Inositol Phosphate Turnover.

COS-7 cells were seeded in 12-well culture dishes 1 day after transfection at a density of 250,000 cells/well and supplemented with 10 μCi [³H]myoinositol/ml (Amersham Pharmacia Biotech). Two days after transfection, cells were washed twice with PI buffer (20 mM HEPES, pH 7.4, with 140 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 1 mM CaCl₂, 10 mM glucose, and 0.05% (w/v) bovine serum albumin) and were incubated in 0.5 ml of PI buffer supplemented with 10 mM LiCl at 37°C for 30 min. After stimulation with increasing concentration of Substance P for 45 min at 37°C, cells were extracted with 10% ice-cold perchloric acid followed by incubation on ice for 30 min. The resulting supernatant was neutralized with KOH in HEPES buffer, and the generated [³H]inositol phosphates were purified on Bio-Rad AG 1-X8 anion exchange resin (Berridge et al., 1983).

Binding Experiments.

Monoiodinated¹²⁵I-Bolton Hunter (BH)-labeled Substance P was prepared and purified by high performance liquid chromatography (Gether et al., 1993). Transfected COS-7 cells were transferred to culture plates 1 day after transfection. The number of cells seeded per well was determined by the expression efficiency of the individual clones aiming at 5 to 10% binding of the added radioligand. Two days after transfection, cells were assayed by competition binding for 3 h at 4°C using 15 pM ¹²⁵I-BH-Substance P plus variable amounts of unlabeled ligand in 0.5 ml of a 50 mM Tris-HCl buffer, pH 7.4, supplemented with 150 mM NaCl, 5 mM MnCl₂, 0.1% (w/v) bovine serum albumin, 40 μg/ml bacitracin.

Radioligand Binding Analysis.

The starting point for the metal-ion site engineering in the NK₁ receptor was the agonistic site recently constructed between position III:08 and VII:06 in the β₂-adrenergic receptor (Elling et al., 1999). In contrast to the β₂-adrenergic receptor, where AspIII:08 (Asp¹¹³) is highly important for catecholamine binding, the corresponding ProIII:08 (Pro¹¹²) in the NK₁receptor is not involved in peptide agonist binding. Thus, it has previously been demonstrated that Pro¹¹² can be substituted with either Asp or His without affecting Substance P binding or its stimulation of inositol phosphate turnover (Holst et al., 1998). Here, a Cys residue was introduced in position VII:06 in the NK₁ receptor, [M291C]-hNK₁, and whole cell binding experiments performed on transiently transfected COS-7 cells revealed that the affinity for Zn²⁺ was increased significantly (K _i = 59 ± 11) compared with wild-type (240 ± 60 μM) (P < .05) (Table1.). In fact, the affinity for Zn²⁺ was also increased in the [P112D]-hNK₁ construct (K _i = 65 ± 1) (P < .05) (Fig. 2A). A further increase in Zn²⁺ affinity was observed in the double mutants [P112D;M291C]-hNK₁ (Fig. 2A) and [P112H;M291C]-hNK₁ where Zn²⁺ inhibited¹²⁵I-BH-Substance P binding withK _i values of 33 ± 12 μM and 41 ± 8 μM, respectively. This corresponds to an increase in apparent Zn²⁺ affinity of 7 fold compared with the wild-type NK₁ receptor. The metal-ion affinity in the NK₁ double mutants corresponds to the affinity obtained for Zn²⁺ in a number of antagonistic bis-His sites previously constructed between different helices in the NK₁ receptor (Elling and Schwartz, 1996), and the affinity corresponds to the potency for Zn²⁺—91 and 38 μM—determined in the corresponding two metal-ion sites in the β₂-adrenergic receptor (Elling et al., 1999). Not only the free zinc ion but also Zn^{2 +} and Cu²⁺ in complex with the strong metal-ion chelator phenanthroline bound to the [P112D;M291C]-hNK₁ and the [P112H;M291C]-hNK₁ constructs with a rather similar affinity as free Zn²⁺ as demonstrated in the ability of the complexes to compete for¹²⁵I-BH-Substance P binding (Fig. 2) (Table1).1

