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A Gq/11-coupled Mutant Histamine H1 Receptor F435A Activated Solely by Synthetic Ligands (RASSL)

2005; Elsevier BV; Volume: 280; Issue: 41 Linguagem: Inglês

10.1074/jbc.m504165200

1083-351X

Martijn Bruysters, Aldo Jongejan, Atilla Akdemir, Remko A. Bakker, Rob Leurs,

Mast cells and histamine

Recently, G protein-coupled receptors activated solely by synthetic ligands (RASSLs) have been introduced as new tools to study Gαi signaling in vivo (1Redfern C.H. Coward P. Degtyarev M.Y. Lee E.K. Kwa A.T. Hennighausen L. Bujard H. Fishman G.I. Conklin B.R. Nat. Biotechnol. 1999; 17: 165-169Crossref PubMed Scopus (154) Google Scholar, 2Redfern C.H. Degtyarev M.Y. Kwa A.T. Salomonis N. Cotte N. Nanevicz T. Fidelman N. Desai K. Vranizan K. Lee E.K. Coward P. Shah N. Warrington J.A. Fishman G.I. Bernstein D. Baker A.J. Conklin B.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4826-4831Crossref PubMed Scopus (142) Google Scholar). Also, Gαs-coupled G protein-coupled receptors have been engineered to generate Gαs-coupled RASSLs (3Srinivasan S. Vaisse C. Conklin B.R. Ann. N. Y. Acad. Sci. 2003; 994: 225-232Crossref PubMed Scopus (23) Google Scholar, 4Claeysen S. Joubert L. Sebben M. Bockaert J. Dumuis A. J. Biol. Chem. 2003; 278: 699-702Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In this study, we exploited the differences in binding pockets between different classes of H1 receptor agonists and identified the first Gαq/11-coupled RASSL. The mutant human H1 receptor F435A (6.55) combines a strongly decreased affinity (25-fold) and potency for the endogenous ligand histamine (200-fold) with improved affinities (54-fold) and potencies (2600-fold) for 2-phenylhistamines, a synthetic class of H1 receptor agonists. Molecular dynamics simulations provided a mechanism for distinct agonist binding to both wild-type and F435A mutant H1 receptors. Recently, G protein-coupled receptors activated solely by synthetic ligands (RASSLs) have been introduced as new tools to study Gαi signaling in vivo (1Redfern C.H. Coward P. Degtyarev M.Y. Lee E.K. Kwa A.T. Hennighausen L. Bujard H. Fishman G.I. Conklin B.R. Nat. Biotechnol. 1999; 17: 165-169Crossref PubMed Scopus (154) Google Scholar, 2Redfern C.H. Degtyarev M.Y. Kwa A.T. Salomonis N. Cotte N. Nanevicz T. Fidelman N. Desai K. Vranizan K. Lee E.K. Coward P. Shah N. Warrington J.A. Fishman G.I. Bernstein D. Baker A.J. Conklin B.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4826-4831Crossref PubMed Scopus (142) Google Scholar). Also, Gαs-coupled G protein-coupled receptors have been engineered to generate Gαs-coupled RASSLs (3Srinivasan S. Vaisse C. Conklin B.R. Ann. N. Y. Acad. Sci. 2003; 994: 225-232Crossref PubMed Scopus (23) Google Scholar, 4Claeysen S. Joubert L. Sebben M. Bockaert J. Dumuis A. J. Biol. Chem. 2003; 278: 699-702Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In this study, we exploited the differences in binding pockets between different classes of H1 receptor agonists and identified the first Gαq/11-coupled RASSL. The mutant human H1 receptor F435A (6.55) combines a strongly decreased affinity (25-fold) and potency for the endogenous ligand histamine (200-fold) with improved affinities (54-fold) and potencies (2600-fold) for 2-phenylhistamines, a synthetic class of H1 receptor agonists. Molecular dynamics simulations provided a mechanism for distinct agonist binding to both wild-type and F435A mutant H1 receptors. Receptors activated solely by synthetic ligands (RASSLs) 2The abbreviations used are: RASSL, receptor activated solely by synthetic ligands; H1R, histamine H1 receptor; WT, wild-type; HA, histamine; PheHA, 2-phenylhistamine; ClPheHA, 2-(3-chlorophenyl)histamine; Cl2PheHA, 2-(3,4-dichlorophenyl)histamine; dClPheHA, 2-(3,4-dichlorophenyl)histamine; CF3PheHA, 2-(3-trifluoromethyl-phenyl-)histamine; CxHA, 2-cyclohexyl-histamine; MeCxHA, 2-(methylcyclohexyl)-histamine; EthCxHA, 2-(ethylcyclohexyl)-histamine; COS-7, African green monkey kidney cells; GPCR, G protein-coupled receptors; MD, molecular dynamics. have recently been developed as tools to control G protein signaling in vivo (5Coward P. Wada H.G. Falk M.S. Chan S.D. Meng F. Akil H. Conklin B.