Evolution of neuropeptide Y, peptide YY and pancreatic polypeptide

REGULATORY

ELSEVIER

PEPTIDES Regulatory Peptides 62 (1996) 1-11

Review

Evolution of neuropeptide Y, peptide YY and pancreatic polypeptide Dan Larhammar * Department of Medical Pharmacology, Uppsala University, Box 593, S-751 24 Uppsala, Sweden Received 2 October 1995; revised 1 December 1995; accepted l December 1995

Abstract

The neuropeptide Y family of peptides consists of neuropeptide Y (NPY), which is expressed in the central and peripheral nervous systems, and peptide YY (PYY) and pancreatic polypeptide (PP) which are gut endocrine peptides. All three peptides are 36 amino acids long and act on G-protein-coupled receptors. NPY and PYY are present in all vertebrates, whereas PP probably arose as a copy of PYY in an early tetrapod ancestor. ~PY is one of the most conserved peptides during evolution and no gnathostome (jawed) species differs from the ancestral gnathostome sequence at more than five positions. PYY is more variable, particularly in mammals which have nine differences to the gnathostome ancestor. PP may be the most rapidly evolving neuroendocrine peptide among tetrapods with only 50% identity between mammals, birds, and amphibians. Ancestral gnathostome NPY and PYY seem to have differed at only four positions, suggesting that the gene duplication occurred shortly before the appearance of the gnathostomes. The two peptides differ from one another at 9-12 positions in tetrapod species and share at least two receptor subtypes in mammals. In bony and cartilaginous fishes, NPY and PYY have only 5-6 differences which, together with more extensive neuronal localization of PYY, indicate an even greater functional overlap between the two peptides in these animal groups. The emergence of sequence information for several receptor subtypes from various species will shed additional light on the evolution of the functions of the NPY-family peptides. Keywords: Neuropeptide Y; Pepfide YY; Gene duplication

1. Introduction

Few peptide families can count as many sequenced members as the neuropeptide Y (NPY) family (Tables 1-3). The sequence information is analysed here to deduce the evolutionary events that have led to the present members. NPY and peptide YY (PYY) have been found in all vertebrates investigated whereas pancreatic polypeptide (PP) is present only in tetrapods. All NPY-family peptides are 36 amino acids long (with one exception) and all have the key residues required to adopt the so-called PP-fold [1] which is characterized by an extended proline helix with three prolines, a turn, and an alpha helix with two tyrosines interdigitating with the three prolines (Fig. 1). NPY is expressed exclusively in neurons and has been found to influence several physiological parameters, e.g., blood pressure, food intake, sexual behaviour, and circadian rhythms [2,3]. The effects are mediated by G-proteincoupled receptors of at least four separate subtypes called Y1, Y2, Y3 and the hypothalamic feeding or appetite

* Corresponding author. Tel.: +46 18 174 173; fax: +46 18 511 540; E-mail: Dan'Larhammar@ MedFann'UU'SE 0167-0115/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 01 67-01 15(95)001 69-7

receptor [4]. PYY and PP are present in endocrine cells of the intestine and the pancreas of all vertebrates investigated (see [5], and references therein). PYY mRNA is also expressed in a few neuronal cell populations in rat [6,7] and in many neurons in the lamprey CNS [8,9]. PYY-like immunoreactivity has also been observed in gut neurons of several mammals [10]. PYY and PP are released in response to meals and inhibit gastric and pancreatic secretion as well as gastric and intestinal motility (see [11,12] for references). The role of PYY in neurons is still unclear. PYY may act on YI and Y2 receptors [4] as well as a separate PYY receptor [4] whereas PP was recently found to have a distinct receptor distantly related to Y1 [13,14]. The branching order for the major vertebrate groups is well known [15] and allows determination of the likely time points for gene duplications as well as amino acid replacements in the NPY family. Ancestral sequences can be deduced by comparing different lineages. For instance, if mammals, amphibians and bony fishes share one residue that differs in birds, one assumes that a replacement has occurred in the bird lineage and that the other animal groups have retained the ancestral residue. Comparisons are sometimes complicated by parallel identical changes in

2

D. Larhammar / Regulatory Peptides 62 (1996) 1-11

separate lineages. Such parallelisms can be informative with regard to peptide-receptor interactions because they imply that only two or a few amino acids can be tolerated at particular positions. The sequence analyses presented here not only shed light on the evolutionary events that have led to the present NPY-family peptides, but also suggest that functional overlap between NPY and PYY (and PP) may differ between animal groups and stress the importance of using endogenous peptides when studying physiological effects in different species.

Table 2 PYY and PY sequence references Peptide Species

Seq. obtained from Peptide cDNA Gene

PYY

Pig Human

X X

Cow Rat

X

X X

2. Neuropeptide Y Despite its relatively large size, NPY displays a remarkable degree of sequence conservation (Fig. 2). No neuroendocrine peptide of equal or larger size displays such high identity among vertebrates [88]. Twenty-two positions are identical in all NPY sequences known. Among the 14 variable positions, several have highly conservative replacements; four are Glu-Asp exchanges, others are Leu-

Table 1 NPY sequence references Species

Seq. obtained from Peptide

NPY

Pig

X

Cow Sheep Human

X X X

Rat

X

cDNA

[16] [17] [183 [19] [201

European green frog, Rana ridibunda European common frog, Rarva temporaria African clawed toad, Xenopus laevis Common goldfish, Carassius aura[us Atlantic cod, Gadus morhua Trout, Oncorhynchus mykiss European common dogfish, Scyliorhinus canicula Torpedo marmorata River lamprey, Lampetra fluoiatilis

[21] [19] [22] [231 [24]

X

Rabbit Guinea pig Chicken Alligator

Ref.

Gene

x

x

[25]

X

[26] [19] [191 [27]

X X X X X X

[281 [29] [3o1

X

[311 X X X

X X X

X

[32] [33] [27] [34] [34]

[351 X X

[27] [9]

[36] [37] X X X

Mouse

Peptide

Ref.

PY

Guinea pig Chicken Tree frog, Phyllomedusa bicolor European green frog, Rana ridibunda Trout, Oncorhynchuz" mykiss Coho salmon, Oncorhynchus kisutch Eel, Anguilla rostrata Bowfin, Amia calva Alligator gar, Lepisosteus spatula Skate, Raja rhina Torpedo marmorata Spiny dogfish, Squalus acanthias European common dogfish, Scyliorhinus canicula River lamprey, Lampetra fluoiatilis Sea lamprey, Petromyzon marinus Anglerfish, Lophius americanus

Daddy sculpin, Cottus scorpius Tilapia, Oreochromis nilotica Seminal-Cow plasmin

X X X X

[38] [39] [401 [221 [41] [42] A.B. Leiter, unpubl. [43]

[441 [451

X

[461

X X X

[34] [471 [48]

X X X

[491 [491 [50]

X X X

[49] [511 [52]

X

[531 [91

X

[54]

X X

[55]

X

[57]

X X

[58] [59]

[561 P. Hobart, unpubl.

