Insulin-like Growth Factors Mediate Heterotrimeric G Protein-dependent ERK1/2 Activation by Transactivating Sphingosine 1-Phosphate Receptors
2006; Elsevier BV; Volume: 281; Issue: 42 Linguagem: Inglês
10.1016/s0021-9258(19)84052-8
ISSN1083-351X
AutoresHesham M. El‐Shewy, Korey R. Johnson, Mi‐Hye Lee, Ayad A. Jaffa, Lina M. Obeid, Louis M. Luttrell,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoAlthough several studies have shown that a subset of insulin-like growth factor (IGF) signals require the activation of heterotrimeric G proteins, the molecular mechanisms underlying IGF-stimulated G protein signaling remain poorly understood. Here, we have studied the mechanism by which endogenous IGF receptors activate the ERK1/2 mitogen-activated protein kinase cascade in HEK293 cells. In these cells, treatment with pertussis toxin and expression of a Gαq/11-(305–359) peptide that inhibits Gq/11 signaling additively inhibited IGF-stimulated ERK1/2 activation, indicating that the signal was almost completely G protein-dependent. Treatment with IGF-1 or IGF-2 promoted translocation of green fluorescent protein (GFP)-tagged sphingosine kinase (SK) 1 from the cytosol to the plasma membrane, increased endogenous SK activity within 30 s of stimulation, and caused a statistically significant increase in intracellular and extracellular sphingosine 1-phosphate (S1P) concentration. Using a GFP-tagged S1P1 receptor as a biological sensor for the generation of physiologically relevant S1P levels, we found that IGF-1 and IGF-2 induced GFP-S1P receptor internalization and that the effect was blocked by pretreatment with the SK inhibitor, dimethylsphingosine. Treating cells with dimethylsphingosine, silencing SK1 expression by RNA interference, and blocking endogenous S1P receptors with the competitive antagonist VPC23019 all significantly inhibited IGF-stimulated ERK1/2 activation, suggesting that IGFs elicit G protein-dependent ERK1/2 activation by stimulating SK1-dependent transactivation of S1P receptors. Given the ubiquity of SK and S1P receptor expression, S1P receptor transactivation may represent a general mechanism for G protein-dependent signaling by non-G protein-coupled receptors. Although several studies have shown that a subset of insulin-like growth factor (IGF) signals require the activation of heterotrimeric G proteins, the molecular mechanisms underlying IGF-stimulated G protein signaling remain poorly understood. Here, we have studied the mechanism by which endogenous IGF receptors activate the ERK1/2 mitogen-activated protein kinase cascade in HEK293 cells. In these cells, treatment with pertussis toxin and expression of a Gαq/11-(305–359) peptide that inhibits Gq/11 signaling additively inhibited IGF-stimulated ERK1/2 activation, indicating that the signal was almost completely G protein-dependent. Treatment with IGF-1 or IGF-2 promoted translocation of green fluorescent protein (GFP)-tagged sphingosine kinase (SK) 1 from the cytosol to the plasma membrane, increased endogenous SK activity within 30 s of stimulation, and caused a statistically significant increase in intracellular and extracellular sphingosine 1-phosphate (S1P) concentration. Using a GFP-tagged S1P1 receptor as a biological sensor for the generation of physiologically relevant S1P levels, we found that IGF-1 and IGF-2 induced GFP-S1P receptor internalization and that the effect was blocked by pretreatment with the SK inhibitor, dimethylsphingosine. Treating cells with dimethylsphingosine, silencing SK1 expression by RNA interference, and blocking endogenous S1P receptors with the competitive antagonist VPC23019 all significantly inhibited IGF-stimulated ERK1/2 activation, suggesting that IGFs elicit G protein-dependent ERK1/2 activation by stimulating SK1-dependent transactivation of S1P receptors. Given the ubiquity of SK and S1P receptor expression, S1P receptor transactivation may represent a general mechanism for G protein-dependent signaling by non-G protein-coupled receptors. The insulin-like growth factors type 1 and 2 (IGF-1 and -2) 2The abbreviations used are: IGF, insulin-like growth factor; DMS, dimethylsphingosine; EGF, epidermal growth factor; ERK1/2, extracellular signal-regulated kinases 1 and 2; GPCR, G protein-coupled receptor; G protein, guanine nucleotide-binding protein; GFP, green fluorescent protein; h, human; M6P, mannose 6-phosphate; PDGF, platelet-derived growth factor; PMA, phorbol myristate acetate; siRNA, small interfering RNA; SK, sphingosine kinase; S1P, sphingosine 1-phosphate.2The abbreviations used are: IGF, insulin-like growth factor; DMS, dimethylsphingosine; EGF, epidermal growth factor; ERK1/2, extracellular signal-regulated kinases 1 and 2; GPCR, G protein-coupled receptor; G protein, guanine nucleotide-binding protein; GFP, green fluorescent protein; h, human; M6P, mannose 6-phosphate; PDGF, platelet-derived growth factor; PMA, phorbol myristate acetate; siRNA, small interfering RNA; SK, sphingosine kinase; S1P, sphingosine 1-phosphate. are single chain polypeptides that share structural homology with proinsulin. The actions of IGF-1 and IGF-2 are mediated by binding to two structurally distinct plasma membrane receptors, referred to as the IGF-1 and IGF-2/mannose 6-phosphate (M6P) receptors. Most of the metabolic and mitogenic effects of IGF-1 and IGF-2 are thought to result from binding to the IGF-1 receptor, a receptor tyrosine kinase with structural homology to the insulin receptor. It is composed of two extracellular α subunits and two transmembrane β subunits linked by disulfide bonds (1LeRoith D. Werner H. Beitner-Johnson D. Roberts Jr., C.T. Endocr. Rev. 1995; 16: 143-163Crossref PubMed Scopus (1250) Google Scholar, 2Baserga R. Hongo A. Rubini M. Prisco M. Valentinis B. Biochim. Biophys. Acta. 1997; 1332: F105-F126Crossref PubMed Scopus (485) Google Scholar). 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Subsequent src homology 2 or protein tyrosine-binding domain-dependent recruitment of enzymes with phospholipase, phosphatase, and protein and lipid kinase activity transmits signals intracellularly, including activation of the mitogenic Ras cascade and initiation of phosphatidylinositol 3-kinase (PI3K)/Akt-dependent cell survival. Adding to the diversity, in some cell types IGF-1 stimulates matrix metalloprotease-dependent shedding of epidermal growth factor (EGF) family peptide ligands, such as heparin-binding EGF (7Roudabush F.L. Pierce K.L. Maudsley S. Khan K.D. Lin F.-T. Luttrell L.M. J. Biol. Chem. 2000; 275: 22583-22589Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 8El-Shewy H.M. Kelly F.L. Barki-Harrington L. Luttrell L.M. Mol. Endocrinol. 2004; 18: 2727-2739Crossref PubMed Scopus (40) Google Scholar). Heparin-binding EGF release transactivates ErbB receptors, conferring a mechanism for IGF-1 to produce Ras-dependent signaling in either an autocrine or paracrine manner. In contrast to the IGF-1 receptor, the IGF-2/M6P receptor is a 300-KDa single transmembrane glycoprotein that binds both the nonglycoslyated IGF-2 polypeptide and glycosylated M6P-containing ligands (9Ghosh P. Dahms N.M. Kornfeld S. Nat. Rev. Mol. Cell. Biol. 2003; 4: 202-212Crossref PubMed Scopus (794) Google Scholar, 10Hassan A.B. Am. J. Pathol. 2003; 162: 3-6Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). It contains a large extracellular domain, a single membrane-spanning region, and a short cytoplasmic tail. The IGF-2/M6P receptor lacks intrinsic catalytic activity. Although it may mediate transmembrane signal transduction by IGF-2 under some circumstances, cell surface IGF-2/M6P receptors are thought to function primarily in the capture and lysosomal degradation or processing of extracellular peptide ligands, including IGF-2, transforming growth factor-β1, and proliferin (11Hawkes C. Kar S. J. Comp. Neurol. 2003; 458: 113-127Crossref PubMed Scopus (52) Google Scholar, 12Purchio A.F. Cooper J.A. Brunner A.M. Lioubin M.N. Gentry L.E. Kovacina K.S. Roth R.A. Marqardt H. J. Biol. Chem. 1988; 263: 14211-14215Abstract Full Text PDF PubMed Google Scholar, 13Dennis P.A. Rifkin D.B. Proc. Natl. Acad. Sci., U. S. A. 1991; 88: 580-584Crossref PubMed Scopus (460) Google Scholar, 14Groskopf J.C. Syu L.J. Saltiel A.R. Linzer D.I. Endocrinology. 