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Genes to Cells (2009) 14, 911-923. doi:10.1111/j.1365-2443.2009.01319.x
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Palmitoylation of the sphingosine 1-phosphate receptor S1P1 is involved in its signaling functions and internalization

 Yusuke Ohno1{dagger}, Ayako Ito2{dagger}, Ren Ogata1, Yuki Hiraga2, Yasuyuki Igarashi3 and Akio Kihara1,*

1 Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12-jo, Nishi 6-choume, Kita-ku, Sapporo 060-0812, Japan
2 Laboratory of Biomembrane and Biofunctional Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12-jo, Nishi 6-choume, Kita-ku, Sapporo 060-0812, Japan
3 Laboratory of Biomembrane and Biofunctional Chemistry, Faculty of Advanced Life Sciences, Hokkaido University, Kita 21-jo, Nishi 11-choume, Kita-ku, Sapporo 001-0021, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The lipid mediator sphingosine 1-phosphate (S1P) regulates several cellular processes through binding to its receptors (S1P1–S1P5), which are heterotrimeric G protein-coupled receptors. Here, we report that all S1P receptors are palmitoylated. In S1P1, three Cys residues in the cytoplasmic tail are palmitoylated. We examined the roles of palmitoylation of S1P1 using model cells in which wild-type S1P1 or a non-palmitoylated mutant S1P1 was overproduced. Compared with wild-type S1P1, the non-palmitoylated S1P1 exhibited binding affinity similar to the natural ligand S1P but lower to the synthetic ligand FTY720 phosphate (FTY720-P), the active form of the immunomodulator FTY720. However, downstream signaling of non-palmitoylated S1P1 was similarly affected by S1P and FTY720-P stimulation. Moreover, upon stimulation with S1P, internalization of the mutant non-palmitoylated S1P1 was retarded, compared with that of the wild-type protein. This effect was much more pronounced with FTY720-P stimulation. Similar differences were observed for the phosphorylation of S1P1 and its mutant. These findings may provide insights into the molecular mechanisms of the pharmacological effects of FTY720. Finally, palmitoylation of wild-type S1P1 increased upon treatment with S1P, suggesting that S1P1 undergoes a palmitoylation/depalmitoylation cycle after stimulation by its ligands.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Many proteins, especially those involved in signal transduction, are modified on their Cys residues by fatty acid acylation (S-acylation) (Resh 1999; Smotrys & Linder 2004). Fatty acids utilized in S-acylation are heterologous, but palmitic acid is the most common. Therefore, S-acylation is often referred to as palmitoylation. Palmitoylation plays a role in regulating protein localization, membrane association, protein–protein interaction, protein–lipid interaction and enzyme activity (Resh 1999; Smotrys & Linder 2004). Unlike other lipid modifications such as N-myristoylation, prenylation and GPI-anchoring, palmitoylation is reversible. Thus, target proteins can be manipulated via a palmitoylation/depalmitoylation cycle.

G protein-coupled receptors (GPCRs) are the most abundant class of cell surface receptors and are involved in a variety of cellular functions. Recent studies have indicated that lipid modifications play important roles in GPCR-associated signaling (Qanbar & Bouvier 2003; Escribáet al. 2007). Most GPCRs are modified by palmitoylation on one or more Cys residues, although G{alpha} proteins are N-myristoylated and/or palmitoylated, and G{gamma} proteins are prenylated (Escribáet al. 2007). In contrast to myristoylation and prenylation, palmitoylation requires no strict motifs; however, in GPCRs, Cys residues positioned 10–14 amino acids downstream of the last transmembrane segment are usually palmitoylated (Qanbar & Bouvier 2003; Escribáet al. 2007). The roles of palmitoylation vary among GPCRs. For example, palmitoylation is required for the cell surface expression of some GPCRs, such as bovine opsin (Karnik et al. 1993), leutropin/choriogonadotropin receptor (Zhu et al. 1995), and H2 histamine receptor (Fukushima et al. 2001). Yet while palmitoylation is necessary for the signaling functions of some GPCRs, including the β2-adrenergic receptor (O’Dowd et al. 1989), it is not involved in the signaling of others, such as the {alpha}2A-adrenergic receptor (Kennedy & Limbird 1993). Palmitoylation also can affect the phosphorylation of GPCRs, due to the close proximity of the respective amino acid residues. Normally, GPCRs are phosphorylated and then internalized during signaling initiated by exposure to their ligands. However, a palmitoylation site mutant of the β2-adrenergic receptor is hyperphosphorylated even without ligand stimulation (Moffett et al. 1993), yet phosphorylation is suppressed in palmitoylation mutants of the V1a vasopressin receptor (Hawtin et al. 2001) and the chemokine receptor CCR5 (Kraft et al. 2001). Therefore, it is important to determine the roles palmitoylation exerts on each individual GPCR.

The GPCRs S1P1–S1P5 recognize the lipid mediator sphingosine 1-phosphate (S1P), which regulates a variety of cellular functions (Spiegel & Milstien 2003; Sanchez & Hla 2004; Kihara et al. 2007). Of these receptors, S1P1 is the most important physiologically, especially in the vascular and immune systems (Spiegel & Milstien 2003; Sanchez & Hla 2004; Kihara et al. 2007). For example, S1P1 performs a pivotal function in lymphocyte egress from the thymus and secondary lymphoid organs (Chiba et al. 2006; Kihara et al. 2007; Kihara & Igarashi 2008). In fact, a novel therapeutic (FTY720) currently undergoing clinical trials as an immunomodulator (Budde et al. 2006; Chiba et al. 2006; Kihara & Igarashi 2008) is actually a prodrug whose active phosphorylated form, FTY720 phosphate (FTY720-P), acts as a ligand for all S1P receptors except S1P2 (Brinkmann et al. 2002; Mandala et al. 2002).

