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Genes to Cells (2008) 13, 747-757. doi:10.1111/j.1365-2443.2008.01202.x
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Attenuation of cell motility observed with high doses of sphingosine 1-phosphate or phosphorylated FTY720 involves RGS2 through its interactions with the receptor S1P

 Takayuki Kohnoa and Yasuyuki Igarashi*

Laboratory of Biomembrane and Biofunctional Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12, Nishi 6, Kita-ku, Sapporo 060-0812, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Sphingosine 1-phosphate (S1P) stimulation enhances cell motility via the G-protein coupled S1P receptor S1P1. This ligand-induced, receptor-mediated cell motility follows a typical bell-shaped dose–response curve, that is, stimulation with low concentrations of S1P enhances cell motility, whereas excess ligand stimulation does not enhance it. So far, the attenuation of the response at higher ligand concentrations has not been explained. We report here that S1P1 interacts with the regulator of G protein signaling (RGS)-2 protein, which is a GTPase-activating protein (GAP) for heterotrimeric G proteins, in a concentration dependent manner. The RGS2–S1P1 complex dissociated at higher ligand concentrations, yet it was unaffected at low concentrations, suggesting that the dissociated RGS2 is involved in the concurrent decrease of cell motility. In RGS2 knockdown cells, the decrease of cell motility induced by high ligand concentrations was rescued. S1P1 internalization was not implicated in the attenuation of the response. Similar results were observed upon stimulation with the phosphorylated form of FTY720 (FTYP), which is an S1P1 agonist. In conclusion, the suppressed response in cell motility induced by excess S1P or FTYP via S1P1 is regulated by RGS2 functioning through a mechanism that is independent of S1P1 internalization.


   Introduction
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 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Chemokine receptor-mediated cell motility follows a typical bell-shaped dose–response curve (Johnston et al. 2003), exhibiting a rise from baseline to a strong response with low concentrations of the ligand, but an attenuated response with higher concentrations. Such attenuation is common for G-protein-coupled receptors (GPCRs), yet it has never been fully explained for these molecules. In addition, the ligand-induced internalization of a GPCR involves desensitization of the receptor and leads to its down-regulation, an essential mechanism for avoidance of long-term and excess ligand stimulation (Pao & Benovic 2005). However, there is no apparent connection between the attenuation of the GPCR response observed with higher ligand concentrations and GPCR desensitization.

Regulator of G-protein signaling (RGS) proteins are negative regulators of GPCR signaling (Hollinger & Hepler 2002). In such signaling, a ligand stimulates a GPCR, and the inactive form of the receptor's G{alpha} protein subunit, G{alpha}-GDP, is converted to the GTP-binding form G{alpha}-GTP. An RGS protein acts as a GTPase-activating protein (GAP), binding to the G{alpha} protein and increasing its intrinsic GTPase activity, thereby terminating signaling. Upon ligand stimulation, both the intracellular levels of G{alpha}-GTP and the GAP activity of RGS proteins are increased, in a dose-dependent manner (Kurose et al. 1986).

RGS proteins are clearly necessary for the abatement of GPCR signaling. However, there is no direct evidence that these proteins are involved in the attenuation of the response of cell motility observed with high ligand concentrations. Studies have shown that cell motility is decreased in a chemokine concentration-independent manner in cells over-expressing RGS proteins (Reif & Cyster 2000). In contrast, though, in RGS1–/– B cells the cell motility is increased (Han et al. 2005).

The bioactive lipid mediator sphingosine 1-phosphate (S1P) is a ligand for S1P receptor 1 (S1P1), a GPCR. S1P-induced, S1P1-mediated signaling is known to be involved in the regulation of cell motility, differentiation and cell growth in several cell types (Lee et al. 1999; Liu et al. 2000; Spiegel & Milstien 2003; Cyster 2004), and is required for the spontaneous circulation of lymphocytes (Allende et al. 2004; Matloubian et al. 2004). Recent studies have shown that a phosphorylated form of the immunosuppressive reagent FTY720 (FTYP) binds to S1P receptors (Brinkmann et al. 2002; Mandala et al. 2002), and, like the physiological ligand, acts as an agonist for S1P1, subsequently inducing MAPK activation and S1P1 internalization (Sanchez et al. 2003). Additionally, FTYP decreases the number of circulating mature lymphocytes by accelerating lymphocyte homing (Singer et al. 2005). An immunosuppressive effect of S1P1 agonist may cause a coordinate interaction between inhibition of lymphocyte migration (Wei et al. 2005) and stabilization of the cell–cell junction in vascular endothelial cells (Paik et al. 2004) and peripheral lymph nodes (Halin et al. 2005). A recent study has shown that FTYP may be useful for the treatment of multiple sclerosis (Kappos et al. 2006).