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Figure 2

Competitions binding experiments. Panel A, affinity for ZnCl₂ in wild-type NK₁ (▪) and after introduction of aspartic acid in TM III:08 [P112D]-NK₁(♦), cysteine in TMVII:06 [M291C]-NK₁ (▴) and the two mutation in combination [P112D;M291C]-NK₁ (●). Panel B, binding of Zn²⁺(phenanthroline)₃ (closed symbol) and Cu²⁺(phenanthroline)₃ (open symbol) in the wild-type NK₁ (▪) receptor and in the metal ion binding site-engineered mutant [P112D;M291C]-NK₁ (●). Whole cell binding experiments were performed in transiently transfected COS-7 cells, using ¹²⁵I -BH-labeled Substance P as a radioligand. Data are mean ± S.E. from three or more separate experiments carried out in duplicate.

Signal Transduction.

None of the single substitutions at either position III:08 or VII:06 affected the EC₅₀ for Substance P in stimulating inositol phosphate turnover in transiently transfected COS-7 cells significantly (P > .05) (Table 2) (Holst et al., 1998). Compared with the β₂-adrenergic receptor, where agonist binding and action was totally eliminated even by the single substitution in position VII:06, the EC₅₀ for Substance P was only increased 6- to 7-fold in the two double mutants in the NK₁ receptor (Table 2). Zn²⁺ did not by itself affect inositol phosphate turnover in COS-7 cells transfected with the wild-type NK₁ receptor (Fig.3). However, in cells transfected with the [P112D;M291C] or the [P112H;M291C] mutant forms of the NK₁ receptor Zn²⁺stimulated inositol phosphate turnover dose dependently and with EC₅₀ values of 83 ±17 and 84 ±19 μM, respectively (Fig. 3).

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Figure 3

Inositol phosphate turnover in response to ZnCl₂ in the wild-type NK₁ receptor (▪), the [P112D;M291C]-NK₁ (●), and the [P112H, M291C]-NK₁ (▴), expressed in COS-7 cells. The ZnCl₂-induced stimulation is expressed as percentage of the maximal Substance P response in the respective constructs. Data are mean ± S.E. from eight (wild-type) or six (mutants) separate experiments carried out in duplicate. For both mutants, the agonist effect of ZnCl₂ concentrations above 10⁻⁵ M were significant (P < .05, two-tailed pairedt test) and for the [P112D,M291C]-NK₁construct the ZnCl₂ stimulation at concentrations above 10⁻⁴ M was highly significant (P < .001).

In the β₂-adrenergic receptor, the construction of the metal-ion site between positions III:08 and VII:06 was deliberately performed as an exchange of crucial parts of the agonist binding site, and accordingly the mutations eliminated both catecholamine binding and action. In contrast, in the NK₁ receptor the agonist, Substance P, was still able to activate the metal-ion site-engineered receptor mutants, which allowed for comparison of efficacies and for studies where the two agonists, Substance P and Zn²⁺, are administered together. As shown in Fig. 4A, Zn²⁺ acted as a 25% partial agonist in the [P112D;M291C] mutant form of the NK₁ receptor compared with Substance P but only as a 5 to 10% agonist in the [P112H;M291C] construct. However, in absolute numbers the measured phosphate inositol turnover induced by Zn²⁺ was rather similar (Table 2). In agreement with basic pharmacological theory the partial agonist, Zn²⁺, also behaved as an antagonist by dose dependently bringing the inositol phosphate turnover down to its own maximal stimulatory level in competition for receptor occupancy against the full agonist, Substance P (Fig. 4A) (Kenakin, 1993). The IC₅₀ for Zn²⁺ inhibition of Substance P induced signaling through the [P112D;M291C]-hNK₁ receptor was 26 ± 5 μM, which corresponds relatively closely to the affinity measured in the binding assay, K _i = 33 ± 12 μM. However, it is interesting that no inhibition of Substance P-induced signaling by Zn²⁺ was observed with the testable concentrations in the two single mutants [P112D] and [M291C], which both showed relative high affinity for Zn²⁺ as determined in competition binding assays (Tables 1 and 2).

