Molecular aspects of the tachykinin receptors

Regulatory Peptides, 43 (1993) 21-35

21

© 1993 Elsevier Science Publishers B.V. All rights reserved 0167-0115/93/$06.00 REGPEP 01253

Review

Molecular aspects of the tachykinin receptors N o r m a P. G e r a r d a'c'd'e, L u B a o b'c'e, H e X i a o - P i n g a'c'e a n d C r a i g G e r a r d b'c'd'e aDepartment of Medicine, Beth Israel Hospital, bDepartment of Pediatrics and clna Sue Perlmutter Laboratory, Children's Hospital, d Center for Blood Research and e Thorndike Laboratory, Harvard Medical School, Boston, MA (USA)

(Received 25 September 1992; accepted 26 September 1992) K e y words: Tachykinin; S u b s t a n c e P; N e u r o p e p t i d e receptor; N e u r o k i n i n A; N e u r o m e d i n K; Seven

t r a n s m e m b r a n e segment receptor

Contents I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

21

II. Structural characterization of the ta~hykinin receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. cDNA and deduced protein structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Gene structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

23 23 27

III. Bioactivity of the cloned tachykinin receptors expressed in transfected cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

30

IV. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

33

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. Introduction The tachykinin receptors, identified here as N K - 1 , N K - 2 a n d N K - 3 receptors, constitute a small group of seven t r a n s m e m b r a n e segment receptors recog-

Correspondence to: N.P. Gerard, Department of Medicine, Beth Israel Hospital, 300 Longwood Avenue, Boston, MA 02215, USA. Abbreviations: PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; CHAPS, 3((3-cholamidopropyl)-dimethylammonio)-l-propanesulfonate; PAF, platelet-activating factor.

nizing peptide neurotransmitters. Structurally, the tachykinin receptors are m e m b e r s o f the m u c h larger family o f sensory receptors w h o s e p a r a d i g m is visual r h o d o p s i n [ 1-5 ]. Various other n o m e n c l a t u r e s identify these molecules as the s u b s t a n c e P r e c e p t o r ( N K - 1 receptor), the substance K, neurokinin A or neurokinin fl receptor ( N K - 2 receptor), and the neur o m e d i n K, neurokinin B or neurokinin ~ receptor ( N K - 3 receptor). Although it is not the authors' intent to cast aside long-standing tradition, a perceived need to unify the n o m e n c l a t u r e in this field has per-

22

suaded us to refer to the respective molecules as described above [6], even though the original publications may have used alternative nomenclature. As detailed below, features common to all members of this family of receptors include the presence of seven hydrophobic protein sequences, each of which is believed to span the cell membrane [4], and interaction with one or more GTP-binding proteins to promote high affinity binding with ligand and transduce intracellular signals [5,7,8]. These receptor molecules as a whole appear to have evolved to promote transcellular communication with the environment. Currently approx. 150 seven transmembrane segment receptors have been characterized, and each with its attendant ligand comprise highly selective combinations responsible for signalling in virtually every organ system in the body. The tachykinin receptors have a relatively wide tissue distribution and interaction with their ligands is associated with such diverse responses as smooth muscle contraction, sensory neurotransmission, immunological responses, nociception, inflammation, sexual behavior and potentially nerve regeneration and wound healing [9-12]. The tachykinin ligands comprise a family of low molecular weight peptide neurotransmitters which share a common C-terminal amino acid sequence, -Phe-X-Gly-Leu-Met-NH 2, which is required for biological activity (Table I) [ 13]. In mammals these peptides include substance P, neurokinin A (substance K, neurokinin ~) and neurokinin B (neuromedin K, neurokinin fl). These peptides are synthesized in nerve cells [ 14] and are re-