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 352-357Crossref PubMed Scopus (225) Google Scholar). RASSLs are G protein-coupled receptors (GPCRs) that no longer respond to their endogenous ligands but can still be activated by synthetic ligands (5Coward P. Wada H.G. Falk M.S. Chan S.D. Meng F. Akil H. Conklin B.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 352-357Crossref PubMed Scopus (225) Google Scholar). Given the biological importance of GPCRs, the ability to stimulate a single G protein pathway in a tissue of choice in vivo is useful for the understanding of subsequent changes in downstream signaling. The use of RASSLs has already provided valuable insight into the effect of Gαi signaling in the heart. Stimulation of a Gαi-coupled RASSL selectively expressed in the murine heart resulted in a strong (3-fold) and rapid (within 1 min after drug administration) decrease in heart rate (1Redfern C.H. Coward P. Degtyarev M.Y. Lee E.K. Kwa A.T. Hennighausen L. Bujard H. Fishman G.I. Conklin B.R. Nat. Biotechnol. 1999; 17: 165-169Crossref PubMed Scopus (154) Google Scholar). In the future, RASSLs may further be applied, e.g. to create reversible models of disease states, or combined with microarrays, to result in gene expression fingerprints of specific G protein pathways (for review, see Ref. 6Scearce-Levie K. Coward P. Redfern C.H. Conklin B.R. Trends Pharmacol Sci. 2001; 22: 414-420Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). There are four major classes of G proteins, named after their respective α-subunit (Gαs, Gαi, Gαq/11, and Gα12). Each class couples to specific signal transduction pathways, which are well characterized in vitro (for a review, see Ref. 7Hamm H.E. J. Biol. Chem. 1998; 273: 669-672Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar). The first described RASSL couples through Gαi proteins (5Coward P. Wada H.G. Falk M.S. Chan S.D. Meng F. Akil H. Conklin B.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 352-357Crossref PubMed Scopus (225) Google Scholar) and thus far is the only RASSL being applied in vivo (1Redfern C.H. Coward P. Degtyarev M.Y. Lee E.K. Kwa A.T. Hennighausen L. Bujard H. Fishman G.I. Conklin B.R. Nat. Biotechnol. 1999; 17: 165-169Crossref PubMed Scopus (154) Google Scholar, 2Redfern C.H. Degtyarev M.Y. Kwa A.T. Salomonis N. Cotte N. Nanevicz T. Fidelman N. Desai K. Vranizan K. Lee E.K. Coward P. Shah N. Warrington J.A. Fishman G.I. Bernstein D. Baker A.J. Conklin B.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4826-4831Crossref PubMed Scopus (142) Google Scholar, 8Baker A.J. Redfern C.H. Harwood M.D. Simpson P.C. Conklin B.R. Am. J. Physiol. 2001; 280: H1653-H1659Crossref PubMed Google Scholar). Also, more recently, several Gαs-coupled RASSLs have been developed (3Srinivasan S. Vaisse C. Conklin B.R. Ann. N. Y. Acad. Sci. 2003; 994: 225-232Crossref PubMed Scopus (23) Google Scholar, 4Claeysen S. Joubert L. Sebben M. Bockaert J. Dumuis A. J. Biol. Chem. 2003; 278: 699-702Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Thus far, there have been no reports about either Gαq/11- or Gα12-coupled RASSLs. The human histamine H1 receptor (H1R) couples through Gαq/11 proteins and thereby activates phospholipase C, resulting in, for example, inositol phosphate hydrolysis and increased concentrations of intracellular Ca2+ (9Gutowski S. Smrcka A. Nowak L. Wu D.G. Simon M. Sternweis P.C. J. Biol. Chem. 1991; 266: 20519-20524Abstract Full Text PDF PubMed Google Scholar, 10Leopoldt D. Harteneck C. Nurnberg B. Naunyn-Schmiedeberg's Arch. Pharmacol. 