Eur. J. Biochem. 204,1025-1033 (1992)

TJ FEBS 1992

The primary structure and gene organization of human substance P and neuromedin K receptors Kenzo TAKAHASHI, Atsuo TANAKA, Masumi HARA and Shigetada NAKANISHI Institutc for Immunology, Kyoto University Faculty of Medicine, Japan (Received October 21, 1991) - EJB 91 1408

The gene organization and amino acid sequences of human substance P and neuromedin K receptors (SPR and NKR, respectively) are reported on the basis of molecular cloning and sequence determination of genomic DNA containing the respective receptor gene. The human SPR and NKR genes, unlike many other genes for G-protein-coupled receptors, (G protein, guanyl-nucleotidebinding-regulatory protein), contain introns which interrupt the protein-coding regions into 5 exons. The human SPR and NKR genes extend over 60 kb and 45 kb, respectively and are considerably larger than the human substance K receptor (SKR) gene consisting of 12 kb. All 4 introns, however, are located at equivalent positions of the three tachykinin receptor genes, suggesting that they evolved from a common ancestoral gene. Human SPR and NKR consist of 407 and 465 amino acid residues, respectively, each possessing structural features characteristic of the members of G-protein-coupled receptors. The human and rat receptors show a common tendency of distinctly segmented sequence conservation and divergence among the three receptors, and the observed sequence conservation and divergence would contribute to the emergence of similar but distinct properties of the three receptors. Furthermore, the amino acid sequences and the gene sizes are more closely related between SPR and NKR than between SKR and NKR, suggesting that the SPR gene evolved from the primordial NKR gene after a gene duplication to form the NKR and SKR genes. neuromedin K receptors (SPR, SKR and NKR) and the characterization of the cloned receptors expressed in Xenopus oocytes and in mammalian cells [5 -91. The three tachykinin receptors belong to the family of G-protein-coupled receptors and mediate the stimulation of both phosphatidylinositol hydrolysis and cyclic AMP cascades [lo]. The three receptors, however, differ in affinities for the three tachykinins and in the expression sites of their mRNA [6 - 111.The varied physiological responses of the three tachykinins thus occur as a result of the selectivity and different distribution of the three receptors. The gene organization of the family of the G-proteincoupled receptors is distinct in that most members of this Family lack introns within their protein-coding regionsIl2,13]. This characteristic feature of the G-protein-coupled receptor family raises an interesting question as to whether the tachykinin receptors have any introns interrupting the protein structures, and if they do, whether there is any correlation between the gene organization and the functional and structural domains of the receptor proteins. The regulatory mechanisms of gene expression of the tachykinin receptors are also -~ interesting, because we have recently demonstrated that the Correspondence to s. Nakanishi, Institute for Immunology, Kyoto expression of the SPR gene is selectively and negatively reguUniversity of Medicine, Yoshida, Sakyo-ku, Kyoto 6O6, lated by glucocorticoids 1141. Recently, the structures of the Japan human SKR and the rat SPR genes have been reported [I 5 Abbreviations. SPR, substance P receptor; NKR, neuromedin K 1717 and it has been shown that the Protein-coding regions Of receptor; SKR, substance K receptor; G protein, guanyl-nucleotidethese receptors are subdivided into 5 exons 115- 171. However, binding regulatory protein. E n q m e s . Restriction endonucledses Suu3A1, HindIII, PstI, ECO- neither the genomic structures of the human SPR and NKR T141, EcoRI (EC 3.1.21.4); reverse transcriplase (EC 2.7.7.49). genes nor their protein sequences have been elucidated yet.

The family of tachykinin peptides represents a typical example of biologically active peptides which exhibit a high degree of functional diversity at the level of both peptide production and peptide reception [l, 21. This system consists of three distinct peptides, substance P, substance K (neurokinin A) and neuromedin K (neurokinin B), and possesses three different types of receptors, each specific for the respective peptide [l, 21. The members of this peptide family are widely distributed both in the central nervous system and in peripheral tissues, and evoke a variety of biological activities, including excitation of neurotransmission, vasodilation, smooth muscle contraction and stimulation of salivary secretion 131. The involvement of tachykinins in the pathology of a variety of human diseases has also been suggested by studies of animal models and humans. Especially, considerable evidence has been presented indicating the role of substance P in diseases involving hypersensitivity reactions in the skin and lung, and in chronic inflammatory disorders such as arthritis [4]. Our group and others have reported the molecular cloning of functional cDNA for the substance P, substance K and

1026 A Exon 1 E

H HE

W

E E

H

-

H E H

1 kb

human SPR

Exon 3

Exon4

Exon5

Exon 1

B

-

1 kb

human NKR 5‘ UTR E

3 UTR

H

Exon2 Exon3 HH

E

H

HhE

Exon4 Exon5 Fig. 1. Restriction mapping of cloned genomic DNA containing the SPR gene (A) and the NKR gene (B). The upper and lower portions indicate the restriction maps of cloned genomic DNA by positioning exons with black boxes; all Hind111 (H) and EcoRI (E) sites are shown in the maps. Between the genomic maps is a schematic structure of the mRNA with the encoded membrane-spanning domains and coding regions which are displayed by large and smaller boxes; S’UTR, 5’-untranslated region; 3’UTR, 3’-untranslated region. Exons in the genomic DNA and their corresponding regions of the mRNA are connected by solid lines. The scale of the genomic DNA is given in kb.

Because of the potential physiological and pathophysiological importance of the human tachykinin receptors and for a more thorough understanding of the regulation of the tachykinin system, we have isolated and determined the genomic structures of the human SPR and NKR genes. We report here the gene organization and the deduced amino acid sequences of these two receptors and discuss the evolution of the mammalian tachykinin system.

Tissues Human tissues were obtained as follows: placenta at the time of natural delivery, brain covering the area from temporal to parietal cortex and submandibular gland by autopsy, stomach and skin at surgical operation. All tissues were stored at - 80 “C until use. Isolation of human tachykinin receptor genes

EXPERIMENTAL PROCEDURES Materials

Materials were obtained from the following sources : ADashII, pBluescript and Gigapack Gold I1 from Stratagene; restriction endonucleases from Takara Shuzo and Toyobo ; SuperscriptTMfrom Bethesda Research Lab. ;oligo(dT)-Latex from Nippon Roche; Genescreen Plus from Du Pont; Biodyne nylon membrane from Pall Ultrafine Filtration Corp. Other reagents were described previously [6].