leased from C-fibers in the skin and viscera following stimulation. Each ligand has one or more corresponding receptors which comprise a high affinity binding interaction. Common sequence motifs among the tachykinins allows them to interact with all of the receptors, albeit with much lower affinities [ 1,2]. The NK-1 receptor is selective for substance P > neurokinin A > neurokinin B; the NK-2 receptor binds neurokinin A > substance P > neurokinin B; and the NK-3 receptor is selective for neurokinin B > neurokinin A > substance P. As a result of this cross reactivity, dissection of the ligand-receptor pair(s) primarily responsible for particular physiological responses in vivo has been somewhat difficult. This phenomonon, in part, provides impetus for cloning the receptors so that they may be studied in more defined systems. Indeed, the recent molecular cloning of the tachykinin receptor genes and cDNAs from several species has provided valuable new information relating to the structure of these molecules. This knowledge, in addition to the development of specific non-peptide antagonists, provides the tools to gain additional information about the ligand binding sites, structurefunction relationships involved in signal transduction, tissue localization, and the regulation of expression of these receptor molecules. The objective of this review is to relate the most recent advances in tachykinin receptor research, much of which is based in molecular biological findings. It is by no means intended to be an exhaustive review of the tachykinin literature, and readers are referred to other

TABLE I Amino acid sequences of the major mammalian tachykinins and their identified receptors The canonical sequence characteristic of tachykinins is indicated in capitals. Ligand

Sequence

Substance P Neurokinin A Neurokinin B

Arg-Pro-Lys-Pro-Gln-Gln-PHE-Phe-GLY-LEU-MET-NH His-Lys-Thr-Asp-Ser-PH E-VaI-GLY-LEU-MET-NH Asp-Met-His-Asp-Phe-PHE-VaI-GLY-LEU-MET-NH

Receptor 2 2 2

NK-1 NK-2 NK-3

23 sources for information about tachykinin physiology and ligand characterization [9,15,16].

II. Structural characterization of the tachykinin receptors II-A. cDNA and deduced protein structure

Studies aimed at the molecular characterization of the tachykinin receptors showed that the human IM9 B-lymphoblastoid cell line is among the richest sources for the NK-1 (substance P) receptor, with reports of 25,000 to 30,000 sites per cell [17]. When radiolabeled substance P was crosslinked to its receptor on these cells with bifunctional crosslinking agents and subjected to SDS polyacrylamide gel electrophoresis, several specifically labeled proteins were revealed, with apparent molecular masses of 116, 78, 58 and 33 kDa [18]. Similar experiments using guinea pig lung membrane preparations revealed a single predominant specifically labeled protein at 76 kDa [8], close to the 78 kDa protein from IM9 cells. Membrane preparations from rat submaxillary glands photoaffinity labeled with a substance P analog yield two proteins with apparent molecular masses of 53 and 46 kDa [ 19]. The NK-2 receptor crosslinked with [ 125I]NKA identifies a protein of 43 kDa in membrane preparations from hamster urinary bladder [20], while a mouse fibroblast cell line transfected with the bovine stomach NK-2 receptor cDNA shows a band at 70 kDa [21]. These findings, coupled with pharmacological evidence outside the scope of this review, have been interpreted by some investigators as evidence for the existence of multiple receptor subtypes. This may, indeed, be the case, as will be discussed below, however, a combination of differential glycosylation and proteolysis could also account for the differences observed. Additional studies showed that when IM9 cell membranes were treated with the detergent CHAPS it was possible to solubilize a form of the molecule which retained high affinity binding, suggesting meth-

odology potentially useful for large scale purification of the receptor protein, or fragments thereof [22], by affinity chromatography, in much the same manner as was done with the alpha adrenergic receptor [23]. An alternative approach to characterizing the tachykinin receptors was under pursuit at the same time by Masu et al. [24,25], who first isolated the c D N A for the bovine NK-2 receptor using techniques of expression cloning in Xenopus oocytes. These authors found a c D N A of 2458 nucleotides, with an open reading frame encoding 384 amino acids. The deduced protein sequence is shown in Fig. 1. Comparison of this sequence with other known sequences showed homologies of 20-25 ~o with other rhodopsin-type receptors. Hydropathy analysis was consistent with assignment to this receptor family, since it showed seven hydrophobic, presumably membranespanning sequences. Pharmacologic evidence further supported assignment to this receptor family since it was known that tachykinin signalling was mediated by activation of GTP-binding proteins [7,8,19,2628]. Although it was not necessarily predictable at the time, the sequence homologies among species as well as among receptor types are high enough that the initial cloning of the bovine NK2 receptor c D N A made possible the subsequent cloning of all of the tachykinin receptors which have been reported since that time. We tackled the cloning of the human NK-2 receptor using an approach based on the polymerase chain reaction (PCR) [29]. We examined the deduced protein sequence of the bovine molecule and generated a partial c D N A probe for the human NK-2 receptor using oligodeoxynucleotide primers based on regions of the molecule which we reasoned should be conserved across species because of their content of cysteine and tryptophan. Sense and antisense primers corresponding to nucleotides 91-108 and 538-555, respectively, were used with human tracheal c D N A as the template and generated a 465 bp fragment of the human NK-2 receptor cDNA. We used this c D N A to probe a human genomic DNA library and isolated the human NK-2 receptor gene,

