(Received for publication, August 18, 1995; and in revised form, September 5, 1995)
From the
The pancreatic polypeptide family includes pancreatic
polypeptide (PP), neuropeptide Y (NPY), and peptide YY (PYY). Members
of the PP family regulate numerous physiological processes, including
appetite, gastrointestinal transit, anxiety, and blood pressure. Of the
multiple Y-type receptors proposed for PP family members, only the Y1
subtype has been cloned previously. We now report the cloning of an
additional Y-type receptor, designated Y4, by homology screening of a
human placental genomic library with transmembrane (TM) probes derived
from the rat Y1 gene. The Y4 genomic clone encodes a predicted protein
of 375 amino acids that is most homologous to Y1 receptors from human,
rat, and mouse (42% overall; 55% in TM). I-PYY binding to
transiently expressed Y4 receptors was saturable (pK
= 9.89) and displaceable by human PP family
derivatives: PP (pK
= 10.25)
PP
(pK
=
10.06) > PYY (pK
= 9.06)
[Leu
,Pro
]NPY (pK
= 8.95) > NPY (pK
= 8.68) > PP
(pK
= 7.13) >
PP
(pK
=
6.46) > PP
free acid (pK
< 5). Human PP decreased [cAMP] and increased
intracellular [Ca
] in Y4-transfected
LMTK
cells. Y4 mRNA was detected by reverse
transcriptase-polymerase chain reaction in human brain, coronary
artery, and ileum, suggesting potential roles for Y4 receptors in
central nervous system, cardiovascular, and gastrointestinal function.
The pancreatic polypeptide family includes pancreatic
polypeptide (PP), ()neuropeptide Y (NPY), and peptide YY
(PYY), all of which are 36 amino acid peptides characterized by a
hairpin loop(1) . PP is released from pancreatic endocrine
cells post-prandially(2) . PP receptors have been characterized
by binding or functional assays in liver(3) ,
intestine(4) , vas deferens(5) , spinal
cord(6) , adrenal gland, superior cervical ganglia(7) ,
brainstem nuclei(8) , and PC-12 cells(9) . PP exerts
regulatory effects on pancreatic exocrine secretion, gall bladder
contraction, gastrointestinal motility, gastric acid secretion, and
corticosterone secretion(2, 10, 11) . PYY is
localized primarily in intestinal endocrine cells, whereas NPY is
localized primarily in the nervous system(1) . NPY and PYY act
similarly in a majority of biological models (e.g. to increase
food intake and vasocontraction), but exceptions have been
noted(1) . Within the PP family, NPY is most conserved among
species, whereas PP is least conserved(1) .
At least five
receptor subtypes have been proposed for PP family members(1) :
1) Y1 binds NPY, PYY, and
[Leu,Pro
]NPY > PP and
COOH-terminal fragments; 2) Y2 binds NPY, PYY, and COOH-terminal
fragments > PP and [Leu
,Pro
]NPY;
3) Y3 binds NPY > PYY; 4) the PP receptor binds PP > NPY, PYY;
and 5) the putative Y1-like feeding receptor is activated by NPY, PYY,
[Leu
,Pro
]NPY, and
NPY
> COOH-terminal fragments. Only the Y1
subtype has been cloned
previously(12, 13, 14, 15, 16) .
Here we describe homology cloning of an additional human Y-type
receptor, Y4, which is functionally activated by PP.
A human genomic placenta library was screened, under reduced stringency conditions, with oligonucleotide probes directed to the first, second, third, fifth, and seventh TM regions of the rat Y1 neuropeptide receptor gene. One positive hybridizing phage clone called hp25a, whose gene product will be referred to in this report as hY4, was characterized by Southern blot analysis to reveal a 1.3-kb PstI fragment and further analyzed by subcloning and sequencing. DNA sequence analysis indicated greatest homology to the rat and human Y1 receptor genes. Since this clone represented a partial intronless gene fragment, the full-length gene was subcloned as a 2.0-kb BamHI/EcoRI hybridizing fragment into an expression vector and sequenced.
The genomic full-length construct
contains an open reading frame of 1125 bp (intronless), with 680 bp
upstream and 205 bp downstream of the coding region. The gene would be
predicted to encode a protein of 375 amino acids, with a predicted
molecular mass of 41 kDa. Hydropathy analysis of the protein is
consistent with a putative topography of seven transmembrane domains,
indicative of the G-protein-coupled receptor family.
Initial sequence analysis revealed that hY4 contained several structural features/residues (see Fig. 1) found among the members of the neuropeptide receptor family, including two glycines and an asparagine in TM1 (positions 55, 58, and 59, respectively), an asparagine, leucine, and aspartic acid in TM2 (positions 82, 83, and 87, respectively), a serine and leucine in TM3 (positions 128 and 132, respectively), a tryptophan and proline in TM4 (positions 164 and 173, respectively), a tyrosine and proline in TM5 (positions 223 and 226, respectively), a phenylalanine, tryptophan, and proline in TM6 (positions 274, 278, and 280, respectively), and a serine, threonine, asparagine, and proline in TM7 (positions 314, 315, 318, and 319, respectively). Other features of hY4 are the presence of three potential sites for N-linked glycosylation in the amino terminus (asparagine residues 2, 19, and 29; Fig. 1) and the presence of several serines and threonines in the carboxyl terminus and intracellular loops, which may serve as sites for potential phosphorylation by protein kinases. It is interesting that the sequence ERH (Glu-Arg-His), which is immediately downstream of TM3, is also contained in the hY1 sequence; this is distinct from the DRY (Asp-Arg-Tyr) sequence which occurs in most members of the G-protein-coupled receptor superfamily. Additionally, two of the three potential N-linked glycosylation sites found in hY4 are also present in the corresponding positions in the hY1 receptor.
