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• W1492615281 abstract "We report the use of thiol chemistry to define specific and reversible disulfide interactions of Cys-substituted NK2 receptor mutants with analogues of neurokinin A (NKA) containing single cysteine substitutions. The NKA analogues wereN-biotinylated to facilitate the rapid detection of covalent analogue-receptor interactions utilizing streptavidin reactivity.N-biotinyl-[Tyr1,Cys9]NKA,N-biotinyl-[Tyr1,Cys10]NKA were both found to reversibly disulfide bond to the NK2 receptor mutant Met297 → Cys. This is consistent with the improved affinities of these particular analogues for the Met297 → Cys receptor as compared with those for the wild-type and Met297 → Leu receptors. In our three-dimensional model, Met297 occupies the equivalent position in helix 7 to the retinal binding Lys296 in rhodopsin. Binding of the NK2receptor antagonist [3H]SR 48968 and of125I-NKA was used to characterize additional receptor mutants. It seems that the aromatic residues Trp99 (helix 3), His198 (helix 5), Tyr266, His267, and Phe270 play an important role in NKA binding as structural determinants. The existence of overlapping SR 48968 and NKA binding sites is also evident. These data suggest that the peptide binding site of the NK2R is at least in part formed by residues buried deep within the transmembrane bundle and that this intramembranous binding domain may correspond to the binding sites for substantially smaller endogenous GPCR ligands. We report the use of thiol chemistry to define specific and reversible disulfide interactions of Cys-substituted NK2 receptor mutants with analogues of neurokinin A (NKA) containing single cysteine substitutions. The NKA analogues wereN-biotinylated to facilitate the rapid detection of covalent analogue-receptor interactions utilizing streptavidin reactivity.N-biotinyl-[Tyr1,Cys9]NKA,N-biotinyl-[Tyr1,Cys10]NKA were both found to reversibly disulfide bond to the NK2 receptor mutant Met297 → Cys. This is consistent with the improved affinities of these particular analogues for the Met297 → Cys receptor as compared with those for the wild-type and Met297 → Leu receptors. In our three-dimensional model, Met297 occupies the equivalent position in helix 7 to the retinal binding Lys296 in rhodopsin. Binding of the NK2receptor antagonist [3H]SR 48968 and of125I-NKA was used to characterize additional receptor mutants. It seems that the aromatic residues Trp99 (helix 3), His198 (helix 5), Tyr266, His267, and Phe270 play an important role in NKA binding as structural determinants. The existence of overlapping SR 48968 and NKA binding sites is also evident. These data suggest that the peptide binding site of the NK2R is at least in part formed by residues buried deep within the transmembrane bundle and that this intramembranous binding domain may correspond to the binding sites for substantially smaller endogenous GPCR ligands. substance P neurokinin A high performance liquid chromatography tachykinin NK2 receptor polyacrylamide gel electrophoresis N-ethylmaleimide G-protein-coupled receptor The undecapeptide substance P (SP)1 and decapeptides neurokinin A (NKA) and neurokinin B (NKB) share a C-terminal motif of Phe-X-Gly-Leu-Met-NH2. Their more variable N termini may play a role in determining the somewhat limited selectivities of these neuropeptides for the mammalian tachykinin NK1, NK2, and NK3 receptors (1Maggi C.A. Schwartz T.W. Trends Pharmacol. Sci. 1997; 18: 351-355Abstract Full Text PDF PubMed Scopus (183) Google Scholar). The tachykinin receptors belong to an extensive family of seven transmembrane G-protein-coupled receptors (GPCRs), which bind an array of extracellular ligands such as biogenic amines, odorant molecules, neuropeptides, and glycoproteins in order to convey their signals to the intracellular environment (2Horn F. Wear J. Beulers M.W. Horsch S. Baurock A. Chen W. Edvardsen O. Campagne F. Vriend G. Nucleic Acids Res. 1998; 26: 275-279Crossref PubMed Scopus (340) Google Scholar). We have constructed a three-dimensional receptor model of the tachykinin NK2 receptor (NK2R) in order to identify putative NKA binding residues (3Donnelly D. Maudsley S. Gent J.P. Moser R.N. Hurrell C.R. Findlay J.B.C. Biochem. J. 1999; 339: 55-61Crossref PubMed Google Scholar). Site-directed mutagenesis was then used to identify a subset of amino acids that participate in NKA binding by virtue of the reduced affinities of receptor mutants containing substitutions of these side chains (4Bhogal N. Donnelly D. Findlay J.B.C. J. Biol. Chem. 1994; 269: 27269-27274Abstract Full Text PDF PubMed Google Scholar, 5Huang R.-R.C. Vicario P.P. Strader C.D. Fong T.M. Biochemistry. 