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 S1P1 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 S1P1 receptor in agonist-activated human umbilical vein endothelial cells and human embryonic kidney 293 cells expressing green fluorescent protein-tagged S1P1. We show that FTY720-P is more potent than S1P at inducing receptor degradation. Pretreatment with an antagonist of S1P1, VPC 44116, prevented receptor internalization and degradation. FTY720-P did not induce degradation of internalization-deficient S1P1 receptor mutants. Further, small interfering RNA-mediated down-regulation of G protein-coupled receptor kinase-2 and β-arrestins abolished FTY720-P-induced S1P1 receptor degradation. These data suggest that agonist-induced phosphorylation of S1P1 and subsequent endocytosis are required for FTY720-P-induced degradation of the receptor. S1P1 degradation is blocked by MG132, a proteasomal inhibitor. Indeed, FTY720-P strongly induced polyubiquitinylation of S1P1 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 S1P1 receptor to the ubiquitinylation and proteasomal degradation pathway may at least in part underlie its immunosuppressive and anti-angiogenic properties. Sphingosine 1-phosphate (S1P), a multifunctional lipid mediator, regulates lymphocyte trafficking, vascular permeability, and angiogenesis by activation of the S1P1 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 S1P1 receptor in agonist-activated human umbilical vein endothelial cells and human embryonic kidney 293 cells expressing green fluorescent protein-tagged S1P1. We show that FTY720-P is more potent than S1P at inducing receptor degradation. Pretreatment with an antagonist of S1P1, VPC 44116, prevented receptor internalization and degradation. FTY720-P did not induce degradation of internalization-deficient S1P1 receptor mutants. Further, small interfering RNA-mediated down-regulation of G protein-coupled receptor kinase-2 and β-arrestins abolished FTY720-P-induced S1P1 receptor degradation. These data suggest that agonist-induced phosphorylation of S1P1 and subsequent endocytosis are required for FTY720-P-induced degradation of the receptor. S1P1 degradation is blocked by MG132, a proteasomal inhibitor. Indeed, FTY720-P strongly induced polyubiquitinylation of S1P1 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 S1P1 receptor to the ubiquitinylation and proteasomal degradation pathway may at least in part underlie its immunosuppressive and anti-angiogenic properties. Sphingosine 1-phosphate (S1P) 2The abbreviations used are: S1P, sphingosine 1-phosphate; siRNA, small interfering RNA; HUVEC, human umbilical vein endothelial cell; HEK, human embryonic kidney; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; GRK2, G protein-coupled receptor kinase 2; SRR, serine-rich region; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. is recognized as a multifunctional bioactive lipid mediator involved in immune cell trafficking, regulation of vascular permeability, and angiogenesis (1Chalfant C.E. Spiegel S. J. Cell Sci. 2005; 118: 4605-4612Crossref PubMed Scopus (363) Google Scholar, 2Chun J. Rosen H. Curr. Pharm. Des. 2006; 12: 161-171Crossref PubMed Scopus (79) Google Scholar). It acts via a family of G protein-coupled receptors referred to as S1Pn receptors (3Rosen H. Goetzl E.J. Nat. Rev. Immunol. 