Research ArticleArticle

Relevance of Aromatic Residues in Transmembrane Segments V to VII for Binding of Peptide and Nonpeptide Antagonists to the Human Tachykinin NK2 Receptor

A. R. Renzetti, R.-M. Catalioto, M. Criscuoli, P. Cucchi, C. Ferrer, A. Giolitti, M. Guelfi, L. Rotondaro, F. J. Warner and C. A. Maggi
Journal of Pharmacology and Experimental Therapeutics August 1999, 290 (2) 487-495;

Abstract

We used membranes from Chinese hamster ovary cells stably transfected with the human tachykinin NK2 receptor, either wild-type or mutated, at four aromatic residues (His198, Tyr266, Phe270, Tyr289) located in transmembrane segments V to VII, to assess the role of these residues in the binding of natural tachykinins and peptide and nonpeptide antagonists. Three radioligands, the agonist [125I]neurokinin A (NKA), the peptide antagonist [3H]MEN 11420, and the nonpeptide antagonist [3H]SR 48968 bound to the wild-type receptor with high affinity (Kd = 2.4 nM, 0.3 nM, and 4.0 nM, respectively). Four of the six mutant receptors tested retained high affinity for at least one of the radioligands. H198A mutation abrogated the binding of NKA but not that of MEN 11420 or SR 48968 (Kd = 4.8 and 11.5 nM, respectively); Y266F mutation abrogated the binding of MEN 11420 but not that of NKA or SR 48968 (Kd = 2.8 nM and 1.2 nM, respectively); F270A mutation abrogated the binding of both NKA and MEN 11420 but not that of SR 48968 (Kd = 1.6 nM); Y289F mutation abrogated the binding of SR 48968 but not that of NKA and MEN 11420 (Kd = 2.0 and 2.9 nM, respectively). Y266A and Y289A mutations abrogated the binding of all radioligands. Among the unlabeled antagonists, the affinity of the nonpeptide GR 159897, at variance with SR 48968, resulted heavily compromised by H198A and Y266F mutations; the peptide antagonists R396 and MEN 10376 essentially followed the binding profile of NKA, but R396 showed markedly increased affinity for the Y289F mutant receptor. Taken together, these results indicate that different, partially overlapping sets of sites may be involved in the binding of agonists and diverse antagonists to the human tachykinin NK2 receptor.

The mammalian tachykinins substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) form a family of peptides that share the common C-terminal sequence Phe-X-Gly-Leu-MetNH2. Tachykinins are found in many regions of the central and peripheral nervous system (Maggio, 1988; Maggi et al., 1993; Otsuka and Yoshioka, 1993) and produce their biological effects by stimulating three distinct G protein-coupled receptors termed tachykinin NK1, NK2, and NK3 receptors (Maggi, 1995, for review).

The tachykinin NK1 receptor has been the object of intense investigations to assess the structural determinants of its interaction with ligands of both peptide and nonpeptide nature, agonists and antagonists, respectively (Schwartz et al., 1995, for review). The site-directed mutagenesis approach has been extensively used to identify residues putatively involved in the binding of agonists and antagonists to the tachykinin NK1receptor. From these studies, it has been proposed that residues forming the binding site of natural agonists on the tachykinin NK1 receptor are quite distinct from those that form the binding site of antagonists, especially those of antagonists of nonpeptide nature (Fong et al., 1992; Gether et al., 1993a; Huang et al., 1994).

Less information is available on the structural determinants of agonist and antagonist binding at the tachykinin NK2receptor. Studies using chimeric tachykinin NK1/NK2 receptors have shown that the binding site of the nonpeptide tachykinin NK2 receptor antagonist SR 48968 is not fully coincident with that of NKA, the natural tachykinin with preferential binding affinity for tachykinin NK2receptors (Gether et al., 1993b). A few site-directed mutagenesis studies have also been performed on the human tachykinin NK2 receptor (hNK2R; Bhogal et al., 1994; Huang et al., 1995). The results of these studies have identified some aromatic residues in transmembrane segments (TM) V to VII of the hNK2R as being involved in the binding of NKA and other natural tachykinins, as well as in the binding of the nonpeptide tachykinin NK2 receptor antagonists SR 48968 (Emonds-Alt et al., 1992) and GR 159897 (Beresford et al., 1995; Fig. 1). In particular, Tyr289 (TM VII), along with Tyr266 and Phe270 (TM VI), were proposed to form part of the SR 48968 binding pocket (Huang et al., 1995). Discrepant results were reported with regard to the role played by His198 (TM V) in determining the binding affinity of SR 48968. Bhogal et al. (1994) and Huang et al. (1995) reported a dramatic fall in the binding affinity of NKA at the H198A mutant tachykinin NK2receptor. However, although Bhogal et al. (1994) reported that this residue is crucial for preserving the binding affinity of SR 48968,Huang et al. (1995) failed to detect any significant change of the affinity with the H198A mutation, although they reported reduced binding affinity for another nonpeptide antagonist, GR 159897. The reasons for this discrepancy have not been found.

