Localization of Agonist and Antagonist Binding Domains of the Human Neurokinin- 1 Receptor*

To identify the molecular determinants of ligand- receptor interactions, the extracellular domain of the human neurokinin- 1 receptor was systematically substituted with the corresponding sequences from the other two neurokinin receptor subtypes. Three residues within the first extracellular segment and 2 resi- dues of the second segment are required for the optimal binding of all three natural peptide agonists. The di- vergent nature of 4 of the 5 residues supports the hypothesis that the peptide binding site on the neuro-kinin-1 receptor is not highly conserved in the other two receptor subtypes. In contrast, substitution of part of the third extracellular segment and the fourth extracellular segment with the corresponding amino acids of the human neurokinin-3 receptor results in an increase in neurokinin B affinity without affecting substance P binding, suggesting that the two peptides do not interact with the same set of functional groups on the receptor. Among the four extracellular regions, only parts of the third and fourth segments affect the binding of the quinuclidine antagonist L-703,606, and these two regions may partially account for the neurokinin- 1 receptor subtype specificity of this non-pep- tide antagonist. These studies demonstrate that both the extracellular and transmembrane domains of the neurokinin-1 receptor are involved in the binding of substance P and related peptides. The peptide neurotransmitters (SP),’ neurokinin and neurokinin B (NKB) are characterized the common C-terminal

To identify the molecular determinants of ligandreceptor interactions, the extracellular domain of the human neurokinin-1 receptor was systematically substituted with the corresponding sequences from the other two neurokinin receptor subtypes. Three residues within the first extracellular segment and 2 residues of the second segment are required for the optimal binding of all three natural peptide agonists. The divergent nature of 4 of the 5 residues supports the hypothesis that the peptide binding site on the neurokinin-1 receptor is not highly conserved in the other two receptor subtypes. In contrast, substitution of part of the third extracellular segment and the fourth extracellular segment with the corresponding amino acids of the human neurokinin-3 receptor results in an increase in neurokinin B affinity without affecting substance P binding, suggesting that the two peptides do not interact with the same set of functional groups on the receptor. Among the four extracellular regions, only parts of the third and fourth segments affect the binding of the quinuclidine antagonist L-703,606, and these two regions may partially account for the neurokinin-1 receptor subtype specificity of this non-peptide antagonist. These studies demonstrate that both the extracellular and transmembrane domains of the neurokinin-1 receptor are involved in the binding of substance P and related peptides.
The peptide neurotransmitters substance P (SP),' neurokinin A (NKA), and neurokinin B (NKB) are characterized by the common C-terminal sequence FXGLM-NH2. The biological actions of neurokinins are mediated by three subtypes of the neurokinin receptor (NKR). These receptors are members of the G-protein coupled receptor family that is characterized by seven putative transmembrane helices (1-5). The rank order of potency of the neurokinin agonists for the * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Previous studies have led to a message-address hypothesis for peptide-receptor interaction. This hypothesis envisions the conserved C-terminal portion of the peptides as an activation message that can be recognized by all three receptor subtypes, whereas the divergent N-terminal portion of the peptides acts as a recognition address to determine receptor subtype selectivity (6-8). Analysis of the amino acid sequences of the three NKR subtypes has revealed a high level of sequence similarity within the transmembrane domains, while the extracellular and cytoplasmic loops are more divergent (9,10). Therefore, it can be postulated that the common Cterminal half of the peptides interacts with the conserved transmembrane domains of the receptors, while the unique N-terminal half of the peptides recognizes the more divergent extracellular domains.
Studies on other G-protein coupled receptors whose endogenous agonists are small molecules have demonstrated that the binding sites for these ligands are located within the transmembrane domains of the receptors (11-13). Because the neurokinin peptides are larger than these non-peptide ligands, it seems likely that the extracellular domains of the NKlR might comprise part of the ligand binding site. We have tested this hypothesis by systematically substituting the extracellular segments of the human NKlR with the corresponding sequence from the human NK3R. If the divergent extracellular sequences of the receptors determine the rank order of potency of peptide agonists, then substitution of the extracellular sequences in the NKlR by the homologous sequences from the NK3R should cause an increase in the affinity of NKB with a concomitant decrease in SP affinity.
The present results confirm the contribution of the extracellular domains of the NKlR to peptide binding. However, the data also suggest that the two peptides do not necessarily interact with the same set of residues on the NKlR. Therefore, the peptide binding domains in the neurokinin receptors are more complicated than what the simple message-address model would predict.