1997; 356: 216-224Crossref PubMed Scopus (91) Google Scholar). We previously demonstrated that the H1 receptor also activates NF-κB, both in a ligand-induced as well as in a constitutive manner, via Gαq/11 proteins (11Bakker R.A. Schoonus S.B. Smit M.J. Timmerman H. Leurs R. Mol. Pharmacol. 2001; 60: 1133-1142Crossref PubMed Scopus (234) Google Scholar). Although several Gαq-coupled GPCRs also activate Gα12 (for a review, see Ref. 12Riobo N.A. Manning D.R. Trends Pharmacol Sci. 2005; 26: 146-154Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), the H1R does not seem to couple to Gα12 (10Leopoldt D. Harteneck C. Nurnberg B. Naunyn-Schmiedeberg's Arch. Pharmacol. 1997; 356: 216-224Crossref PubMed Scopus (91) Google Scholar, 11Bakker R.A. Schoonus S.B. Smit M.J. Timmerman H. Leurs R. Mol. Pharmacol. 2001; 60: 1133-1142Crossref PubMed Scopus (234) Google Scholar, 13Lutz S. Freichel-Blomquist A. Yang Y. Rumenapp U. Jakobs K.H. Schmidt M. Wieland T. J. Biol. Chem. 2005; 280: 11134-11139Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). By mutational analysis, we previously characterized several amino acids that are involved in the binding of histamine and several synthetic agonists, such as the histaprodifens (a new class of specific H1R agonists) (14Bruysters M. Pertz H.H. Teunissen A. Bakker R.A. Gillard M. Chatelain P. Schunack W. Timmerman H. Leurs R. Eur. J. Pharmacol. 2004; 487: 5563Crossref Scopus (43) Google Scholar). Mutation of Phe435 (6.55) to Ala in TM6 (transmembrane domain 6) resulted in a strong decrease in affinity and potency of histamine, whereas only marginally affecting affinities and potencies of histaprodifens. In this study, we showed that for another class of H1R agonists, the substituted 2-phenyl-histamines (PheHAs, Ref. 15Leschke C. Elz S. Garbarg M. Schunack W. J. Med. Chem. 1995; 38: 1287-1294Crossref PubMed Scopus (64) Google Scholar), mutation of F435A results in strongly increased affinities and potencies, thereby identifying the first Gαq/11-coupled RASSL. Materials—pNF-κB-Luc was obtained from Stratagene (La Jolla, CA). ATP disodium salt, bovine serum albumin, chloroquine diphosphate, DEAE-dextran (chloride form), histamine dihydrochloride, mepyramine (pyrilamine maleate), glycerol, Triton X-100, and polyethyleneimine were purchased from Sigma. d-Luciferin was obtained from Duchefa Biochemie BV (Haarlem, The Netherlands). Cell culture media, penicillin, and streptomycin were obtained from Invitrogen. Fetal bovine serum was obtained from Integro B. V. (Dieren, The Netherlands). Cell culture plastics were obtained from Corning Costar (Corning, NY). [3H]Mepyramine (20 Ci/mmol) was purchased from ICN Biomedicals B. V. (Zoetermeer, The Netherlands). 2-(3-Chlorophenyl)histamine, 2-(3,4-dichlorophenyl)histamine, 2-(cyclohexyl)-histamine, 2-(methylcyclohexyl)-histamine, and 2-(ethylcyclohexyl)-histamine were synthesized at the Vrije Universiteit Amsterdam, The Netherlands. Gifts of 2-(3-trifluoromethyl)phenylhistamine dihydrogenmaleate (Dr. W. Schunack), pcDEF3 (Dr. J. Langer, Ref. 16Goldman L.A. Cutrone E.C. Kotenko S.V. Krause C.D. Langer J.A. BioTechniques. 1996; 21: 1013-1015Crossref PubMed Scopus (152) Google Scholar), and the cDNA encoding the human H1 receptor (Dr. H. Fukui, Ref. 17Fukui H. Fujimoto K. Mizuguchi H. Sakamoto K. Horio Y. Takai S. Yamada K. Ito S. Biochem. Biophys. Res. Commun. 