affini ty than HS [K-d = 10.9 (+/-3.5) mu M] may provide an opportunity to re examine the mechanism of action of this sulfated oligosaccharide and o ther related inhibitors of angiogenesis currently under investigation for the treatment of breast cancer and AIDS-related Kaposi sarcoma.