(Received for publication, June 14, 1995; and in revised form, September 7, 1995)
From the
Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic
polypeptide (PP) are structurally related peptides found in all higher
vertebrates. NPY is expressed exclusively in neurons, whereas PYY and
PP are produced primarily in gut endocrine cells. Several receptor
subtypes have been identified pharmacologically, but only the NPY/PYY
receptor of subtype Y1 has been cloned. This is a heptahelix receptor
that couples to G proteins. We utilized Y1 sequence information from
several species to clone a novel human receptor with 43% amino acid
sequence identity to human Y1 and 53% identity in the transmembrane
regions. The novel receptor displays a pharmacological profile that
distinguishes it from all previously described NPY family receptors. It
binds PP with an affinity (K) of 13.8
pM, PYY with 1.44 nM, and NPY with 9.9 nM.
Because these data may identify the receptor as primarily a PP
receptor, we have named it PP1. In stably transfected Chinese hamster
ovary cells the PP1 receptor inhibits forskolin-stimulated cAMP
synthesis. Northern hybridization detected mRNA in colon, small
intestine, pancreas, and prostate. As all three peptides are present in
the gut through either endocrine release or innervation, all three
peptides may be physiological ligands to the novel NPY family receptor
PP1.
Pancreatic polypeptide (PP) ()forms a family of
36-amino acid peptides together with neuropeptide Y (NPY) and peptide
YY (PYY) (Larhammar et al., 1993). PP was the first of these
to be discovered (Kimmel et al., 1968), but in evolutionary
terms it seems to be the most recent member and probably arose by
duplication of the PYY gene in early tetrapods (Larhammar et
al., 1993). PP is exclusively localized to subsets of endocrine
cells in the pancreas and inhibits pancreatic secretion, gall bladder
contraction, and gut motility (see Hazelwood (1993) for review). PYY is
expressed in PP cells and in somatostatin cells
(Böttcher et al., 1993; Upchurch et
al., 1994) as well as in endocrine cells of the large intestine
(El-Salhy et al., 1983; Lundberg et al., 1982), has
similar actions to PP, and, in addition, redistributes blood flow in
gut vessels (see Laburthe(1990) for review). Both peptides are released
into the circulation in response to a meal (see Hazelwood(1993)). In
contrast, NPY is present in the central nervous system but is involved
in gastrointestinal function through potent induction of feeding in the
hypothalamus.
The NPY family peptides exert their actions via heptahelix (seven-transmembrane region) receptors coupled to G-proteins. Several receptor subtypes have been defined by their ability to bind NPY, PYY, PP, and derivatives of these peptides (Gehlert, 1994). Both NPY and PYY bind to the Y1 and Y2 receptors, while the Y3 receptor binds only NPY. The hypothalamic feeding receptor seems to be distinct from all of these (see Gehlert(1994)). An additional receptor has been described that displays a preference for PYY over NPY and is found in the rat small intestine (Laburthe et al., 1986) and in dog adipocytes (Castan et al., 1992), where it mediates reduction of lipolysis. PP does not bind to any of these subtypes but seems to have a unique receptor in dog intestinal mucosa (Gilbert et al., 1988; Gilbert et al., 1986), rat phaeochromocytoma PC12 cells (Schwartz et al., 1987), and rat adrenal cortex and medulla (Whitcomb et al., 1992) as well as in rat vas deferens (Jørgensen et al., 1990) and rat brain area postrema (Whitcomb et al., 1990). Finally, there is a PP-fold-recognizing receptor located in the distal colon in rabbit (Ballantyne et al., 1993) that binds all three peptides. While the discovery of selective peptide agonists has allowed a preliminary receptor classification, the lack of specific receptor antagonists has made functional studies difficult. For instance, it is unclear which receptor mediates the feeding induction reported for human PP in rats (Clark et al., 1984) and dogs (Inui et al., 1991).
To date only the Y1 receptor has been cloned. Cloning of additional receptor subtypes would be helpful to determine their preferences for the three endogenous peptides and to distinguish their physiological roles. The object of the present investigation was to isolate DNA clones encoding additional members of the NPY receptor subfamily. For this purpose we designed degenerate PCR primers based upon the Y1 receptor sequences from human (Herzog et al., 1992; Larhammar et al., 1992), rat (Eva et al., 1990), mouse (Eva et al., 1992), and Xenopus laevis (Blomqvist et al., 1995). This approach allowed the cloning of a human receptor that has a higher degree of amino acid sequence identity to the Y1 receptor than to other heptahelix receptors. We also describe functional expression of this receptor to identify it as a PP-preferring receptor, hence named PP1.
