A murine fibroblast growth factor (FGF) receptor expressed in CHO cells is activated by basic FGF and Kaposi FGF.

Abstract

We have cloned a murine cDNA encoding a tyrosine kinase receptor with about 90% similarity to the chicken fibroblast growth factor (FGF) receptor and the humanfms-like gene (FLG) tyrosine kinase. This mouse receptor lacks 88 amino acids in the extracellular portion, leaving only two immunoglobulin-like domains compared to three in the chicken FGF receptor. The cDNA was cloned into an expression vector and transfected into receptor-negative CHO cells. We show that cells expressing the receptor can bind both basic FGF and Kaposi FGF. Although the receptor binds basic FGF with a 15-to 20-fold higher affinity, Kaposi FGF is able to induce down-regulation of the receptor to the same extent as basic FGF. The receptor is phosphorylated upon stimulation with both FGFs, DNA synthesis is stimulated, and a proliferative response is produced in cells expressing the receptor, whereas cells expressing the cDNA in the antisense orientation show none of these responses to basic FGF or Kaposi FGF. Thus this receptor can functionally interact with two growth factors of the FGF family.The mitogenic effect ofgrowth factors requires binding to and activating specific cell surface receptors, many ofwhich have tyrosine kinase activity (1,2 done/0.02% Ficoll/0.02% bovine serum albumin.) The filters were then washed at 650C with 2x SSC/0.1% SDS for 30 min and with lx SSC/0.1% SDS for 45 min. The cDNAs were subcloned into pBluescript (Stratagene) and sequenced by the dideoxynucleotide chain-termination method (9).Cell Lines and Transfections. CHO-DG44 cells, which are dihydrofolate reductase-negative (DHFR-), were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (vol/vol) fetal calf serum, 5 mM hypoxanthine, 2 mM thymidine, and 10 mM proline. Cells were transfected by calcium phosphate precipitation with cDNA 4A in p91023B vector (10), which carried DHFR as a selectable marker. Positive clones were selected in medium lacking hypoxanthine and thymidine.Scatchard Analysis. Recombinant bFGF was iodinated as described (11) (specific activity = 552 cpm/fmol). CHO clone 3-3 and clone 4-1 cells were incubated at 40C with DMEM containing 0.15% gelatin, 25 mM Hepes (pH 7.4), and various concentrations of '2"I-labeled bFGF from 0.3 to 7 ng/ml.After 2 hr, the medium was removed, the cells were washed once with ice-cold isotonic phosphate-buffered saline (PBS), and 1251I-labeled bFGF bound to the extracellular matrix was removed by washing twice with 2 M NaCl/20 mM Hepes, pH 7.4 (11). Radioactive bFGF bound to high-affinity receptors was released with two subsequent washes with 2 M NaCl/20 mM sodium acetate, pH 4.0. Radioactivity in the combined medium and PBS wash (free fraction) and in the combined 2 M NaCl washes buffered at pH 4 (bound fraction) was quantitated on a Beckman y scintillation counter. The cells of parallel cultures were treated with trypsin and quantitated using a Coulter particle counter.Competition Assays. To investigate the ability of K-FGF to displace 1251-labeled bFGF, CHO cells were incub...

Activation of FGF receptors by mutations in the transmembrane domain

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Cell Release of Bioactive Fibroblast Growth Factor 2 by Exon 6-encoded Sequence of Vascular Endothelial Growth Factor

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