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MINIREVIEW

Recent Developments in Our Understanding of the Physiological Role of PP-Fold Peptide Receptor Subtypes¹

Eli Lilly and Company, Lilly Research Laboratories, Indianapolis, Indiana 46285

	Abstract

Top Abstract Introduction Structure(s) of PP-fold... PP Function. PYY Function. NPY Function. PY. Receptors for the NPY... Other Proposed Receptors Recent Findings on Distribution... Studies of Genetically... Recent Developments in Selective... Small Molecule NPY Medicinal... Structure and Mutagenesis... Overview Conclusion References

 
The three peptides pancreatic polypeptide (PP), peptide YY (PYY), and neuropeptide Y (NPY) share a similar structure known as the PP-fold. There are four known human G-protein coupled receptors for the PP-fold peptides, namely Y1, Y2, Y4, and Y5, each of them being able to bind at least two of the three endogenous ligands. All three peptides are found in the circulation acting as hormones. Although NPY is only released from neurons, PYY and PP are primarily found in endocrine cells in the gut, where they exert such effects as inhibition of gall bladder secretion, gut motility, and pancreatic secretion. However, when PYY is administered in an experimental setting to animals, cloned receptors, or tissue preparations, it can mimic the effects of NPY in essentially all studies, making it difficult to study the effects of PP-fold peptides and to delineate what receptor and peptide accounts for a particular effect. Initial studies with transgenic animals confirmed the well-established action of NPY on metabolism, food-intake, vascular systems, memory, mood, neuronal excitability, and reproduction. More recently, using transgenic techniques and novel antagonists for the Y1, Y2, and Y5 receptors, NPY has been found to be a key player in the regulation of ethanol consumption and neuronal development.

Key Words: GPCR • knockout animal • distribution • mutagenesis • ligand binding

	Introduction

Top Abstract Introduction Structure(s) of PP-fold... PP Function. PYY Function. NPY Function. PY. Receptors for the NPY... Other Proposed Receptors Recent Findings on Distribution... Studies of Genetically... Recent Developments in Selective... Small Molecule NPY Medicinal... Structure and Mutagenesis... Overview Conclusion References

 
The PP-fold family of peptides consists of neuropeptide Y (NPY) and the two gut hormones, peptide YY (PYY) and pancreatic polypeptide (PP). The neuronal and hormonal functions of the PP-fold peptides are intermixed because all three members are found in the bloodstream and can act at several G-protein-coupled receptors (GPCRs). Furthermore, all known PP-fold peptide receptors are found both in nerve cells of the central and peripheral nervous systems as well as in non-neuronal tissues and can bind at least two of the three PP-fold peptides. Thus, it is possible for the peptides to modulate or potentially assume the function of one another. In addition, although NPY is strictly localized in neurons, PYY and PP are mainly found in the gut. However, several studies have found PYY in neurons. Therefore, PYY may exhibit neurotransmitter functions as well. This potential redundancy is important to bear in mind, especially when discussing results from gene-targeting experiments (i.e., knockout and transgenic animals).

This article is an update of previous reviews on PP-fold peptides and receptors in this journal (1, 2). Therefore, we will focus on developments that have occurred since 1998. Since the last review, no new PP-fold peptides or PP-fold receptor subtypes have been cloned from mammals. In fact, knowledge about the full sequence of the human genome will end the complete discovery of new mammalian genes. Nevertheless, new receptors have been cloned from lower vertebrates and invertebrates. However, these will only be discussed briefly in this review when binding data and sequence comparisons can be extrapolated to the mammalian receptors. The cloning of PP-fold receptors during the first 7 years of the 1990s has given us the tools to explore the function of the PP-fold peptides and their receptors both in vivo and in vitro. Therefore, we will discuss advances in our understanding of the role of PP-fold peptides using genetic techniques such as knockout or overexpressing rodents. By comparison, at the time of the latest review, there were only a few Y1-selective nonpeptide antagonists available, and peptides previously thought to be selective were found to be nonselective. Since then, several subtype-selective ligands, both agonists and antagonists, have been discovered that provide better tools to dissect what receptor is responsible for a particular effect.

	Structure(s) of PP-fold Peptides.

Top Abstract Introduction Structure(s) of PP-fold... PP Function. PYY Function. NPY Function. PY. Receptors for the NPY... Other Proposed Receptors Recent Findings on Distribution... Studies of Genetically... Recent Developments in Selective... Small Molecule NPY Medicinal... Structure and Mutagenesis... Overview Conclusion References

 
NPY, PYY, and PP, together with the fish peptide Y (PY), share a common hairpin-like three-dimensional structure called the PP-fold (3). All four peptides are 36 amino acids long (except chicken PYY, which is 37) with an amidated carboxy-terminus. The general structure of the PP-fold peptides has been established using x-ray crystallography of avian PP (4) and confirmed in several studies using nuclear magnetic resonance, most recently for PYY (5) and the synthetic analog [Leu³¹,Pro³⁴]NPY (6). Amino acid residues 1–8 form a type II proline helix followed by a loop. Residues 15–32 form an -helix, and the four most carboxy-terminal residues are in a flexible loop conformation. NPY is one of the most evolutionary conserved peptides known. Only two of the 36 amino acids of NPY are variable between mammals. PYY has evolved at a higher rate than NPY and has eight variable amino acids between different orders of mammals, whereas the third member of the PP-fold family of peptides, PP, has evolved very rapidly and is one of the least-conserved peptides known (see Ref. 7 for review). However, despite the low degree of conservation in amino acid sequence between PP from different species as well as between PP and PYY and NPY, the general three-dimensional structure seems to be conserved in all PP-fold peptides (4–6).

There are several reports on endogenous circulating amino terminally truncated fragments of NPY (and PYY), such as NPY2-36 and NPY3-36 (8) and PYY3-36 (9). These fragments are intermediate degradation products from peptidergic breakdown of NPY and PYY or result from specific cleavage by aminopeptidases (8, 10) and may have a physiological role resulting from the affinity and selectivity of amino terminally truncated NPY fragments for Y2 and Y5 receptors.

	PP Function.

