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Originally published In Press as doi:10.1074/jbc.M610318200 on January 21, 2007

J. Biol. Chem., Vol. 282, Issue 12, 9082-9089, March 23, 2007
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Immunosuppressive and Anti-angiogenic Sphingosine 1-Phosphate Receptor-1 Agonists Induce Ubiquitinylation and Proteasomal Degradation of the Receptor*Formula

Myat Lin Oo{ddagger}, Shobha Thangada{ddagger}, Ming-Tao Wu{ddagger}, Catherine H. Liu{ddagger}, Timothy L. Macdonald§, Kevin R. Lynch, Chen-Yong Lin||, and Timothy Hla{ddagger}1

From the {ddagger}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

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
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 beta-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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
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 S1Pn receptors (3). The prototypical receptor, S1P1 was originally isolated as an inducible gene from vascular endothelial cells (4). Knock out of S1P1 resulted in embryonic lethality due to a vascular maturation defect (5). We recently showed that S1P1 function in endothelial cells is needed for proper endothelial-pericyte interaction, a critical event in vascular maturation (6). In addition, we demonstrated previously that S1P1 is needed for the assembly of vascular endothelial-cadherin-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 S1P1 in T-cells led to inhibition of lymphocyte egress from lymph nodes and the thymus (9, 10). 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 (11, 12). 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 (13–15). The prodrug form of FTY720 is phosphorylated by sphingosine kinase-2 into FTY720-P (16, 17), a potent nanomolar agonist on S1P1, S1P3, S1P4, and S1P5 (13, 14). 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 (18). Both S1P and FTY720-P induce receptor internalization into the endosomal pathway (19, 20). However, in FTY720-treated cells, the S1P1 receptor does not recycle to the plasma membrane (20, 21). 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 (10, 18).

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 (18, 22). Vascular effects of FTY720 include the inhibition of vascular permeability and angiogenesis (16, 23). 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 (24–26). A variety of explanations were proposed, including functional antagonism and vascular gate closure (3, 12). However, systematic examination of the fate of S1P1 when activated by agonists has not been conducted.

In this report, we show that FTY720-P is a potent activator of the ubiquitinylation of the S1P1 receptor. This modification results in rapid and quantitative degradation of S1P1. We suggest that the ability of pharmacologic agonists to target S1P1 degradation may underlie, at least in part, their immunosuppressive and anti-angiogenic properties.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Chemicals and Reagents—Sphingosine and sphingosine 1-phosphate were purchased from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA). FTY720, FTY720-P, and (R)-AFD were kindly provided by Novartis Pharma, Basel, Switzerland. SEW 2871 was purchased from Maybridge. VPC 01211 and VPC 44116 were synthesized as described (27, 28). Fatty acid-free bovine serum albumin, beta-glycerophosphate, beta-actin antibody, and MG132 were from Sigma, ubiquitin monoclonal antibody MMS-258R was from Covance, and ubiquitin monoclonal antibody P4D1 was from Santa Cruz Biotechnology.

S1P1 (E49) Monoclonal Antibody—Escherichia coli-derived human S1P1 full-length antigen (29) was used to develop a murine monoclonal antibody using established procedures (30). Anti-S1P1 monoclonal IgG was purified from hybridoma cell supernatants using protein A-Sepharose.

Cell Culture and Transfection—HEK293 cells stably expressing S1P1-GFP wild type and its various mutants (19) were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 50 units/ml penicillin, and 50 µg/ml streptomycin (Invitrogen) and maintained at 37 °C in a water-saturated atmosphere of 5% CO2 in air. Human umbilical vein endothelial cells (HUVEC) (p4–9; Clonetics) were cultured in M199 medium supplemented with 10% fetal bovine serum and heparin-stabilized endothelial growth factor, as previously described (4). HEK293 cells were transfected with the indicated expression plasmids using the calcium phosphate-mediated method (31) or Lipofectamine 2000 transfection reagent or Oligofectamine (Invitrogen) according to the manufacturer's instructions.

DNA and siRNA Constructs—S1P1-GFP (wild type, {Delta}I, {Delta}III, S5A mutants) in the vector pCDNA3 were either generated by polymerase chain reaction-based mutagenesis or described previously (19). In short, the {Delta}I and {Delta}III constructs were terminated at residues 334 and 362, respectively, and fused with the GFP polypeptide. siRNAs for GRK2 (bases 267–288 from the start codon; target sequence (5'-AAGAAGUACGAGAAGCUGGAG-3')), non-silencing control sequence (5'-AAGUGGACCCUGUAGAUGGCG-3') (32), beta-arrestin 1, beta-arrestin 2, and both (33) were prepared by the Silencer siRNA construction kit (Ambion) according to the manufacturer's protocol or purchased from Dharmacon Research (Lafayette, CO).