After ligand binding, S1P1 is internalized, desensitizing the stimulus (Liu et al. 1999). When the ligand is S1P, the S1P1 receptor is recycled back to the cell surface. However, when the stimulus is FTY720-P, S1P1 is not recycled to the plasma membrane but is degraded, resulting in a loss of the receptor at the cell surface (Gräler & Goetzl 2004; Matloubian et al. 2004; Jo et al. 2005). To date, the molecular mechanism causing the differences between S1P and FTY720-P in internalization of S1P1 has remained unclear.

Several post-translational modifications have been reported for S1P1, including phosphorylation (Watterson et al. 2002), N-glycosylation (Kohno et al. 2002), sulfation (Fieger et al. 2005) and ubiquitinylation (Oo et al. 2007). However, palmitoylation of S1P1 and its role have not been previously examined. In the present study, we have demonstrated that three Cys residues in the C-terminal tail of S1P1 are palmitoylated. We also report that palmitoylation of S1P1 is involved in high-affinity binding to FTY720-P but not to S1P, and in the downstream signaling of S1P1, irrespective of S1P and FTY720-P stimulation. Palmitoylation also plays a role in the internalization of S1P1, especially following FTY720-P stimulation. Thus, palmitoylation affects several post-stimulation processes involving S1P1, sometimes differently following S1P stimulation versus FTY720-P stimulation. The differential effects of palmitoylation on internalization may provide important clues for understanding the molecular mechanism of the pharmacological effects of FTY720.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
All S1P receptors are palmitoylated

Like other GPCRs, S1P receptors contain one to three Cys residues proximal to the last transmembrane in the C-terminal tail (Fig. 1a). To investigate whether S1P receptors are palmitoylated, each receptor was cloned and transiently expressed in human embryonic kidney (HEK) 293T cells as a C-terminally 3xFLAG-tagged protein. All S1P receptors were detected using immunoblotting as several bands, typically a broad upper band and sharp lower band(s) (Fig. 1b). Different protein bands of each S1P receptor presumably represent proteins carrying different forms of glycosylation, as all S1P receptors contain potential N-glycosylation sites at their N-termini, and the N-glycosylation of S1P1 has already been reported (Kohno et al. 2002). To examine whether the S1P receptors carried high mannose- or complex-type N-glycosylation, we treated each protein sample with Endoglycosidase H (Endo H), which removes only high mannose-type N-glycosylation, and peptide : N-glycosidase F (PNGase F), which can remove both high mannose and complex types. The lower sharp bands were shifted to lower molecular weight bands upon treatment with Endo H (Fig. 1b), indicating that these proteins are modified with high mannose-type glycosylation. On the other hand, the upper broad bands were shifted to low molecular weight bands only by treatment with PNGase F (Fig. 1b), indicating that they were modified with complex-type glycosylation. Proteins destined to the plasma membrane, including GPCRs, are synthesized in the ER, where high mannose-type N-glycosylation occurs, are then transported to the Golgi, where the glycosylation is modified to the complex type and, finally, are delivered to the plasma membrane. Therefore, high mannose-type glycosylated S1P receptors may localize in the ER, while complex-type glycosylated S1P receptors may localize in the plasma membrane (or possibly in the Golgi). Detecting such substantial levels of ER-localized receptors may be due to the transient expression system, in which protein synthesis begins to increase ~24 h after transfection. The cells were transfected 24 h before use, and so the observed S1P receptor proteins might have been newly synthesized.


Figure 1
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  		Figure 1  All S1P receptors are palmitoylated. (a) Amino acid sequences of the C-terminal hydrophilic regions of human S1P receptors are shown. Cys residues that may be potential sites for palmitoylation are highlighted in boxes. TM7, transmembrane 7. (b–d) HEK 293T cells were transfected with plasmids encoding S1P1–S1P5, each C-terminally tagged with 3xFLAG. (b) Twenty-four hours after transfection, total cell lysates were prepared, treated with nothing (–), Endo H (E) or PNGase F (P) and separated using SDS-PAGE, followed by immunoblotting with anti-FLAG antibodies. (c) Twenty-four hours after transfection, the culture medium was changed to serum-free DMEM, and after a 1-h incubation at 37°C, cells were labeled with 0.2 mCi [3H]palmitic acid for 2 h at 37°C. Total cell lysates were prepared, and S1P receptor proteins were immunoprecipitated with anti-FLAG M2 agarose beads. The precipitates were separated using SDS-PAGE and detected using autoradiography (upper panel) or using immunoblotting with anti-FLAG antibodies (lower panel). (d) Twenty-four hours after transfection, total cell lysates were prepared and analyzed using ABE assay. Lysates were treated with NEM to block non-palmitoylated Cys residues, and were subjected to immunoprecipitation with anti-FLAG M2 agarose. The precipitates were incubated in the presence or absence of the thioester linkage-cleaving reagent hydroxylamine (NH2OH), which removes palmitic acid and with EZ-linkTM Biotin-HPDP, to biotinylate free thiol groups. Aliquots of samples were subjected to immunoblotting with anti-FLAG antibodies (lower panel), while the remaining proteins were precipitated with avidin beads then detected using immunoblotting with anti-FLAG antibodies (upper panel). IB, immunoblotting; AvP, avidin precipitation.

 
HEK 293T cells transiently expressing each S1P receptor were then metabolically labeled with [3H]palmitic acid. As shown in Fig. 1c, all S1P receptors were radiolabeled, although the intensities of the label varied among the receptors. Labeling efficiency was the highest for S1P1 and S1P5, followed by S1P4. The number of potential palmitoylated Cys residues in the receptor was at least partly responsible for the high labeling efficiency (S1P1, S1P4 and S1P5 each have three); however, this did not always correlate with labeling efficiency. For example, S1P3, which contains two potential palmitoylated Cys residues, was less efficiently labeled with [3H]palmitic acid than was S1P2, which contains only one. Therefore, a higher population of S1P3 may exist in a non-palmitoylated state compared with the other S1P receptors, or S1P3 may be depalmitoylated more rapidly. Both high mannose- and complex-type glycosylated S1P receptors were labeled, indicating that palmitoylation, at least partly, already occurs in the ER, although the possibility that the S1P receptors are further palmitoylated or re-palmitoylated after depalmitoylation at other organelles cannot be excluded.

