From the $Garvan Institute of Medical Research, St. Vincent's Hospital, Darlinghurst, Sydney, ... This work was supported by the National Health and Medical.
Vol. 268, No. 9,Issue of March 25, pp. 6703-6707, 1993 Printed in U.S.A.
CHEMISTRY THEJOURNAL OF BIOLOGICAL
0 1993by The American Society for Biochemistry and Molecular Biology Inc.
Genomic Organization, Localization, andAllelic Differences in the Gene for the Human Neuropeptide Y Y1 Receptor* (Received for publication, November 2, 1992)
Herbert HerzogSB, Martina Baumgartnerll, Christina ViveroS, Lisa A. SelbieS, Bernhard AuerlI, and John Shine* From the $Garvan Institute of Medical Research, St. Vincent’s Hospital, Darlinghurst, Sydney, New South Wales 2010, Australia and the llnstitut f u r Biochemie, Uniuersitat Innsbruck, Peter Mayr Strasse la, 6020 Innsbruck, Austria
A 14-kilobase pair (kb) region of genomic DNA en- at residues 31 and 34 (Led1, Pro34) (3). The Y2 receptor coding the human neuropeptide Y Y1-receptor gene subtype is mainly found in the central nervous system and is including 3’- and 5’-flanking sequenceshasbeen defined on the basis of its affinity for NPY peptide derivatives cloned and the human gene localized to chromosome containing carboxyl-terminal fragments (4,5). NPY Y1 recep4q(31.3-32). In contrast to the contiguous structure tors of have been identified in a variety of tissues, including most G protein-coupled receptor genes, the NPY Y1 brain, spleen, small intestine, kidney, testis,placenta,and receptor gene is divided into three exons. A small 5‘- aortic smooth muscle (1).In addition, Y1 receptors have been exon of the mRNAuntranslated regionis separated by reported in a number of rat and human cell lines (6). a 6-kb intron from the second exon. The coding regionIn peripheral tissues, the Y1 receptor is found predomiof the receptor is interrupted by a small intron, con- nantly at sympathetic postjunctional sites in blood vessels, taining an in-frame stop codon, shortlyafter the pro- especially small cardiac and renal arterioles, and is associated posed fifth transmembrane domain. In the5“flanking with vascular smooth muscle contraction, both directly and region a potential CAMP-responseelement and an AP2 site, inadditiontoaTATA-likesequenceand a indirectly by potentiating the action of other pressure agents typical CAAT, box are present. A single point mutation such as noradrenaline (7). NPY receptors inthecentral within the 6-kb intron generates a PstI polymorphic nervous system are also involved in the central regulation of site with a highly informative allele frequency of blood pressure acting in a vasodepressor way. Other potent effects of NPY, such as stimulation of food intake and regu54:46%in the population. lation of hormone secretion, are also mediated by the Y1 type of receptor (1). Molecular cloning of the NPY Y1 receptor from human (8, 9) and rat(10) tissues has shown that thereceptor is a member Neuropeptide Y (NPY)’ receptors are activated by one of of the G protein-coupled receptor superfamily. To determine the most abundant peptides in the mammalian nervous system and subsequent!y influence a diverse range of important the molecular organization and regulation of the human NPY physiological parameters, including effects on psychomotor Y1 receptor gene, we isolated several genomic clones, subactivity, food intake; central endocrine secretion, and potent cloned and sequenced the exons and intron/exon boundaries, vasoactive effects on the cardiovascular system. Within the mapped the transcription start site, and identified a common central nervous system, NPY is co-localized with noradrena- PstI polymorphism in the first intron of the gene. By using line in discrete areas known to be essential for the mainte- high resolution fluorescence in situ hybridization, we were nance of bloodpressure. Within the periphery, NPY is present also able to localize the position of the single human NPY Y1 in postganglionic sympathetic nerves being co-localized and receptor gene to chromosome 4q(31.3-32). co-released with catecholamines. Particularly high concentraMATERIALS ANDMETHODS tions of NPY are found in the sympathetic nerves supplying Genomic Library Screening-A human peripheral blood genomic the coronary, cerebral, and renal vasculature and when infused into these vascular beds, NPY causes a prolonged vaso- DNA library (Clontech) was screened with a 32P-labeled2.3-kb fragment (nucleotides 14-2327) of the human NPY Y1 receptor cDNA constriction (reviewed in Refs. 1 and 2). (8).Bacteriophage DNA wastransferred to Hybond N’ filters (AmerTwo major NPY receptor subtypes (Y1 and Y2) have been sham Corp.) and hybridized with the probe in a solution containing defined by pharmacological criteria, with the Y1 receptor 6 X SSC, 5 X Denhardt’s, and 0.1% SDS at 65 “C for 16 h. Filters having the ability to respond to an analog of NPY modified were washed twice 15 min in 2 x SSC (1 X SSC: 0.15 M NaCl, 0.015 * This work was supported by the National Health and Medical Research Council (Australia). 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 solelyto indicate this fact. The nucleotide sequence(s)reported in this paperhas been submitted to the GenBankTM/EMBLData Bank with accession number(s) LO7614 and LO7615. 5 Fellow of the E. Schrodinger Foundation (Austria). To whom correspondence should be sent: Garvan Institute of Medical Research, 384 Victoria St., Darlinghurst NSW 2010, Sydney, Australia. Tel.: 61-2-361-2050;Fax: 61-2-332-4876. The abbreviations used are: NPY, neuropeptide Y; kb, kilobase pair; bp, base pair.