Top Abstract Introduction Structure(s) of PP-fold... PP Function. PYY Function. NPY Function. PY. Receptors for the NPY... Other Proposed Receptors Recent Findings on Distribution... Studies of Genetically... Recent Developments in Selective... Small Molecule NPY Medicinal... Structure and Mutagenesis... Overview Conclusion References

 
PP was the first of the PP-fold peptides to be identified and sequenced. It was found as a contaminant in extracts of chicken insulin (11, 12). Fish lack PP, indicating that PP probably arose as a local gene duplication of the PYY gene in early tetrapod evolution (7). The genes for PP and PYY are located only 10 kb apart from one another on chromosome segment 17q21.1 in humans (13). PP is almost exclusively expressed in endocrine pancreas and is released in response to meals (14). The effects of PP, including inhibition of pancreatic secretion, gall bladder activity, and intestinal motility, are mainly located in the gastrointestinal (GI) tract (1). Recently, PP has been found to inhibit ileum contractions (15) and to stimulate colon contractions (16). In addition, PP affects metabolic functions, including glycogenolysis, and decreases fatty acid levels (2). However, binding sites for PP have been found in several rat brain regions including the interpeduncular nucleus, hypothalamus, and brainstem (17–19), suggesting that PP may also have direct effects of brain function. This may explain why intracerebroventricular injection of PP has been found to stimulate feeding in several different species of experimental animals (20–22).

	PYY Function.

Top Abstract Introduction Structure(s) of PP-fold... PP Function. PYY Function. NPY Function. PY. Receptors for the NPY... Other Proposed Receptors Recent Findings on Distribution... Studies of Genetically... Recent Developments in Selective... Small Molecule NPY Medicinal... Structure and Mutagenesis... Overview Conclusion References

 
PYY was first isolated from porcine intestine using a method that captured peptides with an amidated carboxy terminus (23, 24). The peptide had tyrosines in both ends and was therefore named peptide YY after the single letter abbreviation for tyrosine (Y). PYY is released in the GI tract in response to meals. Many of the GI effects described for PP, including inhibition of gall bladder secretion, gut motility, and pancreatic secretion, can also be provoked by PYY (1). Other peripheral effects produced by PYY are the inhibition of fluid and electrolyte secretion in the intestinal tract (25) and vasoconstriction (26). PYY has also been found in neurons indicating that PYY may also have a neuronal function (see Ref. 27 for review).

The chromosome segment that harbors the genes for PYY and PP (17q21.1 in humans; Ref. 13) has been duplicated one more time in primates to generate two new genes, PYY2 and PP2 (28). Specific mutations have changed the processing of the protein generating a product with very low structural similarity to the PP-fold peptides. The function of these gene products is presently unknown.

	NPY Function.

Top Abstract Introduction Structure(s) of PP-fold... PP Function. PYY Function. NPY Function. PY. Receptors for the NPY... Other Proposed Receptors Recent Findings on Distribution... Studies of Genetically... Recent Developments in Selective... Small Molecule NPY Medicinal... Structure and Mutagenesis... Overview Conclusion References

 
NPY was first isolated from pig brain using the same method as for PYY (29, 30). It is widely distributed throughout the mammalian brain but it is also found in many peripheral areas. NPY is one of the most potent orexigenic peptides known. The feeding effect of NPY appears to be very well conserved because NPY has been found to induce food intake with a preference for carbohydrate-rich food when injected intracerebroventricularly in several species, including snake (31), goldfish (32, 33), chicken (34), rat (20, 35), mouse (36, 37), rabbit (38), guinea pig (39), sheep (40), dog (41), pig (42), and rhesus monkey (43). When administered centrally, antisense oligonucleotides against NPY reduce feeding (44, 45). Interestingly, a synthetic fatty acid synthesis inhibitor, C75, dramatically decreases hypothalamic mRNA levels for NPY and causes an almost total inhibition of feeding in both lean and obese mice (46, 47). Centrally administered NPY also regulates metabolism by decreasing energy expenditure (48). Other centrally mediated effects of NPY are decreased thermogenesis (49), anticonvulsant activity (50, 51), inhibition of sedation (52, 53), mood, and memory (see Refs. 2, 54, and 55 for reviews). Recently, it was found that NPY-deficient mice have an impaired development of neurons in the olfactory epithelium (56, 57), which suggests a role in neuronal development. NPY is involved in regulation of reproduction because it stimulates luteinizing hormone-releasing hormone (LHRH) release (58), an effect that can be inhibited by NPY antisense oligonucleotides (59). Furthermore, when NPY is injected into the suprachiasmatic nucleus, it inhibits cell firing (60) and induces a forward shift in circadian rhythms (60–62), possibly through reduction of Per1 and Per2 mRNA levels. NPY is colocalized with noradrenaline in sympathetic nerves and acts to enhance noradrenaline-mediated vasoconstriction, especially upon strong stimulation (see Ref. 63 for review). Contrary to this, centrally administered NPY reduces arterial blood pressure and heart tone shown in both rats and dogs (64). Another vascular effect is stimulation of smooth muscle proliferation (65). In addition, NPY can act as an antinociceptive peptide (66), probably through inhibition of substance P release in the dorsal horn of the spinal cord. NPY and the Y2 receptor are also dramatically upregulated after axotomy of the sciatic nerve in the dorsal root ganglion. This provides further evidence for the involvement of NPY in pain modulation (67–69).

Several disorders and pathological conditions are associated with altered NPY function. Based on the strong orexigenic effect of NPY, the most obvious are various eating and metabolic disorders. NPY levels are changed in all conditions involving a disturbed energy balance, such as anorexia, bulimia neurosa, and diabetes (2). Moreover, several cardiovascular dysfunctions as well as some tumor diseases (70) are associated with increased plasma levels of NPY.

