Identification of Determinants of Ligand Binding Affinity and Selectivity in the Prostaglandin D2 Receptor CRTH2
2005; Elsevier BV; Volume: 280; Issue: 37 Linguagem: Inglês
10.1074/jbc.m502563200
ISSN1083-351X
AutoresAaron N. Hata, Terry P. Lybrand, Richard Breyer,
Tópico(s)Receptor Mechanisms and Signaling
ResumoThe chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) is a G protein-coupled receptor that mediates the pro-inflammatory effects of prostaglandin D2 (PGD2) generated in allergic inflammation. The CRTH2 receptor shares greatest sequence similarity with chemoattractant receptors compared with prostanoid receptors. To investigate the structural determinants of CRTH2 ligand binding, we performed site-directed mutagenesis of putative mCRTH2 ligand-binding residues, and we evaluated mutant receptor ligand binding and functional properties. Substitution of alanine at each of three residues in the transmembrane (TM) helical domains (His-106, TM III; Lys-209, TM V; and Glu-268, TM VI) and one in extracellular loop II (Arg-178) decreased PGD2 binding affinity, suggesting that these residues play a role in binding PGD2. In contrast, the H106A and E268A mutants bound indomethacin, a nonsteroidal anti-inflammatory drug, with an affinity similar to the wild-type receptor. HEK293 cells expressing the H106A, K209A, and E268A mutants displayed reduced inhibition of intracellular cAMP and chemotaxis in response to PGD2, whereas the H106A and E268A mutants had functional responses to indomethacin similar to the wild-type receptor. Binding of PGE2 by the E268A mutant was enhanced compared with the wild-type receptor, suggesting that Glu-268 plays a role in determining prostanoid ligand selectivity. Replacement of Tyr-261 with phenylalanine did not affect PGD2 binding but decreased the binding affinity for indomethacin. These results provided the first details of the ligand binding pocket of an eicosanoid-binding chemoattractant receptor. The chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) is a G protein-coupled receptor that mediates the pro-inflammatory effects of prostaglandin D2 (PGD2) generated in allergic inflammation. The CRTH2 receptor shares greatest sequence similarity with chemoattractant receptors compared with prostanoid receptors. To investigate the structural determinants of CRTH2 ligand binding, we performed site-directed mutagenesis of putative mCRTH2 ligand-binding residues, and we evaluated mutant receptor ligand binding and functional properties. Substitution of alanine at each of three residues in the transmembrane (TM) helical domains (His-106, TM III; Lys-209, TM V; and Glu-268, TM VI) and one in extracellular loop II (Arg-178) decreased PGD2 binding affinity, suggesting that these residues play a role in binding PGD2. In contrast, the H106A and E268A mutants bound indomethacin, a nonsteroidal anti-inflammatory drug, with an affinity similar to the wild-type receptor. HEK293 cells expressing the H106A, K209A, and E268A mutants displayed reduced inhibition of intracellular cAMP and chemotaxis in response to PGD2, whereas the H106A and E268A mutants had functional responses to indomethacin similar to the wild-type receptor. Binding of PGE2 by the E268A mutant was enhanced compared with the wild-type receptor, suggesting that Glu-268 plays a role in determining prostanoid ligand selectivity. Replacement of Tyr-261 with phenylalanine did not affect PGD2 binding but decreased the binding affinity for indomethacin. These results provided the first details of the ligand binding pocket of an eicosanoid-binding chemoattractant receptor. Prostaglandin D2 (PGD2) 3The abbreviations used are: PGD2, prostaglandin