Proc. Nat!. Acad. Sci. USA Vol. 89, pp. 5794-5798, July 1992 Cloned human Y couples to two different second messenger systems HERBERT HERZOG, YVONNE J. HORT, HELEN J. BALL, GILLIAN HAYES, JOHN SHINE, AND LISA A. SELBIE Garvan Institute of Medical Research, St. Vincent's Hospital, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia Communicated by W. J. Peacock, March 12, 1992 (receivedfor review December 12, 1991)

ABSTRACT Neuropeptide Y (NPY) is one of the most We report here the molecular cloning of a cDNA sequence abundant in the mammalian and encoding a human NPY receptor,* which exhibits the char- exhibits a diverse range of important physiological activities, acteristic specificity ofa Y1 receptor. When expressed including effects on psychomotor activity, food intake, regula- in different cell lines, the receptor couples via pertussis tion ofcentral endocrine secretion, and potent vasoactive effects toxin-sensitive G to different second messenger on the cardiovascular system. Two major subtypes of NPY systems. receptor (Y1 and Y2) have been defined by pharmaclgical criteria. We report here the molecular cloning of a cDNA MATERIAL AND METHODS sequence encoding a human NPY receptor and the corrected Nucleotide Sequence Determination. Total RNA (3 pug) from sequence for a rat homologue. Analysis ofthis sequence confirms rat was used as a template to synthesize random primed that the receptor is a member ofthe G -coupled receptor single-stranded cDNA. The cDNA was used in a polymerase superfamily. When expressed in Chinese hamster ovary (CHO) chain reaction (PCR) together with the oligonucleotide prim- or human embryonic kidney (293) cells, the receptor exhibits the ers, which correspond to positions 672-584 and 48-78 in the characteristic ligand specificity ofa Y1 type ofNPY receptor. In rat cDNA clone FC5 (12). PCR conditions: 30 cycles at 950C the 293 cell line, the receptor is coupled to a pertussis toxin- for 1 min, 630C for 2 min, and 720C for 1 min. The reaction sensitive that mediates the inhibition of cyclic AMP product was digested with EcoRI and Pst I, gel purified, and accumulation. In the CHO cell line, the receptor is coupled not subcloned for sequencing in the Bluescript vector (Strata- to the inhibition of adenylate cyclase but rather to the elevation gene) for confirmation of the sequence. of intraceflular . These results demonstrate that second A cDNA libraries derived from human fetal brain (P. See- messenger coupling ofthe NPY-Y1 receptor is cell type specific, burg, University ofHeidelberg) and human adult hippocampus depending on the specific repertoire of G proteins and effector (Stratagene) (9 X 105 plaque-forming units each) were systems present in any cell type. screened with 32P-labeled rat cDNA as a probe under stringent hybridization conditions. Three strongly hybridizing clones Neuropeptide Y (NPY), a 36-amino acid peptide, is an impor- were isolated and the cDNA inserts were subcloned for tant regulator in both the central and peripheral nervous sequencing in Bluescript vectors. The largest cDNA [HY1, 2.5 systems (1). NPY is highly conserved in primary structure kilobases (kb)] contains an open reading frame of 384 amino between species, as the sequences of human, rat, rabbit, and acids encoding the human NPY receptor subtype Y1. The two guinea pig are identical and differ from the porcine sequence other clones (F5 and F13) are truncated versions of the same by only a single amino acid (2). NPY also shares close cDNA with 100%o nucleotide identity in the overlapping re- sequence homology and a common tertiary structure with a gions (positions 664-1555 and 670-1925), respectively. family of peptides which include peptide YY (PYY) and Binding Assay. The coding region of the NPY-Y1 receptor pancreatic polypeptide (PP) (3). Studies