THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 2fi.5, No. 33, Issue of November 25. pp. 20455-20462 1990 <Q 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in Lf S. A.

The Human Neurokinin A (Substance K) Receptor MOLECULAR CLONING OF THE GENE, CHROMOSOME LOCALIZATION, AND ISOLATION OF cDNA FROM TRACHEAL AND GASTRIC TISSUES*

(Received for publication, May 30, 1989)

Norma P. GerardS$nR, Roger L. Eddy, Jr.**, Thomas B Shows**, and Craig Gerard$ll$$§$ . From the $Department of Medicine, Beth Israel Hospital, the $$Department of Pediatrics and the §lna Sue Perlmutter Laboratory, Chldren’s Hospital, and the VThorndike Laboratory, Harvard Medical School, Boston, Massachusetts 02215 and the **Department of Human Genetics, Roswell Park Memorial Institute, Buffalo, New York 14263

Neurokinin A (substance K) is a peptide neurotrans- mitter of the tachykinin family with potential as a major mediator in human airway and gastrointestinal tissues. Neurokinin A acts via a receptor (the NK-2 receptor) believed to be localized on smooth muscle cells and pharmacologically coupled to a GTP-binding protein. To characterize the human NK-2 receptor, we prepared a partial cDNA from human tracheal RNA using the polymerase chain reaction with oligonucleo- tide primers derived from the bovine NK-2 receptor cDNA sequence (Masu, Y., Nakayama, K., Tamaki, H., Harada, Y., Kuno, M., Nakanishi, S. (1987)iVuture 329, 836-838). This partial human NK-2 receptor cDNA was used to screen a human genomic DNA li- brary and yielded a clone, NGNK-2, of approximately 20 kilobases. Analysis of NGNK-2 indicates that it contains the entire coding sequence of the NK-2 recep- tor as well as 5’- and 3’-flanking sequences. The gene is organized with five exons interrupted by four in- trons. The complete sequence of the exons and the intron-exon junctions was determined, as were the transcription initiation site and the 3’-polyadenylation signal. Analysis of EcoRI digests of genomic DNA from human-mouse cell hybrids indicates a single gene for the human NK-2 receptor localized to chromosome 10. Sequence analysis of exons 1 and 5, where major dif- ferences occur between the human and animal species, provided information for polymerase chain reaction primers which allowed us to prepare full-length cDNA for the human NK-2 receptor. The protein predicted from the gene sequence is extended by 14 amino acids at the COOH terminus compared to the bovine and 9 residues compared to the rat molecules. The seven membrane-spanning regions are encoded by exons l- 4 and none is interrupted by introns. These regions are highly conserved among the species studied, suggesting stringent evolutionary control over these molecules.

* This work was supported in part by National Institutes of Health (NIH) Grant HL41587. 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 505680.

11 Recipient of NIH Research Career Development Award HL01777. To whom correspondence should be addressed: Dept. of Medicine, Beth Israel Hospital, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-735-7616.

§§ Recipient of a Faculty Scholars Award from R. J. R. Nabisco.

Neurokinin A (formerly known as substance K) is a member of a family of peptide neurotransmitters known as tachykinins (1). These peptides are associated with the central and pe- ripheral nervous systems and display a wide tissue distribu- tion (2-4). Tachykinins share the COOH-terminal structure Phe-X-Gly-Leu-Met-NH,. The best known members of this family are substance P and neurokinin A or substance K. Tachykinins were first recognized for their spasmogenic effect on smooth muscle-containing tissues (5); however, the full physiologic role for these peptides is still under active inves- tigation. Substance P has been identified as a prominent candidate in sensory neurotransmission (6, 7), and additional evidence suggests effects on immune cells as well (8, 9). Neurokinin A is particularly associated with smooth muscle- containing tissues found in the gastrointestinal, respiratory, genitourinary, and vascular systems (10). The molecular char- acterization of the tachykinins reveal that they arise from common precursor molecules known as preprotachykinins, by proteolytic processing. Three forms of message (o(, 6, y) arise by alternative splicing events (11-13). The /3 and y forms of preprotachykinins encode both substance P and neurokinin A, while the O( form contains only the substance P sequence. Additionally, an amino-terminally extended form of neuro- kinin A, termed neuropeptide K or NpK, is present in the p form.