D2; C5aR, C5a anaphylatoxin receptor; CRTH2, chemoattractant receptor-homologous molecule expressed on Th2 cells; DK-PGD2, 13,14-dihydro-15-keto-PGD2; DP, D prostanoid; EC, extracellular loop domain; FPR, formyl peptide receptor; GPCR, G protein-coupled receptor; NSAID, nonsteroidal anti-inflammatory drug; TM, transmembrane domain; IP, prostacyclin receptor; HA, hemagglutinin; PE, phycoerythrin; ELISA, enzyme-linked immunosorbent assay; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; BSA, bovine serum albumin; FBS, fetal bovine serum. is the predominant prostanoid species produced by allergen-activated mast cells (1Lewis R.A. Soter N.A. Diamond P.T. Austen K.F. Oates J.A. Roberts II, L.J. J. Immunol. 1982; 129: 1627-1631PubMed Google Scholar, 2Murray J.J. Tonnel A.B. Brash A.R. Roberts II, L.J. Gosset P. Workman R. Capron A. Oates J.A. N. Engl. J. Med. 1986; 315: 800-804Crossref PubMed Scopus (410) Google Scholar) and has been implicated in the pathogenesis of allergic diseases such as allergic asthma and atopic dermatitis (1Lewis R.A. Soter N.A. Diamond P.T. Austen K.F. Oates J.A. Roberts II, L.J. J. Immunol. 1982; 129: 1627-1631PubMed Google Scholar, 3Barr R.M. Koro O. Francis D.M. Black A.K. Numata T. Greaves M.W. Br. J. Pharmacol. 1988; 94: 773-780Crossref PubMed Scopus (40) Google Scholar). Increased production or exposure to PGD2 leads to elevated Th2-type cytokines and eosinophilic inflammation in murine asthma models (4Fujitani Y. Kanaoka Y. Aritake K. Uodome N. Okazaki-Hatake K. Urade Y. J. Immunol. 2002; 168: 443-449Crossref PubMed Scopus (207) Google Scholar, 5Honda K. Arima M. Cheng G. Taki S. Hirata H. Eda F. Fukushima F. Yamaguchi B. Hatano M. Tokuhisa T. Fukuda T. J. Exp. Med. 2003; 198: 533-543Crossref PubMed Scopus (105) Google Scholar). However, the molecular mechanism of PGD2 action in the pathogenesis of allergic disease remains only partially characterized. PGD2 exerts its effects through two G protein-coupled receptors (GPCRs), the D prostanoid receptor (DP) and the recently discovered chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). DP receptor signaling has been linked to NF-κB activation (6Mandal A.K. Zhang Z. Ray R. Choi M.S. Chowdhury B. Pattabiraman N. Mukherjee A.B. J. Exp. Med. 2004; 199: 1317-1330Crossref PubMed Scopus (90) Google Scholar) and may influence dendritic cell function leading to skewing of the T cell response toward a Th2 phenotype (7Gosset P. Bureau F. Angeli V. Pichavant M. Faveeuw C. Tonnel A.B. Trottein F. J. Immunol. 2003; 170: 4943-4952Crossref PubMed Scopus (136) Google Scholar). Mice deficient in the DP receptor display reduced Th2-mediated airway inflammation in the ovalbumin-induced asthma model (8Matsuoka T. Hirata M. Tanaka H. Takahashi Y. Murata T. Kabashima K. Sugimoto Y. Kobayashi T. Ushikubi F. Aze Y. Eguchi N. Urade Y. Yoshida N. Kimura K. Mizoguchi A. Honda Y. Nagai H. Narumiya S. Science. 2000; 287: 2013-2017Crossref PubMed Scopus (681) Google Scholar), suggesting that PGD2 signaling through the DP receptor plays a pro-inflammatory role in settings of allergic inflammation. On the other hand, PGD2 has been hypothesized to exert anti-inflammatory effects by inhibiting dendritic cell migration and T cell activation (9Hammad H. de Heer H.J. Soullie T. Hoogsteden H.C. Trottein F. Lambrecht B.N. J. Immunol. 2003; 171: 3936-3940Crossref PubMed Scopus (163) Google Scholar). The role of PGD2 signaling through the CRTH2 receptor in allergic disease is less well established. In humans, the CRTH2 receptor is expressed on Th2 cells, eosinophils, basophils, and monocytes (7Gosset P. Bureau F. Angeli V. Pichavant M. Faveeuw C. Tonnel A.B. Trottein F. J. Immunol. 