M sodium citrate), 0.1% SDS at 65 “C followed by a 15-min wash in 0.1 X SSC, 0.1% SDS and exposed to x-ray film (Kodak, X-Omat) using an intensifying screen a t -70 “C for 16 h. Positive plaques were
purified and DNAwas isolated using a standard lysate procedure (11).h DNA was digested with EcoRI, HindIII, BamHI, Sad, XhoI, or combinations of these enzymes to generate subsequently ordered subclones in the Bluescript SK vector (Stratagene) covering the entire region of the NPY Y1 receptor gene. Nucleotide Sequence Determination-Supercoiled plasmid DNA was alkaline-denatured and sequenced by the dideoxy chain termination method using T7 polymerase (Promega). Oligonucleotide primers were initially from the flanking region of the vector and internal to thecDNA sequence. Additional primers were synthesized based on intron sequence obtained. Southern Blotting-Human genomic DNA (8 pg) wasdigested with
6103
6704
Genomic Organization
ofHuman the
restriction enzymes, electrophoresed on 1%agarose gels, alkalinedenatured (0.4 M NaOH), capillary-transferredusing 0.4 M NaOH to Hybond N' membranes, and hybridized as described above. In Situ Hybridization-Lymphocyte cultures from normal donors were treated as described previously (12). Slides were kept at -20 "C until the day of hybridization. Slides were treated for 1h with RNase (100 pglml, 2 X SSC) at 37 "C and rinsed three times for 10 min in 2 X SSC, then dehydrated in 50,70, and 100% ethanol. Chromosomes were denatured in 70% formamide, 2 X SSC (pH 7) at 70 "C for 2 min, rinsed at 4 "C for 2 min in 2 X SSC, then 50, 70, and 100% ethanol. The slides were incubated overnight at 42 "C with 18 pl of hybridization buffer (50% (v/v) deionized formamide, 10% (w/v) dextran sulfate, 2 X SSC, 40 mM sodium phosphate, 0.1% SDS, 1 X Denhardt's solution, pH 7) containing5 or 10 ng/pl biotinylated probe, which had been denatured previously for 5 min at 100 "C. Immunofluorescence Detection-The slides were rinsed twice in 50% formamide, 2 X SSC, three times for 2 min in 2 X SSC at 42 " C , and 5 min in modified bicarbonate buffer (BN, 0.1 M NazCO3,pH 8, 0.05% Nonidet P-40,5% fat free milk powder). Slides were incubated 20 min at 37 'C with 100 pl of anti-biotin antibody (Sigma) in a dilution of 1:2500 in modified BN. After 5 min wash in modified BN the slides were incubated for 45 min at 37 "C with 100 p1 of avidinfluorescein isothiocyanate (Vector Laboratories) diluted 1:20 in modified BN. After washing three times for 2 min in BN at 45 "C the slides were incubated for 20 min at 37 "C with 100 pl of biotinylated anti-avidin (Vector Laboratories) diluted 1:IOO in BN 5% goat serum. After the same washing as before and incubation for 5 rnin in BN, avidin-fluorescein isothiocyanate was applied for a second time. The slides were stained with propidium iodide at a final concentration of 0.5-1 pg/ml for 3 min and briefly rinsed with phosphate-buffered saline. Slides were mounted with p-phenyldiamine, observed, and photographed using the Zeiss Photomicroscope 111 with the filter combinations 487701 and 487709. R Banding-Before using the slides for the in situ hybridization they were briefly rinsed in distilled water and stained with Hiichst 33258 (0.1 pglpl) for 25 min, washed with 2 X SSC, and exposed to UV light (365 nm) for 25 min. The slides were dehydrated in an ethanol series and air dried before using for fluorescence in situ hybridization.