Figure 1
Figure 1

Chemical structures of the tachykinin NK2receptor antagonists MEN 11420, SR 48968, and GR 159897.

In this study, we used Chinese hamster ovary (CHO) cell lines stably transfected with the wild-type and mutant tachykinin NK2 receptors to more precisely assess the role played by aromatic residues in TM V (His198), TM VI (Tyr266 and Phe270), and TM VII (Tyr289; Fig.2) in the binding of natural agonists and peptide (including both linear and cyclic structures) and nonpeptide antagonists. We performed saturation and competition binding experiments with CHO cell membranes by using [125I]NKA, [3H]SR 48968, and [3H]MEN 11420, a new tachykinin NK2 receptor-selective ligand (Catalioto et al., 1998, Renzetti et al., 1998; Fig. 1) as radioligands. In particular, we aimed at verifying the hypothesis of the highly variable, flexible mode of interaction of the receptor with the different classes of ligands that could be inferred from the previous work of Bhogal et al. (1994)and Huang et al. (1995).

Figure 2
Figure 2

Schematic of the tachykinin NK2 receptor showing the putative localization of the residues subjected to mutation.

Materials and Methods

Chemicals

Penicillin, streptomycin, and trypsin were purchased from GIBCO (Grand Island, NY). Dialyzed fetal calf serum (FCS) was obtained from BioWhittaker (Walkersville, MD). Minimum essential Eagle’s medium α-modification (α-MEM) and GTP were purchased from Sigma Chemical Co. (St. Louis, MO). Flasks and Petri dishes were purchased from Falcon (Becton Dickinson, Milan, Italy). Protein binding dye was from Bio-Rad (Richmond, CA). NKA, NKB, and SP were obtained from Neosystem Laboratoire (Strasbourg, France). SR 48968 [(S)-N-methyl-N[4-(4-acetyl-amino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide] was kindly provided by Drs. X. Emonds-Alt and G. Le Fur (Sanofi Recherche, Montpellier, France). MEN 11420 (c{[(β-d-GlcNAc)Asn-Asp-Trp-Phe-Dpr-Leu]c(2β-5β)}), MEN 10376 ([Tyr5,d-Trp6,8,9,Arg10]NKA (4–10), and GR 159897 [(R)-1-[2-(5-fluoro-1H-indol-3-yl)ethyl]-4-methoxy-4[(phenylsulfinyl)-methyl]piperidine] were synthesized at the Chemistry Department of Menarini Ricerche (Firenze, Italy). R396 (Ac-Leu-Asp-Gln-Trp-Phe-Gly-NH2) was a gift from Prof. D. Regoli.

[3H]MEN 11420 (specific activity, 62 Ci/mmol) was synthesized by SibTech Inc. (Elmsford, NY). [125I]NKA (specific activity, 2000 Ci/mmol) was purchased from Amersham International (Buckinghamshire, UK). [3H]SR 48968 (specific activity, 25.7 Ci/mmol) was purchased from Dupont-NEN Life Sciences (Boston, MA). All other reagents, available from commercial sources, were of analytical grade.

Site-Directed Mutagenesis of hNK2R cDNA.

Plasmid pBS/hNK2R (kindly provided by Dr. J. E. Krause, Washington University, St. Louis, MO) contained a 1.2-kb cDNA for the ileum hNK2R cloned in theSmaI site of the pBlueScript II SK(−) phagemid. Site-directed mutagenesis of the tachykinin NK2receptor cDNA was performed according to the phosphorothioate technique of Eckstein (Taylor et al., 1985; Nakamaye and Eckstein, 1986) using single-stranded DNA of the tachykinin NK2receptor cDNA in pBlueScript II SK(−) and an in vitro mutagenesis kit (Sculptor; Amersham International) according to the manufacturer’s instructions. Wild-type cDNA was isolated fromHindIII/XbaI-digested pBS/hNK2R and cloned in pmCMVβSV1dhfr, partially digested with HindIII, between theHindIII and XbaI sites of the polylinker region, under the transcriptional control of the murine cytomegalovirus major immediate-early promoter. The resulting vector was designated as pmCMVβSV1dhfr-hNK2R. Expression vectors carrying the mutated cDNAs (H198A, Y266A, Y266F, F270A, Y289A, Y289F) were constructed by exchanging the 1.2-kbEcoRV/Xbal wild-tachykinin NK2 receptor cDNA in pmCMVβSV1dhfr-hNK2R with theEcoRV/Xbal mutated cDNAs excised from pBS/hNK2R. pmCMVβSV1 was constructed by removing G-CSF cDNA sequences from XbaI-digested pmCMVβG-CSFSV1dhfr (Rotondaro et al., 1997). The structure of all constructs was confirmed by restriction analysis. The complete coding sequence of wild-type and mutated tachykinin NK2 receptor cDNAs cloned into the pmCMVβSV1dhfr expression vector was confirmed by DNA sequencing.

