The crystal structure of a dimeric 2:2:2 FGF:FGFR:heparin ternary complex at 3 A resolution has been determined. Within each 1:1 FGF:FGFR complex, heparin makes numerous contacts with both FGF and FGFR, thereby augmenting FGF-FGFR binding. Heparin also interacts with FGFR in the adjoining 1:1 FGF:FGFR complex to promote FGFR dimerization. The 6-O-sulfate group of heparin plays a pivotal role in mediating both interactions. The unexpected stoichiometry of heparin binding in the structure led us to propose a revised model for FGFR dimerization. Biochemical data in support of this model are also presented. This model provides a structural basis for FGFR activation by small molecule heparin analogs and may facilitate the design of heparin mimetics capable of modulating FGF signaling.

The fibroblast growth factor (FGF) 19 subfamily of ligands, FGF19, FGF21, and FGF23, function as hormones that regulate bile acid, fatty acid, glucose, and phosphate metabolism in target organs through activating FGF receptors (FGFR1-4). We demonstrated that Klotho and ␤Klotho, homologous single-pass transmembrane proteins that bind to FGFRs, are required for metabolic activity of FGF23 and FGF21, respectively. Here we show that, like FGF21, FGF19 also requires ␤Klotho. Both FGF19 and FGF21 can signal through FGFR1-3 bound by ␤Klotho and increase glucose uptake in adipocytes expressing FGFR1. Additionally, both FGF19 and FGF21 bind to the ␤Klotho-FGFR4 complex; however, only FGF19 signals efficiently through FGFR4. Accordingly, FGF19, but not FGF21, activates FGF signaling in hepatocytes that primarily express FGFR4 and reduces transcription of CYP7A1 that encodes the rate-limiting enzyme for bile acid synthesis. We conclude that the expression of ␤Klotho, in combination with particular FGFR isoforms, determines the tissue-specific metabolic activities of FGF19 and FGF21.

The mammalian fibroblast growth factor (FGF) family comprises 18 polypeptides (FGF1 to FGF10 and FGF16 to FGF23) which participate in a myriad of biological processes during embryogenesis, including but not limited to gastrulation, body plan formation, somitogenesis, and morphogenesis of essentially every tissue/organ such as limb, lung, brain and kidney (3,30). FGFs execute their biological actions by binding to, dimerizing, and activating FGF receptor (FGFR) tyrosine kinases, which are encoded by four distinct genes (Fgfr1 to Fgfr4). Prototypical FGFRs consist of an extracellular domain composed of three immunoglobulin-like domains, a single-pass transmembrane domain, and an intracellular domain responsible for the tyrosine kinase activity (16). The number of principal FGFRs is increased from four to seven due to a major tissue-specific alternative splicing event in the second half of the immunoglobulin-like domain 3 of FGFR1 to FGFR3, which creates epithelial lineage-specific b and mesenchymal lineage-specific c isoforms (16, 21). Generally, the receptor-binding specificity of FGFs is divided along this major alternative splicing of receptors whereby FGFRb-interacting FGFs are produced by mesenchymal cells and FGFRc-interacting FGFs are produced by epithelial cells (21). These reciprocal expression patterns of FGFs and FGFRs result in the establishment of a paracrine epithelial-mesenchymal signaling which is essential for proper organogenesis and patterning during development as well as tissue homeostasis in the adult organism.Based on phylogeny and sequence identity, FGFs are grouped into seven subfamilies (21). The FGF core homology domain (approximately 120 amino acids long) is flanked by Nand C-terminal sequences that are highly variable in both length and primary sequence, particularly among different FGF subfamilies. The core region of FGF19 shares the highest sequence identity with FGF21 (38%) and FGF23 (36%), and therefore, these ligands are considered to form a subfamily. However, the degree of identity within the FGF19 subfamily is only 2 to 3% greater than that between FGF19 subfamily members and members of other FGF subfamilies, making this subfamily the most divergent one. FGF19 subfamily members regulate diverse physiological processes uncommon to classical FGFs, namely, energy (32) and bile acid homeostasis (FGF19) (5,8,13), glucose and lipid metabolism (FGF21) (10), and phosphate and vitamin D homeostasis (FGF23) (27). Moreover, unlike classical FGFs, FGF19 subfamily members achieve their unconventional activities in an endocrine fashion.To date, only a single structure from the endocrine-acting FGF19 subfamily has been reported (4), whereas there are crystal structures available for eight classical, paracrine-acting FGFs (2,20,22,37,38,40). The structures from the paracrine class of FGFs (FGF1, show that the core homology region folds into a globular domain composed of 12 antiparallel ␤-strands (␤1 to ␤12) known as the * Corresponding author. Mailing address:

Fibroblast growth factor 21 (FGF21) is a liver-derived endocrine factor that stimulates glucose uptake in adipocytes. Here, we show that FGF21 activity depends on ␤Klotho, a single-pass transmembrane protein whose expression is induced during differentiation from preadipocytes to adipocytes. ␤Klotho physically interacts with FGF receptors 1c and 4, thereby increasing the ability of these FGF receptors to bind FGF21 and activate the MAP kinase cascade. Knockdown of ␤Klotho expression by siRNA in adipocytes diminishes glucose uptake induced by FGF21. Importantly, administration of FGF21 into mice induces MAP kinase phosphorylation in white adipose tissue and not in tissues without ␤Klotho expression. Thus, ␤Klotho functions as a cofactor essential for FGF21 activity.Klotho ͉ glucose ͉ adipocyte ͉ siRNA ͉ GLUT1

Angiogenesis, the sprouting of new blood vessels from pre-existing ones, is an essential physiological process in development, yet also plays a major role in the progression of human diseases such as diabetic retinopathy, atherosclerosis and cancer. The effects of the most potent angiogenic factors, vascular endothelial growth factor (VEGF), angiopoietin and fibroblast growth factor (FGF) are mediated through cell surface receptors that possess intrinsic protein tyrosine kinase activity. In this report, we describe a synthetic compound of the pyrido[2,3-d]pyrimidine class, designated PD 173074, that selectively inhibits the tyrosine kinase activities of the FGF and VEGF receptors. We show that systemic administration of PD 173074 in mice can effectively block angiogenesis induced by either FGF or VEGF with no apparent toxicity. To elucidate the determinants of selectivity, we have determined the crystal structure of PD 173074 in complex with the tyrosine kinase domain of FGF receptor 1 at 2.5 Å resolution. A high degree of surface complementarity between PD 173074 and the hydrophobic, ATP-binding pocket of FGF receptor 1 underlies the potency and selectivity of this inhibitor. PD 173074 is thus a promising candidate for a therapeutic angiogenesis inhibitor to be used in the treatment of cancer and other diseases whose progression is dependent upon new blood vessel formation.