Mapping Pathways Downstream of Sphingosine 1-Phosphate Subtype 1 by Differential Chemical Perturbation and Proteomics
2007; Elsevier BV; Volume: 282; Issue: 10 Linguagem: Inglês
10.1074/jbc.m610581200
ISSN1083-351X
AutoresPedro J. Gonzalez‐Cabrera, Timothy Hla, Hugh Rosen,
Tópico(s)Cellular transport and secretion
ResumoSphingosine 1-phosphate subtype 1 (S1P1) receptor agonists alter lymphocyte trafficking and endothelial barrier integrity in vivo. Among these is the potent, non-selective agonist, FTY720-P, whose mechanism of action has been suggested to correlate with S1P1 down-regulation. Discovery of the in vivo active S1P1-selective agonist, SEW2871, has broadened our understanding of minimal requirements for S1P1 function while highlighting differences regarding agonist effect on S1P1 fate, because SEW2871 does not degrade S1P1. To further understand the mechanism of agonist-induced S1P1 down-regulation, we compared signaling and fate of human S1P1-green fluorescent protein (GFP) in stable 293 cells, using AFD-R, a chiral analog of FTY720-P, SEW2871, and S1P. Although all agonists acutely internalized S1P1 to late endosomal vesicles and activated GTPγS35 binding and pERK to similar maxima, only AFD-R led to significant S1P1 down-regulation, as shown by GFP immunoprecipitation studies. Down-regulation was time- and concentration-dependent, was partially blocked by proteasomal inhibition and reversed by chloroquine and an antagonist to S1P1. All agonists induced a receptor-associated increase in ubiquitination, with AFD-R inducing 3-fold more accumulation than S1P and being 3–4 logs more potent than SEW2871. The formation of AFD-R-receptor ubiquitin complex was inhibited by antagonist and chloroquine and was enhanced by proteasomal inhibition. Identification of proteins by PAGE liquid chromatography-tandem mass spectrometry in cells treated with AFD-R confirmed the co-migration of ubiquitin peptides with those of S1P1 and GFP, relative to vehicle alone. These data suggest that the hierarchy of ubiquitin recruitment to S1P1 (AFD-R > S1P > SEW2871) correlates with the efficiency of lysosomal receptor degradation and reflects intrinsic differences between agonists. Sphingosine 1-phosphate subtype 1 (S1P1) receptor agonists alter lymphocyte trafficking and endothelial barrier integrity in vivo. Among these is the potent, non-selective agonist, FTY720-P, whose mechanism of action has been suggested to correlate with S1P1 down-regulation. Discovery of the in vivo active S1P1-selective agonist, SEW2871, has broadened our understanding of minimal requirements for S1P1 function while highlighting differences regarding agonist effect on S1P1 fate, because SEW2871 does not degrade S1P1. To further understand the mechanism of agonist-induced S1P1 down-regulation, we compared signaling and fate of human S1P1-green fluorescent protein (GFP) in stable 293 cells, using AFD-R, a chiral analog of FTY720-P, SEW2871, and S1P. Although all agonists acutely internalized S1P1 to late endosomal vesicles and activated GTPγS35 binding and pERK to similar maxima, only AFD-R led to significant S1P1 down-regulation, as shown by GFP immunoprecipitation studies. Down-regulation was time- and concentration-dependent, was partially blocked by proteasomal inhibition and reversed by chloroquine and an antagonist to S1P1. All agonists induced a receptor-associated increase in ubiquitination, with AFD-R inducing 3-fold more accumulation than S1P and being 3–4 logs more potent than SEW2871. The formation of AFD-R-receptor ubiquitin complex was inhibited by antagonist and chloroquine and was enhanced by proteasomal inhibition. Identification of proteins by PAGE liquid chromatography-tandem