Figure 1: Nucleotide sequence and deduced amino acid sequence of the human PP1 receptor gene. The predominantly hydrophobic segments assumed to penetrate the cell membrane are underlined with dotted lines. Four potential sites for N-linked glycosylation are underlined, three in the amino-terminal part and one in extracellular loop 2.
Figure 2: Amino acid sequence alignment. The human PP1 receptor serves as master sequence in alignment with the human Y1 receptor (Larhammar et al., 1992) and the dog gastrin receptor (Kopin et al., 1992). In the two latter sequences only positions that differ from the PP1 sequence are shown, while dots mean identities. Dashes represent gaps introduced to optimize alignment. The hydrophobic segments assumed to be embedded in the cell membrane are underlined. Four tripeptides in extracellular parts underlined with dotted lines conform to the consensus sequence for N-linked glycosylation. Diamonds show four extracellular cysteines and one intracellular cysteine.
The receptor protein deduced from the nucleotide sequence displays many of the characteristic features of heptahelix receptors ( Fig. 1and Fig. 2). The amino terminus has three potential glycosylation sites, and a fourth is present in the second extracellular loop (as in the Y1 receptor). Four extracellular cysteines, one in the amino-terminal region and one in each of the three extracellular loops, presumably form two disulfide bridges (again like the Y1 receptor). A cysteine in the cytoplasmic tail probably serves as an attachment site for palmitate inserted into the cell membrane.
The sequence similarity to Y1 is most prominent in the transmembrane regions, but the loops also show blocks of resemblance. The sequenced portion of the gene extends 180 base pairs beyond the termination codon, but no polyadenylylation signal was found in agreement with the large size of the mRNA (see below).
Figure 3:
Northern hybridizations. A Northern blot
of the human organ panel is shown. Each lane contains 2 µg
of poly(A) RNA.
Figure 4:
Saturation and Scatchard (inset)
analyses of I-pPYY binding to membranes prepared from
COS1 cells transfected with the PP1 expression plasmid Hubert-pTEJ.
Results shown are the average of three experiments performed in
quadruplicate. Nonspecific binding was defined by 1 µM hPP.
Figure 5:
Inhibition of I-pPYY binding
to membranes from COS1 cells transfected with the PP1 expression
plasmid Hubert-pTEJ. Competition data are expressed as a percentage of
binding in the absence of competitor peptide. Data represent the mean
± S.E. for four experiments performed in duplicate. Nonspecific
binding was defined as binding in the presence of 1 µM hPP.
Figure 6:
Inhibition of forskolin-stimulated
adenylyl cyclase activity by hPP and hPYY in Chinese hamster ovary
cells transfected with the hPP1 receptor clone Hubert-pTEJ. hPP
(IC = 7 nM) and hPYY (IC
= 95 nM) produced a dose-dependent inhibition of
cAMP accumulation.
Binding studies to different tissue preparations and cell lines have demonstrated the existence of several distinct receptor subtypes that bind NPY family peptides and peptide analogues. The molecular and physiological characterization of these receptors requires access to molecular clones that can be used for functional expression in cell lines and design of specific DNA and RNA probes. So far only the Y1 receptor has been cloned. We have used molecular biology approaches to find clones for additional receptor subtypes related to Y1 and describe here one such clone that displays greater homology to the Y1 receptor than to any other G-protein-coupled receptor. Because the novel receptor preferentially binds PP among the NPY family peptides, we call the receptor PP1.
The human PP1 receptor consists of 375 amino acids with 53% identity to the human Y1 receptor in the TM regions. This degree of identity is similar to that between different subtypes of tachykinin or somatostatin receptors in the TM regions. The overall identity to hY1 is 43%. The PP1 receptor shares several features with Y1 such as three amino-terminal glycosylation sites and four extracellular cysteines. Intracellular loops 1 and 2 have multiple identical positions; loop 1 has seven out of ten identities, and in loop 2 the first nine amino acids are identical. This motif, ERHQLIINP, is also conserved among all four known Y1 sequences (Blomqvist et al., 1995). It will be interesting to see whether other NPY family receptor subtypes have the same motif. The sequence similarity in the intracellular loops is consistent with the finding that the PP1's messenger response, namely inhibition of forskolin-stimulated cAMP synthesis (Fig. 6), is similar to that of Y1.