2005; 5: 560-570Crossref PubMed Scopus (623) Google Scholar). The prototypical receptor, S1P1 was originally isolated as an inducible gene from vascular endothelial cells (4Hla T. Maciag T. J. Biol. Chem. 1990; 265: 9308-9313Abstract Full Text PDF PubMed Google Scholar). Knock out of S1P1 resulted in embryonic lethality due to a vascular maturation defect (5Liu Y. Wada R. Yamashita T. Mi Y. Deng C.X. Hobson J.P. Rosenfeldt H.M. Nava V.E. Chae S.S. Lee M.J. Liu C.H. Hla T. Spiegel S. Proia R.L. J. Clin. Investig. 2000; 106: 951-961Crossref PubMed Scopus (996) Google Scholar). We recently showed that S1P1 function in endothelial cells is needed for proper endothelial-pericyte interaction, a critical event in vascular maturation (6Paik J.H. Skoura A. Chae S.S. Cowan A.E. Han D.K. Proia R.L. Hla T. Genes Dev. 2004; 18: 2392-2403Crossref PubMed Scopus (221) Google Scholar). In addition, we demonstrated previously that S1P1 is needed for the assembly of vascular endothelial-cadherin-based adherens junctions on vascular endothelial cells (7Lee M.J. Thangada S. Claffey K.P. Ancellin N. Liu C.H. Kluk M. Volpi M. Sha'afi R.I. Hla T. Cell. 1999; 99: 301-312Abstract Full Text Full Text PDF PubMed Scopus (877) Google Scholar). This event is needed for regulation of paracellular permeability, a model system vascular leak syndrome (8Garcia J.G. Liu F. Verin A.D. Birukova A. Dechert M.A. Gerthoffer W.T. Bamberg J.R. English D. J. Clin. Investig. 2001; 108: 689-701Crossref PubMed Scopus (756) Google Scholar). In the immune system, selective deletion of S1P1 in T-cells led to inhibition of lymphocyte egress from lymph nodes and the thymus (9Allende M.L. Dreier J.L. Mandala S. Proia R.L. J. Biol. Chem. 2004; 279: 15396-15401Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 10Matloubian M. Lo C.G. Cinamon G. Lesneski M.J. Xu Y. Brinkmann V. Allende M.L. Proia R.L. Cyster J.G. Nature. 2004; 427: 355-360Crossref PubMed Scopus (2091) Google Scholar). However, function of S1P1 in efferent lymphatics may also be important for the regulation of lymphocyte egress, as activation of this receptor may lead to “gate closure” at mesenteric lymphatics leading to “logjamming” of lymphocytes (11Wei S.H. Rosen H. Matheu M.P. Sanna M.G. Wang S.K. Jo E. Wong C.H. Parker I. Cahalan M.D. Nat. Immunol. 2005; 6: 1228-1235Crossref PubMed Scopus (256) Google Scholar, 12Singer I. Tian M. Wickham L.A. Lin J. Matheravidathu S.S. Forrest M.J. Mandala S. Quackenbush E.J. J. Immunol. 2005; 175: 7151-7161Crossref PubMed Scopus (89) Google Scholar). These studies point to the crucial role played by the S1P1 receptor in the regulation of vascular and immune physiology. The immune modulator FTY720, which is undergoing phase III clinical trials for multiple sclerosis and allograft rejection, is known to interact with and modulate the function of S1P1 receptor (13Mandala S. Hajdu R. Bergstrom J. Quackenbush E. Xie J. Milligan J. Thornton R. Shei G.J. Card D. Keohane C. Rosenbach M. Hale J. Lynch C.L. Rupprecht K. Parsons W. Rosen H. Science. 2002; 296: 346-349Crossref PubMed Scopus (1443) Google Scholar, 14Brinkmann V. Davis M.D. Heise C.E. Albert R. Cottens S. Hof R. Bruns C. Prieschl E. Baumruker T. Hiestand P. Foster C.A. Zollinger M. Lynch K.R. J. Biol. Chem. 2002; 277: 21453-21457Abstract Full Text Full Text PDF PubMed Scopus (1320) Google Scholar, 15Kappos L. Antel J. Comi G. Montalban X. O'Connor P. Polman C.H. Haas T. Korn A.A. Karlsson G. Radue E.W. N. Engl. J. Med. 2006; 355: 1124-1140Crossref PubMed Scopus (949) Google Scholar). The prodrug form of FTY720 is phosphorylated by sphingosine kinase-2 into FTY720-P (16Sanchez T. Estrada-Hernandez T. Paik J.H. Wu M.T. Venkataraman K. Brinkmann V. Claffey K. Hla T. J. Biol. Chem. 2003; 278: 47281-47290Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 17Kharel Y. Lee S. Snyder A.H. Sheasley-O'Neill S.L. Morris M.A. Setiady Y. Zhu R. Zigler M.A. Burcin T.L. Ley K. Tung K.S. Engelhard V.H. Macdonald T.L. Pearson-White S. Lynch K.R. J. Biol. Chem. 2005; 280: 36865-36872Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), a potent nanomolar agonist on S1P1, S1P3, S1P4, and S1P5 (13Mandala S. Hajdu R. Bergstrom J. Quackenbush E. Xie J. Milligan J. Thornton R. Shei G.J. Card D. Keohane C. Rosenbach M. Hale J. Lynch C.L. Rupprecht K. Parsons W. Rosen H. Science. 