A human genomic library was constructed by using the LDash cloning system under the conditions recommended by the vendor. This library was a collection of a recombinant phage that carried human placenta DNA fragments (1 8 25 kb) generated by partial digestion with Sau3AI [18]. Approximately 1.2 x lo6 phage clones were screened with appropriate probes derived from either the rat SPR cDNA clone (prTKR2) [6] or the rat NKR cDNA clone (prTKR3) [8], or both. The cDNA probes were labeled with [cx-~’P]~CTP by the random primer method [8]. For the initial screening, a mixture of the 1991-bp Hind111- PstI fragment derived from

1027 prTKR2 and the 1370-bp EcoT141 fragment derived from prTKR3 was used for hybridization under the reduced stringency condition; hybridization was carried out at 65°C in a solution containing 1 M NaCl, 10% dextran sulfate and 1 % SDS, and filter washing was performed at 50°C in 0.5 x NaC1/ Cit (1 x NaCl/Cit contains 150 mM NaCl and 15 mM sodium citrate). Hybridization-positive clones were isolated by repeated plaque purification. These clones were classified by restriction enzyme and blot-hybridization analyses of the cloned genomic DNA; the hybridization analysis was carried out separately with the cDNA probe representing each of the three rat tachykinin receptors at 65 “C in the above hybridization solution, and filter washing was performed at 65°C in 0.2 x NaCl/Cit. Restriction DNA fragments which were expected to contain an exon or exons on the basis of blothybridization analysis were extracted after gel electrophoresis and subcloned into pBluescript SIC(+) [18]. DNA sequencing was carried out on both strands by the chain-termination method [19].

cDNA probe representing each of the three rat tachykinin receptors under high-stringency conditions. This hybridization analysis indicated that 24 (group l), 5 (group 2) and 5 clones (group 3) hybridized specifically and strongly with the cDNA probes for rat SPR, NKR and SKR, respectively. Each group of cDNA clones was analyzed by hybridization with more limited fragments of the cDNA for each of the three receptors. The genomic clones of groups 1, 2 and 3 were further divided into 4, 3 and 1 subgroups, respectively. The hybridization-positive restriction fragments were isolated from each representative of the 8 subgroups and were subcloned into pBluescript SK(+). The sequences of parts of the hybridized portions of the genomic DNA fragments were determined, and comparisons of the genomic sequences determined with the cDNA sequences reported for the three rat tachykinin receptors indicated that groups 1,2 and 3 represent the human SPR, NKR and SKR genes, respectively. The sequence determination of each of the genomic subclones was further extended to cover the sequences of exons and their surrounding regions.

RNA blot-hybridization and primer-extension analyses Poly(A)-rich RNA was isolated from human brain, submandibular gland, stomach and skin as described [8]. A genomic fragment containing the exon 1 sequence of the human gene isolated in this investigation, together with the above cDNA fragment encoding the rat SPR or NKR, was used as a probe for RNA blot hybridization; the rat cDNA probe was included to increase the intensity of the hybridization signal of this analysis. For primer-extension analysis, two oligodeoxynucleotides, 5’-GGGCCCATTGCAGCGCGACCTGTAAAAGTTC-3‘ and 5’-TAAGGGGCAACAGCTGCACTTTCTCAGAGG-3’, which are the sequences complementary to the 5‘ portions of the human SPR and NKR mRNA, respectively, were chemically synthesized; the former and the latter sequences correspond to nucleotides 437 - 467 and nucleotides 171- 200 upstream from the translation initiation site of the respective mRNA. The synthetic oligodeoxynucleotides were 32P-labeled at the 5’ ends (2.05.0 x lo4 cpm), hybridized to poly(A)-rich RNA (5 pg) at 30°C for 4 h and subjected to reverse transcriptase reaction as described previously [l11. The primer-extended cDNA products were separated by electrophoresis on a 7 M urea/6% polyacrylamide gel ; the same oligodeoxynucleotide primer was used for the synthesis of DNA products from the corresponding genomic DNA as a template by the chain-termination method. The synthesized DNA products were run as a marker parallel with the primer-extended products. RESULTS

Cloning of the human tachykinin receptor genes Genomic clones containing each of the three human tachykinin receptor genes were isolated and characterized as follows. A human genomic DNA library consisting of approximately 1.2 x lo6 phage clones was screened by hybridization in situ with a mixture of 32P-labeled cDNA fragments derived from the rat SPR and NKR cDNA clones. More than 40 plaques were found to hybridize strongly with the mixture of these cDNA Drobes under low-strinpencv conditions. 34 independent clones were isolated by repeated plaque purification and classified by restriction enzyme mapping and DNA blot hybridization of the cloned genomic DNA. The plaque hybridization was carried out separately this time by using a Y

Structural organizations of the human SPR and NKR genes Comparison of the human genomic sequences with the cDNA sequences for the rat tachykinin receptors enabled us to locate exons of the SPR and NKR genes in the genomic sequences, as illustrated in Fig. 1A and B. However, the genomic DNA isolated for SPR and NKR did not completely overlap with one another, indicating that the human SPR and NKR genes extend over 60 kb and 45 kb, respectively. In contrast, the whole exons of the SKR gene were included in a single genomic clone and the human SKR gene was determined to consist of approximately 12 kb (unpublished results). [15, 161. Figure 2A and B show the nucleotide sequences of exons, exon-intron boundaries and the 5’-flanking and 3‘flanking regions of the SPR and NKR genes, respectively. Both genes consist of 5 exons interrupted by 4 introns. All introns are located within the protein-coding regions and the sequences of the exon-intron boundaries all possess the consensus splicing signal conforming to the GT/AG rule [20]. The positions of 4 introns are identical between not only the SPR and NKR genes but also the SKR gene (unpublished results), [IS, 161 (see also Fig. 5). Exon 1 encodes the entire 5’untranslated region and the protein-coding region from the amino-terminal extracellular domain to the third transmembrane segment. Exon 2 covers the second cytoplasmic loop up to the second extracellular loop. Exon 3 contains the fifth transmembrane segment and the third cytoplasmic loop. Exon 4 starts with the sixth transmembrane segment and ends at the seventh transmembrane domain. Exon 5 includes the carboxyl-terminal tail and the 3’-untranslated region. Exons 1 5 of the SPR gene consist of 977, 195, 151,197 and approximately 3300 bp, respectively, while exons 1- 5 of the NKR gene are composed of 853, 189, 151, 197 and approximately 2900 bp, respectively (for the assignment of the 5’ and 3’ ends of the genes, see the next section). The sizes of introns A - D of the SPR genes are > 22 kb, > 26 kb, > 7 kb and approximately 2.2 kb, respectively, while those of the corresponding introns of the NKR gene are > 22 kb, approximately 2 kb, > 15 kb and approximately 1 kb, respectively.