Analysis of S1P1 Degradation by Immunoblotting—HUVEC (4 x 105) were cultured in M199 medium growth medium containing serum for 3 days. Cells were serum-starved in M199 medium without serum and other growth factors for 2 h and then incubated in the same medium with S1P, FTY720-P, or their analogs for the indicated times. Cells were washed with phosphate-buffered saline and lysed by addition of SDS sample buffer and by sonication, followed by heating at 95 °C for 5 min. Protein concentrations were determined by Bio-Rad assay (Bio-Rad protein dye reagent). Equal amounts of proteins were separated on a 10% polyacrylamide gel and transblotted onto nitrocellulose membrane. Blots were incubated with an anti-S1P1 monoclonal antibody (E49) and analyzed by chemiluminescence (Amersham Biosciences). Blots were then stripped and probed using beta-actin primary antibody (Sigma). Films were scanned and normalized for total protein using the beta-actin blots.

HEK293 stable clones of S1P1 wild type and its mutant variants were grown to 50–65% confluency in 6-well plates. The next day cells were incubated in 2% charcoal-stripped serum for 2 days and allowed to grow for an additional 2 h in Dulbecco's modified Eagle's medium. Cells were then treated with the indicated various ligands and analyzed as described above for HUVEC.

Immunofluorescence Confocal Microscopy—2 x 105 cells were plated in fibronectin-coated 35-mm glass-bottom Petri dishes. One day later for HEK293, cells were incubated in 2% charcoal-treated fetal bovine serum for 2 days, washed, and serum-starved 3 h prior to treatment. Then cells were washed with ice-cold phosphate-buffered saline and fixed and examined using a confocal microscope. For immunofluorescence analysis, S1P1 (E49) monoclonal antibody was applied and antibody staining was visualized with Alexa Fluor 488 donkey anti-goat for monoclonal (1:2000) IgG (Molecular Probes). Confocal microscopy was conducted on a Zeiss LSM 510 laser-scanning confocal microscope. Fluorescence was excited using a 488-nm argon laser, and emitted light was detected with a 505-nm long-pass filter.

Detection of Ubiquitinated Receptors—HEK293 cells stably expressing S1P1-GFP (90% confluency) were starved in 2% charcoal-stripped serum for 2 days. Then they were incubated for an additional 2 h in Dulbecco's modified Eagle's medium with the proteasomal inhibitor 20 µM MG132. Cells were incubated with receptor agonists and antagonists and lysed in 50 mM Tris, pH 7.4, 1% Triton, 500 mM NaCl, 10 mM MgCl2, 50 mM beta-glycerophosphate, 0.5 mM Na3VO4, 0.1 mM Na2MoO4, 10 mM NaF, 20 mM CHAPS. Cell lysates were immunoprecipitated with anti-S1P1 (E49) IgG beads or ubiquitin (P4D1) IgG beads for overnight. S1P1 or ubiquitin-bound proteins were separated in 10% SDS-PAGE gel and probed with ubiquitin antibody or E49 antibody and reprobed by E49 or ubiquitin antibody.


Figure 1
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  		FIGURE 1.
Detection of S1P1 receptor in HUVEC and transfected cells. A, Chinese hamster ovary (CHO) cells or HEK293 cells were transiently transfected with S1P1 or S1P1-GFP constructs, and cell lysates were analyzed by SDS-PAGE and immunoblotted with the anti-S1P1 (E49) monoclonal antibody as described. B, HEK293 cells transfected with either vector or S1P1-GFP plasmid were analyzed by an immunoblot assay as described with the indicated antibodies E49 anti-S1P1 or anti-GFP or anti-beta-actin. Some filters were incubated with the E. coli-derived competitor antigen and the E49 antibody as indicated. S1P1 antigen and GST were used at 10 µg/ml. C, HUVEC were analyzed by confocal immunofluorescence microscopy as described. Some cells were treated with vehicle, 100 nM S1P, or 10 nM FTY720-P for 30 min and imaged. Other cells were washed after the treatments, incubated with S1P-free medium for 2 h in the presence of 15 µg/ml cycloheximide, and imaged as described. Note that S1P1 receptor recycles in S1P-treated cells but not in FTY720-P-treated cells.

 


Figure 2
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  		FIGURE 2.
S1P1 agonists induce degradation of the S1P1R. A, HUVEC were incubated for 30 min with the indicated concentrations of S1P, sphingosine, FTY720-P, FTY720, SEW2871, and R-AFD, and equal amounts of cell lysates were analyzed by SDS-PAGE and immunoblot using a S1P1 (E49) monoclonal antibody. The membrane was stripped and reprobed with the beta-actin monoclonal antibody as loading control. B, quantification of S1P1 degradation by densitometric scanning of immunoblots. Results shown represent the mean ± S.E. of three independent experiments.