1995; 34: 10048-10055Crossref PubMed Scopus (76) Google Scholar). Loss-of-function mutagenesis of this kind is commonly used to define the functional determinants within GPCRs. However, the resultant data often require careful interpretation as it may be difficult to distinguish receptor sites that participate in direct ligand interactions from those sites that influence receptor conformation. This is particularly significant for the larger flexible peptides that appear to make a greater number of receptor contacts than smaller endogenous GPCR ligands. Peptide agonist binding sites have been more reliably mapped by affinity and photoaffinity labeling methods (6Girault S. Sagan S. Bolbach G. Lavielle S. Chassaing G. Eur. J. Biochem. 1996; 240: 215-222Crossref PubMed Scopus (55) Google Scholar, 7Servant G. Laporte S.A. Leduc R. Escher E. Guillemette G. J. Biol. Chem. 1997; 272: 8653-8659Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 8Phalipou S. Seyer R. Cotte N. Breton C. Barberis C. Hibert M. Mouillac B. J. Biol. Chem. 1999; 274: 23316-23327Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). It is also possible to confer wild-type receptor binding characteristics upon receptor mutants by chemically modifying the ligand, thus distinguishing binding residues from conformational determinants in the receptor. This concept of positive complementation was utilized by Strader et al.(9Strader C.D. Candelore M.R. Hill W.S. Sigal I.S. Dixon R.A.F. J. Biol. Chem. 1989; 262: 16439-16443Abstract Full Text PDF Google Scholar) to demonstrate the interaction of serine residues in helix 5 of the adrenergic receptors with the hydroxyl groups of adrenalin. Here we describe a technique that incorporates aspects from both affinity labeling and positive complementation studies by using cysteine-substituted NK2R mutants and NKA analogues, to define NKA-NK2R interactions. The NK2R cDNA was the kind gift of Glaxo/Wellcome. 2-[125I]iodohistidyl neurokinin A (125I-NKA) and [benzamide-4-3H]SR 48968 ([3H]SR 48968; (10Emonds-Alt X. Advenier C. Croci T. Manara L. Neliat G. Poncelet M. Proietto V. Santucci V. Soubrie P. Van Broeck D. Vilain P Le Fur G. Breliere J.C. Regul. Pep. 1993; 46: 31-36Crossref PubMed Scopus (32) Google Scholar)) were obtained from PerkinElmer Life Science Products. pFastBac1, CELLFECTINTM,Escherichia coli DH10BAC cells and TC-100 medium were purchased from Life Technologies Inc. Protein chemical reagents were obtained from Bio-Rad or Roche Molecular Biochemicals. The synthesis and purification of the cysteine-substituted NKA analogues has been described previously (11Labrou N.E. Mello L.V. Rigden D.J. Keen J.N. Findlay J.B.C. Peptides. 1999; 20: 795-801Crossref PubMed Scopus (6) Google Scholar). Peptide N-biotinylation was performed by adding 5.0 μmols of biotinyl N-hydroxysuccinimide ester dropwise to 2.5 μmols of each peptide in 0.2 ml of 0.05 mNaHCO3, pH 8.2 and terminated after 30–45 min by the addition 0.2 ml of 5% (v/v) acetic acid. Labeling was assessed using analytical ascending TLC on precoated plastic sheets with silica gel 60 (0.2 mm; Merck) using butan-1-ol/acetic acid/H2O, 4:2:3 as mobile phase. Labeled peptides were visualized using UV light or 0.1% (w/v) ninhydrin, extracted with 5% (v/v) acetic acid, and precipitated. Precipitants were washed three times with 0.5 ml of 1% (v/v) acetic acid and twice with acetone and finally dissolved in 1 ml of DMF containing 0.1 mm β-mercaptoethanol prior to reverse-phase HPLC with a water/acetonitrile linear gradient (0–60% acetonitrile in 30 min). Peptide fractions were subjected to amino acid sequencing, lyophilized, and reconstituted. Final peptide concentrations were determined using 3 mm5,5-dithio-bis-(-2-nitrobenzoic) acid in the presence of 100 mm potassium phosphate buffer, pH 7.6 (12Labrou N.E. Clonis Y.D. Arch. Biochem. Biophys. 1997; 337: 103-114Crossref PubMed Scopus (30) Google Scholar). Cysteine mutations were introduced by the method reported in Ref. 13Kunkel T.A. Bebenek K. McClary J. Methods Enzymol. 1991; 204: 125-139Crossref PubMed Scopus (633) Google Scholar and confirmed by double-stranded DNA sequencing. Wild-type and mutant NK2Rs were expressed in Sf9 insect larval cells using the Bac-to-Bac expression system (Life Technologies Inc.). Briefly, an XbaI-BamHI restriction fragment of the NK2R cDNA was subcloned into pFastBac1. Recombinant bacmid DNAs isolated from transformedE. coli DH10BAC cells were used to transfect Sf9 cells in accordance with the manufacturer's recommendations. Transfected Sf9 cells were maintained at 27 °C in a TC-100 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum, 50 IU/ml penicillin, and 50 μg/ml streptomycin for 5 days before recombinant viruses containing the NK2Rs were recovered and used to further infect Sf9 cells. Typically, 2 × 106 Sf9 cells/ml were infected and harvested 75 h post-infection for radioligand binding and affinity labeling studies. Prior to radioligand binding, cells were washed twice with TC-100 medium and resuspended at 2 × 106 cells/ml in TC-100 medium containing 0.1% (w/v) bovine serum albumin and 100 μg/ml bacitracin (binding buffer). For competition assays, 105cells were incubated in binding buffer with 0.1 or 1 nm125I-NKA and varying concentrations of each competing peptide for 90 min at room temperature. Nonspecific binding was determined using 0.1 μm NKA. Samples were then microcentrifuged at 13,000 rpm for 2 min, overlaid with 2:1 (v/v) dibutylphthalate/dinonylphthalate, microcentrifuged, and snap frozen on dry ice. The tip of each tube were counted in a Wallac 1470 Wizard γ-counter. For antagonist binding assays, the cells were incubated for 60 min at room temperature in binding buffer containing 5 nm [3H]SR 48968 and varying concentrations of SR 48968. In this case, nonspecific binding was determined using 5 μm SR 48968. Tritiated samples were counted in a Tricarb 1900 TR Packard liquid scintillation counter after dissolution in Emulsifier SafeTM (Packard). Data were analyzed by computerized non-linear regression using GraphPAD software (GraphPAD, San Diego). 