I

Structural

of human SpR and NKR mRNA

RNA blot-hybridization analysis of human brain poly(A)rich RNA gave rise to a single band for both SPR and NKR

1028

A ggatccaatttttgcccggcataagtgtatagt -1333 aaatttcccagccttaaagcacttcccgagagatgctttgagcgctcgcggtaccagtgcgtaaacgccgctccccggctggcgcgggtgtgcgccaactccaacctgcgcgcaagtctg -1213 ccggtgcgcgctccagtcccacagctccgagtccccgcagtgaaaggagg~tgcaccggggtagatgggcccctgaggactcccggggttcagttttccgcggctgccaagaggg -1093 ccaagttggacagtggcagggtcctgaagcagatcagcaacaaccgcaagtgctccagccccaggtcctcagacacggaggaaaacgacaagaggcggacacacaacgtcttggaac9tc -973 agaggaggaacgagctgaagcgcagcttttttgccctgcgtgaccagatccctgaattggaaaacaacgaaaaggcccccaaggtagtgatcctcaaaaaagccaccgcctacatcctgt -853 ccattcaagcagacgagcacaagctcacctctgaaaaggacttattgaggaaacgacgagaacagttgaaacacaaactcgaacagcttcgaaactctggtgcataaactgacctaactc -733 gaggaqgagctggaatctctcgtgagagtaaggagaacggttccttctgacagaactqatgcgctggaattaaaat~catqctcaaagcctaacctcacaaccttgqctggggctttggg -613 actgtaagcttagagactgtcacttcccaggtgaatcagctagccaggtaactgagctagatattttgtggqqqtqtttcctaaacacagcctcaggaaagttgttttcgggacacctgg -493 accagggagtcgtcgcctctggcttctcggtagctggagcgcggcccggagcgcggcgctggcacatcgcccccacacatgaccgtttcccattgccacaggcaagccgcctctgcagag -373 ctgtctcagggctctgggcttcattccctggaagttgattgtcctccactccagctgtttcccaaatccttccttcctcccagcacccctcgtgcaacgacgattccaqctqcqqaccgc -253 a tcrg t g t cag t t act tccaagccacct actgciccitcgcggag t gcg tggggctcccggct cgcagactcccacggcaag tagcaagcagcaaaaggcg t g g ta c t gcggcgg tgga -133 -13 a~qaqacagrtgrcaacagctggcgc~cgtgccgccgtgcgcacc~ggactggcgagtacgcagcccaggtactgcccct tcccagtqacqtcrctgcaggggg&a.agcctcgtg cgcagct aac t ~ C G A G C T G A G C A A C C C G ~ c c G A G A G G T G c c c G c G ~ c T G c A G G c G G C G G C A G C G G C A G C ~ G A G A A G G ~ T C T C C A G C T G G A T A C G A A G C T C C A G A A T108 CCT GGCCATAGGCTCAGAACTTTTACAGGTCGCGCTGCAATGGGCCCCCACTTCGCTCCTAAGTCCTCACGCAGCACAGGGCTTTGCCTTTCCCTGCGGAGGMGGAGAAATAGGAGTTGCAG 228 GCAGCAGCAGGTGCATAAATGCGGGGGATCTCTTGCTTCCTAGAACTGTGACCGGTGGAATT”CTTTCCCTTTTTCAGTTTACCGCAAGAGAGATGCTGTCTCCAGACTTCTG~CTCAA 348

ACGTCTCCTGAAGCTTGATGGAGGAATTCAGAGCCACCGCGGGCAGGCGGGCAGTGCATCCAGAAGCGTTTATATTCTGAGCGCCAGTTCAGCTTTC~GAGTGCTGCCCAG~468 AAAGCCTTCCACCCTCCTGTCTGGCTTTAGMGGACCCTGAGCCCCAGGCGCCAGCCACAGGACTCTGCTGCAGAGGGGGGTTGTGTACAGATAGTAGGGCTTTACCGCCTAGCTTCGM 588

Exon 1

M~tASpASnValLeuProValAspSerAspLeuSerProAsnIleSerThrAsnThrSerGluProAsnGlnPheValGlnProAlaTrpGl~:leValLeuTrpAlaAlaAlaTyrT~.r 40 ATGGATMCGTCCTCCCGGTGGACTCAGACCTCTCCCC~CATCTCCACTMCACCTCGGMCCCAATCAGTTCGTGCMCCAGCCTGGC~TTGTCCTTTG~CAGCTGCCTACACG 708 ~ a ~ ~ ~ e V a ~ V a ~ T h r ~ e r V a ~ V a l G l y A s n V a l V a l V a l M e ~ T r p I l e I l e L e u A l a H i s L y s A r g M e ~ A r g T h r V a l T h r A s n T y r P h e L e u V a l A s n L e u A l a P h e A l a G l ~ 80 AlaSer GTCATTGTGGTGACCTCTGTGGTGGGCMCGTGGTGGTAGTGATGTGGATCATCTTAGCCCAC~GAATGAGGACAGTGACGAACTATTTTCTGGTG~CC~GGCCTTCGCGGAGGCCTCC 828 ~~tAlaAlaPheASnThrValValAsnPheThrTyrAlaValHisAsnGluTrpTyrTyrGlyLeuPheTyrCysLysPheHisAsnPhePheProIleAlaAlaValPheAlaSerIle 120 A~GGCTGCATTCAATACAGTGGTGAACTTCACCTATGCTGTCCACAACGAATGGTACTACGG~CTGTTCTACTGCAAGTTCCAC~CTTCTTCCCCATCGCCGCTGTCTTCGCCAGTA~r948 TyrSerMetThrAlaValAlaPheAspAr A TyrMetAlaIleIleHisProLeu 138 TACTCCATGACGGCTGTGGCCTTTGATAI$tgagat tagcct t tgtgaa -In t Ton A ( > 2 2 kbp)t tct t tct tctctgt t cca$TACATGGCCATCATACATCCCCTC 1002

Exon 2 GlnProArgLeuSerAlaThrAlaThrLysVIlValValIleCysValIleTrpValLeuAlaLeuLeuLeuAlaPheProGlnGlyTy~TyrSerThrThrGluThrMetProSerArgVal

178

CAGCCCCGGCTGTCAGCCACAGCCACCAAAGTGGT~TCATCTGTGTCATCTGGGTCCTGGCTCTCCTGCTGGCCTTCCCCCAGGGCTACTACTCAACCACAGAGACCATGCCCAGCAGAGTC 1122

ValCysMetIleGluTrpProGluHisProAsnLysIleTyrGluLysV GTGTGCATGATCGAATGGCCAGAGCATCCGAACAAGATTTATGAGAAAG$

V

tgag tagaga tgact cccca -1nt

ron B

- t t t cctg t t t acct tgc t g t a g

( >26 kbp)

195 1173

Exon 3 TyrHisIleCysValThrValLeuIleTyrPheLeuProLeuLeuValIleGlyTyrAlaTyrThrValValGlyIleThrLeuTrpAlaSerGluIleProGlyAspSerSerAspArg

235 TACCACATCTGTGTGACTGTGCTGATCTACTTCCTCCCCCTGCTGGTGATTGGCTATGCATACACCGTAGTGGGAATCACACTATGGGCCAGTGAGATCCCCGGGGACTCCTCTGACCGC 1293 TyrHisGluGlnValSerAlaLysArgLy alValLyeMetMetIleValVa1 253 TACCACGAGCAAGTCTCTGCCAAGCGC~~tgagcaggggacaggcaga -1ntronF v n nCA (>7 kbp)cctgtctCaccCtcttgcca$TGGTCAAAATGATGATTGTCGTG 1347

_--.. .