with peptide frag- was subcloned in the pcDNA NEO expression vector (In- ments of NPY have indicated that multiple NPY receptor vitrogen, San Diego) to generate pHz3O and transfected into subtypes exist (4). The two major receptor subtypes have been the Chinese hamster ovary (CHO) K1 (American Type Cul- designated Y1 and Y2, with the Y1 receptor having the ability ture Collection CCL 61) and the human embryonic kidney to respond to an analog ofNPY modified at residues 31 and 34 293 (American Type Culture Collection CRL 1573) cell lines ([Leu3l,ProM4]NPY) (5). The Y2 receptor subtype is defined on by using a modified calcium phosphate transfection method the basis of its affinity for the NPY peptide carboxyl-terminal (13). CHO cells were maintained under 5% CO2 in Dulbecco's fragment NPY-(13-36) (4). NPY binding sites have been modified Eagle's medium (DMEM)/Ham's F-12 medium identified in a variety oftissues, including brain (6), spleen (7), (1:1) and 293 cells were grown in minimal essential medium intestinal membranes, and aortic cells (8). In (MEM) with Hanks' salts, and both media contained 2 mM addition, binding sites have been reported in a number of rat glutamine, 100 international units of penicillin, streptomycin and human cell lines (9). Both the Y1 and Y2 receptors are at 100 ,ug/ml, and 10%o fetal calf serum. Human neuroblas- members of the G protein-coupled receptor superfamily and toma SK-N-MC cells (American Type Culture Collection act by transducing the signal caused by the binding ofNPY to HTB10) were cultured in DMEM/Ham's F-12 (1:1) with the an intracellularly located protein complex that binds GTP. above additions and including 0.1 mM MEM nonessential This G protein complex in turn activates a variety of second amino acids. All media and materials for tissue culture were messenger systems, including adecrease in cyclic AMP and an from Cytosystems (Castle Hill, N.S.W., Australia). Stably increase in intracellular calcium (10). However, there are transfected cells were selected with neomycin and tested for reports of NPY receptors coupled to phosphoinositol metab- the ability to bind NPY/PYY peptide analogues. Transfected olism, suggesting the existence ofadditional receptor subtypes cells (1 x 106) were incubated in 0.5 ml ofassay buffer [50 mM and/or multiple functions for the Y1 and Y2 subtypes (6, 11). Abbreviations: NPY, neuropeptide Y; PYY, peptide YY; PP, pan- creatic polypeptide. The publication costs of this article were defrayed in part by page charge *The nucleotide sequence of the human neuropeptide Y-Y1 receptor payment. This article must therefore be hereby marked "advertisement" cDNA clone HY1 has been deposited in the GenBank data base in accordance with 18 U.S.C. §1734 solely to indicate this fact. (accession no. M84755) and will be published elsewhere. 5794 Downloaded by guest on September 24, 2021 Biochemistry: Herzog et al. Proc. Natl. Acad. Sci. USA 89 (1992) 5795

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P F Q N V T L D A Y K D K Y VHF DQ Fr S[ H R - - - YT T L L L VWQ ]GrL]C F IH I C Y I R K P F Q N V S L A A F KD K Y VCI F DK FIP S D SIH R - - - LISL S YT T L LLVIL|Q|Y F|G|P LC F I|F I C YIF KIIIY I R L K R R Q- --G A T K CV V A W[JE G G K T L L JH L V V I AI Y F|L|P L|A V MFV AL JS G L T LW JRR TNIVI "Wft N N M M D K M R D N K YRS S E TKR I N IML L S IV A -A V|C W L P L T I F N TVF D W N HQII A T CN HN LL F NN M M D K I R D S K Y R S S E T K R I N V|M L L S I V V A -| F A VC W L P L T I F N T VAFD W N H MHI|IA THDNC N L L F A V P G H Q A H G A N L RJH L Q AK K KF VK T M V LV i L T A I|C W L Pi Y H L Y F I LJG S F Q E D I Y C H K F I BQ Q V Y TMVII L L C H L TAH IST C V|N P IF Y G FL N K N F 0 R D L Q F F F N F CD F R S R DD D Y E T I A M S TM H T D V S K T S L LA L WTALA I S TMC V N P I FIY G F|L N|KNH F R D L Q FL FRN F|C D F R S R D D Y E T I A M S |TM H T D V S K T S L L A L F W L AHS STM Y|N P I ILSY H R FR S G F R L A FR C CP W V T P T K ED K L E LT P T T S L S T R VN R C