Three classes of tachykinin receptors have been identified by bioassay and radioligand binding (14-16). While the COOH-terminal consensus sequence of the tachykinins con- trols biological activity, the divergent amino-terminal se- quences determine receptor affinity. Thus, each tachykinin recognizes the three receptor types, but with varying avidity. The NK-1 receptor preferentially binds substance P, the NK- 2 receptor prefers neurokinin A and the NK-3 receptor rec- ognizes neurokinin B. The latter is a tachykinin first found in the porcine spinal cord and brain, and arises from a gene distinct from that for substance P and neurokinin A (17). Synthetic tachykinin analogs act as competitive inhibitors with relative selectivity for each of the three neurokinin receptors (E-20).

Within the respiratory system, tachykinins have a number of important physiologic effects. These include bronchocon- striction of large airways, enhancement of vascular permea- bility, and stimulation of mucus secretion (21-24). Character- ization of these responses using tachykinins and structural antagonist analogs have indicated that the NK-2 receptor, which is selective toward neurokinin A (substance K), pre- dominates in animal and human airways (21, 24).

Masu et al. (25) and Sasai and Nakanishi (26) reported the cDNA and deduced protein sequences for the NK-2 receptors from bovine and rat stomach, respectively, showing the NK-

20455

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20456 Human Neurokinin A Receptor

2 receptor to be a member of the rhodopsin superfamily. This multigene family is characterized by the presence of seven hydrophobic sequences which are believed to represent mem- brane-spanning regions, and are coupled to GTP-binding proteins as signal transducers (27,28). Because of the poten- tial importance of the neurokinin A receptor in human res- piratory biology, a thorough understanding of the structure of these molecules and their regulation is indicated. In the present study we report the molecular organization of the gene for the human NK-2 receptor.

ing to nucleotides -191 to -159 in the 5’ untranslated region. The restriction map was determined from the ladder of partial digestion products and confirmed by complete digestion with the same enzymes. All mapping distances are accurate to within 100 bp.

Sequence Analysis-PstI or EcoRI fragments hybridizing with the cDNA corresponding to nucleotides 91-555 or synthetic oligonucleo- tides based on the bovine cDNA sequence were isolated by agarose gel electrophoresis, purified with GeneClean and ligated to pBluescript. Clones were expanded in Escherichia coli XL-lblue, purified by alkaline lysis and centrifugation through cesium chloride as described elsewhere (35), and subjected to double-stranded se- quencing with DNA polymerase and [?S]dATP (Sequenase, United States Biochemical Corp., Cleveland, OH). All of the exons, as well as the 5’- and 3’-flanking regions were sequenced extensively on both strands; intron-exon junctions were assigned by homology with the bovine cDNA sequence and subsequently confirmed by the human cDNA sequence.

MATERIALS AND METHODS

Preparation of RNA-Human tracheal tissue was obtained at au- topsy (approximately 4 h postmortem) from an individual with cystic fibrosis and stored at -80 “C. RNA was extracted from 3-4-z seg- ments of trachea with guanidinium thiocyanate after pulveri&g Fn liquid nitrogen (29,30) and purified by centrifugation through cesium chloride as described elsewhere (31). Poly(A+) RNA was then purified by passing the material over oligo(dT)-Sepharose (32) and transcribed into cDNA by the method of Gubler and Hoffman (33). Human gastrointestinal RNA from resected tissue was the generous gift of Dr. Chris Stevens, Beth Israel Hospital, Boston. Poly(A+) RNA and cDNA were prepared from human stomach RNA as described above.