Receptor Expression in CHO Cells.

Large-scale preparation of vector DNA for transfection experiments was carried out using a Qiagen maxipreparation column (Qiagen, Hilden, Germany). Wild-type and (H198A, Y266A, Y266F, F270A, Y289A, or Y289F) mutated tachykinin NK2 receptor cDNAs in pmCMVβSV1dhfr were introduced by lipofection as described previously (Rotondaro et al., 1997) into dihydrofolate reductase (DHFR)-deficient CHO DUKX-B11 cells (Urlaub and Chasin, 1980; referred to as CHOdhfr−). Stable DHFR+ transformants were selected in nucleoside-free α-MEM containing 5% dialyzed FCS; 12 to 14 days after transfection, more than 100 individual DHFR+ clones were pooled, grown to mass culture, and used for ligand-binding studies. CHOdhfr− cells and stable CHO transfectants were grown in α-MEM containing ribonucleosides and deoxyribonucleosides and supplemented with 5% FCS, in a humidified atmosphere of 5% CO2/95% air at 37°C until slightly confluent.

Binding Assays.

Confluent cells were harvested in PBS, pelleted by centrifugation at 200g (4°C), and homogenized using a Polytron PT3000 (Kinematica, Lucerne, CH) at 13,000 rpm for 15 s in 20 ml of 50 mM Tris, pH 7.4, containing bacitracin (100 μg/ml), chymostatin (10 μg/ml), leupeptin (5 μg/ml), and 10 μM thiorphan (buffer A). The homogenate was centrifuged for 1 h at 25,000g (4°C), and the pellet was resuspended in the binding buffer (pH 7.4) composed of buffer A supplemented with 150 mM NaCl, 5 mM MnCl2, and 0.1% BSA at a protein concentration of about 0.6 mg/ml. The membranes (50–90 μg protein/assay) were incubated for 30 min ([125I]NKA and [3H]SR 48968) or 60 min ([3H]MEN 11420) at 20°C. In saturation experiments, either increasing concentrations (0.09–15.0 nM) of [3H]SR 48968 or [3H]MEN 11420 (hot experiments) or a fixed concentration (150 pM) of [125I]NKA with various concentrations of unlabeled NKA (0.01 nM to 1 μM; cold experiment) was used. For competition experiments, 0.4 to 1.0 nM [3H]MEN 11420, 0.6 nM [3H]SR 48968, or 150 pM [125I]NKA was used, with or without varying concentrations (0.001 nM to 10 μM) of the competing compounds in a final volume of 0.5 ml. Then, 1 μM unlabeled MEN 11420, SR 48968, or NKA was used to define nonspecific binding, depending on the radioligand. The reaction was terminated by the addition of 4 ml of ice-cold Tris (50 mM, pH 7.4) followed by rapid filtration through Whatman GF/B filter sheets (presoaked in 0.3% polyethylenimine for [3H]MEN 11420 or 0.5% BSA for [3H]SR 48968 and [125I]NKA, for at least 3 h using a Brandel (Montreal, Quebec, Canada) cell harvester. Filters were washed three times with 4 ml of ice-cold 50 mM Tris buffer, pH 7.4. The trapped radioactivity was determined by liquid scintillation using a β-scintillation counter (2200 CA; Packard, Milan, Italy) or using a gamma counter (Cobra, Packard).

Analysis of Binding Data

Saturation and competition data were processed according toMunson and Rodbard (1980) by the step-wise application of EBDA and LIGAND programs in the KELL software for Macintosh (Biosoft, Cambridge, UK). The maximal binding (Bmax), equilibrium dissociation constant (Kd), and equilibrium inhibition constant (Ki) values were calculated. They are reported as mean values with approximate S.E.s as estimated by LIGAND. The goodness of fit, according to the single- or multiple-site binding models, was evaluated by the F ratio.

Statistical comparisons were made by using one-way ANOVA followed by Tukey’s test where appropriate.

Results

Saturation Experiments

The specific binding of [125I]NKA, [3H]SR 48968, and [3H]MEN 11420 was directly proportional to the membrane concentration (data not shown). At protein concentrations between 50 and 90 μg/assay, as used in competition experiments, the specific binding represented approximately 80 to 90% of the total binding for each radioligand, respectively. Scatchard transformation of the saturation experiments data showed a monophasic interaction with the wild-type NK2 receptor withKd values of 2.4 ± 0.43, 0.3 ± 0.08, and 4.0 ± 0.44 nM andBmax values of 206 ± 28, 3274 ± 1489, and 1664 ± 416 fmol/mg protein for [125I]NKA, [3H]SR 48968, and [3H]MEN 11420, respectively (n = 5–6; Table 1).