One of the most important developments in NPY research is the recent finding that NPY regulates ethanol consumption. In a genetic study comparing the ethanol preferring and nonpreferring rats, the locus that harbors the gene for NPY was identified (71), suggesting that dysfunction in the gene locus contributes to ethanol preference in these rats. The role of NPY as a potential regulator of alcohol consumption has also been investigated in transgenic mice (72) where an inverse correlation between NPY levels and drinking has been shown (see also transgenic section). At this point, it is unclear how these findings relate to the well-established anxiolytic actions of NPY (73). Furthermore, a point mutation (leucine⁷ to proline) in the pre-pro NPY gene has been associated with higher alcohol consumption in humans (74). This mutation has also been linked to higher cholesterol, serum lipids, and progression of carotid atherosclerosis (75, 76). NPY appears to be involved in regulation of neuronal excitability as mice lacking NPY are more susceptible to seizures (50), and people with temporal lobe epilepsy have increased NPY expression in CA3 regions as well as prominent rearrangements in receptor distribution (77).

NPY is regulated by several other neuropeptides and hormones. Two hormonal signals that act on NPY neurons are ghrelin and leptin (78). Ghrelin is a 28 amino acid peptide that is released from the gut and binds to a specific GPCR to signal release of growth hormone from the pituitary (79, 80). Both intravenous and intracerebroventricular injections of ghrelin result in a dose-dependent increase in growth hormone levels in plasma (see Ref. 80 for review). Interestingly, this peptide is present in neurons of the hypothalamic arcuate nucleus where ghrelin acts to increase the hypothalamic levels of NPY and agouti-related protein mRNA levels (81) and feeding and body weight in rats (81, 82). In contrast, the adipose hormone leptin acts as a satiety factor possibly by inhibiting NPY release in the hypothalamus (83). Another peptide system, the melanocortins, has been found to interact with NPY (84–86). When co-injected intracerebroventricularly, the endogenous agonist -melanocyte-stimulating hormone dose-dependently inhibits NPY-induced feeding (86). It is likely there are additional neuropeptides and endocrine hormones that regulate or are regulated by NPY. Research over the next 5 years should help unravel these undoubtedly complicated interrelationships.

	PY.

Top Abstract Introduction Structure(s) of PP-fold... PP Function. PYY Function. NPY Function. PY. Receptors for the NPY... Other Proposed Receptors Recent Findings on Distribution... Studies of Genetically... Recent Developments in Selective... Small Molecule NPY Medicinal... Structure and Mutagenesis... Overview Conclusion References

 
Fish lack PP but have a fourth PP-fold family peptide, PY, which can be found in the pancreas of several bony fishes (87). This peptide is equally identical to mammalian NPY as to mammalian PYY but displays higher identity to fish PYY than to fish NPY. Thus, PY is most likely the result of an independent evolutionary event in the acantomorph fish lineage (87). Recently, the genes for NPY, PYY, and PY were cloned from sea bass providing strong evidence that the PY gene is the result of a unique gene duplication in fish, most likely of the PYY gene (87). Therefore, it is unlikely that a mammalian ortholog of PY exists.

	Receptors for the NPY Family of Peptides

Top Abstract Introduction Structure(s) of PP-fold... PP Function. PYY Function. NPY Function. PY. Receptors for the NPY... Other Proposed Receptors Recent Findings on Distribution... Studies of Genetically... Recent Developments in Selective... Small Molecule NPY Medicinal... Structure and Mutagenesis... Overview Conclusion References

 
Structural and General Features of PP-fold Receptors.
NPY, PYY, and PP bind to a large and very heterogeneous family of GPCRs belonging to the rhodopsin like superfamily (class 1) of receptors. Five PP-fold family receptors have been cloned from mammals (Y1, Y2, Y4, Y5, and y6; see Table I for cloning references and ligand binding preferences) and several additional receptors have been postulated from pharmacological profiles using various tissue preparations (55). However, no additional mammalian receptor subtypes have been identified since 1996. The y6 receptor has been given the lower case designation (IUPAR nomenclature; Ref. 54) because it encodes for a truncated receptor in most mammals, including humans (54). Cloned Y1, Y2, Y4, Y5, and y6 receptors have all been shown to couple to inhibitory G-proteins (G_i) and thus mediate inhibition of cAMP synthesis (88–92). The Y1 receptor has also been shown to activate mitogen-activated protein kinase in gut epithelial cells (IEC-6; Ref. 93). Very recently, it was shown that activation of PP-fold receptors involves pertussis toxin-sensitive phosphorylation of extracellular signal-regulated protein kinase 1 and 2 in Chinese hamster ovary cells, confirming that these receptors couple to Gi/Go (94). Furthermore, whereas protein kinase C seems to be necessary for Y5 receptor signaling, a protein kinase C-independent pathway may also be involved in Y1, Y2, and Y4 signaling (94). Y1, Y2, Y4, and Y5 receptors can also couple to phospholipase C to provoke release of Ca²⁺ from intracellular stores (88, 89, 91, 95).

View larger version (72K): [in this window] [in a new window]   Figure 4. Summary of structural and mutagenesis studies performed on the mammalian Y1 receptor. Amino acids were mutated to alanine except in three positions. Those are indicated by a shaded amino acid next to the original sequence. Red indicates a loss in affinity >2.5-fold. Upper left: effects on NPY/PYY binding, lower right: antagonist. White: no effect. Black: not done. Special cases: (a) Mutation affects binding of BIBP3226 but not 1229U91 and J-104870, (b) an increase in affinity was detected, (c) when Gln120 was mutated to tyrosine, no binding of NPY was detected while mutation to glutamine abolished BIBP3226 binding, (d) triple mutant [N2Q, N11Q, N17Q] could not be expressed whereas the double mutant [N2Q, N17Q] bound 3H-NPY with the same affinity as the wild-type receptor. Deletion of the carboxy-terminus did not affect binding (black scissor) whereas deletion of the amino-terminus abolished binding of NPY (red scissor). A star (*) indicates that there are differences between the data from different mutagenesis studies at that particular amino acid.