 

   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
S1P1 receptor undergoes rapid internalization via the endosomal pathway upon agonist stimulation (19). The fate of endocytosed S1P1 after stimulation by various agonists is not well characterized. Therefore, we examined whether endogenously expressed S1P1 is endocytosed in HUVEC using the monoclonal antibody E49 to detect the S1P1 antigen. As shown in Fig. 1A, E49 detected the S1P1 protein specifically in transfected Chinese hamster ovary and S1P1-GFP fusion protein in HEK293 cells. Heterogeneity of the immunoreactive bands most likely represents post-translationally modified receptor species, such as glycosylation and/or phosphorylation. In addition, lower Mr bands could be due to proteolysis. The immunoreactivity was competed by the E. coli-derived S1P1 antigen (29) but not by the irrelevant glutathione S-transferase antigen (Fig. 1B). Immunofluorescence analysis of HUVEC indicated that the receptor was localized on the punctate regions of the plasma membrane, in particular in cell-cell contact areas in confluent HUVEC. The epitope detected by E49 is localized on the extracellular surface, because strong membrane staining was observed in non-permeabilized cells (data not shown). Treatment with S1P or FTY720-P strongly induced receptor endocytosis. However, when the ligands were washed away and cells incubated with cycloheximide to block new protein synthesis (19), S1P1 recycled back to the plasma membrane within 2 h in S1P-treated cells whereas it remained in the endosomal structures in the FTY720-P-treated cells (Fig. 1C). Thus, endogenously expressed S1P1 behaves similarly to the behavior of S1P1-GFP fusion protein in HEK293 cells (19–21). However, in HUVEC, native S1P1 appears in a punctate pattern compared with the HEK293 cell, in which a smooth membrane pattern is seen.


Figure 3
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  		FIGURE 3.
Kinetics of FTY720-P down-regulation of S1P1 receptor. A, time course of the effect of FTY720-P and FTY720 on HUVEC. B, FTY720-P induces degradation of S1P1-GFP in HEK293 stably transfected cells. HEK293 S1P1-GFP cells were treated with FTY720-P (10 nM) for the indicated times. Data represent a typical experiment that was repeated three times.

 
We determined the expression level of S1P1 in HUVEC after treatment with various agonists. As shown in Fig. 2. the physiological ligand S1P only modestly reduced receptor levels whereas sphingosine did not have an effect. Interestingly, FTY720-P caused a dramatic reduction in endogenous S1P1 expression. The precursor, FTY720, was inactive. A chiral analog of FTY720-P, (R)-AFD, behaved similarly. SEW2871, a lower potency S1P1-specific agonist (EC50 ~13.8 + 8.3 nM versus 0.4 + 0.24 nM for S1P), did not induce suppression of receptor expression at lower concentrations (10–250 nM) (Fig. 2). Higher concentrations of SEW2981 (1–2.5 µM) suppressed receptor expression ~30%, which is equivalent to the suppression achieved by 250 nM S1P (data not shown). The time course experiment showed that FTY720-P decreased receptor levels within 0.25–1 h after treatment in HUVEC (Fig. 3A). In HEK293 cells expressing S1P1-GFP receptor, sustained suppression (0.25–12 h) of receptor expression was observed. These data suggest that pharmacological agonists and supraphysiological concentrations of the natural ligand S1P suppress S1P1 receptor expression levels in HUVEC and HEK293 cells.


Figure 4
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  		FIGURE 4.
Effect of the S1P1 receptor antagonist VPC 44116 on S1P- and FTY720-P-induced receptor internalization. HEK293 cells stably expressing S1P1-GFP were preincubated for 30 min with the indicated concentrations of VPC 44116. The cells were then treated for 30 min with 100 nM S1P or 10 nM FTY720-P, fixed, and imaged by confocal microscopy as described.

 


Figure 5
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  		FIGURE 5.
S1P1 antagonist blocks FTY720-P-induced S1P1 down-regulation. HUVEC were incubated in 10 nM FTY720-P for the indicated times (A) with or without VPC 44116 (500 nM) and various doses of S1P1 antagonist and FTY720-P (B) for 30 min. C, HUVEC were treated with 100 nM VPC 44116 for the indicated time points. S1P1 receptor levels were determined by immunoblot analysis with the E49 antibody as described.

 
That FTY720-P and (R)-AFD are the most potent ligands at down-regulation of receptor expression is surprising because such agonists have similar affinities and are capable of acutely desensitizing the S1P1 receptor as the natural ligand, S1P (13, 14). It is possible that the phosphate moiety of S1P is more labile to the action of plasma membrane- or endosome-associated phosphatases. However, SEW2981, which does not contain a phosphate moiety, was not effective at inducing receptor degradation, suggesting that FTY720-P and (R)-AFD may induce a unique perturbation of receptor conformation and/or oligomerization so as to down-regulate the S1P1 receptor.

