Gangliosides inhibit platelet-derived growth factor-stimulated growth, receptor phosphorylation, and dimerization in neuroblastoma SH-SY5Y cells

Abstract

SH-SY5Y is a thrice cloned cell line originally derived from the human neuroblastoma cell line SK-N-SH. It grows well in serum-containing medium and undergoes neuritogenesis in response to several trophic factors. Because it has been reported that this clonal line does not have receptors for platelet-derived growth factor (PDGF), it has been unclear what the major mitogenic factor in serum is for these cells. In competitive binding studies using radiolabeled PDGF-BB, we found that SH-SY5Y cells specifically bind PDGF with a KD = 0.14 +/- 0.06 nM and Bmax = 7.3 +/- 2.3 pM. Functionality of these receptors was demonstrated by an increased [3H]-thymidine incorporation in response to PDGF (stimulation index = 2.5). At concentrations of PDGF-BB between 5 and 100 ng/ml, maximum stimulation occurred with 20 ng/ml. Maximum DNA synthesis occurred after 12-24-h exposure to PDGF. Gangliosides GM3 and GT1b greatly inhibited [3H]thymidine incorporation, which was also inhibited to a lesser extent by GM1. Phosphorylation on tyrosine of a 170-kDa protein in response to PDGF stimulation of intact cells was demonstrated by western blot analysis probing with anti-phosphotyrosine antibody. Immunoprecipitation with anti-PDGF beta-receptor antibody and visualization on a western blot with an anti-phosphotyrosine antibody also revealed a 170-kDa protein. Maximum phosphorylation of the 170-kDa protein occurred after 5-min exposure to 20 ng/ml PDGF. This phosphorylation was inhibited by gangliosides GM1, GM2, GD1a, and GT1b but not by GM3. Receptor dimerization was also inhibited by GM1. These results show that SH-SY5Y cells have specific receptors for PDGF-BB that are functional, and can be modulated by gangliosides.