The tissue expression of the Y1 receptor can be regulated differently because of the use of different promoters regulated by alternative splicing (98). The gene for Y1 is located in a cluster together with Y2 and Y5 on human chromosome 4q31. Contrary to the other PP-fold receptors, the coding region of the Y1 gene harbors an intron of about 100 bp in all species explored. This intron has been shown to enhance the expression of the Y1 and Y5 receptors in vitro (99). Interestingly, two in vivo-expressed splice variants of the mouse Y1 receptor have been found (100). The short form (307 amino acids) of the Y1 receptor ends a few amino acids after the third extracellular loop, yielding a receptor with an incomplete TM7. NPY binds to this short form of the Y1 receptor with similar affinity as to the complete 384 amino acid protein. However, the signaling of the short form of the receptor was impaired, indicating that the TM7 and the carboxy-terminal tail are not essential for ligand interaction but rather for G-protein activation. Like many GPCRs, the Y1 receptor can be internalized together with the ligand upon agonist stimulation shown by radioligand binding (101), confocal microscopy with fluorescent ligands (102), as well as by tagging the receptor with green fluorescent protein (103). Upon stimulation with PYY, the Y1 receptor was rapidly internalized into endosomes and recycled to the surface within 60 min (103).

Most of the vascular (65, 104–106) and antinociceptive effects (107, 108) of NPY are transduced via the Y1 receptor. For example, the Y1-selective antagonists SR120819A (109), BIBP3226 (110; Fig. 1), BIBO3304 (111), and H394/84 inhibit NPY-induced vasoconstriction in a variety of species (106, 109, 112, 113). Interestingly, the centrally induced vascular effects of NPY (reduced blood pressure and heart rate) are also signaled mainly through Y1 (64), as are many of the psychological functions of NPY, such as decreased anxiety and depression (114–118). Y1 is involved in the feeding response of NPY (43, 111, 119–123). Hypothalamic Y1 mRNA levels decrease during fasting (124). Although intracerebroventricular injection of Y1-selective agonists increase feeding (125), Y1 antagonists can inhibit NPY-induced feeding (37, 43, 126). Surprisingly, Y1-antisense oligonucleotides have been found to increase food intake in energy-deprived rats (127). The Y1 receptor is also involved in the cross-talk between NPY and another orexigenic peptide in the hypothalamus as the feeding response of melanin concentrating hormone is attenuated by administration of Y1 receptor antagonists (128).

View larger version (27K): [in this window] [in a new window]   Figure 1. Novel nonpeptide analogs with selectivity for the Y1 receptor.

The Y2 Receptor.
Originally, the Y2 receptor was identified using vascular preparations and defined by the activity of amino terminally truncated fragments of NPY and PYY, such as NPY3-36 and NPY13-36, that are full agonists with similar potency as the native peptides (Table I; Ref. 131). When positions 31 and 34 of NPY or PYY (Ile and Gln) are replaced by the corresponding amino acids in PP, Leu, and Pro, respectively, the resulting peptides do not bind to Y2 (132), although this peptide remains a potent full agonist at the other PP-fold receptors. It was later shown that only the Pro³⁴ substitution was essential for preventing Y2 receptor binding (133). When the Y1 receptor was cloned, it was assumed that the Y2 receptor would display a high degree of sequence identity to Y1. When homology screening failed for the Y2 receptor, several research groups finally turned to various expression cloning approaches and found cDNA clones coding for proteins with PYY-binding abilities (134–136). The Y2 receptor gene codes for a 381 amino acid protein and is located close to the Y1 gene on chromosome 4 (137). Like Y1, the Y2 receptor is highly conserved between species with more than 90% identity between orders of mammals (105, 134–136, 138, 139) and about 80% identity when comparing mammalian and chicken Y2 (140). Surprisingly, the Y2 receptor was only about 30% identical to the Y1 and Y4 receptors explaining the failure of homology screening approaches. Unlike the Y1 receptor, Y2 does not appear to internalize after prolonged agonist stimulation (101, 103) or does so very slowly.

The Y2 receptor is mainly located presynaptically where it acts as an autoreceptor, inhibiting further release of neurotransmitter (131, 141, 142). This may explain why agonists specific for the Y1 receptor are anxiolytic (114, 115) whereas Y2 agonists like NPY13-36 and C2-NPY appear to be anxiogenic (114, 143). The same opposing relationship between Y1 and Y2 is evident for the central effects of NPY on blood pressure as Y2 specific agonists increase blood pressure whereas activation of central Y1 receptors decreases it (64). The Y2 receptor is also directly involved in some of the vascular effects of NPY (144). For instance, in pig spleen, a Y2-specific agonist, evoked potent vasoconstriction (105) that could be inhibited by a Y2-selective antagonist, BIIE0246, (145; Fig. 2). In addition, Y2 is involved in NPY-induced angiogenesis (65) and NPY-induced effects on circadian rhythms (60, 146, 147). In addition, centrally administered Y2 agonists delay gastric emptying (148, 149). Knockout studies of the Y2 receptor have shown that also this receptor may be involved in the feeding response to NPY (150) as well as in bone formation (151).

View larger version (14K): [in this window] [in a new window]   Figure 2. Novel nonpeptide analogs with selectivity for the Y2 receptor.

The pharmacological profiles of the rat and human Y4 receptors differ in that the affinity for the rat receptor increases when position 34 of NPY or PYY is replaced by proline (152, 154, 155) whereas the human receptor is unaffected by this change (90, 95). In addition, the difference in affinity between the preferred ligand, PP, compared with NPY and PYY is much greater in rat Y4 than in the human Y4 receptor (90, 152, 155–157). Determination of the pharmacological profile of the Y4 receptor is complicated by the fact that ¹²⁵I-PYY appears to recognize only a fraction of the receptor population recognized by ¹²⁵I-PP (157, 158), which may explain some of the differences in pharmacological profiles of cloned Y4 receptors.

As PP is the preferred ligand at the Y4 receptor, it is likely that this receptor mediates many of the GI effects produced by PP like rabbit ileum contractions (15). Centrally located Y4 receptors may be involved in the regulation of reproduction as the Y1 antagonist/Y4 agonist 1229U91 (159, 160) induced release of luteinizing hormone when injected intracerebroventricularly (161).

