title: Basic fibroblast growth factor binding and processing by human glioma cells.
abstract: Tumor cells of glial origin express high levels of basic fibroblast growth factor (bFGF) which stimulates their proliferation in an autocrine manner. In the present study we examined bFGF receptor (FGFR) expression and 125I-bFGF binding and processing in a human glioma cell line. RT-PCR demonstrated the co-expression of bFGF and FGFR mRNAs in five glioma cell lines examined. The high-affinity FGFR was visualized in U87-MG glioma cells by crosslinking with 125I-bFGF and by Western blotting with anti-receptor antisera. Both techniques identified a discrete 110-kDa moiety associated with the cell membrane, consistent with the reported size of one of the FGFR-1 isoforms. Western blotting also identified an intracellular receptor pool which was not accessible with exogenous 125I-bFGF. Suramin treatment induced a 2-fold increase in immunoreactive FGFR and a 1.5-fold increase in 125I-bFGF binding sites, indicating that FGFRs are chronically down-regulated by endogenous bFGF in U87-MG cells. Removal of extracellular bFGF with heparin resulted in a rapid, cycloheximide-sensitive increase in high-affinity bFGF binding sites. At 37 degrees C, receptor-bound 125I-bFGF was internalized and subjected to limited proteolytic cleavage over 12 h. U87-MG cells also contained abundant low-affinity bFGF binding sites which were removed by digestion with heparinase III but not by chondroitinase ABC. The presence of heparin (25 micrograms/ml) in the binding reaction eliminated the association of 125I-bFGF with the heparin-like sites but did not prevent binding to the high-affinity receptor. Scatchard binding analysis in the presence of heparin revealed a single class of high-affinity sites in U87-MG cells (Kd = 4.9 +/- 0.9 pM; 10-12 x 10(3) sites per cell). Neither heparin nor heparinase digestion prevented the binding of 125I-bFGF to the detergent-extractable high-affinity receptor, although both treatments significantly reduced the extent of 125I-bFGF association with the receptor. These findings indicate that in U87-MG cells, heparan sulfate proteoglycans may be involved in presentation of bFGF to the high-affinity receptor, but are not essential for high-affinity binding to occur.
authors.affiliation: Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: P R
authors.initials: PR
authors.lastname: Murphy
authors.forename: R S
authors.initials: RS
authors.lastname: Knee
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D003432:Cross-Linking Reagents; D017931:DNA Primers / Q000235:genetics; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D005910:Glioma / Q000235:genetics / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism / Q000494:pharmacology; D006497:Heparan Sulfate / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D017468:Receptors, Fibroblast Growth Factor / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D013498:Suramin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D003432:Cross-Linking Reagents; D017931:DNA Primers; D016222:Fibroblast Growth Factor 2; D006493:Heparin; D006497:Heparan Sulfate; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D017468:Receptors, Fibroblast Growth Factor; D013498:Suramin