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J. Biol. Chem., Vol. 282, Issue 10, 7254-7264, March 9, 2007

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Mapping Pathways Downstream of Sphingosine 1-Phosphate Subtype 1 by Differential Chemical Perturbation and Proteomics^*

Pedro J. Gonzalez-Cabrera, Timothy Hla, and Hugh Rosen¹

From the Department of Immunology, The Scripps Research Institute, La Jolla, California 92037 and the Center for Vascular Biology, University of Connecticut Health Center, Farmington, Connecticut 06030-3501

Sphingosine 1-phosphate subtype 1 (S1P₁) receptor agonists alter lymphocyte trafficking and endothelial barrier integrity in vivo. Among these is the potent, non-selective agonist, FTY720-P, whose mechanism of action has been suggested to correlate with S1P₁ down-regulation. Discovery of the in vivo active S1P₁-selective agonist, SEW2871, has broadened our understanding of minimal requirements for S1P₁ function while highlighting differences regarding agonist effect on S1P₁ fate, because SEW2871 does not degrade S1P₁. To further understand the mechanism of agonist-induced S1P₁ down-regulation, we compared signaling and fate of human S1P₁-green fluorescent protein (GFP) in stable 293 cells, using AFD-R, a chiral analog of FTY720-P, SEW2871, and S1P. Although all agonists acutely internalized S1P₁ to late endosomal vesicles and activated GTPS³⁵ binding and pERK to similar maxima, only AFD-R led to significant S1P₁ down-regulation, as shown by GFP immunoprecipitation studies. Down-regulation was time- and concentration-dependent, was partially blocked by proteasomal inhibition and reversed by chloroquine and an antagonist to S1P₁. All agonists induced a receptor-associated increase in ubiquitination, with AFD-R inducing 3-fold more accumulation than S1P and being 3–4 logs more potent than SEW2871. The formation of AFD-R-receptor ubiquitin complex was inhibited by antagonist and chloroquine and was enhanced by proteasomal inhibition. Identification of proteins by PAGE liquid chromatography-tandem mass spectrometry in cells treated with AFD-R confirmed the co-migration of ubiquitin peptides with those of S1P₁ and GFP, relative to vehicle alone. These data suggest that the hierarchy of ubiquitin recruitment to S1P₁ (AFD-R > S1P > SEW2871) correlates with the efficiency of lysosomal receptor degradation and reflects intrinsic differences between agonists.

Received for publication, November 14, 2006 , and in revised form, December 22, 2006.

^* This work was supported by National Institutes of Health Grants RO1 AI055509 and NIMH 074404 (to H. R.) and Grant HL70694 (to T. H.) and a fellowship from the Genomics Institute of the Novartis Research Foundation (to P. J. G.-C.). 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 Fig. S1.

¹ To whom correspondence should be addressed: Dept. of Immunology, The Scripps Research Institute, ICND118, 10550 N. Torrey Pines Road, La Jolla, CA 92037. Tel.: 858-784-2396; Fax: 858-784-2988; E-mail: hrosen{at}scripps.edu.

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