EXPERIMENTAL PROCEDURES
All mutant receptors were constructed from the human NKlR by either the polymerase chain reaction method (Perkin-Elmer Cetus) or the uracil substitution method of site-directed mutagenesis (Bio-Rad). All mutated sequences and any sequence that was derived from polymerase chain reaction were confirmed by DNA sequencing (United States Biochemical Corp.). All receptors were expressed in COS cells to determine the ligand binding affinity. Some mutant receptors were also expressed in Xenopus oocytes to determine the functional activity (10, 14).
The binding affinities of various ligands for the NKlR and its mutants were determined using '251-Bolton-Hunter labeled SP (BHSP) or ['251]L-703,606, which is an iodinated analog of the NK1specific antagonist 345 (15,16), in the presence of varying concentration of unlabeled ligands. lZ5I-BHSP was used when the Kd value of SP was smaller than 10 nM, ['251]L-703,606 was used when the Kd value of S P was larger than 10 nM, and '251-Bolton-Hunter labeled eledoisin (BHE) was used for the human NK3R and the E3 mutant of the human NKlR. The final concentration of radiolabeled ligand was 0.2 nM. Intact COS cells were used in the '"I-BHSP or T -B H E binding assay, whereas plasma membranes were used in the in the high affinity state, KH is the agonist affinity for the high affinity state, and K L is the agonist affinity for the low affinity state. The IC5o value was then solved numerically from the fitted curve.