1994; 201: 894-901Crossref PubMed Scopus (130) Google Scholar) are greatly acknowledged. Cell Culture and Transfection—COS-7 African green monkey kidney cells were maintained at 37 °C in a humidified 5% CO2, 95% air atmosphere in Dulbecco's modified Eagle's medium containing 50 IU/ml penicillin, 50 μg/ml streptomycin, and 5% (v/v) fetal bovine serum. COS-7 cells were transiently transfected using the DEAE-dextran method as described previously (11Bakker R.A. Schoonus S.B. Smit M.J. Timmerman H. Leurs R. Mol. Pharmacol. 2001; 60: 1133-1142Crossref PubMed Scopus (234) Google Scholar). Site-directed Mutagenesis—The cDNA encoding the human H1R (17Fukui H. Fujimoto K. Mizuguchi H. Sakamoto K. Horio Y. Takai S. Yamada K. Ito S. Biochem. Biophys. Res. Commun. 1994; 201: 894-901Crossref PubMed Scopus (130) Google Scholar) was subcloned in the pAlter plasmid (Promega), and point mutations were created according to the manufacturer's protocol (Altered Sites® II, Promega). cDNA of all mutant and wild-type receptors were subcloned into the expression plasmid pcDEF3 (16Goldman L.A. Cutrone E.C. Kotenko S.V. Krause C.D. Langer J.A. BioTechniques. 1996; 21: 1013-1015Crossref PubMed Scopus (152) Google Scholar). Mutations in the cDNA were verified by DNA sequencing using the dideoxy chain termination method. NF-κB Reporter Gene Assay—Cells transiently co-transfected with pNF-κB-Luc (125 μg/1 · 107 cells) and pcDEF3 containing mutant or wild-type human H1R cDNA (25 μg/1 · 107 cells) were seeded in 96-well white plates (Costar) in serum-free culture medium and incubated with drugs. After 48 h, cells were assayed for luminescence by aspiration of the medium and the addition of 25 μl/well luciferase assay reagent (0.83 mm ATP, 0.83 mm d-luciferin, 18.7 mm MgCl2, 0.78 μm Na2H2P2O7, 38.9 mm Tris (pH 7.8), 0.39% (v/v) glycerol, 0.03% (v/v) Triton X-100, and 2.6 μm dithiothreitol). After 30 min, luminescence was measured for 3 s/well in a Victor2 (Wallac). Histamine H1R Binding Studies—The transfected COS-7 cells used for radioligand binding studies were harvested after 48 h and homogenized in ice-cold 50 mm Na2/K phosphate buffer (pH = 7.4) (binding buffer). The COS-7 cell homogenates were incubated for 30 min at 30 °C in H1R binding buffer in 200 μl with 3 nm [3H]mepyramine. The nonspecific binding was determined in the presence of 1 μm mianserin. The incubations were stopped by rapid dilution with 3 ml of ice-cold H1R binding buffer. The bound radioactivity was separated by filtration through Whatman GF/C filters that had been treated with 0.3% polyethylenimine. Filters were washed twice with 3 ml of H1R binding buffer, and radioactivity retained on the filters was measured by liquid scintillation counting. Binding data were evaluated by a non-linear, least squares curve-fitting procedure using GraphPad Prism® (GraphPad Software, Inc., San Diego, CA). Analytical Methods—Protein concentrations were determined according to Bradford (18Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (222621) Google Scholar), using bovine serum albumin as a standard. All data shown are expressed as means ± S.E. Statistical analyses were carried out by non-paired Student's t test. p values < 0.05 were considered to indicate a significant difference (a, p < 0.05; b, p < 0.01; c, p < 0.001). Molecular Modeling—Our H1R homology model was obtained using the bovine rhodopsin crystal structure (Protein Data Bank entry 1L9H, Ref. 19Okada T. Fujiyoshi Y. Silow M. Navarro J. Landau E.M. Shichida Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5982-5987Crossref PubMed Scopus (664) Google Scholar) as the template. The third intracellular loop that connects transmembranes 5 