Selective activation of G alpha i mediated signalling of S1P3 by FTY720-phosphate

The immune modulator FTY720 is phosphorylated in vivo to FTY720 phosphate (FTY-P), which activates four sphingosine 1-phosphate (S1P) receptors including S1P3. Upon activation with S1P, S1P3 couples to Gi- and Gq-protein-dependent signalling pathways. Here we show that FTY-P selectively activates the S1P3-mediated and Gi-coupled inhibition of adenylyl cyclase. Contemporaneously, it antagonizes the S1P-induced activation of Gq via S1P3 in intracellular calcium flux measurements, GTP-binding experiments, and flow cytometric analyses of activation-induced receptor down-regulation. In contrast to S1P, pre-treatment with FTY-P did not desensitize S1P-induced calcium flux or chemotaxis via S1P3. The lack of receptor desensitization prevented S1P3-mediated migration to FTY-P. Human umbilical vein endothelial cells express S1P1 and S1P3, and respond to S1P and FTY-P by ERK1/2 phosphorylation and by intracellular calcium release in a pertussis toxin-sensitive manner. But whereas a mixture of S1P and FTY-P was not affecting ERK1/2 phosphorylation, the intracellular calcium flux was hampered with increasing amounts of FTY-P, which points to a cross-talk between S1P1 and S1P3. FTY-P is therefore one of the rare ligands which bind to a receptor that couples multiple G-proteins but selectively activates only one signalling pathway.