Structural diversity of receptors for neuropeptide Y, peptide YY and pancreatic polypeptide

E L S E V I E R Regulatory Peptides 65 (1996) 165-174

REGULATORY

PEPTIDES

R e v i e w

Structural diversity of receptors for neuropeptide Y, peptide YY and pancreatic polypeptide

D a n L a r h a m m a r *

Department of Medical Pharmacology, Uppsala UniversiO,', Box 593, S-751 24 Uppsala, Sweden

Received 14 February 1996; revised 4 June 1996; accepted 4 June 1996

Abstract

The NPY (neuropeptide Y) family of neuroendocrine peptides consists of NPY, PYY (peptide YY) and PP (pancreatic polypeptide). Several receptors have been characterized pharmacologically of which three have now been cloned. All three belong to the superfamily of receptors that couple to G proteins and all three cause inhibition of cAMP accumulation. Receptor subtypes Y1 and Y2 bind both NPY and PYY. Surprisingly, Y1 and Y2 share only 31% overall sequence identity, the lowest percentage reported for receptors that bind the same peptide ligand. Nevertheless, each subtype is 94% identical between human and rat, suggesting a slow rate of change. These observations suggest that Y1 and Y2 started to diverge from one another very long ago, possibly before the origin of vertebrates. The PP receptor, called PP1 or Y4, is 42% identical to the Y1 receptor (57% in the transmembrane regions) and is one of the most rapidly evolving receptors with only 75% overall identity between man and rat. Interestingly, this receptor's preferred ligand, PP, also evolves extremely rapidly. The PP receptor also differs between man and rat in tissue distribution and binding properties. The YI and PP receptors bind to both termini of their ligands whereas Y2 mainly interacts with the C-terminal part. Thus, within the same family there are highly conserved receptors and peptide ligands as well as one rapidly evolving receptor and ligand.

Keywords: Neuropeptide Y; Peptide YY; Receptor subtype Y1; Receptor subtype Y2; Evolution

1. Introduct ion

The three members of the NPY (neuropeptide Y) family of neuroendocrine peptides have a large variety of effects in the nervous system, the circulatory system, and the gastrointestinal tract (for review, see Refs. [1,2]). NPY is abundant in the nervous system of all mammals and among its more prominent effects are stimulation of food intake, anxiolysis and influence on release of pituitary hormones and circadian rhythms. In peripheral blood ves- sels NPY induces vasoconstriction. PYY (peptide YY) and PP (pancreatic polypeptide) are produced in gut endocrine cells and released in response to meals. They inhibit gastrointestinal motility and pancreatic exocrine secretion. In lower vertebrates, also PYY is widespread in the central nervous system [3,4].

Several receptor subtypes for the NPY-family peptides

* Corresponding author. Tel.: (46-18) 174-173; Fax: (46-18) 511-540; e-mail: dan.larhammar @ medfarm.uu.se

have been described pharmacologically using organ prepa- rations from different species (for reviews, see Refs. [1,2,5,6]). The best characterized are the Y1 and Y2 receptors, both of which bind to NPY as well as PYY. The Y3 receptor has preference for NPY over PYY [7,8], whereas a separate subtype binds most strongly to PYY [9]. Also PP has been reported to have a distinct receptor [10,11]. The feeding response seems to be mediated by a receptor with unique properties [12,13]. Finally, there is a report on a receptor that binds all three peptides, therefore called the 'PP-fold ' receptor [14] to relate to the characteristic three- dimensional structure of the endogenous ligands. Thus, the total number of receptor subtypes may be as large as seven.

For detailed characterization of receptor structure, pharmacology and tissue distribution it is crucial to have access to molecular clones. Although clones for the N P Y / P Y Y receptor of subtype Y1 were identified a few years ago [15-17], the remaining receptor subtypes have long re- sisted all cloning attempts. Recently, however, DNA clones for two more subtypes have been reported, namely the Y2 receptor and a PP receptor. Their surprisingly different

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166 D. Larhammar / Regulatory Peptides 65 (1996) 165-174

structures and evolutionary divergence rates are discussed in this review along with their pharmacological properties.

2. NPY/PYY receptor Y1

Receptor subtype Y1 was initially cloned in rat by homology screening [18] and subsequently in man, mouse and Xenopus laevis (see Table 1 for references). The cloning procedures have been reviewed elsewhere [19]. The Y1 structure has the characteristic features of G-protein coupled receptors that bind peptide ligands (Fig. 1).

Potential glycosylat ion sites are present in the N-terminal portion and the second extracellular loop. Four extracellular cysteines presumably form two disulfide loops, and the single intracellular cysteine in the C-terminal cytoplasmic portion is probably used for attachment of palmitate in- serted into the cell membrane. The two rodent proteins are 98% identical (Table 2) and both are 94% identical to the human sequence. The frog sequence is 80% identical to the three mammals.

The human, rat and mouse Y1 clones have been expressed in cultured cells for pharmacological and functional studies. They all bind to NPY and PYY, as well as

Table 2 Percent sequence identity for entire coding region

rY 1 mY 1 xY 1 hPP 1 rPP 1 mPP 1 hY2 rY2

hY 1 94 94 81 42 42 42 3 l 3 l rYl 98 80 hPPI 75 75 31 3l rPP 1 92 hY2 94

the Y 1-selective analogue [Leu 31 ,Pro 34 ]NPY with affinities

in the 1 -10 nM range in competit ion with iodinated PYY [15-17,20]. The N-terminally-truncated analogues N P Y 2 - 36, N P Y 1 3 - 3 6 and NPY18-36 , as well as PP have significantly lower affinities. This gives the typical Y1 rank order of potency: PYY >__ NPY > [Leu 31,Pro34]NPY > > N P Y 2 -

36 > > hPP > N P Y 1 3 - 3 6 > NPY18-36 . Functionally, the YI receptor inhibits forskolin-stimulated cAMP accumulation and increases Ca 2÷ influx in response to NPY.