Many researchers have used S1P1-over-expressing CHO cells to study S1P1-mediated signaling, and S1P-induced cell motility has been shown in these cells (Kon et al. 1999; Okamoto et al. 2000; Yamaguchi et al. 2003; Sanna et al. 2004). Here, we report that S1P1-mediated cell motility responds to S1P with a typical bell-shaped dose curve, and that the GAP RGS2 is involved in the attenuation of the response at higher ligand concentrations. FTYP elicits a similar response from S1P1, suggesting that RGS2 is also involved in FTYP-mediated inhibition of cell motility.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
RGS2 interacts with S1P1 but dissociates at high ligand concentrations

The attenuation of the response of common GPCRs at higher ligand concentrations has not been explained, although RGS proteins are known to be negative regulators of GPCR signaling (Hollinger & Hepler 2002). To examine whether RGS proteins are involved in S1P1-mediated cellular signaling, we analyzed S1P1 interactions with the RGS family proteins RGS1, RGS2, RGS4 and RGS5. As shown in Fig. 1A, S1P1 was found to be associated with GST–RGS2 in the absence of S1P, but no such association was apparent with GST–RGS1, GST–RGS4 or GST–RGS5. Interestingly, this association was disrupted in the presence of S1P. The amount of GST–RGS fusion proteins was determined by Coomassie Blue staining (Fig. 1B). The S1P1–RGS2 interaction was confirmed in vivo by co-immunoprecipitation assay. Myc-tagged RGS2 was transiently expressed in S1P1-expressing CHO cells (S1P1-CHO) or MOCK-CHO cells, immunoprecipitated with anti-Myc antibody, and analyzed by Western blotting using anti-HA antibody and anti-Myc antibody. As shown in Fig. 1C, S1P1-HA was found in the anti-Myc immunoprecipitate from S1P1-CHO cells transfected with Myc-RGS2. No interaction was observed with S1P1-HA between GST–RGS1, GST–RGS4 or GST–RGS5 (data not shown).


Figure 1
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  		Figure 1  RGS2 interacts with S1P1 but dissociates at high ligand concentrations. (A) Membrane proteins (20 µg) from CHO cells expressing S1P1-HA (S1P1-CHO) or MOCK (CHO) cells were incubated for 10 min in the absence or presence of 1 µM S1P. Each sample was then incubated for 60 min at 4 °C with the indicated purified GST fusion protein and glutathione Sepharose 4B. The precipitates were collected and resuspended in Laemmli sample buffer, and the proteins were separated on SDS-PAGE and analyzed by Western blotting with an anti-HA antibody to detect S1P1 (pull-down). Total membrane proteins from S1P1-CHO cells were separated on SDS-PAGE and analyzed by Western blotting using anti-HA antibody (lower panel). Two separate experiments gave similar results. (B) GST protein or GST fusion proteins, GST–RGS1, GST–RGS2, GST–RGS4 and GST–RGS5, expressed in E. coli BL21 (DE3) cells were solubilized with Laemmli sample buffer. The proteins were separated on SDS-PAGE and stained by CBB. Protein molecular weight standards (in kDa) are indicated. (C) S1P1- or MOCK-CHO cells were transfected with a plasmid encoding Myc-RGS2 or with empty vector as indicated. After serum starvation for 24 h, the cells were lysed in lysis buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MgCl2, 0.1% NP40 and protease inhibitors). The clarified cell lysate was then subjected to immunoprecipitation with anti-Myc antibody. The precipitate was subjected to SDS-PAGE (10% or 12% polyacrylamide gel), followed by Western blotting with anti-HA or anti-Myc antibody (upper panels). As a control for equal protein loading, the amount of S1P1-HA and endogenous actin in cell lysates were determined by immunoblotting using anti-HA or anti-actin antibody (lower panels). Two separate experiments gave similar results. (D) Membrane proteins (20 µg) from S1P1-CHO cells were incubated for 10 min in the indicated concentrations of S1P (lanes 2–7,11,12) or sphingosine (Sph, lanes 9, 10), then GST–RGS2 pull down assay was carried out as in (A). Two separate experiments gave similar results. The arrows indicate S1P1. C, no ligand stimulated control (lanes 1, 8).

 
We further examined whether the S1P-induced dissociation was affected by ligand concentration and found that RGS2 was dissociated with S1P1 in proportion to S1P concentration increases (Fig. 1D, lanes 1–7). In contrast, little dissociation was observed upon treatment with sphingosine, the dephosphorylated form of S1P, at any concentration (lanes 8–10). Together, these results show that RGS2 interacts, directly or indirectly, with S1P1, and that this interaction is sensitive to increased ligand concentrations. The amount of G{alpha}-GTP is known to be increased in S1P1-expressing cells in response to ligand in a dose-dependent manner (Windh et al. 1999). This suggest that the S1P1–RGS2 interaction might be sensitive to the increase in G{alpha}-GTP content.

