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• W2023277555 abstract "Sphingosine 1-phosphate (S1P), a naturally occurring sphingolipid mediator and also a second messenger with growth factor-like actions in almost every cell type, is an endogenous ligand of five G protein-coupled receptors (GPCRs) in the endothelial differentiation gene family. The lack of GPCR crystal structures sets serious limitations to rational drug design and in silico searches for subtype-selective ligands. Here we report on the experimental validation of a computational model of the ligand binding pocket of the S1P1 GPCR surrounding the aliphatic portion of S1P. The extensive mutagenesis-based validation confirmed 18 residues lining the hydrophobic ligand binding pocket, which, combined with the previously validated three head group-interacting residues, now complete the mapping of the S1P ligand recognition site. We identified six mutants (L3.43G/L3.44G, L3.43E/L3.44E, L5.52A, F5.48G, V6.40L, and F6.44G) that maintained wild type [32P]S1P binding with abolished ligand-dependent activation by S1P. These data suggest a role for these amino acids in the conformational transition of S1P1 to its activated state. Three aromatic mutations (F5.48Y, F6.44G, and W6.48A) result in differential activation, by S1P or SEW2871, indicating that structural differences between the two agonists can partially compensate for differences in the amino acid side chain. The now validated ligand binding pocket provided us with a pharmacophore model, which was used for in silico screening of the NCI, National Institutes of Health, Developmental Therapeutics chemical library, leading to the identification of two novel nonlipid agonists of S1P1. Sphingosine 1-phosphate (S1P), a naturally occurring sphingolipid mediator and also a second messenger with growth factor-like actions in almost every cell type, is an endogenous ligand of five G protein-coupled receptors (GPCRs) in the endothelial differentiation gene family. The lack of GPCR crystal structures sets serious limitations to rational drug design and in silico searches for subtype-selective ligands. Here we report on the experimental validation of a computational model of the ligand binding pocket of the S1P1 GPCR surrounding the aliphatic portion of S1P. The extensive mutagenesis-based validation confirmed 18 residues lining the hydrophobic ligand binding pocket, which, combined with the previously validated three head group-interacting residues, now complete the mapping of the S1P ligand recognition site. We identified six mutants (L3.43G/L3.44G, L3.43E/L3.44E, L5.52A, F5.48G, V6.40L, and F6.44G) that maintained wild type [32P]S1P binding with abolished ligand-dependent activation by S1P. These data suggest a role for these amino acids in the conformational transition of S1P1 to its activated state. Three aromatic mutations (F5.48Y, F6.44G, and W6.48A) result in differential activation, by S1P or SEW2871, indicating that structural differences between the two agonists can partially compensate for differences in the amino acid side chain. The now validated ligand binding pocket provided us with a pharmacophore model, which was used for in silico screening of the NCI, National Institutes of Health, Developmental Therapeutics chemical library, leading to the identification of two novel nonlipid agonists of S1P1. Sphingosine 1-phosphate (S1P) 3The abbreviations used are: S1P, d-erythro-sphingosine-1-phosphate; FACS, fluorescence-activated cell sorting; GPCR, G protein-coupled receptor; GTPγS, guanine-γ-thiotriphosphate; TM, transmembrane domain; WT, wild type; EDG, endothelial differentiation gene; LPA, lysophosphatidic acid; BSA, bovine serum albumin. (see Fig. 1) is a naturally occurring sphingolipid mediator and also a second messenger with growth factor-like actions in almost every cell type (1Hla T. Pharmacol. Res. 2003; 47: 401-407Crossref PubMed Scopus (241) Google Scholar, 2Spiegel S. Milstien S. Nat. Rev. Mol. Cell. Biol. 2003; 4: 397-407Crossref PubMed Scopus (1758) Google Scholar, 3Hla T. Semin. Cell. Dev. Biol. 2004; 15: 513-520Crossref PubMed Scopus (347) Google Scholar). S1P plays fundamental physiological roles in vascular stabilization (4Liu Y. Wada R. Yamashita T. Mi Y. Deng C.