2003; 170: 4943-4952Crossref PubMed Scopus (136) Google Scholar, 10Nagata K. Tanaka K. Ogawa K. Kemmotsu K. Imai T. Yoshie O. Abe H. Tada K. Nakamura M. Sugamura K. Takano S. J. Immunol. 1999; 162: 1278-1286PubMed Google Scholar, 11Nagata K. Hirai H. Tanaka K. Ogawa K. Aso T. Sugamura K. Nakamura M. Takano S. FEBS Lett. 1999; 459: 195-199Crossref PubMed Scopus (283) Google Scholar), which are known to play a role in the pathogenesis of allergic diseases such as asthma (12Hamid Q. Tulic M.K. Liu M.C. Moqbel R. J. Allergy Clin. Immunol. 2003; 111: 5-17Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Polymorphisms in the 3′-untranslated region of the CRTH2 receptor gene that confer greater mRNA stability have been linked to increased asthma severity (13Huang J.L. Gao P.S. Mathias R.A. Yao T.C. Chen L.C. Kuo M.L. Hsu S.C. Plunkett B. Togias A. Barnes K.C. Stellato C. Beaty T.H. Huang S.K. Hum. Mol. Genet. 2004; 13: 2691-2697Crossref PubMed Scopus (92) Google Scholar), and increased numbers of circulating T cells expressing the CRTH2 receptor have been correlated with severity of atopic dermatitis (14Cosmi L. Annunziato F. Galli M.I.G. Maggi R.M.E. Nagata K. Romagnani S. Eur. J. Immunol. 2000; 30: 2972-2979Crossref PubMed Scopus (222) Google Scholar, 15Iwasaki M. Nagata K. Takano S. Takahashi K. Ishii N. Ikezawa Z. J. Investig. Dermatol. 2002; 119: 609-616Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). CRTH2 receptor activation stimulates chemotaxis of human Th2 cells, eosinophils, and basophils both in vitro and in vivo (16Hirai H. Tanaka K. Yoshie O. Ogawa K. Kenmotsu K. Takamori Y. Ichimasa M. Sugamura K. Nakamura M. Takano S. Nagata K. J. Exp. Med. 2001; 193: 255-261Crossref PubMed Scopus (981) Google Scholar, 17Shiraishi Y. Asano K. Nakajima T. Oguma T. Suzuki Y. Shiomi T. Sayama K. Niimi K. Wakaki M. Kagyo J. Ikeda E. Hirai H. Yamaguchi K. Ishizaka A. J. Pharmacol. Exp. Ther. 2005; 312: 954-960Crossref PubMed Scopus (113) Google Scholar), suggesting that the CRTH2 receptor may directly mediate recruitment of inflammatory cells in response to PGD2 generated in settings of allergic inflammation and thus a pro-inflammatory role (18Hata A.N. Breyer R.M. Pharmacol. Ther. 2004; 103: 147-166Crossref PubMed Scopus (700) Google Scholar). Recently it was reported that ramatroban, a thromboxane receptor antagonist used clinically for the treatment of allergic rhinitis, also exhibits CRTH2 antagonist activity and inhibits PGD2-stimulated eosinophil migration (17Shiraishi Y. Asano K. Nakajima T. Oguma T. Suzuki Y. Shiomi T. Sayama K. Niimi K. Wakaki M. Kagyo J. Ikeda E. Hirai H. Yamaguchi K. Ishizaka A. J. Pharmacol. Exp. Ther. 2005; 312: 954-960Crossref PubMed Scopus (113) Google Scholar, 19Sugimoto H. Shichijo M. Iino T. Manabe Y. Watanabe A. Shimazaki M. Gantner F. Bacon K.B. J. Pharmacol. Exp. Ther. 2003; 305: 347-352Crossref PubMed Scopus (154) Google Scholar). Consistent with this finding, ramatroban had been observed previously to inhibit antigen-induced mucosal eosinophilia in sensitized guinea pigs (20Narita S. Asakura K. Kataura A. Int. Arch. Allergy Immunol. 1996; 109: 161-166Crossref PubMed Scopus (88) Google Scholar). Ramatroban shares structural similarity with indomethacin, an arylacetic acid class nonselective cyclooxygenase inhibitor and widely used nonsteroidal anti-inflammatory drug (NSAID) that has also been shown to be a potent CRTH2 agonist (21Hirai H. Tanaka K. Takano S. Ichimasa M. Nakamura M. Nagata K. J. Immunol. 2002; 168: 981-985Crossref PubMed Scopus (134) Google Scholar). We recently performed structure-activity relationship analysis of arylacetic acid NSAIDs that revealed play structural features of indomethacin and ramatroban that are required for binding to the mouse CRTH2 receptor (22Hata A.N. Lybrand T.P. Marnett L.J. Breyer R.M. Mol. Pharmacol. 