24

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25 the structure of which is described below. Sequence information generated from the 5' and 3' ends of the coding sequence, then allowed us to synthesize fulllength human c D N A by P C R from human stomach and lung [29]. Kris et al. [30] used a similar approach to clone the human receptor from a jejunal c D N A library. The deduced amino acid sequence is identical to our reported sequence with the single exception that they find a histidine at position 375, where we find an arginine. The significance of this is not yet clear, it is likely an allelic variation in a region of the molecule which has no effect on function. Hybridization of the bovine NK-2 receptor c D N A with a rat stomach c D N A library under reduced stringency yielded the c D N A from the third species [ 31 ]. The mouse N K - 2 receptor was subsequently cloned using a similar approach [32]. The deduced amino acid sequences for all four species of NK-2 receptor are shown in Fig. 1, aligned for maximal sequence identity. The overall homology among species is greater than 85~o, with the greatest differences observed at the N- and C-termini. The rat and mouse sequences are most closely related with only 19 amino acid replacements. The human and bovine molecules have two N-linked glycosylation sites in their putative extracellular N-terminal regions, whereas the rat and mouse have only one. The predicted human molecule is 14 amino acids longer than the rat and mouse receptors and 9 amino acids longer than the bovine. The sequence extension is located at the C-terminus, and the biological significance of length heterogeniety in this position is presently unknown. The NK-1 (substance P) receptor was independently cloned as c D N A s from rat brain [33] and small intestine [34], in both instances by low strin-

gency hybridization with rat N K - 2 receptor probes. We cloned the human NK-1 receptor gene and c D N A from human lung and IM9 lymphoblasts [ 35 ], again using P C R to generate partial length c D N A probes from conserved regions based on the rat sequence. Takeda et al. [36], also cloned the human NK-1 receptor c D N A from the IM9 cell line and Hopkins and coworkers isolated the same molecule from human lung [37], using almost identical approaches. The guinea pig NK-1 receptor c D N A and the mouse NK-1 receptor gene have also been reported, and the deduced amino acid sequences of all four species are shown in Fig. 2. The sequence identities are even higher among species for the NK-1 receptor than they are for the NK-2 receptor. Among the species known, less than 10~o of the amino acids are different, with 23 amino acid substitutions between the human and rat sequences and only 9 between rat and mouse. As with the NK-2 receptor, two N-linked glycosylation sites are evident in the extracellular N-terminal sequence. This molecule also has an Asn-Phe-Thr sequence at position 91, in the beginning of the first extracellular loop, between M2 and M3. It is not yet known whether this site is glycosylated. The rat and mouse NK1 receptors have a fourth potential glycosylation site at position 190 in the second extracellular loop between M4 and M5. This site does not appear in the human or guinea pig NK1 receptor sequences and its absence may explain some of the pharmacologic differences among species. By analogy, the human platelet-activating factor (PAF) receptor, which has no N-linked glycosylation site in its amino-terminus, has an Asn-Val-Thr sequence in the second extracellular loop where the rat and mouse NK-1 receptors have Asn-Arg-Thr sequences [38,39]. Other than the possibility for some subtle pharmacological differences, the signif-

Fig. 1. Comparison of the deduced amino acid sequences of the NK-2 (neurokinin A) receptors from human, bovine, rat and mouse. Differences between human and other species are indicated. Putative membrane spanning sequences are indicated by the horizontal lines above the sequences. The human moleculeis 14 amino acids longer than the bovine or mouse and and 9 longer than the rat sequence. Potential sites for N-linked glycosylationare underlined.