-X. Hobson J.P. Rosenfeldt H.M. Nava V.E. Chae S. -S. Lee M.-J. Liu C.H. Hla T. Spiegel S. Proia R.L. J. Clin. Invest. 2000; 106: 951-961Crossref PubMed Scopus (993) Google Scholar), heart development (5Kupperman E. An S. Osborne N. Waldron S. Stainier D.Y.R. Nature. 2000; 406: 192-195Crossref PubMed Scopus (345) Google Scholar), lymphocyte homing (6Brinkmann V. Cyster J.G. Hla T. Am. J. Transplant. 2004; 4: 1019-1025Crossref PubMed Scopus (418) Google Scholar), and cancer angiogenesis (7Chae S.S. Paik J.H. Furneaux H. Hla T. J. Clin. Invest. 2004; 114: 1082-1089Crossref PubMed Scopus (205) Google Scholar). S1P elicits its biological effects through the activation of G protein-coupled receptors (GPCR) (8Tigyi G. Parrill A.L. Prog. Lipid Res. 2003; 42: 498-526Crossref PubMed Scopus (157) Google Scholar, 9Parrill A.L. Sardar V.M. Yuan H. Semin. Cell Dev. Biol. 2004; 15: 467-476Crossref PubMed Scopus (36) Google Scholar, 10Taha T.A. Argraves K.M. Obeid L.M. Biochim. Biophys. Acta. 2004; 1682: 48-55Crossref PubMed Scopus (164) Google Scholar) and through yet undefined intracellular targets (11Ghosh T.K. Bian J. Gill D.L. Science. 1990; 248: 1653-1656Crossref PubMed Scopus (328) Google Scholar, 12Ghosh T.K. Bian J. Gill D.L. J. Biol. Chem. 1994; 269: 22628-22635Abstract Full Text PDF PubMed Google Scholar, 13Meyer zu Heringdorf D. Liliom K. Schaefer M. Danneberg K. Jaggar J.H. Tigyi G. Jakobs K.H. FEBS Lett. 2003; 554: 443-449Crossref PubMed Scopus (89) Google Scholar, 14Itagaki K. Hauser C.J. J. Biol. Chem. 2003; 278: 27540-27547Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 15Olivera A. Rosenfeldt H.M. Bektas M. Wang F. Ishii I. Chun J. Milstien S. Spiegel S. J. Biol. Chem. 2003; 278: 46452-46460Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The endothelial differentiation gene (EDG) family of GPCR encodes eight highly homologous receptors. Five of these receptors, designated S1P1–S1P5, are specific for S1P, and the other three, LPA1–LPA3, are specific for the related lysophospholipid mediator lysophosphatidic acid (LPA) (16Chun J. Goetzl E.J. Hla T. Igarashi Y. Lynch K.R. Moolenaar W. Pyne S. Tigyi G. Pharmacol. Rev. 2002; 54: 265-269Crossref PubMed Scopus (447) Google Scholar). FTY-720, an immunosuppressive prodrug presently in phase 3 clinical trials, has attracted a lot of interest due to its effective inhibition of kidney transplant rejection and attenuation of autoimmune diseases, including multiple sclerosis (6Brinkmann V. Cyster J.G. Hla T. Am. J. Transplant. 2004; 4: 1019-1025Crossref PubMed Scopus (418) Google Scholar, 17Brinkmann V. Pinschewer D.D. Feng L. Chen S. Transplantation. 2001; 72: 764-769Crossref PubMed Scopus (156) Google Scholar, 18Brinkmann V. Lynch K.R. Curr. Opin. Immunol. 2002; 14: 569-575Crossref PubMed Scopus (254) Google Scholar). In vivo, FTY-720 becomes phosphorylated by sphingosine kinase type 2, and FTY-720-P is a high affinity ligand of all EDG family S1P receptors with the exception of S1P2 (19Mandala S. Hajdu R. Bergstrom J. Quackenbush E. Xie J. Milligan J. Thornton R. Shei G. Card D. Keohane C. Rosenbach M. Hale J. Lynch C.L. Rupprecht K. Parsons W. Rosen H. Science. 2002; 296: 346-349Crossref PubMed Scopus (1435) Google Scholar). In atrial myocytes, FTY-720-P, similarly to S1P (20Buenemann M. Brandts B.K. Pott L. Liliom K. Tseng J.-L. Desiderio D.M. Sun G. Miller D. Tigyi G. EMBO J. 1996; 15: 5524-5537Google Scholar, 21Liliom K. Sun G. Bunemann M. Virag T. Nusser N. Baker D.L. Wang D.A. Fabian M.J. Brandts B. Bender K. Eickel A. Malik K.U. Miller D.D. Desiderio D.M. Tigyi G. Pott L. Biochem. J. 2001; 355: 189-197Crossref PubMed Scopus (144) Google Scholar), activates an inwardly rectifying K+ conductance through the activation of the S1P3 receptor, which in turn elicits unwanted bradycardia (22Sanna 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 (538) Google Scholar). The immunosuppressive effects of FTY-720-P are mediated by the S1P1 receptor (23Cinamon G. Matloubian M. Lesneski M.J. Xu Y. Low C. Lu T. Proia R.L. Cyster J.G. Nat. Immunol. 