2005; 67: 640-647Crossref PubMed Scopus (27) Google Scholar). The CRTH2 receptor does not share significant sequence homology with the DP or other prostanoid receptors but instead exhibits greatest sequence similarity to peptide chemoattractant receptors such as the formyl peptide receptor (16Hirai H. Tanaka K. Yoshie O. Ogawa K. Kenmotsu K. Takamori Y. Ichimasa M. Sugamura K. Nakamura M. Takano S. Nagata K. J. Exp. Med. 2001; 193: 255-261Crossref PubMed Scopus (981) Google Scholar). Residues that have been identified as playing a role in prostanoid ligand binding by the DP and other prostanoid receptors are not conserved in the CRTH2 receptor sequence, suggesting that the CRTH2 receptor binds its prostaglandin ligand in a manner distinct from the other prostanoid receptors. In addition to peptide chemoattractant receptors, the CRTH2 receptor is related to several eicosanoid-binding chemoattractant receptors such as the leukotriene B4 and lipoxin A4 receptors; however, little is known about how these receptors interact with their respective ligands. To investigate the structure of the mouse CRTH2 receptor ligand binding pocket, we performed site-directed mutagenesis of putative ligand-binding residues, and we evaluated the effects of these mutations on ligand binding and receptor function. These studies reveal that PGD2 likely binds in the CRTH2 binding pocket with an orientation that is distinct from that proposed for other prostanoid receptors. Furthermore, these data demonstrate that PGD2 and indomethacin interact with distinct but overlapping sets of residues within the ligand binding pocket and suggest specific ligand-receptor interactions that may play a role in determining ligand binding affinity and selectivity. Materials—[3H]PGD2 was purchased from Amersham Biosciences, and unlabeled prostaglandin ligands were from Cayman Chemical (Ann Arbor, MI). Indomethacin, forskolin, isobutylmethylxanthine, and sodium butyrate were from Sigma. Ramatroban was a kind gift from K. Bacon (Bayer AG, Kyoto, Japan). DMEM and Opti-MEM were from Invitrogen. FBS was obtained from Atlanta Biologicals (Lawrenceville, GA). G418 was purchased from Mediatech (Herndon, VA). l-Glutamine and penicillin/streptomycin were from BioWhittaker (Walkersville, MD). The 262K monoclonal anti-HA antibody was purchased from Cell Signaling Technologies (Beverly, MA); the PE-conjugated goat anti-mouse antibody was from Jackson ImmunoResearch (West Grove, PA); the 3F10 rat anti-HA antibody was from Roche Applied Science, and the horseradish peroxidase-conjugated goat anti-rat antibody was from Amersham Biosciences. Construction and Expression of HA-tagged Wild-type mCRTH2 and HA-mCRTH2 Mutants—The HA-mCRTH2 expression plasmid was generated by ligation of fragments containing the HA epitope tag (SnaBI/NdeI) from the 77AHA pRc/CMV plasmid (23Audoly L. Breyer R.M. Mol. Pharmacol. 1997; 51: 61-68Crossref PubMed Scopus (44) Google Scholar) and the mCRTH2 coding region (NdeI/XbaI) from the pRc/CMV/mCRTH2 plasmid (24Hata A.N. Zent R. Breyer M.D. Breyer R.M. J. Pharmacol. Exp. Ther. 2003; 306: 463-470Crossref PubMed Scopus (64) Google Scholar) into pRc/CMV (SnaBI/XbaI). HA-mCRTH2 mutants were generated by using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) with the HA-mCRTH2 pRc/CMV plasmid as template. Mutagenic oligonucleotides used were as follows (sense, 5′ to 3′ with mutagenic changes