We next tested the effect of VPC 44116, a potent S1P1 antagonist (27, 28), on receptor internalization and FTY720-P-induced down-regulation of the S1P1 receptor. As shown in Fig. 4, VPC 44116 alone did not have an effect on the localization of S1P1-GFP in HEK293 cells. However, at 100–500 nM, VPC 44116 potently inhibited the ability of S1P (100 nM) and FTY720-P (10 nM) to induce receptor internalization, as expected of a potent antagonist. Similar findings were observed with the endogenously expressed S1P1 receptor in HUVEC (data not shown).

In HUVEC, VPC 44116 treatment rescued FTY720-P-induced S1P1 receptor suppression in a time- and dose-dependent manner (Fig. 5, A and B). Similarly, VPC 44116 prevented FTY720-P-induced S1P1-GFP degradation in HEK293 cells (data not shown). These findings provide evidence that receptor agonism is required but not sufficient for FT720-P-induced down-regulation. Interestingly, treatment of HUVEC with VPC 44116 enhanced the basal level of receptor expression within 60 min, suggesting that autocrine signaling of S1P1 in HUVEC induces receptor down-regulation under steady-state conditions (Fig. 5C). It is known that HUVEC produce significant quantities of S1P and are capable of secreting this lipid mediator into the extracellular environment (34). In addition, HUVEC express the sphingosine kinase-1a isoform, which can act as an ectokinase (34, 35). Therefore, endogenously produced S1P may interact with the S1P1 receptor to signal in an autocrine manner. Such an autocrine signaling module in the vascular endothelial cells may have functional consequences, as S1P1 is important in the inhibition of vascular permeability (7, 8), regulation of actin- and microtubule-based cytoskeleton (6, 7, 26), suppression of the inflammatory response (36, 37), and regulation of the angiogenic response (25).


Figure 6
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  		FIGURE 6.
Inability of the S1P1-S5A-GFP receptor to undergo agonist-induced internalization. HEK293 cells stably transfected with wild-type S1P1-GFP or S1P1-S5A-GFP constructs were treated with either vehicle, S1P (100 nM), or FTY720-P (10 nM) for 30 min, fixed, and imaged with confocal fluorescence microscopy as described.

 
Next we determined whether receptor internalization is needed for FTY720-P-induced S1P1 degradation (19). We used stable HEK293 cells expressing various C-terminal mutants of S1P1 receptor. Previous studies from our laboratory have shown that successive deletion of the C-terminal of S1P1 receptor caused inhibition of ligand-induced internalization (19). Such mutants were used in the present study to determine whether FTY720-P is capable of inducing S1P1 degradation in the absence of GPCR internalization. In addition, Watterson et al. (38) have shown that a conserved serine-rich region (SRR) in the C-terminal tail of S1P1 is phosphorylated by GRK2 and protein kinase-C. Our previous data suggest that the SRR domain is essential for S1P-induced receptor internalization (19). We therefore prepared a mutant, S1P1-S5A-GFP, in which the SRR motif (351SRSKSDNSS359) is altered so that all the serines are changed to alanines (351ARAKADNAA359), making it resistant to phosphorylation. As shown in Fig. 6, the resulting mutant, S1P1-S5A-GFP, was expressed in HEK293 cells and transported to the plasma membrane. S1P and FTY720-P induced extracellular signal-regulated kinase (ERK) phosphorylation, Akt phosphorylation, and cell migration in transfected Chinese hamster ovary cells in an indistinguishable manner when the wild type and the S5A mutant receptors were compared (data not shown). These results suggest that ligand binding and acute signaling of the S1P1-S5A-GFP mutant are not altered by the C-terminal mutation.


Figure 7
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  		FIGURE 7.
SRR phosphorylation consensus site of S1P1 is required for FTY720-P-induced S1P1 degradation. A, schematic representation of S1P1-GFP mutants and the wild-type receptor. The internalization data are from Ref. 19 and Fig. 6. B, S1P1-{Delta}III-GFP is sensitive to FTY720-P-induced S1P1 degradation whereas S1P1-{Delta}I-GFP and S1P1-S5A-GFP are resistant. HEK293 cells stably transfected with S1P1-{Delta}III-GFP, S1P1-S5A-GFP, and S1P1-{Delta}I-GFP cells were incubated with FTY720-P (10 nM) for indicated times. All membranes were immunoblotted with E49 antibody and reprobed with the beta-actin antibody. C, quantification of S1P1 degradation by densitometric scanning of immunoblots. Results shown represent the mean ± S.E. of three independent experiments.

 
When HEK293 cells stably transfected with the C-terminal mutants were treated with FTY720-P, receptor degradation was not observed in S1P1-{Delta}I-GFP and S1P1-S5A-GFP (Fig. 7). In contrast, S1P1-{Delta}III-GFP was degraded in a similar manner as the wild-type receptor. Thus, there is strict correlation between agonist-induced receptor internalization and suppression of receptor expression. Therefore, the SRR phosphorylation consensus site of S1P1 receptor is necessary not only for FTY720-P-induced receptor endocytosis but also for down-regulation of receptor expression.