105 infected Sf9 cells were incubated with the appropriateN-biotinylated cysteine-substituted peptide for 90 min at room temperature in 25 mm Tris-HCl buffer, pH 7.4, 140 mm NaCl, 5 mm MgCl2, 0.1% (w/v) bovine serum albumin, 100 μg/ml bacitracin, 10 μg/ml leupeptin, and 10 μg/ml aprotonin ( 1 mm final concentration). Cu2+-phenanthroline was then added, and the cells were incubated for a further 60 min, pelleted in a microcentrifuge at 12,000 rpm for 1 min, resuspended in an 0.2 ml of ice-cold binding buffer, repelleted, and boiled for 1 min in SDS-PAGE sample buffer (60 mm Tris-HCl, pH 6.8, 2% SDS, 10% glycerol) containing 25 mm NEM. Proteins were resolved on 10% SDS-PAGE gels under denaturing conditions and electroblotted onto 0.2-μm polyvinylidene difluoride membranes. Specific ligand-receptor interaction was visualized using horseradish peroxidase-conjugated Streptavidin (Roche Molecular Biochemicals), or alternatively, a mouse anti-His6 monoclonal antibody/POD detection system (Roche Molecular Biochemicals) was used to demonstrate protein expression. 2 × 105 cells were washed with phosphate-buffered saline (PBS), pH 7.0 and incubated for 90 min with 4 nm125I-NKA in the absence and presence of 20 μm NKA. The cells were pelleted, washed twice with PBS, and incubated with 200 μm ice-cold 5 mm 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/PBS, pH 7.0 (30 min on ice followed by 30 min at room temperature). Samples were then subjected to SDS-PAGE. Radiolabeled proteins were visualized by autoradiography following exposure of X-Ograph Blue film (X-Ograph Ltd.) to Coomassie-stained SDS-PAGE gels for 14 days at −70 °C. Table I shows the binding profiles of Cys-substituted NK2R mutants for 125I-NKA and the non-peptide antagonist [3H]SR 48968. The receptor mutants Trp99 → Cys, His198 → Cys, and Tyr266 → Cys demonstrate no detectable binding activity. The mutants Thr24 → Cys and Phe26 → Cys in the N-terminal tail, Asn86 → Cys (in helix 2), Asn97 → Cys (in extracellular loop 1), and His267 → Cys and Phe270 → Cys (in helix 6), despite their inability to bind 125I-NKA retain wild-type affinities for [3H]SR 48968. A final subset of mutants (Gln109 → Cys and Asn110 → Cys in helix 3 and Ile202 → Cys in helix 5) displays approximately wild-type affinities for both ligands.Table IThe affinities of NK2R mutants for NKA and SR 48968ReceptorIC50nm ± S.E.NKA1-aIC50(nm) ± S.E. values determined by competition with 0.1 nm125I-NKA.SR 489681-bIC50 (nm) ± S.E. values determined by competition with 5.0 nm[3H]-SR 48968. The numbers of individual experiments performed in triplicate are indicated in parentheses.Wild type5.6 ± 0.2 (6)8.2 ± 0.6 (3)Thr24 → CysNB1-cNB, no detectable binding.14.1 ± 0.8 (3)Phe26 → CysNB16.2 ± 3.2 (3)Asn86→ CysNB14.5 ± 2.3 (3)Asn97 → CysNB10.2 ± 1.2 (3)Trp99 → CysNBNBGln109 → Cys4.9 ± 0.7 (3)12.5 ± 2.1 (3)Asn110 → Cys15 ± 3.2 (3)9.5 ± 0.8 (3)His198 → CysNBNBIle202 → Cys9.2 ± 0.9 (3)9.1 ± 1.2 (3)Tyr266 → CysNBNBTyr266 → Phe6.1 ± 0.1 (3)6.6 ± 0.3 (3)His267 → CysNB13.2 ± 2.1 (3)Met297 → Cys34 ± 2.8 (3)7.2 ± 0.9 (3)Phe270 → CysNB16.1 ± 1.3 (3)Phe270 → Tyr8.3 ± 0.4 (3)6.4 ± 0.8 (3)Met297 → Leu48 ± 2.5 (3)8.6 ± 0.7 (3)1-a IC50(nm) ± S.E. values determined by competition with 0.1 nm125I-NKA.1-b IC50 (nm) ± S.E. values determined by competition with 5.0 nm[3H]-SR 48968. The numbers of individual experiments performed in triplicate are indicated in parentheses.1-c NB, no detectable binding. Open table in a new tab Interestingly, Met297 → Cys demonstrates a 6-fold lower affinity for NKA, but not for SR 48968, than the wild-type receptor (Fig. 1, A and B). These data are comparable for those obtained for a receptor mutant in which Met297 is substituted with Leu, where a selective 8-fold reduction in NKA affinity is observed (TableII). Further competition studies were performed in order to determine the affinities of the wild-type receptor and Met297 → Cys and Met297 → Leu mutants for the Cys-substituted NKA analogues (Table II). Cysteine substitution of amino acids at positions 1–8 within NKA appear to have similarly detrimental consequences on ligand affinity for both the wild-type and Met297 → Cys receptors; the presence of a tyrosine residue at position 0 or 1 of NKA or of Cys substitutions at His1 or Lys2 has little effect