ValCysThrPheAlaIleCysTrpLeuProPheHisIlePhePheLeuLeuProTyrIleAsnProAspLeuTyrLeuLys~ysPheIleGlnGlnValTyrLeuAlal:eMetTrpLe~ GTGTGCACCTTCGCCATCTGCTGGCTGCCCTTCCACATCTTCTTCCTCCTGCCCTACATC~CCCAGATCTCTACCTGAAGAAGTTTATCCAGCAGGTCTACCTGGCCATCATGTGGCTG AlaMetSerSerThrMetTyrAsnProIleIleTyrCysCysLeuAsnAspAr GCCATGAGCTCCACCATGTACAI\CCCCATCATCTACTGCTGCCTCAATGACA~tgaggat cccaaccccacg -1 n t r on D ( - 2 . 2 k bp) t C t tca t Ctg Cr Cg Ctctcc

-

293 1467 311 1520

Exon 5 PheArgLeuGlyPheLysHisAlaPheArgCysCysProPheIleSerAlaGlyAspTyrGluG~yLeuGluMeCLysSerThrArgTyrLeuGln~~.rGlnGlySerValT~rLys 350

A a~TTCCGTCTGGGCTTCAAGcATGccTTCCGGTGCTGcccCTTCATCAGcGCCGGCGACTATGAGGGtiCTGGAAAT~~TCCACCCGGTATCTCCAGACCCAGGGCAGTGTGTAC~ 1638 ~~~~~~~rgLeuGluThrThrIleSerThrValValGlyAlaHisGluGluGluProGluAspGlyProLysAlaThrProSerSerLeuAspLeuThrSerAsnCysSerSerArgSe~ 390 GTCAGCCGCCTGGAGACCACCATCTCCACAGTC-GTGGGGGCCCACGAGGAGGAGCCAGAGGACGGCCCCAAGGCCACACCCTCGTCCCTGGACCTGACCTCCAACTGCTC~TCACGAAGI 1758 AspSerLysThrMerThrGluSerPheSerPheSerSerAsnValLeuSer 407 GACTCCAAGACCATGACAGAGAGCTTCAGCTTCTCCTCCTCCAATGTGCTCTCCTAGGCCACAGGGCCTTTGGCAGG~CAGCCCCCACTGCCTTTGACCTGCCTCCCTTCATGCATGGAAA~ 1878 1998 2118 2238 2358 2478 2598 2718 2838 2958 3078 3198 3318 3438 3558 3678 3798 3918 4038 4158 4278 4398 4518 4638 4758 4878 4998 5118 5238 5358

5410

Fig. 2.

mRNA with estimated sizes of approximately 4.8 and 4.3 kilonucleotides, respectively (Fig. 3). An identical size of the SPR mRNA was identified in the hybridization of human stomach poly(A)-rich RNA, whereas no appreciable amount of the NKR mRNA was detected in the analysis of stomach poly(A)-rich RNA (data not shown). Primer-extension analysis was carried out to define the 5’ termini of the SPR and NKR mRNA by using human brain poly(A)-rich RNA. Because none of the SPR or NKR mRNA was detected in the blot-hybridization analysis of human skin poly(A)-rich RNA (Fig. 3), we used this poly(A)-rich RNA preparation together with yeast tRNA as a control. In ad-

dition, the SPR mRNA was analyzed in submandibular poly(A)-rich RNA which was expected to contain a high amount of the SPR mRNA [ll]. The results of the primerextension analyses are presented in Fig. 4. A major primerextended product was seen for both analyses of the SPR mRNA and NKR mRNA. Furthermore, submandibular gland and brain poly(A)-rich RNA yielded an identical size of the product in the analysis of the SPR mRNA. On the basis of the genomic sequences, the 5’ termini of the SPR and N K R mRNA were assigned at 588 bp and 305 bp upstream from the mRNA translation initiation sites, respectively. RNase protection analysis was attempted in order to confirm these

1029

B ctgagaaactatacatataattatactgtaaatgagatttctttagaaactcatttttaaatgacaagagaggttcaaacagcttcacgtatatcc agtaqgttgctaaatatatatttaaccctaaatttatacacatgcccccaqqatgctaacaaaggagtttcggctctggaacctcttgctaaatttcctgagattggtctaagcacagac tatgtccttccagcttgcaatccaccgagcactttagctcacttattctcaagcaaagctcagaaatcaggtcctgcctgtcttcccggaaaacccaagtatttgttcagttacggtatt ctcatcagtgtqtcctcatggtcaagccctgaaaactaaggcaccatatatccagaaccctgtaaccaagagcgtgtattcacagtgcttactggctacgttcaacccagggaaatgtag

,1073 -953 -833 -713 agatctgtctcgttgtcgtgtttcaagaaaatgtactccttgggatttgcgatttacccttagtgtgctatgacattccaacctgagcagaatcctccaggagaatccagagtctccagc -593 tgcctgctgggaatggggtcagagggacggatttctagtgatagaggtacaggaagagagaaaatgggtggatatggaaagtgggagcgagaggaaaaggaatctgagatgagagaatat -473 aaacaccagagcagaaaagttgaatgaaattcaaaacccagggacacctaaaaacgtttgcggctgactgtgcccacagcctgcagcagtctacactcatgcactctccgaaaaatcaac -353 agqatcttgcccttqcctcatqaqtqaatggttaatttgtgcaggaaggaatggtccccaatacatgtttgagaacagggacctcagcaagatatcctgaagttaaaagaaggcaaacaa -233 tcttaaaaaaattttaattacaactaaaaaaatacaaccctcccttacacaqqtcaqcttqccaaqctccaqctqcttqcaqcqaatgaatgaaatgcgaag~c~~gatggagggagcg -113

8 128 CCCCTTAGCCCCAGCTGCATCCCCTAACCAGCAGGGATTGCAGTATCTTT~GCTTCCAGTCTTATCTGAAGACTCCGGCACCAAAGTGACCAGGAGGCAGAGAAGAACTTCAGAGG*GT 248 ..~. ~~~. ~~