Figure 8
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  		FIGURE 8.
Requirement for GRK2 and beta-arrestins in FTY720-P-induced S1P1 degradation. A, GRK2 siRNA can rescue FTY720-P-induced S1P1 degradation. Control siRNA and GRK2 siRNA were transfected into HEK293-S1P1-GFP stable cells and incubated in 2% charcoal-treated serum for 2 days, and FTY720-P 10 nM was applied in Dulbecco's modified Eagle's medium. Immunoblot analysis was conducted with S1P1 (E49), beta-actin, and GRK2 antibodies. B, beta-arrestin-dependent internalization is required for S1P1 degradation by FTY720-P. HEK293-S1P1-GFP stable cells were transfected with control siRNA, beta-arrestin 1, beta-arrestin 2, and beta-arrestin-1 and -2 siRNA and incubated with 10 nM FTY720-P for 30 min and 1 h. Cell lysates were separated on SDS-PAGE and immunoblotted with S1P1 (E49), beta-actin, and beta-arrestin-1 and -2 antibodies.

 
To test whether GRK2, an essential kinase involved in the agonist-induced phosphorylation of GPCRs, is needed for S1P1 degradation, we suppressed GRK2 expression by a specific siRNA (32). As shown in Fig. 8A, degradation of S1P1 was not detected in GRK2 siRNA-treated cells.

Next, we tested the role of beta-arrestins, which bind to phosphorylated motifs on activated GPCRs to induce receptor desensitization and facilitate endocytosis. As shown in Fig. 8B, co-administration of siRNA for beta-arrestin-1 and -2 rescued the receptor from FTY720-P-induced degradation. Together, these results suggest that GRK2-mediated receptor phosphorylation and beta-arrestin-dependent receptor internalization is required for FTY720-P-induced S1P1 degradation. These experiments further confirm that receptor endocytosis machinery, i.e. kinases that phosphorylate the receptor and the adaptor proteins that induce endocytosis of receptor-containing endosomes, is essential for FTY720-P-induced receptor down-regulation.

After internalization, GPCRs can have several fates; some are recycled back to the plasma membrane and some are targeted to be degraded in proteasomes and/or lysosomes (39, 40). Both of these processes appear to require the ubiquitinylation of GPCRs (41, 42). First, we tested the effect of MG132, an inhibitor of protein degradation in proteasomes (43). Treatment with MG132 profoundly inhibited FTY720-P-induced receptor degradation in both S1P1-GFP-expressing HEK293 cells and HUVEC (Fig. 9). These data suggested that S1P1 is degraded in proteasomes following FTY720-P treatment of HUVEC and HEK293 cells. However, we did not observe a defect in receptor internalization after the cells were treated with MG132 (data not shown), suggesting that proteasomal activity is not required for receptor internalization.


Figure 9
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  		FIGURE 9.
The proteasomal inhibitor MG132 rescues FTY720-P-induced S1P1 degradation in HEK293-S1P1-GFP stable cells and in HUVEC. Indicated cells were incubated with or without MG132 (20 µM) for 2 h before 10 nM FTY720-P was administered for the indicated times. S1P1 levels were determined by immunoblot analysis as described.

 
Ubiquitination of GPCRs is a critical post-translational modification implicated in endosomal trafficking as well as sorting to proteasome/lysosomal degradation pathways (41, 44). Because S1P and FTY720-P regulate different receptor fates, we tested whether they regulate S1P1 ubiquitinylation differentially. Ubiquitinylation of S1P1 was detected in HEK293 cells expressing S1P1-GFP fusion protein by immunoprecipitation with the anti-S1P1-specific monoclonal antibody E49 followed by immunoblot analysis with the anti-ubiquitin antibody. As shown in Fig. 10A, FTY720-P induced strong polyubiquitinylation of S1P1 even though S1P was also capable of this modification, albeit to a lesser extent. The monoubiquitinylated species was also induced by FTY720-P and S1P. Importantly, polyubiquitinylation of S1P1-GFP was completely blocked by the receptor antagonist, VPC 44116. In contrast, the monoubiquitinylated species was not affected. Analysis of total ubiquitinylated proteins indicated that FTY720-P is capable of inducing a stronger pattern of multiple ubiquitinylated species only in HEK293 cells expressing S1P1-GFP (supplemental Figs. S2 and S3). A reciprocal immunoprecipitation experiment, in which anti-ubiquitin antibody was used, followed by an immunoblot analysis with the E49 antibody (Fig. 10B), showed also that FTY720-P induced strong ubiquitinylation of S1P1-GFP. Immunoprecipitation with anti-ubiquitin followed by immunoblot with the same antibody also showed that FTY720-P induced strong polyubiquitinylation. These data suggest that increased polyubiquitinylation of S1P1 may be critical for sorting into the proteasomal degradative pathway. Similar experiments in HUVEC to detect endogenous S1P1 were not successful due to the low level expression of the receptor in HUVEC (data not shown). These data strongly suggest that FTY720-P-induced polyubiquitinylation of S1P1 is responsible for the preferential targeting of the receptor into the proteasomal degradative pathway.