The Y5 Receptor.
NPY and NPY2-36 are equally potent in producing a large increase in feeding after intracerebroventricular administration (162), suggesting the receptor mediating the feeding response to NPY differs from Y1, Y2, and Y4. In addition, NPY with position 32 replaced with D-tryptophan ([D-Trp³²]NPY) selectively inhibited NPY-induced feeding (163) though it had relatively low affinity for Y1 and Y2. Thus, a feeding receptor was proposed with the profile NPY=PYY=NPY2-36>NPY3-36[D-Trp³²]NPY (Table I). Expression cloning from a hypothalamic rat cDNA library resulted in a gene coding for a 446 amino acid protein (91, 164). The Y5 receptor protein is much larger than the other NPY receptors because of the extended third cytoplasmic loop with about 100 amino acids more than the other PP-fold receptors. However, the carboxy-terminal tail of the Y5 receptor is much shorter than in Y1, Y2, and Y4. Interestingly, in the mouse Y5 receptor gene, the 63 nucleotides encoding amino acids 15–35 have been duplicated in tandem, yielding a receptor that is 21 amino acids longer than the otherwise highly identical rat receptor (165, 166). However, this addition does not appear to affect the pharmacology or signaling properties of the receptor (166). The gene for Y5 is located on human chromosome 4 (4q31) and overlaps with Y1 but is transcribed in the opposite direction. In fact, one of the alternative promoters and 5' exons of the Y1 gene is located within the coding sequence of the Y5 gene, suggesting at least partially coordinated transcriptional regulation (167).

[D-Trp³²]NPY was found to be a modestly selective agonist at Y5 expressed in HEK293 cells acting to inhibit cAMP synthesis but with a lower potency than NPY, PYY and NPY2-36 (91). Although the Y5 receptor is very well conserved (88–90% overall amino acid identity and 95–98% when the third intracellular loop is not accounted for; Refs. 166, 168), between orders of mammals there may be species differences in the endogenous ligand for the Y5 receptor. One interesting feature about the Y5 receptor is that the rat PP binds with very low affinity to the Y5 receptor from various species whereas PP from human and other species has much higher affinity (166). Thus, it is possible that PP is involved in Y5 signaling in humans but most likely not in rats. This may indicate a difference of potential importance when extrapolating effects produced by PP-fold peptides in rodents to physiological and behavioral effects in humans.

The role of the Y5 receptor in NPY-induced feeding has been confirmed by studies involving antisense knockdown (169–172), knockout animals (173), and Y5-selective agonists (48, 91, 174–176). The selective Y5 antagonist CGP71683A (177; Fig. 3) was reported to antagonize NPY induced feeding; however, recent findings suggest that it inhibits feeding by a non-Y5 mechanism (178). Activation of the Y5 receptor also results in a decrease in energy expenditure (48). Other effects of NPY that are mediated by the Y5 receptor are reproduction through inhibition of luteinizing hormone release (179) and regulation of brain excitability and seizures (180, 181). Furthermore, it has been shown that the Y5 selective agonist [D-Trp³²]NPY inhibited neuronal activity in the suprachiasmatic nucleus without generating a phase-shift (60) indicating that Y5 may also be indirectly involved in regulation of circadian rhythms (60, 182).

View larger version (15K): [in this window] [in a new window]   Figure 3. Novel nonpeptide analogs with selectivity for the Y5 receptor.

Messenger RNA for the y6 receptor is expressed in the hypothalamus and in the kidney of the mouse (185). However, because of the lack of pharmacological tools to distinguish the y6 receptor from Y1 and Y5, it has not been conclusively proven that the y6 receptor protein is indeed expressed in the animals where it is not frameshifted. The fact that mice have a functional y6 receptor is also a concern for the interpretation of results from receptor knockout studies.

Receptors from Nonmammalian Species.
Y1, Y2, and Y5 have been cloned in chicken (140, 189) and the Y1 receptor has also been cloned from the frog Xenopus laevis (190). In addition, there are currently five cloned and functionally expressed Y1-/Y4-/y6-like PP-fold receptors from fish, zebrafish (z) Ya (191), Yb (192), Yc (193), and cod Yb (194), as well as a receptor from the river lamprey (195). The fish Yb/c receptors display Y1-like pharmacology with a gradual loss of affinity by progressive amino-terminal truncation of the NPY molecule as well as recognition of Pro³⁴-substituted analogs. In contrast, amino terminally truncated peptides like NPY3-36 and NPY13-36 as well as Pro³⁴-substituted analogs bind with the same affinity as NPY to the zYa receptor. However, neither the two Y1-selective antagonists BIBP3226 (110) and SR120819A (109) nor the Y2-selective BIIE0246 (196) bind to any of the fish receptors (193–195, 197). Several proposed PP-fold like receptors have been cloned from various invertebrate species (198, 199), but most of them display very low sequence identity to the cloned vertebrate PP-fold receptors. In fact, most of the invertebrate receptors display as low amino acid identity to PP-fold receptors (20–30%) as to other peptide receptors. However, the amino acid sequence of the Y1, Y2, and Y5 receptors are also only 25–30% identical to each other. Also, a few invertebrate peptides with PP-fold receptor like sequence have been reported (198, 200). A mutation in a PP-fold like receptor in Caenorhabditis elegans was found to change the feeding behavior of this organism (199), suggesting that the role of the PP-fold peptides and receptors in the regulation of feeding is not limited to vertebrates.

	Other Proposed Receptors

Top Abstract Introduction Structure(s) of PP-fold... PP Function. PYY Function. NPY Function. PY. Receptors for the NPY... Other Proposed Receptors Recent Findings on Distribution... Studies of Genetically... Recent Developments in Selective... Small Molecule NPY Medicinal... Structure and Mutagenesis... Overview Conclusion References

 
The Y3 Receptor.
The existence of an NPY-preferring receptor, often referred to as the Y3 receptor, has been inferred from pharmacological studies of a variety of tissues (2, 201). The typical rank order of peptide potency of the receptor is NPY > NPY13-36 > PYY. Despite numerous attempts, no cloned receptor has been identified with a Y3-like pharmacology. Recently, it was shown that bovine chromaffin cells, which display typical Y3-like pharmacology, express high levels of the Y1 receptor (202). One explanation for the lack of cloning success with the Y3 receptor is that Y1 receptors display a different pharmacological profile in these areas or that the Y3 binding profile is in fact the result of a mix of several of the cloned receptors.