Palmitoylation of the S1P receptors was further confirmed using an acyl-biotinyl exchange (ABE) assay (Drisdel & Green 2004), in which palmitic acid is removed via hydroxylamine-mediated cleavage of the palmitoyl–thioester bond and the remaining thiol group is labeled with a thiol-specific biotinylation reagent. Non-palmitoylated Cys residues were first protected by the thiol modifier N-ethyl maleimide (NEM), and so do not react with the thiol-specific biotinylation reagent. The biotinylated proteins were recovered by avidin–agarose and detected using immunoblotting. Using this method, all S1P receptors were again determined to be palmitoylated proteins (Fig. 1d).

S1P1 is palmitoylated on three Cys residues in the C-terminal tail

We focused on the palmitoylation of S1P1, as it is the physiologically most important S1P receptor. To examine whether the Cys residues are palmitoylated as expected, we constructed S1P1(3CA)-3xFLAG, in which Cys-328, -329 and -331 were all changed to Ala. HEK 293T cells expressing S1P1-3xFLAG or S1P1(3CA)-3xFLAG were metabolically labeled with [3H]palmitic acid. In contrast to wild-type S1P1, no labeling was detected in S1P1(3CA)-3xFLAG (Fig. 2a), indicating that S1P1 is palmitoylated on these Cys residues.


Figure 2
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  		Figure 2  S1P1 is palmitoylated on three Cys residues in the C-terminal tail. (a) HEK 293T cells transfected with pCE-puro 3xFLAG-4 (vector; Vec), pCE-puro S1P1-3xFLAG (wild type; WT) or pCE-puro S1P1(3CA)-3xFLAG [S1P1(3CA); 3CA] were labeled with 0.2 mCi of [3H]palmitic acid for 3 h. The S1P1 receptors were immunoprecipitated from total cell lysates with anti-FLAG M2 agarose and separated using SDS-PAGE, followed by autoradiography (upper panel) or by immunoblotting with anti-FLAG antibodies (lower panel). (b) HEK 293T cells were transfected with pCE-puro 3xFLAG-4 or its derivatives encoding wild type or the indicated Cys-to-Ala substituted mutant of S1P1-3xFLAG. Twenty-four hours after transfection, total cell lysates were prepared and analyzed using ABE assay as in Fig. 1. Lysates were treated with NEM, and subjected to immunoprecipitation with anti-FLAG M2 agarose. The immunoprecipitates were incubated in the presence or absence of hydroxylamine (NH2OH) and biotinylated with EZ-linkTM Biotin-HPDP. Aliquots of samples were subjected to immunoblotting with anti-FLAG antibodies (lower panel), whereas the remaining proteins were precipitated with the avidin beads then detected using immunoblotting with anti-FLAG antibodies (upper panel). IB, immunoblotting; AvP, avidin precipitation.

 
We also created single and double Cys-to-Ala substituted mutants (C328A, C329A, C331A, C328/329A, C329/331A and C328/331A) and subjected them to the ABE assay. Wild-type S1P1-3xFLAG and all of the single and double Cys-substituted mutants were detected as palmitoylated proteins, although the triple Cys-substituted mutant [S1P1(3CA)-3xFLAG] was not (Fig. 2b). The band intensities were not dependent on the number of palmitoylated Cys residues. This result is reasonable, as biotinylated proteins are logically recovered by avidin beads with nearly equal efficiency, regardless of the number of biotinylated Cys residues, and we detected them with an anti-FLAG antibody, rather than an anti-biotin antibody. In conclusion, S1P1 is palmitoylated on Cys-328, Cys-329 and Cys-331 residues.

Palmitoylation is not involved in cell surface expression of S1P1

Palmitoylation is required for the surface expression of some GPCRs (Escribáet al. 2007). Therefore, we examined the intracellular localization of the wild-type (S1P1-3xFLAG) and non-palmitoylated [S1P1(3CA)-3xFLAG] forms of S1P1. Chinese hamster ovary (CHO) cells stably expressing wild-type (S1P1-CHO) and non-palmitoylated S1P1 [S1P1(3CA)-CHO] were subjected to indirect immunofluorescent microscopy using anti-FLAG antibodies. Both wild-type and non-palmitoylated S1P1 were observed in the plasma membrane (Fig. 3a), indicating that palmitoylation is not required for proper localization of S1P1. Both S1P1-CHO and S1P1(3CA)-CHO cells expressed S1P1 to similar levels judging from immunoblotting (Fig. 3b) and flow cytometry (Fig. 3c).


Figure 3
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  		Figure 3  Palmitoylation is not required for cell surface expression of S1P1. (a) S1P1-CHO and S1P1(3CA)-CHO cells were fixed with 3.7% formaldehyde, permeabilized with 0.1% Triton X-100, and immunostained with anti-FLAG antibodies. The stained cells were analyzed using a fluorescence microscope Axioskop 2 PLUS (lens x63/1.40 OIL; ocular x10). Calibration bar, 10 µm. (b) Total lysates were prepared from Vector-CHO, S1P1-CHO and S1P1(3CA)-CHO cells. Proteins (3 µg) were separated using SDS-PAGE and subjected to immunoblotting with anti-FLAG antibodies. Uniform protein loading was demonstrated using immunoblotting with anti-GAPDH antibodies. (c) Vector-CHO (grey peaks in both left and right panels), S1P1-CHO (left panel) and S1P1(3CA)-CHO (right panel) cells were incubated for 12 h with F-12 medium containing charcoal-treated FBS, then for another 12 h with F-12 medium. Cells were then stained with anti-S1P1 antibodies and subjected to flow cytometry. Vec, vector; WT, wild type S1P1; 3CA, S1P1(3CA).