K Q A S P V A F KK I N NN D D N E K I * 384

K Q A S P V A F KK I S M N D - N E K I * 382 H T K E T L F M A G D T A P S E A T S G E A G R P Q D G S G L W F G Y G L L A P T K T H V E I * 398

FIG. 1. Alignment of amino acid sequences for human NPY-Y1 receptor (hNPY-Ylr), rat NPY-Y1 receptor (rNPY-Ylr) (12), and human substance K receptor (hSKr) (16). The boxed amino acids represent residues that are identical in all three sequences. Dashes show deletions of the amino acid residues compared with the other sequences. Putative transmembrane segments (TM I-VII) of the receptor are indicated by bold frames, with the termini of each segment tentatively assigned on the basis of a hydrophobicity analysis. Arrows indicate potential N-glycosylation sites. Sequence identity within the first 350 amino acids between hNPY-Ylr and hSKr is 30%o. Tris-HCl, pH 7.4/2 mM CaCl2/5 mM KCI/120 mM NaCl/1 for 15 min, and neutralization and sample dilution in 50 mM mM MgCI2/0.1% bovine serum albumin (Sigma)] in the sodium acetate, pH 6.2. Cyclic AMP was quantitated by presence of 0.05 nM 125I-labeled NPY (125I-NPY; Dupont/ using a radioimmunoassay (Dupont/NEN). NEN) and increasing concentrations of human NPY or related peptides (Auspep, Melbourne, Victoria, Australia). Cells were incubated with the radiolabeled peptide and RESULTS AND DISCUSSION competitor peptides for 1 hr at room temperature and pelleted Molecular Cloning of the Human NPY-Y1 Receptor cDNA. in a microcentrifuge for 4 min. Radioactivity was measured Recently, a cDNA sequence encoding a putative member of for 1 min in a y counter. Calcium Measurements. Cells transfected with the human 120- NPY-Y1 receptor or SK-N-MC cells were suspended in loading medium (modified RPMI 1640 medium/10 mM Hepes/1% newborn calf serum) and incubated in a spinner 100 flask at 37°C for 2.5 hr at 1 x 106 cells per ml. Cells were then treated with 1 ,uM Fura-2 acetoxymethyl ester (fura-2 AM; - 80-

Molecular Probes) for 30 min at 37°C, washed twice with :5._ loading medium, and resuspended at 5 x 106 cells per ml. zC, 60- Immediately before fluorescence spectroscopy, cells were W_ recovered by centrifugation at 1000 rpm and resuspended at .5 a) 40. 1 x 106 cells per ml in a modified Krebs buffer (135 mM C20 NaCl/4.7 mM KCl/1.2 mM MgSO4/1.2 mM KH2PO4/5 mM NaHCO3/1 mM CaCl2/2.8 mM /10 mM Hepes, pH 20- 7.4) containing 1 mM sulfinpyrazone. Bombesin was pur- chased from Sigma and Auspep. Fluorescence recordings were made on a Hitachi fluorescence spectrometer (F4010) at 10-11 10-10 10-9 10-8 10-7 10-6 340 nm (excitation) and 505 nm (emission) over 10 min with Competitor, M slit widths of 5 nm and a response time of 2 sec. Intracellular calcium was quantitated by using equations described by FIG. 2. Inhibition of porcine 1251-PYY binding with various Grynkiewicz et al. (14). NPY-related peptides. Increasing concentrations of human NPY (o), Cyclic AMP Assay. Cyclic AMP was assayed in whole cells human [Leu3l,Pro34]NPY (i), porcine PYY (A), PYY-(13-36) (0), treated for 15 min at 37°C with 100 ,uM isobutylmethylxan- and human PP (A) were tested for their ability to inhibit the binding of 125I-PYY. Results are expressed as a percentage of the maximal thine (IBMX; Sigma). Transfected 293 cells or CHO cells (1 specifically bound radiolabeled PYY. Standard errors of triplicate x 106/0.5 ml reaction) were incubated with 10 ,uM forskolin samples are shown. Similar values for NPY binding were obtained and various concentrations of [Leu31,Pro34]NPY or PYY- with transfected 293 cells and with the human neuroblastoma SK- (13-36) for 15 min at 37°C. Reactions were terminated with N-MC cell line. Untransfected CHO and 293 cells showed no specific the addition of HCl to 0.1 M, incubation at room temperature binding of NPY. Downloaded by guest on September 24, 2021 57% Biochemistry: Herzog et al. Proc. Natl. Acad. Sci. USA 89 (1992)