X

[40]

Met, Lys-Arg, Ser-Thr, and Leu-Val replacements. Only one position seems to tolerate more than two alternative amino acids, namely position three. Four positions have undergone parallel replacements, i.e., the same change in separate lineages (positions 7, 11, 17 and 19). The strong conservation of NPY is partly due to constraints imposed to allow adoption of the characteristic 'PP-fold' structure. However, this feature is also present in

D. Larhammar ~Regulatory Peptides 62 (1996) 1-11

3

Table 3 Pancreatic polypeptide sequence references Peptide Species

Seq. obtained from

Ref.

Peptide eDNA Gene PP

x x Cow x Sheep x Przewalski's horse x Mountain zebra, Equus zebra x White rhinoceros, x

[60] [61] [62] [62] [63] [63] [63]

Pig

Ceratotherium simum Mountain tapir, Tapirus pinchaque Dog Cat Human

Rabbit Rat

X

[63] [62] [64] [65] [66] [67] [681 [69] [70] [71] [72] [72] [73] [74] [75] [76] [77]

X X X X X

X Mouse Hedgehog Guinea pig

X X X X

Chinchilla Chicken

X X X

[78] X

X

[79]

Turkey Herring gull Magpie, Pica pica Jay, Garrulus glandarius

X X X X

[80]

Hooded crow, Corvus corone Rook, Corvusfrugilegus Jackdaw, Corvus monedala Goose Ostrich Alligator American bullfrog, Rana catesbeiana European common frog, Rana temporaria Three-toed amphiuma (salamander), Amphiuma tridactylum

X X X X X X X

[82] [82] [82] [83] [84] [85] [86]

X

[31]

X

[87]

[811

[821 [82]

the highly variable PP (see below) and primarily involves the interdigitation of proline and tyrosine side chains between the two helices (Fig. 1). Therefore, many if not most positions in NPY are more likely to be kept conserved due to binding to multiple receptor subtypes that differ in their points of interaction with the ligand. (It is also possible that some NPY residues are: conserved to prevent binding to certain receptor subtypes that prefer PYY or PP.) It is

Fig. 1. Schematic structure of PP-fold peptides adapted from Ref. [1]. The interdigitating pralines and tyrosines are shaded.

well documented that receptor subtypes Y1 and Y2 differ markedly in that Y1 requires both ends of the NPY or PYY molecule for binding, whereas Y2 only sees the carboxyterminal part (see [89] and [4] for reviews). Furthermore, Y2 has stricter requirements on the carboxyterminal portion because replacement of the invariant positions Ile-31 and Gin-34 in NPY or PYY (to Leu and Pro, respectively) abolishes Y2 binding but still allows Y1

I 10 20 30 36 I I I I I HPY g n a t h o s t o n YPSKPDNPGEDRPAEDLRKYYSRLAHYINLITRQRY HPY human MR NPY rat MR HPY rabbit MR HPY pig R HPY cow R NPY sheep D R HPY guinea pig MR HPY chicken S fl A HPY alligator M R HPY Rona ridibundo M ffPY Rono tomporaria M HPY Xenopus laevis M HPY gsnopus Ioevls D M HPY goldfish T G E NPY R t l a n t i c cod I E DE NPY trout U E T E T HPY Torpedo morm, G HPY Eur, dogfish G HPY r i v e r lampre~ F H S R L U HPY constant ,P.KP..PG.,AP . . . . R.Y.,R.RHYIHLITRQRY HPY 9nathostome YPSKPDHPOEDflPflEDLAKYYSRLRHYIHLITRQRY

Fig. 2. Alignment of NPY sequences. Only positions that differ from the top sequence, the deduced ancestral gnathostome sequence, are shown. At position 11, it is possible that the gnathostome ancestor had G instead of D. Two distinct Xenopus laeuis sequences have been determined that presumably correspond to separate loci (Xenopus laevis is tetraploid). For references see Table 1.

4

D. Larhammar / Regulatory Peptides 62 (1996) 1-11 1 I PYY PYY PYY PYY PYY PYY

gnothostome rat mouse pig cow human PYY guinea pig PYY chicken

10 I

20 I

R R A R I S

R

R R A OR H R RSS S DR S

S S S $ O $

$ S S

PYY t r o u t

salmon eel bowfln got Rojo rhino Torpedo morm, spiny dogfish Eur, dogfish river lamprey sea lamprey constant gnothostome onglerflsh daddy sculpin tiloplo cow

36 I

origin or PY because of unclear relationships. These difficulties in correlating fish pancreatic peptides with mammalian peptides is due to a more rapid divergence of tetrapod and particularly mammalian PYY (Fig. 3) and to unique replacements among a subgroup of fishes called acanthomorphs (see below). Unambiguous identification of PYY has been possible in the European common dogfish where both a gut peptide and a CNS peptide have been sequenced. The dogfish CNS peptide differs from human NPY at only three positions, whereas the gut peptide differs at six positions. This comparison identifies the dogfish CNS peptide as NPY and, consequently, the dogfish gut peptide should be the homologue of mammalian PYY although these differ at nine positions (dogfish NPY and mammalian PYY differ even more; 13 positions). All gut peptides from cartilaginous and bony fishes resemble one another closely (except acanthomorph PY described below) and define the ancestral gnathostome sequence (Fig. 3), One feature that is shared by all fish gut peptides is a Pro at position 14, whereas NPY has Ala at this position in all species (except trout which has Thr). All tetrapod PYY sequences also have Pro-14, thus supporting the conclusion that the fish gut peptides are, indeed, the counterpart (orthologue) of PYY. PYY has evolved more rapidly than NPY in tetrapods. The two frog PYY sequences known have six and seven differences to the gnathostome ancestor, whereas chicken and mammals have nine differences. The increased evolutionary rate for tetrapod PYY may be a consequence of the appearance of the PP gene as a copy of the PYY gene in the tetrapod ancestor (see below). Recently, another copy of the PYY gene (in addition to PP) was discovered in cow [40]. This gene is expressed in bull semen, hence its product was named seminalplasmin. The gene displays > 95% identity to the PYY gene over 2,000 bp, but the protein has diverged considerably and lacks many of the PYY features such as the 'PP-fold'. It will be interesting to see whether seminalplasmin binds to a receptor with similarity to the NPY-family receptors.

YPPKPENPGEDRPPEELRKVYSALRHYINLITRQRY

PYY tree frog PYY Rang ridibundo PYY PYY PYY PYY PYY PYY PYY PYY PYY PYY PYY PYY PY PY PY spl

30 I

S

0

SR AS SR RS $R AS NR T$ NR R$ RRS I QF MN LT MT LT T T Q T R

LL D D

U U U U U

F

U U V

R

L F M

L L L L L LU

ON S SP $

YL

OM R KR U $ ML U N

.P,KP..P..,A,P . . . . . Y . . . . R,Y.NL,TRQR, YPPKPENPGEDRPPEELAKYYSRLRHYIHLITRQRY O --S

T SH S 6N S S QPSD K

S gg S Off U S DW HA U S DW HA U S DKHHRF--S SR

V U U AK AN --L

Fig. 3. Alignment of PYY and PY sequences as well as the bovine PYY copy called seminalplasmin (spl). Only positions that differ from the top sequence, the deduced ancestral gnathostome sequence, are shown. Dashes are gaps introduced to optimize alignment. For references, see Table 2. Note that the chicken sequence is one amino acid longer at the amino terminus and that the Torpedo sequence has been disregarded for calculation of the gnathostome ancestral sequence due to its many unique replacements.

binding [90,91]. The Y3 and feeding receptors are likely to have distinct requirements on the NPY structure.