Polymeruse Chain Reactions-Polymerase chain reaction was car- ried out initially using primers based on the cDNA sequence reported for the bovine NK-2 recentor (25). A sense mimer from nucleotides 91-108, 5’-AATGAATTCTGGCAGCTGGCACTGTGG-3’, and an antisense mimer from nucleotides 538-555. B’-AATGAATTCCC- AGGCCACCACGCACTT-3’, with EcoRI restriction sites at their 5’ ends, were synthesized using standard cyanoethyl phosphoramidite chemistry (Anulied Biosvstems model 318A DNA Synthesizer), and used with human tracheal cDNA and recombinant Taq DNA polym- erase (Perkin-Elmer) through 25 cycles consisting of 1 min at 95 “C, 2 min at 37 “C!, and 3 min at 72 ‘C with a final extension period of 7 min at 72 “C. The PCR’ uroduct was nurified following aaarose gel electrophoresis using GeneClean (Bid 101, LaJolla, CA); digested with EcoRI, repurified following a second electrophoresis, and ligated to pBluescript SK+ (Stratagene). Full-length cDNA for the human NK-2 receptor was prepared from human gastrointestinal cDNA using mimers determined from the gene sequence, and corresponded to nucleotides 1-18 and 1173-1196. These primers also contained EcoRI restriction sites at their 5’ ends. The PCR reactions utilized 5 cycles consisting of 1 min at 95 “C, 1.5 min at 45 “C, and 3 min at 72 “C, followed by 25 cycles of 1 min at 95 “C, 1.5 min at 55 “C, and 3 min at 72 “C and a final extension of 7 min at 72 “C. Ten percent of the reaction mixture was subjected to secondary PCR using the same primers and the same cycling conditions as were used for the initial reaction. The material obtained was purified. digested with EcoRI, repurified, and ligated to plasmid vector as described above for the partial cDNA.

Genomic Library Screening-A human placental genomic DNA library (generously provided by Dr. S. Orkin, Children’s Hospital and Harvard Medical School. Boston) was screened with the 32P-labeled 465-bp fragment of the human NK-2 receptor cDNA generated by PCR. Bacteriophage DNA was transferred to nitrocellulose filters in duplicate and hybridized with the probe in 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50% formamide, containing 20 mM Tris- HCl, pH 7.5, 1 X Denhardt’s solution, 10% dextran sulfate, and 0.1% SDS, for 16 h at 42 ‘C. Filters were washed 10 min in 2 X SSC, 0.1% SDS at 22 “C with three changes followed by 30 min at 68 ‘C in 0.2 x SSC, 0.1% SDS, and exposed to x-ray film (Kodak, X-Omat) at -70 “C.

Restriction Mapping-Localization of exons within the genomic clone was determined by digesting with PstI or EcoRI, electrophores- ing on 1% agarose TAE (0.04 M Tris acetate, pH 7.6, 1 mM EDTA) gels, blotting to nylon membranes (GeneScreen, Du Pont) and prob- ing with 32P-labeled cDNA corresponding to nucleotides 91-555 gen- erated by PCR, or with synthetic oligonucleotides based on the bovine cDNA sequence. Partial digests were generated with PstI or EcoRI (34), blotted as above, and probed with the 32P-labeled PstI fragment containing exon 4, or a 32P-labeled oligonucleotide primer correspond-

1 The abbreviations used are: PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; bp, base pair; HEPES, 4-(2-hydroxy- ethyl)-l-piperazineethanesulfonic acid.

Primer Extension-A synthetic oligonucleotide corresponding to nucleotides -191 to -159 in the 5’-flanking region of the gene was labeled with [Y-~*P]ATP, and 2 X lo5 cpm were annealed to 2 pg of human stomach poly(A+) RNA for 16 h at 65 “C in 30 ~1 of aqueous hybridization buffer containing 1 M NaCl, 167 mM HEPES, pH 7.5, and 0.3 mM EDTA. The annealed RNA/primer mixture was precipi- tated with ethanol from 0.3 M sodium acetate, redissolved in RT 1 buffer (Boehringer Mannheim), containing 70 units of RNasin, 20 mM dNTPs and extended with 5 units of avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim) for 90 min at 42 “C (36, 37). Remaining RNA was digested with DNase-free RNase A, extracted with phenol/chloroform (38), ethanol-precipitated, and re- dissolved in 5 mM Tris-HCl, pH 7.5, containing 48% formamide, 10 mM EDTA, 0.025% bromphenol blue, and 0.025% xylene cyan01 FF. The reaction products were analyzed by electrophoresis on a poly- acrylamide sequencing gel beside a sequencing reaction conducted with the same primer.