The Y1 mRNA distribution in rat brain is widespread and includes cerebral cortex, hippocampus and thalamus [18,21], as well as hypothalamus [21,22]. A subset of cells in rat dorsal root ganglia express Y1 mRNA, probably prejunctionally [23]. In mouse the Y1 mRNA was found in

Table 1 GenBank accession codes for sequences

Subtype Species Code for DNA Code for protein Reference

Y1 Man M88461 cDNA P25929 [17] M84755 cDNA P25929 [15] L07614 prom + exon 1 [27] L07615 exon 2 + 3 L47167 prom + exon la [61] L47168 prom + exon lb L47169 prom + exon lc

Rat Z11504 cDNA P21555 X95507 prom + exon 1

Mouse Z18280 exons Q04573 Z18281 exon 1 Z18282 exon 2 z18283 exon 3 D63818 cDNA [20] D63819 cDNA

Xenopus laevis L25416 cDNA P34922 [60]

Man Y2

PP1/Y4

Rat Cow

Man

Rat

Mouse

U32500 cDNA [33] U36269 cDNA [32] U42766 cDNA [31 ] U50145 exon 1 [34] U50146 exon 2 gene [36] U50144 cDNA [34]

Z66526 gene [38] U35232 gene [37] Z68180 gene [40] gene [39] U40189 gene [41]

[18], corrected in [16] Lundell and Larhammar, unpubl. [26]

D. Larhammar / Regulatory Peptides 65 (1996) 165-174 167

TMI I I TM~ I I I I I ~ I I I hY] MN-~TkF~Q~ENH~VH$NF5EKN~QLLRFEN~C~LPL~FTLAL~Y~A~L~V~NL~L~LK£KEMRN~TN~L~NLSF$~LL~R~MCLPF~F~Y~LMDHW~FG 109

, Y : . , - . . . . . . R . . . v . _ . . v . , _ , z . , _ . - . sPv . . . . . . . . . . . . . u . . . . . . . . . . . . . . . . . . . . . . . . . . I . . . . . . . . . . . I . . . . . . . . . . . . . . u . . . . . . . . . ] . . . . . . . . t o e mYl , ~ - , ~ , , . K , , ~ I . Y . A . , - , S P . . . . . . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . . . . . . . . . . . . . . . . . . 108

TM3 TM4 T~5

hY1 EAMCKLNPFVQCVS~TV~F~LVL~RVERHQL~NPR6WRPNNRHAYVG~RV~WVLRVA~LPFL~YQVMTDEPFQNVT~DAYKDKYV~F~QFP~DS~RL~YTTLLLVLQ 219 rY1 ,T .......................................... I..T . . . . . . . V. ,IL ~ A F K . ........... 218 mYl .T ............................................. I,.T ............. V,.,IL ...... ~_.A,F ........ K ..... 4 ............ 218

xY1 ,V. I .... EYI .... V ........... I l ................. I,.CF,.T,.,GF,M.C,T.LMM,SI, L ..... K,IS_.,S,IG .... LED,,EK~ ~ ........ FI,, 214

TM6 TM7

hYl YFGP~CFIF~CYFK~Y~RLKRAHNMMDKMRDNKYR~ETKR~N~MLL~VVAFAVCWLPLT~FNTVFDWNHQ~ATCNH~LLFLLCHLTRM~STCUNP~FYGFqNKNFQA 3 2 9

rY1 ............................ l,,S ........... V .................................................................. 328 q

or1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . i . . s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ; ;::; i;;;;;::J:;;;;;j 3 2 0

x Y 1 , L . . . . . . . V , . T , , F L . . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . G , . L . . . . F F , , , L . . . . . . E A V . . . . . . . . . . I . . . . . 3 2 4

~ ' 1 I I I I

hYl DLQFFFNFCDFRSRDDDYE T I flMSTMHTDUSKTSLI(QRSPVAFKK I NNNDDi'IEK I 384

rYl .............................................. SM, ,- .... 382

mYl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SM. , - . . ,V 382

x Y I . . . . . . . . . . . . . . E . . . . . . . . . . . . . . . . . . . . . . . . . I , 3 6 6

Fig. 1. Alignment of Y1 sequences. Dots mark residues identical to the top sequence. Regions within boxes correspond to the transmembrane regions as defined by the generic numbering system [54]. The exact boundaries in individual receptors are not known. Potential glycosylation sites are underlined. Cysteines presumably forming disulfide loops are marked with stars as is the cysteine in the cytoplasmic portion that is likely to be palmitoylated. Dashes mark gaps introduced to optimize alignment. For references and accession codes see Table 1.

several peripheral organs including heart, kidney, spleen, skeletal muscle, and lung but not in liver and testis [20]. Developmentally, the earliest Y1 mRNA was detected around day 14 in rat, both in diencephalon and spinal cord [24]. An in situ hybridization study in man reported widespread distribution of Y1 mRNA [25] but the size of the mRNA differed from previous reports why the probe's specificity may be questioned.

The Y1 gene has a single intron in the coding region after TM5 [26,27] and three distinct promoters seem to exist, each with its own first 5'-untranslated exon [61]. Additional complexity is suggested by an alternative 3' exon in the mouse gene that results in a different sequence from amino acid 303 at the beginning of TM7 [20]. This leads to a truncated receptor with incomplete TM7. A1-

though the binding specificites are identical to the full- length receptor, no second messenger responses could be detected why the truncated receptor was proposed to func- tion in NPY internalization [20].

3. N P Y / P Y Y receptor Y2

The Y2 receptor has been reported to be fairly abundant in rat brain, particularly in hippocampus [28-30]. Never- theless, it proved difficult to identify Y2 clones by homology approaches using Y1 probes. The reason for this is the remarkably low degree of sequence identity between Y2 and Y1 as revealed by Y2 clones recently reported by four different laboratories. Three of these reports used expres-

TMI TM2 I I I I I I I I I I

ov2o : . . L . . . . . . ? ? Z - , : - u : : : L , , s o P . . . . : i l b i i i i i : . . . . . i l ; : : : : i i i : : i : , , , i . : : u . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I o9

I I I

TM3 TM4 TM5

hY2A Y-~LMGEWKMGPU~CH~VPYAQGLRVQVST~TLTV~A~DRHRC~YHLEsK~KR~FL~GLAWG~A~LA~PLA~F~EYsL~E~PDFE~VACTEKWPGEEK~1GTVY 219 hY2B ........................ R ..................................................................................... 219

,V2b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 . . . . . . . . . . U . . . . . . . .i.v 2,g