mass spectrometry in cells treated with AFD-R confirmed the co-migration of ubiquitin peptides with those of S1P1 and GFP, relative to vehicle alone. These data suggest that the hierarchy of ubiquitin recruitment to S1P1 (AFD-R > S1P > SEW2871) correlates with the efficiency of lysosomal receptor degradation and reflects intrinsic differences between agonists. Trafficking of agonist-stimulated G protein-coupled receptors (GPCRs) 2The abbreviations used are: GPCR, G protein-coupled receptor; S1P, sphingosine 1-phosphate; GTPγS, guanosine 5′-3-O-(thio)triphosphate; GFP, green fluorescent protein; M6P, mannose 6-phosphate; ERK, extracellular signal-regulated kinase; pERK, phosphorylated ERK; CHX, cycloheximide; PBS, phosphate-buffered saline; HA, hemagglutinin; MS/MS, tandem mass spectrometry; LC, liquid chromatography. 2The abbreviations used are: GPCR, G protein-coupled receptor; S1P, sphingosine 1-phosphate; GTPγS, guanosine 5′-3-O-(thio)triphosphate; GFP, green fluorescent protein; M6P, mannose 6-phosphate; ERK, extracellular signal-regulated kinase; pERK, phosphorylated ERK; CHX, cycloheximide; PBS, phosphate-buffered saline; HA, hemagglutinin; MS/MS, tandem mass spectrometry; LC, liquid chromatography. classically proceeds through time-dependent steps starting by acute (within minutes) internalization of receptors from plasma membrane into cytoplasmic vesicular compartments and followed by receptor sorting into either recycling or degradative pathways, which usually take place within hours of agonist treatment. The long term effects of agonists on GPCR fate are dependent on both the nature of the agonist as well as the cellular environment and are believed to be important determinants of agonist effectiveness. Several mechanisms implicated in early GPCR trafficking have been described and involve phosphorylation of agonist-stimulated receptor by GPCR kinases and subsequent binding of arrestin proteins to phosphorylated receptor sites and to adapter proteins such as clathrin and AP-2, which form coated-pit receptor complexes (reviewed in Refs. 1Prossnitz E.R. Life Sci. 2004; 75: 893-899Crossref PubMed Scopus (43) Google Scholar and 2Luttrell L.M. Methods Mol. Biol. 2006; 332: 3-49PubMed Google Scholar). Based on the stability of the arrestin-receptor complexes, GPCRs have been separated into class A receptors, which favor the recycling pathway, and class B receptors, which recycle slowly and are instead destined for degradation (3Oakley R.H. Laporte S.A. Holt J.A. Caron M.G. Barak L.S. J. Biol. Chem. 2000; 275: 17201-17210Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar).The ubiquitin-proteasome pathway has been shown to be involved in the trafficking and degradation of some GPCRs (reviewed in Refs. 4Wojcikiewicz R.J. Trends Pharmacol. Sci. 2004; 25: 35-41Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar and 5Marchese A. Benovic J.L. Methods Mol. Biol. 2004; 259: 299-305PubMed Google Scholar). Ubiquitin, a 76-amino acid protein, is known to conjugate via a conserved three-step enzymatic reaction to lysine residues of proteins that are destined for proteasomal degradation (6Ciechanover A. Exp. Biol. Med. 2006; 231: 1197-1211Crossref PubMed Scopus (60) Google Scholar). As such, ligand-dependent GPCR ubiquitination has been shown to impact the down-regulation of CXCR4, protease-activated receptor-2, V2 vasopressin receptor, and β2-adrenergic receptors, in some cases, by functioning as a sorting signal for lysosomal receptor targeting (7Marchese A. Benovic J.L. J. Biol. Chem. 2001; 276: 45509-45512Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 8Jacob C. Cottrell G.S. Gehringer D. Schmidlin F. Grady E.F. Bunnett N.W. J. Biol. Chem. 2005; 280: 16076-16087Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 9Shenoy S.K. McDonald P.H. Kohout T.A. Lefkowitz R.J. Science. 