1997; 138: 2835-2840Crossref PubMed Scopus (65) Google Scholar). It has been known for some time that a subset of the cellular signals transmitted by insulin and IGFs involve heterotrimeric G protein activation. In cultured myocytes, insulin-stimulated processing of the inositol phosphate glycan anchors of membrane proteins requires both regulated proteolysis and pertussis toxin-sensitive G protein-dependent activation of a phosphatidylinositol-specific isoform of phospholipase C (15Saltiel A.R. Fox J.A. Sherline P. Cuatrecasas P. Science. 1986; 233: 967-972Crossref PubMed Scopus (271) Google Scholar, 16Romero G. Luttrell L. Rogol A. Zeller K. Hewlett E. Larner J. Science. 1988; 240: 509-511Crossref PubMed Scopus (163) Google Scholar, 17Luttrell L.M. Hewlett E.L. Romero G. Rogol A.D. J. Biol. Chem. 1988; 263: 6134-6141Abstract Full Text PDF PubMed Google Scholar, 18Luttrell L. Kilgour E. Larner J. Romero G. J. Biol. Chem. 1990; 265: 16873-16879Abstract Full Text PDF PubMed Google Scholar). In Rat1 fibroblasts, IGF-1-stimulated activation of the ERK1/2 mitogen-activated protein kinase cascade is largely pertussis toxin-sensitive and can be blocked by expressing a Gβγ subunit sequestrant peptide derived from the C terminus of G protein-coupled receptor kinase 2 (GRK2ct) (19Luttrell L.M. Van Beisen T. Hawes B.E. Koch W.J. Touhara K. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 16495-16498Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Similar pertussis toxin-sensitive ERK1/2 activation has been reported in undifferentiated 3T3-L1 cells (20Uehara T. Tokumitsu Y. Nomura Y. FEBS J. 1999; 259: 801-808Google Scholar), human intestinal smooth muscle cells (21Kuemmerle J.F. Murthy K.S. J. Biol. Chem. 2001; 276: 7187-7194Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), primary rat cerebellar granule neurons, and NG-108 neuronal cells (22Hallak H. Seiler A.E.M. Green J.S. Ross B.N. Rubin R. J. Biol. 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IGF-2 reportedly mediates pertussis toxin-sensitive activation of a calcium-permeable cation channel in Balb/c 3T3 cells by binding to the IGF-2/M6P receptor (31Nishimoto I. Murayama Y. Katada T. Ui M. Ogata E. J. Biol. Chem. 1989; 264: 14029-14038Abstract Full Text PDF PubMed Google Scholar). Despite the evidence that heterotrimeric G proteins play significant roles in IGF signaling, the mechanism of G protein activation by such nonclassical G protein-coupled receptors as the IGF-1 and IGF-2 receptors remains poorly understood. Two principal mechanisms have been advanced to account for the phenomenon. The first proposal is that the receptors possess the ability to directly catalyze heterotrimeric G protein guanine nucleotide exchange (32Patel T.B. Pharmacol. Rev. 2004; 56: 371-385Crossref PubMed Scopus (86) Google Scholar). Consistent with this, several groups have reported that heterotrimeric G protein subunits co-precipitate with the IGF-1 and insulin receptors (22Hallak H. 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However the evidence that this might represent a general model of GPCR transactivation is presently limited. In this study, we have examined the mechanism whereby endogenous IGF receptors activate the ERK1/2 cascade in HEK293 cells, a system in which IGF-stimulated ERK1/2 activation is almost entirely heterotrimeric G protein-dependent. We found that IGF-1 and IGF-2 stimulated rapid SK activation, S1P production, and S1P receptor activation. Moreover, inhibiting SK activity either pharmacologically or using small interfering RNA (siRNA) against SK1 blocked IGF receptor-mediated S1P receptor and ERK1/2 activation, suggesting that SK-dependent transactivation of endogenous S1P receptors is sufficient to account for G protein-dependent responses to IGF in these cells. Given the structural diversity of the non-GPCRs that have been shown to activate heterotrimeric G proteins