Chromosome Localization-Genomic DNA was prepared from mouse liver and from human leukocytes as previously described (39, 40). Aliquots of 7 pg each were digested with EcoRI, PstI or Hind111 (Boehringer Mannheim), products separated on a 1% agarose TAE gel and the DNA blotted to a nylon membrane (GeneScreen, Du Pont). The blots were hybridized with “P-labeled NK-2 receptor cDNA 91-555 obtained by PCR for 16 h at 42 “C in 50% formamide, 1% SDS, 1 M NaCl 10% dextran sulfate, containing 100 rg/ml salmon sperm DNA. Nonspecifically bound probe was washed by incubating 2 x 5 min in 2 x SSC at room temperature followed by 2 x 30 min at 65 “C in 2 x SSC, 1% SDS and the blots were exposed to x-ray film at -70 “C (Kodak, X-Omat). The restriction fragments hybrid- izing to the NK-2 receptor probe generated by EcoRI and PstI were different for human and mouse DNA. Chromosome localization for the NK-2 receptor gene was accomplished using DNA from 40 cell hybrids involving 18 unrelated human cell lines and 4 mouse cell lines (41-43). The hybrids were characterized by karyotypic analysis and by mapped enzyme markers (41, 43, 44). The human NK-2 receptor cDNA 91-555 was hybridized to Southern blots containing EcoRI-digested DNA from the human-mouse hybrids as described above. Scoring was determined by the presence or absence of human bands in the hybrids on the blots.

RESULTS

Isolation of a Partial cDNA for the Human NK-2 Receptor- Synthetic oligonucleotide primers were designed based on the sequence reported for the bovine cDNA (25) from regions of the molecule that contained sequences enriched in tryptophan or cysteine residues. One such pair encoded protein residues 30-35 and 180-185 of bovine sequence. When these primers were used in polymerase chain reaction with cDNA prepared from human trachea obtained at autopsy, they generated a 465-bp cDNA fragment, as shown in Fig. 1. The PCR product was subcloned into pBluescript for subsequent studies. Se- quence analysis confirmed the identity of this cDNA as a portion of the human NK-2 receptor by homology with the bovine molecule (data not shown).

Isolation of a Genomic Fragment Encoding the Human NK- 2 Receptor-A human genomic DNA library was probed with the 465-bp cDNA, encoding a fragment of the NK-2 receptor

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UN -

.-- .-‘.

*?A-

0

FIG. 1. Polymer

1635 1016

516

NK-2 receptor. A primer pair corresponding to nucleotides 91-108 and 538-555 of the bovine neurokinin A receptor (25) was used in PCR with -100 ng of cDNA prepared from human tracheal RNA. Conditions for PCR are detailed under “Materials and Methods.” A 465-bp fragment was specifically amplified; sequence analysis of this PCR product revealed >90% identity with the bovine structure.

E PE p E EEE? Epp " '

Exon I2 3 4 5

Ikb FIN. 2. Restriction map of the human NK-2 receptor gene.

DNA from clone NGNK-2, isolated from a human genomic library in EMBL 3, was digested with EcoRI (E) or PstI (I’) and analyzed by hybridization with appropriate cDNA or oligonucleotide probes as detailed under “Materials and Methods.” The five exons encoding the NK-2 receptor coding sequence are indicated by the thich line.

obtained from PCR. Of approximately 1 X 10” bacteriophage plaques screened, four positive clones were obtained, three of which yielded identical restriction patterns with PstI and EcoRI. One of the latter bacteriophage clones, NGNK-2, was subjected to extensive characterization. The restriction map of this clone is shown in Fig. 2 for PstI and EcoRI. Exon- containing fragments were identified by hybridization of Southern blots prepared following digestion with either of these restriction enzymes with cDNA generated by PCR or with oligonucleotide probes based on the bovine sequence. Fragments hybridizing with the tracheal cDNA probe identi- fied exons 1 and 2. Exons 4 and 5 were identified using oligonucleotides encoding the cysteine-rich sequences (CCLNHR, bovine residues 308-313 for exon 4; CCPWVT, bovine residues 324-329 for exon 5). Exon 3 was identified using an oligonucleotide probe patterned after the M5 hydro- phobic sequence, LIVIAL (bovine residues 199-205). Frag- ments identified by hybridization were then subcloned into pBluescript for double-stranded sequencing. The DNA and deduced amino acid sequences are shown in Fig. 3. The coding sequence is interrupted by four introns which vary in size from 1 to 4 kilobases. Although the overall sequence identity is approximately 90%, the human coding sequence is 42 nucleotides longer than the bovine and 30 nucleotides longer than the rat (25, 26).