2001; 294: 1307-1313Crossref PubMed Scopus (706) Google Scholar, 10Martin N.P. Lefkowitz R.J. Shenoy S.K. J. Biol. Chem. 2003; 278: 45954-45959Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar).Sphingosine 1-phosphate (S1P) is a secreted lipid that binds with nanomolar affinity to a family of five GPCRs, referred to as S1P1–5. Receptor coupling for S1P subtypes includes inhibition of adenylyl cyclase, activation of the small G proteins Rac and Rho, and activation p42/p44 mitogen-activated protein and AKT kinases and calcium release (reviewed in Ref. 11Chun J. Rosen H. Curr. Pharm. Des. 2006; 12: 161-171Crossref PubMed Scopus (78) Google Scholar). The physiological functions of S1P extend to multiple systems, including cardiovascular, lymphoid, and auditory, and are being unveiled through both genetic and pharmacological approaches (11Chun J. Rosen H. Curr. Pharm. Des. 2006; 12: 161-171Crossref PubMed Scopus (78) Google Scholar, 12MacLennan A.J. Benner S.J. Andringa A. Chaves A.H. Rosing J.L. Vesey R. Karpman A.M. Cronier S.A. Lee N. Erway L.C. Miller M.L. Hear. Res. 2006; 220: 38-48Crossref PubMed Scopus (70) Google Scholar). A well known outcome of S1P1 receptor agonist administration in vivo is the inhibition of lymphocyte egress from lymph node and thymus. Originally described for FTY720 (13Mandala S. Hajdu R. Bergstrom J. Quackenbush E. Xie J. Milligan J. Thornton R. Shei G.J. Card D. Keohane C. Rosenbach M. Hale J. Lynch C.L. Rupprecht K. Parsons W. Rosen H. Science. 2002; 296: 346-349Crossref PubMed Scopus (1419) Google Scholar), this pro-drug, once converted into its active phosphate-ester form (FTY720-P) by cellular sphingosine kinase 2, acts as a potent and non-selective agonist (activates S1P receptors 1, 3, 4, and 5), which induces and maintains blockade of lymphocyte egress. Studies in the S1P1 genetic knock-out mouse (14Matloubian M. Lo C.G. Cinamon G. Lesneski M.J. Xu Y. Brinkmann V. Allende M.L. Proia R.L. Cyster J.G. Nature. 2004; 427: 355-360Crossref PubMed Scopus (2033) Google Scholar) and the discovery of S1P1-selective agonists (15Sanna M.G. Liao J. Jo E. Alfonso C. Ahn M.Y. Peterson M.S. Webb B. Lefebvre S. Chun J. Gray N. Rosen H. J. Biol. Chem. 2004; 279: 13839-13848Abstract Full Text Full Text PDF PubMed Scopus (534) Google Scholar, 16Forrest M. Sun S.Y. Hajdu R. Bergstrom J. Card D. Doherty G. Hale J. Keohane C. Meyers C. Milligan J. Mills S. Nomura N. Rosen H. Rosenbach M. Shei G.J. Singer I.I. Tian M. West S. White V. Xie J. Proia R.L. Mandala S. J. Pharmacol. Exp. Ther. 2004; 309: 758-768Crossref PubMed Scopus (296) Google Scholar), later singled out the S1P1 receptor as the primary mediator of S1P signals that alter lymphocyte recirculation. In fact, we have provided evidence that administration of the selective S1P1 agonist, SEW2871, discovered from high throughput screening, induces dose-dependent and reversible lymphopenia in mice, with onset kinetics and magnitude similar to the FTY720-P chiral analog, AFD-R (15Sanna M.G. Liao J. Jo E. Alfonso C. Ahn M.Y. Peterson M.S. Webb B. Lefebvre S. Chun J. Gray N. Rosen H. J. Biol. Chem. 2004; 279: 13839-13848Abstract Full Text Full Text PDF PubMed Scopus (534) Google Scholar). Additional studies, intended to compare S1P1 agonist signaling in cell lines stably expressing S1P1, determined that SEW2871 recapitulates S1P effectors signaling and overlaps with S1P for key S1P1 pocket interactions, although at lower potency (17Jo E. Sanna M.G. Gonzalez-Cabrera P.J. Thangada S. Tigyi G. Osborne D.A. Hla T. Parrill A.L. Rosen H. Chem. Biol. 2005; 