Figure 10
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  		FIGURE 10.
FTY720-P induces S1P1 polyubiquitinylation. A, HEK293-S1P1 cells pretreated with 20 µM MG132 were incubated with S1P (100 nM)or FTY720-P (10 nM) for 15 min in the presence or absence of VPC 44116 (500 nM). Solubilized S1P1 receptor was immunoprecipitated by E49 monoclonal IgG beads and immunoblotted with the ubiquitin IgG. In the lower panel, immunoprecipitates were reacted with the GFP antibody that primarily detected the non-intensively modified S1P1-GFP polypeptide. B, solubilized S1P1 receptor was immunoprecipitated with the ubiquitin monoclonal IgG (P4D1; Santa Cruz Biotechnology) beads and immunoblotted with the E49 IgG (left panel). The membrane was reprobed with the same IgG (middle panel). Input cell lysates were detected by direct immunoblot with S1P1 (E49) (right panel, top) and beta-actin antibody (right panel, bottom).

 
Studies in yeast and lymphocytes have shown that FTY720 treatment modifies the cellular ubiquitin pathway (45, 46). In a Saccharomyces cerevisiae screen, Movva and co-workers (46) noted that enhanced ubiquitination is involved in the growth inhibition mediated by FTY720. In lymphocytes treated with FTY720, proteins involved in the ubiquitin system were modulated. Thus, FTY720-P in our system may modulate the conformation and/or oligomerization of the S1P1 receptor in a manner such that it is preferentially targeted for the degradative pathway. It is of interest to note that only very high concentrations of S1P (≥250 nM) induced significant receptor degradation. Although plasma S1P levels are high, free or bioactive S1P in plasma is in the low nM range due to the sequestration of plasma chaperones such as high density lipoprotein and albumin (47–49). Indeed, in situations where high local production of S1P is observed, significant receptor degradation may occur upon autocrine and/or paracrine signaling. Thus, we speculate that under physiological conditions S1P1 receptor may follow the recycling pathway primarily. However, under conditions where high S1P levels are present, for example during platelet activation and immune cell activation, etc., the receptor signaling may be attenuated by targeting of the S1P1 receptor to the degradative pathway.

In summary, the present study establishes a novel link between ubiquitination and degradation of S1P1 induced by FTY720-P. We show that GPCR internalization is required for degradation of S1P1. However, receptor internalization is not sufficient for receptor degradation, as S1P treatment that efficiently internalizes S1P1 is not efficiently degraded. Exaggerated ubiquitinylation induced by FTY720-P targets the receptor to the proteasomal degradative pathway. Because FTY720-P is immunosuppressive and anti-angiogenic, we speculate that ubiquitinylation and degradation of S1P1 may at least in part be responsible for the pharmacological action of FTY720-P.


