Characterization of the Human Type 2 Neuropeptide Y Receptor Gene (NPY2R) and Localization to the Chromosome 4q Region Containing the Type 1 Neuropeptide Y Receptor Gene

GENOMICS

38, 392–398 (1996) 0642

ARTICLE NO.

Characterization of the Human Type 2 Neuropeptide Y Receptor Gene (NPY2R) and Localization to the Chromosome 4q Region Containing the Type 1 Neuropeptide Y Receptor Gene DAVID A. AMMAR,* DEBORAH M. EADIE,† DEBORAH J. WONG,† YEN-YING MA,‡ LEE F. KOLAKOWSKI, JR.,§ TERESA L. YANG-FENG,‡ AND DEBRA A. THOMPSON*,†,1 †Department of Ophthalmology and *Department of Biological Chemistry, the University of Michigan Medical School, Ann Arbor, Michigan 48105; §Department of Pharmacology, the University of Texas Health Science Center, San Antonio, Texas 78284; and ‡Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510 Received August 13, 1996; accepted October 14, 1996

Neuropeptide Y (NPY) signals through a family of G-protein-coupled receptors present in the brain and sympathetic neurons. To further our understanding of the genetic elements involved in the regulation of NPY receptor expression, we have cloned and characterized the human gene encoding the type 2 NPY receptor (Y2 receptor, HGMW-approved symbol NPY2R).2 The transcript spans 9 kb of genomic sequence and is encoded on two exons. As in the type 1 NPY receptor (Y1 receptor) gene, the 5*-untranslated region of the Y2 receptor is interrupted by an intervening sequence (Ç4.5 kb). However, the Y2 receptor gene does not contain an intron analogous to that present in the coding region of the Y1 receptor. The predicted transcript size (Ç4.5 kb) is consistent with the size observed by Northern analysis. The 381-amino-acid sequence deduced from the open reading frame is identical to that encoded by the cDNA. The Y2 receptor gene maps to human chromosome 4q31, the same region containing the Y1 receptor locus, suggesting that these subtypes may have arisen by gene duplication despite their structural differences. q 1996 Academic Press, Inc.

INTRODUCTION

Neuropeptide Y (NPY) is a 36-amino-acid peptide hormone widely distributed throughout the central and peripheral nervous systems. The most abundant neuropeptide in brain, NPY is present at high levels in the basal ganglia, amygdala, nucleus accumbens, and hySequence data from this article have been deposited with the GenBank/EMBL Data Libraries under Accession Nos. U50145 and U50146. 1 To whom correspondence should be addressed at University of Michigan Medical School, Kellogg Eye Center, 1000 Wall Street, Ann Arbor, MI 48105-0714. Telephone: (313) 936-9504. Fax: (313) 6470228. E-mail: [email protected]. 2 The HGMW-approved symbol for the gene described in this paper is NPY2R.

0888-7543/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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pothalamus (Tatemoto et al., 1982; Adrian et al., 1983). In the periphery, NPY is associated with postganglionic sympathetic neurons supplying the vasculature (Lundberg et al., 1982, 1983). NPY functions include regulation of blood pressure, appetite, mood, and circadian rhythms. Aberrations in NPY signaling pathways are predicted to result in hypertension, eating disorders, and anxiety (Grundemar and Ha˚kanson, 1994). An example is the genetically obese mouse (ob/ob), in which a truncated form of the ‘‘satiety factor,’’ leptin, fails to activate its receptor. Downstream effects include a failure to inhibit NPY synthesis and release in response to feeding (Stephens et al., 1995). NPY signals through a heterogeneous population of receptors differing in tissue distribution and specificity of ligand binding. In early pharmacological assays, three NPY receptor subtypes (Y1, Y2, Y3) were defined by their affinity for NPY analogs and carboxy-terminal fragments and the related peptides, peptide YY (PYY) and pancreatic polypeptide (PP) (Wahlestedt et al., 1991). A number of additional NPY receptor subtypes are now known as the result of molecular cloning studies. The Y1 receptor was the first to be characterized, when the expression pattern of an orphan receptor was recognized to overlap with the distribution of NPY in brain (Larhammar et al., 1992; Herzog et al., 1992). Subsequently, cDNAs encoding two additional NPY receptors were identified using the Y1 receptor sequences for low-stringency screening: a Y4 receptor exhibiting highest affinity for PP (also called the PP1 receptor) (Bard et al., 1995; Lundell et al., 1995) and a mouse Y5 receptor exhibiting Y1 receptor-like pharmacology (Weinberg et al., 1996). Homology screening did not result in the identification of Y2 or Y3 receptor sequences. However, Y2 receptor cDNAs have been identified using a number of different expression cloning strategies (Rose et al., 1995; Gerald et al., 1995; Gehlert et al., 1996; Rimland et al., 1996). This approach also resulted in the identification of cDNAs encoding rat and human Y5 receptors (distinct from the mouse Y5