+
RESULTS AND DISSCUSION
Isolation of the Human NPY Y1 Receptor Gene-A human genomic DNA library constructed in X GEM 11phage vector (Clontech) was screened with a 32P-labeledcloned NPY Y1 receptor cDNA (nucleotides 14-2327) isolated from human hippocampal cDNA (8). Two positive clones were obtained from 7.5 X lo5bacteriophage plaques. Clone XC contained an insert of approximately 14 kb and clone XD contained an insert of approximately 11 kb. The insert in XD was subsequently shown to be completely contained within the longer 5.1 kb
NPY Yl Receptor
clone. The restriction map for SacI, XhoI, BamHI, and EcoRI of the XC clone is shown in Fig. 1. The insert of this clone was digested with different enzymes and the fragments subcloned into theBluescript SK vector for sequencing. The exon sequence of the NPY Y 1 receptor gene is identical to that of the human hippocampal cDNA previously reported (8). The human NPY Y1 receptor gene consists of 3 exons. This is in contrast to many of the other G protein-coupled receptor genes, which are intronless. The overall sequence of the gene consists of approximately 10 kb (Fig. 1).The first 57 nucleotides of the 5"untranslated sequence of the human hippocampal NPY Y1 receptor mRNA are separated by a 6-kb intron from the second exon. The second intron (97 bp), containing an in-frame stop codon, is located exactly after the proposed fifth transmembrane domain at nucleotide 908 corresponding to the cDNA sequence (Fig. 2). Introns in several other Gcoupled receptor genes (human substance K, human rhodopsin) tend to be positioned the same way, shortly after or in front of a transmembrane domain (13). This organization is also consistent with suggestions that the following third cytoplasmatic loop of the receptor forms a specific domain involved in determination of the specificity of coupling to different G proteins(14). The nucleotide sequences of the two introns adjoining the splice junctions (Fig. 2) are consistent with the recognized consensus sequence GT/AG (15). Putative Regulatory Sequence Elements-Identification of the transcriptional initiation sitewas carried out with primer extension, using a 21-mer primer corresponding to nucleotides 38-18. The primer when extended on mRNA derived from the NPY Y1 receptor-specific expressing human neuroblastoma cell line SK-N-MC revealed a transcription start site at 210 nucleotides upstream from the initiation start codon (data notshown). This position is also identical with a type 1 cap site (CCATTC) (16) and is accompanied 35 bp upstream by a TATA box-like motif (AAATAC)(17), a typical CAAT box (TCAATCT) (18) 60 bp upstream, a CAMP-response element (CGACGTCA) (19) 124 bp upstream, and a AP-2 recognition site (GCGAGCCC) (20) 451 bp upstream (Fig. 2). No other typical transcription factor binding sites are found in the orientation in this region. A potential polyadenylation site (ATTAAA) was found at position 2670 of the cDNA sequence. The approximate 2.7-kb sequence of the cDNA is consistent with the size of the NPY Y1 receptor
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FIG.1. Organization of the human NPY Y 1 receptor gene. A schematic representation of the gene for the human hippocampus NPY Y1 receptor is shown (A). The corresponding positions of the 3 exons with respect to the hippocampus NPY Y1 cDNA are shown as boxes above the cDNA ( B ) . The positions of the translation initiation site (ATG) and termination codons are indicated. Restriction sites indicated E (BamHI), E (EcoRI), S (SacI),X (XhoI), P (PstI),and P* (polymorphic PstI site).
6705
Genomic Organizationof the HumanNPY Y1 Receptor AARTATTCAGCTCATGGTTCTCCCCTAAAAAAACTAAAAAGCAGCTAAGCGCTGGGAAC~TCTGACTTATTGCATTTTCTCAGTGGGCC~G~GGAGGGCCGATT~CTGCTTTG-590
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FIG. 2. Coding region and 6'-and 3"flanking sequences of the human NPY Y 1 receptor gene. The 5 ' - and 3"flanking sequences of the first exon of the human NPY Y1 receptor gene are shown. The major start transcription site identified by primer extension is indicated by +1. The numbering is according to the cDNA sequence. The potential recognition sequences for various transcription factors are boxed as indicated (CRE, cyclic AMP-response element). The sequence for exon I is underlined. The shown intron sequences are dotted underlined. A potential polyadenylation site is double underlined.