2004; 5: 713-720Crossref PubMed Scopus (346) Google Scholar). FTY-720-P is an agonist of S1P1 and causes a long lasting desensitization of this receptor subtype, which appears to be the mechanism responsible for the inhibition of lymphocyte egress from the secondary lymphoid organs and the lymphopenia and the sequestration and eventual death of T lymphocytes (24Graeler M. Goetzl E.J. FASEB J. 2002; 16: 1874-1878Crossref PubMed Scopus (192) Google Scholar, 25Graeler M.H. Kong Y. Karliner J.S. Goetzl E.J. J. Biol. Chem. 2003; 278: 27737-27741Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Therefore, S1P1-selective antagonists or functional antagonists would probably be devoid of cardiac side effects and retain the immunosuppressive effect. Given the simple and highly flexible structure of S1P and its high similarity to LPA combined with the high degree of homology in the EDG receptor family, identification of novel nonlipid drug candidates with FTY-720-P-like effects is a fundamental challenge in the field. Development of receptor subtype-selective pharmacophores could aid rational drug design and lead optimization as well as identification of novel molecular scaffolds through in silico searches of large chemical libraries. However, the lack of crystal structures of GPCR sets serious limitations on this effort. Our groups have embarked on a computational modeling-driven mutagenesis approach to delineate agonist recognition by S1P1 at the atomic level. This effort has enabled us to identify S1P receptor residues that make essential interactions with the charged phosphate and amino moieties of the S1P pharmacophore. We have identified three basic amino acids, Arg-3.28, Lys-5.38, and Arg-7.34 in S1P1 and S1P4 that form salt bridges with the phosphate group of S1P and are essential for ligand binding in one or both receptors (26Parrill A.L. Wang D. Bautista D.L. Van Brocklyn J.R. Lorincz Z. Fischer D.J. Baker D.L. Liliom K. Spiegel S. Tigyi G. J. Biol. Chem. 2000; 275: 39379-39384Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 27Inagaki Y. Pham T.T. Fujiwara Y. Kohno T. Osborne D.A. Igarashi Y. Tigyi G. Parrill A.L. Biochem. J. 2005; 389: 187-195Crossref PubMed Scopus (46) Google Scholar). Furthermore, we have pinpointed position 3.29, conserved as glutamine in LPA- and glutamate in S1P-specific members of the EDG family, as the single locus that determines ligand specificity for S1P versus LPA through required ion pairing between glutamate and the ammonium moiety of S1P (28Wang D.A. Lorincz Z. Bautista D.L. Liliom K. Tigyi G. Parrill A.L. J. Biol. Chem. 2001; 276: 49213-49220Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The Gln/Asn-3.29 residue also plays an essential role in ligand binding, because substitution to alanine results in a loss of S1P and LPA binding and receptor activation. We also succeeded in elucidating differences between S1P1 and S1P4, as in the latter subtype Lys-5.38 and Trp-4.64 together compensate for the lack of a cationic residue at position 7.34 as in S1P1 (27Inagaki Y. Pham T.T. Fujiwara Y. Kohno T. Osborne D.A. Igarashi Y. Tigyi G. Parrill A.L. Biochem. J. 2005; 389: 187-195Crossref PubMed Scopus (46) Google Scholar). These polar head group interactions are essential for ligand binding, activation, and specificity. However, the hydrophobic tail constituting the bulk of S1P has not been assigned a function, and its interaction with the ligand binding pocket has not been elucidated. In the present study, we set out to identify the residues of S1P1 that interact with the aliphatic part of S1P, which we designate the hydrophobic binding pocket of the receptor. In this context, we examined the role of these hydrophobic interactions in ligand binding and receptor activation. Starting with our previously published S1P1-S1P complex, we hypothesized that 15 residues line the hydrophobic binding pocket. Combined characterization of mutations at these 15 positions and work done in parallel on S1P4 revealed that the orientation of transmembrane helix 5 (TM5) was incorrect in the original model. We developed a new S1P1 model that predicted an additional six residues in TM5 constituting the hydrophobic binding pocket and resolved the apparent inconsistencies between our experimental findings and the original S1P1 model (supplemental Fig. 1). The new theoretical model predicts that the hydrophobic tail of S1P interacts with 20 residues in TM3, TM5, and TM6. We evaluated the impact of mutations that changed the size and/or