2002; 296: 346-349Crossref PubMed Scopus (1443) Google Scholar, 14Brinkmann V. Davis M.D. Heise C.E. Albert R. Cottens S. Hof R. Bruns C. Prieschl E. Baumruker T. Hiestand P. Foster C.A. Zollinger M. Lynch K.R. J. Biol. Chem. 2002; 277: 21453-21457Abstract Full Text Full Text PDF PubMed Scopus (1320) Google Scholar). Acute agonism of S1P1 with FTY720-P leads to similar cellular effects as S1P, including intracellular calcium rise, adenylate cyclase inhibition, mitogen-activated protein kinase activation, vascular endothelial-cadherin assembly, and cell migration (18Brinkmann V. Cyster J.G. Hla T. Am. J. Transplant. 2004; 4: 1019-1025Crossref PubMed Scopus (422) Google Scholar). Both S1P and FTY720-P induce receptor internalization into the endosomal pathway (19Liu C.H. Thangada S. Lee M.J. Van Brocklyn J.R. Spiegel S. Hla T. Mol. Biol. Cell. 1999; 10: 1179-1190Crossref PubMed Scopus (168) Google Scholar, 20Graler M.H. Goetzl E.J. FASEB J. 2004; 18: 551-553Crossref PubMed Scopus (462) Google Scholar). However, in FTY720-treated cells, the S1P1 receptor does not recycle to the plasma membrane (20Graler M.H. Goetzl E.J. FASEB J. 2004; 18: 551-553Crossref PubMed Scopus (462) Google Scholar, 21Jo E. Sanna M.G. Gonzalez-Cabrera P.J. Thangada S. Tigyi G. Osborne D.A. Hla T. Parrill A.L. Rosen H. Chem. Biol. 2005; 12: 703-715Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). The significance of this finding is not clear, even though it was hypothesized that the ability of FTY720 to down-regulate the receptor from the plasma membrane may underlie its immunosuppressive action (10Matloubian M. Lo C.G. Cinamon G. Lesneski M.J. Xu Y. Brinkmann V. Allende M.L. Proia R.L. Cyster J.G. Nature. 2004; 427: 355-360Crossref PubMed Scopus (2091) Google Scholar, 18Brinkmann V. Cyster J.G. Hla T. Am. J. Transplant. 2004; 4: 1019-1025Crossref PubMed Scopus (422) Google Scholar). In vivo, FTY720 exerts profound effects on immune and vascular systems. In the immune system, it induces rapid yet sustained lymphopenia, dendritic cell migration, B-cell trafficking, and hematopoietic stem cell homing (18Brinkmann V. Cyster J.G. Hla T. Am. J. Transplant. 2004; 4: 1019-1025Crossref PubMed Scopus (422) Google Scholar, 22Cyster J.G. Annu. Rev. Immunol. 2005; 23: 127-159Crossref PubMed Scopus (722) Google Scholar). Vascular effects of FTY720 include the inhibition of vascular permeability and angiogenesis (16Sanchez T. Estrada-Hernandez T. Paik J.H. Wu M.T. Venkataraman K. Brinkmann V. Claffey K. Hla T. J. Biol. Chem. 2003; 278: 47281-47290Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 23LaMontagne K. Littlewood-Evans A. Schnell C. O'Reilly T. Wyder L. Sanchez T. Probst B. Butler J. Wood A. Liau G. Billy E. Theuer A. Hla T. Wood J. Cancer Res. 2006; 66: 221-231Crossref PubMed Scopus (254) Google Scholar). The apparent antagonistic nature of the in vivo effects of FTY720 were surprising in light of the fact that it is an agonist for the S1P1 receptor, which is a pro-angiogenic and pro-migratory receptor (24Saba J.D. Hla T. Circ. Res. 2004; 94: 724-734Crossref PubMed Scopus (229) Google Scholar, 25Chae S.S. Paik J.H. Furneaux H. Hla T. J. Clin. Investig. 