View this table:
Table 1

Saturation analysis-derived binding constants of the three diverse radioligands for wild-type and mutant human tachykinin NK2receptors expressed in CHO cells

Y266A and Y289A mutant receptors did not bind any of the radioligands (Table 1). For the other mutant receptors, the specific binding of [125I]NKA, [3H]SR 48968, and [3H]MEN 11420 was directly proportional to the membrane concentration (data not shown). Saturation experiments always produced data congruent with monophasic ligand-receptor interaction. NeitherKd norBmax values for [125I]NKA changed with the mutant receptors, which maintained the ability to interact with the agonist ligand (Table1). The Bmax values for [3H]SR 48968 and [3H]MEN 11420, considerably higher than that measured for [125I]NKA, were not statistically different from each other. They, too, despite an apparent variability, did not change in a significant manner throughout the responsive receptors; the only exception was the H198A mutant, which showed both lower expression and reduced affinity for the antagonist ligands (Table 1).

Competition Experiments

Wild-Type.

Among the tachykinins, only NKA competed with high affinity for the binding of the three radioligands to the wild-type hNK2R. When using either [3H]SR 48968 or [3H]MEN 11420 as tracers, the competition of unlabeled NKA was biphasic, probably reflecting its binding to the high-affinity (i.e., G protein-coupled) and low-affinity (i.e., G protein-uncoupled) states of the tachykinin NK2 receptor (Fig.3). The rank order of affinity of the receptor antagonists, which was independent from the radioligand used to label tachykinin NK2 receptors, was as follows: SR 48968 > GR 159897 > MEN 11420 > MEN 10376 ≫ R396 (Table 2). The curves describing the displacement of [3H]SR 48968 and [3H]MEN 11420 by the prototype antagonists SR 48968, GR 159897, and MEN 11420 are shown in Figs.4A and 5A.

Figure 3
Figure 3

Homologous and heterologous displacements of [125I]NKA (▴), [3H]SR 48968 (▪), and [3H]MEN 11420 (●) by unlabeled NKA in membrane preparations of CHO cells transfected with wild-type nNK2R. Data shown represent the mean of at least three separate determinations in duplicate. Curves were fitted by the simultaneous analysis of all data sets to a one-site (for [125I]NKA) or two-site (for [3H]SR 48968 and [3H]MEN 11420) binding model by GraphPAD Prism.

View this table:
Table 2

Binding affinity of tachykinins and selective antagonists for wild-type and mutant human tachykinin NK2 receptors expressed in CHO cells

Figure 4
Figure 4

Displacement curves of [3H]SR 48968 binding to CHO cell membranes transfected with wild-type (A), H198A (B), Y266F (C), and F270A (D) hNK2R by GR 159897 (▪), MEN 11420 (▴), and SR 48968 (●). Data shown represent the mean of at least three separate determinations in duplicate. Curves were fitted by the simultaneous analysis of all data sets to a one-site binding model by GraphPAD Prism.

H198A.

This mutation completely abrogated the binding of [125I]NKA to the hNK2R and reduced by about 16-fold the binding affinity of [3H]SR 48968, whereas binding affinity of [3H]MEN 11420 was reduced by about 3-fold (Table 1). Because a sizable binding affinity was measured with [3H]SR 48968 and [3H]MEN 11420, these tracers were used in competition experiments with natural tachykinins and antagonists of both peptide and nonpeptide nature (Table 2, Fig. 4B).

The affinity of NKA for the H198A tachykinin NK2 receptor was then estimated to be decreased by about 143- and 159-fold using [3H]SR 48968 and [3H]MEN 11420 as tracers, respectively (Table 2), whereas no binding affinity could be estimated for NKB or SP (Table 2). Confirming the results of Huang et al. (1995), the binding affinity of GR159897 was also markedly decreased as measured by displacing either [3H]SR 48968 or [3H]MEN 11420 (66- and 25-fold, respectively; Table 2). No sizable binding affinity was measured for the two linear peptide antagonists, MEN 10376 and R396, with either tracer (Table 2).

Y266F.

This mutation did not affect the binding of [125I]NKA, slightly affected (4-fold decrease) the binding of [3H]SR 48968, and abrogated the binding of [3H]MEN 11420 to the hNK2R (Table 1). The binding affinity of unlabeled MEN 11420 at the mutant receptor was decreased by about 12-fold when using either [125I]NKA or [3H]SR 48968 as tracers (Table 2, Fig. 4C). The binding affinity of NKB, MEN 10376, and R396 was not substantially altered by the Y266F mutation (Table 2), whereas the binding affinity of GR 159897 was markedly decreased, by about 185- and 149-fold when using [125I]NKA or [3H]SR 48968 as tracers, respectively (Table 2, Fig. 4C).

F270A.

This mutation completely abrogated the binding of [125I]NKA and [3H]MEN 11420 with only a minor effect on the binding of [3H]SR 48968 (Table 1). When using [3H]SR 48968 as a tracer, a 66-fold decrease in the binding affinity of MEN 11420 was estimated (Table 2, Fig. 4D), whereas no competition was exerted by NKA up to 10 μM (Table 2). Likewise, no binding affinity of NKB and SP could be estimated using [3H]SR 48968 as a tracer. The binding affinity of GR 159897 was, if any, slightly increased (5-fold) by the F270A mutation (Table 2, Fig. 4D), whereas the binding affinity of the linear peptide antagonists was decreased (MEN 10376; 17-fold) or abolished (R396; Table 2).

