SPHINGOSINE 1-PHOSPHATE REGULATES MELANOMA CELL MOTILITY THROUGH A RECEPTOR-COUPLED EXTRACELLULAR ACTION AND IN A PERTUSSIS TOXIN-INSENSITIVE MANNER

Our previous work showed that sphingosine l-phosphate (Sph-1-P) inhibi ts the cell motility of mouse melanoma B16/F10, and other types of cel ls at 10-100 nM concentrations. In the present paper, we have identifi ed and characterized specific cell surface binding sites for Sph-l-P i n F10 cells. Sph-l-P immobilized on controlled pore glass beads inhibi ted the motility of F10 cells, suggesting that Sph-l-P acts on the cel ls from the outside. Binding assays with [H-3]Sph-l-P revealed the pre sence of specific cell surface binding sites for Sph-l-P in F10 cells. Scatchard analysis demonstrated a single class of binding sites for S ph-l-P. The binding of [H-3]Sph-l-P to F10 cells was inhibited by the addition of excess unlabeled Sph-l-P but not other natural sphingolipi ds. The specific binding was also sensitive to treatment with a protea se. Using Sph-1-P-immobilized affinity chromatography, we, for the fir st time, identified 41-kDa and 79-kDa Sph-l-P binding proteins on the melanoma cell surface, although the 41-kDa protein was less specific t o Sph-l-P. We demonstrated that pertussis toxin (PTX) treatment did no t abolish the motility inhibition by Sph-l-P, suggesting that no PTX-s ensitive G-protein is involved in the signaling. Furthermore, Sph-l-P was found to be. specifically released from mouse BALB/3T3 clone A31 c ells and F10 cells. Collectively, these results strongly suggest that Sph-l-P regulates melanoma cell motility through an extracellular acti on by specific binding to cell surface receptor protein(s), which is i ndependent of PTX-sensitive G-protein.