on peptide affinity, whereas substitutions at Thr3, Asp4, Ser5, Val7, Gly8, Leu9, and Met10 have more notable effects on peptide binding and [Tyr1,Cys6]NKA has little binding activity. However, whereas the affinity of [Tyr1,Cys9]NKA for the wild-type receptor is 286-fold lower than that of NKA, this analogue displays a similar affinity to NKA for the Met297 → Cys mutant. The Met297 → Leu mutant more closely resembles the wild-type receptor in this respect as its affinity for [Tyr1,Cys9]NKA is 82-fold lower than its affinity for NKA. The difference between the Met297 → Cys and Met297 → Leu mutants is further emphasized by the 5-fold greater affinity of the Met297 → Cys receptor as compared with a 141-fold lower affinity of the Met297 → Leu mutant for [Tyr1,Cys10]NKA than for NKA.Table IIAffinities of the wildtype, Met297 → Cys and Met297→ Leu NK2Rs for Cys-substituted NKA analoguesNKA analogue2-aThe sequences of these analogues are given in Ref. 11.Kinm ± S.E.Wild-type2-bKi (nm) ± S.E. values determined by competition with 0.1 nm125I-NKA (wild type column) or 1 nm[125I]-NKA (Met297 mutants) using the equationKi = IC50 (nm)/(1 + [125I-NKA]/Kd where Kd= IC50 (from Table I) [125I-NKA (nm)].2-cData taken from Ref. 11. Numbers of individual experiments performed in triplicate are indicated in parentheses.Met297→ Cys2-bKi (nm) ± S.E. values determined by competition with 0.1 nm125I-NKA (wild type column) or 1 nm[125I]-NKA (Met297 mutants) using the equationKi = IC50 (nm)/(1 + [125I-NKA]/Kd where Kd= IC50 (from Table I) [125I-NKA (nm)].Met297 → Leu2-bKi (nm) ± S.E. values determined by competition with 0.1 nm125I-NKA (wild type column) or 1 nm[125I]-NKA (Met297 mutants) using the equationKi = IC50 (nm)/(1 + [125I-NKA]/Kd where Kd= IC50 (from Table I) [125I-NKA (nm)].NKA5.5 ± 0.2 (6)35 ± 1.4 (6)50 ± 2.6 (6)[Tyr1]NKA5.0 ± 0.2 (3)45 ± 1.2 (3)ND2-dND, not determined.[Tyr0,Cys1]NKA3.4 ± 0.2 (3)56 ± 3.2 (3)ND[Tyr1,Cys2]NKA3.5 ± 0.3 (3)48 ± 7.3 (3)ND[Tyr1,Cys3]NKA28 ± 1.5 (3)211 ± 7.7 (3)ND[Tyr1,Cys4]NKA124 ± 5.7 (3)1781 ± 216 (3)ND[Tyr1,Cys5]NKA30 ± 1.8 (3)466 ± 22 (3)ND[Tyr1,Cys6]NKA>10,000 (3)NB2-eNB, no detectable binding.ND[Tyr1,Cys7]NKA182 ± 8.5 (3)1435 ± 148 (3)ND[Tyr1,Cys8]NKA442 ± 17 (3)2532 ± 123 (3)ND[Tyr1,Cys9]NKA1571 ± 68 (3)41 ± 3.4 (3)4121 ± 343 (3)[Tyr1,Cys10]NKA481 ± 17 (3)7.0 ± 0.8 (3)7065 ± 764 (3)2-a The sequences of these analogues are given in Ref. 11Labrou N.E. Mello L.V. Rigden D.J. Keen J.N. Findlay J.B.C. Peptides. 1999; 20: 795-801Crossref PubMed Scopus (6) Google Scholar.2-b Ki (nm) ± S.E. values determined by competition with 0.1 nm125I-NKA (wild type column) or 1 nm[125I]-NKA (Met297 mutants) using the equationKi = IC50 (nm)/(1 + [125I-NKA]/Kd where Kd= IC50 (from Table I) [125I-NKA (nm)].2-c Data taken from Ref. 11Labrou N.E. Mello L.V. Rigden D.J. Keen J.N. Findlay J.B.C. Peptides. 1999; 20: 795-801Crossref PubMed Scopus (6) Google Scholar. Numbers of individual experiments performed in triplicate are indicated in parentheses.2-d ND, not determined.2-e NB, no detectable binding. Open table in a new tab The presence of a biotin group at the N-terminal end of NKA does not appear to alter the affinity of NKA for the wild-type NK2R (Fig. 1C). Therefore, the cysteine-substituted NKA analogues were N-biotinylated to facilitate the rapid detection of covalent ligand-receptor interaction using streptavidin affinity chemistry. Disulfide bond formation was catalyzed by the addition of Cu2+-phenanthroline and assessed using a Streptavidin-peroxidase system. In the case of cells expressing the Met297 → Cys receptor, a 45-kDa protein was labeled by both N-biotin-[Tyr1,Cys9]NKA andN-biotin-[Tyr1,Cys10]NKA but not with the remaining analogues (Fig. 2). Labeling was not evident for uninfected or Sf9 cells expressing the wild-type receptor cDNA (Fig.3).Figure 3Specificity of Met297 → Cys labeling with N-biotin-[Tyr1, Cys9] NKA and N-biotin-[Tyr1, Cys10] NKA. The labeling of Met297 → Cys receptors withN-biotin-[Tyr1,Cys9]NKA (lane 1) andN-biotin-[Tyr1,Cys10]NKA (lane 2) is reduced in the presence of 1 μmNKA (lanes 3 and 4, respectively) and abolished in the presence of 10 μm NKA (lanes 5 and6, respectively). Neither cells expressing the wild-type receptor (lanes 7 and 9) nor uninfected cells (lanes 8 and 10) were labeled withN-biotin-[Tyr1,Cys9]NKA (lanes 7 and 8) orN-biotin-[Tyr1,Cys10]NKA (lanes 9 and 10). Molecular mass (kDa) is indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Furthermore, labeling of the Met297 → Cys receptor byN-biotin-[Tyr1,Cys9]NKA andN-biotin-[Tyr1,Cys10]NKA was inhibited by NKA (Fig. 3), reversed by 100 mmβ-mercaptoethanol (Fig. 4), and abolished by NEM pretreatment of the Cys-containing NKA analogues (Fig. 4). TheN-biotin-[Tyr1,Cys9]NKA- andN-biotin-[Tyr1,Cys10]NKA-labeled 45-kDa species correspond to an anti-His6 antibody immunoreactive protein detected in Sf9 cells expressing C-terminally His6-tagged NK2Rs and to a protein expressed in Sf9 cells transfected with the wild-type NK2R cDNA, which cross-links to 125I-NKA