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Figure 4

Inositol phosphate turnover in response to ZnCl₂ (panel A), Zn(phenanthroline)₃ (panel B), or Cu(phenanthroline)₃ (panel C) on the wild-type NK₁ receptor (▪) and the [P112D,M291C]-NK₁(●) construct in COS-7 cells. Curves with open symbols represent inhibition experiments where the compounds are administered together with a submaximal dose of Substance P (30 nM). The dotted line in panel A marks the 25% stimulation induced by zinc ions in the metal binding site-engineered receptor. Data are mean ± S.E. in duplicate from three or more experiments.

The metal-ion chelator complexes, Zn²⁺- and Cu²⁺-phenanthroline, which competed for Substance P binding with similar affinity as the free zinc-ion (Fig. 2), were in contrast to Zn²⁺ not able to activate the metal-ion site-engineered NK₁ receptor (Fig. 4, B and C). This lack of agonist activity of the chelator complexes is surprising because the chelators in fact stimulated signaling more efficaciously in the corresponding sites in the β₂-adrenergic receptor than the free metal-ions did. In accordance with the fact that they did bind to the mutated NK₁ receptor with high affinity (Fig. 2B), these metal-ion complexes acted as antagonists of the Substance P-induced inositol phosphate turnover in the [P112D;M291C]-hNK₁ receptor (Fig. 4, B and C) as well as in the [P112H;M291C] construct (Table 2). However, where chelation of Zn²⁺ with phenanthrolineincreased the affinity approximately 10-fold in the corresponding metal-ion site-engineered β₂-adrenergic receptor, it had the opposite effect in the metal-ion site-engineered NK₁receptor, where the IC₅₀ value for Zn²⁺-phenanthroline inhibition of Substance P signaling was almost 10-fold lower than the IC₅₀ value for free Zn²⁺(Fig. 4, Table 1). Moreover, no inhibition of Substance P-induced signaling by metal-ion chelator complexes with the testable concentrations in the two single mutants [P112D] and [M291C], which both showed relative high affinity for these complexes as determined in competition binding assays (Tables 1 and 2).

No “Common Lock for All Keys” in 7TM Receptors.

It has generally been believed that there should exist a common active site or “lock” that all agonist “keys” should fit into or touch to activate 7TM receptors. The relatively well characterized and rather small catecholamine binding site was originally thought to represent this common lock (Hibert et al., 1993). However, based on the significantly different maps of binding sites, which subsequently were generated for peptide and monoamine agonists, respectively, it was argued that there may not be a common lock for all agonist keys in 7TM receptors (Schwartz and Rosenkilde, 1996; Holst et al., 1998). Such a common lock was not even needed in the various allosteric models for 7TM receptor function. It has, however, been difficult to prove this point, although mapping of different binding sites in different receptors by site-directed mutagenesis has supported this notion (Schwartz et al., 1995). For example, in the angiotensin AT₁ receptor, which can be stimulated both by the endogenous peptide agonist angiotensin and by nonpeptide, biphenyl-imidazole agonists, mutational analysis has generated substantially different maps of supposed molecular interactions for these two types of agonists (Hjorth et al., 1994; Perlman et al., 1995,1997). But, unfortunately no definitively clear picture of the binding site for the nonpeptide agonists was obtained. Importantly, in the zinc site-engineered NK₁ receptor of this study, Zn²⁺ most certainly activates the receptor through binding at the introduced zinc switch located deep between TM-III and -VII. In contrast, mutational mapping—including steric hindrance mutagenesis—as well as cross-linking experiments indicates that Substance P activates this receptor through binding to extracellular epitopes as well as the most exterior parts of TM-III, -VI, and VII (Fong et al., 1992a,b; Strader et al., 1994; Boyd et al., 1996; Kage et al., 1996). Thus, the present study in which two chemically different agents, a metal-ion and a peptide, activates a common receptor through interactions at distinct binding sites is a strong argument in favor of the notion that there is no common lock for all agonist keys in 7TM receptors (Schwartz and Rosenkilde, 1996).

