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J. Biol. Chem., Vol. 282, Issue 12, 9082-9089, March 23, 2007

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 282/12/9082    most recent M610318200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Oo, M. L. Articles by Hla, T. Search for Related Content PubMed PubMed Citation Articles by Oo, M. L. Articles by Hla, T. Social Bookmarking                      What's this?

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

Myat Lin Oo, Shobha Thangada, Ming-Tao Wu, Catherine H. Liu, Timothy L. Macdonald, Kevin R. Lynch^¶, Chen-Yong Lin^||, and Timothy Hla¹

From the Center for Vascular Biology, University of Connecticut Health Center, Farmington, Connecticut 06030-3501, Departments of Chemistry and ^¶Pharmacology, University of Virginia, Charlottesville, Virginia 22908, and ^||Lombardi Cancer Center, Georgetown University School of Medicine, Washington, D. C. 20007

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 -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.

Received for publication, November 6, 2006 , and in revised form, January 11, 2007.

^* This work was supported by National Institutes of Health Grants PO1-HL70694 and R37-HL67330 (to T. H.), RO1-GM67958 (to K. R. L.), and R01-CA096851 and R01-CA104944 (to C. Y. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3.

¹ To whom correspondence should be addressed. Tel.: 860-679-4128; E-mail: hla{at}nso2.uchc.edu.

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