RGS2 is involved in the decrease of cell motility at higher concentrations of the ligand

S1P activates cell motility via the S1P receptor S1P1 in various cell type (Kon et al. 1999; Wang et al. 1999). We examined the effect of S1P at concentrations from 10–10 M to 10–5 M on cell motility via S1P1 in S1P1-CHO cells using a modified Boyden chamber assay. As shown in Fig. 2A, S1P1-CHO cells exhibited enhanced cell motility after treatment with S1P at concentrations < 10–8 M in a dose-dependent manner. In contrast, a decrease of cell motility was observed at concentrations from 10–7 M to 10–5 M. Similar results were observed in a second S1P1-CHO clone (data not shown). No migration was observed in vector-transfected cells (data not shown).


Figure 2
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  		Figure 2  RGS2 is involved in the decrease of cell motility at higher concentrations of the ligand. (A) S1P1-CHO cells were incubated in serum-free medium for 4 h. Cells (5 x 105) were added to the upper chamber of a Transwell filter plate, and the indicated concentration of S1P was placed into the lower chamber. The cells were allowed to transmigrate for 4 h. Transmigrated cells were counted as described in Experimental procedures. These data represent the average of four independent experiments with error bars indicating the SD. C, no ligand treatment control. (B) S1P1-CHO cells (5 x 105) were transfected with siRNAs encoding RGS2 (RGS2-siRNA-1 and -2) or control siRNA. After a 48 h transfection, the RGS2 protein in the cell lysate was analyzed by Western blotting with an anti-RGS2 antibody (upper left panel). The blot was subsequently reprobed with an anti-HA antibody to detect S1P1 as a control for equal protein loading (lower left panel). The relative amount of RGS2 protein was quantified using the NIH Image program (right graph). The data represent the average of four independent experiments with error bars indicating the SD. Statistical significance: *P < 0.001. S1P1-CHO cells were transfected with control siRNA (C), RGS2-siRNA-1 (D), or RGS2-siRNA-2 (E) as in (B). S1P-induced migration assay was carried out as in (A). These data represent the average of six independent experiments with error bars indicating the SD. C, no ligand stimulated control. Statistical significance: *P < 0.001; ns, not significant.

 
To identify the essential role of RGS2 in the attenuation of the response of cell motility, RNA interference was employed to specifically knockdown the RGS2 protein. Two siRNAs against hamster RGS2 were generated and transfected into S1P1-CHO cells. As shown in Fig. 2B, RGS2 protein expression was significantly reduced in each RGS2-siRNA-transfected cell line compared with the control siRNA-transfected cells, yet the siRNA had no significant effect on the S1P1. RGS2 expression was reduced by approximately 50% in siRNA-RGS2-1 transfectants, and 80% in siRNA-RGS2-2 transfectants. Thus, siRNA is an effective way to accomplish RGS2 knockdown in S1P1-CHO cells.

To address whether decreased RGS2 expression might affect S1P-induced cell motility, S1P1-CHO cells were transfected with siRNAs and examined using a modified Boyden chamber. At concentrations of S1P below 10–7 M, the control siRNA-transfected cells exhibited a dose-dependent motility, whereas at 10–6 M S1P, the cell motility was decreased (Fig. 2C). In contrast, in siRNA-RGS2-1-transfected cells, which exhibited a 50% decrease in RGS2 expression, less suppression in cell motility was significantly observed at 10–6 M ligand as compared to control siRNA-transfected cells (P < 0.001), and little effect on cell motility was observed at concentrations below 10–7 M (Fig. 2D). Moreover, in siRNA-RGS2-2-transfected cells, which exhibited an 80% decrease in RGS2 expression, no suppression in cell motility was observed in the presence of 10–6 M ligand; instead, the cells displayed further enhanced cell motility, and little effect on cell motility was observed in cells stimulated at concentrations below 10–7 M (Fig. 2E). These results clearly show that RGS2 is closely involved in the attenuation of the response of cell motility observed with higher concentrations of the ligand.

Phosphorylation-negative S1P1 mutant exhibits no internalization but shows a decrease of cell motility at high ligand doses

The ligand-induced internalization of a GPCR involves desensitization of the receptor and leads to its down-regulation (Pao & Benovic 2005). Multiple serine clusters in the β-adrenergic receptor are known to be phosphorylated upon ligand stimulation (Fredericks et al. 1996), and this phosphorylation is indispensable for ligand-induced receptor internalization and subsequent desensitization (Sibley et al. 1987). We also found that S1P1 was phosphorylated upon metabolic labeling of the S1P1-CHO cells with 32P (data not shown). Phosphoamino acid analysis further revealed that S1P stimulation induced phosphorylation in S1P1 on the serine residues but not on threonine or tyrosine (Fig. 3A). To establish the specific residues that are phosphorylated and identify a phosphorylation-ablated mutant of S1P1, we generated a series of mutants targeting the serine residues (Fig. 3B). The mutants and wild-type S1P1 were each expressed transiently in CHO cells, and the cells were labeled with 32P, then stimulated with S1P. The receptor phosphorylation was analyzed by autoradiography. As shown in Fig. 3C, the IM3-4SA mutant was phosphorylated similarly to S1P1, indicating that the serine residues at the third intracellular loop (S-cluster-1) are generally not phosphorylated in response to S1P stimulation. In contrast, the S-cluster-2 (CT-5SA), S-cluster-3 (CT-2SA) and S-cluster-4 (CT-3SA) mutants each exhibited decreased S1P-induced phosphorylation. The CT-7SA and CT-8SA mutants, in which alanine replaced the serine residues of two clusters (clusters 2 and 3 and clusters 2 and 4, respectively), displayed further decreased phosphorylation. The CT-10SA mutant, in which the serine residues in all the S-clusters at the cytoplasmic tail were replaced with alanine residues, had no receptor phosphorylation upon S1P stimulation. These results revealed that multiple serine clusters in the cytoplasmic tail of S1P1 are phosphorylated upon S1P stimulation.


