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Type 1 sphingosine 1-phosphate G protein-coupled receptor signaling of lymphocyte functions requires sulfation of its extracellular amino-terminal tyrosines

Claudia B. Fieger^*,1, Mei-Chuan Huang^,1, James R. Van Brocklyn and Edward J. Goetzl^,2

Departments of
^* Anatomy,
Medicine and Microbiology-Immunology, University of California, San Francisco, California, USA; and
Department of Pathology, Ohio State University, Columbus, Ohio, USA

² Correspondence: University of California, Room UB8B, UC Box 0711, 533 Parnassus at 4th Ave., San Francisco, CA 94143-0711, USA. E-mail: egoetzl{at}itsa.ucsf.edu

SPECIFIC AIMS

The lysosphingolipid mediator sphingosine 1-phosphate (S1P) affects many functions of different types of cells through a family of five G protein-coupled receptors (GPCRs) designated S1P₁ to S1P₅. T lymphocytes express predominantly S1P₁ and S1P₄ that are down-regulated by immune activation. S1P₁ transduces signals from S1P that are required for thymocyte emigration, T cell transmigration of lymph nodes, and regulation of T cell chemotaxis in tissues. Alterations in expression of functional S1P₁ receptors by T cells are the major mechanism controlling T cell responses to S1P and were thought to result solely from changes in the balance between cell-surface down-regulation and insertion of recycled and newly synthesized S1P₁ receptors. Studies thus were designed to investigate roles of functionally relevant posttranslational modifications of S1P₁ receptors in their expression and activities in T cells.

PRINCIPAL FINDINGS

1. Human and mouse S1P₁ receptors have tyrosines in their amino-terminal sequences that are susceptible to sulfation
The amino-terminal amino acid sequences of human and mouse S1P₁ contain two tyrosine residues at positions 19 and 22, which are flanked by aspartic acid, and are typical sites of protein sulfation. In contrast, S1P₂ has one tyrosine with a single adjacent glutamic acid, which has not been a site for sulfation, and none of the other S1P GPCRs has a tyrosine with a neighboring aspartic acid or glutamic acid. Similar tyrosine motifs with flanking aspartic acid residues also have been observed in four different chemokine GPCRs.

2. The level of T cell-surface expression of S1P₁ GPCRs is independent of amino-terminal tyrosine sulfation in studies of a tyrosine 19,22 to phenylalanine 19,22 mutant S1P₁ GPCR
Similar levels of total expression of wild-type and mutant (Y19,22F)S1P₁ were detected in Jurkat T cell transfectants, as assessed by real-time PCR quantification of mRNA encoding S1P₁ and Western blot analyses of extracted cmyc-tagged S1P₁ protein. S1P₁ protein was at similar plateau levels for at least 48 h after transfection in both the wild-type and mutant (Y19,22F) S1P₁ transfectants. The levels of Jurkat T cell surface S1P₁ also were similar in the wild-type and mutant S1P₁ transfectants, as assessed by Western blots of cell membrane protein-selective extracts and flow cytometric detection of surface S1P₁ protein (Fig. 1 ). Thus, biochemical and functional differences between the wild-type and mutant (Y19,22F)S1P₁ Jurkat T cell transfectants are not attributable to dissimilar levels of surface expression of these different S1P₁ GPCRs.

View larger version (30K): [in this window] [in a new window]   Figure 1. Demonstration of equal cell-surface expression of human wild-type and Y19,22F mutant S1P1 GPCRs in Jurkat T cell transfectants. Flow cytometric quantification of expression of wild-type (top) and Y19,22F mutant (bottom) S1P1 receptors in Jurkat T cell transfectants at 48 h after nucleofection. The dark line tracing in each frame depicts results from incubation of transfectant with rabbit anti-S1P1 antibody plus Alexa-Fluor 488-labeled goat anti-rabbit IgG , and the other lighter line tracings are from untransfected Jurkat T cells incubated with both antibodies (second largest rightward shift), and J-WT and J-MUT transfectants incubated with only Alexa-Fluor 488-labeled second antibody.

3. Tyrosines 19 and 22 of wild-type S1P₁ GPCRs are sulfated in T cells
Sulfation of Y19 and Y22 in wild-type S1P₁ of Jurkat S1P₁ transfectants and mouse splenic CD4 T cells was demonstrated by uptake of ³⁵SO4 specifically into a 42–48 kDa protein, that was precipitated by two anti-S1P₁ GPCR antibodies of different specificities. This specific sulfation was suppressed in both types of T cells by sodium chlorate inhibition of sulfotransferases and arylsulfatase-catalyzed de-sulfation and was absent in Jurkat T cell mutant (Y19,22F)S1P₁ transfectants.