2004; 114: 1082-1089Crossref PubMed Scopus (207) Google Scholar, 26Paik J.H. Chae S. Lee M.J. Thangada S. Hla T. J. Biol. Chem. 2001; 276: 11830-11837Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). A variety of explanations were proposed, including functional antagonism and vascular gate closure (3Rosen H. Goetzl E.J. Nat. Rev. Immunol. 2005; 5: 560-570Crossref PubMed Scopus (623) Google Scholar, 12Singer I. Tian M. Wickham L.A. Lin J. Matheravidathu S.S. Forrest M.J. Mandala S. Quackenbush E.J. J. Immunol. 2005; 175: 7151-7161Crossref PubMed Scopus (89) Google Scholar). However, of the fate of S1P1 activated by not In this we show that FTY720-P is a potent of the ubiquitinylation of the S1P1 receptor. This in rapid and degradation of S1P1. We suggest that the ability of to target S1P1 degradation may at least in immunosuppressive and anti-angiogenic properties. and and sphingosine 1-phosphate were from FTY720, FTY720-P, and were by was from VPC and VPC were as M.D. Macdonald T.L. Lynch K.R. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar, Snyder A.H. Davis M.D. M. M.D. Lynch K.R. Macdonald T.L. Med. Chem. Scopus Google Scholar). and were from was from and was from S1P1 human S1P1 M.J. Thangada S. Paik J.H. Ancellin N. Chae S.S. Wu M. M. Hla T. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google was used to a Lin M.L. Liu C.H. Lo Wang W.T. W. Lin Lin Lin S. T. Wang E. Wang J.L. Wu R. Wu C. S. J. Cell Sci. PubMed Google Scholar). was from cell protein Cell and cells expressing and its (19Liu C.H. Thangada S. Lee M.J. Van Brocklyn J.R. Spiegel S. Hla T. Mol. Biol. Cell. 1999; 10: 1179-1190Crossref PubMed Scopus (168) Google were in with and and at in a of in umbilical vein endothelial cells were in with and endothelial as previously (4Hla T. Maciag T. J. Biol. Chem. 1990; 265: 9308-9313Abstract Full Text PDF PubMed Google Scholar). cells were with the the calcium C. H. Mol. Cell. Biol. PubMed Scopus Google to the and in the were by previously (19Liu C.H. Thangada S. Lee M.J. Van Brocklyn J.R. Spiegel S. Hla T. Mol. Biol. Cell. 1999; 10: 1179-1190Crossref PubMed Scopus (168) Google Scholar). In the and were at and and with the for from the target W. F. Science. 2004; PubMed Scopus Google Scholar), and D. M. E. S. Wang S. A.E. Cowan C.L. J. Biol. 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However, in HUVEC, S1P1 appears in a with the in which a membrane is We the of S1P1 in with in the ligand S1P receptor whereas sphingosine did not an FTY720-P a in S1P1 The FTY720, was A of FTY720-P, a agonist for did not induce of receptor at concentrations concentrations of receptor which is to the by S1P not The showed that FTY720-P receptor in In cells expressing receptor, sustained of receptor was These data suggest that and concentrations of the natural ligand S1P S1P1 receptor in and of FTY720-P down-regulation of S1P1 receptor. of the of FTY720-P and FTY720 on FTY720-P induces degradation of in cells were with FTY720-P for the a that was FTY720-P and are the potent at down-regulation of receptor is surprising similar and are of the S1P1 receptor as the natural S1P (13Mandala S. Hajdu R. Bergstrom J. Quackenbush E. Xie J. Milligan J. Thornton R. Shei G.J. Card D. Keohane C. Rosenbach M. Hale J. Lynch C.L. Rupprecht K. Parsons W. Rosen H. Science. 