26 M1 25

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27

icance of these glycosylation sites is not yet known. This subject has been addressed for the beta adrenergic receptor as described by Rands et al. [40]. The c D N A for the third tachykinin receptor, the NK-3 (neuromedin K) receptor was cloned from rat and human brain [41,42], and the human NK-3 receptor gene was isolated [43,44]. These molecules also have a high degree of sequence identity (Fig. 3), with only 12~o of the amino acids differing between the two. Both are considerably longer than the NK- 1 and N K2 receptors; alignment of the transmembrane sequences indicates extensions of 40-50 amino acids at the amino terminus for both species. The human NK-3 receptor is 13 amino acid residues longer than the rat molecule and contains three N-linked glycosylation sites where the rat has four, all in the amino terminal sequence. Comparison of the three human receptors indicates somewhat closer relationship between NK-1 and NK-3, with 51~o sequence identity, compared with 41 ~o and 47 ~o identity between NK-2 and NK-3 or between NK-1 and NK-2, respectively. The membrane-spanning segments average 70~o identity for all three receptors and the cytoplasmic sequences 75 ~o, with the greatest identity in the first cytoplasmic loop. The significance of the latter observation is questionable since this loop is small and has not been implicated in signal transduction for other members of the G-protein coupled receptor family. In contrast, changes in the other cytoplasmic segments have been associated with alterations in function for other seven transmembrane segment receptors. By analogy with rhodopsin, these molecules are thought to be arranged in the cell membrane as diagramed in Fig. 4. In three-dimensional space, transmembrane sequence 1 is thought to be juxtaposed next to transmembrane sequence 7, so that the molecule forms a cylindrical pore or horseshoe, and, by

analogy with the adrenergic receptor [45], binds ligand deeply inside the pore. One might assume that the amino acid replacements among species have no significant effect on ligand binding since the reported binding constants for these species are all quite similar. II-B. Gene structure

When we initially embarked on the cloning of the human NK-2 receptor gene, we expected it to be intronless, like most other G protein receptor genes [46]. Indeed, our choice in screening a genomic library instead of a cDNA library was based on this assuption and on the knowledge of the rarity of tachykinin receptor mRNA in tissues available for c D N A libraries. We reasoned that a genomic library should contain more positive clones since it would not be biased by negative transcriptional control mechanisms. We found, to our surprise, that the NK2 receptor gene is encoded in not one, but five, exons spanning some 12 kb. Its organization is shown in Fig. 5. The NK-1 (substance P) and NK-3 receptor genes have a similar structure [35,43]; the introns interrupt the coding sequence in analogous positions, making the exons the same size, but the introns for these genes are considerably larger. The NK-1 receptor gene spans ~ 60 kb of DNA and the NK-3 receptor ~45 kb. Chromosome localization for these genes shows the NK-1 receptor is on chromosome 2 [35]; the NK-2 receptor is on the q23-pter region of chromosome 10 [29], and to our knowledge the NK-3 receptor gene has not yet been mapped. In contrast, the ligands are encoded as preprotachykinins on two genes, each containing multiple exons [47-49]. Alternative splicing of the preprotachykinin A gene yields three distinct mRNAs encoding different pre-

Fig. 2. Comparison of the deduced amino acid sequences of the NK-1 (substance P) receptors from human, rat, mouse and guinea pig (GP). Differences between h u m a n and other species are indicated. Putative membrane spanning sequences are indicated by the horizontal lines above the sequences. Consensus sequences for N-linked glycosylation are underlined.

28 25 Human NK-3R Rat NK-3R

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Fig. 3. Comparison of the deduced amino acid sequences for the h u m a n and rat NK-3 receptors. Differences between h u m a n and other species are indicated. Putative membrane-spanning sequences are indicated by the horizontal lines above the sequences. Potential sites for N-linked glycosylation are underlined.