 
Palmitoylation of S1P1 is required for efficient S1P signal transduction

We next investigated the role of palmitoylation in S1P signaling. S1P1 couples to Gi and transduces the S1P signal to several downstream pathways including the Ras/ERK, cAMP/protein kinase A and phosphatidylinositol 3-kinase/Akt/Rac pathways (Spiegel & Milstien 2003; Sanchez & Hla 2004; Kihara et al. 2007). We first examined the binding affinities of the wild-type and 3CA mutant S1P1 to S1P using a binding assay and [32P]S1P. We found that the wild-type and mutant proteins exhibited similar Kd values (39.4 and 31.4 nM respectively).

We next investigated the signaling activities of the wild-type and non-palmitoylated mutant S1P1 by measuring the phosphorylation levels of ERK upon treatment with various concentrations of S1P. Maximal activation of ERK was observed in S1P1-CHO cells at 5 nM S1P (Fig. 4a,b) and the EC50 value was estimated to be 1.4 nM. Although the EC50 value of the 3CA mutant was similar (2.4 nM), the maximal p-ERK/ERK value was determined to be 32% of that observed for the wild-type S1P1, after subtracting background levels (those of the vector-transfected cells). These results suggest that palmitoylation affects the signaling activity of S1P1 without affecting its binding to S1P.


Figure 4
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  		Figure 4  Palmitoylation of S1P1 is required for efficient activation of the S1P signaling pathways. (a–f) Vector-CHO, S1P1-CHO and S1P1(3CA)-CHO cells were incubated for 12 h with F-12 medium containing charcoal-treated FBS, and for another 12 h with F-12 medium. The cells were then stimulated with the indicated concentrations of S1P (a,b) or FTY720-P (c,d) for 5 min, or with 200 nM S1P for the indicated time (e,f). Total cell lysates were prepared, and proteins (5 µg) were separated using SDS-PAGE, followed by immunoblotting with anti-p-ERK (upper panel) and anti-ERK (lower panel) antibodies. The intensities of the bands in (a), (c) and (e) were quantified using IMAGEJ software (http://rsb.info.nih.gov/ij/) and are expressed as a ratio of p-ERK levels to ERK levels in (b), (d) and (f) respectively. Values presented in (b) and (d) represent a mean ± SD from three independent experiments. Statistically significant differences are indicated (**P < 0.01 and *P < 0.05; Student’s t-test). (g) Vector-CHO, S1P1-CHO and S1P1(3CA)-CHO cells suspended in F-12 medium containing 0.1% fatty acid-free BSA were added to the upper chambers of a Transwell filter plate, and 200 nM S1P was placed into each lower chamber. The cells were allowed to transmigrate for 4 h at 37°C and the migrated cells were counted. Values presented are the number of migrated cells relative to that observed for Vector-CHO cells, and represent a mean ± SD from three independent experiments. Statistically significant differences are indicated (P < 0.05; Student’s t-test). Vec, vector; WT, wild type S1P1; 3CA, S1P1(3CA).

 
When FTY720-P, the active form of the novel immunomodulating agent FTY720 (Brinkmann et al. 2002; Mandala et al. 2002), was used for stimulation instead of S1P, wild-type S1P1 fully activated ERK even at 1 nM (EC50, 0.49 nM; Fig. 4c,d). However, such a full activation was observed for the 3CA mutant only at 50 nM (EC50, 5.2 nM; Fig. 4c,d). Moreover, the maximal p-ERK/ERK value was ~1/2 of that of the wild-type S1P1. Thus, although palmitoylation affects the signaling activity of S1P1 similarly after S1P or FTY720-P stimulation, it affects only the high-affinity binding of S1P1 to FTY720-P, and has little or no apparent effect on S1P1 binding to S1P.

We also examined the kinetics of ERK activation upon treatment with S1P. Phosphorylation of ERK reached maximal levels within 3 min in S1P1-CHO cells and then gradually decreased (Fig. 4e,f). Non-palmitoylated S1P1 also activated ERK with similar kinetics, but p-ERK levels in the S1P1(3CA)-CHO cells were lower than those in the S1P1-CHO cells until 20 min post-stimulation (Fig. 4e,f).

Stimulated S1P1 induces cell migration toward S1P by activating the small GTPase Rac, via phosphatidylinositol 3-kinase (Okamoto et al. 2000). We next investigated the ability of non-palmitoylated S1P1 to promote cell migration, using a modified Boyden chamber. Inclusion of S1P in the lower chamber significantly stimulated the cell migration of the S1P1-CHO cells, but non-palmitoylated S1P1 was less active (Fig. 4g). The number of migrated S1P1(3CA)-CHO cells was ~25% that of the S1P1-CHO cells (Fig. 4g). These results also suggest that palmitoylation is needed for efficient signaling of S1P1.

Palmitoylation affects internalization of S1P1

After stimulation, S1P1 is rapidly internalized (Liu et al. 1999). We compared the internalization of wild-type and non-palmitoylated S1P1 using flow cytometry. The overall amount of wild-type S1P1 internalized was greater than that of non-palmitoylated S1P1 at any time point examined, although a statistically significant difference was observed only at 60 min post-stimulation (Fig. 5a). We also investigated the effect of palmitoylation on the internalization of S1P1 induced by FTY720-P. As reported previously (Gräler & Goetzl 2004; Matloubian et al. 2004; Jo et al. 2005), FTY720-P stimulated the internalization of S1P1 more efficiently than did S1P (Fig. 5b). The effect of stimulation on non-palmitoylated S1P1 versus S1P1 was likewise more pronounced with FTY720-P (Fig. 5b).