TM6 TM7 I I I I I I

hY2B hY2R SL~LL~LYVLPLG~SFSY~TR~WSK~KNHV~PGAANDHYHQRRQKTTKILVCVVVVFAUsW~PLHAFQLAVD~DSQVLDLKEYK~FTVFH~AMC~TFANP~LY5WMN...~`~...'....................'..`....'~.....~....'.~.'..~.~.........~......''`............`.... ............ 329 329

~v2o r . . . . . . . . . . . . . . . . . , s...... , . . . . . . . . . . . . . . . . . . . H . . . ........... 329

I ¢r I I I I hY2fl SNYRKflFLSAFRCEQflLDfl I HSEUSUTFKflKKNLEURKNSGPNDSFTEflTNU 381

hY2B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381 rY2a ......................... ii .......... K, ,i'l. LT,. ,S ..... 381

rY2b ......................... ii .......... K..N,LT, , ,S ..... 381

Fig. 2. Alignment of Y2 sequences. The two human sequences hY2A [31,32] and hY2B [33] are probably allelic, as are probably the two rat sequences [36]. Symbols as in Fig. 1.


Table 3 Percent sequence identity for TM regions

rYl mYl XYI hPPI rPP1 mPPI hY2 rY2 Dm C. el. GCRC NK3

hYl 98 98 81 56 57 57 40 40 33 37 38 37 rY1 100 81 hPP1 85 85 42 41 rPP 1 97 hY2 98 36 40 43 29

Calculated for TM regions as defined by Schwartz et al. [54] (see Fig. 1), in total 169 positions. For Y1, PP1 /Y4 and Y2 references see Table 1. Dm is the Drosophila melanogaster proposed NPY receptor (Genbank accession No. P25931), C. el. is the Caenorhabditis elegans Yl-like receptor (U41028), GCRC is a mouse orphan receptor (P30731), and NK3 is the human neurokinin-3 receptor (P29371).

sion screening with PYY as radioligand to identify human Y2 clones [31-33], whereas the fourth Y2 clone was found in a bovine retina library in a search for G-protein coupled receptors [34].

The human Y2 receptor was found to bind NPY and PYY with affinities in the low nanomolar range in competition with ~25I-PYY. The truncated analogues NPY2-36, NPY13-36 and NPY18-36 also competed in the low nanomolar range, but with slightly lower affinities; NPY18-36 had about 6:fold lower affinity as compared to intact NPY (1.8 nM vs. 0.30 nM) [31]. The Yl-selective analog [Leu 3j,Pro34]NPY, in contrast, had a K i of about 1 IxM, and the Yl-selective antagonist BIBP3226 [35] was relatively inactive [31]. The pharmacological properties of the cloned rat Y2 receptor agree well with the human receptor [36]. Like the Y1 and PP1/Y4 receptors, the Y2 receptor was found to inhibit forskolin-stimulated cAMP accumulation [32,33].

The Y2 receptor is a typical G-protein coupled receptor (Fig. 2) but has only 31% overall identity to Y1 and P P l / Y 4 (Fig. 4, Table 2) and 40-42% identity in the TM regions (Table 3). Two extracellular cysteines presumably form a single disulfide bridge (Y1 and PP1/Y4 probably have two such bridges) and a single consensus site for N-linked carbohydrate is present (the other two receptor subtypes each have four sites). Another interesting differ-

ence is that Y2 has the sequence motif DRY after TM3 instead of ERH found in Y1 and PP1/Y4. Despite the drastic sequence differences between Y2 and Y1, both receptors seem to evolve slowly; rat and human Y2 are 94% identical (like Y1) and the bovine and human proteins are 94.5% identical [34]. A single amino-acid difference between the published human sequences probably repre- sents allelic variability (Fig. 2). Similarly, the two reported rat clones [36] display allelic differences at two amino-acid positions (Fig. 2).

The human Y2 mRNA was detected by Northern hybridization in several brain subregions [31-33], but surprisingly it could not be detected in most peripheral organs investigated. Southern hybridization to genomic human DNA with a Y2 probe detected no additional genes, suggesting that any other (peripheral) Y2-1ike receptor gene must be fairly divergent from the cloned Y2 receptor gene [31-33].

4. Pancreatic polypeptide receptor

Clones encoding a novel receptor with sequence similarity to Y1 have been isolated from human [37,38], rat [39,40] and mouse [41] DNA libraries using PCR primers or oligonucleotide probes based upon Y1 sequences. The receptor encoded by these clones has greater amino-acid sequence identity to Y1 than to any other receptor. Fur- thermore, this receptor shares with Y1 the unusual sequence motif ERH following TM3. Upon expression this receptor was found to bind preferentially to PP, hence it was named PP1 [38,40]. Other investigators gave the same receptor the designation Y4 because of its similarity to Y1 [39,37].

Interestingly, the PP1/Y4 receptor seems to differ between species in its selectivity for ligands. The human PP1/Y4 receptor binds human PP with a K i of 0.014 nM [38] and ICs0 of 0.056 nM [37] in competition with porcine PYY. High affinity was also observed for PYY and NPY (1-10 nM range), implying that all three peptides may

hPPI/Y4 rPPI/Y4 mPPI/Y4




TMI TM2 i i i i f i i i i i

MNTS~LL~LLLPK$PQ6EHRS~PL~TPY~Fs~HCQ~s~M~F~T~S~ET~VG~LG~LCLM~T~RQKEK~H~T~L~L~F~FLMCLLCQPLT~T~M~W~FG 110 ..... M. S. S. R F L.. K~_CL.T_N.. D S L..~L_~ G .... A , L L p , . I . T . . U . . . L . . . . . . . . IF..~ ..... S .... ~ . . . . . . . . . . . . . . I ..... VT . . . . . . . . . . 110 ..... F..P.F.G.L..K.GTH..DS.....pG .... AELL~..I.T ..... IL ........ IF..~ ..... S .... ~ . . . . . . . . . . . . . . I ..... VT,. I ........ 110

,TM3 TM4 I* TM5 I I I I I I

E~LCKM~RF~QCM~VTVSIL~LVLUALERHQL~NPTGWKP~SQAYLG~UL~W~CVL$LPFLANS~LEN~FHKNH~LEFLRDKV~CTE~WPLAHHIRT~YTTF~LL 220 .V .... LT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V..F.S F ...... NDL,.Y~.UU..,E ..... FV..SSD...L ........ 220

S.F . . . . . . . . . IT.NDL.,Y~-.UV...E ..... FU..SSD...L. ° I ................... I ................ ....... o _

TM6 TM7 I I

I I i I I * I FLVCHLLRMASTCVNPFIYGFLNTHFK 330 FHKGTYSLRAGHMKQVNVIVLVUMVVAFAVLWLPLHVFNSLEDIWHHEAI CHG FOYCLPLGFILVCYARIYRRLQRQGRV P . . . . . . . . . . . . . . . . . . . . . . . . . . . . ' ' " / ' O ' " . . . . . . . . , . . .