underlined): H38A, GTCGGTGCTGTTGGCCGGGCTGGCCTC; T87A, CCTGCCTTTCTTCGCCTACTTCCTGGCAG; H106A, CACTACCTTCTGCAAGCTAGCATCCTCGGTCTTCTTCCTCA; S107A, TTCTGCAAGCTACATGCCTCGGTCTTCTTCCTC; S108A, TGCAAGCTACATTCCGCGGTCTTCTTCCTCAAC; F110A, GCAAGCTACATTCCTCGGTCGCATTCCTCAACATGTTTGCCAG; R178A, CGCGGCTTGATGGCGCCATCATGTGCTACTACA; R178K, CGCGGCTTGATGGCAAGATCATGTGCTACTACA; R178H, CGCGGCTTGATGGCCACATCATGTGCTACTACA; L205A, CGCCAGAAGGCCGCGGCGGTCAGCAAATT; S208A, GCCCTGGCGGTCGCCAAATTCCTGCTGGC; K209A, CCTGGCGGTCAGCGCATTCCTGCTGGCCTTC; K209R, GGCGGTCAGCAGATTCCTGCTGGCC; Y261A, TCTGCTGGGGGCCCGCACACATCTTCAGTCTGC; Y261F, GCTGGGGGCCCTTCCACATCTTCAGTCTG; S265A, GCCCTACCACATCTTCGCACTGCTGGAGGCGCG; E268A, CAGTCTGCTGGCGGCGCGTGCCC; E268Q, CAGTCTGCTGCAGGCGCGTGCCC; E268D, CAGTCTGCTGGACGCGCGTGCCC; and S290A, GCTGCCCTTTGTCACCGCACTGGCCTTCTTCAACAGC. The coding regions of two independent clones of each mutant were verified by sequencing to ensure that only the intended mutation had been introduced into the expression construct. Expression of HA-mCRTH2 and Mutant Receptors in HEK293 Cells— Two independently derived plasmids encoding the HA-mCRTH2 and each mutant mCRTH2 receptor construct were transiently transfected into HEK293 cells using Lipofectamine 2000 (Invitrogen). Stable cell lines (HA-mCRTH2, H106A, K209A, K209R, and E268A) were selected in media containing 600 μg/ml G418, and clones were isolated by manual colony isolation using cloning rings. For the Y261F mutant, a stably transfected polyclonal population was generated. Cells were maintained at 37 °C in humidified air containing 5.5% CO2 in DMEM supplemented with 10% FBS, 2 mm l-glutamine, 100 units ml-1 penicillin, 100 μg and ml-1 streptomycin. Expression of HA-mCRTH2 and mutant receptors in both transiently and stably expressing cells was enhance incubation with 5 mm sodium butyrate for 24 h prior to all experiments. Flow Cytometric Analysis of Receptor Expression—HA-mCRTH2 and mutant receptor expression in transiently and stably transfected HEK293 cells was monitored by flow cytometry. After brief trypsinization, cells were resuspended in media containing 10% FBS and immediately placed on ice. Cells were incubated with 1:100 of the 262K monoclonal anti-HA antibody for 1 h at 4 °C, washed twice with ice-cold PBS, and incubated with 1:100 of a PE-conjugated goat anti-mouse antibody for 30 min at 4 °C. Cells were washed once with ice-cold PBS, resuspended in PBS, and analyzed for PE fluorescence on a FACScan flow cytometer (BD Biosciences). Cell Surface ELISA Analysis of Receptor Expression—Cells stably expressing HA-mCRTH2 or mutant receptors were plated in poly-d-lysine-coated 96-well plates (BD Biosciences) at a density of 4 × 105/well 2 days prior to experiment to ensure confluency at the time of ELISA. Receptor expression was enhanced by addition of sodium butyrate (5 mm) 24 h prior to the ELISA. Cells transiently expressing HA-mCRTH2 or mutant receptors were plated at a density of 6 × 105/well in the presence of sodium butyrate 1 day prior to experiment. Cells were fixed by incubation with 4% paraformaldehyde containing 0.12 m sucrose in PBS containing 1 mm MgCl2 and 0.5 m CaCl2 (PBS-CM) for 20–30 min at room temperature. Cells were washed twice with PBS-CM and incubated with 3% BSA in PBS-CM for 30 min at 37 °C. Cells were incubated with 1:500 of the 3F10 rat anti-HA antibody in 3% BSA in PBS-CM for 1 h at 37 °C, washed three times with PBS-CM for 5 min each, and incubated with 1:100 of a horseradish peroxidase-conjugated goat anti-rat antibody in 3% BSA in PBS-CM for 1 h at 37 °C. Cells were washed three times with PBS-CM, and the chromogenic substrate o-phenylenediamine dihydrochloride (1 mg/ml, Pierce) was added. After color development (20–30 min), the reaction was stopped with the addition of an equal volume of 