29

humaNnK-r Ieceptor

~

~~% ~ ~ ~ ~

membrane inside

Fig. 4. Schematicmodel of the putative structure of the tachykinin receptors, as illustrated by the human NK-1 receptor. Circles indicate individual amino acids, horizonal lines indicate the membrane bilayer, with membrane spanning sequences between. The N-terminal sequence and three extracellular loops lie above the cell membrane, while the C-terminus and three cytoplasmicloops are within the cell. The filled circles indicate positions of non-identity between the human and rat sequences.

cursors, two of which include both substance P and neurokinin A. Primer extension experiments using R N A from IM9 cells, brain, and submandibular gland for the human NK-1 receptor show identical size 5' untranslated regions of 588 bp for this gene [35,43]. The human NK-2 receptor gene has a transcriptional initiation site 282 bp from the translational initiation site based on primer extension using R N A from human stomach [29]. The 5' untranslated region for the bovine NK-2 receptor is somewhat longer; the c D N A clone isolated had 447 bp of sequence before the translational initiation site [24]. Primer extension for the human NK-3 receptor shows 305 bp for this

5' untranslated region using R N A from brain [43]. All three genes have canonical T A T A A sequences 25-30 bp 5' to the identified transcriptional initiation sites, consistent with T F I I d binding sites. A number of D N A regulatory elements are also identified in the NK-1 receptor gene, including a c A M P response element, and sites for AP-1, NF-KB, AP-2, AP-4, OCT-2 and SP-1 [35,43]. The NK-2 receptor contains a possible modified c A M P response element and G C box [29], and the NK-3 receptor gene has sites for AP-1, AP-2, Sp-1 and a c A M P response element. Ihara and Nakanishi showed that expression of the rat NK-1 receptor was inhibited by dexametha-

30

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Fig. 5. Gene structure of the human NK-1 and NK-2 receptors indicating the size and relative positions of exons 1-5 by the heavy lines. Restriction sites for Eco RI (E) and Pst I (P) are indicated for the NK-2 receptor gene. Some of the Hind III (H) and Pst I (P) sites are shown for the NK-1 receptor gene. The NK-2 receptor is located on chromosome 10 and spans ~ 12 kb. The NK-1 receptor is located on chromosome 2 and spans ~ 60 kb; the distance between exons is not shown for this gene.

sone in pancreatic acinar AR42J cells [50]. We demonstrated similar inhibition of NK-1 receptor expression in the human system using IM9 lymphoblasts [ 35 ], consistent with the presence of a glucocorticoid response element. Treatment of these cells with dibutyryl-cAMP did not significantly alter NK-1 mRNA levels, suggesting that IM9 cells may not express the regulatory protein required. Si-nilar studies of the regulation of expression for NK-2 and NK-3 receptors are somewhat impractical because of the rarity of their mRNAs in any established cell line. We have begun to analyze the function of each of these promoters using plasmid DNA constructs containing the gene for bacterial chloramphenicol acetyl transferase (CAT). A ,~ 5 kb fragment of the NK-1 receptor 5' flanking region containing the TATAA box, ~ 400 bp of the 5' untranslated region and additional upstream sequence strongly suppresses CAT activity when transfected into human neuroblastoma SK-N-SH cells. When 3.2 kb of DNA is removed from the 5' end the promoter is functional, and if an additional ~ 900 bp is deleted, the expression is increased approx. 2-fold (X-P. He and N.P. Gerard, unpublished data). A 2.5 kb fragment of the NK-2

receptor gene containing the TATAA box is also a strong silencer of CAT expression in SK-N-SH cells, and current efforts are focused toward identifying the regulatory sequences involved. A major stumbling block in this regard is again the lack of a cloned cell line expressing NK-2 receptors. Optimum promoter analysis requires a 'permissive' transfectable host cell.

IlL Bioactivity of the cloned tachykinin receptors expressed in transfected cells The work of Buck and Burcher showed that the tachykinins have individual receptor specificity, with differences in apparent binding affinity and responsiveness varying from 50- to 1000-fold, depending on the tissue source and the ligand of interest. Because of ligand-receptor cross reactivity, discerning the relative contribution of each tachykinin was always confounded by the uncertainties of receptor distribution and abundance. Transfected cell systems offer the possibility of studying each of the individual receptors in the absence of others. This is particularly