Genomic Organizationof the Human NPY Yl Receptor
6706
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FIG. 4. Fluorescence in situ hybridizationofthehuman neuropeptide Y 1 receptor. Y a, clone XC (yellow dots) hybridizes to the long arm of chromosome 4,corresponding to the region q31.3q32. At the right the same detail of the metaphase where R banding can be seen after filter change is shown. b, single chromosome 4 with two hybridization signals. c, histogram of the distribution of the fluorescent spots in 44 metaphases hybridized with the human NPY Y1 receptor gene.
9 9
B
FIG. 3. Southern M&& of genomic DNA encodingthe human NPY Y1 receptor gene. A, restriction enzyme analysis of DNA from two individuals heterozygote for a PstI polymorphism. Genomic DNA was digested with restrictionendonucleases SacI, PstI, BamHI, and EcoRI and subjected to electrophoresis in 1% agarose gel. B, PstI polymorphism in five unrelated individuals. a and b, homozygote 5.115.1;c, heterozygote 5.113.8; d and e, homozygote 3.81 3.8. Southern analysis was performed as described under "Materials and Methods" using the *P-labeledcDNA fragment (nucleotides 142327). HindIII digested X DNA was used as molecular marker.
TABLEI NPY Yl receptor gene PstI polymorphism frequency Allele Genotypes 3.813.8 5.115.1 3.815.1
3.8
Samples (69)
22
30
17
kequency
31.9%
43.4%
74 24.6% 53.6% 46.4%
5.1
64
mRNA demonstrated in Northern analysis of placental and kidney mRNA (21). Characterization of a PstI Polymorphism in the Human NPY Y1 ReceptorGene-Southern blot analysis was per-
formed usingthe 32P-labeledcDNA fragment (nucleotides 142327) as a probe (Fig.3A). The restriction patterns obtained with BamHI, EcoRI, and Sac1 correspondexactly to the fragments found in the human genomic clones. However,the pattern for the restriction enzyme PstI shows an additional 5.1-kb band, which suggests the presence of a polymorphic site within the first intron of the gene. Southern blot analysis of genomic DNA samples from 69 normal individuals confirmed this suggestion (Fig. 3B) and demonstrated that the allele frequency for the PstI polymorphism in this population is 54%:46% (Table I). Sequence analysis revealedasingle point mutation in the recognition site for the restriction enzyme PstI, changing the first cytidine to a thymidine. Chromosomal Localization-High resolution fluorescencein situ hybridization was used to determine the chromosomal location of the human NPY Y1 receptor gene. The genomic clone XC was labeled by standard nick translation with biotin11-dUTP and hybridized to human metaphase spreads as described under "Materials and Methods." Hybridization of the biotinylated probe was detected with fluoresceinisothiocyanate-conjugated avidin and biotinylated anti-avidin. The exact location sites of the spots on the chromosomes were revealed by changingthe filter to make the R banding of the chromosome visible. Analysis of 44 metaphase spreads revealed a total of 208 fluorescent signals (4.7/metaphase). Of the total fluorescent spots recorded 43 (21%) were localized at the region q31.3932 of chromosome 4, as identified from R banding pattern (Fig. 4a). In 41% of these metaphase spreads, specific signals were present at both chromatids of chromosomes 4. There was no other significant signal cluster on any other chromosome. Localization to chromosome4 was confvmed with polymerase chain reaction analysis of somatic cellhybrid DNA using two different sets of NPY Y1 receptor-specific oligonucleotides (data not shown). This result demonstrates