Figure 3
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  		Figure 3  The phosphorylation-negative S1P1 mutant exhibits no internalization but shows a decrease of cell motility at high ligand doses. (A) S1P1-CHO cells (1 x 106) were incubated for 16 h at 37 °C in serum free medium. The cells were metabolically labeled with [32P]-orthophosphate (40 µCi/mL), then stimulated with 1 µM S1P for 10 min. Cells were lysed, and the lysates were immunoprecipitated using an anti-HA antibody then deglycosylated with PNGase F. The deglycosylated [32P]-labeled S1P1 was subjected to phosphoamino acid analysis. The hydrolyzed samples were analyzed by two-dimensional thin layer chromatography at pH 1.9 and 3.5 in the presence of unlabeled phosphotyrosine (p-Tyr), phosphothreonine (p-Thr) and phosphoserine (p-Ser) standards. The circles on each panel indicate the relative migration of the ninhydrin-stained standards visible in the right panel. Three separate experiments gave similar results. (B) A schematic representation of the amino acid sequences of the mutant S1P1 used in this study. Four serine clusters, S-cluster-1 to S-cluster-4, are indicated. IM3 indicates the third intracellular loop region, and CT the cytoplasmic tail of S1P1. (C) CHO cells transiently expressing S1P1 or a mutant were metabolically labeled with [32P]-orthophosphate (75 µCi/mL), then stimulated with 1 µM S1P or sphingosine (SPH) for 10 min. Cells were lysed, and the lysates were immunoprecipitated with an anti-HA antibody then deglycosylated with PNGase F. The phosphorylated receptors were analyzed by SDS-PAGE and autoradiography. The arrows indicate the phosphorylated form of the S1P1 or mutant protein. Protein molecular weight standards (in kDa) are indicated on the left. (D) CHO cells were transfected with cDNA encoding S1P1 (a, b) or CT-10SA mutant (c, d). Twenty-four hours after serum starvation, the cells were incubated for 15 min in the absence (a, c) or presence (b, d) of 1 µM S1P, and then fixed, permeabilized, and immunostained with an anti-HA antibody to detect S1P1. Cellular localization of S1P1 was observed by confocal microscopy (left panels) with corresponding differential interference contrast (DIC) imaging (right panels). Scale bar, 20 µm. (E) S1P1 (open circles), CT-10SA (closed circles) or an empty vector (triangles) was transiently expressed in CHO cells. S1P-induced cell migration assay was carried out as in Fig. 2A. These data represent the average of six independent experiments with error bars indicating the SD. C, no ligand treatment control.

 
To examine the relationship between S1P1 internalization and its phosphorylation upon S1P stimulation, S1P1 and the CT-10SA mutant were expressed transiently in CHO cells, and the cells were immunostained in the absence or presence of S1P. As shown in Fig. 3D, most of the transiently expressed S1P1 was internalized from the cell surface (a) to an intracellular compartment upon S1P stimulation (b). In contrast, in CT-10SA-expressing cells (Fig. 3D, panels c and d) no internalization was observed upon ligand stimulation (d).

We next studied whether S1P1 internalization might be involved in the decrease of cell motility in CHO cells expressing the internalization-deficient mutant CT-10SA or wild-type S1P1. CT-10SA-expressing CHO cells exhibited enhanced cell motility after treatment with 10–8 M S1P, but a decrease of cell motility at concentrations from 10–7 M to 10–5 M (Fig. 3E, closed circles). A similar dose–response curve was observed in S1P1-expressing CHO cells (open circles). Although the S1P1 mutant CT-10SA inhibited desensitization of S1P-induced MAPK and Akt activation (data not shown), it did exhibit a decrease of cell motility at by high ligand concentrations with rates similar to those observed for the wild-type S1P1. These results clearly show that the inhibitory signal for cell migration mediated by higher concentrations of the ligand is independent of S1P1 internalization and desensitization.