3. Peptide YY and fish PY PYY was originally purified from pig intestine [36] and subsequently from additional mammals. However, the gut peptides extracted from bony and cartilaginous fishes were found to be equally similar to mammalian NPY as to mammalian PYY, and were therefore classified as either NPY or PYY, or sometimes PP because of pancreatic

Gar Bowfin Eel Salmon, trout PYY

Anglerfish

J

Daddy sculpin] ~ PY

250

200

150

1 O0

Tilapia

50

J~

J o E

[

Million years before present

Fig. 4. Evolutionary tree for PYY and PY in bony fishes. PYY has been sequenced in gar, bowf'm, eel, salmon and trout. The PY peptides have only been found in the three species belonging to Acanthomorph fishes and probably constitute the orthologue of PYY. Numerous branches have been left out for clarity. Information for the branches between the salmonid fishes and anglerfish (dotted lines) should be helpful in determining the time point for the rapid divergence of PY. Time points for branch divergencies should be considered approximate; however, the relative branch order is generally agreed upon. See [92] for references.

D. Larhammar / Regulatory Peptides 62 (1996) 1-11

PP PP PP PP PP PP PP PP

PP

pig cow sheep horse mountain zebra rhinoceros mountain tapir

human rabbit

I

I0

20

39

36

I

I

I

I

I

PP PP

mouse hedgehog PP guinea-pig PP PP PP PP PP PP PP

chinchilla chicken turkey herring gull crow (5 sp.) goose

P

EVD

N Y H R ETQ Y ETQ H H O n N M

M M U G SQ T G SQ T 6 UQ T AO R G SQ T G RQ T

ostrich PP alligator T Q K S HH PP Rana catssb. S HH PP Runs temporaria K EH PP salooander PP constant PP tetrapod

pancreatic peptides are most probably the Acanthomorph orthologue of PYY, because if they were the result of a separate duplication of the PYY gene, the true PYY peptide should also have been found in these species. Peptide sequence information for species representing the branches between the salmonid fishes and anglerfish (dotted lines in Fig. 4) should be helpful in determining the time point for the rapid divergence of PY.

RPLEPVYPGDDATPEQMRQYflRELRRY IHMLTRPRY E H S E H M H M N $ H E H h O

PP dog PP cot PP rat

PU PU PU PU N PU PU G PU

0

T T

4. Tetrapod

DLIRFYND OO L ug H DLIRFYND OO L UV H DLVRFYHD OO L UU H DLLRFYND OO L UU DLXRFYDN OO RLNVF H DLURFYHD OO L UU H DLI FYHD OO L UU

O L

YSD YQ TFI

Q QD L YSD YQ TFU S LEK YQD FQ IFI . . . . P,,PG,,R . . . . . . . . . . . . . . Y . . . . . R.R, APXEPXXPGDDRTPEXLMQYYXDLXQYIXXXTRPRY

Fig. 5. Alignment of PP sequences. Only positions that differ from the top sequence are shown. For references, see Table 3.

Peptides isolated from l~e pancreas of three species of bony fish, anglerfish, daddy sculpin, and tilapia, share several unique replacements as compared to NPY as well as PYY of other fish species (Fig. 3) and the first of these peptides was named anglerfish peptide YG, abbreviated APY [55]. As all three of these fishes belong to Acanthomorpha (but no other species does among those whose peptides have been sequenced), the sequences probably reflect unique evolutionary events in this lineage (Fig. 4). The stronger sequence similarity between tilapia and daddy sculpin is in agreement with their closer evolutionary relationship as both belong to Percomorpha [92]. These

NPY Eur. PYY Eur.

dogfish dogfish

5

pancreatic

polypeptide

Pancreatic polypeptide has only been identified in tetrapods. The sequence variability is much greater than for NPY and PYY with only 50% identity between mammals, birds, and amphibians (Fig. 5). In fact, mammalian and chicken PP are no more similar to each other than either is to NPY or PYY. The alligator PP sequence is clearly most similar to the bird sequences and the salamander is closest to the frog sequences in agreement with the general view from palaeontology and comparative anatomy on the relationships between these groups [93]. However, sequence information for several other proteins have previously given ambiguous results [94]. The close fit of the PP sequences with palaeontology may be because PP was in rapid evolutionary flux during the period when these animal groups diverged from one another. Subsequently, PP seems to have stabilized in the various lineages. Therefore, PP sequences may be exceptionally powerful to resolve the branching order in early tetrapod evolution and sequence information for turtles, snakes and lizards is eagerly awaited. The rapid divergence of the PP amino acid sequences seems to be accompanied by changes in the exon organization of the gene. The PP gene in man [68] has the same

I

10

20

30

36

I

I

I

I

I

Diff.

YPSKPDNP6EGRPREDLAKYYSALRHYINLITRQRY P E D P E

5

NPY human PYY human

YPSKPDNPGEDRPAEDMARYYSRLRHYINLITRQRY I EA $P ELN AS L V

12

NPY pig PYY pig

YPSKPDNPGEDRPAEDLARYYSALRHYINLITRQRY R ER SP E S AS L V

11

NPY chicken

YPSKPDSPGEDRPAEDMARYYSALRHYINLITRQRY

PYY c h i c k e n

A

P

E

DR SP El

O F

U

11+I

NPY Rana ridibunda PYY Rana ridlbunda

YPSKPDNP6EDAPAEDMAKYYSALRHYINLITRQRY P E SP E T LT U

9

NPY trout PYY trout

YPUKPEHPGEDAPTEELAKYYTALRHYINLITRQRY P P

2

FPNKPDSP6EDAPREDLRRYLSRURHYIHLITRQRY P H DN SP QM KR

10

NF'Y river PYY river

lamprey lamprey

Fig. 6. Comparison of NPY and PYY within species. For references, see Table 1 and 2.

6

D. Larhammar / Regulatory Peptides 62 (1996) 1-11

PYY. Cloning of the peptide precursors should resolve this issue as prepro-NPY and prepro-PYY differ extensively in the carboxyterminal extensions, The degree of sequence identity between NPY and PYY within each species is of course crucial for various receptor subtypes to distinguish or accept the two peptides. Although Y 1 and Y2 receptors bind both NPY and PYY in mammals, the situation may be different in other tetrapods. Furthermore, the NPY-preferring and PYY-preferring receptor subtypes described in mammals (see [4]) may not be able to distinguish the two ligands in cartilaginous and bony fishes.

exon organization as PYY and NPY. However, the other four PP nucleotide sequences known, namely rat, mouse, guinea pig, and chicken (see Table 3) reveal changes in exon organization that, in all four cases, result in dramatically altered structure of the carboxyterminal extension of prepro-PP [75,79]. As each species has unique structural changes, these mutation events are comparatively recent, at least in rat and mouse.

