The Glycine Box:  A Determinant of Specificity for Fibroblast Growth Factor

Abstract

Acidic fibroblast growth factor (FGF-1), keratinocyte growth factor (FGF-7), and FGF-10 are homologues with distinct specificity. In the presence of heparin, FGF-1 binds and activates in vitro all FGFR subtypes, while FGF-7 exhibits absolute specificity for the IIIb splice variant of FGFR2. FGF-10 exhibits a similar specificity but also binds the FGFR1IIIb isoform. Neither FGF-7 nor FGF-10 will bind to IIIc isoforms of FGFR. Molecular models of FGF, heparin, and the FGFR ectodomain suggested that sequences between beta-strands 10 and 12 of FGF may be important for the interaction of FGF with the heparin-FGFR ectodomain duplex. Site-directed mutants of FGF-7 and FGF-10 were prepared to test whether this domain might underlie failure of FGF-7 and FGF-10 to bind to the FGFRIIIc isoforms. Constructions with substitution of FGF-1 sequences spanning the entire C-terminus encoded in exon 3 or only C-terminal sequences spanning beta-strands 10 through 12 conferred ability on FGF-7 to bind to and activate FGFRIIIc without a significant loss in binding to or activation of FGFR2IIIb. A series of twelve different substitutions of shorter segments of FGF-1 sequences into the C-terminal portion of FGF-7 or FGF-10 revealed that substitution of GSCKRG for GIPVRG or the tri-peptide sequence KKN for NQK just N-terminal to it conferred dual activities on both the FGF-7 and FGF-10 backbones. The results suggest that the combined sequence domain, which we call the FGF glycine box (G-box), is a major determinant for the specificity of the binding of FGF to heparan sulfate-FGFR duplexes.

Maintenance of surfactant protein A and D secretion by rat alveolar type II cells in vitro

“…However, the difference between KGF and FGF-10 was surprising. Both are thought to recognize the same receptor (20,29). Presumably, there are costimulatory molecules or other subtle differences in the receptor signaling that are presently not known that account for this difference.…”

“…1, KGF was the only growth factor that stimulated the production and secretion of significant amounts of SP-A or SP-D. This was surprising, because FGF-10 is very similar to KGF, binds the same FGF receptor splice variant (FGFR-2IIIb) (20,29), and stimulates type II cell proliferation in vivo. The morphology of type II cell cultures without KGF was that of a flat monolayer, whereas the cultures with KGF had areas of refractile cuboidal cells.…”

Directionally Specific Paracrine Communication Mediated by Epithelial FGF9 to Stromal FGFR3 in Two-Compartment Premalignant Prostate Tumors

“…The product of Ser34-FGF9 exhibited a single band of M r 21,000 on SDS-PAGE and the single N-terminal sequence ser-asp-his-leu-gly expected of mature homogenous Ser34-FGF9. The yield of high specific activity radiolabel grade FGF9 by this procedure was 5-10% and 25% of that reported for FGF1 and FGF7, respectively (24).…”

“…Because efficient production and recovery of FGF7 depended on the length of the NH 2 terminus upstream of the minimal active core, we tested three constructions fused to GST at the NH 2 terminus (GST-Leu4-FGF9, GSTVal13-FGF9, and GST-Ser34-FGF9) for highest yield and recovery of fulllength active product after removal of the fusion partner (10,11,24). GSTSer34-FGF9 (residues 34 -208) was superior and used throughout the study.…”

“…GSTSer34-FGF9 (residues 34 -208) was superior and used throughout the study. Initial purification, recovery, and immobilization of product from bacterial lysates onto heparin-agarose, followed by controlled trypsin treatment to remove the GST part resulted in highest recovery of total FGF9, but purity was unsatisfactory and the product was unstable on storage (24). Therefore, we immobilized the GST-Ser34-FGF9 from the lysate, removed the GST part by treatment of the column with thrombin, and subsequently purified the eluate on fast protein liquid chromatography heparin-agarose.…”