High doses of either S1P or FTYP decrease S1P1-mediated cell motility

The phosphorylated form of FTY720 (FTYP) is known to bind to S1P1 (Brinkmann et al. 2002; Mandala et al. 2002) and to inhibit S1P-activated cell motility via S1P1 (Graler & Goetzl 2004). However, the function of FTYP on cell motility has not been examined. We found that FTYP-induced cell motility followed a typical bell-shaped dose–response curve, that is, high concentrations of FTYP did not enhance cell motility as well as the high doses of S1P did (data not shown). To examine whether stimulation with high doses of FTYP would suppress the enhanced cell motility observed with low ligand doses, cell motility in S1P1-CHO cells was analyzed upon co-stimulation with varying concentrations of the two compounds. As shown in Fig. 4, in cells stimulated with 10–8 M S1P, the normally enhanced cell motility was significantly suppressed by co-stimulation with 10–6 M FTYP. Conversely, the enhanced cell motility observed with a low dose of FTYP was also suppressed by co-stimulation with the high dose of S1P. These results may suggest that FTYP and S1P exhibit equivalent effects on cell motility as agonists for S1P1.


Figure 4
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  		Figure 4  High doses of either S1P or FTYP decrease S1P1-mediated cell motility. S1P1-CHO (2 x 106) cells were added to the upper chamber of a Transwell filter, and the indicated concentration of S1P and/or FTYP was placed into the lower chamber. Transmigrated cells were determined as described in Fig. 2A. These data represent the average of four independent experiments with error bars indicating the SD. Statistical significance: *P < 0.001 vs. 10–8 M S1P, {dagger}P < 0.001 vs. 10–8 M FTYP.

 
RGS2 is involved in excess FTYP-mediated attenuation of the response of cell motility

We also studied whether S1P1 internalization might be involved in the decrease of cell motility upon FTYP treatment. CT-10SA-expressing CHO cells exhibited enhanced cell motility after treatment with 10–8 M to 10–7 M FTYP, but did not enhance the cell motility at concentrations > 10–6 M (Fig. 5A, closed circles). A similar dose–response curve was observed in S1P1-expressing CHO cells (open circles), and no migration activity was observed in vector-transfected cells (triangles). We found RGS2 is involved in the decrease of cell motility observed with higher S1P doses (Fig. 2C–E). To examine whether RGS2 is involved in FTYP-mediated cellular signaling, we analyzed S1P1 interactions with GST–RGS2. The S1P1–RGS2 complex was stable upon stimulation with lower FTYP concentrations, whereas the complex dissociated at concentrations > 10–6 M (Fig. 5B). To address whether decreased RGS2 expression might affect FTYP-induced cell motility, the siRNA transfectants tested in Fig. 2B were again used, as shown in Fig. 5C. At 10–8 M FTYP, the control siRNA-transfected S1P1-CHO cells exhibited enhanced cell motility, whereas at 10–6 M FTYP the cell motility was decreased. In contrast, in siRNA-RGS2-1 transfected cells, less suppression in cell motility was observed at 10–6 M FTYP as compared to control siRNA-transfected cells, and little effect on cell motility was observed at concentration at 10–8  M. Moreover, in siRNA-RGS2-2-transfected cells, no suppression in cell motility was observed in the presence of 10–6 M FTYP; instead, the cells again displayed slightly enhanced cell motility. We further examined the effect of co-stimulation with high and low doses of the ligand on cell motility in CHO cells expressing the internalization-deficient mutant CT-10SA or wild-type S1P1, and found that in each cells stimulated with 10–8 M S1P, enhanced cell motility was significantly suppressed by co-stimulation with 10–6 M FTYP (Fig. 5D). Conversely, the enhanced cell motility in each cells observed with 10–8 M FTYP was also suppressed by co-stimulation with 10–6 M S1P (data not shown). These results clearly show that S1P1-mediated cell motility is decreased with high doses of either of the S1P1 ligands, S1P or FTYP, and that this response acts independently of S1P1 internalization. Moreover, RGS2 is involved in the attenuation of the response of cell motility observed with higher doses of either ligand.