5. NPY-PYY within species Given that the ancestral gnathostome presumably had four differences between NPY and PYY, it becomes interesting to see how many differences exist between the two peptides in living species. Both of the peptide sequences are known in several species as shown in Fig. 6. The cartilaginous representative, European common dogfish, has five differences between NPY and PYY which suggests that virtually no changes have occurred throughout the evolution of this lineage. Zebrafish NPY and PYY (not shown) differ at six positions (S~Sderberg et al., unpublished). The tetrapods show 9-12 differences between the two peptides which are primarily due to accumulation of replacements in PYY, whereas NPY has remained virtually identical. Also the river lamprey has a similar number of differences between NPY and PYY that seem to be largely due to divergence of PYY (Figs. 2 and 3). The trout sequences, in contrast, are actually more similar to one another than the ancestral gnathostome sequences (Figs. 6 and 7A). Two alternative hypotheses may account for this finding. One possibility is that both of the sequences are actually PYY, as salmonid fishes have undergone an additional tetraploidization and may have retained expression of both of the PYY gene copies. If so, the two PYY genes may have acquired different tissue expression as one peptide was purified from gut and the other from brain [34]. The other possibility is that there has been sequence convergence so that the trout NPY sequence has undergone mutations rendering it more like

6. Gene localization The human NPY gene has been localized by in situ hybridization to chromosome 7p15.1 [95] whereas the PYY and PP genes are located only 10 kb apart in tandem on chromosome 17q21.1 [96]. The finding that the NPY gene is located adjacent to the HOX1 cluster of antennapaedia-like homeobox genes, and that the PYY gene flanks the HOX2 cluster, supports the possibility that NPY and PYY arose from a common ancestral gene in the two genome duplications (tetraploidizations) that occurred in early vertebrate evolution [97]. Chromosomal mapping is underway with the zebrafish NPY and PYY genes (SSderberg et al., unpublished) to see if they reside adjacent to the orthologous HOX clusters. These observations open the possibility that additional NPY/PYY-like genes may be found in the corresponding position adjacent to the other two HOX gene clusters HOX3 and HOX4. The close proximity of the PYY and PP genes [96] is consistent with a more recent separate duplication of PYY to generate PP. An additional separate gene duplication seems to have occurred in cow where the PYY gene was duplicated to generate the seminalplasmin gene [40] which has since undergone rapid divergence.

NPY gnathostome

I 10 20 3Q 36 I I I I I YPSKPDHPGEDAPREDLAKYYSALRHYINLITRQRY

PYY 9nathostome

YPPKPENPGEDRPPEELRKYYSRLRHYINLITRQRY

PP

RPXEPXXPGDDATPEXLMQYYXOLXQYIXXXTRPRY

A

O

tetrapod

B

I

NPY constant PYY constant

I I I I I Constant ,P,KP, ,PG. ,AP .....A,Y, ,A,RHYINLITRQRY 22 .P,KP,.P,,,A.P ..... Y .... R,Y,NL.TRQR, 15

PP

....

constant

10

P,,PG,.A

20

..............

30

Y .....

36

R,R.

?

Fig. 7. (A) Ancestral NPY-familysequences as deduced in Fig. 2, Fig. 3 and Fig. 5. Dots mark differencesbetween the NPY ancestor and the PYY ancestor. The diamond marks identity of the PP ancestor with the PYY ancestor at the PYY-diagnostic position Pro-14. (B) Constant positions in NPY-familysequencesas deducedin Fig. 2, Fig. 3 and Fig. 5.

D. Larhammar / Regulatory Peptides 62 (1996) 1-11

7. NPY family evolution Comparisons of the NPY-family peptide sequences give a fairly clear picture of their evolution. This contrasts with the situation for many other peptide families such as oxytocin/vasopressin [98] and GnRH [99] which despite ample sequence information remain complicated, probably because of parallel identical changes in separate lineages and additional gene duplications in certain lineages. The deduced ancestral gnathostome NPY and PYY sequences and the ance~;tral tetrapod PP sequence are shown in Fig. 7A. Constant positions for each of the three peptides among all species known are shown in Fig. 7B. The major events leading to the present NPY-family peptides are displayed in the chordate evolutionary tree shown in Fig. 8. As outlined above, the small differences between ancestral gnathostome NPY and PYY, along with the location of the NPY and PYY genes at similar positions near HOX gene clusters, strongly argue for an ancestral gene duplication as a result of the tetraploidizations that occurred in early vertebrate or craniate evolution [97]. Amphioxus has a single HOX cluster [100] and should therefore be expected to have a single NPY/PYY gene. It is still a matter of debate exactly when the genome duplications occurred; a recent suggestion is that one tetraploidization occurred between amphioxus and hagfish whereas the second took place between lamprey and the gnathostomes (jawed vertebrates) [101]. Cloning of N P Y / P Y Y genes in amphioxus and hagfish will therefore be crucial to test this hypothesis. Whether the previously described invertebrate peptides NPF and PYF share a common ancestor with N P Y / P Y Y is still an open question as they lack the PP-fold structure found in all vertebrate sequences (see [12]). The other major event in NPY-family evolution was the

7

appearance of PP, presumably in an ancestral tetrapod (Fig. 8) as PP has only been found in tetrapods (Fig. 5). Several lines of evidence argue that the PP gene arose as a copy of the PYY gene: (i) sequence comparisons suggest that the ancestral tetrapod PP sequence had the PYY-diagnostic proline at position 14 [12]; (ii) the PP gene has small introns like the PYY gene whereas the NPY gene has large introns [88]; (iii) the PP gene is located adjacent to the PYY gene in the human genome [96]; (iv) PP and PYY are both expressed in pancreatic islets [42,102]. The relatively rapid evolution of PYY among tetrapods, as opposed to the slow rate in cartilaginous and most bony fishes, may be a consequence of the gene duplication that generated PP, which also has undergone rapid evolution. Another rapidly evolving branch seems to be PYY (PY) in acanthomorph fishes (Fig. 4). The differences in evolutionary rate between lineages make experimental use of peptides across species exceedingly difficult to interpret. For instance, studies with avian PP in mammals or vice versa are equally, if not more, likely to elicit responses mediated by NPY- or PYY-preferring receptors rather than PP receptors. On the other hand, some such studies may be informative with regard to receptor-ligand interaction sites provided peptide and receptor sequences are known in the respective species. In addition to understanding how and when NPY and PYY became separate entities in the early vertebrate or craniate, it will be interesting to try to unravel how NPY and PYY differ in distribution and function in the major vertebrate groups, (This can only be accomplished with certainty using DNA or RNA probes to detect mRNA because antibodies have a high risk of detecting both of the peptides - furthermore cross-reactivity may differ between species due to sequence differences.) The expression pattern of the ancestral N P Y / P Y Y gene may be informaAmphioxus Hagfishes Lampreys Cartilaginous fishes Bony fishes Acanthomorph fishes

Amphibians Single Etnceet hal N PYIPYY gene

Reptiles and birds } i

Duplication generating N P Y and P Y Y

pp

Mammals 600

500

400

300

200

100

Million years before present

Fig. 8. Probablegene duplication events in the NPY familyshown in a schematic chordateevolutionarytree. Both NPY and PYY were present in the ancestral vertebrateas both peptides have been found in lamprey.PP probablyarose early in tetrapodevolutionby duplication of the PYY gene. PY is probablya divergedform of PYY and has been foundonly in acanthomorphfishes which are a subgroupamong the bony fishes.Time points for branches are from [15] and [92].