12: 703-715Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). Interestingly, fate of the receptor was significantly different with different agonists, and while stimulation with the physiological ligand S1P or SEW2871 supported S1P1-GFP recycling, FTY-720-P-treated cells did not lead to recycling, suggesting the existence of ligand-dependent differences in receptor fate within the same cellular environment. In the present study, we have investigated whether differences in agonist-induced receptor ubiquitination could account for this discrepancy in fate.Here, we provide biochemical and proteomic evidence that S1P1 agonists induce recruitment of ubiquitin to S1P1, although by different magnitudes depending on the choice of agonist and despite similar ligands' efficacy at acute receptor activation. We found that there is enhanced efficacy of receptor ubiquitination by AFD-R relative to SEW2871 or S1P, and this is strongly associated with receptor sorting to lysosomes and receptor down-regulation. Our model proposes that the extent of ubiquitin recruitment by distinct ligands, and thus receptor degradation, represents a ligand-regulated step in determining the fate of S1P1.EXPERIMENTAL PROCEDURESMaterials—GTPγS35 was obtained from PerkinElmer Life Sciences. S1P was obtained from Biomol. The selective S1P1 agonist, SEW2871, was from Maybridge. The S1P receptor agonist, AFD-R (the phosphate-ester of the prodrug amino alcohol AAL(R)) and AAL(S) were gifts from Dr. Volker Brinkmann (Novartis Pharma). Anti-GFP antibodies and the mannose 6-phosphate (M6P) receptor antibody were from Abcam, anti-ubiquitin P4D1 antibody from Santa Cruz Biotechnology (Santa Cruz, CA), and ERK antibodies from Cell Signaling. The proteasomal inhibitors (MG132 and lactacystin) were obtained from Calbiochem. Chloroquine and cycloheximide (CHX) were from Sigma-Aldrich.Cell Culture—HEK293 cells stably expressing the GFP-tagged human S1P1 receptor (293-S1P1-GFP) and 293-vector-GFP cells (18Liu C.H. Thangada S. Lee M.J. Van Brocklyn J.R. Spiegel S. Hla T. Mol. Biol. Cell. 1999; 10: 1179-1190Crossref PubMed Scopus (166) Google Scholar) were maintained in regular growth medium consisting of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin solution and selected with 500 μg/ml G418 (Invitrogen). Prior to every experiment, the growth medium was replaced with 0.2% charcoal-stripped fetal bovine serum (cs-FBS, HyClone) medium, and the cells were incubated overnight.Detection of S1P1-GFP and Evaluation of Ligand-dependent Down-regulation—Confluent cells expressing S1P1-GFP or vector-GFP control grown in 6-well plates were washed twice in ice-cold PBS, and lysates were obtained by incubation in radioimmune precipitation assay buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton x-100) containing 1 mm NaVO4, 1 mm NaF, 0.5 m β-glycerol phosphate and protease inhibitor mixture (Roche Applied Science). Cellular lysates were cleared by centrifugation (16,500 × g, 15 min), and the protein concentration of lysates supernatants was determined by the BCA (Pierce) method. Equal amounts of protein lysates were incubated overnight at 4 °C with a monoclonal GFP antibody (1 μg of antibody per 400 μg of protein), followed by incubation (2 h, 4 °C) with protein-A-Sepharose beads. The beads were recovered by centrifugation (10,000 × g, 1 min) and washing: three times in radioimmune precipitation assay buffer: PBS (1:1) without protease inhibitors and twice in PBS. The beads were suspended in Laemmli buffer containing 2-mercaptoethanol and boiled for 10 min, and proteins in the beads were separated by SDS-PAGE using either 4–12% gradient NuPage gels (Invitrogen) or 10% linear gels. The gels were