A recent study of the human Y1 receptor by site-directed mutagenesis suggested four acidic residues as points of interaction with basic side chains in NPY, namely Asp-104, Asp-194, Asp-200, and Asp-287 (Walker et al., 1994). We have previously shown that three of these positions are negatively charged also in the Xenopus laevis Y1 receptor (Blomqvist et al., 1995), whereas the position corresponding to Asp-194 is a glycine. The PP1 receptor presented here has negatively charged side chains in all four corresponding positions, namely Asp-105, Asp-197, Glu-203, and Asp-289. The similar pattern in negatively charged residues might indicate that PP and PYY bind this receptor in a manner similar to NPY binding to Y1. However, because NPY binds less strongly to PP1 than to Y1, there clearly must be additional structural aspects that diminish NPY binding to the PP1 receptor.
While many heptahelix receptor genes lack introns in their coding regions, the Y1 gene was found to have a small intron immediately after the segment encoding TM5 (Eva et al., 1992; Herzog et al., 1992; Larhammar et al., 1992). The human PP1 receptor gene described here lacks this intron as does the rat genomic fragment generated with PCR that was used to isolate the human PP1 gene. Evolutionary studies may show whether the intron was present in the ancestral NPY family receptor gene. Southern hybridizations to human genomic DNA at high stringency suggest a single PP1 receptor gene.
The functional
expression binding studies of the PP1 receptor revealed a high affinity
for hPP with a K of only 13.8 pM. The PP1
receptor also exhibits high affinity for hPYY (1.44 nM) and
hNPY (9.9 nM). No previous reports in the literature have
described a human PP receptor. When comparing the pharmacological
profile of the human PP1 receptor with PP-preferring receptors
described for other species in the literature, some important
differences emerge.
I-bPP has been reported to bind to a
receptor on rat PC12 cells that differs in pharmacology to the Y1
receptor also found on these cells (Schwartz et al., 1987).
However, while the PC12 receptor has high affinity for bPP, it exhibits
very low affinity for NPY (>1000 nM) whereas the human PP1
receptor binds NPY with a K
of 9.9 nM. We
have recently cloned the rat ortholog of PP1, (
)and this
receptor, too, binds NPY with higher affinity than the PC12 receptor.
In the rat vas deferens, both PP and NPY mediate an inhibition of the
electrically evoked twitch response with similar IC
values
(Jørgensen et al., 1990); however, this effect is
probably mediated by separate receptor populations. A previously
reported PP receptor in the basolateral membranes of the canine
intestine (duodenum, jejunum, ileum, and colon) (Gilbert et
al., 1988; Gilbert et al., 1986) displayed high binding
to bPP. However, because it had very low affinity for PYY and NPY and
these were almost equal to one another, this receptor seems to be
distinct from the PP1 receptor described here. The PP-fold receptor
found in rabbit distal colon was reported to bind all three peptides
with almost equal affinity (Ballantyne et al., 1993).
Naturally, some of these differences in pharmacology may be due to
species differences.
The selectivity (but not the affinity) of NPY
for the Y1 receptor can be improved by replacing the amino acids found
in positions 31 and 34 with those found in PP, Leu, and Pro,
respectively. In the present study, we found that
h[Leu,Pro
]NPY has a 2-fold lower
affinity with a K
of 21.2 nM, whereas NPY
has 9.9 nM. Thus, in this respect our novel receptor is
reminiscent of, but distinct from, Y1. However, the Y1 as well as the
Y2 receptor has low affinity for PP (Schwartz et al., 1990).
The Y3 receptor in bovine adrenal chromaffin cells has high affinity
for NPY but relatively lower affinity for PYY and PP (Wahlestedt et
al., 1992).
The presence of mRNA for the human PP1 receptor in
colon, small intestine, and pancreas (Fig. 3) is consistent with
the known effects of PP. The faint mRNA band in medulla (not shown) may
indicate a relationship to the binding sites for I-bPP
that have been localized in the nucleus of the solitary tract in rat
(Whitcomb et al., 1990). Our recently cloned rat PP1 receptor
will allow investigation of this possibility.
Thus, the novel human receptor PP1 has pharmacological properties that are consistent with a PP receptor but distinguish it from all pharmacologically characterized receptor subtypes for PP, PYY, and NPY.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z66526[GenBank].