following EDC treatment (Fig. 5).Figure 5Confirmation of the 45-kDaN-biotin-[Tyr1, Cys9] NKA - and N-biotin-[Tyr1, Cys10]NKA-labeled proteins as the NK2R. The figure shows anti-His6 antibody immunoreactive proteins detected in Sf9 cells expressing C-terminally His6-tagged Met297 → Cys (lanes 3and 4) and wild-type NK2Rs (lane 5) but not in cells expressing the untagged receptors (lanes 6and 7; Met297 → Cys NK2Rs,lane 8; wild-type NK2R). The proteins are comparable in mobility to those labeled byN-biotin-[Tyr1,Cys9]NKA (lane 1) andN-biotin-[Tyr1,Cys10]NKA (lane 2) in cells expressing the Met297 → Cys receptor cDNA. Equivalent species were detected in Sf9 cells expressing the wild-type receptor cDNA by EDC cross-linking with [125I]NKA in the absence (lane 9) but not the presence (lane 10) of 20 μm NKA. Molecular mass (kDa) is indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The molecular basis of receptor-ligand interaction is of fundamental importance for the design of highly specific therapeutic agents. It is generally accepted that small conformationally constrained ligands interact with a limited number of receptor sites whereas the binding of larger peptides or proteins involves more extensive receptor contacts. Ligand binding may be influenced by both the local and global conformations of the receptor and the conformation of the ligand itself. As such, the binding sites of small chemical compounds, which typically contain few rotatable bonds, are more readily deduced than those of larger peptide ligands. Here we describe the identification of NKA binding residues within the human NK2R by exploiting the ability of Cu2+-phenanthroline to promote disulfide bond formation between two spatially proximal thiol groups; one within the peptide ligand and the second within the receptor. The affinities of ten NKA analogues, each containing a single cysteine substitution for the wild-type NK2R (11Labrou N.E. Mello L.V. Rigden D.J. Keen J.N. Findlay J.B.C. Peptides. 1999; 20: 795-801Crossref PubMed Scopus (6) Google Scholar) are consistent with data from previous structure-activity studies (12Labrou N.E. Clonis Y.D. Arch. Biochem. Biophys. 1997; 337: 103-114Crossref PubMed Scopus (30) Google Scholar, 13Kunkel T.A. Bebenek K. McClary J. Methods Enzymol. 1991; 204: 125-139Crossref PubMed Scopus (633) Google Scholar, 14Buck S.H. Shatzer S.A. Life Sci. 1988; 42: 2701-2708Crossref PubMed Scopus (75) Google Scholar, 15Guthrie D.J.S. Abushanab A. Allen J. Irvine B. Mcferran N. Walker B. Biochem. Soc. Trans. 1990; 18: 1323-1325Crossref PubMed Scopus (3) Google Scholar). Our data suggest that neither the presence of a bulky tyrosine residue nor of a biotin moiety at the extreme N terminus of NKA affect ligand affinity (Fig. 1C). Single Cys substitutions of His1 and Lys2 also appear to be of little consequence. However, Cys substitutions made at the remaining positions within NKA, particularly of the conserved residues within the C-terminal motif Phe-X-Gly-Leu-Met-NH2, significantly reduce peptide affinity for 125I-NKA binding sites (Table II). This is indicative of a NKA binding site that leaves the extreme N terminus of NKA exposed to receptor regions that are less constrained than the transmembrane bundle, such as the extracellular N terminus and loops. Molecular dynamic simulations of NKA and the cysteine-substituted analogues were used to examine their global structures and essential motions in solution (11Labrou N.E. Mello L.V. Rigden D.J. Keen J.N. Findlay J.B.C. Peptides. 1999; 20: 795-801Crossref PubMed Scopus (6) Google Scholar). Interestingly, although the Cys5analogue displays only a 5-fold lower affinity for the wild-type receptor than NKA, this peptide was structurally more dissimilar from NKA than the other analogues. In contrast, Cys6 displays an average structure that most closely resembled that of NKA yet displays little binding activity. As the [Cys6]NKA analogue is more conformationally flexible than [Cys5]NKA, this suggests that even substantial changes in the overall average structure and/or modest changes in the conformational flexibility of the NKA analogues can be tolerated. This is supportive of a model of peptide binding in which there is an induced fit of the ligand into a conformationally adjustable binding site. These data also suggest a role for the highly conserved Phe6 of NKA in receptor interaction. Using solution-based molecular dynamics simulations, we predict Phe6 clusters with Leu9 and Met10 (11Labrou N.E. Mello L.V. Rigden D.J. Keen J.N. Findlay J.B.C. Peptides. 1999; 20: 795-801Crossref PubMed Scopus (6) Google Scholar). Cysteine substitutions of Leu9 or Met10 were found to alter the average structure of the peptide less than the Cys5 but more than the Cys6 substitutions (11Labrou N.E. Mello L.V. Rigden D.J. Keen J.N. Findlay J.B.C. Peptides. 