and 6 was omitted due to its large size. Side chains were added using the homology module of InsightII (20Canutescu A.A. Shelenkov A.A. Dunbrack Jr., R.L. Protein Sci. 2003; 12: 2001-2014Crossref PubMed Scopus (882) Google Scholar). Water molecules present in the crystal structure were not incorporated. The initial model was refined by a steepest descent energy minimization. The minimized model was placed in a dodecahedral box filled with simple point charge water (21Vangunsteren W.F. Berendsen H.J.C. Angew. Chem. Int. Ed. Engl. 1990; 29: 992-1023Crossref Scopus (1354) Google Scholar), and a second minimization step using steepest descent was performed. Hereafter, a 20-ps molecular dynamics (MD) run was performed with positions restraints (1000 kJ · mol-1 nm-2) on all heavy protein atoms. Finally, the model was refined by a 100-ps MD run. In this structure, Phe435 was changed to Ala with InsightII, thus creating a model of the F435A H1R. Histamine and 2-(3-chlorophenyl)histamine (ClPheHA) were docked in the WT and F435A models using AutoDock 3.0.0 (22Morris 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 (9521) Google Scholar) applying default parameters. The monocationic ligands were assigned Löwdin atomic charges obtained after a single point ab initio restricted Hartree-Fock calculation using 6-31Gr* with the GAMESS US package (23Schmidt M.W. Baldridge K.K. Boatz J.A. Elbert S.T. Gordon M.S. Jensen J.H. Koseki S. Matsunaga N. Nguyen K.A. Su S.J. Windus T.L. Dupuis M. Montgomery J.A. J. Comput. Chem. 1993; 14: 1347-1363Crossref Scopus (19613) Google Scholar). The protein was assigned with KOLLUA partial charges using the SYBYL program (version 6, Tripos, St. Louis). Only essential hydrogens were taken into account. The obtained ligand-receptor complexes were minimized using the steepest descent method. These ligand-receptor complexes were placed in a dodecahedral box, with a minimum distance of 7 Å between protein and the box. The system was solvated with simple point charge water, and the protein partial charges were assigned by GROMACS. Again, a steepest descent procedure was performed on the system. Subsequently, a set of MD runs with position restraints (1000 kJ· mol-1nm-2) was applied to the system in which a controlled release of the restraints was performed. A run for 50 ps with position restraints on all heavy protein atoms and all ligand atoms was performed, with the protein hydrogens and all water molecules unrestrained. Consecutively, a run was performed for 20 ps with position restraints on all Cα-atoms, all heavy atoms of residues Asp107 and Asn198, and all ligand atoms. Finally, a MD run was performed for 1 ns with only position restraints on Cα-atoms. During the last 500 ps of this run, the presence of hydrogen bonds was analyzed using cutoff distance between heavy atoms of 3.5 Å and a cutoff angle between acceptor donor and hydrogen of 60°. All minimizations and MD simulations were performed using the GROMACS software package and the GROMOS 43a1 force field (24Lindahl E. Hess B. Van der Spoel D. J. Mol. Model. 2001; 7: 306-317Crossref Google Scholar, 25Berendsen H.J.C. Van der Spoel D. Van Drunen R. Comput. Phys. Commun. 1995; 91: 43-56Crossref Scopus (7714) Google Scholar) and LINCS constraints (26Hess B. Bekker H. Berendsen H.J.C. Fraaije J.G.E.M. J. Comput. Chem. 1997; 18: 1463-1472Crossref Scopus (12662) Google Scholar) on all bonds. An NPT ensemble was generated using both the Berendsen thermostat and the barostat with default settings for 300 K and 1 bar (25Berendsen H.J.C. Van der Spoel D. Van Drunen R. Comput. Phys. Commun. 