   FOOTNOTES
 
* 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. Back

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

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

2 The 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. Back


   ACKNOWLEDGMENTS
 
We thank Novartis Pharma for the kind gift of FTY720 and related compounds.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Chalfant, C. E., and Spiegel, S. (2005) J. Cell Sci. 118, Pt. 20, 4605-4612[Abstract/Free Full Text]
  2. Chun, J., and Rosen, H. (2006) Curr. Pharm. Des. 12, 161-171[CrossRef][Medline] [Order article via Infotrieve]
  3. Rosen, H., and Goetzl, E. J. (2005) Nat. Rev. Immunol. 5, 560-570[CrossRef][Medline] [Order article via Infotrieve]
  4. Hla, T., and Maciag, T. (1990) J. Biol. Chem. 265, 9308-9313[Abstract/Free Full Text]
  5. Liu, 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., and Proia, R. L. (2000) J. Clin. Investig. 106, 951-961[Medline] [Order article via Infotrieve]
  6. Paik, J. H., Skoura, A., Chae, S. S., Cowan, A. E., Han, D. K., Proia, R. L., and Hla, T. (2004) Genes Dev. 18, 2392-2403[Abstract/Free Full Text]
  7. Lee, M. J., Thangada, S., Claffey, K. P., Ancellin, N., Liu, C. H., Kluk, M., Volpi, M., Sha'afi, R. I., and Hla, T. (1999) Cell 99, 301-312[CrossRef][Medline] [Order article via Infotrieve]
  8. Garcia, J. G., Liu, F., Verin, A. D., Birukova, A., Dechert, M. A., Gerthoffer, W. T., Bamberg, J. R., and English, D. (2001) J. Clin. Investig. 108, 689-701[CrossRef][Medline] [Order article via Infotrieve]
  9. Allende, M. L., Dreier, J. L., Mandala, S., and Proia, R. L. (2004) J. Biol. Chem. 279, 15396-15401[Abstract/Free Full Text]
  10. Matloubian, M., Lo, C. G., Cinamon, G., Lesneski, M. J., Xu, Y., Brinkmann, V., Allende, M. L., Proia, R. L., and Cyster, J. G. (2004) Nature 427, 355-360[CrossRef][Medline] [Order article via Infotrieve]
  11. Wei, S. H., Rosen, H., Matheu, M. P., Sanna, M. G., Wang, S. K., Jo, E., Wong, C. H., Parker, I., and Cahalan, M. D. (2005) Nat. Immunol. 6, 1228-1235[CrossRef][Medline] [Order article via Infotrieve]
  12. Singer, I., Tian, M., Wickham, L. A., Lin, J., Matheravidathu, S. S., Forrest, M. J., Mandala, S., and Quackenbush, E. J. (2005) J. Immunol. 175, 7151-7161[Abstract/Free Full Text]
  13. Mandala, 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., and Rosen, H. (2002) Science 296, 346-349[Abstract/Free Full Text]
  14. Brinkmann, 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., and Lynch, K. R. (2002) J. Biol. Chem. 277, 21453-21457[Abstract/Free Full Text]
  15. Kappos, L., Antel, J., Comi, G., Montalban, X., O'Connor, P., Polman, C. H., Haas, T., Korn, A. A., Karlsson, G., and Radue, E. W. (2006) N. Engl. J. Med. 355, 1124-1140[Abstract/Free Full Text]
  16. Sanchez, T., Estrada-Hernandez, T., Paik, J. H., Wu, M. T., Venkataraman, K., Brinkmann, V., Claffey, K., and Hla, T. (2003) J. Biol. Chem. 278, 47281-47290[Abstract/Free Full Text]
  17. Kharel, 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., and Lynch, K. R. (2005) J. Biol. Chem. 280, 36865-36872[Abstract/Free Full Text]
  18. Brinkmann, V., Cyster, J. G., and Hla, T. (2004) Am. J. Transplant 4, 1019-1025[CrossRef][Medline] [Order article via Infotrieve]
  19. Liu, C. H., Thangada, S., Lee, M. J., Van Brocklyn, J. R., Spiegel, S., and Hla, T. (1999) Mol. Biol. Cell 10, 1179-1190[Abstract/Free Full Text]
  20. Graler, M. H., and Goetzl, E. J. (2004) FASEB J. 18, 551-553[Abstract/Free Full Text]
  21. Jo, E., Sanna, M. G., Gonzalez-Cabrera, P. J., Thangada, S., Tigyi, G., Osborne, D. A., Hla, T., Parrill, A. L., and Rosen, H. (2005) Chem. Biol. 12, 703-715[CrossRef][Medline] [Order article via Infotrieve]
  22. Cyster, J. G. (2005) Annu. Rev. Immunol 23, 127-159[CrossRef][Medline] [Order article via Infotrieve]
  23. LaMontagne, 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., and Wood, J. (2006) Cancer Res. 66, 221-231[Abstract/Free Full Text]
  24. Saba, J. D., and Hla, T. (2004) Circ. Res. 94, 724-734[Abstract/Free Full Text]
  25. Chae, S. S., Paik, J. H., Furneaux, H., and Hla, T. (2004) J. Clin. Investig. 114, 1082-1089[CrossRef][Medline] [Order article via Infotrieve]