The PYY-Preferring Receptor.
The presence of a PYY-selective receptor has been reported using tissue preparations from the GI tract (see Ref. 2 for review) and also electrophysiology of the rat dorsal vagal complex (203). Very recently, the rat intestinal PYY-preferring receptor was found to be identical to the rat Y2 receptor expressed in peripheral organs (204).

Peripheral Y2 Receptor.
When the human Y2 receptor was cloned, it was striking how little of the mRNA that could be detected in peripheral organs (134, 136, 205). However, high levels of Y2-like binding have been detected in several organs, for instance, kidney (105, 206). This discrepancy might be explained by the existence of another not yet cloned receptor with a Y2-like binding profile in the periphery. However, such a receptor has not been identified to date. Considering the presynaptic localization of the Y2 receptor (see Y2 section), one possible explanation could be that the peripheral Y2-like binding may be caused by the already cloned Y2 receptor presynaptically located on peripheral nerve terminals.

	Recent Findings on Distribution of PP-fold Peptide Receptors

Top Abstract Introduction Structure(s) of PP-fold... PP Function. PYY Function. NPY Function. PY. Receptors for the NPY... Other Proposed Receptors Recent Findings on Distribution... Studies of Genetically... Recent Developments in Selective... Small Molecule NPY Medicinal... Structure and Mutagenesis... Overview Conclusion References

 
General.
NPY as well as all mammalian PP-fold receptors are expressed both in the brain and in the periphery, and there is an overlap in the distribution of the different receptors in the brain. Furthermore, determination of the receptor distribution is complicated because there are extensive species differences in the distribution of the receptors between orders of mammals as well as within the rodent and primate families for all PP-fold receptor subtypes (2, 207, 208). Thus, it appears that although the functions of the PP-fold peptides are very well conserved throughout evolution, the receptor system involved in a particular effect can vary dramatically between closely related species.

There are also discrepancies between the distribution of mRNA and protein (209). For example, although the highest mRNA concentrations in the human hypothalamus were found to be Y5, the binding profile of human hypothalamic homogenate identified Y2 as being the predominant receptor in this brain region (210). This has also been observed using autoradiography (211). A possible explanation for these discrepancies could be that Y2-expressing neurons have their nuclei outside the hypothalamus and Y2 binding in this tissue is to presynaptic sites. Another explanation for the discrepancies between mRNA and protein distribution are differences in mRNA stability and protein breakdown in post-mortem tissues (212). We therefore chose to discuss expression of the gene and protein separately. For more details, especially on the distribution of the peptides, see the previous reviews in this journal as well as others (1, 2, 27, 54) for reviews.

Receptors: mRNA.
The distribution of the PP-fold receptor mRNAs in the brain differs dramatically between species. There are even reports about differences between different primate species (211) as well as differences between rat and mouse (213). By reverse-transcription polymerase chain reaction (RT-PCR), the relative levels of mRNA for Y1, Y2, and Y5 in the human hypothalamus were determined (210). The Y5 mRNA levels were 400 and 200 times higher than for the Y1 and Y2 receptors, respectively. Interestingly, Y5 receptor mRNA appears to be consistently colocalized with the Y1 mRNA in the rat (214) and mouse (213) brain whereas Y1 has a much broader distribution and is expressed in many areas without the presence of Y5.

Messenger RNA for Y1 has been found in brain areas important for the regulation of feeding, especially the arcuate nucleus of the hypothalamus where it is sometimes but not exclusively localized on pro-opiomelanocortin-expressing neurons (215). Y1 mRNA is also highly concentrated throughout the cerebral cortex and cerebellum in human brain with moderate levels in pyramidal cells of CA1-3 (212).

In human post mortem brain, the highest levels of Y2 mRNA was found in dentate gyrus (216). High levels were also found throughout the cerebral cortex as well as in lateral geniculate nucleus, amygdala, substantia nigra, hypothalamus, cerebellum, and choroid plexus (216). Furthermore, in the cerebral cortex, hippocampus, striatum, and amygdala Y2 (but not Y1) mRNA was found on NPY-positive cells suggesting that Y2 is an autoreceptor in these areas (217). In the arcuate nucleus of the rat hypothalamus, Y2 mRNA is mainly found in NPY expressing neurons in agreement with a presynaptic function (215). In the rat, a similar distribution is observed with the highest levels of Y2 mRNA in hippocampal areas (218, 219) and in the arcuate nucleus of the hypothalamus (218). In the intestinal tract of the rat, Y1 mRNA is exclusively found in non-epithelial colon while Y2 is found in all crypt cells, villus, colon epithelium, and jejunal epithelium (220). RT-PCR detected Y1 as the predominant receptor subtype in human adipocytes although Y4 and Y5 transcripts were also detected (221).

The Y4 receptor is highly variable across species both with regards to pharmacology and distribution, which may explain why its exact role may differ between species (90, 152). In humans, Y4 mRNA is found in prostate, colon, pancreas, and small intestine (90) as well as skeletal muscle (154). Lower levels of human Y4 mRNA was also found in brain by RT-PCR (95, 220) and Northern blot (154). A high level of Y4 mRNA in the rat was only found in the testis (152). In a more detailed study using RT-PCR, rat Y4 mRNA was found in all intestinal tissues examined with the highest levels localized to colon epithelium (220). Y4 mRNA has also been found in rat hypothalamus by RT-PCR (220) and in situ hybridization (214). Furthermore, in situ hybridization has also detected high levels of Y4 mRNA in neurons of the rat dorsal vagal complex, area postrema, and nucleus of the solitary tract (222). Very recently, the presence of the Y4 receptor in several colon adenocarcinoma cell lines was reported (223).

By in situ hybridization, Y5 mRNA is found in many brain regions of the rat. High levels can be found in many areas, including the paraventricular and arcuate nuclei of the hypothalamus, lateral hypothalamus, medial thalamus, suprachiasmatic nucleus, and hippocampus (91, 219, 224, 225). A similar distribution was found in the human brain (225). Low levels of Y5 mRNA are present throughout the cerebral cortex of the rat (224). However, compared to rat and human, the overall levels of Y5 mRNA appear to be very low in mouse brain (213). The Y5 receptor is also found in several organs in the periphery. The highest level of Y5 expression in the periphery has been found in the testis (91). By RT-PCR, rat Y5 mRNA has been detected in colon crypt cells, non-epithelial colon (220), spleen, and pancreas (210).