Location of the Transcription Initiation Site-Analysis of

Human Neurokinin A Receptor 20457

exon 1 revealed a single open reading frame which initiated with a methionyl residue analogous to the bovine and rat sequences (25, 26). Inspection of -500 bp of sequence up- stream from the methionine revealed a putative TATA box (nucleotide -307) and a GC-like box (nucleotides -356 to -366). The transcription initiation site was determined by primer extension using a synthetic oligonucleotide corre- sponding to nucleotides -191 to -159 in the 5’-untranslated region. This region was chosen because of the proximity to the putative TATA and GC boxes, within 200 bp upstream of the primer. No other characterized transcriptional control signals were observed within 500 bp of the presumed initiating methionine. The results of this experiment are shown in Fig. 4 and locate this position at nucleotide -282.

Molecular Cloning of NK-2 Receptor cDNA Using PCR- An oligonucleotide corresponding to the 5’ end of the cDNA was prepared by analyzing the genomic sequence of exon 1 for ATG sites in the reading frame displaying some homology with the bovine sequence. An 18-nucleotide sense oligomer, containing a nested 5’-EcoRI site, was synthesized. At the 3’ end, sequence analysis of exon 5 revealed extensive homology with the bovine sequence through the M7 membrane-span- ning sequence. Sequences diverged at the genomic region corresponding to the 3’-end of the bovine coding sequence, and the stop codon occurred 42 bp downstream from the bovine homolog. An antisense oligomer, complementary to the genomic sequence proximal to the putative human stop codon, and containing a nested EcoRI site, was synthesized. As shown in Fig. 5, these primers amplified a message of approximately 1.2 kilobases from human stomach RNA, which hybridized specifically with the tracheal cDNA clone on Southern analysis (data not shown).

Chromosome Assignment-Preliminary data indicated that EcoRI and PstI restriction digests of mouse and human ge- nomic DNA yielded different patterns of hybridization with the tracheal NK-2 receptor cDNA fragment (not shown). Southern blots of EcoRI-digested mouse-human hybrid cell DNAs probed with human NK-2 receptor cDNA 91-555 and analyzed by concordance/discordance ratios indicate that the receptor is encoded on human chromosome 10 (Table I). The hybrid XTR-SBSAGB with the lOq-, lOpter+lOq23: (and no intact chromosome lo), would localize the NK-2 receptor to be on the pter-+q23 region of human chromosome 10.

DISCUSSION

We generated a cDNA fragment of the human NK-2 recep- tor molecule by reverse transcriptase-polymerase chain reac- tion using RNA from human tracheal tissue and a primer pair derived from the bovine structure (25). Sequence analysis of this partial cDNA, which spans protein residues 31-155 of the bovine molecule, was remarkable for the high degree of sequence identity between the human and bovine structures over this region (Fig. 6). Subsequent attempts to generate full-length cDNA for the human molecule were unsuccessful when primers based on the bovine sequence were used. Ret- rospectively, this is not surprising, since the 5’ and 3’ ends of the molecule are quite divergent.

The preliminary Southern blot analyses with restriction digests of genomic DNA suggested a single copy gene for the human NK-2 receptor. DNA from human-mouse hybrid cells mapped the gene to the p23-pter region of chromosome 10 (Table I). No evidence was found for cross-hybridization with related genes or pseudogenes under conditions of medium stringency. The chromosome localization of other members of the rhodopsin superfamily are largely unknown, although

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20458

FIG. 3. Nucleotide sequences of NK-2 receptor exons and intron- exon junctions. The nucleotide and de- duced amino acid sequences are shown for each exon. The numbering of nucle- otides following exon 1 is relative to the proposed coding sequences. The putative TATA and GC boxes are underlined, as is the presumed polyadenylation signal.

the olz-adrenergic receptor gene has also been mapped to several identical clones. One of these, NGNK-2, was charac- chromosome 10 (45). terized in detail. The partial cDNA provided the identification

The tracheal NK-2 receptor cDNA was used to screen a of exons 1 and 2, and allowed for preliminary mapping exper- human genomic DNA library, resulting in the isolation of iments. In order to determine the coding sequence for the

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Human Neurokinin A Receptor

2031 163!