2.5 m sulfuric acid, and the absorbance at 490 nm was determined. In some experiments, cells were permeabilized by incubation with 0.2% Triton X-100 for 15 min prior to blocking with BSA. Radioligand Binding—Membranes for radioligand binding experiments were harvested from HEK293 cells expressing HA-mCRTH2 wild-type or mutant receptors as described (24Hata A.N. Zent R. Breyer M.D. Breyer R.M. J. Pharmacol. Exp. Ther. 2003; 306: 463-470Crossref PubMed Scopus (64) Google Scholar). Membranes (30 μg of membrane protein) were incubated with [3H]PGD2 and unlabeled ligands for 1.5 h at 4 °C in binding buffer (25 mm HEPES (pH 7.4), 1 mm EDTA, 5 mm MgCl2, 140 mm NaCl, 5 mm KCl). These conditions were sufficient to achieve apparent equilibrium of binding while ensuring ligand stability and minimizing nonspecific binding. The binding reaction was terminated by the addition of 3 ml of ice-cold binding buffer and rapidly filtered under vacuum over Whatman GF/F filters. Filters were washed three times with 3 ml of ice-cold binding buffer, dried and counted in 4 ml of Ultima Gold scintillation fluid (Packard Biosciences, Groningen, The Netherlands). For saturation isotherm experiments, the [3H]PGD2 concentration ranged from 2.5 to 30 nm. Specific binding did not exceed 5% of total radioligand concentration present in the binding reaction. For competition experiments, 3 nm [3H]PGD2 was used. [cAMP]i Assay—HEK293 cells stably expressing HA-mCRTH2 or mutant receptors were plated at a density of 9 × 105/well in 6-well plates 2 days prior to the experiment, and 5 mm sodium butyrate was added for the final 24 h. Thirty minutes prior to addition of ligands, media were replaced with Opti-MEM I containing 0.5 mm isobutylmethylxanthine. Cells were incubated with ligands for 10 min and washed once with PBS, and the reaction was terminated by the addition of 0.1 m HCl. Cells were scraped free, and the resulting cell suspension was centrifuged for 10 min at 1000 × g. Supernatants were assayed for protein content by BCA assay (Pierce). After normalization to protein content, [cAMP]i levels were determined by an enzyme-linked immunoassay according to the manufacturer's instructions (Cayman Chemical). Transwell Migration Assay—Cells stably expressing HA-mCRTH2 or mutant receptors were incubated with 5 mm sodium butyrate prior to harvesting. Cells were trypsinized, washed three times in PBS, and resuspended in DMEM. Cells (1 × 105) were added to the upper chamber of 24-well 8.0-μm polycarbonate transwell inserts (Costar, Cambridge, MA) that had been previously treated overnight with 5 μg/ml Matrigel (BD Biosciences) in PBS at 4 °C and blocked in the presence of 2% BSA in PBS for 1 h at 37°C. Ligands were diluted in DMEM and added to the lower chamber. After incubating for 4 h at 37°C, inserts were removed, and cells adhering to the top of the membrane were removed with a cotton swab. Cells on the bottom of the membrane were fixed with 3.7% formaldehyde for 1 h, washed twice with PBS, and stained overnight with crystal violet. For each insert, five independent fields were counted in blinded fashion at ×200 magnification. Molecular Modeling—The mCRTH2 model was constructed as described previously (22Hata A.N. Lybrand T.P. Marnett L.J. Breyer R.M. Mol. Pharmacol. 