1995; 91: 43-56Crossref Scopus (7714) Google Scholar). Mutation F435A Differentially Modulates Affinities of Several Classes of H1R Agonists—At the H1R, several H1R agonists show a higher affinity than the natural agonist histamine. The histamine analogs 2-(3-trifluoromethyl-phenyl)histamine (CF3PheHA), histaprodifen, suprahistaprodifen, and the newly characterized H1R agonist 8R-lisuride bind 9-1000-fold better to the H1R as compared with histamine (TABLE ONE). Previously, we characterized Phe435 (6.55), as a specific interaction point for histamine (14Bruysters M. Pertz H.H. Teunissen A. Bakker R.A. Gillard M. Chatelain P. Schunack W. Timmerman H. Leurs R. Eur. J. Pharmacol. 2004; 487: 5563Crossref Scopus (43) Google Scholar). Mutation of this residue into alanine resulted in a 25-fold decrease in histamine affinity without drastic alterations in the affinity of the agonists histaprodifen and suprahistapodifen (TABLE ONE) (Ref. 14Bruysters M. Pertz H.H. Teunissen A. Bakker R.A. Gillard M. Chatelain P. Schunack W. Timmerman H. Leurs R. Eur. J. Pharmacol. 2004; 487: 5563Crossref Scopus (43) Google Scholar). In this study, we further studied the role of Phe435 (6.55) in agonist binding to the H1R. TABLE ONE shows that, similar to histaprodifen and suprahistaprodifen, affinities of pyridylethylamine as well as 8R-lisuride are comparable for WT and F435A mutant H1Rs. Unexpectedly, for F435A H1R, the affinity of CF3PheHA was increased 54-fold as compared with WT H1R (Fig. 1 and TABLE ONE). Thus, the F435A mutant H1R has an affinity for CF3PheHA that exceeds the affinity of histamine more than 10,000-fold.TABLE ONEAffinities of H1R agonists for wild-type and Phe435 Ala mutant H1Rsa Affinities are determined by [3H]mepyramine displacement. All data are calculated as the mean ± S.E. of at least three experiments, each performed in triplicate.b p < 0.01.c p < 0.001 vs. WT H1R.FIGURE 1Displacement of [3H]mepyramine binding to wild-type H1R (closed symbols, solid lines) and to mutant receptor H1R F435A (open symbols, dotted lines) receptors by histamine (triangles), and 2-(3-trifluoromethyl-phenyl)histamine (CF3PheHA, circles). A representative experiment is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) a Affinities are determined by [3H]mepyramine displacement. All data are calculated as the mean ± S.E. of at least three experiments, each performed in triplicate. b p < 0.01. c p < 0.001 vs. WT H1R. Basic Characterization of Phe435 Mutant H1Rs—Phenylalanine at position 6.55 is conserved among all known H1Rs (27Horn F. Vriend G. Cohen F.E. Nucleic Acids Res. 2001; 29: 346-349Crossref PubMed Scopus (158) Google Scholar) and is implicated in ligand binding in both human and guinea pig H1Rs (14Bruysters M. Pertz H.H. Teunissen A. Bakker R.A. Gillard M. Chatelain P. Schunack W. Timmerman H. Leurs R. Eur. J. Pharmacol. 2004; 487: 5563Crossref Scopus (43) Google Scholar, 28Wieland K. Laak A.M. Smit M.J. Kuhne R. Timmerman H. Leurs R. J. Biol. Chem. 1999; 274: 29994-30000Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The F435A mutation removes an aromatic ring structure from the binding pocket, and therefore, potential interactions with either ligands or other amino acids in the H1R-binding pocket may be lost. However, exchanging the bulky Phe435 (6.52) with the smaller alanine may also increase the size of the ligand-binding pocket and thus create space for the trifluoromethyl-phenyl moiety of CF3PheHA, resulting in an increased affinity for this ligand. To address the latter possibility, we created additional mutant H1Rs (F435V and F435L) that vary in the size of the side chain at position 6.55. The generated mutant receptors are expressed at comparable levels (Bmax = 13.6-20.1 pmol/mg of protein) in transiently transfected COS-7 cells and bind the H1 R radioligand [3H]mepyramine with virtually unchanged affinity (Kd = 1.5-5.7 nm), as determined by radioligand saturation binding experiments (TABLE TWO). Although the expression levels are similar, there is a clear difference in constitutive GPCR activities of the tested H1R mutants as measured by an NF-κB-driven reporter gene assay (Fig. 2, white bars). Although the basal activity of H1R F435L H1R is comparable with the WT H1R, the level of constitutive NF-κB activation of H1R F435V is twice as high, whereas for mutant H1R, F435A constitutive activity is hardly detectable. Although the levels of constitutive NF-κB activation vary between the mutant H1Rs, their agonist-induced responses are comparable (Fig. 2, black bars), indicating that G protein-coupling of the mutant H1Rs is not impaired.TABLE TWOCharacteristics of [3H]mepyramine binding to WT and mutant H1R The values are determined by saturation radioligand binding assays. All data are calculated as the mean ± S.E. of at least three experiments, each performed in triplicate.a p < 0.01 vs. WT receptor.FIGURE 2Basal H1R activation (white bars) and maximal histamine-induced NF-κB activation (black bars) in COS-7 cells transfected with cDNA of WT, mutant H1Rs, or empty vector (mock). Basal activity of WT H1R is put at 100%. Values are determined by NF-κB-driven reporter gene assays.View Large Image Figure ViewerDownload Hi-res image Download (PPT) a p < 0.01 vs. WT receptor. Binding Analysis of WT and Phe435 Mutant H1Rs—Similar to the mutant F435A H1R (pKi = 2.7), the mutant H1R F435V (pKi = 2.9) and F435L (pKi = 2.9) exhibit a strong decrease in their affinity for histamine as compared with the WT receptor (pKi = 4.1) (TABLE THREE). Again, these mutant receptors exhibit an increased affinity for CF3PheHA. The exchange of Phe435 for leucine, valine, or alanine results in mutant H1Rs with a 47-, 124-, and 54-fold increased affinity for CF3PheHA respectively, as compared with the WT receptor (pKi = 5.0) (TABLE THREE). For these mutant H1Rs, the affinity of CF3PheHA greatly exceeds their affinity of the endogenous ligand histamine, the mutant F435V H1R exhibiting the greatest selectivity for the synthetic agonist CF3PheHA over histamine (17,000-fold).TABLE THREECharacterization of H1R agonists on wild-type and mutant H1Rs Potencies of agonists (pEC50) are determined by an NF-κB-driven reporter gene assay. Affinities are determined by [3H]mepyramine displacement. All data are calculated as the mean ± S.E. of at least three experiments, each performed in triplicate. ND indicates that the value could not be determined.a p < 0.05.b p < 0.01.c p < 0.001 vs. WT receptor. a p < 0.05. b p < 0.01. c p < 0.001 vs. WT receptor. We tested two other substituted PheHAs, ClPheHA and 2-(3,4-dichlorophenyl)histamine (Cl2PheHA), on the mutant H1Rs as well. For the WT H1R, the affinity of ClPheHA (pKi = 5.3) is slightly higher then than that of CF3PheHA (pKi = 5.0). An (additional) para-chloro substituent is not favorable for H1R affinity (pKi = 4.6), fitting previous findings for both human and rodent H1Rs (29Seifert R. Wenzel-Seifert K. Burckstummer T. Pertz H.H. Schunack W. Dove S. Buschauer A. Elz S. J. Pharmacol. Exp. Ther. 2003; 305: 1104-1115Crossref PubMed Scopus (79) Google Scholar). Similar to CF3PheHA, the affinity of ClPheHA is increased upon mutation of Phe435 into leucine (pKi = 6.8), valine (pKi = 7.3), or alanine (pKi = 6.7). Although for Cl2PheHA, H1R affinities are lower and increases in affinity are smaller, the same trend upon mutation is observed (pKi values, 4.6, 5.3, 5.8, and 5.4, respectively). Similar to CF3PheHA, for ClPheHA and Cl2PheHA, mutation F435V also results in the largest increase in affinity, as compared with the