8

D. Larhammar/Regulatory Peptides 62 (1996) 1-I1

tive and might be expected to display both neuronal and endocrine expression because PYY is expressed in neurons as well as endocrine cells in taxa as remotely related as lampreys [8,9] and rats [10,6,7]. Studies of PYY in lamprey [8,9] and zebrafish (unpublished) show a much more widespread neuronal expression than in rats and suggest that this was the ancestral distribution with subsequent loss of much of the neuronal PYY expression in rat and presumably other mammals. The division of labour between PYY and its copy PP will also be an interesting topic for future studies and may be expected to differ between tetrapod groups due to the rapid evolution of PP. Studies of receptor gene duplications and the ligand repertoire for each receptor subtype will help resolve these issues. In fact, preliminary evidence for the novel PP receptor PP1 suggests differences with regard to endogenous ligand preferences even between rat and human [ 13,14]. Thus, the NPY family of peptides includes one of the most conserved peptides known, namely NPY itself, and one of the most variable neuroendocrine peptides, pancreatic polypeptide. With three peptides in the same family evolving at such different rates, it will be very interesting to see how receptor evolution correlates with peptide evolution. The Y 1 receptor has been cloned in three mammals and Xenopus laevis [103] and displays a high degree of sequence conservation. The PP1 receptor, in contrast, seems to be the most divergent receptor known between different orders of mammals [14] in accordance with its rapidly evolving ligand. The extensive sequence differences between Y1 and PP1 on the one hand and the Y2 receptor on the other [104] constitute and intriguing puzzle for evolutionary and functional studies. Undoubtedly the comparative approach will be very useful to unravel the secrets of the NPY family and its many receptors.

Acknowledgements This work was supported by a grant from the Swedish Natural Science Research Council. I thank Adrew B. Leiter for communication of unpublished results.

References [1] Fuhlendorff, J., Langeland Johansen, N., Melberg, S.G., Th0gersen, H. and Schwartz, T.W., The antiparallel pancreatic polypeptide fold in the binding of neuropeptide Y to Yi and Y2 receptors, J. Biol. Chem., 265 (1990) 11706-11712. [2] Dumont, Y., Martel, J.-C., Fouruier, A., St-Pierre, S. and Quirion, R., Neuropeptide Y and neuropeptide Y receptor subtypes in brain and peripheral tissues, Prog. Neurobiol., 38 (1992) 125-167. [3] McDonald, J.K., NPY and related substances, CRC Crit. Rev. Neurobiol., 4 (1988) 97-135. [4] Gehlert, D.R., Subtypes of receptors for neuropeptide Y: Implications for the targeting of therapeutics, Life Sci., 55 (1994) 551-562.

[5] B~ttcher, G., Sjbberg, J., Ekman, R., H~tkanson, R. and Sundler, F., Peptide YY in the mammalian pancreas: immunocytochemical localization and immunochemical characterization, Regul. Pept,, 43 (1993) 115-130. [6] Jazin, E.E., Zhang, X., SiSderstrbm, S., Williams, R., H~kfelt, T., Ebendal, T. and Larhammar, D., Expression of peptide YY and mRNA for the NPY/PYY receptor of the Y1 subtype in dorsal root ganglia during rat embryogenesis, Dev. Brain Res., 76 (1993) 105-113. [7] Pieribone, V.A., Brodin, L., Friberg, K., Dahlstrand, J., Siklerberg, C., Larhammar, D. and HiSkfelt, T., Differential expression of mRNAs for neuropeptide Y-related peptides in rat nervous tissues: possible evolutionary conservation, J. Neurosci., 12 (1992) 33613371. [8] Brodin, L., S~derberg, C., Pieribone, V. and Larhammar, D., Peptidergic neurons in the vertebrate spinal cord: evolutionary trends. In Nyberg, F., Sharma, H.S. and Wiesenfeld-Hallin, Z. (Eds.), Progress in Brain Research, Vol. 104, 'Neuropeptides in the Spinal Cord'. Elsevier, Amsterdam, 1995, pp. 61-74. [9] SiSderberg, C., Pieribone, V.A., Dahlstrand, J., Brodin, L. and Larhammar, D., Neuropeptide role of both peptide YY and neuropeptide Y in vertebrates suggested by abundant expression of their mRNAs in a cyclostome brain, J. Neurosci. Res., 37 (1993) 633-640. [10] BSttcher, G., Ekblad, E., Ekman, R., H~lkanson, R. and Sundler, F., Peptide YY: a neuropeptide in the gut. Immuuocytochemical and immunocbemical evidence, Neuroscience, 55 (1993) 281-290. [11] Hazelwood, R.L., The pancreatic polypeptide (PP-fold) family: Gastrointestinal, vascular, and feeding behavioral implications., Proc. Soc. Exp. Biol. Med, 202 (1993) 44-63. [12] Larhammar, D., Blomqvist, A.G. and S&lerberg, C., Evolution of neuropeptide Y and its related peptides, Comp. Biocbem. Physiol., 106C (1993) 743-752. [13] Lundell, I., Blomqvist, A.G., Berglund, M.M., Schober, D.A., Johnson, D., Stamick, M., Gadski, R.A., Gehlert, D.R. and Larhammar, D., Cloning of a human receptor of the NPY receptor family with high affinity for pancreatic polypeptide and peptide YY, J, Biol. Chem. 270 (1995) 29123-29128. [14] Lundell, I., Statnick, M.A., Johnson, D., Schober, D.A., Starb~ick, P., Gehlert, D.R. and Larhammar, D., Cloning of a rat pancreatic polypeptide receptor related to the neuropeptide Y receptor Y l, Proc. Natl. Acad. Sci. USA (1996) in press. [15] Benton, M.J., Vertebrate palaeontology, Unwin Hyman, London, 1990. [16] Tatemoto, K., Neuropeptide Y: Complete amino acid sequence of the brain peptide, Proc. Natl. Acad. Sci. USA, 79 (1982) 54855489. [17] Tatemoto, K., Neuropeptide Y: Isolation, structure and function. In Mutt, V., Fuxe, K., Hbkfelt, T. and Lundberg, J.M. (Eds.), Neuropeptide Y, Raven Press, New York, 1989, pp. 13-21. [18] Sillard, R., Agerberth, B., Mutt, V. and Jbrnvall, H., Sheep neuropeptide Y. A third structural type of a highly conserved peptide, FEBS Lett., 258 (1989) 263-265. [19] O'Hare, M.M.T., Tenmoku, S., Aakerlund, L., Hilsted, L., Johnsen, A. and Schwartz, T.W., Neuropeptide Y in guinea pig, rabbit, rat and man. Identical amino acid sequence and oxidation of methionine-17, Regul. Pept., 20 (1988) 293-304. [20] Minth, C.D., Bloom, S.R., Polak, J.M. and Dixon, J.E., Cloning, characterization, and DNA sequence of a human cDNA encoding neuropeptide tyrosine, Proc. Natl. Acad. Sci. USA, 81 (1984) 4577-4581. [21] Minth, C.D., Andrews, P.C. and Dixon, J.E., Characterization, sequence, and expression of the cloned human neuropeptide Y gene, J. Biol. Chem., 261 (1986) 11974-11979. [22] Corder, R., Galliard, R.C. and BiShlen, P., Isolation and sequence of rat peptide YY and neuropeptide Y, Regul. Pept., 21 (1988) 253-261.