~

~

~~

~

~

~

~

~

~

.~ .~~~~~ ~

~

~

~

~

~

Exonl

21 368 61 488 101 608 141 728 GGAAATCTCATCGTCATCTGATCATCCTGGCCCACAAGCGCATGAGGACTGTCACCAACTACTTCCTTGTGAACCTGGCTTTCTCCGACGCCTCCATGGCCGCCTTCAACACGTTGGTC 181 AsnPheIleTyrAlaLeuHisSerGluTrpTyrPheGlyAlaAsnTyrCysArgPheGlnAsnPhePheProIleThrAlaValPheAlaSerIleTyrSerMetThrAlaIleAlaVal RRTTTCATCTACGCGCTTCATAGCGAGTGGTACTTTGGCGCCAACTACTGCCGCTTCCAGAACTTCTTTCCTATCACAGCTGTGTTCGCCAGCATCTACTCCA~ACGGCCATTGCGGTG 848 AspAr A TyrMetAlaIleIleAspProLeuLysProArgLeuSerAlaThrA1a 199 G A C A d tgaggagaggacagacaga -1 nt ron A ( >22 kbp ) - ttttg t t tg tttttatata~TATATGGCTATTATTGATCCCTTGAAACCCAGACTGTCTGCTACAGCA 902 MetAlaThrLeUProAlaAlaGluThrTrpIleAspGlyGlyGlyGlyValGlyAlaAspAla

CTCGTCTTGGGCTGCCCGTGGGTGAGTGGGAGGGTCCGGGACTGCAGACCGGTGGCGATGGCCACTCTCCCAGCAGCAGAAACCTGGATAGACGGGGGTGGAGGCGTGGGTGCAGACGCC ValAsnLeuThrAlaSerLeuAlaAlaGlyAlaAlaThrGlyAlaValGluThrGlyTrpLeuGlnLeuLeuAspGlnAlaGlyAsnLeuSerSerSerProSerAlaLeuGlyLeuPra GTGAACCTGACCGCCTCGCTAGCTGCCGGGGCGGCCACGGGGGCAGTTGAGACTGGGTGGCTG~CTGCTGGACCAAGCTGGCAACCTCTCCTCCTCCCCTTCCGCGCTGGGACTGCCT ValAlaSerProAlaProSerGlnProTrpAlaAsnLeuThrAsnGlnPheValGlnProSerTrpArgIleAlaLeuTrpSerLeuAlaTyrGlyValValValAlaValAlaValLeu GTGGCTTCCCCCGCGCCCTCCCAGCCCTGGGCCAACCTCACCAACCAGTTCGTGCAGCCGTCCTGGCGCATCGCGCTCTGGTCCCTGGCGTATGGTGTGGTGGTGGCAGTGGCAGTTTTG GlyAsnLeuIleValIleTrpIleIleLeuAlaHisLysArgMetArgThrValThrAsnTyrPheLeuValAsnLeuAlaPheSerAspAlaSerMetAlaAlaPheAsnThrLeuVal

Exon 2

ThrLysIleValIleGlySerIleTrpIleLeuAlaPheLeuLeuAlaPheProGlnCysLeuTyrSerLysThrLysValMetProGlyArgThrLeuCysPheValGlnTrpProGlu

239

1022 ACCAAGATTGTCATTGGAAGTATTTGGATTCTAGCATTTCTACTTGCCTTCCCTCAGTGTCTTTATTC~CCAAAGTCATGCCAGGCCGTACTCTCTGCTTTGTGCAATGGCCAGAA GlyProLysGlnHisPheT T TyrHisIleIleValIleIleLeuValTyrCys 257 1076 GGTCCCAAACAACATTTCA%taag ttaattc tc ta tta t -1nt ron B ( -2 k bp ) - tgttt tt cttattttt cata$?TACCATATTATCGTCATTATACTGGTGTACTGT

Exon 3

296 PheProLeuLeuIleMetGlyIleThrTyrThrIleValGlyIleThrLeuTrpGlyGlyGluIleProGlyAspTh~CysAspLysTyrHisGluGlnLeuLysAlaLysArgLys 1193 TTCCCATTGCTCATCATGGGTATTACATACACCATTGTTGGAATTACTCTCTGGGGAGGAGAAATCCCAGGAGATACCTGTGACAAGTATCATGAGCAGCTAAAGGCC-GTTAAR~ta 315 alValLysMetMetIleIleValValMetThrPheAlaIleCysTrpLeuProTyr ctggtccatgttgttta -1ntron C (>15 kbp)atgactttttttctttatag TTGTCAAAATGATGATTATTGTTGTCATGACATTTGCTATCTGCTGGCTGCCCTAT 1250 Exon 4 HisIleTyrPheIleLeuThrAlaIleTyrGlnGlnLeuAsnArgTrpLysTyrIleGlnGlnValTyrLeuAlaSerPheTrpLeuAlaMetSerSerThrMetTyrAsnProIleI~~ 355 131 0 CATATTTACT’PCRTTCTCACTGCRRTCTATCAAC~CTAAATAGATGG~TACATC~GCAGGTCTACCTGGCTAGCTTTTGGCTGGCAATGAGCTCAACCATGTA~TCCCATCATC TyrCysCysLeuAsnLysAr A PheArgAlaGlyPheLysArgAlaPheArg 372 1421 TACTGCTGTCTGAATA A A A gtaaaaacaaaactacgaaat g -1 n t ron D ( -1 k bp ) - tctg tggcctgct t t t cc t CaaTTTCGAGCTGGCTTCAAGAGAGCATTTCGC

Exon,5

TrpCysProPheIleLysValSerSerTyrAspGluLeuGluLeuLysThrThrArgPheH~sProAsnArgGlnSerSerMetTyrThrValThrArgMetGluSerMetThrValVal 412 1541 TGGTGTCCTTTCATCAAAGTTTCCAGCTATGATGAGCTAGAGCTCAAGACCACCAGGTTTCATCCAAACCGGCAAA~CAGTATGTACACCGTGACCAGAATGGAGTCCATGACAGTCGTG 452 PheAspProAsnAspAlaAspThrThrArgSerSerArgLysLysArgAlaThrProArgAspProSerPheAsnGlyPheSerArgArgAsnSerLysSerAlaSe~AlaThrSerSer TTTGACCCCAACGATGCAGACACCACCAGGTCCAGTCGGAAGAAAAGAGCAACGCCAAGAGACCCAAGTTTCAATGGGTTCTCTCGCAGG~TTCCAAATCTGCCTCCGCCACTTC~GT 1661 465 1781 1901 2021 2141 2261 2381 2501 2621 2141 2861 2981 3101 3221