Y289F.

This mutation completely abrogated the binding of [3H]SR 48968 without affecting the binding of [125I]NKA and [3H]MEN 11420 (Table 1). In competition experiments, a 2741- and 3088-fold decrease in binding affinity of SR 48968 was estimated when using [125I]NKA and [3H]MEN 11420 as tracers, respectively (Table2, Fig. 5B). The binding affinity of NKB and SP was unaffected by the Y289F mutation (Table 2). The binding affinity of GR 159897 was totally abrogated (Table 2, Fig. 5B), whereas that of MEN 10376 was unaffected when using either [125I]NKA or [3H]MEN 11420 as tracers (Table 2). Interestingly, the binding affinity of the linear antagonist R396 was consistently increased, by about 18-fold, by the Y289F mutation, when using either radioligand (Table 2).

Figure 5
Figure 5

Displacement curves of [3H]MEN 11420 binding to CHO cell membranes transfected with wild-type (A) and Y289F (B) hNK2R by GR 159897 (▪), MEN 11420 (▴), and SR 48968 (●). Data shown represent the mean of at least three separate determinations in duplicate. Curves were fitted by the simultaneous analysis of all data sets to a one-site binding model by GraphPAD Prism.

Discussion

In the attempt to overcome problems that may be related to transient transfection and variable levels of expression of the receptor, we prepared cell lines stably transfected with wild-type and mutant hNK2Rs. This approach enabled us to produce a systematic pharmacological analysis of the mutant receptors by using three different radioligands and a panel of agonists and antagonists.

The even Bmax values found with the agonist [125I]NKA generally represent only a fraction of the receptor density estimated with the antagonist radioligands, which are not able to discriminate between G protein-coupled and -uncoupled states of the NK2receptor. This is likely due to the fact that transfected receptors are expressed in excess relative to the G protein-coupling capability of the CHO. In the case of the H198A mutant, a significant reduction in the Bmaxvalue accompanied the decrease in affinity for the recognized antagonists.

It is interesting to note that three of the four aromatic residues mutated in this study (His198, Phe270, and Tyr289 in the sequence of the hNK2R) are fully conserved at equivalent positions in the sequence of the various mammalian tachykinin receptors cloned so far; the fourth residue (Tyr266 in the sequence of the hNK2R) is conserved in the mammalian tachykinin NK2 and NK3 receptors and is replaced by Phe at the equivalent position of the mammalian tachykinin NK1 receptor. Due to their lipophilic nature, these conserved residues are likely to play a structural role in the correct positioning of the TMs. From the present data, we have evidence that each one of the H198A, Y266F, F270A, and Y289F mutant receptors maintained high-affinity binding for at least one of the three radioligands used. This can be considered as a kind of positive control (i.e., it allows us to exclude the possibility that these mutations produced a marked alteration in the gross structure of the receptor or that the mutant receptor protein was synthesized but not expressed at the cell membrane level). Therefore, the observed changes in the affinity of a given ligand for each of these mutant receptors can be confidently assumed as indications that the corresponding residues represent a true point of drug-receptor interaction or, alternatively, that the mutation has modified the geometry of the ligand-binding pocket, thus indirectly influencing the affinity of the ligand.

With regard to the binding of natural tachykinins, our data demonstrate that both His198 and Phe270are crucial for maintaining the high-affinity binding of NKA (see Huang et al., 1995) and that these residues are also of relevance for the binding of NKB and SP to the wild-type tachykinin NK2 receptor.

His198, putatively located on TM V of the hNK2R, corresponds to His197 in the sequence of the hNK1R. In the latter, the Phe→Ala replacement abolished the binding of several nonpeptide receptor antagonists without affecting the binding of SP (Fong et al., 1993). On the other hand, SP displayed a weak but sizable binding to the wild-type tachykinin NK2 receptor (Ki = 1.8 μM against [125I]NKA), which was lost in the H198A mutant (Ki> 10 μM). This observation, along with the data of Fong et al. (1993), implies that the mode of interaction of SP with the tachykinin NK1 and NK2 receptors are different (i.e., the same tachykinin interacts with different regions of the tachykinin NK1 and NK2 receptors).

Our data also indicate that the —OH function of Tyr266 and Tyr289 is not involved in determining the binding affinity of natural tachykinins to the hNK2R. On the other hand, the actual role of the aromatic function of these two Tyr residues in the binding of tachykinins could not be definitely assessed; indeed, Ala replacement of Tyr266 and Tyr289completely abolished the binding affinity for each of the three radioligands tested. Unfortunately, this also means that no positive control (as defined above) was any more available, which hampered any speculation on the relevance of these data.

