Molecular basis for the species selectivity of the neurokinin-1 receptor antagonists CP-96,345 and RP67580.

Two non-peptide substance P antagonists exhibit opposite rank orders of potency for the human and rat neurokinin-1 receptors. CP-96,345 shows selectivity for the human receptor, whereas RP67580 shows selectivity for the rat receptor. Amino acid sequence comparison of the two receptors reveals 22 divergent residues. To elucidate the molecular basis for the species selectivity of these antagonists, divergent residues in the human neurokinin-1 receptor were substituted by the rat homologs. Analysis of mutant receptors revealed that substitution of 2 residues (V116L and I290S) in the transmembrane domain of the human neurokinin-1 receptor is both necessary and sufficient to reproduce the antagonist binding affinities of the rat receptor. The nature of these substitutions and the magnitude of the changes in binding affinity suggest that residues 116 and 290 do not interact directly with the antagonist molecules. The present results support a model in which phylogenetically conserved residues interact directly with the antagonists, while phylogenetically divergent residues affect the local helical packing of the receptor. Such a change in local structure would lead to increased binding affinity for one class of antagonists and decreased affinity for another.

Two non-peptide substance P antagonists exhibit opposite rank orders of potency for the human and rat neurokinin-1 receptors. CP-96,345 shows selectivity for the human receptor, whereas RP67580 shows selectivity for the rat receptor. Amino acid sequence comparison of the two receptors reveals 22 divergent residues. To elucidate the molecular basis for the species selectivity of these antagonists, divergent residues in the human neurokinin-1 receptor were substituted by the rat homologs. Analysis of mutant receptors revealed that substitution of 2 residues (V116L and 1290s) in the transmembrane domain of the human neurokinin-1 receptor is both necessary and sufficient to reproduce the antagonist binding affinities of the rat receptor. The nature of these substitutions and the magnitude of the changes in binding affinity suggest that residues 116 and 290 do not interact directly with the antagonist molecules. The present results support a model in which phylogenetically conserved residues interact directly with the antagonists, while phylogenetically divergent residues affect the local helical packing of the receptor. Such a change in local structure would lead to increased binding affinity for one class of antagonists and decreased affinity for another.
The neurotransmitter substance P (SP)' plays an important role in pain, neuroimmune interactions and neuromodulation (Helke et al., 1990;Iversen et al., 1987;Maggio, 1988;Nakanishi, 1991;Payan, 1989). It binds to the neurokinin-1 receptor (NKlR) to elicit various biological responses. Antagonists of the NKlR have been proposed for the treatment of diseases involving SP, and several potent and specific NKlR antagonists have recently been described (Garret et al., 1991;Snider et al., 1991). However, subsequent studies have demonstrated that the quinuclidine antagonist CP-96,345 has a higher affinity for the human NKlR than the rat NKlR, whereas the perhydroisoindole antagonist RP67580 shows the reverse species specificity (Beresford et al., 1991;Garret et al., 1991).
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Thus, it has been postulated that there may be two subtypes of NKlR within the same species (Watling, 1992). An analogous hypothesis was also proposed for the existence of two NK2 receptor subtypes based on tissue preparations from two species (Patacchini et al., 1991). Understanding the cause of the pharmacological difference observed for species variants of the NKlR will not only clarify the definition of receptor subtypes, but also provide a rational basis for the selection of animal models in drug development.
Molecular cloning and expression of the NKlR from several species have demonstrated that the NKlR belongs to the G protein-coupled receptor family (Fong et al., 1992;Hershey and Krause, 1990;Yokota et al., 1989). Sequence comparison of the human and rat NKlR reveals 22 divergent residues ( Fig. 1). It is possible that some of the phylogenetically divergent residues in the human NKlR could interact directly with CP-96,345, thus conferring upon it a higher affinity than that for the rat NKlR. To test whether these residues are directly involved in CP-96,345 and RP67580 binding, divergent residues in the human NKlR were substituted by the rat homologs. Analysis of these mutant receptors revealed that two conservative substitutions within the transmembrane domain are necessary and sufficient to reverse the species selectivity of the two antagonists. The direct involvement of phylogenetically conserved residues in antagonist binding and indirect effects of phylogenetically divergent residues are discussed.