3341 3461 3581 3701 3821

3941 4061 4181 4301 4405

Fig. 2. The nucleotidc sequences of exons and their surrounding regions of the human SPR (A) and NKR (B) genes. The nuclcotide sequence of the messagc strand togethcr with the deduced amino acid sequence of SPR or NKR is indicated.The large and small letters represent the exon sequence,and the 5’-flankingand intron sequences,respectively.The precise 3’-terminiof the two mRNA remain to be determined and the nucleotidesequencebeyond the presumed 3’-terminusof the respectivegene (aroundresidue 4800 in the SPR gene and 4300 in the NKR gene) is indicated with large Ictters.The nucleotide sequence is numbered for the exon sequence with + 1 at the transcription initiationsite defined as described in the text, while the protein sequence is numbered by starting with the translation initiation site as + 1 . The canonicalTATA and AATAAA sequences are encloscd by a solid line. The DNA sequenceshomologous to those of promoter,enhancer and regulatory DNA clementsand the ATTTA scqucnce are marked by lines under the nucleotide sequences.

transcription initiation sites. W e failed in this confirmation, because the limited availability of human poly(A)-rich preparationsdid not allow us to perform serial protection analyses under different conditions.However,examination of the gene sequences 5’to the above transcription initiation sitesrevealed that a TATA box is located at 24-27 bp and 32-35 bp preceding the putative transcription initiation sites of the SPR dnd NKR genes, respectively.The TATA box is involved in

the polymerase nucleotideselectionto initiate transcriptionat a specific site 1211. Accordingly,on the basis of the primer-extensionstudies together with the presence of the TATA box at reasonable positions in both genes, w e tentatively defined the transcription initiation sites at 588 bp and 305 bp upstream from the translation initiation sitesof the SPR and NKR genes,respectively.There are many potential promoter,enhancer and reg-

1030

Fig. 3. RNA blot-hybridization analyses of the SPR and NKR mRNA. Poly(A)-rich RNA (5 pg each) isolated from the brain and skin were analyzed in lanes 1 and 2, respectively. The probe used was a genomic fragment containing the exon 1 sequence of the human SPR or NKR gene and the cDNA fragment encoding the corresponding rat receptor. Human ribosomal RNA (28s and 18s) was used as a marker. The autoradiographicexposure for the SPR and NKR mRNA was performed for 2 and 10 days, respectively.

ulating DNA elements in the 5’-flanking regions of both genes [22]. These include TGACGTCT (cyclic AMP responsive element), TGAGACA (AP-I), GGGTGTTTCC (NF-KB), CCCCAGGCG (AP-2), CCAGCTGCGGA (AP-4), AAATGCATG (OCT-2) and GGGCGG (Sp-I) in the SPR gene, and CCCCAGGAT (AP-2), TGTCCTCA (cyclic AMP responsive element), TGzETgA (AP-1) and GGGCGG (Sp-I) in the NKR gene (Fig. 2). From the results of the size determination and the 5’terminal assignment of the two mRNA, we calculated the lengths of the 3’-untranslated regions of the SPR and NKR mRNA to be approximately 3100 and 2500 nucleotides, respectively. Consistent with this calculation, there is a potential pol yadenylation/processing signal, AAUAAA [23],near and upstream from the putative 3’ ends of these two mRNAs (Fig. 2). There are many AUUUA sequences in the 3’-untranslated regions of both mRNA (Fig. 2). This sequence has been shown to govern the instability of eukaryotic mRNA which is the least stable, having half-lives of 15 - 30 min [24].

Comparison of amino acid sequences of human SPR, NKR and other tachykinin receptors The amino acid sequences of human SPR and NKR were deduced from the nucleotide sequences determined for the genomic clones containing the respective receptor gene (Fig. 2). The nucleotide sequences surrounding the initiation codon in both human receptor genes agree reasonably well with the consensus sequence 1253. The human SPR consists of 407 amino acids with a relative molecular mass of 46 248 Da, and the number of amino acid residues of the human SPR is identical with that of the rat counterpart [6]. Human and rat SPR shows an overall amino acid identity of 94.6%. The human NKR is composed of 465 amino acids with a relative molecular mass of 52268 Da and the human receptor is 13 amino acid residues longer than the rat NKR at the aminoterminal extracellular domain [8]. The overall identity between these two proteins is 88.2% in amino acids. Figure 5 shows the amino acid sequence alignment of human SPR, NKR and five other tachykinin receptors including three rat receptors 16-91 and human and bovine SKR [5,

Fig. 4. Primer-extension analyses of the 5’-termini of the SPR and NKR mRNA. The 32P-labeledsynthetic oligodeoxynucleotide complementary to the 5‘ portion of the SPR or NKR gene was hybridized to poly(A)-rich RNA ( 5 pg each) and subjected to reverse transcriptase reaction. The same oligodeoxynucleotide primer was used for the DNA synthesis of the corresponding genomic DNA by the chaintermination method and the synthesized DNA products were run as a marker parallel with the primer-extended products. For the SPR mRNA, the following RNA was analyzed: lane 1, brain poly(A)-rich RNA; lane 2, submandibular poly(A)-rich RNA; lane 3, skin poly(A)rich RNA; lane 4, yeast tRNA. For the NKR mRNA, the following RNA was analyzed: lane 1 , brain poly(A)-rich RNA; lane 2, skin poly(A)-rich RNA; lane 3, yeast tRNA. In both analyses, the DNA ladder indicating the genomic sequence around the 5’-tcrminus of the respective gene is shown on the left of the primer-extended products; TATAA; the position of the TATA box. A major primer-extended product is indicated by an arrow. In the analysis of the N K R mRNA, some faint bands observed in the brain poly(A)-rich RNA were also detected for the skin poly(A)-rich RNA at the corresponding position5 after prolonged autoradiographicexposure.

15, 16, 261. Human SPR and NKR, like the other tachykinin receptors, possess structural characteristics common to the. members of G-protein-coupled receptors [2, 12, 271: the presence of seven hydrophobic segments which would represent seven membrane-spanning domains; three potential Nglycosylation sites at the extracellular domains [28]; two cysteine residues situated at the first and second extracellular loops, which are probably involved in the formation of a disulfide linkage between these two loops [12]; one conserved cysteine residue immediately following the seventh membranespanning domain; which would participate in anchoring the receptor to the plasma membrane by palmitoylation [29]. Several interesting structural features are also apparent for human SPR and NKR and most of the these features are conserved in the rat counterpart [l, 21: there are many serine and threonine residues at the third cytoplasmic loops and the carboxyl-terminal tails of both SPR and NKR, which may serve as phosphorylation sites and thus cause desensitization in response to repeated application of agonists [12]; most of the G-protein-coupled receptors contain an aspartic acid in

