Fibroblast growth factor (FGF) receptor (FGFR) gene family consists of at least four receptor tyrosine kinases that transduce signals important in a variety of developmental and physiological processes related to cell growth and differentiation. Here we have characterized the binding of different FGFs to FGFR‐4. Our results establish an FGF binding profile for FGFR‐4 with aFGF having the highest affinity, followed by K‐FGF/hst‐1 and bFGF. In addition, FGF‐6 was found to bind to FGFR‐4 in ligand competition experiments. Interestingly, the FGFR‐4 gene was found to encode only the prototype receptor in a region where both FGFR‐1 and FGFR‐2 show alternative splicing leading to differences in their ligand binding specificities and to secreted forms of these receptors. Ligands binding to FGFR‐4 induced receptor autophosphorylation and phosphorylation of a set of cellular polypeptides, which differed from those phosphorylated in FGFR‐1‐expressing cells. Specifically, the FGFR‐1‐expressing cells showed a considerably more extensive tyrosine phosphorylation of PLC‐gamma than the FGFR‐4‐expressing cells. Structural and functional specificity within the FGFR family exemplified by FGFR‐4 may help to explain how FGFs perform their diverse functions.
We studied the androgen regulation of fibroblast growth factor (FGF) receptors (FGFRs) in the Shionogi 115 (S115) mouse mammary tumor cell line and its genetic variant Clone 22. In S115 cells, androgen maintains a transformed morphology, rate of proliferation, and serum and anchorage independence. Similar effects were induced by treatment of the cells with FGF-2 or a heparin-binding growth factor (HBGF) fraction prepared from the medium conditioned by the cells. The effects of androgen and FGF-2 could be partly reversed with a specific anti-FGF-2 immunoglobulin G or by suramin, which inhibits binding of FGFs to their high affinity receptors. Testosterone and FGF-2 increased the expression of FGFR-1 messenger RNA (mRNA) and, to a lesser extent, FGFR-3 mRNA, but down-regulated FGFR-2 mRNA in S115 cells. No FGFR-4 mRNA was detected. FGF-2 also down-regulated the expression of syndecan-1, a heparan sulfate proteoglycan that binds FGF with low affinity. The binding of radiolabeled FGF-2 to FGFRs was lower in the cells cultured with testosterone or in the presence of the HBGFs from androgen-treated cells, presumably because of the autocrine production of FGF-like factors. In Clone 22 cells, FGFRs and syndecan-1 responded to androgen as in S115 cells, but they were less sensitive to FGF-2. Androgen or FGF-2 could not induce morphological transformation, although both stimulated proliferation. Androgen-increased proliferation was not, however, decreased by anti-FGF-2 immunoglobulin G in Clone 22 cells. These data suggest that of the HBGFs produced, FGF-2 is required in androgen induction of morphological change, whereas the effect on proliferation involves other factors as well (perhaps mostly FGF-8). The results show that androgen differentially regulates the expression of the high and low affinity FGF receptors, which could mediate androgen induction of the transformed phenotype in S115 cells by an autocrine mechanism. The differential responses of the Clone 22 variant cells to androgen and FGF-2 suggest that the pathways of steroid induction of different parameters of the transformed phenotype, such as transition to fibroblastic morphology and stimulation of proliferation, are divergent.
Fibroblast growth factors (FGFs) transduce a variety of biological signals via four distinct tyrosine kinase receptors. We have characterized the phosphorylation of FGF receptor 4 (FGFR-4) and its association with a putative substrate, p85, using transfected L6 myoblast and NIH3T3 fibroblast cell lines. FGFR-4 was phosphorylated in vivo and in vitro mainly on serine and threonine residues in several peptides and to a lower degree on tyrosine residues. When analyzed further by in-gel kinase assay, immunoprecipitates of ligand-activated FGFR-4 contained a serine autophosphorylated polypeptide doublet of 85 kDa. Analysis of the major autophosphorylation site Y754F mutant of FGFR-4 showed that binding of p85 and its serine phosphorylation were independent of receptor autophosphorylation at this site. Okadaic acid treatment increased the basal autophosphorylation activity of p85 but decreased FGFR-4 tyrosine phosphorylation. In contrast, orthovanadate treatment increased the tyrosine phosphorylation of FGFR-4. These data show that a serine kinase is associated with activated FGFR-4 and suggest a role for serine phosphorylation in FGFR-4 function.
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