Identification of receptor and heparin binding sites in fibroblast growth factor 4 by structure-based mutagenesis.

Fibroblast growth factors (FGFs) comprise a large family of multifunctional, heparin-binding polypeptides that show diverse patterns of interaction with a family of receptors (FGFR1 to -4) that are subject to alternative splicing. FGFR binding specificity is an essential mechanism in the regulation of FGF signaling and is achieved through primary sequence differences among FGFs and FGFRs and through usage of two alternative exons, IIIc and IIIb, for the second half of immunoglobulin-like domain 3 (D3) in FGFRs. While FGF4 binds and activates the IIIc splice forms of FGFR1 to -3 at comparable levels, it shows little activity towards the IIIb splice forms of FGFR1 to -3 as well as towards FGFR4. To begin to explore the structural determinants for this differential affinity, we determined the crystal structure of FGF4 at a 1.8-A resolution. FGF4 adopts a beta-trefoil fold similar to other FGFs. To identify potential receptor and heparin binding sites in FGF4, a ternary FGF4-FGFR1-heparin model was constructed by superimposing the FGF4 structure onto FGF2 in the FGF2-FGFR1-heparin structure. Mutation of several key residues in FGF4, observed to interact with FGFR1 or with heparin in the model, produced ligands with reduced receptor binding and concomitant low mitogenic potential. Based on the modeling and mutational data, we propose that FGF4, like FGF2, but unlike FGF1, engages the betaC'-betaE loop in D3 and thus can differentiate between the IIIc and IIIb splice isoforms of FGFRs for binding. Moreover, we show that FGF4 needs to interact with both the 2-O- and 6-O-sulfates in heparin to exert its optimal biological activity.

Structural basis for fibroblast growth factor receptor 2 activation in Apert syndrome.

Apert syndrome (AS) is characterized by craniosynostosis (premature fusion of cranial sutures) and severe syndactyly of the hands and feet. Two activating mutations, Ser-252 --> Trp and Pro-253 --> Arg, in fibroblast growth factor receptor 2 (FGFR2) account for nearly all known cases of AS. To elucidate the mechanism by which these substitutions cause AS, we determined the crystal structures of these two FGFR2 mutants in complex with fibroblast growth factor 2 (FGF2). These structures demonstrate that both mutations introduce additional interactions between FGFR2 and FGF2, thereby augmenting FGFR2-FGF2 affinity. Moreover, based on these structures and sequence alignment of the FGF family, we propose that the Pro-253 --> Arg mutation will indiscriminately increase the affinity of FGFR2 toward any FGF. In contrast, the Ser-252 --> Trp mutation will selectively enhance the affinity of FGFR2 toward a limited subset of FGFs. These predictions are consistent with previous biochemical data describing the effects of AS mutations on FGF binding. Alterations in FGFR2 ligand affinity and specificity may allow inappropriate autocrine or paracrine activation of FGFR2. Furthermore, the distinct gain-of-function interactions observed in each crystal structure provide a model to explain the phenotypic variability among AS patients.

Crystal structure of fibroblast growth factor 9 reveals regions implicated in dimerization and autoinhibition.

Fibroblast growth factors (FGFs) constitute a large family of heparin-binding growth factors with diverse biological activities. FGF9 was originally described as glia-activating factor and is expressed in the nervous system as a potent mitogen for glia cells. Unlike most FGFs, FGF9 forms dimers in solution with a K(d) of 680 nm. To elucidate the molecular mechanism of FGF9 dimerization, the crystal structure of FGF9 was determined at 2.2 A resolution. FGF9 adopts a beta-trefoil fold similar to other FGFs. However, unlike other FGFs, the N- and C-terminal regions outside the beta-trefoil core in FGF9 are ordered and involved in the formation of a 2-fold crystallographic dimer. A significant surface area (>2000 A(2)) is buried in the dimer interface that occludes a major receptor binding site of FGF9. Thus, we propose an autoinhibitory mechanism for FGF9 that is dependent on sequences outside of the beta-trefoil core. Moreover, a model is presented providing a molecular basis for the preferential affinity of FGF9 toward FGFR3.

Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization.

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.

Crystal structure of an angiogenesis inhibitor bound to the FGF receptor tyrosine kinase domain.

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 A 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.

Induction of P-glycoprotein functional activity and multidrug resistance by a soluble factor(s) produced by some mammalian cells.

In the previous study we have found that Djungarian hamster fibroblasts with high levels of multidrug resistance (MDR) (colchicine-resistance index RI of 1000 to 42000) produce soluble factor(s) communicating MDR to the drug-sensitive cells of the same species by elevating the functional activity of P-glycoprotein (Pgp). Here we have shown that these cells can influence human tumor cells in the same fashion. Rat hepatoma McA RH7777 cells and their colchicine-resistant derivatives are shown to produce a factor with similar effects (induction of MDR and Pgp functional activity in the drug-sensitive cells). These effects seem to depend on the drug resistance level of the donor cells. Our results show that induction of the Pgp-mediated MDR is not species-specific and the tumor cells with intrinsic MDR (arising from the tissue with a high level of Pgp expression) can produce a factor(s) communicating this type of drug resistance to the sensitive cells.

Alterations of melanin synthesis in human melanoma cells selected in vitro for multidrug resistance.

Previous data showing the correlation of multidrug resistance (MDR) and differentiation in tumor cell populations (Melloni et al. 1988; Stavrovskaya et al. 1990) suggest that: 1) isolation of MDR cells by cytostatic drugs leads to the selection of more differentiated cell variants and 2) in more differentiated cell variants the activity of MDR-related P-glycoprotein (Pgp) is more prominent than in less differentiated cells. Here we used human melanoma cell line mS and two variants selected from mS population: a) MDR variant of mS selected by colchicine (mS-0.5) and b) mS-trRAR/2--variant obtained by introduction of expressing retinoic acid receptor RAR-alpha cDNA into mS cell. The differentiation status, expression of MDR1 gene and Pgp functioning were compared in wild-type cells and mS variants. Electron microscopic examination of melanosomes showed that the mS-0.5 subline comprised more differentiating cells in the population than parental mS cultures and that these cells were at later stages of melanogenesis. The increase in the degree of differentiation in mS-0.5 population coincided with MDR1 gene overexpression, occurrence of Pgp molecules on the cell membrane and acceleration of Pgp-mediated Rhodamine 123 (Rh123) efflux. mS-trRAR/2, proved to be more differentiated than mS cells. The MDR1 mRNA level and Rh123 efflux were not elevated in mS-trRAR/2 cells, however, retinoic acid (RA) treatment increased both the degree of differentiation and Rh123 efflux in mS-trRAR/2 to a greater extent than in mS cultures. Thus, the data obtained in this study are in favor of the suppositions mentioned above. The mechanisms of coordinated alterations of differentiation and Pgp activity in MDR cells are discussed.

Colchicine-resistance and enhancement of P-glycoprotein activity after co-cultivation of drug-sensitive cells with multidrug resistant variants.

The role of cellular interactions in the resistance of Djungurian hamster cells to colchicine (CH) and in the efficiency of P-glycoprotein function was studied. Mixtures of CH-resistant and CH-sensitive cells as well as control unmixed cells were propagated for 3 days and the sensitivity of the cells to CH was measured by colony forming assay. Identification of individual subpopulations was possible due to genetic marker (6TG-resistance). The data show that the survival of CH-sensitive cells in CH-supplemented medium increased after co-cultivation with CH-resistant counterparts. To measure Pgp activity the fluorescent dye RH123 and FACScan analysis were used. Pgp-mediated RH123 efflux increased after co-cultivation of CH-sensitive and CH-resistant cells.