Figure 5
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  		Figure 5  RGS2 is involved in FTYP-mediated attenuation of the response of cell motility. (A) S1P1 (open circles), CT-10SA (closed circles) or an empty vector (triangles) was transiently expressed into CHO cells. The cells (5 x 105) were added to the upper chamber of a Transwell filter plate, and the indicated concentration of FTYP was placed into the lower chamber. Transmigrated cells were determined as described in Fig. 2A. These data represent the average of six independent experiments with error bars indicating the SD. C, no ligand treatment control. (B) Membrane proteins (20 µg) from S1P1-CHO cells were incubated for 10 min in the indicated concentrations of FTYP, then GST–RGS2 pull down assay was carried out as in Fig. 1D. Two separate experiments gave similar results. The arrows indicate S1P1. C, no ligand stimulated control. (C) S1P1-CHO cells were transfected with RGS2-siRNA-1, RGS2-siRNA-2, or control siRNA as in Fig. 2B. FTYP-induced migration assay was carried out as in (A). These data represent the average of six independent experiments with error bars indicating the SD. Statistical significance: *P < 0.001; ns, not significant. (D) S1P1 or CT-10SA was transiently expressed into CHO cells. The cells (5 x 105) were added to the upper chamber of a Transwell filter plate, and the indicated concentration of S1P and/or FTYP was placed into the lower chamber. Transmigrated cells were determined as described in (A). These data represent the average of four independent experiments with error bars indicating the SD. Statistical significance: *P < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
We conclude that there is an RGS2-mediated inhibitory mechanism involved in cell migration that is induced through the activation of S1P1 by high concentrations of its ligand, and this mechanism acts independently of S1P1 internalization and desensitization. At least 30 genes encoding RGS proteins have been predicted in the human genome (Hepler 2003), and the corresponding proteins have been classified into six subfamilies based on structure and function (Hollinger & Hepler 2002). RGS2 belongs to the B/R4 family, whose members have a simple structure among RGS superfamilies. We found that RGS2 and RGS5 were expressed predominantly in CHO cells among B/R4 family (T Kohno and Y Igarashi, unpubl. data). In cells over-expressing other B/R4 family members, RGS1, RGS3, RGS4 or RGS5, an inhibition of MAPK activation is observed upon S1P stimulation (Cho et al. 2003a,b), however little is known about the interaction between these RGS proteins and S1P receptors, or their response to ligand doses. Interestingly, although the structure of RGS4 is similar to that of RGS2 (Heximer et al. 1999), RGS4 did not interact with S1P1 (Fig. 1). Our findings provide the first evidence for an interaction, direct or indirect, between S1P1 (or a S1P1–G{alpha}–GDP complex) with RGS2, an interaction that is sensitive to ligand concentrations. S1P1 interacts dominantly with the G{alpha}i protein, over other G{alpha} members such as G{alpha}q, G{alpha}s and G{alpha}12/13; a characteristic that differs from other S1P receptors (Lee et al. 1996). Reportedly, RGS2 interacts dominantly with G{alpha}q rather than G{alpha}i in vitro (Heximer et al. 1999). However, there were many reports providing a role for RGS2 in G{alpha}i signaling (Takesono et al. 1999). Clearly, further studies will be necessary to elucidate the precise molecular mechanism of the dissociation and association between RGS2 and S1P1. Furthermore, S1P1 may also interact with other RGS proteins in vivo, as RGS protein expression varies among cell types.

S1P2, which is expressed endogenously in CHO cells, inhibits cell motility (Okamoto et al. 2000). In parental CHO cells, IGF-I-induced cell motility is, in fact, inhibited by co-stimulation with 10–6 M S1P (T Kohno and Y Igarashi, unpubl. data). In contrast, a similar bell-shaped S1P dose–response curve was obtained for S1P1-over-expressing HEK293 cells, which lack endogenous S1P2 (T Kohno and Y Igarashi, unpubl. data), suggesting that S1P2-independent signaling is involved in the decrease of cell motility. In the present study, we evaluated cell motility at 4 h post-stimulation, by which time the amount of S1P1 expression had decreased by approximately 20% (data not shown). However, the CT-10SA mutant, which prevent internalization-dependent receptor degradation, had no significant effect on the decrease of cell motility (Figs 3E, 5A), and knockdown of RGS2 rescued the suppression of cell motility normally observed upon stimulation with high ligand concentrations (Figs 2D,E, 5C). This suggests that decreased S1P1 expression had no effect on the decrease of cell motility in our experimental conditions.

In this study, we showed that FTYP and S1P exhibited equivalent effects on cell motility as agonists for S1P1. Analysis of SPHK2 knockout mice showed that in blood FTY720 is constitutively modified by phospho–dephospho conversion (Kharel et al. 2005). That data shows that down-regulation of S1P1 is not involved in the inhibition of lymphocyte motility. FTYP can exist in blood at higher concentrations, because the kinetics of FTYP metabolism is slower than that for S1P (Mandala et al. 2002). S1P concentrations in blood remain low, because most is adsorbed to plasma lipoproteins (Okajima 2002). Mouse lymphocytes express a large number of RGS proteins including RGS1, RGS2, RGS10, RGS13, RGS14, RGS16 and RGS19 (Kehrl 2006). Previous studies using mice with a conditional knockout of T cells expressing S1P1, showed that spontaneous circulation of lymphocytes is required for the activation of S1P1 by physiological concentrations of S1P and subsequent signaling (Allende et al. 2004; Matloubian et al. 2004), suggesting that low concentrations of S1P are involved in lymphocyte motility. We showed that stimulation with low concentrations of S1P1 ligand enhances cell motility, whereas excess ligand stimulation suppresses it. Taken together, we believed that inhibition of lymphocyte motility by higher doses of FTYP might be related to an RGS2-mediated inhibition of cellular signaling.


   Experimental procedures
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 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Cell culture and transfection

Chinese hamster ovary (CHO) cells were cultured in Ham's F-12 medium (Sigma, St Louis, MO) containing 10% fetal bovine serum (FBS; Iwaki, Chiba, Japan), 100 U/mL penicillin and 100 µg/mL streptomycin (Sigma) at 37 °C in a humidified 5% CO2 atmosphere. Transfections were carried out using a LipofectAMINE plus kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Stable transfected clones were selected with 400 µg/mL Geneticin (G-418 sulfate, Invitrogen). C-terminal HA-tagged S1P1 and mutant receptor transfectants were cultured in media containing 10% charcoal (C-4386, Sigma)-stripped FBS and antibiotics as above.