During mouse embryo development, Y1, Y2, and Y5 are turned on at about embryonic day 12-15 and the expression remains rather stable with relative levels Y1>Y2>Y5. In cerebellum, however, Y1 and Y2 expression is reduced after post-natal day 4 (213).

Receptors: Protein.
Some initial successes have been reported using antibodies directed against PP-fold receptors. In general these studies have used synthetic peptides corresponding to unique regions of the receptors to obtain antisera from rabbits. Antibodies directed against the rat Y1 detected the highest levels in the subiculum of the hippocampal formation with lower levels found in the dentate gyrus and the CA2 region (226). Strong Y1-ir was also detected in striatum, claustrum, piriform cortex, arcuate nucleus of the hypothalamus, interpeduncular nucleus, paratrigeminal nucleus, and lamina II of the spinal trigeminal nucleus as well as in the entire spinal cord of the rat (227). In the hypothalamus of the mouse, Y1-ir was found to be colocalized with thyrotropin-releasing hormone and CART in parvocellular neurons (228). In the periphery, Y1-ir has been detected on NPY- and vasoactive intestinal peptide (VIP)-positive neurons throughout the intestine of the rat. Y1-ir can also be found in endothelium and on some endocrine cells (229), as well as in arterial smooth muscle in testis (230). The use of subtype selective antagonists to inhibit PYY-evoked vasoconstriction revealed the existence of Y1 and Y2 receptors in pig spleen (26). To our knowledge, there are no reports yet on successful use of antibodies raised against the Y2 receptor protein.

Fewer studies are available using antisera directed against the other PP-fold receptors. Using immunohistochemistry, high levels of Y5 were detected in the rat hypothalamus, where the highest levels were found in the magnocellular neurons of the paraventricular hypothalamus, the supraoptic nucleus, and in the arcuate nucleus (172). Scattered immunopositive neurons were observed in the thalamus, including paraventricular nucleus of the thalamus, habenula, mediodorsal thalamic nucleus, and zona incerta. Furthermore, high levels of Y5-ir were found throughout the hippocampus and cortex of the rat (172). Using double-label immunofluorescence, Y5-ir was colocalized with corticotropin-releasing hormone (CRH) and gamma-aminobutyric acid (GABA) in brainstem (231). In the preoptic area, the Y5 receptor is present on about 55% of the gonadotropin-releasing hormone (GnRH) neurons (172). Y5-ir was also found on CRH-, neurophysin-, and GABA-positive neurons in the hypothalamus (172, 231).

Until recently, autoradiographic studies of the PP-fold receptors have been hampered by the lack of specific tools to delineate receptor subtypes. All peptide agonists used (¹²⁵I-PYY, ¹²⁵I-PYY3-36, ¹²⁵I-[Leu³¹,Pro³⁴]PYY/NPY, ¹²⁵I-hPP, and ¹²⁵I-1229U91) bind to several receptor subtypes with high affinity. The recent development of highly specific antagonists will undoubtedly help future studies of receptor distribution using this method in that it will be possible to selectively mask unwanted binding. However, consistent with previous studies using less selective radioligands, the Y1 and Y2 receptors appear to be the predominant receptor subtypes in rat brain when using autoradiography.

When ¹²⁵I-[Leu³¹,Pro³⁴]PYY binding was compared with the distribution of ¹²⁵I-PYY3-36 binding (Y2-like) in human postmortem brain (211), high levels of Y2 binding was found throughout the cortex and in the hypothalamus whereas ¹²⁵I-[Leu³¹,Pro³⁴]PYY only detected moderate levels of receptors in the dentate gyrus of the hippocampal formation and in the caudate nucleus confirming the low abundance of Y1 and Y5 receptors in the human brain. However, ¹²⁵I-[Leu³¹,Pro³⁴]PYY and ¹²⁵I-[Leu³¹,Pro³⁴]NPY bind to all PP-fold receptors except Y2. Dumont and co-workers identified low densities of BIIE0246 (Y2-selective antagonist, see Fig. 2 and Ref. 196) -insensitive ¹²⁵I-PYY3-36 binding sites, most likely Y5, in olfactory tubercle of the rat and several hippocampal areas of the rat, monkey (marmoset), and human (232). A similar approach used BIBO3304 (Y1-selective antagonist; Ref. 111; Fig. 1) and CGP71683A (proposed Y5 antagonist; Ref. 177; Fig. 3) to compete with ¹²⁵I-[Leu³¹,Pro³⁴]PYY binding to rat brain homogenate. In these studies, it was found that the Y1-like binding outnumbered Y5 by 3:1 (233). Y5-like binding (BIBO3304 insensitive) was found mainly in the olfactory bulb and hippocampal areas with very little in the hypothalamus (233).

When comparing Y1-like and Y2-like binding in human and rat, it has been found that Y2 is the predominant receptor in human brain whereas Y1 dominates in the rat (2, 211) and mouse (213) brain. However, in another study, homogenate binding of mouse forebrain detected similar amounts of binding sites for ¹²⁵I-[Leu³¹,Pro³⁴]PYY (Y1, Y4, and Y5) as for ¹²⁵I-PYY3-36 (Y2 and possibly Y5) (234). Using autoradiography, the highest concentrations of ¹²⁵I-[Leu³¹,Pro³⁴]PYY sites were found in the islands of Calleja and dentate gyrus of the mouse whereas ¹²⁵I-PYY3-36 binding displayed a much broader distribution (234). Dumont and colleagues only detected low levels of BIBP3226-insensitive binding sites for ¹²⁵I-[Leu³¹,Pro³⁴]PYY (i.e., Y5 or possibly Y4) in the rat hypothalamus (235). Instead, the highest Y5-like binding was detected in CA3 area of the hippocampus, lateral septum, olfactory bulb, the nucleus tractus solitarius, and the area postrema (235).