Interestingly, the most “deeply” located interaction points for Substance P, residues III:04 (His¹⁰⁸) and VII:02 (Tyr²⁸⁷), are located exactly one helical turn “above” the two positions—residues III:08 and VII:06, respectively—at which the activating zinc site was introduced (Figs. 1and 5). Thus, although the two agonist “keys” fit into two different locks in the zinc site-engineered NK₁ receptor, the location of supposedly crucial residues of each of these locks above each other at the interface between TM-III and -VII indicates that, although the locks are clearly different, a common mechanism of activation of 7TM receptors does exist (Fig. 5).

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Figure 5

Low resolution model of the NK₁receptor as viewed from outside the cell and a helical wheel diagram of the most extracellularly located 18 residues of each helix. The as yet identified supposed interaction points for the endogenous agonist, Substance P, are indicated in white on green (Fong.et al., 1992b;Strader et al., 1994; Boyd et al., 1996; Kage et al., 1996). ProIII:08 (Pro¹¹²) and MetVII:06 (Met²⁹¹), which in the present study are substituted by either Asp or His or by Cys, respectively, making an activating metal-ion site, are indicated in white on red. The helical wheel diagram has been constructed in accordance with the general model of Baldwin and coworkers (Baldwin, 1993), and the helices are arranged counter-clockwise as viewed from the outside-in according to previous studies using interhelical, inhibitory bis-His sites (Elling et al., 1996).

Full versus Partial Agonism.

Electron paramagnetic resonance studies of site-directed spin-labeled rhodopsin have shown that during receptor activation the most conspicuous conformational change appears to occur between TM-III and -VI (Farrens et al., 1996). Several other studies using various methodological approaches support this notion (Sheikh et al., 1996; Gether et al., 1997). Both electron paramagnetic resonance studies of rhodopsin (Sieving, 1995) as well as the substituted cysteine accessibility method (Engelman et al., 1980) and general fluorescence spectroscopy (Gether et al., 1995) combined with, for example, constitutively active mutants have previously been used to address the issue of partial versus full agonism. A major advantage in using a metal-ion as an agonist is that its binding mode is very well defined as opposed to the larger and chemically much more complicated monoamine or peptide agonists. In our case, it is clear that Zn²⁺ must activate the receptor through binding to residues III:08 and VII:06. However, Zn²⁺ acts as a 25% partial agonist in the NK₁ receptor (Fig. 4), which probably is also the case in the mutated β₂-receptor (Elling et al., 1999). In view of the biophysical observation of Farrens and coworkers (1996) that receptor activation is associated with mainly a movement of TM-III and -VI relative to each other, it is very interesting that the full agonist, Substance P, in addition to its interaction points in TM-III and -VII, also has apparent interactions with the extracellular end of TM-VI, i.e., residue VI:20 (Phe²⁶⁸) (Holst et al., 1998). The full agonists for the β-receptor, isoproterenol has also been shown to interact with residue VI:20 (Asn²⁹³) in addition to interactions with the earlier recognized more deeply located residueS VI:16 (Phe²⁹⁰) and VI:17 (Phe²⁹¹) (Schwartz, 1994; Wieland et al., 1996) Thus, we would suggest that the reason Zn²⁺ is only a relatively weak partial agonist is that it is only able to stabilize the right conformation between TM-III and TM-VII and that the metal-ion lacks key interactions in TM-VI, which are required for stabilizing a conformation associated with full agonism.