Our findings provide a clear demonstration that the binding of peptide antagonists to the hNK2R is partially overlapping but not coincident to that of nonpeptide antagonists SR 48968 and GR 159897. Moreover, differences in binding behavior also emerged between linear and cyclic peptide antagonists. In general, the observed changes in the binding affinity of linear peptide antagonists MEN 10376 (Maggi et al., 1991) and R396 (Dion et al., 1990) for the mutant tachykinin NK2 receptors follow quite closely those observed for NKA and agonists. In fact, Ala substitution of His198 and Phe270 almost completely abrogated the binding of MEN 10376 and R396, whereas the Tyr→Phe replacement at position 266 was irrelevant for both antagonists and, for MEN 10376, the Tyr→Phe replacement at position 289 had no effect on binding affinity. Our data for MEN 11420, a glycosylated bicyclic peptide (Catalioto et al., 1998), indicate that in common with NKA, Phe270 is relevant for determining the high-affinity binding of this antagonist to the hNK2R. On the other hand, although His198 is clearly relevant for the binding of NKA, MEN 10376, and R396, this residue is apparently less important for the binding of MEN 11420.

Huang et al. (1995) described some pharmacological properties of Tyr266 mutants of the hNK2R. They reported that although the Y266F mutant maintains a full binding affinity for both NKA and SR 48968, the binding affinity was largely lost when Tyr266 was mutated to Ser or Ala (findings confirmed by the present results). Because a functional response (phosphoinositol formation) to NKA was preserved in the Y266S mutant, the derived affinity estimates for NKA and SR 48968 could be computed and found to be obviously low (0.3 and 3.4 μM, respectively; Huang et al., 1995). From this, they concluded that the aromatic group of Tyr266 is involved, directly or indirectly, in the binding of NKA and SR 48968 to the hNK2R. In this study, we observed that it is sufficient to remove the —OH function of Tyr266to abrogate the high-affinity binding of [3H]MEN 11420 to the hNK2R. This indicates that the binding site of MEN 11420 on the hNK2R is partly different from that of both NKA and SR 48968. The drop in affinity of MEN 11420, as estimated from competition experiments against [125I]NKA or [3H]SR 48968, is relatively small (about 10-fold) but consistent and evidently sufficient to prevent the detection of a specific binding at the low concentrations of the radioligand used in saturation experiments. This same mutation did not affect the binding affinity of the linear peptides MEN 10376 and R396, nor that of SR 48968, but decreased the binding affinity of GR 159897. This may suggest that the —OH function of Tyr266 directly interacts with some moiety present in both MEN 11420 and GR 159897 but not in SR 48968. An hydrogen-bond donor/acceptor interplay between the —OH function of Tyr266 and the indole amino group present in both MEN 11420 and GR 159897 could be postulated (Fig.6).

Figure 6
Figure 6

Molecular models of the binding of the tachykinin NK2 receptor antagonists MEN 11420 (top) and SR 48968 (bottom) to the tachykinin NK2 receptor. Presented view is from outside the cell membrane, along the α-helix axis. TM I and TM II have been omitted for clarity.

With regard to the binding of SR 48968 at the H198A mutant receptor, we found a sizable but limited decrease in affinity (about 16-fold): in this respect, our data differ from those of Huang et al. (1995), who reported that the binding of SR 48968 is unaffected by the H198A mutation, and from those of Bhogal et al. (1994), who reported that binding of SR 48968 is abrogated by the H198A mutation. Because both previous studies were performed on transiently transfected cell lines, it is possible that variable levels of receptor expression had influenced the results. With regard to the other residues, our data confirm the observation (Huang et al., 1995) that the removal of the —OH function of Tyr289 almost totally eliminated the binding affinity of SR 48968. On the other hand, the F270A mutation, which impaired the binding of most other ligands tested (with the exception of GR 159897), also had little effect on the binding of SR 48968 to the hNK2R. The equivalent residue (Phe268) in the sequence of the hNK1R has been recently proposed to be partially involved, directly or indirectly, in the binding of both SP and various nonpeptide antagonists to the tachykinin NK1receptor, with the exception of SR 140333 (Holst et al., 1998).

On the whole, the two nonpeptide antagonists appear to have quite different sensitivities to residue mutation: the binding affinity of GR 159897 was heavily compromised by mutations at sites (His198 and Tyr266), which have a weaker effect on the binding of SR 48968. This strongly suggests that similar to the tachykinin NK1 receptor (Holst et al., 1998), nonpeptide ligands of different chemical classes possess different modes of interaction with the tachykinin NK2 receptor.