electrostatic properties of these residues on ligand activation and binding. Among these mutants, we found six mutations that showed no or greatly reduced ligand-dependent activation yet maintained [32P]S1P binding similar to the wild type (WT) S1P1. The lack of activation in the presence of ligand binding suggests that these residues play an important role in the conformational transition of S1P1 to its activated state. The theoretical model was used as the basis for in silico screening of the NCI, National Institutes of Health, Developmental Therapeutics Database library for novel scaffolds that might produce selective ligands of the S1P1 receptor using the Enhanced NCI Database Browser. Two novel nonlipid lead compounds were experimentally confirmed as partial agonists of S1P1. Additionally, comparison of residues at analogous positions in other S1P receptors suggests modifications that will lead to selective agonists of each receptor. All reagents were of analytical purity obtained from Sigma unless specified otherwise. S1P was purchased from Avanti Polar Lipids (Alabaster, AL). SEW2871 (Fig. 1) was a generous gift from Dr. Hugh Rosen (Scripps Research Institute, San Diego, CA). Amino acids in the TM domains of S1P1 can be assigned index positions to facilitate comparison between GPCRs with different numbers of amino acids, as described by Ballesteros and Weinstein (29Ballesteros J.A. Weinstein H. Conn P.M. Sealfon S.C. Methods in Neurosciences. Academic Press, Inc., San Diego1995: 366-428Google Scholar). An index position is in the format x.xx. The first number denotes the TM domain in which the residue appears. The second number indicates the position of that residue relative to the most highly conserved residue in that TM domain, which is arbitrarily assigned position 50. E3.29, then, indicates the relative position of this glutamate in TM3 relative to the highly conserved arginine 3.50 in the (E/D)RY motif (29Ballesteros J.A. Weinstein H. Conn P.M. Sealfon S.C. Methods in Neurosciences. Academic Press, Inc., San Diego1995: 366-428Google Scholar). S1P1—A model of human S1P1 (GenBank™ accession number AFP23365) was developed by homology to a model of rhodopsin (Protein Data Bank entry 1boj) in a manner described in our previous publications (26Parrill A.L. Wang D. Bautista D.L. Van Brocklyn J.R. Lorincz Z. Fischer D.J. Baker D.L. Liliom K. Spiegel S. Tigyi G. J. Biol. Chem. 2000; 275: 39379-39384Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 30Bautista D.L. Baker D.L. Wang D. Fischer D.J. Van Brocklyn J. Spiegel S. Tigyi G. Parrill A.L. J. Mol. Struct. (Theochem.). 2000; 529: 219-224Crossref Scopus (13) Google Scholar). Briefly, the rhodopsin model was used to generate TM1–TM6, whereas the structure for the seventh TM was based on TM7 of the dopamine D2 receptor model (31Konvicka K. Ballesteros J.A. Weinstein H. Biophys. J. 1998; 75: 601-611Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The preliminary model was further refined by converting all cis amide bonds to the trans configuration and by manually rotating side chains at polarity-conserved positions to optimize hydrogen bonding between TMs. The AMBER94 force field (32Cornell W.D. Cieplak P. Bayly C.I. Gould I.R. Merz K.M.J. Ferguson D.M. Spellmeyer D.C. Fox T. Caldwell J.W. Kollman P.A. J. Am. Chem. Soc. 1995; 117: 5179-5197Crossref Scopus (11590) Google Scholar) was utilized to optimize the receptor to a 0.1 kcal/mol·Å root mean square gradient. A corrected model was constructed using the previous model as the template with a manual realignment of TM5 to move each residue back one position in the alignment. The corrected model was refined and minimized using the same protocol. S1P1 Single/Double Point Mutants—Mutant models of S1P1 were developed by homology to the corrected S1P1 model. Using the MOE software package, the appropriate mutation was constructed by side chain replacement. Nonpolar hydrogen atoms were added to the mutated amino acid side chain, and the model was subsequently geometry-optimized. The AMBER94 force field (32Cornell W.D. Cieplak P. Bayly C.I. Gould I.R. Merz K.M.J. Ferguson D.M. Spellmeyer D.C. Fox T. Caldwell J.W. Kollman P.A. J. Am. Chem. Soc. 1995; 117: 5179-5197Crossref Scopus (11590) Google Scholar) was utilized