and the ubiquity of SK and S1P receptor expression, our data suggest that SK-dependent activation of S1P receptors may represent a general mechanism for G protein-dependent signaling by non-GPCRs. Materials—Tissue culture medium, fetal bovine serum, and penicillin/streptomycin were from Invitrogen. FuGENE 6 was from Roche Diagnostics. Double-stranded siRNAs were purchased from Xeragon (Germantown, MD). GeneSilencer™ transfection reagent was from Gene Therapy Systems (San Diego, CA). Primers for real-time PCR were from Integrated DNA Technologies (Coralville, IA). RNeasy kits were from Qiagen Corp. (Valencia, CA), and iScript cDNA synthesis kits and iQ™ SYBR® Green Supermix kits were from Bio-Rad Laboratories. Pertussis toxin was from List Biological Laboratories (Campbell, CA). IGF-1, IGF-2, and phorbol 12-myristate 13-acetate (PMA) were from Sigma. VPC23019 was from Avanti Polar Lipids (Alabaster, AL). d-erythro-Sphingosine 1-phosphate and dimethylsphingosine (DMS) were provided by the MUSC Lipidomics Core (Dept. of Biochemistry, Medical University of South Carolina, Charleston). Rabbit polyclonal anti-ERK1/2 and phospho-ERK1/2 IgG were from Cell Signaling Technology (Beverly, MA). Horseradish peroxidase-conjugated donkey anti-rabbit IgG was from Amersham Biosciences. Rabbit polyclonal anti-human SK1 antiserum was prepared as described previously (38Johnson K.R. Becker K.P. Facchinetti M.M. Hannun Y.A. Obeid L.M. J. Biol. Chem. 2002; 277: 35257-35262Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). cDNA Constructs—The pEGFP-N1-myc-S1P1R plasmid encoding a myc epitope-tagged S1P1 receptor fused in-frame to the N terminus of green fluorescent protein (GFP) was a generous gift from Dr. Timothy Palmer (Inst. of Biomedical and Life Sciences, University of Glasgow, UK) (39Watterson K.R. Johnston E. Chalmers C. Pronin A. Cook S.J. Benovic J.L. Palmer T.M. J. Biol. Chem. 2002; 277: 5767-5777Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The pEGFP-N1-ERK2 plasmid encoding GFP-tagged ERK2 was generously provided by Dr. Nigel Bunnett (University of California, San Francisco) (40DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (686) Google Scholar). The pcDNA3-hSK1 and pEGFP-C3-hSK1 plasmids encoding human SK1 (hSK1) and GFP-tagged hSK1, respectively, were prepared as described previously (38Johnson K.R. Becker K.P. Facchinetti M.M. Hannun Y.A. Obeid L.M. J. Biol. Chem. 2002; 277: 35257-35262Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). The pRK5-Gαq (305–359) minigene plasmid encoding the C-terminal 55 amino acids of Gαq/11 was as described (41Akhter S.A. Luttrell L.M. Rockman H.A. Lefkowitz R.J. Koch W.J. Science. 1998; 280: 574-577Crossref PubMed Scopus (393) Google Scholar). Cell Culture and Transfection—HEK293 cells were obtained from the American Type Culture Collection and maintained in minimum essential medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. Prior to experimentation, subclones were isolated and screened for pertussis toxin-sensitive ERK1/2 activation in response to IGF-1. Cells plated at low density in 15-cm dishes were grown for 7 days after which colonies representing the progeny of single cells were collected by sterile pipetting and seeded into individual wells of a 96-well culture plate for expansion. Subclones were tested for pertussis toxin-sensitive ERK1/2 activation as described below. Sensitivity ranged from 30 to 50% for individual subclones, and the most sensitive isolates were employed in all subsequent experiments. Transient transfection of 50–60% confluent cultures of HEK293 cells was performed in 10-cm dishes using a ratio of 3 μl of FuGENE 6/μg of plasmid DNA according to the manufacturer's protocol. Empty pcDNA3.1 vector DNA was added to each transfection as needed to keep the mass of DNA constant. Transfected cells, split into multi-well plates as appropriate, were serum-deprived overnight in growth medium supplemented with 0.5% fetal bovine serum and 10 mm HEPES, pH 7.4, prior to stimulation. All assays using transiently transfected cells