MATERIALS AND METHODS
The cDNA clones of the human NKlR and rat NKlR were obtained as described (Fong et al., 1992;Yokota et al., 1989). Chimeric mutants between the human and rat NKlR were constructed by restriction endonuclease cleavage followed by ligation of the appropriate DNA fragments. The StuI site at residue 79 and the BglII site at residue 276 were utilized in constructing the chimeric mutants. Point mutations were constructed from the human NKlR template, and they were introduced by the uracil selection method of sitedirected mutagenesis (Bio-Rad). Double or triple mutations were constructed by recombining the appropriate single point mutations. All mutations were confirmed by DNA sequencing.
The wild type and mutant receptors were expressed in COS cells. The ['251]Bolton-Hunter labeled SP (BHSP) binding assay was carried out using intact COS cells as described (Fong et al., 1992). Briefly, 0.02 ml of 2.5 nM ["51]BHSP, 0.02 ml of unlabeled ligand at various concentration, and 0.2 ml of cell suspension were incubated for 1 h prior to GF/C filtration to determine the amount of bound [ " ' I ] BHSP. The data were fitted by the equation (cpm(L)cpm(1 pM and cpm(0) represent the amount of bound ["51]BHSP in the presence or in the absence of unlabeled ligand, respectively; L represents the concentration of unlabeled ligand, and ICs0 represents the concentration of unlabeled ligand that causes 50% inhibition of specifically bound [Iz5I]BHSP.

RESULTS
The non-peptide antagonist CP-96,345 has a higher affinity for the human NKlR (IC50 = 0.5 nM) but a lower affinity for the rat NKlR (ICbo = 35 nM). In contrast, the binding affinity of RP67580 for the rat NKlR (IC6o = 4 nM) is 5-fold higher than that for the human NKlR (IC50 = 20 nM; Fig. 2). TO identify regions of the NKlR that contribute to the species selectivity of these two antagonists, chimeric receptors were constructed between the human and rat NKlRs. Substitution of residues 1-276 or residues 80-276 of the human NKlR with the analogous region of the rat NKlR (designated as h(l-276) and h(80-276), respectively) resulted in a decreased affinity for CP-96,345 and an increased affinity for RP67580. The binding affinity of RP67580 for both mutants was identical to that of the rat NKlR. However, the binding affinity of CP-96,345 for both mutants was intermediate between those of the human NKlR and the rat NKlR (Fig. 3). These data indicate that some of the divergent residues in the region 80-276 are necessary to account for the differential antagonist binding affinities of the NKlR from these two species.

Species
When residues 1-79 and 277-407 or 277-407 alone of the human NKlR were substituted by the rat NKlR sequence (h(1-79, 277-407) and h(277-407) mutants in Fig. 3), the binding affinities of CP-96,345 for both mutants were inter- mediate between those of the human and rat NKlRs. The similarity of binding affinities of these two mutants again argues against a role for the divergent residues in the 1-79 region in determining species selectivity. The data also indicate that some residues in the 277-407 region are important for the species selectivity. The h(290) mutant in which the isoleucine-290 of the human NKlR was substituted by the rat homolog serine also exhibited properties similar to those of the h(277-407) mutant. Moreover, when residues 1-290 of the human NKlR were substituted by the rat NKlR sequence (the h(1-290) mutant), the binding affinities of both antagonists were identical to those of the rat NKlR (Fig. 3). These data rule out any contribution from the C-terminal tail of the NKlR to the species selectivity of antagonists, and indicate that residue 290 is critical in this respect. Therefore, some of the residues in the region of 80-276 as well as residue 290 are necessary and sufficient to explain the differential antagonist binding properties of the human and rat NKlRs.
Within the 80-276 region, there are 8 divergent residues between the human and rat NKlR. Combinations of point mutations a t positions 80,152,190,191,195, and 266 with the h(290) mutant yielded mutant receptors with binding affinities similar to those of the h(290) mutant (Fig. 3), ruling out any significant contribution from these 6 residues to the species selectivity of antagonist binding. In contrast, the binding affinities of both antagonists for the rat NKlR can be reproduced in the double mutant h(116, 290) in which valine-116 and isoleucine-290 of the human NKlR were substituted by the rat homologs leucine and serine (Fig. 3).