again to optimize each mutant receptor to a 0.1 kcal/mol·Å root mean square gradient. A computational model of S1P was built using the MOE software package. The phosphate group was modeled with a net –1 charge, and the amine moiety was modeled with a net +1 charge. S1P was geometry-optimized using the MMFF94 force field (33Halgren T.A. J. Comp. Chem. 1996; 17: 490-519Crossref Scopus (4331) Google Scholar). Using the AUTODOCK 3.0 software package (34Morris G.M. Goodsell D.S. Halliday R.S. Huey R. Hart W.E. Belew R.K. Olson A.J. J. Comput. Chem. 1998; 19: 1639-1662Crossref Scopus (9210) Google Scholar), S1P was docked into S1P1 and the S1P1 mutant receptor models, and these complexes were evaluated based on final docked energy as well as visual analysis of electrostatic and other nonbonded interactions between the ligand and receptor. Docking parameters were set to default values with the exception of the number of energy evaluations (2.5 × 109), number of generations (30,000), local search iterations (3000), and number of runs (15Olivera A. Rosenfeldt H.M. Bektas M. Wang F. Ishii I. Chun J. Milstien S. Spiegel S. J. Biol. Chem. 2003; 278: 46452-46460Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The complexes exhibiting the best interactions based on either final docked energy or visual analysis were geometry-optimized using the MMFF94 force field (33Halgren T.A. J. Comp. Chem. 1996; 17: 490-519Crossref Scopus (4331) Google Scholar) and were subjected to critical qualitative analysis. SEW2871 was docked into the S1P1 receptor model using the same parameters and evaluation criteria. The docked positions of S1P and SEW2871 in the S1P1 receptor model were superposed and used to derive pharmacophore features sharing common location in both structures. Distances between these common pharmacophore features comprise the pharmacophore. The pharmacophore was used to search the Enhanced NCI Database Browser (available on the World Wide Web at 129.43.27.140/ncidb2/) for novel lead compounds. A trifluoromethylphenyl group was used for the anionic bioisostere, and carbon atoms were used to represent the hydrophobic functionality at other pharmacophore points. Hits from the search were evaluated based on their superposition onto the S1P and SEW2871 conformations from the S1P1 complexes. Hits were categorized as good, marginal, or negative based on these superpositions. Hits were considered negative if they exceeded the volume occupied by S1P or SEW2871 due to likely steric interactions with receptor atoms. The N-terminal FLAG epitope-tagged S1P1 receptor construct (GenBank™ accession number AF233365) was provided by Dr. Timothy Hla. Site-specific mutations were generated using the ExSite™ mutagenesis kit (Stratagene, La Jolla, CA) as described previously (26Parrill A.L. Wang D. Bautista D.L. Van Brocklyn J.R. Lorincz Z. Fischer D.J. Baker D.L. Liliom K. Spiegel S. Tigyi G. J. Biol. Chem. 2000; 275: 39379-39384Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 28Wang D.A. Lorincz Z. Bautista D.L. Liliom K. Tigyi G. Parrill A.L. J. Biol. Chem. 2001; 276: 49213-49220Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). S1P1 and the generated mutants were subcloned into pcDNA3.1 vector (Invitrogen). The sequence information of the mutants is listed in supplemental Table 1. Clones were verified by complete sequencing of the inserts. RH7777 and HEK293 cells (ATCC, Manassas, VA) were maintained in Dulbecco's modified minimal essential medium containing 10% fetal bovine serum (Hyclone, Logan, UT). Cells (2 × 106) were transfected with 2 μg of plasmid DNA with Effectene (Qiagen, Valencia, CA) according to the manufacturer's instructions for 24 h. Before ligand binding and receptor activation assays, the cells were washed twice with serum-free Dulbecco's modified minimal essential medium and serum-starved for at least 6 h. Western blot analysis of the FLAG epitope-tagged receptor construct was performed in transiently transfected RH7777 cells using a protocol described earlier (27Inagaki Y. Pham T.T. Fujiwara Y. Kohno T. Osborne D.A. Igarashi Y. Tigyi G. Parrill A.L. Biochem. J. 2005; 389: 187-195Crossref PubMed Scopus (46) Google Scholar). Anti-FLAG M2 and anti-β-actin antibody were purchased from Sigma. Goat anti-mouse antibody