Immunosuppressive and anti-angiogenic sphingosine 1-phosphate receptor-1 agonists induce ubipuitinylation and proteasomal degradation of the receptor

Sphingosine 1-phosphate (SIP), a multifunctional lipid mediator, regulates lymphocyte trafficking, vascular permeability, and angiogenesis by activation of the SIP1 receptor. This receptor is activated by FTY720-P, a phosphorylated derivative of the immunosuppressant and vasoactive compound FTY720. However, in contrast to the natural ligand SIP, FTY720-P appears to act as a functional antagonist, even though the mechanisms involved are poorly understood. In this study, we investigated the fate of endogenously expressed SIP1 receptor in agonist-activated human umbilical vein endothelial cells and human embryonic kidney 293 cells expressing green fluorescent protein-tagged SIP1. We show that FTY720-P is more potent than SIP at inducing receptor degradation. Pretreatment with an antagonist of S1P(1), VPC 44116, prevented receptor internalization and degradation. FTY720-P did not induce degradation of internalization-deficient SIP1 receptor mutants. Further, small interfering RNA-mediated down-regulation of G protein-coupled receptor kinase-2 and beta-arrestins abolished FTY720-P-induced SIP1 receptor degradation. These data suggest that agonist-induced phosphorylation of SIP1 and subsequent endocytosis are required for FTY720-P-induced degradation of the receptor. S1P(1) degradation is blocked by MG132, a proteasomal inhibitor. Indeed, FTY720-P strongly induced polyubiquitinylation of SIP1 receptor, whereas SIP at concentrations that induced complete internalization was not as efficient, suggesting that receptor internalization is required but not sufficient for ubiquitinylation and degradation. We propose that the ability of FTY720-P to target the S1P(1) receptor to the ubiquitinylation and proteasomal degradation pathway may at least in part underlie its immunosuppressive and anti-angiogenic properties.