WT receptor. Additionally, we tested a series of 2-cyclohexyl-histamines (CxHAs), varying in spacer length between the imidazole ring and the cyclohexyl moieties: CxHA, 2-(methyl-cyclohexyl)-histamine (MeCxHA), and 2-(ethyl-cyclohexyl)-histamine (EthCxHA). The affinities of the CxHAs are approximately equal to the affinity of histamine (TABLE THREE). Again, mutation of Phe435 into alanine, valine, or leucine results in increased affinities as compared with the WT H1R (TABLE THREE). The increases in affinity, however, are smaller (maximum 10-fold) than those observed for the PheHAs (maximum 120-fold). Although changes in affinity are less prominent, for EthCxHA, we observe a correlation between the space available in the binding pocket (Ala → Val → Leu → Phe) and the affinities for the mutant receptors (F435A, pKi 4.9; F435V, pKi = 4.6; F435L, pKi = 4.3; and WT, pKi = 4.1, TABLE THREE). For both CxHA and MeCxHA, the highest affinities are observed for the F435V mutant H1R, following the trend observed for the PheHAs. Functional Analysis of WT and Phe435 Mutant H1Rs—Using an NF-κB-driven reporter gene assay, we evaluated the potencies of several 2-phenylsubstituted histamine analogs for mutant and WT H1Rs. For the WT H1R, these analogs are less potent than the endogenous ligand histamine (pEC50 = 6.9) (TABLE THREE). Fig. 3 clearly illustrates that as compared with the WT H1R, the potency of HA at the H1R F435A is decreased 200-fold, whereas the potency of ClPheHA is increased 2,600-fold. Also, for CF3PheHA and Cl2PheHA, potencies were increased upon the mutation F435A; again ClPheHA (pEC50 = 9.4) and CF3PheHA (pEC50 = 9.2) are equipotent, and Cl2PheHA is clearly less potent (pEC50 = 7.9) (TABLE THREE). For mutant H1Rs F435L and F435V, similar trends are observed, with PheHA potencies for H1R F435V being slightly higher than for F435A and F435L H1Rs. For mutant H1Rs, F435A, F435V, and F435L ClPheHA and CF3PheHA are agonists with subnanomolar potencies, which exceed the potency of histamine 70,000-280,000-fold. Molecular Modeling of WT and F435A Mutant H1Rs—Our model of the H1R, based on the bovine rhodopsin crystal structure, was used to visualize the interactions of either histamine or ClPheHA with the WT and F435A mutant H1R. Models of both WT and F435A H1Rs were optimized by molecular dynamics simulations. Hereafter, histamine and ClPheHA were automatically docked in both WT and F435A mutant H1R models. Finally, a second round of molecular dynamics was used to optimize the receptor-ligand complexes. In the WT H1R, both histamine and ClPheHA show an ionic interaction between the conserved Asp107 (3.32) in TM3 (transmembrane domain 3) and their ethylamine group over distances of 2.6 and 3.0 Å, respectively (Fig. 4, A and C). As expected, differences between histamine and ClPheHA can be observed for interaction with Phe435. In binding histamine, Phe435 has an indirect role, keeping Phe432 in an edge-to-face orientation toward the imidazole moiety of histamine (Fig. 4A). In this orientation, a hydrogen bond can be formed between the distal nitrogen of the imidazole ring of histamine and Asn198 at a distance of 2.7 Å and is observed during 47% of the MD simulation. In binding ClPheHA, Phe435 directly interacts with the ligand by π-π stacking with the phenyl moiety of ClPheHA (Fig. 4B). By this interaction, Phe435 prevents hydrogen bonding of the imidazole ring with Asn198 since the distance exceeds 5 Å. The difference in interaction with Phe435 between histamine and ClPheHA is further illustrated by observed changes in receptor-ligand interactions upon the mutation F435A. For histamine, the mutation F435A releases Phe432, thereby allowing histamine to shift toward Asp