1999; 20: 795-801Crossref PubMed Scopus (6) Google Scholar). Hence, the reduced affinities of [Tyr1,Cys9]NKA and [Tyr1,Cys10]NKA may only be partly attributed to conformational differences in ligand structure and flexibility. This, in turn, implicates disruption of specific receptor-ligand interactions as the primary cause of the reduction in the affinity of C-terminally Cys-substituted NKA analogues. Consistent with the loss in affinity of [Tyr1,Cys8]NKA for the wild-type receptor, Gly8 is unlikely to participate in direct receptor interaction. Instead, this residue may stereochemically influence Leu9 and Met10 within the binding crevice. We targeted putative NKA binding residues within the transmembrane bundle of the NK2R for site-directed cysteine substitution mutagenesis. Residues in the equivalent positions in the extracellular loop regions and N terminus to NK1R, which influence SP binding (16Munekata E. Kubo K. Tanaka H. Osakada F. Peptides. 1987; 8: 169-173Crossref PubMed Scopus (28) Google Scholar, 17Saviano G. Temussi P.A. Motta A. Maggi C.A. Rovero P. Biochemistry. 1991; 30: 10175-10181Crossref PubMed Scopus (20) Google Scholar, 18Zoffmann S. Gether U. Schwartz T.W. FEBS Lett. 1993; 336: 506-510Crossref PubMed Scopus (34) Google Scholar) were also targeted. The ternary complex model of ligand binding to GPCRs (19Samama P. Cotecchia S. Costa T. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 4625-4636Abstract Full Text PDF PubMed Google Scholar) implies that there are at least two possible receptor conformations, R and R*. Agonists bind with highest affinity to and stabilize receptors in the active R* conformation and classical antagonists highlight both R* and inactive R sites without altering the R-R* interchange. Therefore, as illustrated for the NK1R, (16Munekata E. Kubo K. Tanaka H. Osakada F. Peptides. 1987; 8: 169-173Crossref PubMed Scopus (28) Google Scholar, 17Saviano G. Temussi P.A. Motta A. Maggi C.A. Rovero P. Biochemistry. 1991; 30: 10175-10181Crossref PubMed Scopus (20) Google Scholar, 20Huang R.-R.C. Yu H. Strader C.D. Fong T.M. Biochemistry. 1994; 33: 3007-3013Crossref PubMed Scopus (136) Google Scholar), the inability of an agonist to compete for radiolabeled antagonist binding sites (predominantly R) but not agonist binding sites (predominantly R*) may result from disruption of the R-R* interchange. For this reason, the agonist and antagonist binding characteristics of the mutant receptors were determined using125I-NKA and [3H]SR 48968, respectively (Table I). Consistent with the effect of alanine substitutions, Cys substitutions of Thr24, Phe26 (in the N terminus), Asn86, Asn97 (in helix 2), His267, or Phe270 (in helix 6) abolish 125I-NKA but not [3H]SR 48968 binding (5Huang R.-R.C. Vicario P.P. Strader C.D. Fong T.M. Biochemistry. 1995; 34: 10048-10055Crossref PubMed Scopus (76) Google Scholar). Only those residues at the equivalent positions in the N-terminal tail play a similar role in SP binding to the NK1R (16Munekata E. Kubo K. Tanaka H. Osakada F. Peptides. 1987; 8: 169-173Crossref PubMed Scopus (28) Google Scholar, 17Saviano G. Temussi P.A. Motta A. Maggi C.A. Rovero P. Biochemistry. 1991; 30: 10175-10181Crossref PubMed Scopus (20) Google Scholar, 18Zoffmann S. Gether U. Schwartz T.W. FEBS Lett. 1993; 336: 506-510Crossref PubMed Scopus (34) Google Scholar). This illustrates that although there may be some similarities among the agonist binding sites of these tachykinin receptor subtypes, evolutionary divergence of the NK1R and NK2R and/or SP and NKA has resulted in the development of different modes of receptor-ligand recognition (21Rosenkilde M.M. Cahir M. Gether U. Hjorth S.A. Schwartz T.W. J. Biol. Chem. 1994; 269: 28160-28164Abstract Full Text PDF PubMed Google Scholar). The mutant receptors Gln109 → Cys and Ile202→ Cys appear to display wild-type affinities for both125I-NKA and [3H]SR 48968. Ile202lies close to the NKR-conserved His198, a known NKA binding residue (4Bhogal N. Donnelly D. Findlay J.B.C. J. Biol. Chem. 1994; 269: 27269-27274Abstract Full Text PDF PubMed Google Scholar, 17Saviano G. Temussi P.A. Motta A. Maggi C.A. Rovero P. Biochemistry. 1991; 30: 10175-10181Crossref PubMed Scopus (20) Google Scholar). In our three-dimensional receptor model, both these residues face His267, the second conserved His (helix 6). Surprisingly, the substitution of Ile202 with Cys but not with Val (4Bhogal N. Donnelly D. Findlay J.B.C. J. Biol. Chem. 1994; 269: 27269-27274Abstract Full Text PDF PubMed Google Scholar) is tolerated. Gln109 occupies the equivalent position to Glu113, the Schiff's base counterion of rhodopsin (22Zhukovsky E.A. Oprian D.D. Science. 1989; 246: 928-930Crossref PubMed Scopus (429) Google Scholar, 23Zhukovsky E.A. Robinson P.R. Oprian D.D. Biochemistry. 1992; 31: 10400-10405Crossref PubMed Scopus (48) Google Scholar, 24Sakmar T.R Franke R.R. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8309-8313Crossref PubMed Scopus (602) Google Scholar). Its substitution with the equivalent residue in the NK1R (His), but not with Ala, has been shown to reduce the affinity of NKA binding (4Bhogal N. Donnelly D. Findlay J.B.C. J. Biol. Chem. 1994; 269: 27269-27274Abstract Full Text PDF PubMed Google Scholar, 5Huang R.