RESULTS
To probe the role of the extracellular loops of the NK receptors in agonist and antagonist binding, these regions of the human NKlR were systematically replaced with the analogous regions from the human NK3R (Fig. 1). The resulting mutant receptors were transiently expressed in COS cells, and their ability to bind peptide agonists and a non-peptide quinuclidine antagonist was assessed. Substitution of the second extracellular loop of the NKlR (E2, residues 96-108) with the analogous region of the NK3R resulted in undetectable binding of lZ5I-BHSP at 0.2 nM. In contrast, the nonpeptide antagonist ['251]L-703,606 bound to both the E2 mutant and the wild type N K l R with the same affinity (Fig. 2). The binding affinities of both SP and NKB for the E2 mutant were greatly reduced (Fig. 2). However, G-protein activation by the E2 mutant was normal. In Xenopus oocytes expressing the E2 mutant, agonists elicited an oscillating calcium-activated chloride current that is characteristic of activation of the phospholipase C-mediated phosphatidylinositol pathway (14). As would be expected from the reduced binding affinities of agonists for the E2 mutant receptor, the dose-response curves for SP and NKB were shifted to the right compared to the wild type receptor (Fig. 3B).
Substitution of the entire third extracellular domain (E3) of the NKlR with the corresponding sequence of the NK3R resulted in a mutant receptor with no detectable binding of lZ5I-BHSP or ['251]L"703,606. In addition, the E3 mutant did not bind lZ5I-BHE, which has high affinity for the NK3R. Because the separation of free and bound radioactive ligands by filtration is possible if the dissociation rate constant is smaller than 0.5 s" (equivalent to K d < 20 nM), a lower limit of IC,, > 20 nM was assigned to the E3 mutant for all ligands. However, the E3 mutant receptor was fully functional in Xenopus oocytes (Fig. 3C), indicating that the receptor was correctly processed. The EC50 values for the E3 mutant were also consistent with reduced peptide binding affinities compared to the wild type. Three smaller substitutions in the E3 region were constructed in order to analyze the contribution of this extracellular loop to ligand binding. Substitution of residues 170-174 by the homologous NK3R residues (E3a mutant) resulted in a change in the rank order of agonist potency, with S P > NKB > NKA (Table I; Fig. 2). The affinity of SP for the E3a mutant was similar to that of the wild type NKlR. However, the affinity for NKB was increased 3-f0ld, suggesting that this region of the NK3R might be important for NKB binding. A second substitution in this region, in which residues 176-183 were replaced with the NK3R sequence (E3b mutant), resulted in a reduction in the binding affinities for all three peptide agonists, while the affinity for the antagonist L-703,606 was not affected (Table  I; Fig. 2). Finally, when residues 187-195 of the NKlR were substituted with the analogous NK3R residues (E3c mutant), the binding affinities for the three peptides were not significantly affected. In contrast, the binding affinity of the antagonist L-703,606 was reduced 200-fold (Table I; Fig. 2). In the wild type NK3R, the Kd of L-703,606 was greater than 1 KM.
Substitution of the fourth extracellular loop of the NKlR (E4 mutant, residues 271-280) with the corresponding NK3R Ligand Binding Site of NKlR sequence did not significantly affect SP binding affinity (Table I). However, increases in binding affinity for the E4 mutant compared to the wild type NKlR. A parallel decrease in affinity of the NK1 selective antagonist L-703,606 was observed for this mutant receptor (Fig. 2). In addition, a point mutation in helix 7 was constructed to replace methionine 291 with the NK3R homolog phenylalanine. A %fold increase in NKB affinity was also observed for the M291F mutant (Table I).
The systematic replacement of the extracellular loops of the NKlR described above implicates the E2 loop in the binding of all three peptide agonists. Our previous analysis of the N-terminal domain (El region) also indicated that residues 21-29 are important for high affinity peptide binding (17). To elucidate the precise role of individual residues in these segments in peptide binding, point mutations were analyzed. The three non-conserved residues at positions 21, 22, and 29 in the E l region of the human NKlR can be substituted with the corresponding amino acids from the NK3R without any effect on agonist or antagonist binding ( Table I). Likewise, substitution of the conserved proline 28 and tryptophan 30 with alanine did not affect the ligand interactions. In contrast, substitution of the conserved phenylalanine 25 with alanine resulted in a dramatic decrease in affinity for all three neurokinin peptides. L-703,606 binding was not affected by this substitution. Asparagine 23 and glutamine 24 are conserved between the NKlR and NK3R, while the NK2R contains threonine and alanine, respectively. Substitution of asparagine 23 and glutamine 24 also resulted in a large reduction in the affinities of all three peptides for the NKlR. These substitutions did not affect the binding of the antagonist L-703,606. In the second extracellular loop, 6 residues are divergent between the NKlR and NK3R (Fig.  1). Substitution of the non-conserved asparagine 96 and histidine 108 with their NK3R homologs (serine and glutamine, respectively) resulted in a substantial reduction in the affinities for all three agonist peptides, although the antagonist affinity was not affected by these point mutations (Table I).

DISCUSSION
The present study was designed to identify the contribution of the extracellular domains of the NKlR to agonist and antagonist binding. None of the extracellular loop replacements described here resulted in a reversal in the relative affinities of SP and NKB. Therefore it is probable that some residues in the transmembrane domain of the receptor may confer SP specificity on the NKlR. On the other hand, the present results indicate that several residues in the N-terminal domain (N23, Q24, F25), loop E2 (N96, H108), and loop E3 (176-183) are required for the high affinity binding of all three peptides. However, these mutations do not affect the rank order of potency of the three neurokinin peptide agonists, and they are not required for the binding of the NK1selective antagonist L-703,606. The absence of a decrease in the affinity of the antagonist argues against any effect of these mutations on the overall conformation of the receptor, although local conformational effects within the loops cannot be determined a t present. The data suggest that these residues