Figure 1:
Amino acid sequence alignment of human
Y4 (hp25a clone) with human Y1 and Y2 receptors. The deduced amino acid
sequence of the human Y4 receptor (first line) is aligned with the
human Y1 receptor (12) and the human Y2 receptor clone (see
accompanying paper(36) ). Periods represent added spaces
necessary for proper alignment. Gray shading indicates
residues in receptors which are identical to Y4. Numbers correspond to amino acid positions of Y4 (M). The seven
putative transmembrane domains are boxed and numbered I-VII. Black circles indicate potential N-linked glycosylation sites in the NH-terminal
extracellular part of human Y4; arrows correspond to protein
kinase C consensus sites.
A comparison of nucleotide and peptide sequences of hY4 with sequences contained in the GenBank(TM)/EMBL data bases reveals that the clone is most related to the rat, mouse, Xenopus, and human Y1 receptor genes and proteins. At the nucleotide level there is 58% identity between hY4 and hY1; at the amino acid level there is 42% identity overall, 55% in TM domains, with the greatest identity of 71% in TM7 (Fig. 1). A similar comparison of hY4 with the cloned human Y2 (hY2) gene (see accompanying paper by Gerald et al.(36) ) reveals lower homology; hY4 versus hY2 nucleotide identity is 57% and amino acid identity is 34% overall, with 43% in TM domains (see also Fig. 1). Southern blot analysis on human genomic DNA suggest that the genome contains a single Y4 gene (data not shown).
Human Y4 mRNA was detected by reverse
transcriptase-PCR using specific hY4 primers in a broad range of human
tissues (Fig. 2). Relatively intense hybridization signals were
detected in total brain (including the hypothalamus), coronary artery,
and ileum. Liver failed to express hY4 mRNA, whereas pancreas and
kidney exhibited very low levels of expression. Interestingly, the
endometrium and myometrium of the uterus displayed differential
expression, with the former containing higher levels of Y4 mRNA than
the latter. No signal was observed when either the RNA was absent
(distilled H0 control) or reverse transcriptase was omitted
from the first strand cDNA conversion (data not shown); the latter
suggests that the signals observed not due to any genomic DNA
contaminating the RNA. We also demonstrated that equal amounts of cDNA
from the different tissues were assayed for NPY expression by
conducting control reverse transcriptase-PCR with primers for the
moderately high level constitutively expressed gene,
glyceraldehyde-3-phosphate dehydrogenase (Clontech) (Fig. 2).
Figure 2:
Tissue distribution of Y4 receptor mRNA in
various human tissues. Localization data reflect PCR-based
amplification of human Y4 cDNA derived from RNA extracts of human
tissues. Total RNA (250 ng) was assayed for all tissues except
hippocampus and hypothalamus, where poly(A) RNA (5 ng)
was used. Southern blots of the PCR products were prepared and
hybridized with
P-labeled oligonucleotide probes selective
for the Y4 receptor subtype or glyceraldehyde-3-phosphate dehydrogenase (G3PDH).
I-PYY (0.06 nM) bound specifically to
membranes from hY4-transfected COS-7 cells (but not from
mock-transfected cells) with an observed association rate (K
) = 0.12 ± 0.02
min
, t
= 6 min, and
100% complete equilibrium binding within 50 min at 30 °C (n = 3). Human Y1-transfected COS-7 cell membranes, when
studied under the same conditions, yielded a K
= 0.06 ± 0.02 min
, t
= 12 min, and 100% complete equilibrium
binding within 90 min (n = 3). Subsequent
I-PYY binding assays involving both hY1 and hY4 receptors
were conducted for 120 min.
I-PYY binding to the
transiently expressed hY4 receptor was specific and saturable at
I-PYY concentrations ranging from 0.003 to 2.5
nM. Binding data were fit to a one-site model with an apparent
pK
= 9.89 ± 0.04 (0.13 nM)
and B
= 1.9 ± 0.3 pmol/mg membrane
protein (n = 8). The transiently expressed hY1 receptor
bound
I-PYY with an apparent pK
= 10.19 ± 0.04 (0.065 nM) and B
= 4.0 ± 0.7 pmol/mg membrane
protein (n = 9).