2002; 296: 346-349Crossref PubMed Scopus (1443) Google Scholar, 14Brinkmann V. Davis M.D. Heise C.E. Albert R. Cottens S. Hof R. Bruns C. Prieschl E. Baumruker T. Hiestand P. Foster C.A. Zollinger M. Lynch K.R. J. Biol. Chem. 2002; 277: 21453-21457Abstract Full Text Full Text PDF PubMed Scopus (1320) Google Scholar). It is that the of S1P is more to the action of plasma However, which does not a was not at inducing receptor suggesting that FTY720-P and may induce a of receptor as to down-regulate the S1P1 receptor. We the of VPC 44116, a potent S1P1 antagonist M.D. Macdonald T.L. Lynch K.R. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar, Snyder A.H. Davis M.D. M. M.D. Lynch K.R. Macdonald T.L. Med. Chem. Scopus Google Scholar), on receptor internalization and FTY720-P-induced down-regulation of the S1P1 receptor. in VPC did not an on the of in However, at VPC the ability of S1P and FTY720-P to induce receptor as of a potent were with the endogenously expressed S1P1 receptor in not In HUVEC, VPC FTY720-P-induced S1P1 receptor in a and A and VPC prevented FTY720-P-induced degradation in cells not These that receptor agonism is required but not sufficient for of with VPC the of receptor suggesting that of S1P1 in induces receptor down-regulation It is known that of S1P and are of this lipid mediator into the N. C. J. N. Chae S.S. S. Liau G. Hla T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus (256) Google Scholar). In addition, the sphingosine which act as an N. C. J. N. Chae S.S. S. Liau G. Hla T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus (256) Google Scholar, K. Thangada S. J. M.L. Y. Lee Wu M. F. Proia R.L. Hla T. J. 2006; PubMed Scopus Google Scholar). endogenously S1P may interact with the S1P1 receptor to in an an in the vascular endothelial cells may functional as S1P1 is important in the inhibition of vascular permeability (7Lee M.J. Thangada S. Claffey K.P. Ancellin N. Liu C.H. Kluk M. Volpi M. Sha'afi R.I. Hla T. Cell. 1999; 99: 301-312Abstract Full Text Full Text PDF PubMed Scopus (877) Google Scholar, J.G. Liu F. Verin A.D. Birukova A. Dechert M.A. Gerthoffer W.T. Bamberg J.R. English D. J. Clin. Investig. 2001; 108: 689-701Crossref PubMed Scopus (756) Google Scholar), regulation of and (6Paik J.H. Skoura A. Chae S.S. Cowan A.E. Han D.K. Proia R.L. Hla T. Genes Dev. 2004; 18: 2392-2403Crossref PubMed Scopus (221) Google Scholar, M.J. Thangada S. Claffey K.P. Ancellin N. Liu C.H. Kluk M. Volpi M. Sha'afi R.I. Hla T. Cell. 1999; 99: 301-312Abstract Full Text Full Text PDF PubMed Scopus (877) Google Scholar, 26Paik J.H. Chae S. Lee M.J. Thangada S. Hla T. J. Biol. 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Cell. 1999; 10: 1179-1190Crossref PubMed Scopus (168) Google Scholar). were used in the to FTY720-P is of inducing S1P1 degradation in the of In addition, K.R. E. C. A. J.L. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google that a serine-rich in the of S1P1 is phosphorylated by and protein data suggest that the is for receptor internalization (19Liu C.H. Thangada S. Lee M.J. Van Brocklyn J.R. Spiegel S. Hla T. Mol. Biol. Cell. 1999; 10: 1179-1190Crossref PubMed Scopus (168) Google Scholar). We a in which the is that the are to it to in the was expressed in cells and to the plasma S1P and FTY720-P induced kinase and cell migration in cells in an the and the receptors were not These suggest that ligand and of the are not by the cells with the were with FTY720-P, receptor degradation was not in and In was in a similar as the receptor. is agonist-induced receptor internalization and of receptor the phosphorylation of S1P1 receptor is not for FTY720-P-induced receptor endocytosis but also for down-regulation of receptor GRK2, an kinase involved in the agonist-induced phosphorylation of is needed for S1P1 we by a W. F. Science. 