-R.C. Vicario P.P. Strader C.D. Fong T.M. Biochemistry. 1995; 34: 10048-10055Crossref PubMed Scopus (76) Google Scholar). In the NK2R, the imidazole ring of His but not the smaller side chains of Ala and Cys presumably sterically hinders ligand binding. Within the NK1R the equivalent amino acid to Gln109 (His108) has also been shown to participate in high affinity agonist binding (25Fong T.M. Yu H. Strader C.D. J. Biol. Chem. 1992; 267: 25668-25671Abstract Full Text PDF PubMed Google Scholar). This implies that both Gln109 and Ile202 lie close to residues that interact with NKA. Gln109 packs against Met297 in helix 7, which occupies the equivalent position to the retinal binding Lys296 of rhodopsin (26Cohen G.B. Yang T. Robinson P.R. Oprian D.D. Biochemistry. 1993; 32: 6111-6115Crossref PubMed Scopus (218) Google Scholar). The substitution of Met297 with either Cys or Leu reduces NKA affinity. The Met297 → Cys mutant displayed a rank order of affinity for the Cys-substituted analogues, which was similar to that of the wild-type receptor except that whereas [Tyr1,Cys9]NKA and [Tyr1,Cys10]NKA have substantially lower affinities for the wild-type receptor than NKA, [Tyr1,Cys9]NKA demonstrates an affinity similar to NKA, and [Tyr1,Cys10]NKA has a 5-fold higher affinity than NKA for Met297 → Cys (TableII). This mutation-induced recovery of analogue affinity was not evident for the Met297 → Leu mutant or for other mutant receptors, which displayed 125I-NKA binding. Specific disulfide interactions between Cys297 in the mutant receptor and Cys9- or Cys10-substituted NKA analogues were confirmed by Cu2+-phenanthroline-catalyzed disulfide bond formation between Met297 → Cys receptorsN-biotin-[Tyr1,Cys9]NKA andN-biotin-[Tyr1,Cys10]NKA (Fig. 2). The 45-kDa proteins visualized using Streptavidin-peroxidase correspond to proteins detected n Sf9 cells expressing His-tagged wild-type or Met297 → Cys receptors with a monoclonal His6 antibody (Fig. 5). Furthermore, labeling is reductant-reversible and NEM-sensitive (Fig. 4) and is inhibited by excess NKA (Fig. 3). These observations indicate labeling of NK2Rs and illustrate the proximity of Cys297 to the extreme C terminus of the NKA analogues and therefore to NKA. Fig. 6 shows the possible transmembrane disposition of NKA (4Bhogal N. Donnelly D. Findlay J.B.C. J. Biol. Chem. 1994; 269: 27269-27274Abstract Full Text PDF PubMed Google Scholar, 5Huang R.-R.C. Vicario P.P. Strader C.D. Fong T.M. Biochemistry. 1995; 34: 10048-10055Crossref PubMed Scopus (76) Google Scholar, 6Girault S. Sagan S. Bolbach G. Lavielle S. Chassaing G. Eur. J. Biochem. 1996; 240: 215-222Crossref PubMed Scopus (55) Google Scholar, 7Servant G. Laporte S.A. Leduc R. Escher E. Guillemette G. J. Biol. Chem. 1997; 272: 8653-8659Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 8Phalipou S. Seyer R. Cotte N. Breton C. Barberis C. Hibert M. Mouillac B. J. Biol. Chem. 1999; 274: 23316-23327Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 9Strader C.D. Candelore M.R. Hill W.S. Sigal I.S. Dixon R.A.F. J. Biol. Chem. 1989; 262: 16439-16443Abstract Full Text PDF Google Scholar, 10Emonds-Alt X. Advenier C. Croci T. Manara L. Neliat G. Poncelet M. Proietto V. Santucci V. Soubrie P. Van Broeck D. Vilain P Le Fur G. Breliere J.C. Regul. Pep. 1993; 46: 31-36Crossref PubMed Scopus (32) Google Scholar) within the NK2R. We have shown that NKA adopts an extended loop-like conformation within which the side chains on Asp4, Phe6, Val7, Leu9, and Met10 are exposed (11Labrou N.E. Mello L.V. Rigden D.J. Keen J.N. Findlay J.B.C. Peptides. 1999; 20: 795-801Crossref PubMed Scopus (6) Google Scholar). As Phe6, Leu9, and Met10 appear to cluster together and as Leu9 and Met10 lie close to Met297 in the receptor, this would place Phe6, and perhaps Val7, within the transmembrane bundle. Previous data (3Donnelly D. Maudsley S. Gent J.P. Moser R.N. Hurrell C.R. Findlay J.B.C. Biochem. J. 1999; 339: 55-61Crossref PubMed Google Scholar, 4Bhogal N. Donnelly D. Findlay J.B.C. J. Biol. Chem. 1994; 269: 27269-27274Abstract Full Text PDF PubMed Google Scholar, 5Huang R.-R.C. Vicario P.P. Strader C.D. Fong T.M. Biochemistry. 1995; 34: 10048-10055Crossref PubMed Scopus (76) Google Scholar) suggest the NKA binding site is formed partly by residues in helices 3, 5, 6, and 7 of the receptor. The absence of covalent labeling of Asn86 in helix 2 and Gln109 in helix 3 suggests that these residues may not participate in direct NKA interaction but rather may influence the local conformation around Met297. Similarly, the substitution of His198 and His267 with aliphatic amino acids appears to compromise the binding of or the subsequent intracellular response to NKA (4Bhogal N. Donnelly D. Findlay J.B.C. J. Biol. Chem. 1994; 269: 27269-27274Abstract Full Text PDF PubMed Google Scholar, 5Huang R.-R.C. Vicario P.P. Strader C.D. Fong T.M. Biochemistry. 