2005; 67: 640-647Crossref PubMed Scopus (27) Google Scholar). Briefly, the transmembrane-spanning α-helical bundle of mCRTH2 was constructed with homology modeling methods, using a β2-adrenergic receptor model as a template (25Furse K.E. Lybrand T.P. J. Med. Chem. 2003; 46: 4450-4462Crossref PubMed Scopus (60) Google Scholar). The extracellular and cytosolic loops were generated de novo by attaching the loops as extended polypeptides to the appropriate helix and applying weak harmonic constraints during low temperature (30 K) molecular dynamics to connect appropriate loop segments with a trans-peptide bond. A putative disulfide cross-link between the extracellular region of transmembrane helix III and extracellular loop II was generated by applying additional constraints during the generation of extracellular loop II. The N and C termini were generated using similar techniques in the absence of constraints. To simplify model construction, the C-terminal tail was truncated at Val-321. The intact mCRTH2 receptor model was then refined further with limited energy minimization and low temperature molecular dynamics simulation to relieve any peptide backbone conformational strain or residual bad steric interactions. Ligands were docked into the putative ligand-binding site using both manual (PGD2) and automated (indomethacin) ligand docking algorithms. Receptor-ligand complexes that were consistent with mutagenesis data were refined by using limited energy minimization and low temperature molecular dynamics simulations in which weak harmonic positional restraints were placed on backbone atoms to prevent overcompaction of the receptor. All structural refinement calculations were performed in vacuo with a distance-dependent dielectric model, using standard AMBER all-atom potential functions. Energy minimization and molecular dynamics calculations were performed with the AMBER package. Automated ligand docking was performed using the automated docking module in MOE (Chemical Computing, Inc.). To predict putative CRTH2 receptor ligand-binding residues, we performed multiple sequence alignments to compare the transmembrane domains of the human and mouse CRTH2 receptors with those of related chemoattractant receptors, including the formyl peptide receptor (FPR), the C5a anaphylatoxin receptor (C5aR), and the leukotriene B4 receptors (BLT1 and BLT2). The ligand binding pockets of the FPR and C5a receptors have been extensively studied and ligand-binding residues identified (26Quehenberger O. Pan Z.K. Prossnitz E.R. Cavanagh S.L. Cochrane C.G. Ye R.D. Biochem. Biophys. Res. Commun. 1997; 238: 377-381Crossref PubMed Scopus (30) Google Scholar, 27Miettinen H.M. Mills J.S. Gripentrog J.M. Dratz E.A. Granger B.L. Jesaitis A.J. J. Immunol. 1997; 159: 4045-4054PubMed Google Scholar, 28Siciliano S.J. Rollins T.E. DeMartino J. Konteatis Z. Malkowitz L. Van Riper G. Bondy S. 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We reasoned that CRTH2 receptor residues with the highest probability of interacting with ligand are as follows: 1) those that are conserved between the CRTH2 and chemoattractant receptors that have been shown to play a role in chemoattractant ligand binding or 2) those charged or polar residues that lie within the regions of conservation but are not themselves conserved. Based on these criteria (TABLE ONE), an initial set of candidate residues (His-106, Ser-107, Ser-108, Leu-205, Ser-208, and Lys-209) was selected and individually mutated to Ala. Additionally, Ser-290 was selected because its position corresponds to that of a highly conserved arginine that has been shown to play a role in ligand binding for members of the prostanoid receptor GPCR subfamily. The wild-type and mutant mCRTH2 receptor constructs possessed an N-terminal hemagglutinin (HA) epitope tag, which had ligand binding and signaling indistinguishable from that observed for the wild-type untagged receptor (data not shown).TABLE ONERationale for selection of mCRTH2 residues to mutateResidueRationaleTM IHis-38Based on mCRTH2 receptor model (22Hata A.N. Lybrand T.P. Marnett L.J. Breyer R.M. Mol. Pharmacol. 