Species Selectivity of NK1 Antagonists
Therefore, residues 116 and 290 are necessary and sufficient t o account for the species selectivity of CP-96,345 and RP67580 binding to the NKlR.
However, the antagonist binding affinities of the single mutant h( 116) were very similar t o those of the wild type human receptor, indicating a cooperative effect of substitutions a t residues 116 and 290 on the binding properties of NKlR.
The endogenous agonist SP exhibited a %fold difference in binding affinity for the human and rat NKlRs. For all the mutants reported here, the binding affinities for S P fell within this 3-fold range (Fig. 4), indicating that there is no large scale structural rearrangement of the receptor due to the mutations.

DISCUSSION
The present study was designed to elucidate the molecular basis of the observed species selectivity of antagonists for the NKlR. To test whether divergent residues at different positions could lead to the observed species selectivity of the two antagonists, chimeric and point mutants of the human NKlRs were analyzed. These results demonstrate that the Nterminal sequence (1-54), the intracellular C-terminal tail (291-407), and the divergent extracellular residues (190,191,195) do not contribute to the species selectivity of antagonist binding. In the transmembrane region, 2 divergent residues at positions 80 and 152 possess chemical characteristics (see Fig. 1) that might be expected to contribute to additional binding energy for CP-96,345 in the human NKlR or for RP57680 in the rat NKlR. However, substitution of either of these 2 residues in the human NKlR does not enhance the binding affinity of RP67580 nor reduce the affinity of CP-96,345. In contrast, substitution of valine-116 and isoleucine-290 of the human NKlR by the rat homologs leucine and serine resulted in a mutant human receptor where both CP-96,345 and RP67580 exhibited binding affinities very similar t o those of the wild type rat NKlR (Fig. 3). Because the two antagonists change their affinities in opposite directions in this double mutant, the effect is specific and does not seem t o be a result of abnormal folding of the receptor. In addition, the single mutant h(116) does not significantly change the antagonist affinities, whereas the single mutant h(290) binds the antagonists with affinities that are intermediate between the rat and human NKlRs. Therefore, the 2 residues at positions 116 and 290 are both necessary and sufficient to account for the species selectivity of the two antagonists. The observation that the affinity of RP67580 for the rat NKlR is higher than that for the human receptor could be the result of a favorable interaction with residues 116 and 290 in the rat receptor or an unfavorable contact (such as repulsive force or steric hindrance) with the 2 residues in the human receptor (and uice uersa for CP-96,345). Comparison of affinity measurements on several mutants, however, argues against a direct interaction of residues 116 and 290 with the antagonists and supports an indirect effect of substituting the 2 residues on antagonist binding for the following reasons. First, substitution of residue 116 alone does not significantly affect the antagonist binding affinities. Thus, either residue 116 does not interact directly with the antagonists or the direct interaction is too weak to be measurable. Second, the potential hydrogen bonding capability of serine 290 in the rat NKlR does not explain the higher affinity of RP67580 for the rat NKlR because the h(l-276), h(80-276), and h(1-290) mutants have the same affinity for RP67580 as the wild type rat NKlR. In other words, serine 290 is not absolutely required for the higher affinity binding of RP67580. This result also indicates that steric hindrance of isoleucine 290 in the human NKlR does not explain the reduced affinity of RP67580 for the human NKlR. Third, binding energy calculations suggest that van der Waals interactions involving hydrocarbon side chains (such as isoleucine) usually contribute less than 1 kcal/ mol of binding energy, which is equivalent to a less than 5fold change in binding