Cell migration assay

Chemotactic cell migration was carried out as described previously with minor modifications (Kohno et al. 2003). Cells were trypsinized and added to the upper chambers of the Transwell filters, and medium containing sphingosine 1-phosphate (S1P, Matreya, Pleasant Gap, PA), FTY720 (Sankyo Co., Tokyo, Japan), FTYP (enzymatically generated from FTY720 using recombinant sphingosine kinases), or recombinant human insulin-like growth factor (IGF)-I (Peprotech EC, London, UK) was added to the lower chamber, then the cultures were incubated at 37 °C in a humidified CO2 incubator. After 4 h, cells remaining on the upper surface of the filter were removed with a cotton swab. Cells on the lower side of the Transwell filter were fixed with cold methanol and stained with 1% crystal violet in 2% ethanol. Cell images were digitally captured from an Olympus IX70 microscope (Olympus, Tokyo, Japan) with a CCD camera (Sony, Tokyo, Japan). The number of cells was determined using the "analyze particles method" of the NIH Image program.

Construction of the mutant receptor cDNAs

Seven mutants of S1P1, in which serine residues were replaced by alanine residues, were amplified from pcDNA3-S1P1 (Kohno et al. 2002) by PCR using the KOD-plus DNA polymerase (TOYOBO, Tokyo, Japan) and oligonucleotides, for IM3-4SA-F (5'-GCGCTGCTGAGAAGGCTCTGGCCTTGCTGAAG-3'), IM3-4SA-R (5'-GAGCGGCCTTGGAGATGTTCTTGCG GAAG-3', CT-2SA-F (5'-GTCAATTCTTCTTCGGATCCC TACCCATAC-3'), CT-2SA-R (5'-GTTTCCAGCCGCCAT AATGGTCTCTGGGTTG-3'), CT-3SA-F (5'-GTCAATGCT GCTGCGGATCCCTACCCATAC-3'), CT-3SA-R (5'-GTT TCCAGACGACATAATGGTCTCTGGGTTG-3'), CT-5SA-F (5'-GCATGGAATTTGCCCGCGCCAAAGCAGAC-3') and CT-5SA-R (5'-CTGGGATGATGGGCCTCTTGAATTTGCC-3'). For the CT-7SA and CT-8SA mutants, PCR was carried out with the CT-5SA mutant as a template and oligonucleotides (CT-2SA-F/R and CT-3SA-F/R, respectively). For CT-10SA mutants, PCR was carried out with the CT-7SA mutant as a template and oligonucleotides (CT-3SA-F and CT-2SA-R). The antisense primers were treated with a T4 polynucleotide kinase (Takara, Shiga, Japan) in advance. The cycling parameters were an initial denaturation step of 2 min at 94 °C, followed by 25 cycles of denaturation at 94 °C for 20 s, annealing at 65 °C for 3 s, and extension at 74 °C for 3 min. The CT-2SA/3SA mutant was generated by ligating the C-terminal fragment of CT-10SA from a XcmI/XhoI digestion into a pcDNA3-S1P1 that had been cut by XcmI and XhoI. All genes were completely sequenced after mutagenesis. Localization of the mutant proteins was determined by confocal microscopy (Kohno et al. 2003).

Isolation of RGS clones

For RT-PCR, total RNA was isolated from CHO cells, HEK293 cells or NIH3T3 cells using TRIzol reagent (Invitrogen) and the manufacturer's recommended procedures. RT-PCR was carried out using SuperScript One-Step RT-PCR system (Invitrogen) and oligonucleotides, for RGS1-F (5'-ATGCC AGGAATGTTCTTTTCTGCTAGCCC-3'), RGS1-R (5'-TCACTTTAAAGTATTTGCCTGAAGGTCAT-3'), RGS2-F (5'-GGATCCATGCAAAGTGCCATGTTCCTG-3'), RGS2-R (5'-GGATCCGTAGCATGGGGCTCCGTGGTG-3'), RGS4-F (5'-ATGTGCAAAGGACTTGCAGGTCTGC-3'), RGS4-R (5'-TTAGGCACACTGGGAGACCAGGGAA-3'), RGS5-F (5'-ATGTGTAAGGGACTGGCAGCTCTG-3') and RGS5-R (5'-CTACTTGATTAGCTCCTTATAAAATTCAG-3'). The amplified RGS1, RGS4 and RGS5 were introduced into BamHI sites by PCR with oligonucleotides, for RGS1-F2 (5'-GGATC CATGCCAGGAATGTTCTTTTCTGC-3'), RGS1-R2 (5'-GGATCCTTTAAAGTATTTGCCTGAAGG-3'), RGS4-F2 (5'-GGATCCATGTGCAAAGGACTTGCAGGTC-3'), RGS4-R2 (5'-GGATCCGCACACTGGGAGACCAGGGAAG-3'), RGS5-F2 (5'-GGATCCATGTGTAAGGGACTGGCAGC-3') and RGS5-R2 (5'-GGATCCTTGATTAGCTCCTTATAAA ATTC-3'). The PCR products were subcloned into the BamHI site of the pGEX2T vector (Amersham Biosciences, Piscataway, NJ), or pcDNA3-Myc vector (Kohno et al. 2003). GST-fusion proteins were expressed in Escherichia coli BL21 (DE3) and purified with affinity column chromatographies.

