Figure 6.

Effect of exogenous gangliosides on the binding of ¹²⁵I-FGF-2 to FGFRs and HSPGs and its internalization in endothelial cells. (A and B) Subconfluent cultures of GM 7373 cells were incubated for 2 h at 4°C with 10 ng/ml ¹²⁵I-FGF-2 in the presence of the indicated concentrations of asialo-GM₁(○), GM₁ (•), GD_1b (▵), GT_1b (□), and sulfatide (▪). At the end of incubation, ¹²⁵I-FGF-2 bound to FGFRs (A) and to HSPGs (B) was evaluated as described in MATERIALS AND METHODS and expressed as percentage of the radioactivity measured in cell cultures incubated in the absence of ganglioside. Each point is the mean of three determinations in duplicate. SEM never exceeded 9% of the mean value. (C) Subconfluent cultures of GM 7373 cells were incubated for 2 h at 4° C with 10 ng/ml ¹²⁵I-FGF-2 with no addition (open bars) or in the presence of asialo-GM₁ (gray bars) or of GT_1b (black bars), both at 30 μM. At the end of incubation, cell cultures were shifted at 37 °C and incubated at this temperature for the indicated periods of time. At the end of incubation, cell surface-associated ¹²⁵I-FGF-2 was removed, and the amount of internalized ¹²⁵I-FGF-2 was measured. The data are representative of two independent experiments in duplicate. (D) GM 7373 cells were incubated for 72 h at 37°C in the absence (•, ▴) or in the presence (○, ▵) of GM₁ (100 μM). At the end of the incubation, cell cultures were washed extensively and incubated for 2 h at 4° C with increasing concentrations of ¹²⁵I-FGF-2 in the absence of free ganglioside. At the end of incubation, ¹²⁵I-FGF-2 bound to FGFRs (circles) and to HSPGs (triangles) was evaluated as described in MATERIALS AND METHODS. The data are representative of four independent experiments in duplicate.