D. Larhammar/Regulatory Peptides 62 (1996) 1-11 [23] Allen, J., Novotny, J., Martin, J. and Heinrich, G., Molecular structure of mammalian neuropeptide Y: Analysis by molecular cloning and computer-aidecl comparison with crystal structure of avian homologue, Proc. N.atl. Acad. Sci. USA, 84 (1987) 25322536. [24] Higuchi, H., Yang, H.-Y.'I'. and Sabol, S.L., Rat neuropeptide Y precursor gene expression, .I. Biol. Chem., 263 (1988) 6288-6295. [25] Larhammar, D., Ericsson, A. and Persson, H., Structure and expression of the rat neuropeptide Y gene, Proc. Natl. Acad. Sci. USA, 84 (1987) 2068-2072. [26] Allen, J.M., Molecular structure of neuropeptide Y and regulation of expression of its gene. In Mutt, V., Fuxe, K., H~kfelt, T. and Lundberg, J.M. (Eds.), Neuropeptide Y, Raven Press, New York, 1989, pp. 33-41. [27] Blomqvist, A.G., S&lerbelg, C., Lundell, I., Milner, R.J. and Larhammar, D., Strong evolutionary conservation of neuropeptide Y: Sequences of chicken, goldfish, and Torpedo marmorata DNA clones, Proc. Natl. Acad. Sci. USA, 89 (1992) 2350-2354. [28] Wang, Y. and Conlon, J.M., Neuroendocrine peptides (NPY, GRP, VIP, somatostatin) from the brain and stomach of the alligator, Peptides, 14 (1993) 573-579. [29] Parker, D.B., McRory, J.E., Fischer, W.H., Park, M. and Sherwood, N.M., Primary structure of neuropeptide Y from brains of the American alligator (Alligator mississippiensis), Regal. Pept., 45 (1993) 379-386. [30] Chattel, N., Conlon, J.M., Danger, J.-M., Foumier, A., Tonon, N.-C. and Vandry, H., Characterization of melanotropin-release-inhibiting factor (melanostatia) from frog brain: Homology with human neuropeptide Y, Proc. Natl. Acad. Sci. USA, 88 (1991) 3862-3866. [31] McKay, D.M., Shaw, c., Thim, L., Johnston, C.F., Halton, D.W., Fairweather, I. and Buchanan, K.D., The complete primary structure of pancreatic polypeptide from the European common frog, Rana temporaria, Regul. Pept., 31 0990) 187-198. [32] van Riel, M.C.H.M., Tuinhof, R., Roubos, E.W. and Martens, G.J.M., Cloning and sequence analysis of hypothalamic cDNA encoding Xenopus preproneuropeptide Y, Biochem. Biopbys. Res. Commun., 190 (1993) 948-951. [33] Griffin, D., Minth, C.D. anti Taylor, W.L., Isolation and characterization of the Xenopus laevis cDNA and genomic homologs of neuropeptide Y, Mol. Cell. Endocrinol., 101 (1994) 1-10. [34] Jensen, J. and Conion, J.M., Characterization of peptides related to enuropeptide Y and peptide 't'Y from the brain and gastrointestinal tract of teleost fish, Eur. J. 13:iochem., 210 (1992) 405-410. [35] Conion, J.M., Bjenning, C. and Hazon, N., Structural characterization of neuropeptide Y from the brain of the dogfish, Scyliorhinus can&ula, Peptides, 13 (1992)493-497. [36] Tatemoto, K., Isolation and characterization of peptide YY (PYY), a candidate gut hormone that inhibits pancreatic exocrine secretion., Proc. Natl. Acad. Sci. USA, 79 (1982) 2514-2518. [37] Tatemoto, K., Nakano, 1., Makk, G., Angwin, P., Mann, M., Schilling, J. and Go, V.L.W., Isolation and primary structure of human peptide YY, Biochem Biophys. Res. Commun., 157 (1988) 713-717. [38] Kohri, K., Nata, K., Yonekura, H., Nagai, A., Konno, K. and Okamoto, H., Cloning and structural determination of human peptide YY cDNA and gene, Biochim. Biophys. Acta, 1173 (1993) 345-349. [39] Herzog, H., Genomic organisation and localisation of the gene for the human peptide YY, Genl3ank accession code L25648., (1993). [40] Herzog, H., Hort, Y., Schneider, R. and Shine, J., Seminalplasmin: Recent evolution of another member of the neuropeptide Y geue family, Proc. Natl. Acad. Sci. USA, 92 (1995) 594-598. [41] Leiter, A.B., Toder, A., Wolfe, HJ., Taylor, I.L., Cooperman, S., Mandel, G. and Goodman, R.H., Peptide YY: Structure of the precursor and expression in e:~ocrine pancreas, J. Biol. Chem., 262 (1987) 12984-12988.