Figure 5
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  		Figure 5  Palmitoylation affects the internalization of S1P1 stimulated by FTY720-P. S1P1-CHO and S1P1(3CA)-CHO cells were incubated for 12 h with F-12 medium containing charcoal-treated FBS, then for another 12 h with F-12 medium. After a 1-h treatment with 10 µg/mL cycloheximide to inhibit protein synthesis, the cells were stimulated for 10 min (arrows) with 200 nM S1P (a) or FTY720-P (b). Cells were washed with F-12 medium containing 4 mg/mL fatty acid-free BSA, suspended in the same medium (t = 0), and incubated for 15, 30 and 60 min at 37°C. Cells were then stained with anti-S1P1 antibodies and subjected to flow cytometry. Values presented are a percentage of the geometric mean values of the fluorescence compared with that at the time ligands were added and represent a mean ± SD from three independent experiments. Statistically significant differences are indicated (*P < 0.05, **P < 0.01; Student’s t-test).

 
Phosphorylation of the C-terminal tail in S1P1 by G protein-coupled receptor kinases (GRKs) facilitates binding of the adaptor protein β-arrestin, which then induces receptor desensitization and endocytosis (Watterson et al. 2002; Oo et al. 2007). To investigate whether retarded internalization of non-palmitoylated S1P1 was due to lowered phosphorylation, we performed [32P]orthophosphate labeling. After a 5-min treatment with S1P, phosphorylation of wild-type S1P1 was increased in a concentration-dependent manner, reaching nearly maximum levels with 10 nM S1P (Fig. 6a,b). A similar dose-dependent curve was observed for non-palmitoylated S1P1 (Fig. 6a,b). However, when FTY720-P was used, the wild-type S1P1 was phosphorylated at the maximum level even at 1 nM, whereas maximal phosphorylation of the 3CA mutant required 10 nM, and its phosphorylation level was ~2/3 of that of wild-type S1P1 (Fig. 6a,b).


Figure 6
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  		Figure 6  Palmitoylation affects the phosphorylation levels of S1P1 in cells stimulated with FTY720-P. S1P1-CHO and S1P1(3CA)-CHO cells were incubated for 12 h with F-12 medium containing charcoal-treated FBS, and for another 12 h with F-12 medium. The culture medium was then changed to phosphate-free DMEM, and the cells were labeled with 75 µCi [32P]orthophosphate for 4 h at 37°C. Cells were then treated with the indicated concentrations of S1P or FTY720-P for 5 min (a,b) or with 200 nM S1P or 200 nM FTY720-P for the indicated times (c,d). S1P1 proteins were immunoprecipitated from total cell lysates with anti-FLAG M2 agarose and separated using SDS-PAGE, followed by autoradiography (a,c; upper panels) or by immunoblotting with anti-FLAG antibodies (a,c; lower panels). The amount of phosphorylated S1P1 in (a) and (c) (upper panels) was quantified using a bioimaging analyzer BAS-2500 to measure the radioactivity of the bands, and the total immunoprecipitated S1P1 in (a) and (c) (lower panels) was quantified using IMAGEJ software to analyze scanned images of the bands. The values are presented as a ratio of phosphorylated S1P1 to S1P1 (b,d). WT, wild type S1P1; 3CA, S1P1(3CA); IB, immunoblotting; pS1P1, phosphorylated S1P1.

 
We also investigated the kinetics of phosphorylation for wild-type and non-palmitoylated S1P1 following stimulation with S1P or FTY720-P. In general, phosphorylation of wild-type S1P1 and non-palmitoylated S1P1 increased over 60 min post-stimulation (Fig. 6c,d). However, following treatment with FTY720-P, although both proteins were phosphorylated, the phosphorylation levels for non-palmitoylated S1P1 were lower than those for wild-type S1P1 (Fig. 6c,d). No difference in phosphorylation levels between wild-type and non-palmitoylated S1P1 were apparent following S1P treatment. This is consistent with the small difference noted in the internalization of the proteins (Fig. 5a), indicating some correlation between the phosphorylation levels and internalized amounts.

Palmitoylation of S1P1 is increased upon treatment with S1P

It has been reported that in some GPCRs the state of palmitoylation is altered upon ligand treatment (Moffett et al. 1993; Hawtin et al. 2001). Therefore, we next performed [3H]palmitic acid labeling in the presence or absence of S1P using S1P1-CHO cells. In the absence of S1P stimulation, the amount of labeled S1P1 was unchanged over time (Fig. 7a). By contrast, labeling was enhanced by increasing the length of S1P treatment (Fig. 7a). Note that most S1P1 in S1P1-CHO cells is already localized in the plasma membrane (Fig. 3a) and has undergone complex-type glycosylation. Therefore, palmitoylation following treatment with S1P occurs in the plasma membrane.


Figure 7
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  		Figure 7  Palmitoylation is increased upon ligand stimulation. (a) S1P1-CHO cells were incubated for 12 h with F-12 medium containing charcoal-treated FBS, and for another 12 h with F-12 medium. Cells were labeled for 2 h with 0.2 mCi [3H]palmitic acid, during which time the cells were treated with 0 or 200 nM S1P for the indicated times. S1P1 was immunoprecipitated from total cell lysates with anti-FLAG M2 agarose, separated using SDS-PAGE, and detected using autoradiography (upper panel) or using immunoblotting with anti-FLAG antibodies (lower panel). (b) S1P1-CHO cells were incubated for 12 h with F-12 medium containing charcoal-treated FBS, and for another 12 h with F-12 medium in the presence or absence of 100 ng/mL pertussis toxin (PTX). Cells were labeled with [3H]palmitic acid as in (a). S1P1 was immunoprecipitated from total cell lysates with anti-FLAG M2 agarose, separated using SDS-PAGE, and detected using autoradiography (upper panel) or using immunoblotting with anti-FLAG antibodies (lower panel). IB, immunoblotting.