.... I. A I Q , KH...AHAC.S...Q..RI.SM.MT..T ............. T .... YQ...' ''' ..M .................... I... 330

KEIKflLVLTCQQSRPLEESEHLPLSTVHTEVSKfiSLRL$GRSHPI 37S .D ........ RCRP.QG.P.P ........ DL .... M,MGSK..UM 375 .D ........ HCR$.QG . . . . . . . . . . . . DL .... M.MGSK..F. 375

Fig. 3. Alignment of P P I / Y 4 sequences. Symbols as in Fig. 1.


TMI TM2 I I I I ~ I I I I l

hY1 MN-ST~FSQVE~zLI:J.~S~.UHSNFS~KNAQLLA ........... FENDDCHLPLAMIFTLALflYGAVIILGUSGNLALIIIILKQKEMRNVTNILIUNLSFSDLLVAIMCLPFTF 98 rY1 ..-,,_...R..,~L~...Y~_.-.SPF..- .......... . .......... V ................................................... V ....... 97

mY1 ..-, .... K ...... I.Y.A..-,SP.,,- .......... . .......... U ...................................................... U ....... 97

TTS..PNZ~- ITFPI SE,.A,..P.. I I xYI ..... ............. .............. L .......... ........... I ............ ~ ....... ~ ~

hPPIIY4 .,_.T_~.LALLLPK,PQGENAS_,PLGTPY ........... NFSSH,ODSVDVMIUFIVTS.SIETVV.,L.,.C,MCUTIUR.,,KA .... IL,,R,.R,,F,MCLL.Q,L.A 99 I

rPPIIY4 ,,T,H.MASLSPaFLQGK~PLDS,Y ........... U.J...~._,G,QDSADLLIAFIITT.SVETU,.,L,,,C..FUTITR,.,K5 .... IL.,A,,A.,.F,MCLI,O,L,V 99

mPPIIY4 ..~_L~FLAPLFPG.LQGKNGT~PLDSPY . . . . . . . . . . . NFS_.G.QDSAELLIAFIITT.SIET . . . . L . . . C . . F U T I T R . . . K S . . . . I L . . A . . R . , . F . M C L I . Q . L . U 99 hY2 •G-P•G•E•D• . •T- •EEMK••QY•-P•TTPRGELUPDPEP•LI • •TKLIE•Q•• • • • • •CS••L•• • • • • •L• •HU•••F•S••T•• • IFF•• .RU• . . . . NTL . . . . . L 108 rY2 .O-PLGREBD,~ .E-VKU,LYGSGPTTPRGELPPDPEP.L .STKLUEUQUV . . . . CS . L . . . U . . S L U . H U U . F . S . . T . . . F F . A . . A U A . . . . ~TL . . . . . L 108

, TM3 TM4 I I I I I I ~¢ I

hy1 V-I~ILMDHWVFGERMCKLNPFUQCUS I TUS I FSLVL I AVERHQI. I I i~P R G WRP N NR HAY VO I FIU I I/U L A V R SSLP F L I YI~UMT D EP FQ NVT_--LDA YK~KYV CFIDIQFPS D$ 206 rY1 ............. T ................................................ I..T . . . . . . . . . . . . . V .... IL ...... ..S--.A.F ........ K ..... 205

I ......... ~I ........................ ', ................. ', ~ ~ ............. o ,,~" ........ ~ - - , ~ ........ ~ ..... mY1 2O5

×vl I., I ..... I,,.u, I .... EYJ .... v ........... q ' ................. ' I, .CF.. T, , ,GF, M. C. T, LMM,SI,L • ..... K~--, ,S. IG .... LED..E.K 201 hPPI/Y4 . . I I . V . I . . . T L I, .MSA. I . .M .U . . . . L . . . . u q . . . . . . . . . r . . K . S I S l Q . . L . . U L . . , r .CUL . . . . . AIiSII LENUFFIK_]:I.~<A. EFLA . .V . .TESW.LAH 209 rPPI/Y4 T, ,II..Y. I,, ,UL I, .MLT, I..M,U .... L .... U,L[ ......... T..K.SIS[Q, ,L. ,V., ,FISCFL ..... FINSIILN.LFHY,HSKUUEFLE. ,U,, ,USWS, ,H 209

mPPI/Y4 T. I I Y i .UL I. .MLT. I . . M . U . . . . L . . . . u q . . . . . . . . . T . . K . S l F I Q . . L . . U . . . F I S C F L . . . . . A N S I T L ~ I . L F H Y ~ K V V E F L E . . U . . . U S W S . . H 209 hY2 T..I.. GE. KM .PULl. H. U. VR. GLAUQ.. T I T. TU.. LID RC. UYHLESK I SK.II SFL I . GLA. G I SALLflS. LA. FRIEYSL IE I I PDFE I UA ....... .TEKW.GEE 211 rY2 T..I.. GE KM. PULl. H. U. YA. GLAV~.. T I T. TV.. LID RC. VYHLESK I SK(~II SFL I . GLA.GVSI~LLAS, LR. FRIEYSL I E I I PDFE I VA ....... .TEKW.GEE 211

hY1

rY1

mY1

xY1 hPPI/Y4

rPPI/Y4

mPPIIY4

hY2 rY2

TM5 TM6 TM? I

H---RLSYTTLLLULQYFGPLCF I F I CYFK I Y I RLKRRNNMMDKMRDNKYRSSETKR I i4 I MLLS I UUAFRVCWLPLT I F N T vLFLDItli'IH Q I I AT C NI-HIi4L LFL L C HL T AM I ST

, ---, ....................................... I , , S ................................... ..I .......................... F---. ....... FI...L ....... U..T.,FL ........... I ...................... G..L .... FF.,.L .... ..1 EAV .......... I ......... . --- , T I . . . F.. LF.. CL.. 6.. LU..IAR.. R.. Q. QGAUF-HKGTYSL. AGHM. QU. VIU. VVM ...... L .... HU.. SLE .~. H. EA. P I . HG..II . . U... L.. A..