Human Y4 bound human PP family
members in I-PYY membrane binding assays with a
distinctive rank order (Table 1): PP > PYY > NPY > NPY
free acid. Human Y4 also bound PP from bovine, rat, salmon, and frog
with a wide range of K
values consistent with PP
species diversity. PYY and NPY binding to hY4 could be enhanced by
inserting PP residues into positions 31 and 34 to make
[Pro
]PYY and
[Leu
,Pro
]NPY. However, the
corresponding modifications of PP to reflect the NPY peptide (e.g. [Ile
,Gln
]PP) had no effect on
binding affinity to hY4 (see Table 1), suggesting that for the
favored ligand PP there are significant contributions to binding
affinity from other peptide regions. Human PP could be truncated to
PP
but further NH
-terminal deletion
was disruptive for hY4 binding. The shortest COOH-terminal fragment
studied (PP
) was rendered inactive by hydrolysis
of the carboxyl amide. A comparison of structure/activity profiles for
hY4 and hY1 suggests a common mechanism of peptide interaction
optimized for either PP or NPY/PYY, respectively.
Incubation of
intact LMTK cells with 10 µM forskolin
produced an average 7-fold increase in cAMP over a 5-min period (n = 20). Simultaneous incubation with human PP decreased the
forskolin-stimulated [cAMP] with an E
of 83 ± 2% in LMTK
cells stably
transfected with hY4 (Fig. 3A), but not in
untransfected cells. The pEC
for human PP (10.13 ±
0.06) was in close agreement with the pK
(10.25
± 0.06) from
I-PYY binding assays. The list of
Y-type receptors negatively coupled to cAMP (previously composed of Y1,
Y2, and Y3) can therefore be extended to include Y4(1) .
Figure 3:
PP-dependent signaling events in intact
LMTK cells stably expressing the hY4 receptor. A, inhibition of forskolin-stimulated [cAMP]. Data
shown are representative of 20 independent experiments. B,
stimulation of intracellular free [Ca
].
Human PP (100 nM) was added at the time indicated by the arrow. Data shown are selected from 10 recordings made in four
independent experiments.
Intracellular free [Ca] was markedly
increased by 100 nM human PP in LMTK
cells
stably transfected with the hY4
(
[Ca
]
= 158
± 32 nM, n = 10) but not in
untransfected cells. The Ca
transient could be
detected within 30 s of PP application (Fig. 3B). These
data add complexity to existing notions of PP-dependent Ca
regulation centered around inhibition of voltage-dependent
Ca
channels in rat superior cervical
ganglia(26, 27) .
We have cloned the gene for a
novel human Y-type receptor, Y4, which is functionally activated by PP.
The human Y4 pharmacological profile is similar to rat PP receptor
binding profiles derived from cell and tissue models. For example, a PP
receptor in rat PC-12 cells was reported to bind PP >
PP(7) and also PP >
[Leu
,Pro
]NPY,
[Ile
,Gln
]PP NPY (5) .
Receptors on PC-12 cells bound bovine PP > rat PP(7) ,
although rat hepatocyte receptors bound both with equally high
affinity(3) . A receptor on intact rat cells from liver,
pancreas, and elsewhere was reported to bind all PP family members with
similar K
values(28) . Whether the rat
receptors described in these reports represent the same gene product
analyzed under different assay conditions or multiple subtypes and what
their relationship is to hY4 is not yet clear.
The human Y4 could
conceivably be designated a PP receptor. We propose the name Y4,
however, as a direct extension of the Y-type nomenclature previously
established for Y1, Y2, and Y3 receptors. Arguments are as follows: 1)
Y4 was cloned by virtue of its homology with the Y1 receptor and is
most similar in sequence with Y1 receptors from several species. As
such, the Y4 is structurally linked to receptors currently entrenched
in Y-type nomenclature; 2) the human Y4 displayed a narrow range of K values for human PP, PYY, and NPY in
I-PYY binding assays (10.25
pK
8.68); 3) The letter ``Y'' encodes the conserved
COOH-terminal Tyr in pancreatic polypeptide family members and is
therefore a unifying symbol for the entire ligand family; and 4) the Y4
designation represents a reasonable nomenclature in the event that
peptide rank order should change with assay conditions or with the
discovery of additional PP family members.
The tissue distribution of the Y4 mRNA is consistent with reports of PP binding and function in brain and peripheral tissues and further suggests potential Y4 involvement in gastrointestinal, cardiovascular, and central nervous system function. As there appears to be an inverse correlation between pancreatic or circulating PP levels and obesity in human, rat, and mouse (i.e. circulating PP levels are increased in anorexia nervosa), one may speculate a role for Y4 in appetite and body weight control(23, 29, 30, 31) . Circulating PP has access to central sites through penetration of fenestrated capillaries (8) and through transport past the blood brain barrier(32) . Additional routes of Y4 receptor activation may also exist, as PP mRNA has been recently identified by PCR in rat brain, and in particular, hypothalamus(33) . Localization of Y4 receptor mRNA in hypothalamus and potential receptor activation are intriguing in that not only NPY and PYY, but also PP, can enhance food intake when injected into rat brain intracerebroventricular(34, 35) . One hypothesis is that the Y4 receptor is involved in hypothalamic control of feeding. This and other aspects of Y4 function can be further explored using techniques based on the Y4 receptor clone, such as expression of species homologs, selective antagonist design, receptor antisense, and transgenic animal models.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U35232[GenBank].