Gangliosides Inhibit Platelet‐Derived Growth Factor‐Stimulated Growth, Receptor Phosphorylation, and Dimerization in Neuroblastoma SH‐SY5Y Cells

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 K D = 0.14 ± 0.06 n M and B max = 7.3 ± 2.3 p M . Functionality of these receptors was demonstrated by an increased [ 3 H]‐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 [ 3 H]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 β‐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.