1031 v v

SPR human rat

N K R human rat

T

I

1

MDNVLPVDSDLSPNISTNTSEPNQFVQPAWQIVLWAAAYTMWII

n

v

MDNVLPMDSDLFPNISTNT~ESNQFVQPTWQIVLWAAAYTVIWTSWGNVWIWII

57 57

MATLPAAF;;WIDGGGGVGADAVPLTASLAAGAATGAV~PGWLQLLDQAGNLSSSPSALGLPVASPAPSQPWA~LTNQ~VQPSWRIALWSLAYGVWAVAVLGNLIVIWII 110

MRSVPRGENWTDGTVEVGTHTGNL--SSALGVTE------WL~--QAGNFSS---ALGLPA~TQAPSQ~NLTNQ~QPSWRIALWSLAYGLWAVAVFGNLIVIWIII 97 Y

SKR human

MGTCDIVTEANI SSGPESNTTGITAFSMPSWQLALWATAYLALVLVAVTGNAIVIWII MGTRAIVSDANILSGLESNATGVTAFSMPGWQLALWATAYLALVLVAVTGNATVIWIII MGACWMTDINISSGLDSNATGITAFSMPGWQLALWTARYLALVLVAVMGNATVIWII

rat bovine

58 58 58

lntron A

a

I 1 1

177 177 230 217

178 178 178

Intrp B

,

lntrpn -vI------r C

7 V I I294 294

345 332

296 296 296

lntrvn D 407 407 465 452

MSSTMYNPIIYCCLNHRPRSGFRLRFRCCPWVTPTEEDRLELTXTPSLSRR~RCHTKETLFMTGDMTHS~TNGQVGSPQDGEPAGPICKAQA MSSTMYNPIIYCCLNHRFRSGFRLAFRCCPWVTPTEEDKMELTYTPSLSTRVNRCHTKEIFFMSGDVAPSEAVNGQAESPQAGVSTEP

398 390 384

Fig. 5. Comparison of the amino acid sequences of human SPR and NKR and five other tachykinin receptors. The amino acid sequences of the seven tachykinin receptors including rat SPR [6], rat NKR [8], rat SKR [7],human SKR [15,16] and bovine SKR [S]are aligned by introducing gaps (dashed lines) to maximize the homology. Positions of the seven transmembrane segments (I--11) and the four introns (arrow-headed lines) are indicated above the amino acid sequences. Two conserved cysteine residues present in the first and second extracellular loops and one conserved cysteine residue located following the seventh transmembrane segment are indicated by black circles and a white circle, respectively. Potential N-glycosylation sites present at the extracellular domains are shown by black and white triangles; white triangles indicate the positions where one of the sequences indicated lacks the consensus sequence for N-glycosylation.

the second membrane-spanning domain [27], but this amino considerably diverge among the three receptors. The selectivitacid is replaced with a glutamic acid in both human and rat ies of the three receptors for three mammalian tachykinin SPR; no other aspartic acid nor glutamic acid is present in peptides are more closely related between NKR and SKR the seven membrane-spanning domains: one histidine residue than between SPR and NKR/SKR [l, 21. The affinity rank each is located in the fifth a n d sixth memhr~ine-spanningdo- orders of the three receptors are as follows: for SPR, substance P > substance K > neuromedin K; for SKR, substance mains. To inspect the sequence conservation and divergence of K > neuromedin K > substance P; for NKR, neuromedin the three tachykinin receptors, we calculated the amino acid K > substance K > substance P. Interestingly, however, the similarity of segmented sequences of the three human and rat sequence comparison of the three receptors clearly indicates receptors according to the receptor topology relative to the that the overall similarity in the core sequences covering the membrane, as summarized in Fig. 6. In this calculation, the seven membrane-spanning domains is higher between SPR sequence immediately following the seventh membrane-span- and NKR than between SKR and NKR in both mammalian ning domain up to the putative palmitoylated cysteine residue species. is included as the fourth cytoplasmic domain. This sequence comparison revealed a striking common tendency in conserDISCUSSION vation and divergence of the segmented sequences between This paper describes the structural organization of the the human and rat receptors. The most conserved regions are the first cytoplasmic loops and the seventh membrane- human SPR and NKR genes, and the amino acid sequences spanning domains of the three receptors, and the second mem- deduced for their coding proteins, on the basis of molecular brane-spanning domain and the second cytoplasmic loop be- cloning and sequence determination of sets of genomic clones tween SPR and NKR. The conservation is also relatively high containing the respective receptor gene. The human SPR and in the third and sixth membrane-spanning domains between NKR genes, unlike many other genes for G-protein-coupled SPR and NKR, and between NKR and SKR; the second receptors [12,13], contain introns which interrupt the proteinmembrane-spanning domains between NKR and SKR, and coding regions into 5 exons. The human SPR and NKR genes between SKR and SPR ;the second cytoplasmic loops between extend over 60 kb and 45 kb, respectively, and are thus conNKR and SKR, and between SKR and SPR; the third cyto- siderably larger than the other tachykinin receptor gene; the plasmic loop between SPR and NKR; and the fourth cytoplas- human SKR gene consists of 12 kb (unpublished results), [15, mic loops between SPR and NKR, and between SKR and 161. However, all 4 introns are located at equivalent positions SPR. In contrast, the second and third extracellular loops not only between the SPR and NKR genes but also withln