transferred to polyvinylidene difluoride membranes, which were blocked in 5% milk and subsequently probed with a polyclonal GFP antibody (1:10,000; 1 h at room temperature) for detection of S1P1-GFP. Horseradish peroxidase-labeled goat anti-rabbit (1:5,000) antibodies were visualized by ECL chemiluminescence (Amersham Biosciences).Agonist-induced down-regulation was measured in cells treated with 15 μg/ml CHX to prevent new receptor synthesis. Cells were exposed to either 10 μm SEW2871, 500 nm AFD-R, 500 nm S1P, or vehicle (0.1% fatty acid-free BSA) for the indicated times, and lysed as described above. S1P1-GFP down-regulation was analyzed by comparing the density of immunoprecipitated S1P1-GFP bands in agonist versus vehicle-treated cells, quantified by scanning densitometry using Kodak-1D Application software.The effect of proteasomal inhibition, chloroquine, or the S1P1 competitive antagonist W146 (19Sanna M.G. Wang S.K. Gonzalez-Cabrera P.J. Don A. Marsolais D. Matheu M.P. Wei S.H. Parker I. Jo E. Cheng W.C. Cahalan M.D. Wong C.H. Rosen H. Nat. Chem. Biol. 2006; 2: 434-441Crossref PubMed Scopus (339) Google Scholar) on AFD-R-stimulated S1P1-GFP down-regulation was studied by comparing intensity of S1P1-GFP bands in cells incubated with the respective agents (5 μm MG132, 10 μm lactacystin, 80 μm chloroquine, and 10 μm W146) for 30 min prior to and during incubation with AFD-R or vehicle for an additional 4 h.Detection of Ubiquitinated S1P1-GFP—Ubiquitinated S1P1-GFP was probed for in the same lysates used for measuring S1P1-GFP down-regulation. Briefly, aliquots of boiled GFP immunoprecipitates were resolved by SDS-PAGE, and membranes were incubated overnight at 4 °C with mouse monoclonal P4D1 antibody (1:200–1:800) and horseradish peroxidase-conjugated goat anti-mouse (1:5000) secondary antibody.AFD-R-stimulated S1P1 ubiquitination was additionally tested in HEK293 cells transiently transfected with N-terminal-tagged hemagglutinin (HA)-S1P1 (purchased from cDNA.org). Cells were transfected in 6-well plates with either 3, 2, or 1 μg of receptor plasmid for 48 h using Lipofectamine 2000. Following transfection, cells were incubated with vehicle or 500 nm AFD-R for 1 h, and cellular lysates were obtained as described above. Receptor expression and AFD-R-stimulated receptor ubiquitination were determined by immunoblotting with an anti-HA (Bio-Rad) antibody at 1:1000 dilution.Acute Receptor Activation Experiments—Activation of ERK phosphorylation (pERK) by S1P1 agonists was determined by incubating cells with either 10 μm SEW2871, 500 nm AFD-R, or 500 nm S1P for the indicated times. The conditions for determining activation of pERK were done as described previously (17Jo E. Sanna M.G. Gonzalez-Cabrera P.J. Thangada S. Tigyi G. Osborne D.A. Hla T. Parrill A.L. Rosen H. Chem. Biol. 2005; 12: 703-715Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). The potency (EC50) and maximal responses of S1P, AFD-R, and SEW2871 in activating GTP binding were determined in S1P1-GFP membranes using GTPγS35. Membrane preparation and conditions for binding were done as reported before (15Sanna M.G. Liao J. Jo E. Alfonso C. Ahn M.Y. Peterson M.S. Webb B. Lefebvre S. Chun J. Gray N. Rosen H. J. Biol. Chem. 2004; 279: 13839-13848Abstract Full Text Full Text PDF PubMed Scopus (534) Google Scholar), using 40-μg membranes per well. Data analysis was performed using GraphPad Prism (San Diego, CA).Imaging—Single S1P1-GFP cells grown in 0.2% gelatin-coated coverslips were used to study ligand-induced localization with the late endosomal M6P-receptor marker. Incubation with agonists (500 nm AFD-R, 500 nm S1P, or 10 μm SEW2871) was terminated by removal of medium and washing with