An interesting further observation is that relative to the ligand-dependent variation of the affinity of GR 159897 for the H198A mutant receptor, the affinity of GR 159897 was about 4-fold higher if measured in competition with [3H]MEN 11420 than with [3H]SR 48968 (Ki = 58 and 214 nM, respectively). A similar (5-fold) difference was observed when SR 48968 was tested in homologous (saturation) or heterologous (competition versus [3H]MEN 11420) binding assays (Kd = 5.2 nM,Ki = 1.0 nM, respectively). These observations suggest that [3H]MEN 11420 and [3H]SR 48968 may label different conformers of the tachykinin NK2 receptor, both unable to signal second messenger activation, and that the His198 residue is relatively less important in the binding energy balance for the ligand-receptor complex formed by [3H]MEN 11420 with the selected receptor conformer.

In conclusion, the present findings provide a detailed pharmacological analysis of the role of some aromatic residues in TM V to VII for binding of different tachykinin receptor agonists and antagonists to the hNK2R. With regard to the residues examined, it appears that the binding of linear peptide antagonists is affected in a similar manner to that of agonists, whereas the chemical modification introduced in the cyclic backbone of MEN 11420 partially changed the regions of the receptor that provide high-affinity binding for this ligand. The binding of agonists and antagonists (of both peptide and nonpeptide nature) to the hNK2R only partly overlaps.

Footnotes

  • Send reprint requests to: Dr. Anna Rita Renzetti, Pharmacology Department, Menarini Ricerche S.p. A., Via Rismondo 12A, I-50131 Florence, Italy.

  • Abbreviations:
    SP
    substance P
    CHO
    Chinese hamster ovary
    NKA
    neurokinin A
    NKB
    neurokinin B
    TM
    transmembrane segment
    α-MEM
    minimum essential medium α-modification
    DHFR
    dihydrofolate reductase
    hNK2R
    human tachykinin NK2receptor
    FCS
    fetal calf serum
    • Received January 29, 1999.
    • Accepted April 6, 1999.