It should be noted that this argument does not address the basic question whether partial agonism is obtained through stabilization of a conformation that is partially active in signaling or through stabilization of only part of the receptors in a fully signaling conformation at a given time. The metal-ion stabilized complex of TM-III and -VII could, for example, simply constitute a good firm scaffold for the final “docking” of TM-VI in a putative, signaling conformation. Importantly, this is still merely speculation, which nevertheless possibly could be experimentally addressed through further attempts in engineering of activating metal-ion sites in this region of the receptors.

Comparison of the Metal-Ion Switch in Two Different Receptors.

Although similar metal-ion affinities were found in the zinc site-engineered β₂ and NK₁ receptors, some interesting differences were observed between the two metal-ion sites. First, in the β₂-receptor, Zn²⁺ was more efficacious in the construct where His was placed at position III:08 than with the natural Asp at this location (Elling et al., 1999). In contrast, in the NK₁ receptor, Zn²⁺ preferred an Asp at position III:08 (Fig.3). Second, in the β₂-adrenergic receptor, the additional binding of small aromatic metal-ion chelators, phenantroline or bipyridine, through a bridging zinc- or copper-ion resulted in both a higher affinity and higher efficacy for the metal-ions (Elling et al., 1999). In contrast, in the NK₁receptor the metal-ion chelator decreased the zinc potency, and the complex was devoid of agonistic properties (Fig. 4). Thus, in the β₂-receptor, the aromatic metal-ion chelator must make some favorable interactions that are not possible in the NK₁ receptor. Several residues could be involved. For example, TrpIII:04 of the β-receptor—which is a His in NK₁—could make an aromatic-aromatic interaction with the chelator. The presence of this potentially metal-ion binding His residue at position III:04 in the NK₁receptor may also be the reason why Zn²⁺ and the metal-ion chelator complexes do bind with increased affinity in the two single mutants where Asp or Cys residues are introduced individually at the closely located III:08 and VII:06 positions, respectively (Figs. 1and 2A and Table 1). Moreover, in the NK₁receptor, PheVI:16 could be imagined to sterically interfere with the binding of the chelator, which in the β-receptor could be favored by the smaller Asn residue. Conceivably, through additional substitutions in the NK₁ receptor along these lines, it should be possible to make the metal-ion chelator complexes bind better and act as agonists also in this receptor.

Implications for Potential Use of Metal-Ion Site-Engineered Receptors in Transgenic Animals.

In this study anactivating metal-ion switch was built into the NK₁ receptor and previouslyinactivating switches have been constructed in various other receptors (Thirstrup et al., 1996; Rosenkilde et al., 1999; Sheikh et al., 1999; Lu Zhi-Liang and Hulme, 2000). In most of these receptors the metal-ion sites function as real silent switches because the endogenous peptide ligand can bind and activate the receptor normally. Receptors with such silent switches could become useful in transgenic animal models aiming at pharmacologically conditioned “knock-out” experiments. Due to the normal binding of the endogenous ligand, problems such as embryologic lethality and up-regulation of compensatory mechanisms could possibly be surmounted, because the introduced receptor would be perceived by the animal as being normal. However, because of general toxicity, free metal-ions cannot be used in vivo to turn on and off the metal-ion switches in the mutated 7TM receptors. For this purpose, the metal-ion chelator complexes are very interesting because the chelators generally render the metal-ions relatively atoxic. As shown in this present study, although the metal-ion sites can be moved relatively freely between rhodopsin-like 7TM receptors, the surrounding residues in the receptor may interfere with the binding and pharmacological properties of the metal-ion chelator complexes. Thus, in vitro optimization of metal-ion sites and chelator complexes needs to be performed in the individual receptor to obtain the desired pharmacological phenotype.