2004; PubMed Scopus Google Scholar). in degradation of S1P1 was not in we the role of which to phosphorylated on activated to induce receptor and in of for and the receptor from FTY720-P-induced degradation. suggest that receptor phosphorylation and receptor internalization is required for FTY720-P-induced S1P1 degradation. These that receptor endocytosis that the receptor and the that induce endocytosis of is for FTY720-P-induced receptor are to the plasma membrane and are to be in M.T. S.K. Circ. Res. 2006; 99: PubMed Scopus Google Scholar, S.K. Science. 2001; PubMed Scopus Google Scholar). Both of to the ubiquitinylation of A. J.L. Mol. Biol. 2004; Google Scholar, A. C. F. J.H. H. J.L. Dev. Cell. 2003; 5: Full Text Full Text PDF PubMed Scopus Google Scholar). we the of MG132, an of protein degradation in S. H. Y. N. M. S. Res. PubMed Scopus Google Scholar). with FTY720-P-induced receptor degradation in cells and These data that S1P1 is in FTY720-P of and However, we did not a defect in receptor internalization the cells were with not suggesting that proteasomal is not required for receptor of is a critical in endosomal as as to degradation A. J.L. Mol. Biol. 2004; Google Scholar, Sci. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). S1P and FTY720-P receptor we S1P1 ubiquitinylation of S1P1 was in cells expressing protein by with the by with the in FTY720-P induced polyubiquitinylation of S1P1 even though S1P was also of this to a The was also induced by FTY720-P and polyubiquitinylation of was blocked by the receptor antagonist, VPC In the was not of that FTY720-P is of inducing a of multiple in cells expressing and A in which was by an with the showed also that FTY720-P induced ubiquitinylation of with by with the also showed that FTY720-P induced These data suggest that polyubiquitinylation of S1P1 may be critical for into the proteasomal in to S1P1 were not due to the of the receptor in not These data strongly suggest that FTY720-P-induced polyubiquitinylation of S1P1 is for the of the receptor into the proteasomal in and lymphocytes that FTY720 the cellular pathway T. K. J. 2005; PubMed Scopus Google Scholar, C.A. S. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). In a and C.A. S. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google that is involved in the inhibition by FTY720. In lymphocytes with FTY720, involved in the system were FTY720-P in system may modulate the of the S1P1 receptor in a that it is for the It is of to that concentrations of S1P induced receptor degradation. plasma S1P are bioactive S1P in plasma is in the due to the of plasma as and T. K. A. H. M. I. M. F. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, N. K. J. H. M. A. M. F. J. 2000; PubMed Scopus Google Scholar, B. H. A. T. S. K. F. K. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). Indeed, in of S1P is receptor degradation may we that S1P1 receptor may the pathway However, S1P are for activation and immune cell activation, the receptor may be by of the S1P1 receptor to the In the a and degradation of S1P1 induced by We show that internalization is required for degradation of S1P1. However, receptor internalization is not sufficient for receptor as S1P that S1P1 is not ubiquitinylation induced by FTY720-P the receptor to the proteasomal FTY720-P is immunosuppressive and we that ubiquitinylation and degradation of S1P1 may at least in part be for the action of We for the of FTY720 and with