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receptor) proposed to be involved in the feeding response (Gerald et al., 1996). The Y3 receptor, specific for NPY and not PYY, has yet to be cloned. The Y2 receptor, pharmacologically defined by its high affinity for carboxy-terminal fragments of NPY, functions in the presynaptic inhibition of neurotransmitter release (Wahlestedt et al., 1986). This subtype is likely to be the predominant form present in brain, being highly enriched in the amygdala, hypothalamus, hippocampus, and frontal lobe (Widdowson, 1993). Y2 receptors are also associated with sympathetic neurons innervating the vas deferens, blood vessels, kidney proximal tubules, and intestinal mucosa (Wahlestedt et al., 1986; Laburthe et al., 1986; Sheikh et al., 1989). The percent sequence identity between the Y2 receptor and the other NPY receptor subtypes is relatively low (30–34%), despite their shared high affinity for NPY, suggesting that relatively small structural domains specify the interaction of ligand with receptor. To explore the structural relationships of the NPY receptor subtypes and their differential expression further, we have cloned the gene encoding the human Y2 receptor.3 The probes used were Y2 receptor cDNAs identified using RT-PCR with degenerate oligonucleotide primers corresponding to G-protein-coupled receptor consensus sequences. We now report the characterization and chromosomal localization of the human Y2 receptor gene. Comparison with the human gene encoding the Y1 receptor reveals a number of similarities and differences, including localization of both genes to the same region of chromosome 4q. MATERIALS AND METHODS RT-PCR of bovine Y2 receptor cDNA. Total RNA was isolated from bovine retinal/retinal pigment epithelial cells by centrifugation through CsCl, and the poly(A) fraction was selected on oligo(dT) cellulose (Chirgwin et al., 1979). First-strand cDNAs were synthesized using 2.5 mg poly(A) RNA, 5 mM random hexamers, 1 mM each dNTP, 50 U RNasin, 1 mM dithiothreitol, and 275 U M-MLV–reverse transcriptase (Gibco/BRL) in the buffer supplied. G-protein-coupled receptor sequences were amplified using oligonucleotide primers A1– A5 and B1–B6 in pairs (Buck and Axel, 1991) in reactions containing 0.5 mM each primer, template from 0.2 mg RNA, 200 mM each dNTP, 1.5 mM MgCl2 , and 2.5 U AmpliTaq polymerase (Perkin–Elmer) in the buffer supplied, in 48 cycles of 45 s at 967C, 2 min at 457C, and 3 min plus 6 s/cycle at 727C. Products were ligated into pCR1000 (Invitrogen), pAMP1 (Gibco/BRL), or SmaI-cut pBluescript II SK(0) (Stratagene). Plasmid DNA from approximately 100 independent clones derived from 18 primer pairs was prepared and sequenced (Sanger et al., 1977). A single clone, 1A2.bn1, derived from primer pair A1B1 contained a 673-bp insert homologous to human Y2 receptor cDNA (see below). Identification of human Y2 receptor genomic DNA. A human genomic library in lambda DASH II (Stratagene) was infected into XL1-Blue MRA (P2) cells and plated on NZYM agar. The insert from bovine cDNA 1A2.bn1 was radiolabeled with [a-32P]dCTP by random primer extension (Rediprime; Amersham) and used as probe. Dupli3 Abbreviations used: NPY, neuropeptide Y; PYY, peptide YY; RTPCR, reverse transcriptase coupled–polymerase chain reaction; SSC, sodium chloride/sodium citrate; Y1–Y5 receptors, types 1–5 neuropeptide Y receptors.