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FIG. 3. Stimulation of intracellular calcium response mediated by the NPY-Y1 receptor. Intracellular calcium levels of fura-2-loaded CHO cells were measured in response to NPY, [Leu3l,Pro34]NPY (Y1), PYY, PYY-(13-36) (13-36), or PP. After successive addition of two of these compounds the intracellular calcium response mediated by the endogenous bombesin receptor was measured with the addition of1 AuM bombesin (Bom) (Auspep). The NPY-Y1 receptor-expressing CHO cell line was stimulated with 100 nM NPY (A), PYY (B), [Leu3l,Pro34]NPY (C), PYY-(13-36) (D), or PP (E). Untransfected CHO cells did not respond to any of the NPY analogues, including 100 nM [Leu31,Pro34]NPY (F). Treatment of stable transfected cells overnight with pertussis toxin at 100 ng/ml abolished the response to 2.5 ILM PYY (H), as compared with untreated cells (G), but did not affect the intracellular response to 1 AM bombesin. The intracellular calcium increase mediated by the NPY-Y1 receptor was dependent on the concentration of [Leu3l,Pro34]NPY and was maximal at 10 nM (J). the G-coupled receptor-family was isolated from rat brain receptor sequences identifies the human substance K recep- RNA (12). The distribution of this mRNA in the central tor (16) as the most closely related sequence, with 30%o nervous system is similar to that predicted for NPY binding identity (Fig. 1). sites (15). We used the PCR to amplify sequences of this Analyses of Ligand Specificity. To examine the ligand cDNA from rat brain RNA as a probe to screen human binding properties and second messenger coupling of the hippocampal and fetal brain cDNA libraries. Several strongly receptor encoded by HY1, the coding region was subcloned hybridizing clones were isolated and sequenced, with the in a mammalian cell expression vector and transfected into longest of these (HY1, 2.5 kb) encoding a complete open CHO and 293 cell lines. Stable transformed cell lines were reading frame of 384 amino acids with high homology to the selected for detailed analysis. Both types ofcell line express- rat receptor sequence (Fig. 1). The sequence shown for the ing the HY1 clone exhibit specific binding ofNPY. Scatchard rat receptor is a corrected version of the published sequence analysis of NPY binding indicates the presence of high- and for the cDNA clone FC5 (12). Sequence analysis of a PCR low-affinity sites for NPY, typical for coupling ofthe receptor fragment (nucleotides 643-1191) encoding the carboxyl- to a G protein or proteins in these cells. The calculated Kd terminal sequence of the rat receptor, obtained from the values for these sites are approximately 5.1 nM and 198 nM, pheochromocytoma cell line PC12 (17), demonstrated three respectively (data not shown). These values are similar to the differences from the published sequence. These introduced binding affinities reported for specific NPY binding to various two frame shifts by deleting G at position 1031 and 1034 and peripheral tissues and cell lines (18, 19), consistent with its inserting G at position 1098. The corrected rat receptor classification as a NPY receptor. NPY receptor subtypes sequence shows 94% identity to the human HY1 sequence. have been pharmacologically defined by their affinity for a Analysis of the amino acid sequence of HY1 shows features range of related peptide analogues. NPY and PYY, members typical of G protein-coupled receptors (10). These include ofthe PP family, have nearly identical affinities for the cloned seven hydrophobic putative membrane-spanning regions as receptor (Fig. 