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 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.
Yamamura
S
S
The Biomembrane Institute, Seattle, Washington 98119, USA.
Yatomi
Y
Y
Ruan
F
F
Sweeney
E A
EA
Hakomori
S
S
Igarashi
Y
Y
D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002352:Carrier Proteins / Q000737:chemistry; D002465:Cell Movement / Q000187:drug effects*; D005110:Extracellular Space / Q000378:metabolism / Q000502:physiology*; D008246:Lysophospholipids; D008545:Melanoma / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D008863:Microspheres; D037342:Pertussis Toxin; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000502:physiology*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D013498:Suramin / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D002352:Carrier Proteins; D008246:Lysophospholipids; D011956:Receptors, Cell Surface; D010566:Virulence Factors, Bordetella; C060506:sphingosine 1-phosphate; D013498:Suramin; D037342:Pertussis Toxin; D013110:Sphingosine