. --- . . I . . . F.. LF.. CU.. A.. LV..IIiR . . Q. , Q. QRRRF-HTHTCSS. UBQM .... G I, . MAM. T .... L .... HU... LE. I, YQEA. PA. HG..II . . M . . . F.. A..

.---.. I . . .F.. LF..C I . .A..LV.. ~R..Q..Q. QKHVF-HAHACSS.RGQM .... S..MTM.T.. .k .... HU...LE.I. YQEA.PA.HG.. I . .M...L..A..

KS I YGTV. SLSS, L I L . VL, . G I . SFS. TR.klSK. ,i'IHVSPGAANDHYHQ. RQK. TK---I.. UCV.. U. .S .... Hfl,QLAVIIDS.VLDLKEYK. I .TVF. I I . .C..

KSUYGTV.SLST.L L.VL.,G .SFS.TR.WSK..NHUSPGRASDHYHQ.RHK.TK---.,VCV..U. .S .... H~,QLRU, l DSHVLDLKEYK..TUF.I ,.C..

313

372

312

308 315 315

315

318 318

I I ~ I I I i hYl ~UNPrFYGFLNKNFORDLQFFFNFCDFRSRDDDYETIRMSTMHTDUSKTSLKQRSPUAFKKI~NND---DHEKI 384 r Y l , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S M . . - - - - . . . . 382 mY1 ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S M . . - - - - . . . U 382 xY1 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . . . . . . . . . . . . . . . . . . . . . . . . . I . 366 hPPIIY4 ~..,FI .... [.T.,KKEIKALULT.QQSAPLEES.HLPL..U..E...G.,RLSGRSNPI 375

rPPI IY4 ~ . . . F I . . . . I . I . . K K . I K A L U L T . R C . P P Q G E P . P L P L . . V . . . L . , G . M R M G . K S N U M 375 mPPI/Y4 ~ . . . F I . . . . I . I . . K K . I K A L U L T . H C . . P Q G E S . H L P L . . U . . . L . . G . M R M G . K S N . I 3?5 bY2 Ffl..LL..WMI, S.YRKRFLSA.R-.EQ,- ......... LDAI.SE..U.FKAKKNLEUB.NSGP..SFTEATNU 381 rY2 FA..LL..WMI. S.YRKRFLSA.R-.EQ.- ......... LDAI.SE..M.FKAKKNLEUK.NNGLT.SFSEBTNU 381

Fig. 4. Alignment of NPY-family receptor sequences. Symbols as in Fig. 1. The six boxed residues in the human YI sequence (YIO0, D194, D200, F286, D287, H298) have been implicated in NPY binding as deduced ~om position-specific mutagenesis [51,50].

serve as ligands at physiological concentrations. N-terminally-truncated peptide analogues displayed lower affinities [37,42] like they do on the Y1 receptor. The rat and mouse PP1 /Y4 receptors seem to be more selective for PP than the human receptor, at least when bovine 125I-PP is used as radioligand in competition experiments. In the study by Lundell et al. [40] rat PP had a K i of 0.017 nM, whereas PYY and NPY had K i values as high as 162 and 192 nM, respectively. However, the rat PP1 /Y4 receptor reported by Bard et al. [39] did not distinguish as clearly between the three peptides with 125I-PYY as radioligand: ICs0 was 0.15 nM for bPP, 0.58 nM for PYY, and 1.7 nM for NPY. The mouse P P I / Y 4 receptor performed like the rat receptor reported by Lundell et al. [40] in competition with rat 125I-PP with an ICs0 of about 0.1 nM while PYY and NPY had values of 500 and 790 nM [41]. With 125I-PYY as radioligand, also the mouse receptor became less selective for PP relative to PYY and NPY with ICs0 values of 0.11, 3 and 3 nM, respectively [41]. However, ~25I-PYY displayed much lower specific binding than 125I- PP, only 50% versus 95%. All of the studies found that the modified NPY analogue [Leu31,Pro 34 ]NPY binds with high affinity to the PP1 /Y4 receptor, thus ruling out this compound as a Yl-selective ligand. The affinity of [Leu3~,Pro34]NpY is much higher than native NPY or

PYY in agreement with the two amino-acid replacements being derived from the PP structure. Functionally, the PP1/Y4 receptor seems to couple like Y1 by inhibition of forskolin-stimulated cAMP accumulation [38,39] and in- creased Ca 2+ influx in response to PP [39].

The PP1/Y4 receptor has 42% overall identity to the human Y1 receptor and 56% identity in the TM regions. It shares several features with Y1 such as two putative disulfide loops (Figs. 3 and 4). Interestingly, the three PP1/Y4 sequences show unusually low sequence identity among themselves with only 75% overall identity (85% in the TM regions) between the human sequence and the two rodent sequences (Tables 2 and 3). The rat and mouse sequences are 92% identical to each other. The orthologous relationship between the genes has been confirmed by Southern hybridizations; i.e., the genes are each other's closest homologoues in the three species.

The PP1 /Y4 receptor has a much more restricted mRNA distribution than Y1. In man, it has been detected by Northern hybridization in colon, small intestine, stom- ach and pancreas as well as in prostate [38,41]. Faint bands were observed in some brain subregions after long expo- sure. In rat, mRNA was found in colon at low level and, surprisingly, in lung and testis [40]. Mouse mRNA was observed in heart and small intestine [41 ], but not in lung.