conjugated with horseradish peroxidase was purchased from Promega (Madison, WI). Cell surface expression of the FLAG-tagged S1P1 and its mutants was determined by flow cytometry (FACS) as described previously (27Inagaki Y. Pham T.T. Fujiwara Y. Kohno T. Osborne D.A. Igarashi Y. Tigyi G. Parrill A.L. Biochem. J. 2005; 389: 187-195Crossref PubMed Scopus (46) Google Scholar). Transfected RH7777 cells were harvested by trypsinization, and upon harvesting, the cells were maintained at 4 °C for the subsequent steps. The cells were washed with ice-cold FACS buffer (phosphate-buffered saline, pH 7.4, and 3% bovine serum albumin (BSA)). After washing once with FACS buffer, the cells were incubated for 30 min in blocking solution (5% BSA and 5% donkey serum (Sigma) in phosphate-buffered saline, pH 7.4). The cells were washed once with FACS buffer, and the cells were subsequently incubated for 60 min in FACS buffer with the anti-FLAG M2 monoclonal antibody (Sigma) (1:200). After washing the cells twice with FACS buffer, the cells were incubated for 30 min in FACS buffer with the Alexa Fluor 488-labeled donkey anti-mouse IgG (Molecular Probes, Inc., Eugene, OR) (1:1600). After washing the cells twice, samples were resuspended in 1% BSA in phosphate-buffered saline, pH 7.4, and analyzed using an LSR II flow cytometer (BD Biosciences). Data were analyzed with the Cell Quest software (BD Biosciences). The S1P binding assays were done essentially as previously described (28Wang D.A. Lorincz Z. Bautista D.L. Liliom K. Tigyi G. Parrill A.L. J. Biol. Chem. 2001; 276: 49213-49220Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Transfected HEK293 cells (5 × 105) were incubated at 4 °C in 20 mm Tris-HCl (pH 7.5) binding buffer containing 100 mm NaCl, 15 mm NaF, protease inhibitor mixture (Sigma), and 0.2 mg/ml essentially fatty acid-free BSA with 1 nm [32P]S1P in 50 nm S1P for 40 min. Cells were centrifuged and washed twice in binding buffer. The final pellet was resuspended in 2:1 CHCl3/MeOH, and the suspension was equilibrated in scintillation fluid overnight. Cell-bound radioactivity was measured by liquid scintillation counting using a Beckman LS5000 TA counter (Beckman Coulter, Irvine, CA). Specific binding was defined as the difference between total binding and nonspecific binding (in the presence of 2–5 μm cold S1P). S.E. values were computed on the basis of triplicate samples from two transfections. For the competition assays, HEK293 cells were used. Briefly, 4 × 105 cells were plated in 24-well dishes and allowed to adhere overnight. The cells were then transfected with 0.4 μg of the cDNA using Lipofectamine 2000 (Invitrogen), and the transfection proceeded for 48 h. After washing the cells twice with ice-cold binding buffer (20 mm Tris-HCl, pH 7.4, and 150 mm NaCl), 0.1 nm [32P]S1P and competing concentrations of cold S1P (1 nm-10 μm), resuspended in binding buffer plus 4% BSA, were applied to the cells and incubated on ice for 30 min. After washing the cells twice with ice-cold binding buffer plus 0.4% BSA, the cells were lysed with 0.5% SDS and equilibrated in scintillation fluid. The triplicate samples were measured. The KD and Bmax values were determined using GraphPad Prism software (San Diego, CA). Receptor functional assays were performed as previously described (28Wang D.A. Lorincz Z. Bautista D.L. Liliom K. Tigyi G. Parrill A.L. J. Biol. Chem. 2001; 276: 49213-49220Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) in transiently transfected RH7777 cells by measuring either S1P- or SEW-activated [35S]GTPγS binding. The significance of differences was determined by one-way analysis of variance, Bonferroni post hoc test, using Prism statistical software (GraphPad, San Diego, CA). Values were considered significantly different at p < 0.05. Mutagenesis Strategy—The previously reported computationally modeled complex of S1P in S1P1 features 15 previously unexplored amino acid residues in TM3, TM4, and TM6 with atoms within 4.5 Å of S1P. In the present study, we pursued a three-pronged replacement strategy of these residues. First, we introduced property-conserving mutations of these residues that either reduced or increased size. The logic behind this approach was to probe the impact of increased or relaxed steric