1995; 34: 10048-10055Crossref PubMed Scopus (76) Google Scholar). This is consistent with the absence of covalent labeling with any of the biotinylated NKA analogues and is supported by the variable degrees to which different substitution of these residues or of surrounding residues (such as Val201, Ile202, and Tyr206) are tolerated. The remaining candidates for NKA interaction reside in helices 6 and 7. The apparent lack of involvement of Leu292(4Bhogal N. Donnelly D. Findlay J.B.C. J. Biol. Chem. 1994; 269: 27269-27274Abstract Full Text PDF PubMed Google Scholar), Phe293, (5Huang R.-R.C. Vicario P.P. Strader C.D. Fong T.M. Biochemistry. 1995; 34: 10048-10055Crossref PubMed Scopus (76) Google Scholar) and Trp294 (unpublished observation) in NKA binding is suggestive of a mode of NKA-receptor interaction where Phe6 and Val7 of NKA lie in a horizontal plane with Leu9 and Met10. This places Phe6 close to Tyr266 and Phe270 on helix 6 of the NK2R. The near wild-type NKA binding properties of receptors retaining the aromatic nature at these sites as compared with the absence of NKA binding to mutants that contain aliphatic residues at these two positions within helix 6 suggest Tyr266 and Phe270 may participate in aromatic-aromatic interactions with the NK-conserved Phe6 of NKA and, as such, may play similar ligand stabilizing roles to the aromatic floor residues in the aminergic receptors (27Donnelly D. Findlay J.B.C. Blundell T.L. Recept. Chann. 1994; 2: 61-78PubMed Google Scholar). Although a region near the extracellular side of helix 7 has been implicated in SP binding to the NK1R (20Huang R.-R.C. Yu H. Strader C.D. Fong T.M. Biochemistry. 1994; 33: 3007-3013Crossref PubMed Scopus (136) Google Scholar, 28Li Y.-M. Marnerakis M. Stimson E.R. Maggio J.E. J. Biol. Chem. 1995; 270: 1213-1220Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), to date there is little evidence for the interaction of SP with more deeply buried residues in this transmembrane segment. Possible differences in the active conformations, and therefore receptor binding sites, of NKA and SP is further highlighted by the absence of roles for the highly conserved helix 5 and 6 histidine residues in SP binding to the NK1R (18Zoffmann S. Gether U. Schwartz T.W. FEBS Lett. 1993; 336: 506-510Crossref PubMed Scopus (34) Google Scholar, 29Fong T.M. Yu H. Cascieri M.A. Underwood D. Swain C.J. Strader C.D. J. Biol. Chem. 1994; 269: 2728-2732Abstract Full Text PDF PubMed Google Scholar). Instead, it has been suggested that the N-and C-terminal residues of SP both interact with receptor regions exposed or close to the extracellular environment (20Huang R.-R.C. Yu H. Strader C.D. Fong T.M. Biochemistry. 1994; 33: 3007-3013Crossref PubMed Scopus (136) Google Scholar, 28Li Y.-M. Marnerakis M. Stimson E.R. Maggio J.E. J. Biol. Chem. 1995; 270: 1213-1220Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 30Werge T.M. J. Biol. Chem. 1994; 269: 22054-22058Abstract Full Text PDF PubMed Google Scholar). We predict that Asp4 of NKA lies close to the receptor N terminus (11Labrou N.E. Mello L.V. Rigden D.J. Keen J.N. Findlay J.B.C. Peptides. 1999; 20: 795-801Crossref PubMed Scopus (6) Google Scholar) where it may interact with Thr24 and Phe26 in the receptor N terminus, Tyr93, Asn97, and Trp99 in the first extracellular loop, or residues in the third extracellular loop (5Huang R.-R.C. Vicario P.P. Strader C.D. Fong T.M. Biochemistry. 1995; 34: 10048-10055Crossref PubMed Scopus (76) Google Scholar). However, the conserved C terminus of NKA appears to interact with the Met297 within the helical bundle. In conclusion, using thiol we have demonstrated the proximity of the two most C-terminal residues of NKA to Met297, a deeply buried site in the seventh transmembrane helix of the NK2R. To date, this is the most convincing evidence to support the role of the transmembrane helices in the binding of peptide agonists to tachykinin receptors. Consistent with other views of peptide agonist binding to GPCRs (31Feng Y.-H. Noda K. Saad Y. Liu X.-P. Husain A. Karnik S.S. J. Biol. Chem. 1995; 270: 12846-12850Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 32Yamano Y. Ohyama K. Chaki S. Guo D.F. Inagami T. Biochem. Biophys. Res. Commun. 1992; 187: 1426-1431Crossref PubMed Scopus (129) Google Scholar, 33Yamano Y. Ohyama K. Kikyo M. Sano T. Nakagomi Y. Inuoe Y. Nakamura N. Morishima I. Hamakubo T. Inagami T. J. Biol. Chem. 1995; 270: 14024-14030Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), it seems that NKA binds partly within the transmembrane bundle but possesses a more hydrophilic N terminus, which may interact with the receptor N terminus and extracellular loop regions (Fig. 6). This strategy of mapping the peptide binding site of the NK2R dispenses with the need for proteolytic cleavage and peptide sequencing associated with more classical affinity labeling methodologies." @default.
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• W1492615281 title "Interaction of Met297 in the Seventh Transmembrane Segment of the Tachykinin NK2 Receptor with Neurokinin A" @default.
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