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cate lifts were hybridized overnight at 657C in 61 sodium chloride/ sodium citrate (SSC), 11 Denhardt’s, and 0.1% SDS (Sambrook et al., 1989). Final stringency washes were at 657C in 0.11 SSC and 0.1% SDS. Phage DNA was prepared from a single third-round positive, 1A2.hg2, and insert DNA was released by NotI digestion, ligated into pGEM-5Zf(0) (Promega), and amplified in JM109 cells. Plasmid DNA was isolated, and a map of the genomic DNA insert was derived from Southern transfers of restriction enzyme digests of the DNA (1–2 mg) transferred to nylon membranes (Southern, 1975). Hybridizations were performed as described above, with the following probes: the bovine cDNA, 1A2.bn1; the 1.6-kb EcoRI fragment of 1A2.hg2 containing the entire open reading frame; a 697-bp EcoRI– BglII fragment of 1A2.hg2 centered over the initiation codon; an 827bp PvuII–PstI fragment of 1A2.hg2 centered over the termination codon; and pGEM-5Zf(0). The sequence of the genomic DNA was determined using a combination of automated and manual sequencing with primers corresponding to internal sequences. Identification of Y2 receptor 5*-untranslated sequence. To amplify sequences corresponding to the 5*-untranslated region of the human Y2 receptor, first-strand cDNAs were synthesized as described above using 100 ng human hippocampal poly(A) RNA (Clontech) and 0.8 mM primer (r2) corresponding to residues 0151 through 0169 in the genomic sequence. Amplification reactions contained template derived from 4 ng RNA and 0.4 mM each primer, corresponding to residues 01630 through 01650 (f1), 01464 through 01481 (f2), 01252 through 01270 (f3), and 01175 through 01192 (f4) for the upstream primers and to residues 0653 through 0670 (r1) for the downstream primer. PCR was performed using 40 cycles of 1 min at 947C, 1 min at 537C, and 2 min at 727C. Human genomic DNA (10 ng) served as a positive control. Products were run on agarose gels, and Southern blots were probed with a 32P-end-labeled primer (Sambrook et al., 1989) corresponding to residues 0926 through 0961 (h1). Hybridizations were at 427C in Rapid Hybe (Amersham) for 1 h, and final washes were at 427C in 0.11 SSC and 0.1% SDS for 15 min. Chromosomal localization—Hybrid cell line screening. Mapping Panel No. 1, consisting of 17 mouse–human and 1 Chinese hamster– human hybrids, was obtained from the NIGMS cell repository. Characterization and human chromosome content in these hybrids are described in detail in the NIGMS catalog. DNA samples were digested with BglII, and Southern transfers were screened by hybridization as described in Yang-Feng et al. (1985) using as probe the 32Plabeled bovine cDNA, 1A2.bn1, or the 350-bp EcoRI/PstI fragment of the genomic clone 1A2.hg2 corresponding to the Y2 receptor 3*untranslated region. In situ hybridization of metaphase chromosomes. The 1.6-kb EcoRI genomic subclone of 1A2.hg2 was nick-translated with biotinylated dUTP and used for hybridization at a concentration of 50 ng/ml. Hybridization to metaphase chromosomes, posthybridization washes, and signal detection with avidin–fluorescein isothiocyanate were carried out essentially as previously described (Shaper et al., 1992). For their identification, chromosomes were counterstained with DAPI (4,6-diamidino-2-phenylindole) and cohybridized with a chromosome 4 centromere probe.

RESULTS

Isolation of cDNA and Genomic Clones A genomic clone, 1A2.hg2, encoding the human Y2 receptor and containing 13.5 kb of insert DNA, was obtained by screening a human genomic library with the bovine cDNA, 1A2.bn1. The bovine probe was obtained using RT-PCR with degenerate oligonucleotide primers corresponding to G-protein-coupled receptor consensus sequences (Buck and Axel, 1991) and is 90.3% identical to published human Y2 receptor cDNAs in the region corresponding to transmembrane regions 2 through 7. Southern blots of the genomic clone

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FIG. 1. Structure of the human gene encoding a type 2 NPY receptor. (A) The genomic map derived from clone 1A2.hg2. Exons are indicated by boxes, with filled areas corresponding to the protein coding region and open areas corresponding to the untranslated regions. (B) The map of the transcribed region, derived from sequence analysis of 1A2.hg2 and published cDNAs (see text). Open and filled boxes are as in (A). The position of the 5*-end of the transcript corresponds to the minimum predicted size. Restriction enzyme cleavage sites are as shown. A, ApaI; B, BamHI; E, EcoRI; H, HindIII; G, BglII; P, PstI; S, ScaI; V, PvuII.