9

[42] Krasinski, S.D., Wheeler, M.B. and Leiter, A.B., Isolation, characterization, and developmental expression of the rat peptide-YY gene, Mol. Endocrinol., 5 (1991) 433-440. [43] Adrian, T.E. and Conlon, J.M., Novel sequence for peptide YY in the guinea pig, Gastroenterology, 100 (1991) A628. [44] Conlon, J.M. and O'Harte, F., The primary structure of a PYY-related peptide from chicken intestine suggests an anomalous site of cleavage of the signal peptide in preproPYY, FEBS Lett., 313 (1992) 225-228. [45] Mor, A., Chartrel, N., Vaudry, H. and Nicolas, P., Skin peptide tyrosine-tyrosine, a member of the pancreatic polypeptide family: Isolation, structure, synthesis, and endocrine activity, Proc. Natl. Acad. Sci. USA, 91 (1994) 10295-10299. [46] Conlon, J.M., Chartrel, N. and Vaudry, H., Primary structure of frog PYY: implications for the molecular evolution of the pancreatic polypeptide family, Peptides, 13 (1992) 145-149. [47] Barton, C.L., Shaw, C., Halton, D.W. and Thim, L., Rainbow trout (Oncorhynchus mykiss) neuropeptide Y, Peptides, 13 (1992) 11591163. [48] Kimmel, J.R., Plisetskaya, E.M., Pollock, H.G., Hamilton, J.W., Rouse, J.B., Ebner, K.E. and Rawitch, A.B., Structure of a peptide from coho salmon endocrine pancreas with homology to neuropeptide Y, Biochem. Biophys. Res. Commun., 141 (1986) 1084-1091. [49] Conlon, M.J., Bjenning, C., Moon, T.W., Youson, J.H. and Thim, L., Neuropeptide Y-related peptides from the pancreas of a teleostean (eel), holostean (bowfin) and elasmobranch (skate) fish, Peptides, 12 (1991) 221-226. [50] Pollock, H.G., Kimmel, J.R., Hamilton, J.W., Rouse, J.B., Ebner, K.E., Lance, V. and Rawiteh, A.B., Isolation and structures of alligator gar (Lepisosteus spatula) insulin and pancreatic polypeptide, Gen. Comp. Endocrinol., 67 (1987) 375-382. [51] Pan, J.-Z., Shaw, C., Halton, D.W., Thim, L. and Whittaker, V.P., Peptide leucine-tyrosine (PLY): A novel neuropeptide Y-related peptide from the intestine of the electric ray (Torpedo marmorata), Regul. Pept., 39 (1992) 271. [52] Pan, J.Z., Shaw, C., Halton, D.W., Thim, L., Johnston, C.F. and Buchanan, K.D., The primary structure of peptide Y (PY) of the spiny dogfish, Squalus acanthias: immunocytochemical localisation and isolation from the pancreas, Comp. Biochem. Physiol., 102 (1992) 1-5. [53] Conlon, J.M., Balasumbramaniam, A. and Hazon, N., Structural characterization and biological activity of a neuropeptide Y-related peptide from the dogfish, Scyliorhinus canicula, Endocrinology, 128 (1991) 2273-2279. [54] Conlon, J.M., Bjcmholm, B., J~rgensen, F.S., Youson, J.H. and Schwartz, T.W., Primary structure and conformational analysis of peptide methionine-tyrosine, a peptide related to neuropeptide Y and peptide YY isolated from lamprey intestine, Eur. J. Biochem., 199 (1991) 293-298. [55] Andrews, P.C., Hawke, D., Shively, J.E. and Dixon, J.E., A nonamidated peptide homologous to porcine peptide YY and neuropeptide YY, Endocrinol., 116 (1985) 2677-2681. [56] Balasubramaniam, A., Andrews, P.C., Renugopalakrishnan, V. and Rigel, D.F., Glycine-extended anglerfish peptide YG (aPY) a neuropeptide Y (NPY) homologue may be a precursor of a biologically active peptide, Peptides, 10 (1989) 581-585. [57] Conlon, J.M., Schmidt, W.E., Galiwitz, B., Falkmer, S. and Thim, L., Characterization of an amidated form of pancreatic polypeptide from the daddy sculpin (Cottus scorpius), Regul. Pept., 16 (1986) 261-268. [58] Cutfield, S.M., Came, A. and Cutfield, J.F., The amino-acid sequences of sculpin islet somatostatin-28 and peptide YY, FEBS Lett., 214 (1987) 57-61. [59] Nguyen, T.M., Wright, J.R.J., Nielsen, P.F. and Conlon, J.M., Characterization of the pancreatic hormones from the Brockmann body of the tilapia: implications for islet xenograft studies, Comp. Biochem. Physiol., 111C (1995) 33-44.

10

D. Larhammar / Regulatory Peptides 62 (1996) 1-11

[60] Chance, R.E., Johnson, M.G., Hoffman, J.A. and Lin, T.-M., Pancreatic polypeptide: a newly recognized hormone. In Baba, S., Kaneko, T. and Yanaihara, N. (Eds.), Proinsulin, Insulin, C-peptide. Excerpta Medica, Amsterdam, 1979, pp. 419-425. [61] Chen, Z.-w., Bergman, T., C)stenson, C.-G., H/SiSg,A., N~islund, J., Norberg, ,g,., Carlquist, M., Efendic, S., Mutt, V. and JiSrnvall, H., A porcine gut polypeptide identical to the pancreatic hormone PP (pancreatic polypeptide), FEBS Lett., 341 (1994) 239-243. [62] Chance, R.E., Moon, N,E. and Johnson, M.G., Human pancreatic polypeptide (HPP) and bovine pancreatic polypeptide (BPP). In Jaffe, B.M. and Behrman, H.R. (Eds.), Methods of Hormone Radioimmunoassay. Academic Press, New York, San Francisco, and London, 1979, pp. 657-672. [63] Henry, J.S., Lance, V.A. and Conlon, J.M., Primary structure of pancreatic polypeptide from four species of Perissodactyla (Przewalski's horse, zebra, rhino, tapir), Gen. Comp. Endocrinol., 84 (1991) 440-446. [64] Nielsen, H.V., Gether, U. and Schwartz, T.W., Cat pancreatic eicosapeptide and its biosynthetic intermediate, Biochem. J., 240 (1986) 69-74. [65] Leiter, A.B., Keutmann, H.T. and Goodman, R.H., Structure of a precursor to human pancreatic polypeptide, J. Biol. Chem., 259 (1984) 14702-14705. [66] Boel, E., Schwartz, T.W., Norris, K.E. and Fiil, N.P., A cDNA encoding a small common precursor for human pancreatic polypep° tide and pancreatic icosapeptide, EMBO J., 3 (1984) 909-912. [67] Takeuchi, T. and Yamada, T., Isolation of a cDNA clone encoding pancreatic polypeptide, Proc. Natl. Acad. Sci. USA, 82 (1985) 1536-1539. [68] Leiter, A.B., Montminy, M.R., Jamieson, E. and Goodman, R.H., Exons of the human pancreatic polypeptide gene define functional domains of the precursor, J. Biol. Chem., 260 (1985) 13013-13017. [69] Marks, N.J., Shaw, C., Halton, D.W., Curry, W.J. and Thim, L., Rabbit pancreatic polypeptide, Comp. Biochem. Physiol., 106B (1993) 883-887. [70] Kimmel, J.R., Pollock, H.G., Chance, R.E., Johnson, M.G., Reeve, J.R., Taylor, I.L., Miller, C. and Shively, J.E., Pancreatic polypeptide from rat pancreas, Endocrinol., 114 (1984) 1725-1731. [71] Yamamoto, H., Nata, K. and Okamoto, H., Mosaic evolution of prepropancreatic polypeptide, J. Biol. Chem., 261 (1986) 61566159. [72] Yonekura, H., Nata, K., Watanabe, T., Kurashina, Y., Yamamoto, H. and Okamoto, H., Mosaic evolution of prepropancreatic polypeptide. II. Structural conservation and divergence in pancreatic polypeptide gene, J. Biol. Chem., 263 (1988) 2990-2997. [73] Marks, N.J., Shaw, C., Halton, D.W. and Thim, L., The primary structure of pancreatic polypeptide from a primitive insectivorous mammal, the European hedgehog (Erinaceous europaeus), Regul. Pept., 47 (1993) 179-185. [74] Eng, J., Huang, C.-G., Pan, Y.-C,E., Hulmes, J.D. and Yalow, R.S., Guinea pig pancreatic polypeptide: Structure and pancreatic content, Peptides, 8 (1987) 165-168. [75] Blackstone, C.D,, Seino, S., Takeuchi, T., Yamada, T. and Steiner, D.F., Novel organization and processing of the guinea pig pancreatic polypeptide precursor, J. Biol. Chem., 263 (1988) 2911-2916. [76] Eng, J., Kleinman, W.A. and Chu, L.-S., Purification of peptide hormones from chinchilla pancreas by chemical assay., Peptides, 11 (1990) 683-685. [77] Kimmel, J.R., Hayden, L.J. and Pollock, H.G., Isolation and characterization of a new pancreatic polypeptide hormone, J. Biol. Chem., 250 (1975) 9369-9374. [78] Marks, N.J., Shaw, C., Halton, D.W. and Thim, L., The corrected primary structure of chicken (avian) pancreatic polypeptide, Regul. Pept., 52 (1994) 159-164. [79] Nata, K., Sugimoto, T., Kohri, K., Hidaka, H., Hattori, E., Yamamoto, H., Yonekura, H. and Okamoto, H., Structure determina-

