The three-dimensional structure of the 146-residue form of human basic fibroblast growth factor (bFGF), expressed as a recombinant protein in yeast, has been determined by x-ray crystallography to a resolution of 1.8 A. bFGF is composed entirely of f-sheet structure, comprising a threefold repeat of a four-stranded antiparallel -meander. The topology of bFGF is Identical to that of interleukin 1fl, showing that although the two proteins share only 10% sequence identity, bFGF, interleukin 1, and their homologs comprise a family of structurally related mitogenic factors. Analysis of the three-dimensional structure in light of functional studies of bFGF suggests that the receptor binding site and the positively charged heparin binding site correspond to adjacent but separate loci on the fl-barrel.

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Abstract. Basic fibroblast growth factor (FGF) is synthesized as a phosphoprotein by both bovine capillary endothelial and human hepatoma cells in culture. Because basic FGF is characterized by its high affinity for heparin and its association in vivo with the extracellular matrix, we examined the possibility that the phosphorylation of this growth factor by purified protein kinase C (PK-C) and the catalytic subunit of cAMP-dependent protein kinase-A (PK-A) can be modulated by components of the extracellular matrix.Heparin and other glycosaminoglycans (GAGs) inhibit the ability of PK-C to phosphorylate basic FGE In contrast, heparin can directly increase the phosphorylation of basic FGF by PK-A. While fibronectin, laminin, and collagen IV have no effect on the ability of PK-C to phosphorylate basic FGE they all can inhibit the effects of PK-A. Thus, there is a differential effect of extracellular matrix-derived proteins and GAGs on the phosphorylation of basic FGE The enhanced phosphorylation of basic FGF that is mediated by heparin is associated with a change in the kinetics of the reaction and the identity of the amino acid targeted by this enzyme. The amino acids that are targeted by PK-C and PK-A have been identified by phosphopeptide analyses as Ser 64 and Thr "2, respectively. In the presence of heparin, basic FGF is no longer phosphorylated by PK-A at the usual PK-A consensus site (Thr"2), but instead is phosphorylated at the canonical PK-C site (SetS4). Accordingly, heparin inhibits the phosphorylation of basic FGF by PK-C presumably by masking the PK-C dependent consensus sequence surrounding Set 64. Thus, when basic FGF is no longer phosphorylated by PK-A in the receptor binding domain (ThrU2), it loses the increased receptor binding ability that characterizes PK-A phosphorylated basic FGEThe results presented here demonstrate three novel features of basic FGE First, they identify a functional effect of the binding of heparin to basic FGE Second, they establish that the binding of beparin to basic FGF can induce structural changes that alter the substrate specificity of protein kinases. Third, and perhaps most important, the results demonstrate the existence of a novel interaction between basic FGF, fibronectin, and laminin. Although the physiological significance of this phosphorylation is not known, these results clearly suggest that the biological activities of basic FGF are regulated by a complex array of biochemical interactions with the proteins, proteoglycans, and glycosaminoglycans present in the extracellular milieu and the cytoplasm. HEPARIN and related glycosaminoglycans (GAGs) ~ appear to be important regulators of the activity and binding of basic FGE It is the discovery that basic FGF has a high affinity for beparin that first established the link between GAGs and this growth factor (20,31). Since that J.-J. Feige is a visiting scientist from INSERM (Unit 244; Grenoble, France).Some of these studies were performed at

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