1A2.hg2 probed with the bovine cDNA revealed a single 1.6-kb cross-hybridizing EcoRI fragment located approximately 9 kb downstream from the 5*-end of the insert DNA. Restriction enzyme mapping and sequence analysis were used to compare the sequences of the genomic clone and human Y2 receptor cDNAs. The restriction map is shown in Fig. 1. The 1.6-kb EcoRI fragment contains the full-length protein coding sequence. The genomic clone contains the entire Y2 receptor transcript, spanning approximately 9 kb and interrupted by a single intron in the 5*-untranslated region (see below). Characterization of the Genomic Sequence Analysis of approximately 4000 bp of sequence surrounding the 1.6-kb EcoRI fragment of genomic clone 1A2.hg2 revealed an uninterrupted 1143-bp open reading frame encoding the full-length Y2 receptor. The sequence of the open reading frame and the 3*-untranslated region to position 3140 (relative to the initiation codon) is identical to that of the cDNA reported by Gehlert et al. (1996), with the exception of a silent C to T substitution at position 585. This substitution is also present in the sequence reported in Gerald et al. (1995), but not in Rose et al. (1995), and may represent a polymorphism occurring in the human population. A polyadenylation signal (ATTAAA) (Sheets et al., 1990) is present at position 3117, and two potential eukaryotic terminator consensus sequences (YGTGTTYY) (McLauchlan et al., 1985) are present at positions 3145 and 3165 (Fig. 2). This analysis and the locations of poly(A) tracts in the published cDNAs indicate that the 3*-untranslated region is 1996–1998 bp and is not interrupted by introns. On the other hand, sequence analysis and restriction enzyme mapping of the Y2 receptor 5*-flanking region revealed the presence of a single, large intron. As shown in Fig. 2, the genomic sequence diverges from the cDNA at position 049 relative to the initiation codon, then matches again approximately 4.5 kb upstream, to position 0489 of the longest

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published 5*-untranslated sequence (Gehlert et al., 1996). The intron boundaries contain the invariant GT (splice donor) and AG (splice acceptor) residues, as well as surrounding sequences in close agreement with consensus splice site sequences (Padgett et al., 1986). A thymidine-rich region is present in the intron just upstream of the 3*-splice site. To determine the approximate location of the transcription start site in the Y2 receptor gene, RT-PCR was used to amplify the 5*-end of the transcript. Firststrand cDNAs were synthesized from hippocampal mRNA using a primer corresponding to residues 0151 through 0169 relative to the initiation codon. Amplifications were performed using primers corresponding to upstream sequences, and the products were analyzed by hybridization with a probe corresponding to sequence within the amplified region. The results of one such experiment are shown in Fig. 3. PCR products of the expected sizes were amplified from first-strand cDNAs using primers corresponding to residues 01252 through 01270 (f3) and 01175 through 01192 (f4) (Fig. 3, lanes 5 and 6), whereas primers corresponding to residues 01630 through 01650 (f1) or 01464 through 01481 (f2) (Fig. 3, lanes 7 and 8) did not reproducibly amplify the expected products. In contrast, all four products were amplified from reactions containing human genomic DNA (Fig. 3, lanes 1–4) and were not seen in the absence of template (Fig. 3, lanes 9–12). These results indicate that transcription begins in the region upstream of position 01270, most likely within the adjacent 200 bp of sequence. More precise localization of the start site has so far been precluded by the low abundance of transcript. Our analysis places the estimated length of the 5*-untranslated region at 1.3– 1.4 kb and the total length of the untranslated regions plus open reading frame at Ç4.5 kb. This size is consistent with the estimated size of the single Y2 receptor transcript detected in Northern analysis of brain-derived RNA (Gerald et al., 1995; Rose et al., 1995). Analysis of the genomic sequence upstream to position

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FIG. 2. The sequence of the genomic region encompassing the 5* and 3*-untranslated regions of a human type 2 NPY receptor. Lowercase letters correspond to intronic sequence. Numbering is with respect to the initiation codon. The partial amino acid sequence is shown in single-letter code below the nucleotide sequence. The genomic DNA does not contain sequence corresponding to the first 6 bp of the cDNA published by Gehlert et al. (1996), which appears to be derived from the 5*-BstXI/EcoRI adapter sequence.