2005; 67: 640-647Crossref PubMed Scopus (27) Google Scholar), oriented toward putative ligand binding pocket region; position corresponds to residues in the DP and IP receptor implicated in ligand recognition (32Kobayashi T. Ushikubi F. Narumiya S. J. Biol. Chem. 2000; 275: 24294-24303Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar)TM IIThr-87Based on mCRTH2 receptor model (22Hata A.N. Lybrand T.P. Marnett L.J. Breyer R.M. Mol. Pharmacol. 2005; 67: 640-647Crossref PubMed Scopus (27) Google Scholar), oriented toward putative ligand binding pocket regionTM IIIHis-106Represents gain of charge/polarity compared with chemoattractant receptors; C5aR tolerates only hydrophobic residues at this position (30Baranski T.J. Herzmark P. Lichtarge O. Gerber B.O. Trueheart J. Meng E.C. Iiri T. Sheikh S.P. Bourne H.R. J. Biol. Chem. 1999; 274: 15757-15765Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar)Ser-107Phenylalanine at this position in FPR is part of a cluster of residues that has been shown to be involved in ligand binding (26Quehenberger O. Pan Z.K. Prossnitz E.R. Cavanagh S.L. Cochrane C.G. Ye R.D. Biochem. Biophys. Res. Commun. 1997; 238: 377-381Crossref PubMed Scopus (30) Google Scholar)Ser-108Threonine at this position in FPR is part of a cluster of residues that has been shown to be involved in ligand binding (26Quehenberger O. Pan Z.K. Prossnitz E.R. Cavanagh S.L. Cochrane C.G. Ye R.D. Biochem. Biophys. Res. Commun. 1997; 238: 377-381Crossref PubMed Scopus (30) Google Scholar)Phe-110Based on mCRTH2 receptor model (22Hata A.N. Lybrand T.P. Marnett L.J. Breyer R.M. Mol. Pharmacol. 2005; 67: 640-647Crossref PubMed Scopus (27) Google Scholar), preliminary ligand docking simulations suggested an interaction between the phenylalanine aromatic ring and the p-chlorobenzoyl moiety of indomethacinEC IIArg-178Based on mCRTH2 receptor model (22Hata A.N. Lybrand T.P. Marnett L.J. Breyer R.M. Mol. Pharmacol. 2005; 67: 640-647Crossref PubMed Scopus (27) Google Scholar), positively charged side chain forms a "lid" on the putative ligand binding pocketTM VLeu-205Arginine in FPR has been shown to be involved in ligand binding (27Miettinen H.M. Mills J.S. Gripentrog J.M. Dratz E.A. Granger B.L. Jesaitis A.J. J. Immunol. 1997; 159: 4045-4054PubMed Google Scholar)Ser-208Represents gain of polarity compared with chemoattractant receptors; alanine substitution for isoleucine in FPR leads to decrease in ligand binding (27Miettinen H.M. Mills J.S. Gripentrog J.M. Dratz E.A. Granger B.L. Jesaitis A.J. J. Immunol. 1997; 159: 4045-4054PubMed Google Scholar)Lys-209Conserved arginine in FPR and C5aR has been shown to be involved in ligand binding (27Miettinen H.M. Mills J.S. Gripentrog J.M. Dratz E.A. Granger B.L. Jesaitis A.J. J. Immunol. 1997; 159: 4045-4054PubMed Google Scholar, 29DeMartino J.A. Konteatis Z.D. Siciliano S.J. Van Riper G. Underwood D.J. Fischer P.A. Springer M.S. J. Biol. Chem. 1995; 270: 15966-15969Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 31Raffetseder U. Roper D. Mery L. Gietz C. Klos A. Grotzinger J. Wollmer A. Boulay F. Kohl J. Bautsch W. Eur. J. Biochem. 1996; 235: 82-90Crossref PubMed Scopus (37) Google Scholar)TM VITyr-261Based on mCRTH2 receptor model (22Hata A.N. Lybrand T.P. Marnett L.J. Breyer R.M. Mol. Pharmacol. 2005; 67: 640-647Crossref PubMed Scopus (27) Google Scholar), oriented toward putative ligand binding pocket regionSer-265Based on mCRTH2 receptor model (22Hata A.N. Lybrand T.P. Marnett L.J. Breyer R.M. Mol. Pharmacol. 2005; 67: 640-647Crossref PubMed Scopus (27) Google Scholar),
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