Abstract
We have investigated the interaction of basic fibroblast growth factor (bFGF) with its receptors and heparan sulfate proteoglycans (HSPG). It has been suggested that in the absence of HSPG, cells are not able to bind bFGF or respond to treatment with bFGF. In our studies, Balb/c3T3 fibroblasts were treated with 50 mM sodium chlorate to completely inhibit (99%) sulfation of proteoglycans. We found that bFGF was able to bind, be internalized, and stimulate DNA synthesis in the absence of HSPG in a dose-dependent manner. bFGF bound to its receptors on chlorate-treated cells with a lower apparent affinity and no change in receptor number. To determine if this decreased affinity bFGF-receptor interaction is functional, we quantitatively analyzed bFGF internalization and stimulation of DNA synthesis in control and chlorate-treated cells. Endocytotic rate constants (ke) for chlorate-treated and control cells were ke = 0. 078 +/- 0.022 min-1 and ke = 0.043 +/- 0.012 min-1, respectively, suggesting that the process of bFGF internalization is not dramatically altered by HSPG. bFGF stimulated DNA synthesis to the same maximal level under both conditions, but chlorate-treated cells were significantly less responsive at low bFGF doses (approximately 10-fold increase in ED50). The differences observed for control and chlorate-treated cells in the dose-response curves for stimulation of DNA synthesis and receptor binding correlated directly, suggesting that receptors are equally capable of eliciting a mitogenic signal under both conditions. It is unlikely that these results are due to residual HSPG since heparinase (I and III) digestion of chlorate-treated cells had little effect. Although the presence of HSPG on the cell surface increases the affinity of bFGF for its receptors, our observations suggest that HSPG are not "absolutely" required for binding, internalization, or stimulation of mitogenic activity.
Highlights
Isolated and best studied members of the large family of heparin-binding growth factors [1,2,3]
Balb/c3T3 fibroblasts were treated with sodium chlorate to completely inhibit sulfation of proteoglycans and binding of basic fibroblast growth factor (bFGF) to heparan sulfate proteoglycans (HSPG). bFGF bound to its receptors on chlorate-treated cells with a lower apparent affinity
There was a considerable amount of specific binding in the receptor fraction from chloratetreated cells (Fig. 1B). To ensure that this binding was not dependent on residual HSPG, or contaminating heparin/heparan sulfate within the binding reaction, chlorate-treated cells and/or the binding buffer were exhaustively digested with heparinase I and/or heparinase III prior to conducting bFGF binding
Summary
Materials—bFGF (human recombinant) (18 kDa) was from SciosNova (Mountain View, CA). [3H]Thymidine, [35S]sulfate, and 125IBolton-Hunter reagent were obtained from DuPont NEN. 125I-bFGF was prepared using a modification of the Bolton-Hunter method [13]. 125I-bFGF was added directly to the binding buffer and the cells incubated at 37 °C for the indicated times. Where [F-R]I and [F-R]S are the concentrations of the bound bFGFreceptor complex in the interior of the cell and on the cell surface, respectively This equation is only valid when there is no degradation of bFGF during the time course of measurement. Western Blot Analyses of FGF Receptor and bFGF—For FGF receptor analysis, control and chlorate-treated cells were washed twice in PBS, and 1 ml of HTG (20 mM HEPES, 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride) was added, and cells were scraped. The chlorate-treated cells contained 99% less sulfated proteoglycan than control cells after the 3-day treatment period
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