Sphingosine 1-phosphate regulates melanoma cell motility through a receptor-coupled extracellular action and in a pertussis toxin-insensitive manner

Biochemistry. 1997 Sep 2;36(35):10751-9. doi: 10.1021/bi970926s.

Abstract

Our previous work showed that sphingosine 1-phosphate (Sph-1-P) inhibits the cell motility of mouse melanoma B16/F10, and other types of cells at 10-100 nM concentrations. In the present paper, we have identified and characterized specific cell surface binding sites for Sph-1-P in F10 cells. Sph-1-P immobilized on controlled pore glass beads inhibited the motility of F10 cells, suggesting that Sph-1-P acts on the cells from the outside. Binding assays with [3H]Sph-1-P revealed the presence of specific cell surface binding sites for Sph-1-P in F10 cells. Scatchard analysis demonstrated a single class of binding sites for Sph-1-P. The binding of [3H]Sph-1-P to F10 cells was inhibited by the addition of excess unlabeled Sph-1-P but not other natural sphingolipids. The specific binding was also sensitive to treatment with a protease. Using Sph-1-P-immobilized affinity chromatography, we, for the first time, identified 41-kDa and 79-kDa Sph-1-P binding proteins on the melanoma cell surface, although the 41-kDa protein was less specific to Sph-1-P. We demonstrated that pertussis toxin (PTX) treatment did not abolish the motility inhibition by Sph-1-P, suggesting that no PTX-sensitive G-protein is involved in the signaling. Furthermore, Sph-1-P was found to be specifically released from mouse BALB/3T3 clone A31 cells and F10 cells. Collectively, these results strongly suggest that Sph-1-P regulates melanoma cell motility through an extracellular action by specific binding to cell surface receptor protein(s), which is independent of PTX-sensitive G-protein.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Binding Sites / drug effects
  • Carrier Proteins / chemistry
  • Cell Movement / drug effects*
  • Extracellular Space / metabolism
  • Extracellular Space / physiology*
  • Lysophospholipids*
  • Melanoma / chemistry
  • Melanoma / metabolism*
  • Melanoma / ultrastructure
  • Mice
  • Microspheres
  • Pertussis Toxin*
  • Receptors, Cell Surface / drug effects
  • Receptors, Cell Surface / physiology*
  • Sphingosine / analogs & derivatives*
  • Sphingosine / chemistry
  • Sphingosine / metabolism
  • Sphingosine / physiology
  • Suramin / pharmacology
  • Tumor Cells, Cultured
  • Virulence Factors, Bordetella / pharmacology*

Substances

  • Carrier Proteins
  • Lysophospholipids
  • Receptors, Cell Surface
  • Virulence Factors, Bordetella
  • sphingosine 1-phosphate
  • Suramin
  • Pertussis Toxin
  • Sphingosine