PBS. Cells were fixed in 3.7% paraformaldehyde in PBS for 10 min, permeabilized in PBS/0.1% Triton X-100 (PBST) for 30 min, and blocked for 30 min in PBST containing 1% BSA and 5% normal goat serum. Primary antibody incubation (1:1000) was performed in blocking buffer overnight at 4 °C. Secondary antibody (goat-anti-mouse Alexafluor-546) incubation was performed in blocking buffer for 30 min at room temperature. Coverslips were washed three times with PBS and mounted onto slides by using Gel Mount (Biomeda Corp.) mounting media. Cells were scanned with an Olympus BX61 scanning confocal fluorescence microscope. For detecting GFP, fluorescence was excited by using an argon laser at a wavelength of 488 nm, and the absorbed wavelength was detected at 510–520 nm. For detecting Alexa Fluor 546, fluorescence was excited by using a helium-neon laser at a wavelength of 522 nm. In experiments using Lysotracker® Red (Cambrex) dye (75 nm) was added to the medium 15 min before the end of agonist incubation. Cells were then washed and fixed with 3.7% paraformaldehyde in PBS for 10 min. For all experiments, images were processed with Adobe Photoshop 6.0.Chromatography and Mass Spectrometry—AFD-R-stimulated ubiquitin recruitment to S1P1-GFP was determined using a scale-up GFP immunoprecipitation using identical conditions as above. Briefly, vehicle and AFD-R lysates isolated from five 150-mm plates per condition (vehicle or 500 nm AFD-R, 1-h incubation) were immunoprecipitated with anti-GFP (400 μg of protein to 1 μg of antibody), separated by SDS-PAGE (4–12% NuPage gels), and the gel was subsequently stained by Colloidal Blue. In-gel stained proteins derived from three separate gel fragments (bottom, 73–85 kDa; medium, 100–115 kDa; top, 150–170 kDa, see Fig. 8) were cut out from either vehicle or AFD-R lanes and sent for LC-MS/MS analysis. The gel fragments were chosen by imaging against a matched AFD-R-induced "ladder-like" ubiquitin receptor complex (run in an adjacent well of the same gel and derived from the same lysates). As controls, corresponding vehicle-only gel fragments were analyzed. The gel bands were excised and treated with 10 mm dithiothreitol to reduce disulfide linkages. Alkylation was performed with 55 mm iodoacetamide (Sigma-Aldrich) before digestion with trypsin (Promega) overnight at 37 °C using an estimated (1:30) enzyme to substrate ratio in 50 mm ammonium bicarbonate. The LC separation was performed on a laser-pulled 100-μm inner diameter C18 column with a tip of <5 μm that is also used as a nanoelectrospray emitter. Gradient elution was used with 0.1% formic acid/acetonitrile as the mobile phases, from 5% to 60% acetonitrile in 90 min, and then maintained for an additional 20 min with flow rates of ∼200 nl/minute. The MS/MS analysis was performed on a linear ion mass spectrometer (LTQ, ThermoFisher Corp.). Data-dependent scanning was used to maximize the number of peptides sequenced in the highly complex mixture. This mode of operation uses preset criteria to select unique peptides on-the-fly for undergoing MS/MS. Over 10,000 MS/MS spectra were obtained during the run. These were searched using Mascot (Matrix Science, Ltd.) and Sequest (University of Washington, WA) search engine software using the NCBInr (non-redundant data base). To improve searching efficiency, the taxonomic category was limited to mammalian proteins. Only peptides producing good quality fragmentation spectra and scoring higher than the threshold required for 95% confidence level for Mascot were used for protein identification.RESULTSWe have shown previously (17Jo E. Sanna M.G. Gonzalez-Cabrera P.J. Thangada S. Tigyi G. Osborne D.A. Hla T. Parrill A.L. Rosen H. Chem. Biol. 