Molecular Cloning of a Human Basic Fibroblast Growth Factor Receptor cDNA and Expression of a Biologically Active Extracellular Domain in a Baculovirus System

Abstract

A cDNA clone encoding a human fibroblast growth factor (FGF) receptor was isolated from a hepatoma cell line cDNA library. The cDNA encodes a three immunoglobulinlike-domain FGF receptor that is similar to a human placental FGF receptor cDNA but lacks two amino acids. The variation observed at these two amino acids, also seen in the two immunoglobulinlike-domain FGF-receptors, can be explained by an alternate splicing mechanism. We have used a baculovirus expression system to produce high levels of a soluble, extracellular domain form of the FGF receptor (EC-FGF receptor). Spodoptera frugiperda (Sf9) insect cells infected with recombinant EC-FGF receptor viruses synthesized and secreted an EC-FGF receptor of apparent Mr = 58,000. The EC-FGF receptor purified from conditioned media of infected Sf9 cells by lentil lectin affinity chromatography was shown to bind basic FGF with high affinity (Kd = 1-5 nM), to inhibit the binding of radioiodinated basic FGF to its high affinity receptor and to inhibit endothelial cell proliferation. Furthermore, binding of basic FGF to the EC-FGF receptor was shown to be significantly enhanced by heparin. The availability of biologically active FGF receptors will allow an analysis of their interaction with members of the FGF family of proteins and viruses of the herpes family that have been shown to use the FGF receptor system for cell entry.

Heparan sulfation is essential for the prevention of cellular senescence

“…The present study explains how FGF signaling is tumor promoting in some contexts, but tumor-suppressive in others. Our results are consistent with previous studies demonstrating that FGFR1 can bind to FGF2 in the absence of heparin 66 and that mutations in the heparinbinding region of FGF2 reduce its affinity for cell-associated HS, but do not affect the affinity for its receptor FGFR1. 67 In conclusion, our findings suggest that the potential to regulate FGF activity through HS-dependent interactions can provide an attractive system for regulating cellular responses to FGF in the extracellular environment.…”

Characterization of a biologically active extracellular domain of fibroblast growth factor receptor 1 expressed in Escherichia coli

“…5). The high doses of XC-FGF-R which are required to obtain this inhibition are in agreement with the apparent Kd of the molecule and are comparable to those reported for glycosylated EC-FGF-R (Kiefer et al, 1991).…”

“…Scatchard analysis of independent binding data yielded a dissociation constant of 5 -10 nM. These values are in the same range as those obtained for a similar soluble FGF-R, EC-FGF-R, produced in a baculovirus system, for which a Kd of 1 -5 nM was reported (Kiefer et al, 1991). Similar Kd was recently reported for soluble extracellular domains of the FGF-R family expressed as fusion proteins secreted in mammalian cells (Yayon et al, 1992;Ornitz et al, 1992).…”

“…Binding of bFGF to soluble receptors produced in eukaryotic systems was shown to be enhanced by heparin (Ornitz et al, 1992;Kiefer et al, 1991). In our conditions, heparin significantly reduced the concentrations of XC-FGF-R necessary to obtain 50% inhibition of lz51-bFGF binding to BHK cells only at 0.5 -1 pg/ml; however, these doses also interfered with bFGF binding in the absence of XC-FGF-R (data not shown).…”

“…These results indicate that the molecule was obtained in a biologically active, highly homogeneous form suitable for in vivo as well as for in vitro studies. This is the first report of a soluble FGF-R produced in a prokaryotic expression system, while a similar protein, named EC-FGF-R, has been expressed recently in a baculovirus system (Kiefer et al, 1991). Interestingly, the amino acid sequence of the two receptors is quite similar, with both molecules corresponding to the three Ig-like structure of FGF-R1 and mainly differing because the receptor produced in E. coli is not glycosylated.…”