 
To determine whether this increase in S1P1 palmitoylation could be attributed to direct activation of the receptor or to an indirect effect mediated by some non-receptor, we performed additional [3H]palmitic acid labeling assays in the presence of pertussis toxin, which inactivates S1P1 by ADP ribosylation of the Gi{alpha} protein. The S1P-dependent increase in the S1P1 palmitoylation was indeed inhibited by pertussis toxin (Fig. 7b). Thus, it is most likely that binding of the ligand to S1P1 induces a conformational change in the structure, rendering it susceptible to palmitoylation.


   Discussion
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 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In the present study, we determined that all S1P receptors are palmitoylated (Fig. 1c,d). Even S1P receptors with high mannose-type glycosylation are palmitoylated (Fig. 1b–d); so, palmitoylation would seem to have already occurred in the ER. However, S1P1, at least, is also palmitoylated in other organelles. Palmitoylation of complex-type glycosylated S1P1, localized in the plasma membrane, was increased upon treatment with S1P (Fig. 7a). Therefore, S1P1 in the plasma membrane seems to be subjected to a palmitoylation/depalmitoylation cycle. This cycle may be regulated during receptor activation, desensitization, internalization and/or recycling.

Palmitoylation does not cause a large change in the affinity of S1P1 toward S1P (wild-type S1P1: Kd = 39.4 nM, EC50 for ERK activation = 1.4 nM; 3CA mutant: Kd = 31.4 nM and EC50 = 2.4 nM). However, S1P1 binding affinities toward FTY720-P seem to be affected greatly by palmitoylation, judging from the EC50 value for ERK activation (wild-type S1P1: 0.5 nM; 3CA mutant: 5.2 nM). It is possible that the insertion into the membrane of the palmitic acid moieties attached to the C-terminal tail of S1P1, which generates an additional intracellular loop in S1P1, may induce a local conformational change in the ligand binding site.

Although many GPCRs are known to be palmitoylated, the roles of their palmitoylation vary (Qanbar & Bouvier 2003; Escribáet al. 2007). For S1P1, palmitoylation affects downstream signaling pathways (Fig. 4) irrespective of whether S1P or FTY720-P is the ligand. The maximum levels of p-ERK were significantly lower in cells overproducing the non-palmitoylated mutant S1P1 compared with those overproducing the wild-type S1P1 (Fig. 4a–d). Heterotrimeric G proteins (G{alpha}β{gamma}) are attached to the membrane via lipid modifications on G{alpha} and G{gamma} (Escribáet al. 2007). It is possible that palmitoylation of S1P1 increases its affinity for the heterotrimeric G proteins through interactions with their lipid moieties. Alternatively, palmitoylation may cause a local conformational change that affects the interactions between S1P1 and heterotrimeric G proteins.

Like other GPCRs, ligand stimulation induces a conformational change in S1P1, which aids not only in the GDP–GTP exchange of the heterotrimeric G protein {alpha}-subunit, but also the availability of the receptor for phosphorylation by GRK and β-arrestin recruitment. Several lines of evidence suggest that S1P-bound S1P1 and FTY720-P-bound S1P1 differ in structure. For example, following treatment with S1P, internalized S1P1 is recycled back to the cell surface, yet treatment with FTY720-P causes ubiquitination of S1P1 and degradation (Gräler & Goetzl 2004; Matloubian et al. 2004; Jo et al. 2005). Therefore, ubiquitin ligase may recognize the structure of S1P1 induced by FTY720-P but not that induced by S1P. These differences are thought to be responsible for the pharmacological effects of FTY720. However, the molecular mechanism behind the difference in the fate of S1P1 following treatment with S1P and FTY720-P has been completely unclear. We found only a small difference in the internalization of the wild-type and non-palmitoylated mutant S1P1 upon treatment with S1P (Fig. 5a), in contrast to significant differences observed with FTY720-P treatment (Fig. 5b). Thus, palmitoylation affects the internalization of S1P1 differently when binding S1P or FTY720-P. Phosphorylation of the mutant S1P1 was also reduced compared with that of wild-type S1P1 following treatment with FTY720-P but not with S1P (Fig. 6). The differential effects of palmitoylation on internalization and phosphorylation provide important clues in understanding the molecular mechanism of the pharmacological effects of FTY720. We speculate that a combination of FTY720-P binding by S1P1 and palmitoylation of S1P1 causes the creation of high-affinity binding sites for GRKs and putative ubiquitin ligases.

Although palmitoylation was first recognized over 30 years ago, identification of the responsible enzymes, palmitoyltransferases (PATs), was only recently achieved. In 2002, two yeast proteins containing the Cys-rich domain and conserved signature motif identifying them as DHHC proteins were determined to be PATs; Erf2 and Akr1 were found to palmitoylate Ras and the casein kinase Yck2 respectively (Lobo et al. 2002; Roth et al. 2002). Studies have shown that yeast and mammals contain 7 and 23 DHHC proteins respectively. Yeast mutant analysis has further indicated that each DHHC protein exhibits PAT activity with an obvious substrate preference, which overlaps among the enzymes (Roth et al. 2006). To date, PATs active toward GPCRs have not been identified. Therefore, future studies are required for identification of S1P1 PATs.