constraints in the hydrophobic binding pocket on ligand-induced activation. In addition, we replaced many of these residues with charged amino acids of similar size to probe whether disruption to hydrophobicity in the putative binding pocket would have an impact on receptor function. Third, in a few cases, we replaced charged residues with noncharged residues of similar size to test the effect of polar interactions between the ligand and the receptor. Theoretical Model of the S1P1-S1P Complex; Revisions of the Previous Model—Discrepancies between model-derived predictions and experimental observations in this and a previous study on S1P4 (27Inagaki Y. Pham T.T. Fujiwara Y. Kohno T. Osborne D.A. Igarashi Y. Tigyi G. Parrill A.L. Biochem. J. 2005; 389: 187-195Crossref PubMed Scopus (46) Google Scholar) involved residues localized at the extracellular end of TM5. The differences we found were not consistent with proposed structural differences between active and inactive GPCR conformations; thus, they were probably an error in our previous S1P1 model (26Parrill A.L. Wang D. Bautista D.L. Van Brocklyn J.R. Lorincz Z. Fischer D.J. Baker D.L. Liliom K. Spiegel S. Tigyi G. J. Biol. Chem. 2000; 275: 39379-39384Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 28Wang D.A. Lorincz Z. Bautista D.L. Liliom K. Tigyi G. Parrill A.L. J. Biol. Chem. 2001; 276: 49213-49220Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). A corrected model of S1P1 was built based on an alternative alignment of TM5 derived from the recently validated S1P4 model (27Inagaki Y. Pham T.T. Fujiwara Y. Kohno T. Osborne D.A. Igarashi Y. Tigyi G. Parrill A.L. Biochem. J. 2005; 389: 187-195Crossref PubMed Scopus (46) Google Scholar). As depicted in supplemental Fig. 1, the corrected model demonstrates that eight residues in TM5 have atoms within 4.5 Å of S1P. One of these residues, Lys-5.38, forms an ion pair with the phosphate group of S1P. This polar interaction was not identified in previous validation of the S1P1 model due to the incorrect positioning of amino acid residues at the top of TM5. Cell Surface Expression of S1P1 Mutants—In order to verify that the WT and mutant constructs were expressed at comparable levels, membrane fractions were prepared and analyzed for expression by Western blot analysis using the N-terminal FLAG epitope present in the constructs (supplemental Fig. 2). The levels of expression on the membrane fractions were comparable with that of the WT receptor with the exception of L3.43E/L3.44E, L6.41G, F5.48Y, F6.44G, and W6.48E. FACS analysis was used to determine if cell surface expression of the N-terminal FLAG epitope was similar for the mutant constructs to that of the WT (Table 1). The WT and the mutant constructs, with the exception of V5.47T and L6.41G, were expressed at the cell surface in similar levels based on immunolabeling for the FLAG epitope. However, the V5.47L and L6.41E mutants were expressed and included in the pharmacological testing. The cell surface expression of L3.43E/L3.44E, F5.48Y, F6.44G, and W6.48E were comparable with that of the WT receptor.TABLE 1Cell surface expression of WT S1P1 and S1P1 mutants with abolished S1P-induced activation in RH7777 cells determined by flow cytometryConstructAnti-FLAG-stained cells%Vector2.5WT23.0M3.32K19.3L3.36E15.2L3.43E/L3.44E10.3L3.43G/L3.44G14.715.41A24.2C5.44D16.0V5.47L22.3V5.47T0.6F5.48G22.1F5.48Y22.1L5.51E17.4L5.52A14.1V6.40L19.6L6.41G5.6F6.44G17.2W6.48A12.6W6.48E23.4 Open table in a new tab The Effects of Mutations of Residues Lining Predicted Hydrophobic Binding Pocket on Ligand-induced Activation of S1P1—In the first round of pharmacological testing, we evaluated the impact of all amino acid replacements on the EC50 and maximal activation (Emax) elicited by S1P. The summary of the pharmacological properties caused by these replacements is presented" @default.
• W2023277555 created "2016-06-24" @default.
• W2023277555 creator A5001837381 @default.
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• W2023277555 date "2007-01-01" @default.
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• W2023277555 title "Identification of the Hydrophobic Ligand Binding Pocket of the S1P1 Receptor" @default.
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