101:

-. _ -- --

-- _ .-_

I B -250

L I ACACTGTGATTTGAATTCTGTGTTT~~A~ATGATATTCGAG~GTCTGGCCGG~GGATGGA

-200 -159 I < I

ATCTGAAATGACAATGGTTCTGGACTGGGCTTTGTGCTCAGCCCAGCTCATCTTTGCCTGAGA

FIG. 4. Primer extension analysis. An antisense olignucleotide corresponding to nucleotides -191 to -159 in the 5’-untranslated region was labeled with [‘“P]dCTP and polynucleotide kinase. The labeled primer was hybridized with -2 pg of human stomach poly(A’) RNA as described under “Materials and Methods,” and extended with avian myeloblastic virus reverse transcriptase. A, dideoxy se- quencing reaction, using the identical primer is shown for comparison. The results indicate a cytosine residue at position -282 as the transcription initiation site. B, diagramatic presentation of primer and assigned cap site, indicated by the triangle.

remainder of the molecule, we relied on the high degree of homology encountered in the initial experiments to design appropriate oligonucleotide probes for the remaining exons. Exons 4 and 5 were identified using oligonucleotides spanning regions containing Cys-Cys sequences in the bovine molecule (nucleotides 922-939 for exon 4 and nucleotides 970-987 for exon 5). We reasoned that these sequences might represent structurally important regions with potential for disulfide bond formation, and hence might be more likely to be con- served between species. Hybridization experiments with P&I- or EcoRI-digested NGNK-2 allowed for the isolation of plas- mid subclones containing these exons. Exon 3 was identified using an oligonucleotide patterned after the M5 membrane- spanning sequence, because we had come to appreciate that these sequences are, in fact, the most conserved among spe- cies. Each exon was sequenced extensively on both strands, as were the intron-exon junction sequences. All intron-exon junctions are compatible with the reported consensus se- quences (46).

Our finding of four introns in the human NK-2 receptor gene is in contrast with the organization of genes for other members of the G-protein coupled receptor family. To date, only bovine rhodopsin (47), and D, dopamine receptor (48) and an unidentified rat molecule (49) contain introns; the

20459

FIG. 5. Polymerase chain reaction for full-length human NK-2 receptor. A primer pair encoding the 5’- and 3’4erminal sequences, as deduced from genomic sequences, was used in PCR with -200 ng of cDNA transcribed from human stomach poly(A’) RNA. Primer sequences and PCR conditions are detailed under “Materials and Methods.” Hybridization and sequence analyses con- firmed the identity of the amplified product.

coding sequences of the other genes in this family are gener- ally intronless (49).

The sequence deduced from analysis of the genomic clone indicated that the human molecule would be longer than either the bovine or the rat sequences. In order to confirm this finding, we synthesized PCR primers encoding the pre- dicted 5’- and 3’-coding sequences and amplified message transcribed from human stomach RNA (Fig. 5). Hybridization and sequence analyses confirmed the identity of the PCR- generated cDNA as the NK-2 receptor.

In analyzing the sequences of the three species (Fig. 6), it is seen that significant variability occurs in the 5’- and 3’- regions of the coding sequences. This is of interest since the model for this class of receptors suggests that the amino- terminal region is part of the extracellular domain, and is potentially part of the ligand-binding site (50). A single site for N-glycosylation is preserved among the three species, at residue 19 of the predicted proteins. Human and bovine, but not rat, have an additional Asn-X-Ser sequence at residues 11-13. Also by analogy with the proposed model for adrenergic receptors, the COOH-terminal segment is the predicted cy- toplasmic tail. Recent mutagenesis studies with the &adre- nergic receptor indicate that the proposed third cytoplasmic loop and the proximal portion of the COOH terminus form an interaction site with the G, protein (50, 51). As previously mentioned, the least structural diversity is seen in the seven hydrophobic domains, which presumably form a pocket or pore within the membrane for ligand binding.