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

HEK 293T cells and CHO cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (D6429; Sigma, St Louis, MO, USA) and in Ham’s F-12 medium (N6658; Sigma), respectively, each containing 10% FBS and supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin. HEK 293T cells were grown in 0.3% collagen-coated dishes. Transfections were performed using Lipofectamine PlusTM Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Plasmids

The pCE-puro 3xFLAG-4 plasmid is a derivative of the pCE-puro vector (Kihara et al. 2003) and is designed to produce a C-terminal triple FLAG (3xFLAG)-tagged protein. Each pCE-puro S1Px-3xFLAG plasmid (with x representing the S1P receptor number) encodes a human S1P receptor tagged with 3xFLAG at its C-terminus. The genes of the S1P receptors were amplified using PCR using appropriate templates [S1P1, human spleen cDNA (Clontech, Takara Bio, Shiga, Japan); S1P2 and S1P3, HEK 293T cDNA; S1P4, human leukocyte cDNA (Clontech, Takara Bio) and S1P5, human lung cDNA (Clontech, Takara Bio)] and primers (for S1P1, 5'-AGGATCCGCCACCATGGGGCCCACCAGCGTCCCG-3' and 5'-AGGATCCCTGGAAGAAGAGTTGACGTTTCCAGAAG-3'; for S1P2, 5'-GGATCCGCCACCATGGGCAGCTTGTACTCGGAGTACC-3' and 5'-GGATCCCTGACCACCGTGTTGCCCTCCAGAAACG-3'; for S1P3, 5'-GCCACCATGG-CAACTGCCCTCCCGCCGCG-3' and 5'-GGATCCCTGTTGCAGAAGATCCCATTCTGAAGTG-3'; for S1P4, 5'-GCCACCATGAACGCCACGGGGACC-3' and 5'-AGATCTCCGATGCTCCGCACGCTGGAGATGCTGG-3'; and for S1P5, 5'-GCCACCATGGAGTCGGGGCTGCTGC-3' and 5'-GGATCCGCGTCTGCAGCCGGTTCTGATACC-3'). Each amplified fragment was first cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA), and then the BamHI–BamHI (S1P1 and S1P2), EcoRI–BamHI (S1P3 and S1P5) or EcoRI–BglII (S1P4) fragments of the resulting plasmids were cloned into the pCE-puro 3xFLAG-4 vector, generating the respective pCE-puro S1Px-3xFLAG plasmids.

The pCE-puro S1P1(C328A)-3xFLAG, pCE-puro S1P1(C329A)-3xFLAG, pCE-puro S1P1(C331A)-3xFLAG, pCE-puro S1P1(C328/329A)-3xFLAG, pCE-puro S1P1(C329/331A)-3xFLAG, pCE-puro S1P1(C328/331A)-3xFLAG, and pCE-puro S1P1(3CA)-3xFLAG plasmids encode Cys-to-Ala-substituted S1P1 mutants. These plasmids were constructed from the pCE-puro S1P1-3xFLAG plasmid by site-directed mutagenesis using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). Two primers with complementary sequences were used for each mutagenesis. The sense primers used were, for pCE-puro S1P1(C328A)-3xFLAG, 5'-CATCCGGATCATGTCCGCCTGCAAGTGCCCGAGCG-3'; for pCE-puro S1P1(C329A)-3xFLAG, 5'-CCGGATCATGTCCTGCGCCAAGTGCCCGAGCGGAG-3'; for pCE-puro S1P1(C331A)-3xFLAG, 5'-CATGTCCTGCTGCAAGCCCCGAGCGGAGACTCTG-3'; for pCE-puro S1P1(C328/329A)-3xFLAG, 5'-CATCCGGATCATGTCCGCCGCCAAGTGCCCGAGCG-3'; for pCE-puro S1P1(C328/331A)-3xFLAG, 5'-CATCCGGATCATGTCCGCCTGCAAGGCCCCGAGCG-3'; for pCE-puro S1P1(C329/331A)-3xFLAG, 5'-CCGGATCATGTCCTGCGCCAAGGCCCCGAGCGGAG-3'; and for S1P1(3CA)-3xFLAG, 5'-CCGGATCATGTCCGCCGCCAAGGCCCCGAGCGGAGACTC-3'.

Production of stable transformants

To obtain stable transformants of the S1P1-3xFLAG or S1P1(3CA)-3xFLAG gene, the pCE-puro S1P1-3xFLAG or pCE-puro S1P1(3CA)-3xFLAG plasmid was transfected into CHO cells. Cells were subjected to puromycin selection at 10 µg/mL. One stable transformant for each plasmid, termed S1P1-CHO and S1P1(3CA)-CHO, expressed the highest level of S1P1-3xFLAG and S1P1(3CA)-3xFLAG, respectively, among the isolated clones. Although we present only the results obtained using S1P1-CHO and S1P1(3CA)-CHO cells here, other clones also tested gave similar results. For use as a control, Vector-CHO cells were similarly obtained using the pCE-puro 3xFLAG-4 vector.

In vivo labeling experiments

[3H]Palmitic acid labeling was carried out as described previously (Kihara et al. 2006) using 0.2 mCi of [3H]palmitic acid (60 Ci/mmol; American Radiolabeled Chemical, St Louis, MO, USA). Prior to [32P]orthophosphate labeling, the culture medium was changed to phosphate-free DMEM (11971-025; Invitrogen). After 1 h, the cells were placed in phosphate-free DMEM containing 75 µCi [32P]orthophosphate (28 kCi/mmol; Perkin-Elmer Life Sciences, Waltham, MA, USA) and 0.1% fatty acid-free BSA. After a 4-h incubation, cells were incubated with 200 nM S1P or 200 nM FTY720-P for various times. Cells were then lyzed with RIPA buffer [50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS and 0.1% sodium deoxycholate], and the resulting total cell lysates were subjected to immunoprecipitation with anti-FLAG M2 agarose beads (Sigma). Beads were washed with RIPA buffer twice and with 10 mM Tris–HCl (pH 8.0) once, then proteins were eluted with 2x SDS sample buffer [125 mM Tris–HCl (pH 6.8), 4% SDS, 20% glycerol, 10% 2-mercapoethanol, and a trace amount of bromophenol blue]. Proteins were separated using SDS-PAGE and detected using autoradiography.