4. T cell binding of [³²P]S1P by wild-type S1P₁ is higher in affinity than binding by mutant (Y19,22F) S1P₁
Specific binding of [³²P]S1P by Jurkat T cells expressing wild-type S1P₁ was half an order-of-magnitude higher in affinity than that by Jurkat T cells expressing mutant (Y19,22F)S1P₁ GPCRs. Computer-assisted analyses of Scatchard plots of binding of [³²P]S1P by wild-type and mutant (Y19,22F)S1P₁ GPCRs revealed respective K_d values of 18 nM and 52 nM.

5. Diminished transduction by mutant (Y19,22F)S1P₁ GPCRs of S1P signals to T cell migration, proliferation, and cytokine generation
S1P elicited significant chemotaxis of Jurkat wild-type S1P₁ transfectants, but not of Jurkat mutant S1P₁ transfectants at 10^–8 M and 10^–7 M. When added to the T cell compartment of chemotactic chambers at 3 x 10^–7 M and 3 x 10^–6 M, S1P significantly inhibited chemotaxis of Jurkat wild-type S1P₁ transfectants to the chemokine CXCL-12 (SDF-1) without altering the responses of Jurkat mutant S1P₁ transfectants. Inhibition of sulfotransferases by sodium chlorate and desulfation by arylsulfatase suppressed S1P-evoked chemotaxis of mouse CD4 T cells and of Jurkat wild-type S1P₁ transfectants. In studies of other T cell functions, S1P suppressed T cell receptor-driven proliferation of Jurkat wild-type S1P₁ transfectants and mouse spleen CD4 T cells, but not of Jurkat mutant S1P₁ transfectants. Pretreatment of mouse T cells with sodium chlorate or arylsulfatase, under conditions that reduced tyrosine sulfation of S1P₁ receptors, eliminated S1P suppression of proliferation. S1P inhibition of IFN- generation by mouse CD4 T cells, also was diminished by preincubation with sodium chlorate or arylsulfatase (Fig. 2 ).

View larger version (44K): [in this window] [in a new window]   Figure 2. Requirement for S1P1 sulfation in S1P suppression of mouse spleen CD4 T cell generation of IFN-gamma. Mouse CD4 T cells were preincubated with sodium chlorate and arylsulfatase, incubated without (100% control) or with S1P, and stimulated with anti-CD3 and anti-CD28 antibodies. Supernates were harvested after 24 h for ELISA assays. Each column and bar is the mean ± SD of 3 sets of results and statistical significance depicted as *P <0.01. Mean control (100%) levels of IFN- were 6.6 ng/mL, 4.8 ng/mL and 5.9 ng/mL, respectively, for control, sodium chlorate-treated, and arylsulfatase-treated T cells.

CONCLUSIONS AND SIGNIFICANCE

Hypotheses proposed recently to explain the capacity of the S1P-S1P₁ GPCR axis to regulate T cell traffic in secondary lymphoid organs have emphasized its roles both in suppressing blood T cell chemotactic migration into lymph nodes in response to chemokines and in stimulating T cell chemotaxis out of lymph nodes into lymph and then blood (Fig. 3 ). The first assumption required is that S1P concentrations are higher in lymph and blood than in lymph nodes, which creates a gradient to stimulate chemotaxis of T cells from lymph nodes into lymph. The second assumption is that T cell-surface levels of S1P₁ are higher in lymph nodes than blood and lymph, so that T cells in lymph nodes sense and respond chemotactically to the higher concentrations of S1P in lymph. The T cells would not then be trapped in lymph by bound S1P because S1P₁ expression diminishes after their egress from lymph nodes. This possible series of events requires S1P₁ up-regulation in lymph nodes and down-regulation in lymph with some recovery after entry into blood. The sulfation data show higher affinity binding of S1P and more efficient signal transduction by sulfated S1P₁ and consequently another mechanism for regulating the effective level of expression of signal-active S1P₁. A high level of S1P₁ sulfation in blood T cells would facilitate S1P-S1P₁ inhibitory control of their responses to lymph node chemokines. Desulfation of T cell S1P₁ after entry into lymph nodes would allow their persistent residence due to lack of sensitivity to S1P in lymph until resulfation or insertion of new sulfated S1P₁ GPCRs restores responsiveness and triggers chemotactic egress from the lymph nodes. Analyses of sulfation of S1P₁ in T cells from these different sites will be required to assess the possible contributions of this mechanism to T cell traffic in lymphoid tissues.

View larger version (23K): [in this window] [in a new window]   Figure 3. Model of possible role for S1P1 sulfation in S1P-S1P1 axis regulation of T cell trans-lymph node migration. S, sulfate; HEV, high endothelial venule; CK, chemokines; zigzag arrow, S1P-S1P1 GPCR inhibition of chemotaxis to a CK.

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4476fje;

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