were performed 48 h after transfection. siRNA Down-regulation of Sphingosine Kinase 1 Expression—SK1 expression was down-regulated using 200 nm hSK1 sequence-specific siRNAs (5′-GAGCUGCAAGGCCUUGCCCdTdT-3′ and 5′-GGGCAAGGCCUUGUUGCAGCUCdTdT-3′). Scrambled siRNA sequences (5′-ACGUGACACGUUCGGAGAAAdTdT-3′ and 5′-UUCUCCGAACGUGUCACGUdTdT-3′) were used as negative controls. HEK293 cells were seeded in collagen coated 10-cm dishes at a density of 2 × 105 cells/dish 24 h before transfection. Cells were transfected using Gene Silencer® siRNA transfection reagent according to the manufacturer's protocol. The efficiency of the knockdown was determined by quantitative real-time PCR for hSK1 mRNA and immunoblotting for hSK1 protein 48 h after transfection. Quantitative Real-time PCR—Total cellular RNA was isolated using the RNeasy kit according to manufacturer's instructions. cDNA was prepared from 1 μg of total RNA with A260/A280 > 1.8 using the iScript cDNA synthesis kit per the manufacturer's instructions. Quantitative real-time PCR was performed with an iCycler 1Q Real-Time Detection System using the iQ™ SYBR® Green Supermix kit. Reactions were performed using hSK1-specific primers (forward primer 5′-CAGACATGACCACCAGAG-3′; reverse primer 5′-ATCTTCACGCTGATG-3′) and β-actin-specific primers (forward primer 5′-ATTGGCAATGAGCGGTTCC-3′; reverse primer 5′-GGTAGTTTCGTCGATGCCACA-3′). Real-time PCR results were analyzed using Softmax® Pro software (Molecular Devices Corp.). hSK1 expression data were normalized to expression of β-actin as an endogenous control. Protein Immunoblotting—Appropriately transfected HEK293 cells were split into poly-d-lysine-coated 12-well plates, serum-deprived overnight, and preincubated in the presence or absence of inhibitors as described in the figure legend (Figs. 1, 5, 6, and 8). Agonist stimulations were for 10 min after which monolayers were washed once in 4 °C phosphate-buffered saline and lysed in 200 μl of Laemmli sample buffer. For the determination of ERK1/2 phosphorylation, samples containing 20 μg of cell protein were resolved by SDS-PAGE, and ERK1/2 and phospho-ERK1/2 were detected by protein immunoblotting using rabbit polyclonal anti-phospho-ERK1/2 IgG with horseradish peroxidase-conjugated polyclonal donkey anti-rabbit IgG as the secondary antibody. Immune complexes were visualized by enzyme-linked chemiluminescence and quantified using a Fluor-S MultiImager. In each experiment, equal loading of ERK1/2 protein was confirmed by probing parallel immunoblots using anti-ERK1/2 antisera.FIGURE 5Activation of ERK1/2 by IGF-1 and IGF-2 is blocked by inhibiting sphingosine kinase activity. Serum-deprived HEK293 cells were pretreated with 20 μm DMS for 30 min prior to stimulation for 10 min with 10 nm IGF-1, 10 nm IGF-2, 100 nm PMA, or 5 nm S1P. Basal (NS, non-stimulated), IGF-1-, IGF-2-, PMA-, and S1P-stimulated ERK1/2 phosphorylation in whole cell lysate samples was determined as described. A representative phospho-ERK1/2 immunoblot is shown above a bar graph depicting the mean ± S.D. of three independent experiments. The change in ERK1/2 phosphorylation is expressed as the -fold increase above the basal level in non-stimulated cells not exposed to DMS. *, less than in vehicle-treated cells; p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6Down-regulation of sphingosine kinase 1 expression by RNA interference inhibits IGF-1- and IGF-2-stimulated ERK1/2 activation. A, HEK293 cells were transfected with control scrambled siRNA (SCR) or siRNA targeted to hSK1 (SK1 siRNA). RNA was isolated 48 h after transfection, and mRNA levels of SK1 and β-actin were determined by quantitative real-time PCR as described under "Experimental Procedures." B, the level of SK1 protein expression in HEK293 cells transfected with control scrambled siRNA or siRNA targeted to hSK1 was determined by immunoblotting whole cell lysates prepared 48 h after transfection. A representative SK1 immunoblot is shown above a bar graph depicting the mean ± S.D. for three independent experiments. C, serum-deprived