The radioligand ¹²⁵I-1229U91 binds with very high affinity to both Y1 and Y4 receptors (159, 160). Using ¹²⁵I-1229U91, binding was detected throughout the rat brain, including the lamina I-III of the cerebral cortex, olfactory tubercle, many thalamic areas, and the dorsal hypothalamus (236, 237). Interestingly, ¹²⁵I-rPP binds to the interpeduncular nucleus and the paraventricular nucleus (PVN) of the rat (208, 236), whereas these areas were not labeled by ¹²⁵I-1229U91, suggesting the presence of an atypical PP binding site in these areas (236). In another study of the rat brain, high levels of BIBO3304-insensitive but PP-sensitive ¹²⁵I-1229U91 binding (Y4-like) sites were detected in area postrema with lower levels in the olfactory bulb and hippocampus (237).

In the periphery, autoradiography studies using rat and rabbit kidney revealed that Y1 is the predominant receptor subtype in the rat kidney, whereas Y2 is the predominant PP-fold receptor in rabbit kidney (238). Human adipose tissue contains a BIBP3226-sensitive receptor that bound ¹²⁵I-[Leu³¹,Pro³⁴]PYY and ¹²⁵I-PYY and thus confirmed the presence of the Y1 receptor and absence of Y2 in adipocytes (221).

	Studies of Genetically Engineered Rodents

Top Abstract Introduction Structure(s) of PP-fold... PP Function. PYY Function. NPY Function. PY. Receptors for the NPY... Other Proposed Receptors Recent Findings on Distribution... Studies of Genetically... Recent Developments in Selective... Small Molecule NPY Medicinal... Structure and Mutagenesis... Overview Conclusion References

 
Overview.
Numerous genetic manipulations have been made to create mice that are deficient in NPY and individual NPY receptor subtypes (Table II). In addition, a line of rats, which exhibit a 3- to 4-fold increase in NPY concentrations in certain tissues, has been created. In general, these animals exhibit very little overt phenotype when raised under standard conditions (for review, see Ref. 239). For instance, the NPY knockout mice eat normally, grow normally, and refeed normally after fasting conditions. Furthermore, the endocrine responses to fasting under normal conditions are within normal parameters. The NPY overexpressing rats exhibit a similar normal phenotype with little change in body weight and resting blood pressure. In general, the receptor knockouts also exhibit a normal phenotype but with a mild late onset obesity in most cases. Given these modest changes in resting phenotype, it has been difficult to discern the role of NPY in normal physiology and in the maintenance of body weight, blood pressure, epilepsy, and in models of certain emotional and psychiatric indications. Now that these tools are available to understand the NPY system, numerous investigators have begun to alter the physiological parameters of these animals to understand the role of NPY in disease pathology.

A more recent study conducted with NPY -/- mice examined the behavioral phenotype created by this mutation (240). Based on data from behavioral models, the NPY -/- mice exhibit increased anxiogenic-like behavior and also appear to be hypoalgesic on the hot plate test of pain. Knockout studies have also suggested NPY to be a neuroproliferator. Mice lacking NPY displayed an impaired development of olfactory neurons (56, 57). Very recently, NPY -/- mice were crossed with mice lacking agouti-related peptide (AgRP; Ref. 244). AgRP acts as an antagonist at melanocortin receptors (see NPY section) and is thus, like NPY, an orexigenic peptide. Interestingly, AgRP is expressed by the same hypothalamic neurons as NPY. Like the NPY -/- mice, these double knockout animals exhibited a normal feeding behavior and weight gain under normal conditions (244).

Y1 Receptor Knockout Mice.
Y1 receptor knockout mice have been generated by numerous investigators (108, 121, 122, 245). Again, under standard vivarium conditions, little overt phenotype is discernible between the knockout and control wild-type animals. Peripherally, the Y1 knockouts exhibit a substantially reduced blood pressure increase after NPY administered intravenously (246), confirming the role of this receptor in the pressor effects of NPY. In initial studies, female Y1 knockout mice display a late onset increase in body weight compared to their wild-type litter mates (121, 122); however, male animals exhibited no difference in body weight when compared with wild-type male litter mates. When NPY was injected intracerebroventricularly, there was a significant reduction in feeding observed in the Y1 knockout mice when compared to the wild-type litter mate controls (121, 245). Therefore, NPY may produce its effects on food consumption at least partially by activation of the Y1 receptor. However, a significant increase in NPY-induced food consumption was still observed in the Y1 knockout animals. This may be the result of the cooperative interaction with other receptor subtypes to produce the full effect of NPY on feeding (see Y5 knockout data below). In a recent report, Naveilhan and colleagues (108) have demonstrated that the Y1 knockout mice develop hypoalgesia to acute thermal, cutaneous, and visceral pain. In addition, these animals exhibit increased mechanical hypersensitivity in pain models. In models of neuropathic pain, there is also an increased response and a complete absence of the pharmacological effects of NPY analgesia. Therefore, it is likely that the Y1 receptor participates in the analgesic effects of NPY centrally and, perhaps, peripherally as well.

In another study (52), NPY was found to potentiate pentobarbital-induced sedation in wild-type animals but not in Y1 knockout animals. This suggests the Y1 receptor plays an important role in mediating the sedation seen with GABAergic compounds. Similar results were obtained for Avertin but not Kealar (NMDA antagonist)-induced sedation, thus reinforcing the proposed GABAergic/NPY hypothesis.

Y2 Receptor Knockout Mice.
In initial studies, the Y2 receptor knockout mice exhibit a small increase in body weight, slightly increased food intake and increased fat deposition (150). The mutant mice also showed a blunted response to leptin but a normal response to NPY-induced food intake and intact regulation of refeeding and body weight after fasting. Therefore, the Y2 receptor may be involved in the regulation of hypothalamic NPY release in a tonic fashion. An absence of the Y2 receptor subtype also appeared to affect the basal control of heart rate in mice but did not affect normal blood pressure (150). The Y2 receptor knockouts exhibit very interesting phenotypes in behavioral studies. In a study that evaluated the role of both the Y1 and Y2 receptors in sedation (53), an increased sensitivity to pentobarbital-induced sedation was observed in the Y2 -/- mice. Therefore the Y2 receptor appears to modulate GABAergic-induced sedation physiologica