[80]

[81]

[82]

[83]

[84]

[85]

[86]

[87]

[88]

[89]

[90]

[91]

[92] [93]

[94]

[95]

[96]

[97]

tion and evolution of the chicken cDNA and gene encoding prepropancreatic polypeptide, Gene, 130 (1993) 183- 189. Blundell, T.L., Pitts, J.E., Tickle, l.J., Wood, S.P. and Wu, C.-W., X-ray analysis (1.4-,g, resolution) of avian pancreatic polypeptide: Small globular protein hormone, Proc. Natl. Acad. Sci. USA, 78 (1981) 4175-4179. Barton, C.L., Shaw, C., Halton, D.W. and Thim, L., Isolation and structural characterisation of herring gull (Larus argentatus) pancreatic polypeptide, Gen. Comp. Endocrinol., 93 (1994) 255-259. Marks, N.J., Shaw, C., Halton, D.W., Maule, A.G., Curry, W.J., Verhaert, P. and Thim, L., Isolation and primary structure of a novel avian pancreatic polypeptide from five species of Eurasian crow, Regul. Pept., 47 (1993) 187-194. Xu, Y., Lin, N. and Zhan, Y., Isolation and sequence determination of goose pancreatic polypeptide, Scientia Sinica (Series B), 27 (1984) 590-592. Litthauer, D. and Oelofsen, W., Purification and primary structure of ostrich pancreatic polypeptide, Int. J. Pept. Protein Res., 29 (1987) 739-745. Lance, V., Hamilton, J.W., Rouse, J.B., Kimmel, J.R. and Pollock, H.G., Isolation and characterization of reptilian insulin, glucagon, and pancreatic polypeptide: Complete amino acid sequence of alligator (Alligator mississippiensis) insulin and pancreatic polypeptide, Gen. Comp. Endocrinol., 55 (1984) 112-124. Pollock, H.G., Hamilton, J.W., Rouse, J.B., Ebner, K.E. and Rawitch, A.B., Isolation of peptide hormones form the pancreas of the bullfrog (Rana catesbeiana), J. Biol. Chem., 263 (1988) 97469751. Cavanangh, E.S., Nielsen, P.F. and Conlon, J.M., Isolation and structural characterization of progucagon-derived peptides, pancreatic polypeptide and somatostatin from the urodele Amphiuma tridactylum, Gen. Comp. Endocrinol., (1995) in press. Larhammar, D., S~:lerberg, C. and Blomqvist, A.G., Evolution of the neuropeptide Y family of peptides. In Wahlestedt, C. Colmers, W.F. (Eds.), The Neurobiology of Neuropeptide Y and Related Peptides. Humana Press, Clifton, N.J., 1993, pp. 1-42. Grundemar, L. and H,~tkanson, R., Neuropeptide Y effector systems: perspectives for drug development., Trends Pharmacol. Sci., 15 (1994) 153-159. Dumont, Y., Fournier, A., St-Pierre, S. and Quirion, R., Characterization of neuropeptide Y binding sites in rat brain membrane preparations using [125l][Leu31, Pro34]peptide y y and [125I]peptide YY3-36 as selective Yl and Y2 radioligands, J. Pharmacol. Exp. Ther., 272 (1995) 673-680. Fuhlendorff, J., Gether, U., Aakerlund, L., Johansen, N.L., Thcgersen, H., Melberg, S.G., Olsen, U.B., Thastrup, O. and Schwartz, T.W., [Leu3t, Pro34]Neuropeptide Y: A specific Yt receptor agonist, Proc. Natl. Acad. Sci. USA, 87 (1990) 182-186. Nelson, J.S., Fishes of the World, 3rd Edn., Wiley, New York, 1994. Kemp, T.S., Haemothermia or Archosauria? The interrelationships of mammals, birds and crocodiles, Zool. J. Linnean Soc., 92 (1988) 67-104. Larhammar, D. and Milner, R.J., Phylogenetic relationship of birds with crocodiles and mammals as deduced from protein sequences, Mol. Biol. Evol., 6 (1989) 693-696. Baker, E., Hort, Y.J., Ball, H., Sutherland, G.R., Shine, J. and Herzog, H., Assignment of the human neuropeptide Y gene to chromosome 7p15.1 by nonisotopic in situ hybridization, Genomics, 26 (1995) 163-164. Hort, Y., Baker, E., Sutherland, G.R., Shine, J. and Herzog, H., Gene duplication of the human peptide YY gene (PYY) generated the pancreatic polypeptide gene (PPY) on chromosome 17q21.1, Genomics, 26 (1995) 77-83. Ohno, S., Evolution by Gene Duplication., Springer-Verlag, Berlin, 1970.

D. Larhammar / Regulatory Peptides 62 (1996) 1-11 [98] Urano, A., Hyodo, S. aJad Suzuki, M., Molecular evolution of neurohypophysial hormone precursors. In Joosse, J., Buijs, R.M. and Tilders, F.J.H. (Eds.), Prog. Brain Res., Vol. 92, Elsevier, Amsterdam, 1992, pp. 39--46. [99] Sherwood, N.M., Lovejoy, D.A. and Coe, I.R., Origin of mammalian gonadotropin-releasing hormone, Endocr. Rev., 14 (1993) 241-254. [100] Garcia-Femhndez, J. and Holland, P.W.H., Archetypal organization of the amphioxus Hox gene cluster, Nature, 370 (1994) 563-566. [101] Holland, P.W.H., Garcia-Fernhndez, J., Williams, N.A. and Sidow, A., Gene duplications and the origins of vertebrate development, Development, Suppl., (1994) 135-133.

11

[102] Upchurch, B.H., Aponte, G.W. and Leiter, A.B., Expression of pcptide YY in all four islet cell types in the developing mouse pancreas suggests a common peptide YY-producing progenitor, Development, 120 (1994) 245-252. [103] Blomqvist, A.G., Roubos, E.W., Larhammar, D. and Martens, G.J., Cloning and sequence analysis of a neuropeptide Y/peptide YY receptor YI cDNA from Xenopus laevis, Biochim. Biophys. Acta, 1261 (1995) 439-441. [104] Rose, P.M., Femandes, P., Lynch, LS., Frazier, S.T., Fisher, S.M., Kodukula, K., Kienzle, B. and Seethala, R., Cloning and functional expression of a cDNA encoding a human type 2 neuropeptide Y receptor, J. Biol. Chem., 270 (1995) 22661-22664.