31 useful for the human NK-2 and NK-3 receptors, which appear to be expressed only in tissues which also express the NK-1 receptor. Masu et al. [24] showed that the bovine NK-2 receptor transfected in Xenopus oocytes responds to NK-A with an EDs0 of 60 nM, and to substance P with an EDs0 of 8.7 #M, a shift of -~ 150-fold. Similarly, the human NK-1 receptor binds substance P with 0.35 nM affinity; neurokinin A and neurokinin B bind with affinities 100- and 500-fold lower, respectively [35]. Demonstration of these differences under conditions where the receptor of interest is known to be the only one present serves, therefore, as an independant confirmation of relative potencies which can be used to further validate studies which have been carried out using synthetic peptide analogs selective for each of the receptors. In a number of tissue preparations, stimulation of tachykinin receptors induces hydrolysis ofphosphatidyl inositol [10,51-53], indicating activation of phospholipase C. In some preparations, but not all, tachykinins also enhance synthesis of cyclic AMP [52,54,55], and in one report cAMP synthesis was inhibited [56]. This suggests tissue and/or cell typespecificity for coupling of the receptors with G-protein subunits, since the Gsc~ subunit stimulates adenylyl cyclase, whereas the Gi~ subunit is associated with inhibition of adenylyl cyclase and with activation of phospholipase C [57]. Stimulation of the tachykinin receptors transfected either transiently in COS-7 cells or stably in CHO or C6-2B rat glioma cells [ 35,58,59] induces the metabolism ofphosphatidyl inositol. In C6-2B cells, the bovine NK-2 receptor also caused an increase in intracellular calcium concentration and inhibition of agonist induced cyclic AMP accumulation [58]. In CHO cells Nakajima et al. [59] showed that all three rat tachykinin receptors stimulated adenylyl cyclase in response to ligand in addition to activating phospholipase C. C6 glioma cells do not express G i l a [60] and CHO cells contain several Gs~ and at least one Gi~ subunits [61]. Further, recent data show that two of the four mammalian adenylyl cyclases are stimulated by the

G-protein fl~ subunits [62]. Thus it seems clear that the tachykinin receptors are capable of interacting with multiple G-proteins and that the signalling observed upon stimulation in transfected systems likely depends on the nature of the host cell and its repertoire of signalling components. Reconstitution studies have also shown a pertussus toxin-insensitive component of the response to substance P in rat salivary glands, further supporting the possibility that the NK-1 receptor interacts with multiple G-proteins in vivo [19]. Another recent development which promises to be useful for binding site determinations as well as pharmacological studies is the discovery of several nonpeptide tachykinin receptor antagonists. Two structurally similar NK-1 receptor antagonists have been reported which are effective in nanomolar concentrations [63,64]. One of these, CP 96,345, is ~ 200fold more potent on the human NK-1 receptor than on the rat or mouse homolog [63], a somewhat surprising finding since the ligand binds with similar affinity on all three species. A second compound, RP 67,580, is roughly equipotent (5-fold better for rat brain than human U373MG astrocytoma cells) [64]. A third compound, SR 48,968, has been reported as a selective NK-2 receptor antagonist [65]. CP 96,345 appears to be highly selective for blocking substance P since micromolar concentrations of drug had no effect on binding of ~25I-labeled neurokinin A in cells transfected with the human NK-2 receptor (N.P. Gerard, unpublished data). In contrast, SR 48,968, which is also effective in nanomolar concentrations for inhibition of binding of radiolabeled neurokinin A to NK-2 receptor transfected cells, has the same potency for blocking [~25I]substance P binding to the NK-1 receptor as neurokinin A (i.e., ~ 150-fold less potent) (N.P. Gerard, unpublished data). In some of the first reported studies of structurefunction relationships for the tachykinin receptors, Fong and Strader [65] replaced the amino-terminal 13 residues of the rat NK-1 receptor with the amino acid sequence, M D Y K D D D D K P W (Flag sequence, for which monoclonal antibodies are commercially