5. Other reported NPY-family receptors

A eDNA clone called PR4 has been isolated from the fruitfly Drosophila melanogaster and was reported to encode a G-protein coupled receptor with functional response (inward current) to PYY and NPY (but not PP) when expressed in Xenopus laevis oocytes [43]. EC50 was approximately 0.3 ~M for PYY. However, no endogenous fly NPY/PYY-like peptide has yet been isolated and sequenced although NPY-like immunoreactivity has been detected in flies as well as several other invertebrates [44]. The Drosophila receptor's sequence identity to human Y1 and Y2 is not greater than to several other mammalian peptide receptors (Figs. 5 and 6). This also applies to the TM regions (Fig. 5): it has lower identity to both YI (33%) and Y2 (36%) (Table 3) than to the murine orphan receptor GCRC (38%) and the human NK3 receptor (36%). Thus, it is still unclear whether PR4 encodes a receptor that binds Drosophila melanogaster homologues of NP¥/VYV.

A G-protein-coupled receptor gene has been sequenced in the nematode Caenorhabditis elegans and was reported to have greater sequence identity to mammalian Y1 than to any other receptor (Fig. 5). In the TM regions, this receptor has 37% identity to Y1 and 40% identity to Y2 (Table 3). However, several gaps have to be introduced to optimize alignment. Like in Drosophila, no C. elegans homologue of NPY or PYY has yet been identified.

A bovine eDNA cloned called LCR1 was previously

% rPP1/Y4

[ " ' - - - - " ~ PP1/Y4

hPP1/Y4

~ - rY2a

hY2A

mGCRC P30731

Dros P25931

hNK3

Fig. 6. Distance tree for receptors that bind NPY-family peptides and a few other receptors. Branch lengths correspond to sequence divergence calculated from the alignment in Fig. 5, using the neighbour joining method [59] of the Lasergene DNASTAR Megalign software. The termini of the sequences have been excluded as they evolve very rapidly. The branching order within the shaded box is uncertain and may vary depending on which sequences are included (sequence information from additional species for these receptors may increase the resolution). Note that the mammalian Y2 sequences and the possible Drosophila NPY receptor do not cluster with the YI and PP1/Y4 sequences, but may be equally if not more similar to tachykinin receptors (hNK3). Note also the very different replacement rates for different branches; Xenopus laevis YI and the mammalian sequences split approx. 350 Myr ago, whereas the equally divergent PPI sequences for human and rodents split only about 80 Myr ago. Thus, the PP1/Y4 sequences have had a fourfold higher replacement rate than the Y1 sequences. For references see Table 1. The human Y2 sequence hY2A is that of Refs. [31,32]. The other receptors included in the analyses are the human neurokinin 3 receptor (hNK3, accession No. P29371), the murine orphan receptor mGCRC P30731 [58], and a Drosophila melanogaster receptor (Dros P25931) with reported binding to mammalian NPY and PYY [43]. The C. elegans receptor U41028 was used as outgroup (i.e., a reference sequence that is the least related to the others).

bY1 hPPI/Y4 hY2A hNK3 mGCRC P30731 Dros P25931 C, el U41028

hY1 hPPI/Y4 hY2A hNK3 mGCRC P30731 Dros P25931 C, el U41028

TMI TM2 ~ TM3 I I I I

~LPLAM~FTLAL~YGAV~LGV~GNLAL~[LKQKEMRNVTNpL~NLSF~DLLVA~MCLPFTFVYT~-~LMDHWuFGEAMCKLNPFVQCVS~TV~FS 130 ,QDSVDUMIVFIVTS.SIETVV..L,,,C,MCUTUR,,,KA .... ~,,R,,A,,,F.MCLL.Q,L,A,,,--I,,Y.I,,.TL..MSA.I.,M.U .... L, 131 STKLIEUQIVV,I,.,CSI,L,..I,,SLV.HVVII,F,S,,T.,.~F.R,,AVA .... NTL ..... LT,.I--,,GE.KM,PVLI. H.V,YA.GLAVQ,.TIT ] 140 FVQ.SWR.IALWS .... V.VAVA.L..~V.W~.AH.R.~T...~FL...A...A~M.~FNTLVN.~'A~--.HSE.Y~'~NY].RFQN.FP~TAVFA..Y. ~ 175 E$QNPTUK{RL,IV,,SFT,UFSLF..VLVCHV,IF,NQR,HSA,S~F .... AVA,IMITLLNT.,,L,RFI--VNST .... KG.I, HVSR,fl,YC,LH..ALT [ 161 MW~YFK1~VYML~P~F.FRL~.~GTVCY~Y~TPR..T~..~F.A~.~G.~.M~FF.E~S~.~L~-F~LNY~P'.L.L~HF~Y~A..VL`~YT 181 SPKEFGYF ,F.FT,MLI,LF,A .,FLT,,VVILNPA..TTR,~F,L,.AL..FF.C.UTR,T,LYTULYMFFT,P,SRTL,, flGSL,GFN,FL.T,, 133

TM4 * TM5 I I I I I I I I

LVL1~UERHQL~NPRGWRPNNRH~YV~A~WVLAVASSLPFL~YQVMTDEPFQ~VT--LDAYKDKY~CFDQFPSDSH---~RL~YTTLLLVLQYF6-- 223 ,..V,L ......... T,,K,SISQ..L.,VL.,,I,CVL ..... ANSILENVFHK,HSKR,EFLR,,V,.TESW,LRR,---~,TI.,,F,,LF.,CL-- I 226 ,TV,.LID,,RC,VYHLESKISK,IISFLI.GLA,GISALLAS,LR,FRIEYSLIEIIPDFEIUA ....... ,TEKW.GEEKSIY~GTV.SLSS.LIL,VL--~ 231 MTA,..D,YMA,.D,LKP,LSAT~TKIV.GS,,I,,FLLAF,QCL.SKTKVM,GRTL ............ ,.V,W.EGPKQ--~HFT.HIIVII.V,CF-- I 259 .TA,,,D..,V,MH,LKP,ISIT~GVIY ...... M,TFF.,,HA,C.KLFTFKYSEDIVRSL ....... ,LPD..-EPRDLF~WKYLDLATFI.L.LL-- I 251 ,,A,SID.Y~A,MW.LKP,ITK.~.TFI,,GU.FI.L,TA,,IP.VSGLDIPMSPWH,KCEK ..... .I,REMW,,R.Q---~EYY,,LS.FR,.FVV-- 271 RS,,,O,YU,.,F,TKRERQQN~SFCFF M,.,FTISL ,RUPLL,A$DLT.VFVEPSCDL.L---.I,NE.NEIWEKMII~KGT.,LAU. T..AFFT 230