01834 indicates that the promoter region of the Y2 receptor gene does not contain canonical TATA- or CAAT-box sequences (Gannon et al., 1979). These sequences are also absent from the Y1 receptor gene promoters (Ball et al., 1995). Chromosomal Localization Hybridization analysis of Southern transfers of human genomic DNA indicates that the gene encoding the Y2 receptor is single copy and distinct from that

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encoding the Y1 receptor (EcoRI fragment sizes are 1.6 and 3.0 kb, respectively) (data not shown). Hybridization analysis of Southern transfers of DNA from 18 rodent–human somatic cell hybrids mapped the Y2 receptor sequence to human chromosome 4 (Table 1). Probes corresponding to the protein coding or 3*-untranslated region detected 12-, 4.1-, and 1.4-kb fragments in HindIII-cleaved DNA from human, Chinese hamster, and mouse, respectively. One hybrid, GM/NA 09931, reported to have no intact chromosome 4 present, was positive for the 12-kb human fragment. As

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Y2 receptor. The Y2 receptor transcript is encoded on two exons separated by approximately 4.5 kb of intervening sequence. Our analysis indicates that the Y2 receptor gene is less structurally complex than the gene encoding the human Y1 receptor. Although the 5*-untranslated regions of both genes contain introns just upstream from the translation initiation codon, the Y1 receptor gene also contains a 97-bp intron in the protein coding region after the fifth transmembrane domain, between exons 2 and 3 (Herzog et al., 1993). In addition, the Y1 receptor gene contains three alternative exon 1 sequences (80, 110, and 106 bp) located 6.4, 18.4, and 23.9 kb upstream of exon 2 (Ball et al., 1995). The mouse Y1 receptor gene has also been shown to contain an alternate exon 4 located over 15 kb downstream of exon 3 (Nakamura et al., 1995). This structural arrangement results in differential transcriptional regulation by three distinct promoters (Ball et al., 1995), as well as two Y1 receptor isoforms with differing C-terminal ends (seventh transmembrane domain and C-terminal tail) (Nakamura et al., 1995). In contrast, in the Y2 receptor gene, only a single large exon 1 is present (Ç1.3–1.4 kb), with the published cDNAs having the same 5*-untranslated sequences immediately upstream of the intron, as well as the same 3*-untranslated sequences. However, all studies to date have examined Y2 receptor transcripts derived from the central nervous system. Transcript abundance in peripheral tissues is apparently very low (Rose et al., 1995; Gerald et al., 1995) and likely to be differentially regulated. Therefore, it will be of future interest to analyze transcripts from the periphery using more sensitive techniques. The Y2 receptor gene maps to human chromosome 4q31, a location as yet unlinked to any human inherited disease (McKusick, 1993). However, this region of chromosome 4 has been previously shown to contain the Y1 receptor locus (4q31.3–q32) (Herzog et al., 1993), and Gerald et al. (1996) suggest that the human Y5 receptor gene also may be present in the same region, in the reverse orientation. The distance between the Y1 and

FIG. 3. Identification of the 5*-untranslated region of the human type 2 NPY receptor gene. Human genomic DNA (lanes 1–4), firststrand cDNAs synthesized from human hippocampal poly(A) RNA (lanes 5–8), or no template controls (lanes 9–12) were subjected to PCR amplification using primers corresponding to Y2 receptor gene sequence upstream of the translation initiation site and intervening sequence. (Lanes 1, 5, and 9) Upstream primer (f1), residues 01630 through 01650. (Lanes 2, 6, and 10) Upstream primer (f2), residues 01464 through 01481. (Lanes 3, 7, and 11) Upstream primer (f3), residues 01252 through 01270. (Lanes 4, 8, and 12) Upstream primer (f4), residues 01175 through 01192. All reactions contain downstream primer (r1), residues 0653 through 0670. Products were electrophoresed on agarose gels, and Southern hybridization was performed using a radiolabeled probe (h1), residues 0926 through 0961. The autoradiograph is shown.