affinity (Andrews, 1986). In the present study, substitution of isoleucine-290 in the human receptor by serine resulted in a 10-fold reduction in the affinity of CP-96,345, arguing against a direct van der Waals interaction between isoleucine 290 and CP-96,345. Fourth, an unfavorable direct contact between CP-96,345 and serine 290 can not explain the reduced affinity of CP-96,345 for the rat NKlR because the affinity of CP-96,345 for the h(290) mutant is similar to that for the h(80-276) and h(1-276) mutants which do not contain serine 290. Taken together, all the present data suggest that residues 116 and 290 do not interact directly with the two antagonists.
The results from the h(116), h(290), and h(116, 290) mutants also indicate a cooperative effect on the binding properties of the NKlR when both residues 116 and 290 are changed. Such a cooperative effect is consistent with a localized conformational change as a result of substituting the 2 residues. Similarly, both the h(116,290) mutant and the h(80-276) mutant reproduce the affinity of RP67580 for the rat NKlR, suggesting that different combinations of amino acid substitutions can lead to the same enhanced affinity for RP67580. Thus, we propose that the phylogenetically divergent residues affect the local helical packing of the receptor (Chothia et al., 1981), and some of the phylogenetically conserved residues must interact directly with the antagonists. Such a local change in helical packing can affect the 3dimensional positions of neighboring residues that are important for antagonist binding.
The discovery of non-peptide antagonists (such as CP-96,345, RP67580, and the heterosteroid antagonists reported by Appell et al. (1992)) and the observation of species differences in antagonist binding affinity for these three classes of antagonists have prompted the speculation that multiple subtypes of NKlR may exist in the same species (Watling, 1992). Based on the present data and all the cDNA and genomic cloning data, however, it seems appropriate to classify the human NKlR and rat NKlR as species variants of the same receptor subtype rather than as two different NKlR subtypes. There is precedent in other systems for two species variants of a protein that give rise to some functional differences. For instance, two species homologs of the serotonin-1B receptor share 96% sequence identity in the transmembrane domain, but they exhibit different pharmacological profiles (Hartig et czl., 1992). Substitution of one phylogenetically divergent residue in the glycine receptor affects the binding of agonists and antagonists (Kuhse et al., 1990). Examination of the xray structures of clam hemoglobin and human hemoglobin has demonstrated that structural divergence can explain the observed differences in cooperative oxygen binding (Royer et al., 1990) * In summary, the binding sites for CP-96,345 and RP67580 in the NKlR are not identical because substituting residues 80-276 in the human NKlR by the rat homologs can reproduce the binding affinity of RP67580, but not CP-96,345, for the rat NKlR. Furthermore, the present results demonstrate a role for residues 116 and 290 of the NKlR as determinants of the differential affinities of CP-96,345 and RP67580 for the NKlR from different species. This conclusion is consistent with the observation that the guinea pig NKlR, which contains the same residues in the transmembrane domain as the human NKlR, has binding affinities for both CP-96,345 and RP67580 similar to those of the human NKlR (Beresford etal., 1991;Garret etal., 1991;Gorbulev etal., 1992). Similarly, the mouse NKlR contains the same residues in the transmembrane domain as the rat NKlR except for residue 266, and both the mouse and rat NKlRs bind CP-96,345 with the same affinity (Beresford et al., 1991;Sundelin et al., 1992). All the present data are consistent with a model in which the residues at 116 and 290 influence the geometry of the antagonist binding pocket. These data further demonstrate that a minor sequence divergence among variants of the same receptor can lead to pronounced pharmacological differences.