2005; 12: 703-715Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar) that stable HEK293 cells expressing C-terminal GFP-tagged human S1P1 differ in trafficking pattern when stimulated with FTY720-P (which induces receptor degradation), compared with S1P or SEW2871 (which induce receptor recycling). To further investigate agonist-induced receptor fate differences, we used the same HEK293-S1P1-GFP cell line (18Liu C.H. Thangada S. Lee M.J. Van Brocklyn J.R. Spiegel S. Hla T. Mol. Biol. Cell. 1999; 10: 1179-1190Crossref PubMed Scopus (166) Google Scholar) to compare three agonists (AFD-R, SEW2871, and S1P) for stimulating 1) receptor down-regulation, 2) acute receptor signaling (GTPγS35 binding and pERK activation), and 3) short- and long-term receptor trafficking. Additionally, we augmented biochemical and pharmacological data with a proteomics identification approach in exploring possible mechanism(s) of agonist-induced S1P1 fate.Agonist-stimulated S1P1-GFP down-regulation was evaluated by immunoprecipitating the receptor with GFP antibodies (Fig. 1A). We could detect S1P1-GFP in transfected S1P1-GFP cells, but not in vector-GFP-expressing cells, as a band that migrated between 64 and 82 kDa, corresponding to the GFP-tagged human S1P1 (44-kDa S1P1 plus 27-kDa GFP). Heavy- and light-IgG chains were visible in both samples, and a GFP band (immediately above the light chain) was detected in vector-GFP-expressing cells.FIGURE 1AFD-R promotes S1P1-GFP down-regulation and enhanced protein ubiquitination. Equal amounts of 293-vector-GFP or 293-S1P1-GFP cell lysates were immunoprecipitated (IP) and immunoblotted (IB) with GFP antibodies to detect S1P1-GFP expression. A, S1P1-GFP expression was detected as a band running between 64 and 82 kDa (lane 2), whereas vector-GFP cells without insert did not express receptor (lane 1). B, vector GFP or S1P1-GFP cells were incubated with S1P1 agonists: AFD-R (500 nm), S1P (500 nm), SEW2871 (10 μm), or vehicle (0.1% fatty acid-free BSA) for 4 h in the presence of CHX. Lower panel, GFP immunoprecipitation-immunoblotting experiments from equivalent protein amounts revealed AFD-R-mediated S1P1-GFP down-regulation versus vehicle (Veh) treatment (n = 3). Upper panel, membranes were subsequently probed with P4D1 antibody for detection of agonist-induced ubiquitination. Note that P4D1 only recognized protein ubiquitination in ligand-stimulated, S1P1-GFP-expressing cells. The positions of molecular mass markers are indicated on the right (in kilodaltons). C, densitometric analysis of S1P1-GFP expression (left graph) and agonist-induced ubiquitinated-receptor complex (right graph) in cells treated with S1P1 agonists for 4 h. *, p < 0.05 relative to vehicle alone (left graph); *, p < 0.05 versus AFD-R treatment (right graph). Bars represent the mean ± S.E. of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Down-regulation was measured as the loss of receptor band to agonist stimulation in experiments in the presence of CHX. Treatment of S1P1-GFP cells with either AFD-R (500 nm), S1P (500 nm), SEW2871 (10 μm), or vehicle (0.1% fatty acid-free BSA) for 4 h led to the finding that AFD-R down-regulated approximately half (44 ± 2%) of total S1P1-GFP expression versus vehicle-treated cells (Fig. 1B, bottom). On the other hand, there were no significant differences in S1P1-GFP expression in cells incubated for the same time with either SEW2871 or S1P.Because protein ubiquitination has been implicated in regulating GPCR trafficking and fate, we employed an antibody that recognizes ubiquitinated substrates (P4D1) and probed the same GFP immunoprecipitates used for determining agonist-induced receptor down-regulation. Fig. 1B shows that