I I I TM6 I I I I TM7 I~ hY1 PLCFIFICY~KIYIRLKRRNNMMDKMRDNKYR$ ........ SETKRINIIMLLSIVVAFA--UCWLPLTIFNTVF~WNHQIIATCNHNLLFLLCHLTAMIS 312 hPPI/Y4 ..G,.LU,,~R,.R,.Q,QGRUF-HKGTYSL.A ........ GHM,QV.VV,UVM ..... --,L .... HV.,SLE~,H,EA,PI,HG.,II,,V,,,L.,A, 314 hY2R ..GI,SFS,~R.WSK,,NHV$PGAANDHYHQ,R ........ QK.TK---,.VCV.,U..--,S .... HA.QLAV~IDS.ULDLKEYK~I,TUF,II..C, 317 hNK3 ..LIMG.T,~IVG.T.WGGEIPG,TCDKYHEQLK ........ RKRKVVK~,MIIU,MT,.--I .... YH,YFILT~IYQ.LNRWKYIQ~UY.ASFWL..S,] 348 m6CRC P30731 ,,FI,SUA.~RUAKK.WLC,TIG.UTTEQYLALR ....... RKK,TTVK,.VLV,.L,,--LU.C.,NCYVLLL~--SKA.H,--N,~,YFAF.WF,,S. 337 Dros P25931 ..GVLIFT,~R,T.,VWAKRPPGEAETNROQ.MA ....... R$KRKMVK,M.TU,IV,T--C .... FN.LQLLL~-DEEFAHWDPLP~UWFAF.WL,,SH 360 C. el U41028 ,.FSLUFA,~R,AH.M,L,FANRNQNVTTNTXTSQRRRSVVERQR,THLL,UCV.AV,,FT,A .... NU.HIFN~FELVNSFSV---~T,SI,.CL,,C. 327

I I hY1 TCVNP I FYGFLINKNFQRDLQFFFNFCDFR 341 hPPI/Y4 ..... F I , . . 'I' T.. KKE I KALVLT. QQ$ 343 hY2A . FR , , LL , . WM I . S , YRKAFLSA . R- , EQ . 345 hNK3 . MY , . , I , CC. I , , R , RflGFKRA , RW , P , I 377 mGCRC P30731 . ,Y. ,FI .CW,I,E..RUE. KRLLSM,-Q, 365 Oros P25931 C,Y. ,, 1 , CYII, AR,RSGFVQLIIHRMPOL 389 e, el U41028 A.L, ,LI ,A.F],I4, ,RIEFMHL,FTDRVG 356

Fig. 5. Alignment of NPY-family receptor sequences and a few additional G-protein coupled receptors. Symbols as in Fig. 1. hNK3 is the human neurokinin 3 receptor (accession No. P29371), mGCRC P30731 is a murine orphan receptor [58], Dros P25931 is a Drosophila melanogaster receptor with reported binding to mammalian NPY and PYY [43], and C. el U41028 is a Caenorhabditis elegans receptor with greater sequence similarity to Y1 than to any other known receptor.


reported to encode a G-protein coupled receptor displaying a pharmacological profile in agreement with subtype Y3 [45]. However, the LCRI receptor was subsequently found not to bind NPY [46], nor did its human orthologue [46,47]. The identification of this clone as Y3 may be due to endogenous expression of a Y3 receptor at low level in the cell line used for functional expression of the clone [46]. No ligand has yet been identified for LCR1 why it remains an orphan receptor. Interestingly, this receptor, named fusin, has recently been found to mediate entry of HIV-1 (human immunodeficiency virus) into CD4 + T lymphocytes [62].

6. Discussion

Two surprising discoveries have been made through the recent molecular cloning of the Y2 and PP1/Y4 receptors. The first concerns the low sequence identity of the Y2 receptor to both Y1 and PP1/Y4. The second surprise is the great variability between species for the pancreatic polypeptide receptor PP1/Y4 in sequence, pharmacology, and tissue distribution.

The Y2 receptor's degree of sequence identity to the Y1 receptor (31% overall, 40% in the TM regions; see Table 2) is exceptionally low. Other ligands such as 5-hydroxy- t ryp tamine (sero tonin) , dopamine , no rad rena - line/adrenaline and histamine also have receptors that differ extensively and do not form structural subfamilies [48], but NPY and PYY may be the peptide ligands with the most divergent receptors yet discovered (the angiotensin II receptors type 1 and type 2 differ almost equally much as the Y 1 and Y2 receptors with 34% overall identity and 43% in the TM regions [49]).

The evolutionary rate for Y2, as well as Y1 seems to be in the slower range for G-protein-coupled receptors as each displays 94% identity between man and rat. A slow rate of evolution in combination with the great sequence divergence between Y1 and Y2 would suggest that the gene duplication that generated Y1 and Y2 from a common ancestral gene took place very long ago, presumably before the origin of vertebrates. Another intriguing possibility to explain the structural difference between Y1 and Y2 would be functional convergence of structurally very different receptors, i.e., Y1 and Y2 have evolved NPY/PYY binding separately (this scenario is possible also for some of the small-ligand receptors mentioned above). This possibility may find some support in the seemingly very different modes of interaction of Y1 and Y2 with their ligands. Whereas Y1 binds to both the N- and the C-terminus of the peptides, Y2 bind almost exclu- sively to the C-terminus. Studies of the cloned Y2 receptor agree well with previous data in that N-terminally-truncated peptide analogues compete almost as efficiently as the intact peptides with the radioligand, giving the rank order of potency PYY > NPY > NPY2-36 > NPY13-36 > > [Leu31,Pro34]NpY > PP [6].

Sequence comparisons between Y1 and Y2 show that the positions that have been identified to interact with ligands in Y1 are unlikely to be the same in Y2. A series of human Y1 mutants [50] identified three Asp residues that were proposed to form salt bridges with NPY, namely D194 and D200 in loop 2 and D287 in loop 3 (Fig. 4). When either of these positions was mutated to Ala, the receptor completely lost NPY binding. (Position D104 in extracellular loop 1 of hY1 was also proposed to form a salt bridge, but was abandoned in a later study [51].) D287 in loop 3 is present in the human and rat Y2 sequences (as well as in PP1/Y4), but loop 2 is very divergent in size as well as sequence and therefore difficult to compare. An- other series of human Y1 mutants proposed interaction of NPY Tyr-36 with a hydrophobic pocket in Y1 formed by Y100, F286 and H298 [51]. Only YI00 is identical in Y2 (Fig. 4). Thus, the proposed points of interaction with NPY are likely to be rather different between Y1 and Y2. Site-directed mutagenesis will shed light on these interesting differences.