described in the NIGMS catalog, this hybrid repeatedly revealed a positive hybridization signal of ADH 3 probe located at 4q21–q23 and likely contains at least a portion of the long arm of chromosome 4. Regional assignment of the gene encoding the Y2 receptor was accomplished by in situ hybridization using an EcoRI fragment of the genomic clone encompassing the protein coding sequence. Each of 20 cells scored revealed hybridization signals on both chromosome 4 homologs, at band 4q31, as shown in Fig. 4. DISCUSSION

The present studies describe the structure and chromosomal localization of the human gene encoding the

TABLE 1 Correlation of Sequences Detected by Y2 Receptor cDNA Probes with Human Chromosomes in Rodent 1 Human Somatic Cell Hybrids Human chromosomes Hybridization Concordant //0 0/0 Discordant //0 0// Discordant clones Informative clonesa a

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

X

Y

4 7

3 4

6 4

9 6

6 4

10 5

8 4

7 3

0 6

4 6

4 5

8 5

4 5

10 4

5 3

1 6

11 3

6 5

5 6

8 5

5 4

5 4

0 5

2 5

7 0 7 18

8 3 11 18

4 3 7 17

1 0 1 16

4 3 7 17

1 2 3 18

2 2 4 16

2 3 5 15

11 1 12 18

3 0 3 13

6 1 7 16

2 2 4 17

5 1 6 15

1 2 3 17

3 4 7 15

9 1 10 17

0 4 4 18

5 2 7 18

3 1 4 15

2 2 4 17

5 3 8 17

5 2 7 16

9 1 10 15

8 1 9 16

Data for chromosomes involved in rearrangement or present at a frequency of 0.1 or less were excluded.

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Mari Hagiwara, Khalia Thomas, and Claudia Figueroa for assistance with sequence analysis. This research was supported in part by grants from the National Eye Institute RO1 EY09193 (D.A.T.) and EY07003 (Vision Core Grant), the National Retinitis Pigmentosa Foundation (T.L.Y.-F.), and the National Center for Research Resources MO1 RR 00042 (U-M General Clinical Research Center).

REFERENCES

FIG. 4. Localization of the gene encoding a type 2 NPY receptor to human chromosome 4q31. Fluorescence in situ hybridization to metaphase chromosomes was performed using the genomic clone 1A2.hg2 and a centromere-specific probe for chromosomes 4 and 9. Arrowheads indicate the positions of hybridization signals on chromosome 4. Chromosomes were identified by DAPI banding and counterstaining with propidium iodide.

Y2 receptor genes is not yet known, nor is it known whether other NPY receptor subtypes map to the same region. Recent studies have shown that the genes encoding a number of other G-protein-coupled receptor subtypes exist as tightly linked pairs, including adrenergic (Yang-Feng et al., 1990), interleukin-8 (Ahuja et al., 1992), prostanoid (Duncan et al., 1995), bradykinin (Chai et al., 1996), and chemokine (Raport et al., 1996) receptor subtypes, as well as a number of others. The localization, homology, and structural similarity of the genes encoding many of these receptor pairs suggest that the subtypes have evolved by gene duplication. The Y1 and Y2 receptor subtypes are significantly less homologous, in comparison, with the percentage identity between them only slightly greater than that between NPY receptors and the next most closely related family, the tachykinin receptors. Nevertheless, the colocalization of the Y1 and Y2 receptor genes to the same region of chromosome 4q suggests the possibility that these subtypes also have a common origin, with their structural differences providing evidence of the extent of their independent evolution. Future studies will focus on the characterization of the Y2 receptor promoter region and transcriptional control elements, with an interest in elucidating the mechanisms involved in the differential expression of the genes encoding the NPY receptor subtypes. ACKNOWLEDGMENTS We thank Dr. Julia E. Richards and Dr. Barry E. Knox for critical discussions, Dr. Paul A. Sieving for gifts of oligonucleotides, and

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