  26. Paik, J. H., Chae, S., Lee, M. J., Thangada, S., and Hla, T. (2001) J. Biol. Chem. 276, 11830-11837[Abstract/Free Full Text]
  27. Davis, M. D., Clemens, J. J., Macdonald, T. L., and Lynch, K. R. (2005) J. Biol. Chem. 280, 9833-9841[Abstract/Free Full Text]
  28. Foss, F. W., Jr., Snyder, A. H., Davis, M. D., Rouse, M., Osuka, M. D., Lynch, K. R., and Macdonald, T. L. (2007) Bioorg. Med. Chem. Lett. 15, 663-677
  29. Lee, M. J., Thangada, S., Paik, J. H., Sapkota, G. P., Ancellin, N., Chae, S. S., Wu, M., Morales-Ruiz, M., Sessa, W. C., Alessi, D. R., and Hla, T. (2001) Mol. Cell 8, 693-704[CrossRef][Medline] [Order article via Infotrieve]
  30. Tang, T. K., Hong, T. M., Lin, C. Y., Lai, M. L., Liu, C. H., Lo, H. J., Wang, M. E., Chen, L. B., Chen, W. T., Ip, W., Lin, C. D., Lin, J. J., Lin, S., Sun, T., Wang, E., Wang, J. L., Wu, R., Wu, C., and Chien, S. (1993) J. Cell Sci. 104, 237-247[Abstract]
  31. Chen, C., and Okayama, H. (1987) Mol. Cell. Biol. 7, 2745-2752[Abstract/Free Full Text]
  32. Chen, W., Ren, X. R., Nelson, C. D., Barak, L. S., Chen, J. K., Beachy, P. A., de Sauvage, F., and Lefkowitz, R. J. (2004) Science 306, 2257-2260[Abstract/Free Full Text]
  33. Gesty-Palmer, D., Chen, M., Reiter, E., Ahn, S., Nelson, C. D., Wang, S., Eckhardt, A. E., Cowan, C. L., Spurney, R. F., Luttrell, L. M., and Lefkowitz, R. J. (2006) J. Biol. Chem. 281, 10856-10864[Abstract/Free Full Text]
  34. Ancellin, N., Colmont, C., Su, J., Li, Q., Mittereder, N., Chae, S. S., Stefansson, S., Liau, G., and Hla, T. (2002) J. Biol. Chem. 277, 6667-6675[Abstract/Free Full Text]
  35. Venkataraman, K., Thangada, S., Michaud, J., Oo, M. L., Ai, Y., Lee, Y. M., Wu, M., Parikh, N. S., Khan, F., Proia, R. L., and Hla, T. (2006) Biochem. J. 397, 461-471[CrossRef][Medline] [Order article via Infotrieve]
  36. Bolick, D. T., Srinivasan, S., Kim, K. W., Hatley, M. E., Clemens, J. J., Whetzel, A., Ferger, N., Macdonald, T. L., Davis, M. D., Tsao, P. S., Lynch, K. R., and Hedrick, C. C. (2005) Arterioscler. Thromb. Vasc. Biol. 25, 976-981[Abstract/Free Full Text]
  37. Whetzel, A. M., Bolick, D. T., Srinivasan, S., Macdonald, T. L., Morris, M. A., Ley, K., and Hedrick, C. C. (2006) Circ. Res. 99, 731-739[Abstract/Free Full Text]
  38. Watterson, K. R., Johnston, E., Chalmers, C., Pronin, A., Cook, S. J., Benovic, J. L., and Palmer, T. M. (2002) J. Biol. Chem. 277, 5767-5777[Abstract/Free Full Text]
  39. Drake, M. T., Shenoy, S. K., and Lefkowitz, R. J. (2006) Circ. Res. 99, 570-582[Abstract/Free Full Text]
  40. Shenoy, S. K., McDonald, P. H., Kohout, T. A., and Lefkowitz, R. J. (2001) Science 294, 1307-1313[Abstract/Free Full Text]
  41. Marchese, A., and Benovic, J. L. (2004) Methods Mol. Biol. 259, 299-305[Medline] [Order article via Infotrieve]
  42. Marchese, A., Raiborg, C., Santini, F., Keen, J. H., Stenmark, H., and Benovic, J. L. (2003) Dev. Cell 5, 709-722[CrossRef][Medline] [Order article via Infotrieve]
  43. Tsubuki, S., Kawasaki, H., Saito, Y., Miyashita, N., Inomata, M., and Kawashima, S. (1993) Biochem. Biophys. Res. Commun. 196, 1195-1201[CrossRef][Medline] [Order article via Infotrieve]
  44. Wojcikiewicz, R. J. (2004) Trends Pharmacol. Sci. 25, 35-41[CrossRef][Medline] [Order article via Infotrieve]
  45. Bohler, T., Budde, K., Neumayer, H. H., and Waiser, J. (2005) Transplantation 79, 492-495[CrossRef][Medline] [Order article via Infotrieve]
  46. Welsch, C. A., Hagiwara, S., Goetschy, J. F., and Movva, N. R. (2003) J. Biol. Chem. 278, 26976-26982[Abstract/Free Full Text]
  47. Kimura, T., Sato, K., Kuwabara, A., Tomura, H., Ishiwara, M., Kobayashi, I., Ui, M., and Okajima, F. (2001) J. Biol. Chem. 276, 31780-31785[Abstract/Free Full Text]
  48. Murata, N., Sato, K., Kon, J., Tomura, H., Yanagita, M., Kuwabara, A., Ui, M., and Okajima, F. (2000) Biochem. J. 352, Pt. 3, 809-815
  49. Zhang, B., Tomura, H., Kuwabara, A., Kimura, T., Miura, S., Noda, K., Okajima, F., and Saku, K. (2005) Atherosclerosis 178, 199-205[CrossRef][Medline] [Order article via Infotrieve]

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