1032 these potential functional domains appear to be encoded by distinct exons of the tachykinin receptor genes. domain human rat human rat human rat The transcription initiation sites of the SPR and NKR genes were tentatively assigned by primer-extension analysis. ( percent homology ) Consistent with this assignment, the initiation sites of both 43 48 61 57 M-1 48 48 genes are preceded at reasonable positions by a TATA box 9 0 90 9 0 90 1 0 0 100 c-1 which functions as an RNA polymerase I1 selection sequence 73 73 7 3 64 82 M-2 91 [21]. Besides this sequence, there are several potential pro50 50 5 0 50 45 E-1 60 moter, enhancer and regulatory DNA elements in the 5’8 2 82 6 8 68 82 77 M-3 flanking regions of the two genes. In our previous study using 80 80 7 0 70 90 c-2 90 rat pancreatic acinar AR42J cells, we showed that the SPR 57 52 57 52 67 M-4 67 gene is negatively and selectively regulated at the transcrip20 16 29 33 33 E-2 38 tional level by glucocorticoids [14]. Glucocorticoid-mediated negative regulation of the human SPR gene was similarly 56 64 40 40 52 52 M-5 observed in the studies of human lymphoblast IM-9 cells 45 41 45 41 76 C-3 76 (unpublished results). However, a typical consensus sequence 77 77 64 64 77 77 M-6 of the glucocorticoid responsive element for negative gene 1 5 15 3 8 38 31 E-3 31 regulation (ATfACNNNNTGATCe) [36] could not be found 9 2 92 9 6 96 96 M-7 92 in either the 5‘4anking region of the human SPR gene report73 73 67 67 73 73 c-4 ed in this paper or that of the rat SPR gene reported by 5 9 59 60 59 overall 69 67 Hershey et al., [17]. Thus, it is possible that, like some other glucocorticoid-responsivegenes [22], the sequence responsible NH2 I E-1 E-2 E-3 for negative regulation by glucocorticoids is present in the further upstream 5’-flanking region or within the intron of the SPR gene. Both the SPR and NKR genes have relatively long 3’untranslated regions and contain a potential polyadenylation/ M-1 M-2 M-3 M-4 M-5 M-6 M-7 \ processing signal, AAUAAA, at appropriate positions preceding the presumed 3’ ends of both mRNA. Another interesting feature of the 3‘-untranslated regions of the two mRNA is the presence of many AUUUA sequences which have been shown to play an important role in governing a rapid turnover of eukaryotic mRNA [24]. In fact, rat SPR mRNA showed a c-3 c-1 c-2 COOH short half-life of approximately 30 min [I41 and it is likely that Fig. 6. Comparison of the amino acid homology of segmented sequences the instability of the SPR mRNA is governed by the AUUUA of the paired receptors. The receptor is segmented as illustrated in a sequences present in the 3’-untranslated regions of human and schematic structure of the receptor and the amino acid sequence rat mRNA. In contrast, the human SKR mRNA lacks the homology of segmented sequences of the paired receptors was calcu- AUUUA sequence in the 3’-untranslated region [IS, 261. It lated; possible palmitoylation is indicated by a zigzag line. The overall homology was calculated for the sequence starting with M-1 and would thus be interesting to test whether a distinct turnover of the mRNA is involved in the regulation of the three tachykinin ending with C-4. receptor genes. A comparison of the amino acid sequences of the three the small SKR gene [lS, 161, and these introns interrupt the tachykinin receptors indicates that the three receptors not protein-coding region near putative membrane-spanning do- only possess several characteristic features in their amino acid main borders. Introns A and D occur immediately following sequences but also show a common tendency in conservation the third and seventh membrane-spanning domains, respec- and divergence of their segmented sequences between the hutively, while introns B and C are present at the amino-terminal man and rat receptors. Most of the segments covering the edges to the fifth and sixth membrane-spanning domains, seven membrane-spanning domains and their extending cytorespectively. Because exons in many eukaryotic genes rep- plasmic loops are highly conserved among the three receptors. resent genetic building blocks that code for discrete structural The sequence conservation is particularly remarkable in the or functional units for their coding proteins [30], the dissection seventh membrane-spanning domains of the three receptors of the tachykinin receptors by intron interruptions may help and this membrane domain may play an important role in to define several important functional units of this receptor interacting with the carboxyl-terminal sequence common to family. Many investigations of the adrenergic and other G- the three tachykinin peptides. In addition, the first and second protein-coupled receptors indicated important structural fea- cytoplasmic loops are well conserved among the three receptures of these receptor proteins, including the second and third tors. All three tachykinin receptors are coupled to the stimumembrane-spanning domains governing agonist/antagonist lation of both phosphatidylinositol hydrolysis and cyclic AMP binding [12,31,32], the third cytoplasmic loop responsible for formation [lo]. The conserved cytoplasmic domains may thus G-protein interaction [ 12, 33, 341, and the carboxyl-terminal be important in the coupling of the receptors to common domain involved in phosphorylation/agonist desensitization cytoplasmic effectors. Interestingly, however, the third cyto[12, 351. Our recent study of a series of chimeric receptors plasmic loops, the domains thought to be primarily involved constructed between rat SPR and SKR also indicated the in the coupling to the G protein [12, 33, 341, are conserved importance of the second-to-fourth membrane-spanning do- between SPR and NKR but diverge between SKR and SPR/ mains in determining the ligand-binding selectivity of the NKR in both of the mammalian species. In contrast to the tachykinin receptors (unpublished results). Thus, many of above conservation, the amino-terminal and carboxyl-terSPR-NKR NKR-SKR SKR-SPR

w

1033 minal regions, and the extracellular loops diverge among the three receptors. Since the tachykinin peptides are larger than many other ligands that interact with receptors in this supcrfamily, the tachykinin receptors may require some extracellular domains for determining the binding specificity to the three peptides. The number and distribution of serine and threonine residues in the third cytoplasmic loops and the carboxyl-terminal tails are well conserved between the human and rat receptors but differ among the three tachykinin receptors. A number of studies of adrenergic and other receptors have shown that the phosphorylation of serine and threonine residues in these cytoplasmic regions is responsible for causing desensitization in response to repeated application of agonists [12, 351. All three tachykinin receptors show desensitization, but the desensitization behavior is different among the three receptors [l];SPR is strongly desensitized to agonists, whereas desensitization occurrs moderately and weakly for NKR and SPR, respectively. It is possible that the phosphorylation may differ in the three receptors and thus cause differing desensitization, due to the sequence divergence and the varied number and distribution of serine and threonine in the cytoplasmic regions of the three receptors. The exact matchng of intron positions of the three tachykinin receptor genes supports the view that the three receptors evolved from a common ancestor gene by successive gene duplication events. This evolution of the receptor proteins is interesting, when the evolution of the three tachykinin peptides is envisaged. The three peptides are derived from two peptide precursor genes termed preprotachykinin A and B genes [37]. The two genes show closely related exon-intron arrangements and possess an exon encoding either substance K or neuromedin K at an equivalent position [38]. This structural resemblance suggests that substance K and neuromedin K were generated as a result of duplication of the peptideprecursor gene followed by a series of nucleotide changes in the peptide-coding genomic segments. Substance P, however, is contained in a distinct genomic sequence preceding the substance-K-coding exon within the preprotachykinin A gene [39]. It is thus conceivable that substance P was generated by intragenic duplication of a genomic segment encoding an ancestral substance K peptide and then acquired its biological activities in response to the concomitant appearance of the corresponding receptor. With respect to the evolution of this receptor, there was an equal chance for either the primordial SKR gene or the NKR gene becoming a new receptor for substance P after gene duplication. Noteworthy here is our present finding that not only the amino acid sequences but also intron sizes are more closely related between SPR and NKR than between SKR and NKR. This finding leads to the assumption that the SPR gene evolved by duplication of the primordial NKR gene rather than the primordial SKR gene. This hypothesis also explains the apparent reversed relationship between the sequence homology and the tachykinin-binding selectivity of the three receptors. Thus, on the basis of the comparison of the gene organizations and the sequence homology of the tachykinin peptides and their receptors, we speculate that substance P and its receptor evolved more recently than the two other peptides and receptors, but this peptide and receptor may have different evolutionary origins, the peptide being derived from substance K and the receptor having originated from NKR. This work was supported in part by research grants from the Ministry of Education, Science and Culture ofJapan, the Yamanouchi Foundation for Research on Metabolic Disorders, the Uehara Memorial Foundation and thc Suzuken Memorial Foundation.

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