2), whereas PP has virtually no affinity at well as potential glycosylation sites (Fig. 1). Several serine physiological concentrations. A NPY analogue, [Leu31, and threonine residues, candidates for phosphorylation, are Prom]NPY, with known selectivity for Y1 receptors (5), has found in the carboxyl-terminal sequence. Amino acid resi- a high affinity for the expressed receptor (IC50 = 10 nM), dues highly conserved among different G protein-coupled while the Y2 selective analogue PYY-(13-36) has almost no receptors are also present (e.g., Arg138) (Fig. 1). Comparison affinity (Fig. 2). This ligand specificity and rank order of of the amino acid sequence with the other G protein-coupled potency-NPY = PYY > [Leu31,Pro&]NPY >> PP > PYY- Downloaded by guest on September 24, 2021 Biochemistry: Herzog et al. Proc. Natl. Acad. Sci. USA 89 (1992) 5797 A SK-N-MC ceols which contains only the Y1 specific NPY receptor subtype 120- (18) (data not shown), as well as in vascular smooth muscle 0 cells (19). However, in the 293 cell line no increase in -5 intracellular calcium was observed after stimulation by NPY, E PYY, or [Leu31,Pro34]NPY. c)13 Inhibition of Adenylate Cyclase Activity in Stably Trans- fected 293 Cells. The majority of NPY receptors in the brain .O and peripheral tissue couple to inhibition of adenylate cyclase c) and hence decrease cyclic AMP (20). As shown in Fig. 4A, NPY decreases forskolin-stimulated cyclic AMP levels in c-) SK-N-MC cells. Interestingly, NPY or PYY had no specific effect on the inhibition of cyclic AMP accumulation (either 10 - basal or stimulated by forskolin) in the HYl-expressing CHO 0C :E cell line. This suggests that either this subtype is not normally -~e 2 m U c c_ coupled to this second messenger system or, more likely, 0 C+ CHO cells lack the appropriate G,/0 isoform. The latter LL explanation is supported by the demonstration that, in con-

0+o trast to CHO cells, transfected 293 cells exhibit a clear inhibition of cyclic AMP accumulation (Fig. 4B). The inhi- bition of the cyclic AMP accumulation was Y1 specific and B transfected 293 cells the effect was maximal at 10 nM NPY. Preincubation with pertussis toxin abolished the effect. Conclusions. On the basis of both receptor pharmacology and the effects on intracellular calcium and cyclic AMP o° 100- accumulation, the sequence encoded by HY1 represents a Y1 -5 E 80-_ subtype of a human NPY/PYY receptor. The demonstration u that this receptor couples to different second messenger C) 60 - systems in a cell type specific manner suggests that the functional diversity exhibited by NPY may result not only <, 40- _ from the presence of different receptor subtypes but also -F :.> }- from the restricted availability of second messenger systems -'F in different cell types. Knowledge of the primary structure 20-_ +>X 00 - and the second messenger coupling of the NPY-Y1 receptor 00 -.+:>' subtype is particularly important given the role of this sub- O-_ type in control of cardiovascular parameters (21). In this C L >- >- context, these results should facilitate the development of therapeutic drugs important in blood-pressure control and other cardiovascular diseases. Using this sequence for iso- 2 + + lation of other related receptor subtypes and evaluation of systems in other cell lines should shed considerable light on the molecular mechanisms involved in the NPY-mediated control of a variety of physiological FIG. 4. [Leu3l,Pro34]NPY inhibition of cyclic AMP accumulation responses. in SK-N-MC (A) cells and 293 cells transfected with the human NPY-Y1 receptor cDNA (B). The cyclic AMP response to We gratefully acknowledge the expert advice of A. P. Czernilofsky [Leu31,Pro34]NPY (Y1) or porcine PYY-(13-36) (13-36) is expressed and T. Biden and the assistance of C. Browne and M. Lui. This work as a