32 available, IBI), and observed a 3-fold decrease in ligand binding affinity. If they replaced the first 27 residues with this sequence, substance P binding was no longer detectable. Studies performed in our laboratory (N.P. Gerard, unpublished data) showed that the same Flag amino acid sequence can be placed at the amino terminus of the human NK-1 receptor with no change in binding affinity, suggesting the effect they observed was the result of deleting residues important for ligand recognition, and was unrelated to addition of the Flag sequence. These investigators also noted a consistently higher affinity of the human NK-1 receptor for substance P binding compared with the rat molecule (0.7 vs. 3.2 nM, respectively). Comparison of the deduced amino acid sequences, as indicated above, shows only 22 amino acid replacements between rat and human NK-1 receptors. One of these, at position 97 in the first extracellular loop of the rat sequence is a valine where in the human it is glutamate. Substitution of a Val codon for a Glu codon in this position did not effect binding of substance P, however, it increased the affinity of this mutated receptor for neurokinin B approx. 2.5-fold [66]. Since the NK-3 receptor has a glutamate in the analogous position, they concluded that the first extracellular loop contains recognition sites for neurokinin B. To our knowledge, the corrolary experiment, to remove the glutamate from the NK-3 receptor at this position and test its ability to bind ligand, has not yet been performed. Fong et al. also identified a c D N A library clone of the human NK-1 receptor which lacks the coding sequence of exon five, yielding a molecule whose sequence is deleted at the end of the seventh transmembrane sequence [67]. Examination of the gene sequence shows an in-frame termination codon in the intron just following exon four and PCR experiments showed that such a molecule was transcribed in human brain. Signal transduction in Xenopus and binding in transfected COS cells indicates 10- to 100-fold lower affinity than the full length receptor. This may represent a NK-1 receptor subtype but requires further study. The NK-2 receptor gene

also contains an in-frame stop codon just after exon four [29], suggesting the possibility of subtypes for this receptor as well. Because these clones can arise by alternative splicing, however, it will be important to prove the existence of the predicted protein product. Using a different approach, we and others have made chimeric receptors between NK-1 and NK-2 or between NK-1 and NK-3. We reasoned that since all three receptors recognize all three ligands, swapping domains of the receptors might yield information about their structural specificity. When we exchange the sequence of the human NK-1 receptor at the end oftransmembrane segment five for the NK-2 receptor sequence, the molecule produced binds substance P with an affinity equal to the native NK-1 receptor, but neurokinin A has an equal affinity, ie, increased in affinity by ~ 150-fold (Gerard et al., unpublished data). The NK-2 receptor antagonist, SR 48,968, is also increased in affinity by an equal amount, suggesting that it binds in the same site as neurokinin A. The potency of the NK-1 receptor antagonist, CP 96,345, is slightly reduced. The chimeric molecule with an NK-2 receptor tail from the middle of transmembrane segment seven to the C-terminus on the human NK-1 receptor has binding properties indistinguishable from the natural receptor, however, its ability to stimulate phosphatidyl inositol hydrolysis is reduced by about 50~o. The corrolary structure, NK-2 receptor from the N-terminus to the end of transmembrane segment five, with NK-1 receptor to the C-terminus binds neither ligand. These data suggest that the specificity of recognition for substance P and neurokinin A reside in different domains of the receptors. The site(s) for substance P requires sequences before transmembrane segment five, consistent with the studies described above which indicate the first extracellular loop as a part of the binding site. The site(s) which dictate specificity to neurokinin A are found in the sequence between the end of transmembrane segment five and the middle of transmembrane segment seven, and likely are in the third extracellular loop.

33 Site directed mutagenesis will confirm these observations. Schwartz et al. adopted a slightly different approach a n d made chimeras between rat N K 1 and N K - 3 receptors since these molecules have slightly higher sequence identity than N K - 1 and N K - 2 receptors. They also used radiolabeled eledoisin as the test ligand since it b o u n d equally well to both receptors, and examined the ability of the N K - 1 receptor antagonist to block binding. They found that exchange of the second extracellular loop through t r a n s m e m b r a n e segment four from the NK-1 receptor to the N K - 3 molecule yielded a protein which could n o longer interact with the antagonist, but could still b i n d ligand (T.W. Schwartz, personal c o m m u nication). This suggests that C P 96,345 interacts with the receptor at a site which is distinct from the ligand binding site but which somehow also prevents binding of ligand.

IV. Conclusions The molecular cloning of the tachykinin receptors, N K - 1 , N K - 2 a n d N K - 3 , will allow detailed investigations in their s t r u c t u r e - f u n c t i o n relationships, a n d insight into how they transduce sensory signals in neural and smooth muscle tissues. The precise anatomic distribution of each receptor, as well as its role in developmental and inflammatory processes can n o w be addressed with transgenic technology, a n d the regulation of receptor gene expression can n o w be addressed with direct measurements. The next several years p o r t e n d these and other advances in our u n d e r s t a n d i n g of the tachykinin system.

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