HEK293 cells transfected with scrambled siRNA or siRNA targeted to hSK1 were stimulated for 10 min with 10 nm IGF-1 or 10 nm IGF-2. Basal (NS, non-stimulated), and IGF-1- and IGF-2-stimulatedERK1/2phosphorylation in whole cell lysate samples was determined as described. A representative phospho-ERK1/2 immunoblot is shown above a bar graph depicting the mean ± S.D. for three independent experiments. The change in ERK1/2 phosphorylation is expressed as the -fold increase above the basal level in non-stimulated control scrambled siRNA-transfected cells. *, less than in control scrambled siRNA-treated cells; p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 8The competitive S1P1/S1P3 receptor antagonist VPC23019 attenuates IGF-1- and IGF-2-stimulated ERK1/2 activation. Serum-deprived HEK293 cells were pretreated with 10 μm VPC23019 (VPC) for 30 min prior to stimulation for 10 min with 10 nm IGF-1, 10 nm IGF-2, or 5 nm S1P. Basal (NS, non-stimulated), IGF-1, IGF-2, and S1P-stimulated ERK1/2 phosphorylation in whole cell lysate samples was determined as described under "Experimental Procedures." A representative phospho-ERK1/2 immunoblot is shown above a bar graph depicting the mean ± S.D. for three independent experiments. The change in ERK1/2 phosphorylation is expressed as the -fold increase above the basal level in non-stimulated cells not exposed to VPC23019. *, less than in vehicle-treated cells; p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Assay of Sphingosine Kinase Activity—SK activity was determined as described previously with minor modifications (42Olivera A. Rosenthal J. Spiegel S. J. Cell. Biochem. 1996; 60: 529-537Crossref PubMed Scopus (92) Google Scholar). After stimulation, cells were collected by centrifugation for 5 min at 3000 × g and resuspended in ice-cold 0.1 m phosphate buffer containing 20 mm Tris-Cl, pH 7.4, 1 mm EDTA, 0.5 mm deoxypyridoxine, 15 mm NaF, 1 mm β-mercaptoethanol, 1 mm sodium orthovanadate, 40 mm β-glycerophosphate, 0.4 mm phenylmethylsulfonyl fluoride, 20% glycerol, 0.1% Triton X-100, and protease inhibitor mixture. After sonication and assay of protein concentration, equal amounts of lysate protein were incubated for 30 min at 37 °C with 50 μm sphingosine (delivered in 4 mg/ml fatty acid-free bovine serum albumin) and [γ-32P]ATP (5 μCi; 50 μm final concentration). The reaction was terminated by the addition of 20 μl of 1 n HCl and 800 μl of chloroform/methanol/HCl (100:200:1) and allowed to stand at room temperature for 10 min. Subsequently, 240 μlof chloroform and 240 μl of 2 m potassium chloride were added, and samples were centrifuged at 3000 × g for 5 min. The aqueous layer was aspirated, and 250 μl of the organic layer was transferred to new glass tubes. The samples were dried and resuspended in chloroform/methanol/HCl (100:200:1). Lipids were resolved on silica thin layer chromatography plates using 1-butanol/methanol/acetic acid/water (80:20:10:20) as the solvent system. Labeled S1P was visualized by autoradiography, and radioactivity corresponding to sphingosine phosphate bands was scraped and counted in a scintillation counter. In Vivo Assay of Sphingosine 1-Phosphate Formation—Serum-deprived HEK293 cells in 2 ml of medium/60-mm dish were pulsed with 300 nm d-[erythro-3H]sphingosine (2.5 μCi) in the presence or absence of stimuli as described in the legend to Fig. 4. Lipids were extracted by the addition of 2.5 volumes of chloroform/butanol/HCl (50:50:1), and the organic layer was dried under nitrogen. Lipids were separated using thin layer chromatography in chloroform/methanol/15 mm calcium chloride (60:35:8). Labeled S1P was visualized by autoradiography. Bands corresponding to S1P standards were scraped and incorporated radioactivity measured by scintillation counting. Confocal Fluorescence Microscopy—For visualization of GFP-SK1 and myc-S1P1-GFP receptor, appropriately transfected HEK293 cells were plated in collagen-coated 35-mm glass-bottom dishes, serum-deprived overnight, preincubated w
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