Analysis of the flanking sequences of the human neurokinin A receptor gene revealed a polyadenylation signal (AATAAA) at a position 329 nucleotides downstream from the TGA stop signal of the coding seqeunce. At the 5’-flanking end, a putative TATA box sequence (ATTTATAA) occurs at nu- cleotide -307 relative to the initiation methionyl codon. A possible modified GC box (GGGCTGGTCCCG) occurs in the 5’-flanking region at position -356 to -366. Inspection for

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1 2

3 4

5 6

7 8

9 10

11

12

13

14

15

16

17

18

19

20

21

22

X

Tran

aloca

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1108

38

L-39

66

0 AT

R-13

23

3 DU

A-3B

SAGA

19

7 DU

A-5B

SAGA

85

9 DU

A-6

186

DUM-

13

1185

GA

R-1

229

ICL-

15CS

BF

389

JSR-

2 44

JSR

-17s

653

JWR-

22H

187

JWR-

26C

1146

NS

L-9

42 R

EW-1

1 17

REW

-15

184

REX-

11BS

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254

REX-

11BS

HF

1162

RS

R-3

390

SIR-

1 1

643

TSL-

1 64

4 TS

L-2

395

VTL-

6 38

7 VT

L-8

407

VTL-

17

1098

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55

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6 84

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64

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534

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1107

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19

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12

- + - + - + - + + - + + - + + - - - - - - + + - + + + + - 4 19

21

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6 22

18

+ - + + - - - + - + + + + + - + t - - 7 23

15

+ + - 8 26

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Human Neurokinin A Receptor 20461

3. Lundberg, J. M., Brodin, E., and Saria, A. (1983) Acta Physiol.

- 225 .'+

LWI*LIYPLP~~WAYS"IGLTL-"~~Q~G~~~*~ FVKTMVLVVLTFAICWLPYH w L G I( R A

I " S

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350 375 I I

ELTPPTSLSTRVNRCHTKETL~G~APSEATSGEAGKTSVEI HP R T NTH N QV S EPAGPICKPIQ YP IF S v VN Q ES A "STEP

FIG. 6. Deduced protein sequence for the human NK-2 receptor. The human sequence is presented continuously with rat and bovine substitutions (25, 26) indicated below. Two potential glycosylation sites are indicated in the amino-terminal region by triangles. Ml through M7 indicates each of the seven hydrophobic regions characteristic of the G-protein-coupled receptors of the rho- dopsin superfamily. Significant variation at the amino and carboxyl termini are apparent.

other regulatory motifs was negative within the region scanned. The transcription initiation site was localized with primer extension analysis to a C residue at position -282, and occurs within 25 nucleotides of the proposed TATA box sequence (Fig. 3).

Comparison of the 5’-flanking sequence of the human gene with the 5’-untranslated sequence reported for the bovine cDNA revealed some interesting findings. First, comparison of the 450 nucleotides of genomic sequence upstream from the putative initiating ATG with the published bovine se- quence reveals a striking sequence identity for a portion of the noncoding structures. The human sequence beginning at position -447 extending through -156 is 65% identical to the bovine sequence from positions l-277 of the 5’-untranslated region. The 156 nucleotides of the human sequence and 135 of the bovine sequence immediately upstream from the initi- ating methionyl residue are not significantly related. This suggests that regulatory elements responsible for neurokinin A gene expression may be contained within the homologous regions. On this score, a 30-nucleotide segment (beginning at nucleotide -280 of the human sequence and nucleotide 1 of the bovine mRNA sequence) has a consensus sequence, CTG(N,)TXAXXTCT(N,)CCACAT. Further study of the regulation of gene expression of the neurokinin receptor genes will be facilitated by the molecular cloning of the human gene.

Acknowledgments-Drs. Stuart Orkin and Chris Stevens gener- ously provided the genomic library and RNA samples essential to this study. The technical assistance of Christine Squassoni is appre- ciated.

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1990, 265:20455-20462.J. Biol. Chem. 

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