Genomic Organization of the Human NPY Y l Receptor that there is a single gene for the human NPY Y1 receptor which is located in the region q31.3-q32 of chromosome 4. The 4q(31.3-32) region has also been shown to contain the genes encoding the mineralocorticoid receptor (22) and the recently described Type I11 growth factor receptor tyrosine kinase (23). Furthermore, loss of heterozygosity of the 4q region has been shown to be involved in development of primary hepatocellular carcinoma (24). The use of the highly informative NPY-Y1 receptor-associated PstI polymorphism should therefore be of use in analysis of this receptor in the regulation of cardiovascular parameters and itspotential role in the pathophysiology of hypertension and a range of other potential metabolic disorders reflecting the role of these closely linked genes, e.g. NPY-Y1 receptor in obesity and behavioral disorders, NPY-Y1 and mineralocorticoid receptors in hypertension and salt balance, NPY-Y1 receptor and the Type I11 tyrosine kinase in tumorgenesis. Acknowledgments-We gratefully acknowledge the expert advice of Prof. M. Hirsch-Kauffmann and the assistance of M. Liu. We thank P. Schofield for critical reading of the manuscript. REFERENCES 1. Dumant, Y., Martel, J. C., Fournier, A., St-Pierre, S. & Quirion, R. (1992)
Prog. Neurobwl. 3 8 , 125-167 2. Heilig, M. & Widerlov, E. (1990) Acta Psychiatr. S c a d . 8 2 , 95-114 3. Fuhlendorff, L., Gether, U., Aakerlund, L., Langeland-Johansen, N., Thogersen, H., Melherg, S. G., Olsen, u. B., Thastrup,0. & Schwartz, W. T. (1990) Proc. Natl. Acad. Sci. U. S. A. 87. 182-186
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4. Wahlestedt, C., Yanaihara, N. & Hakanson, R. (1986) Regul. Pept. 1 3 , 307-318, 5. Beck-Sickmger, A. G., Grouzmann, E., Hoffmann, E., Gaida, W., Van Meir, E. G., Waeber, B. & Jung, G. (1992) Eur. J.Biochern. 206,957-964 6. Aakerlund, L., Gether, U., Fuhlendorff, J., Schwartz, T. W. & Thastrup, 0. (1990) FEBS Lett. 260,73-78 7. Potter, E. K., Fuhlendorff, J. & Schwartz, T.W. (1991) Eur. J. Pharrnacol. 1 9 3 , 15-19 8. Herzog, H., Hort, Y. J., Ball, H. J., Hayes, G., Shine, J. & Selbie, L. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 8 9 , 5794-5798 9. Larhammar, D., Blomqvist, A. G., Yee, F., Jazin, E., Yoo, H. & Wahlestedt, C. (1992) J. Biol. Chern. 267,10935-10938 10. Krause, J., Eva, C., Seeburg, P. H. & Sprengel, R. (1992) Mol. Pharmncol. 41,817-821 11. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring . . HarborLaboratory Press, Cold Spring Harbor,NY 12. Baumgartner, M., Dutrillaux, B., Lemieux, A., Lilienbaum, A., Paulin, D. & Viegas-PB uignot, E. (1991) Cell 64,761-766 13. Prohst, W. C.,%nyder, L. A., Schuster, D. I., Brosius, d . & Sealfon, S. C. (1992) DNA (N. Y . ) 1 1 , l - 2 0 14. Selhie, L. A., Ha es, G. & Shine, J. (1989) D N A ( N .Y . ) 8,683-689 15. Breathnach, R. Chambon, P. (1981) Annu. Reu. Bwchern. 60,349-383 16. Nahon, J. L., Venetianer, A. & Sala-Trepat, J. M. (1987) Proc. Natl. A d . Sci. U. S. A. 84,2135-2139 17. McKni ht S & Kingsbury, R. (1982) Science 217,316-318 18. Chodos%, A., Baldwin, A. S., Carthew, R. W. & Sharp, P. A. (1988) Cell 63,ll-24 19. h e a l e r , W. J., Vandenhark, G. R. & Hanson, R. W. (1988) J. Bid. Chern. 263,9063-9066 20. Imagawa, R., Chiu, R. & Karin, M. (1987) Cell 61,251-260 21. Wharton, J., Gordon, L., Byrne, J., Herzog, H., Selhie, L. A., Moore, K., Sullivan, M. H. F., Elder, M. G., Moscoso, G., Taylor, K. M., Shine, J., and Polak, J. M. (1993) Proc. Natl. Acad. Scr. U. S. A , , in press 22. Morrison. N.. Harrau. S. B.. Arriza.. J. L... Bovd. ~. E. & Connor. J. M. (1990) Hum. Gemit. 8 6 , i30-132 23. Terman, B. I.,Jani-Sait, S., Carrion, M. E. & Shows, T. B. (1992) Cytogenet. Cell Genet. 60, 214-215 24. Buetow, K. H., Murray, J. C., Israel, J. L., London, W. T., Smith,M., Kew, M., Blanquet, V., Brechot, C., Redeker, A., and Govindarajah, S. (1989) Proc. Natl Acad. Sei. U. S. A. 86,8852-8856
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