agonist incubation was associated with increases in the ubiquitination of high (∼115–180 kDa) molecular mass protein(s), which we have referred to as the "ubiquitinated receptor complex." Ubiquitinated receptor complex was dependent upon agonist stimulation in receptor-transfected cells only, because it was not found to be associated with either vehicle-treated S1P1-GFP cells or vector-GFP stimulated (using 500 nm S1P) cells. Interestingly, at the 4-h agonist incubation studied, the ubiquitinated receptor complex was found to differ in magnitude depending on agonist utilized, with AFD-R stimulating significantly (1.8- and 2.5-fold) higher ubiquitination relative to S1P and SEW2871, respectively.A time course (0–6 h) of agonist-induced receptor down-regulation and associated ubiquitinated receptor complex is shown in Fig. 2. Again, depending on choice of agonist, two main differences in the measures were observed. First, only cells receiving AFD-R (500 nm) displayed significant receptor down-regulation relative to untreated cells. Down-regulation by AFD-R was apparent at 4 h (with a loss of approximately half of the total S1P1 expression), and continued at 6 h. In contrast, SEW2871 (10 μm) or S1P (500 nm) incubations did not significantly alter S1P1-GFP expression up to 6 h, relative to unstimulated cells, respectively. Second, and consistent with results from Fig. 1, AFD-R stimulated the highest magnitude of ubiquitinated receptor complex throughout the entire time course, relative to SEW2871 or S1P treatments, respectively. For all agonists, the time courses of ubiquitin receptor complex were biphasic, and of similar onsets (30 min), yet AFD-R induced sustained higher ubiquitination levels throughout most of the study, in comparison to SEW2871 or S1P.FIGURE 2Time course of agonist-stimulated ubiquitin receptor complex and receptor down-regulation. 293-S1P1-GFP cells were incubated for the indicated times with either 500 nm AFD-R, 500 nm S1P, or 10 μm SEW2871 in the presence of CHX. A, equal amounts of protein lysates were immunoprecipitated (IP) with a GFP antibody, and the membranes were immunoblotted (IB) with either anti-GFP (lower panels) to detect receptor-GFP expression or P4D1 (upper panels) for determination of agonist-induced ubiquitinated receptor complex. B, densitometry analysis of receptor-GFP and ubiquitinated receptor complex following either AFD-R (▪), SEW2871 (▴), or S1P (▾) stimulation. Time courses were plotted as the fraction of each agonist maximum, relative to unstimulated (time 0) cells. The graphs are from a representative experiment that was repeated twice with identical conditions.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The time course indicated that the agonist's maximal ubiquitination of cellular substrate(s) occurred at ∼1-h incubation; thus we compared the concentration dependence of agonist-induced ubiquitinated signaling in cells treated for 1 h with each agonist. Fig. 3 shows that AFD-R was 1,000- and 10,000-fold more potent in stimulating ubiquitin receptor complex formation than S1P and SEW2871, respectively, whereas no effect on amount of receptor (aside from the 50 nm S1P lane, which reflects a minor loading defect) was apparent at the 1-h incubation time. The potencies for AFD-R, S1P, and SEW2871 in stimulating ubiquitination at 1 h were 0.5 nm, 0.3 μm, and 2.5 μm, respectively. In addition, Fig. 3B indicates that AFD-R was a full agonist in activating receptor-complex ubiquitination relative to S1P and SEW2871 responses.FIGURE 3Ubiquitination of cellular substrates by distinct agonists displays vast differences in potency. 293-S1P1-GFP cells were i
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