The P P I / Y 4 sequence identity between human and rat is a modest 75% for the complete sequence (85% for the TM regions), one of the lowest percentages reported for orthologous G-protein coupled receptors between different orders of mammals (Figs. 3 and 6). The low PP1/Y4 conservation contrasts with the high identities for Y1 (94%) and Y2 (94%). The rapid evolutionary rate of PP1/Y4 seems to correlate with that of its ligand, PP, which is also quite divergent between rat and man with eight replacements in 36 positions (78% identity). Thus, PP and its receptor seem to co-evolve at a rapid pace. PP is the most recent member of the NPY family and probably arose in an early tetrapod ancestor and has evolved rapidly in all lineages since then, leaving only 50% identity between amphibians, reptiles/birds and mammals. NPY and PYY, in contrast, have been found in all vertebrates investigated including the river lamprey [3], and have changed remarkably little in all vertebrate lineages (see Ref. [52] for review). As rat and mouse PP are the most divergent among all mammalian PP sequences, it will be interesting to see if also their PP1/Y4 sequences are exceptionally divergent among mammals. Indeed, preliminary sequence data for the guinea-pig PP1/Y4 receptor indicates that this has had a slightly lower rate of change than the rat and mouse receptors (H. Eriksson and D. Larhammar, unpublished).

The PP1/Y4 receptor differs between species not only in sequence, but also in ligand-binding properties and tissue distribution. The human and rodent receptors have the same rank order of binding preference, PP > [Leu31,Pro34]NPY > PYY > NPY, and both species bind PP with very high affinity (K i is 0.014 nM in man and 0.017 nM in rat, ICs0 is 0.1 nM in mouse) [37,38,41]. However, the rat and the mouse receptors are much more selective than the human receptor for PP as compared to NPY and PYY when competition is done against ~25I-PP

172 D. Larharnmar / Regulatory Peptides 65 (1996) 165-174

[38,41]. Interestingly, this selectivity is not as strong in competition with 125I-PYY [37,41] in agreement with recent studies on some receptor mutants showing differences in K i depending on which radioligand was used for competition [53,54].

The mRNA distribution of the PP1/Y4 receptor has so far been studied primarily with Northern hybridization. The presence of PP1/Y4 mRNA in different organs of the gastrointestinal tract is in agreement with the major sites of production of PP (pancreas) and PYY (pancreas and colon). Surprisingly, mRNA was also observed in mouse heart, rat lung and testis, and human prostate. The exact sites of expression within these organs need to be investigated with in situ hybridization in order to assess the physiological significance. Also the possible correlation with the reported PP-binding regions in rat brain, namely area postrema and the interpeduncular nucleus [55,56], will have to be explored with in situ hybridization.

Binding experiments with peptide analogues showed that the cloned PP receptor shares two characteristic features with the Y1 receptor as might be expected from their sequence similarity [37,42]. Firstly, PP1/Y4 is dependent on the N-terminal portion of the peptide ligands for opti- mal binding (albeit somewhat less dependent than Y1), and secondly, PP1/Y4 binds the analogue [Leu31,Pro34]NpY that has been regarded to be selective for Y1 (and Y3) relative to Y2. Thus, previous binding experiments with this analogue will have to be reevaluated in the light of these results, although the limited distribution of the PP1/Y4 receptor in both man, rat and mouse suggests that it is unlikely to have interfered significantly with Y1, at least in the brain.

Information on the evolutionary relationships between the NPY-family receptor genes may be provided by features such as positions of introns and chromosomal localization. Of the three cloned genes, only Y1 has an intron in the coding region and it is presently not clear whether Y 1 received this intron after it had diverged from the PP1/Y4 gene or if the latter gene lost this intron by reverse transcription of Y1 mRNA, reinsertion in a chromosome and subsequent sequence divergence. Chromosomal localization may distinguish between these possible scenarios.

Sequences for these receptor subtypes from additional species and sequences for the remaining receptor subtypes, along with pharmacological studies of expressed clones, will shed more light on the many functions of the NPY- family peptides. Interesting prospects are indicated by the absence of expression of the cloned Y2 receptor in peripheral organs, suggesting additional Y2-1ike receptor subtypes, as well as the cloning of three novel NPY-receptor- like genes in zebrafish that seem to represent subtypes distinct from the three cloned mammalian receptors (I. Lundell, P. Starb~ick, M. Ringvall and D. Larhammar, unpublished). This offers fascinating complexity for a receptor family that already harbours the two highly divergent and slowly evolving receptors Y 1 and Y2, as well as

the rapidly evolving PP receptor. Fortunately, the evolution of NPY, PYY and PP seems to have been resolved [44,52,57] to the extent that it will be helpful in the efforts to understand the evolution of the many receptor subtypes and their functions.

7. Note added in proof

After submission of this manuscript, two additional NPY receptors have been reported, unfortunately both with the designation Y5. A mouse NPY/PYY receptor with greatest sequence similarity to Y1 was reported by Wein- berg et al. [63]. A rat NPY/PYY receptor involved in food intake was reported by Gerald et al. [64]. This receptor is as different to both Y1 and Y2 as the latter two are to one another. Remarkably, this Y5 receptor gene overlaps par- tially with a distant promoter of the Y1 gene. These astonishing findings add new twists to the fascinating evolutionary history of the NPY receptor family.

Acknowledgements

This work was supported by a grant from the Swedish Natural Science Research Council. I thank Ingrid Lundell, Paula Starb~ick, Maria Ringvall, Henrik Eriksson (all at Uppsala University), Donald R. Gehlert (Lilly Research Laboratories) and Paul Gregor (Bayer) for communication of unpublished results.

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Structural diversity of receptors for neuropeptide Y, peptide YY and pancreatic polypeptide

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