percentage of the cyclic AMP level produced with 10 /AM was supported by the National Health and Medical Research Council forskolin from three independent experiments. Basal levels were 2.5 and Boehringer Ingelheim (Bender & Co., Austria). H.H. is a fellow ± 0.13 pmol per 106 cells (untransfected 293 cells), 2.26 ± 0.6 pmol of the E. Schrodinger Foundation (Austria). per 106 cells (transfected 293 cells), and 1.8 ± 0.55 pmol per 106 cells (SK-N-MC cells). Cyclic AMP levels in cells stimulated by 10 /AM 1. Heilig, M. & Widerlov, E. (1990) Acta Psychiatr. Scand. forskolin were 121.2 ± 19.8 pmol per 106 cells (untransfected 293 82, 95-114. cells), 139 ± 14.2 pmol per 106 cells (transfected 293 cells), and 137.6 2. Tatemoto, K. & Carlquist, M. (1982) Nature (London) 296,659. ± 1.85 pmol per 106 cells (SK-N-MC cells). 3. Glover,I. D., Barlow, D. J., Pitts, J. E., Wood, S. P., Tickle, I. J., Blundell, T. L., Tatemoto, K., Kimmel, J. R., Wollmer, (13-36)-clearly demonstrate that the HY1 clone encodes a A., Strassburger, W. & Zhang, Y. S. (1985) Eur. J. Biochem. Y1 subtype of the NPY/PYY receptor family. 142, 379-385. Increases in Intracellular Calcium in Stable Transfected 4. Wahlstedt, C., Yanaihara, N. & Hikanson, R. (1986) Regul. CHO Cells. To analyze the functional coupling of the cloned Pept. 13, 307-318. receptor in CHO K1 and 293 cells, we measured the effects 5. Fuhlendorff, J., Gether, U., Aakerlund, L., Langeland-Jo- hansen, N., Thogersen, H., Melberg, S. G., Olsen, U. B., of NPY on intracellular calcium levels and cyclic AMP Thastrup, 0. & Schwartz, T. W. (1990) Proc. Nati. Acad. Sci. accumulation. In the CHO cell line, stimulation with either USA 87, 182-186. NPY or PYY results in an immediate and substantial increase 6. Hinson, L., Rauh, C. & Coupet, J. (1988) Brain Res. 446, in intracellular calcium (Fig. 3). This effect can be abolished 379-382. by preincubation with pertussis toxin, suggesting receptor 7. Lundberg, J. M., Hemsen, A., Larsson,O., Rudeheill, A., interaction with a pertussis toxin-sensitive G protein. The Saria, A. & Fredholm, B. B. (1988) Eur. J. Pharmacol. 145, 21-25. calcium response is receptor specific, being elicited by NPY, 8. Mihara, S., Shigeri, Y. & Fujimito, M. (1989) FEBS Lett. 259, PYY, and the Y1 agonist but not by PP or PYY-(13-36). The 79-82. NPY-induced rise in intracellular calcium is comparable to 9. Aakerlund, L., Gether, U., Fuhlendorff, J., Schwartz, T. W. & that observed in the human neuronal cell line SK-N-MC, Thastrup, 0. (1990) FEBS Lett. 260, 73-78. Downloaded by guest on September 24, 2021 5798 Biochemistry: Herzog et al. Proc. Nat!. Acad. Sci. USA 89 (1992)

10. Strosberg, A. D. (1991) Eur. J. Biochem. 196, 1-10. 16. Gerard, N. P., Eddy, R. L., Shows, T. B. & Gerard, C. (1990) 11. Wahlestedt, C., Hikanson, R., Vaz, C. A. & Zukowska-Gro- J. Biol. Chem. 265, 20455-20462. jec, Z. (1990) Am. J. Physiol. 258, R736-R742. 17. Green, L. A. & Tischler, A. S. (1982) Adv. Cell. Neurobiol. 3, 12. Eva, C., Keinanen, K., Monyer, H., Seeburg, P. & Sprengel, 373-414. R. (1990) FEBS Lett. 271, 81-84. 18. Sheikh, S. P., O'Hare, M., Torotora, 0. & Schwartz, T. W. 13. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Lab., Cold (1989) J. Biol. Chem. 264, 6648-6654. Spring Harbor, NY), 2nd Ed., pp. 16.39-16.40. 19. Shigeri, Y., Mihara, S. & Fujimoto, M. (1991) J. Neurochem. 14. Grynkiewicz, G., Poenie, M. & Tsien, R. Y. (1985) J. Biol. 56, 852-859. Chem. 260, 3440-3450. 20. Wahlestedt, C. (1987) Ph.D. thesis (University of Lund, Swe- 15. Adrian, T. E., Allen, J. M., Bloom, S. R., Ghatei, M. A., den). Rossor, M. N., Roberts, G. W., Crow, T. J., Tatemoto, K. & 21. Potter, E. K., Fuhlendorff, J. & Schwartz, T. W. (1991) Eur. J. Polak, J. M. (1983) Nature (London) 306, 584-586. Pharmacol. 193, 15-19. Downloaded by guest on September 24, 2021