References

  1. ↵
    1. Beresford IJM,
    2. Sheldrick RLG,
    3. Ball DI,
    4. Turpin MP,
    5. Walsh DM,
    6. Hawcock AB,
    7. Coleman RA,
    8. Hagan RM,
    9. Tyers MB
    (1995) GR 159897, a potent non-peptide antagonist at tachykinin NK2 receptors. Eur J Pharmacol 272:241–248.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Bhogal N,
    2. Donnelly D,
    3. Findlay JBC
    (1994) The ligand binding site of the neurokinin 2 receptor. J Biol Chem 269:27269–27274.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Catalioto R-M,
    2. Criscuoli M,
    3. Cucchi P,
    4. Giachetti A,
    5. Giannotti D,
    6. Giuliani S,
    7. Lecci A,
    8. Lippi A,
    9. Patacchini R,
    10. Quartara L,
    11. Renzetti AR,
    12. Tramontana M,
    13. Arcamone F,
    14. Maggi CA
    (1998) MEN 11420 (Nepadutant), a novel glycosylated bicyclic peptide tachykinin NK2 receptor antagonist. Br J Pharmacol 123:81–91.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Dion S,
    2. Rouissi N,
    3. Nantel F,
    4. Jukic D,
    5. Rhaleb NE,
    6. Tousignant C,
    7. Telemaque S,
    8. Drapeau G,
    9. Regoli D,
    10. Naline E,
    11. Advenier C,
    12. Rovero P,
    13. Maggi CA
    (1990) Structure-activity study of neurokinins: Antagonists for the NK2 receptor. Pharmacology 41:184–194.
    OpenUrlPubMed
  5. ↵
    1. Emonds-Alt X,
    2. Vilain P,
    3. Goulaouic P,
    4. Proietto V,
    5. Van Broeck D,
    6. Advenier C,
    7. Naline E,
    8. Neliat G,
    9. Le Fur G,
    10. Breliere JC
    (1992) A potent and selective nonpeptide antagonist of the neurokinin A (NK2) receptor. Life Sci Pharmacol Lett 50:PL101–106.
    OpenUrl
  6. ↵
    1. Fong TM,
    2. Cascieri MA,
    3. Yu H,
    4. Bansal A,
    5. Swain C,
    6. Strader C
    (1993) Amino-aromatic interaction between histidine 197 of the NK1 receptor and CP 96,345. Nature (Lond) 362:350–353.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Fong TM,
    2. Huang R-RC,
    3. Strader CD
    (1992) Localization of agonist and antagonist binding domains of the human NK-1 receptor. J Biol Chem 267:25664–25667.
    OpenUrlAbstract/FREE Full Text
    1. Gether U,
    2. Emonds-Alt X,
    3. Breliere JC,
    4. Fujii T,
    5. Hagiwara D,
    6. Pradier L,
    7. Garret C,
    8. Johansen TE,
    9. Schwartz TW
    (1994) Evidence for a common molecular mode of action for chemically distinct nonpeptide antagonists at the NK1 (substance P) receptor. Mol Pharmacol 45:500–508.
    OpenUrlAbstract
  9. ↵
    1. Gether U,
    2. Johansen TE,
    3. Snider RM,
    4. Lowe JA III,
    5. Nakanishi S,
    6. Schwartz TW
    (1993a) Different binding epitopes on the NK1 receptor for substance P and a nonpeptide antagonist. Nature (Lond) 362:345–348.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Gether U,
    2. Yokota Y,
    3. Emonds-Alt X,
    4. Breliere JC,
    5. Lowe JA III,
    6. Snider RM,
    7. Nakanishi S,
    8. Schwartz TW
    (1993b) Two nonpeptide tachykinin antagonists act through epitopes on corresponding segments of the NK1 and NK2 receptors. Proc Natl Acad Sci USA 90:6194–6198.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Holst B,
    2. Zoffmann S,
    3. Elling CE,
    4. Hjorth SA,
    5. Schwartz TW
    (1998) Steric hindrance mutagenesis vs. alanine scan in mapping of ligand binding sites in the tachykinin NK1 receptor. Mol Pharmacol 53:166–175.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Huang R-RC,
    2. Vicario PP,
    3. Strader CD,
    4. Fong TM
    (1995) Identification of residues involved in ligand binding to the neurokinin 2 receptor. Biochemistry 34:10048–10055.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Huang R-RC,
    2. Yu H,
    3. Strader CD,
    4. Fong TM
    (1994) Interaction of substance P with the II and VII transmembrane domains of the NK-1 receptor. Biochemistry 33:3007–3013.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Maggi CA
    (1995) The mammalian tachykinin receptors. Gen Pharmacol 26:911–944.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Maggi CA,
    2. Giuliani S,
    3. Ballati L,
    4. Lecci A,
    5. Manzini S,
    6. Patacchini R,
    7. Renzetti AR,
    8. Rovero P,
    9. Quartara L,
    10. Giachetti A
    (1991) In vivo evidence for tachykininergic transmission using a new NK-2 receptor selective antagonist, MEN 10376. J Pharmacol Exp Ther 257:1172–1178.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Maggi CA,
    2. Patacchini R,
    3. Rovero P,
    4. Giachetti A
    (1993) Tachykinin receptors and receptor antagonists. J Auton Pharmacol 13:23–93.
    OpenUrlPubMed
  17. ↵
    1. Maggio JE
    (1988) Tachykinins. Annu Rev Neurosci 11:13–28.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Munson PJ,
    2. Rodbard D
    (1980) A versatile computerised approach for the characterisation of ligand binding systems. Anal Biochem 107:220–239.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Nakamaye KL,
    2. Eckstein F
    (1986) Inhibition of restriction endonuclease Ncil cleavage by phosphorothioate groups and its application to oligonucleotidedirected mutagenesis. Nucl Acids Res 14:9679–9698.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Otsuka M,
    2. Yoshioka K
    (1993) Neurotransmitter functions of mammalian tachykinins. Physiol Rev 73:229–308.
    OpenUrlFREE Full Text
  21. ↵
    1. Renzetti AR,
    2. Catalioto R-M,
    3. Criscuoli M,
    4. Cucci P,
    5. Lippi A,
    6. Guelfi M,
    7. Quartara L,
    8. Maggi CA
    (1998) Characterization of [3H]MEN 11420, a novel glycosylated peptide antagonist radioligand of the tachykinin NK2 receptor. Biochem Biophys Res Commun 248:78–82.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Rotondaro L,
    2. Mazzanti L,
    3. Mele A,
    4. Rovera G
    (1997) High-level expression of cDNA for granulocyte colony-stimulating factor in Chinese hamster ovary cells: Effect of 3′-noncoding sequences. Mol Biotechnol 7:231–240.
    OpenUrlPubMed
  23. ↵
    1. Schwartz TW,
    2. Gether U,
    3. Schambye HT,
    4. Hjorth SA
    (1995) Molecular mechanisms of action of nonpeptide ligands for peptide receptors. Curr Pharmacol Des 1:325–342.
  24. ↵
    1. Taylor JW,
    2. Ott J,
    3. Eckstein F
    (1985) The rapid generation of oligonucleotide directed mutations at high frequency using phosphorothioate-modified DNA. Nucl Acids Res 13:8764–8785.
    OpenUrl
  25. ↵
    1. Urlaub G,
    2. Chasin LA
    (1980) Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc Natl Acad Sci USA 77:4216–4220.
    OpenUrlAbstract/FREE Full Text
View Abstract
Back to top

In this issue

Download PDF
Article Alerts
Email Article
Citation Tools

Research ArticleArticle

Relevance of Aromatic Residues in Transmembrane Segments V to VII for Binding of Peptide and Nonpeptide Antagonists to the Human Tachykinin NK2 Receptor

A. R. Renzetti, R.-M. Catalioto, M. Criscuoli, P. Cucchi, C. Ferrer, A. Giolitti, M. Guelfi, L. Rotondaro, F. J. Warner and C. A. Maggi
Journal of Pharmacology and Experimental Therapeutics August 1, 1999, 290 (2) 487-495;
Permalink:
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Jump to section