Immunosuppressive and Anti-angiogenic Sphingosine 1-Phosphate Receptor-1 Agonists Induce Ubiquitinylation and Proteasomal Degradation of the Receptor

Abstract

Sphingosine 1-phosphate (S1P), a multifunctional lipid mediator, regulates lymphocyte trafficking, vascular permeability, and angiogenesis by activation of the S1P 1 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 S1P, 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 S1P 1 receptor in agonist-activated human umbilical vein endothelial cells and human embryonic kidney 293 cells expressing green fluorescent protein-tagged S1P 1 . We show that FTY720-P is more potent than S1P 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 S1P 1 receptor mutants. Further, small interfering RNA-mediated down-regulation of G protein-coupled receptor kinase-2 and ␤-arrestins abolished FTY720-P-induced S1P 1 receptor degradation. These data suggest that agonistinduced phosphorylation of S1P 1 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 S1P 1 receptor, whereas S1P 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.Sphingosine 1-phosphate (S1P) 2 is recognized as a multifunctional bioactive lipid mediator involved in immune cell trafficking, regulation of vascular permeability, and angiogenesis (1, 2). It acts via a family of G protein-coupled receptors referred to as S1P n receptors (3). The prototypical receptor, S1P 1 was originally isolated as an inducible gene from vascular endothelial cells (4). Knock out of S1P 1 resulted in embryonic lethality due to a vascular maturation defect (5). We recently showed that S1P 1 function in endothelial cells is needed for proper endothelial-pericyte interaction, a critical event in vascular maturation (6). In addition, we demonstrated previously that S1P 1 is needed for the assembly of vascular endothelialcadherin-based adherens junctions on vascular endothelial cells (7). This event is needed for regulation of paracellular permeability, a model system vascular leak syndrome (8). In the immune system, selective deletion of S1P 1 in T-cells led to inhibition of lymphocyte egress from lymph nodes and the thymus (9, 10). However, function of S1P 1 in efferent lymphatics may also be important for the regulation of lymphocyte egress, as activation of this receptor may lead to "gate clos...

Size-selective opening of the blood–brain barrier by targeting endothelial sphingosine 1–phosphate receptor 1

“…For this purpose, we used the FDA-approved drug FTY720 (fingolimod), which targets all S1P receptors except S1P 2 (7). FTY720 is a prodrug that is phosphorylated in vivo to form FTY720-P, which works as functional antagonist for the S1P 1 by inducing its internalization and degradation (11,36,37). Indeed, three consecutive days of treatment with FTY720 successfully reduced the expression of S1P 1 in microvascular fragments (Fig.…”

Efficacy and immunomodulatory actions of ONO-4641, a novel selective agonist for sphingosine 1-phosphate receptors 1 and 5, in preclinical models of multiple sclerosis

“…It was reported that S1P receptor agonists such as fingolimod and SEW2871 exert their effects by down-regulating S1P1 on the cell membrane [21][22][23]. We examined whether ONO-4641 also induces S1P1 down-regulation on the cell membrane.…”

“…Fingolimod, a first-in-class S1P receptor agonist, has been approved recently for the treatment of relapsing MS. Fingolimod is a prodrug and phosphorylated reversibly to fingolimod-phosphate, the active moiety [11,12]. Fingolimod-phosphate activates S1P1 on lymphocytes via high-affinity receptor binding and subsequently induces S1P1 down-regulation on the cell membrane [21,22]. It is believed that down-regulation of S1P1 in lymphocytes by fingolimod-phosphate renders them unresponsive to the S1P gradient and prevents the egress from secondary lymphoid tissues [3,27,28].…”

S1P Is Associated with Protection in Human and Experimental Cerebral Malaria

“…FTY720 administration has proved beneficial in experimental disease models (19)(20)(21)(22)(23), and recently in human clinical trials of relapsing multiple sclerosis (24). Both S1P and FTY720 result in S1P1 internalization and recycling (25,26), FTY720 being a more efficient inducer of receptor degradation (27). This functional antagonism of S1P receptors leads to lymphocyte retention in lymphoid tissues, thus reducing the number of activated lymphocytes available to enter sites of inflammation, including the central nervous system (CNS) (28).…”