Deglycosylation of proteins

Endo H and PNGase F were purchased from New England Biolabs (Beverly, MA, USA). Cells were washed with PBS twice, suspended in buffer A [62.5 mM Tris–HCl (pH 6.8), 2% SDS, 10% glycerol and 10% 2-mercaptoethanol] and sonicated. After a 5-min incubation at 37°C, samples were centrifuged at 17 400 g for 5 min at room temperature. The supernatant was subjected to deglycosylation by EndoH or PNGase F according to the manufacturer’s instructions.

Immunoblotting

Immunoblotting was performed as described previously (Kihara et al. 2003). Anti-FLAG M2 (1 µg/mL; Stratagene), anti-extracellular signal-regulated kinase (ERK; p44/42 MAP kinase) (1:1000 dilution; Cell Signaling Technology, Beverly, MA, USA) and anti-phospho-ERK (p-ERK) (200 ng/mL; Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies were used as primary antibodies. HRP-conjugated anti-mouse or anti-rabbit IgG F(ab')2 fragment (both from GE Healthcare Bio-Sciences, Piscataway, NJ, USA, and diluted 1 : 7500) were used as secondary antibodies. Labeling was detected using ECLTM Reagents or an ECL plus System for Western Blotting Detection (both from GE Healthcare).

Immunofluorescence microscopy

Microscopic immunofluorescence analysis was performed as described previously (Ogawa et al. 2003) with anti-FLAG M2 antibodies (0.5 µg/mL) and Alexa Fluor 488 goat anti-mouse IgG (H + L) conjugated antibody (5 µg/mL; Molecular Probes, Invitrogen). The stained cells were analyzed using fluorescence microscopy (Axioskop 2 PLUS; Carl Zeiss, Oberkochen, Germany).

Flow cytometry

Cells were detached from the culture surface by treatment with Trypsin/EDTA (0.25% Trypsin and 0.02% EDTA) solution, washed twice with FACS buffer (0.5% fatty acid-free BSA in PBS) and suspended in FACS buffer containing a human S1P1 antibody (anti-hEDG1; 20 µg/mL; R&D Systems, Minneapolis, MN, USA). After a 30-min incubation at 4°C, cells were washed twice with FACS buffer, then treated with Alexa Fluor 488 goat anti-mouse IgG (H + L) conjugated antibody (10 µg/mL) for 30 min at 4°C. Cells were washed twice with FACS buffer then suspended in FACS buffer, and the intensity of cell fluorescence was determined using a FACScan cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Data were analyzed using CELLQUESTTM 3.0 software (Becton Dickinson).

ABE assay

An ABE assay was carried out as described elsewhere with modifications (Drisdel & Green 2004). Cells were washed twice with PBS and lyzed on ice using RIPA buffer containing 1x CompleteTM protease inhibitor mixture (EDTA free; Roche Diagnostics, Indianapolis, IN, USA), 1 mM phenylmethylsulfonyl fluoride and 10 mM NEM. After sonication, cell debris was removed by centrifuging at 17 400 g for 5 min at 4°C. The resulting supernatant was incubated overnight at 4°C with anti-FLAG M2 agarose by rotating the sample tubes. Beads were washed twice with 1-mL RIPA buffer containing 10 mM NEM, and suspended in buffer B [50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 and 0.1 % SDS] containing 50 mM NEM. After rotating the sample tube for 1 h at room temperature, beads were washed twice with 1 mL buffer B and once with 1 mL 10 mM Tris–HCl (pH 7.4), then suspended in buffer C [10 mM Tris–HCl (pH 7.4), 0.2% Triton X-100, 150 mM NaCl] and 0.2 mM EZ-LinkTM Biotin-HPDP (Pierce, Rockford, IL, USA) containing 1 M hydroxylamine (pH 7.4) or 50 mM Tris–HCl (pH 7.4), and incubated for 1 h at room temperature with rotation. Beads were washed twice with 1 mL buffer B and once with 1 mL 10 mM Tris–HCl (pH 7.4) and bound proteins were eluted with 80-µL elution buffer [50 mM Tris–HCl (pH 7.4), 2% SDS and 5 mM EDTA]. Samples were then diluted with 20 volumes of buffer D [50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA and 1% Triton X-100) and incubated with Immunopure Immobilized Avidin (Pierce) overnight at 4°C with rotation. Beads were washed twice with 1-mL buffer B and once with 1-mL 10 mM Tris–HCl (pH 7.4) and bound proteins were eluted with 2x SDS sample buffer and subjected to immunoblotting with anti-FLAG M2 antibodies.

Cell migration assay

Trypsinized cells were suspended in F-12 medium containing 0.1% fatty acid-free BSA then added to the upper chambers of Transwell filters (polycarbonate membrane, pore size of 8.0 µm; Corning, New York, NY, USA) coated with 10 µg/mL fibronectin. Medium containing 200 nM S1P or FTY720-P was added to the lower chamber. The cultures were incubated for 4 h at 37°C. Cells on the upper surface of the Transwell filter were removed with a cotton swab, and those on the lower side of the filter were fixed with cold methanol and stained with 1% crystal violet in 2% ethanol. Cells within four randomly selected regions were counted under a phase-contrast microscope (IX70-S8F2; Olympus, Tokyo, Japan).


   Acknowledgements
 
We are grateful to Dr E.A. Sweeney for scientific editing of the manuscript. We thank Ayaka Okuda for technical support. This work was supported by a Grant-in-Aid for Exploratory Research (20659007) (A.K.) and by a grant from the Japan Society for the Promotion of Sciences (Y.O.).


   Footnotes
 
Communicated by: Yoshinori Ohsumi Back

{dagger}These authors contributed equally to this work. Back

* kihara{at}pharm.hokudai.ac.jp


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Received: 6 January 2009
Accepted: 30 April 2009





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