RNA interference

Double-stranded RNA (dsRNA) corresponded to the following target sequences: hamster RGS2-siRNA-1 bp 431–455, 5'-CUCC CAAAGAGAUAAACAUAGACUU-3'; hamster RGS2-siRNA-2 bp 567–591, 5'-CUUGGAGUCAGAAUUCUACCAGGAC-3'; and control RNA, 5'-AUUGUCAUUCAUGACGUGGUA AUCA-3'. The dsRNA (400 pmol) was introduced into 1.5 x 106 cells using Lipofectamine2000 reagent (Invitrogen) according to the manufacturer's instructions. Cells were harvested for migration assays and Western blot analysis 48 h post-transfection.

Receptor phosphorylation

Stably transfected CHO cells expressing S1P1 (S1P1-CHO), or transiently transfected CHO cells expressing S1P1 or one of the mutants, were incubated in phosphate-free DMEM (Invitrogen, Carlsbad, CA) for 1 h, then incubated for 4 h with 0.04–0.1 mCi of [32P]-orthophosphate (NEX053; Perkin Elmer Life Sciences, Japan) in fresh phosphate-free DMEM containing 0.1% fatty acid-free BSA (Sigma). A ligand was then added directly to the medium. After incubations of various times, cells were washed three times with cold PBS and lysed with extraction buffer A (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 10 mM MgCl2, 5 mM EDTA, 5 mM sodium orthovanadate, 1% Triton X-100, 0.1% sodium lauryl sulfate, 0.5% sodium deoxycholate and protease inhibitors). The cell lysate was immunoprecipitated using an anti-HA antibody (Y-11, Santa Cruz Biotechnology, Santa Cruz, CA). The immunoprecipitates were then deglycosylated (Kohno et al. 2002) and resolved by SDS-PAGE. The gel was then dried and analyzed using a BAS2500 Bio Imaging Analyzer (Fuji Film, Tokyo, Japan).

GST–RGS binding assay

Cells were grown to confluency on culture dishes, followed by serum starvation for 16 h. The cells were washed once with cold phosphate-buffered saline (PBS) and sonicated in homogenization buffer (20 mM HEPES (pH 7.5), 50 mM NaCl, protease inhibitor cocktail, and 2 mM EDTA). After a centrifugation at 2000 g for 5 min at 4 °C, supernatants were collected and centrifuged at 35 000 g for 30 min at 4 °C. The membrane pellets were washed once with homogenization buffer and resuspended in buffer (50 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM EDTA, and 1 mM MgCl2). The protein concentrations were measured by a Micro BCA kit (Pierce). Varying concentrations of S1P and 20 µg membrane protein were mixed in 100 µL binding buffer (50 mM HEPES (pH 7.5), 100 mM NaCl, 5 mM MgCl2, 30 µM GDP, 1 nM GppNHp, 2 mM DTT and 0.1% fatty acid free BSA) and incubated for 10 min. Then, samples were mixed with extraction buffer B (50 mM HEPES (pH 7.5), 100 mM NaCl, 5 mM MgCl2, 1% NP-40, and a protease inhibitor cocktail). After centrifugation at 20 000 g for 10 min at 4 °C, the supernatants were collected and incubated with a purified glutathione S-transferase (GST)–RGS protein and glutathione Sepharose 4B (Amersham Biosciences, Piscataway, NJ) at 4 °C for 60 min. The amount of GST–RGS proteins was monitored by Coomassie Blue staining. The precipitates were collected by centrifugation at 8000 g for 40 s at 4 °C, washed three times with lubrol buffer, then resuspended in Laemmli's sample buffer. These samples were analyzed by Western blotting using an anti-HA antibody (Y-11).


   Acknowledgements
 
This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (B) (12140201) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

We thank Dr A. Kihara for helpful discussion, and Y. Hiraga (for the laboratory) for providing siRNA constructs. FTY720 was a kind gift of Dr F. Nara (Sankyo Co., Tokyo, Japan).


   Footnotes
 
Communicated by: Yoshinori Ohsumi

aPresent address: Department of Biochemistry, Cancer Research Institute, Sapporo Medical University School of Medicine, Sapporo, Japan. Back

* Correspondence: Email: yigarash{at}pharm.hokudai.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
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Received: 3 March 2008
Accepted: 10 April 2008




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