(Received for publication, August 18, 1995)
From the
The pancreatic polypeptide family includes neuropeptide Y (NPY),
one of the most abundant neuropeptides in the mammalian nervous system,
as well as peptide YY (PYY) and pancreatic polypeptide (PP). This
peptide family is involved in numerous physiological processes such as
memory, pain, blood pressure, appetite, anxiety, and circadian rhythm.
Of the multiple Y-type receptors proposed for PP family members, only
the Y1 subtype was cloned previously. We now report the isolation of a
human Y2 (hhY2) receptor cDNA by expression cloning from a human
hippocampal cDNA library, using a I-PYY binding assay.
hhY2 cDNA encodes a predicted protein of 381 amino acids with low amino
acid identity to the human Y1 receptor (31% overall; 41%
transmembrane).
I-PYY binding to transiently expressed
hY2 receptors was saturable (pK
=
10.17) and displaceable by human PP family members in rank order: PYY
(pK
= 9.47)
NPY
(pK
= 9.27) PP
(pK
< 6) and by peptide analogs:
NPY
(pK
=
8.80)
NPY
(pK
= 8.55)
C2-NPY (pK
= 8.54) > NPY
(pK
= 6.51)
[Leu
,Pro
]NPY (pK
= 6.23). Human PYY decreased [cAMP] and
increased intracellular [Ca
] in
hY2-transfected 293 cells.
The pancreatic polypeptide family includes neuropeptide Y (NPY), ()peptide YY (PYY), and pancreatic polypeptide (PP), all of
which are 36 amino acid peptides characterized by an
NH
-terminal polyproline helix and a COOH-terminal
-helix brought together by a hairpin loop (1) . NPY
functions primarily as a neurotransmitter and is widely distributed
throughout the central and peripheral nervous system, with additional
localization in adrenal gland and certain non-neuronal cells (1, 3) . NPY modulates numerous physiological
processes, including appetite, anxiety, blood pressure, and circadian
rhythm(1, 2) . PYY is localized primarily in
intestinal endocrine cells and functions as a circulating hormone with
increasing levels post-prandially; small amounts are also found in
central and peripheral neurons(1, 2, 4) . PYY
is known to regulate intestinal secretion and motility, as well as
emesis(5, 6) . PYY and NPY act similarly in a majority
of physiological models (e.g. to stimulate feeding and
increase blood pressure), but exceptions have been noted(1) .
PP is localized primarily in endocrine cells of pancreatic islets and
exerts regulatory effects on gastrointestinal processes such as
pancreatic exocrine secretion, gall bladder contraction and gastric
emptying(7, 8) .
NPY and related family members are
proposed to activate at least five receptor
subtypes(1, 2) : 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 >
[Leu
,Pro
]NPY > NPY; and 5) the
putative hypothalamic Y1-like feeding receptor is activated by NPY,
PYY, [Leu
,Pro
]NPY, and
NPY
> COOH-terminal fragments. Only the Y1
subtype was reported previously to be
cloned(9, 10, 11, 12, 13) .
We describe here the expression cloning and pharmacological
characterization of a human hippocampal Y2 receptor.
In order to clone a human NPY/PYY Y2 receptor subtype, we used an expression cloning strategy in COS-7 cells(23, 24, 25) . Because the Y2 receptor is described as a presynaptic receptor, it is difficult to locate cell bodies that actually contain this specific mRNA in restricted brain areas. We reasoned that human hippocampus was a good source of mRNA, because it contains both a large number of interneurons and has been shown to carry a particularly dense population of Y2 receptors(26, 27, 28, 29) . A single clone encoding a Y2 receptor (designated hhY2) was isolated from a human hippocampal cDNA library (see ``Materials and Methods''). The isolated clone carries a 4.2-kb cDNA. This cDNA contains an open reading frame between nucleotides 1003 and 2145 that encodes a 381 amino acid protein. The flanking sequence around the putative initiation codon conforms to the Kozak consensus sequence for optimal translation initiation(30, 31) . The hydrophobicity plot displayed seven hydrophobic, putative membrane-spanning regions which make the human hippocampal Y2 receptor a member of the G protein-coupled superfamily. The deduced hhY2 amino acid sequence and an alignment with the human Y1 and Y4 receptors (see accompanying paper by Bard et al.(46) ) are shown in Fig. 1. The hhY2 receptor presents features common to most members of the GPCR family(32, 33) , including a potential N-linked glycosylation site and multiple potential phosphorylation sites in the putative intracellular loop regions. In addition, it carries transmembrane amino acid residues highly conserved within the neuropeptide receptor family(34) . Interestingly, the two amino acid residues ``RY'' downstream of transmembrane domain 3, found in almost all known GPCR sequences(32) , are replaced by ``RH'' in all three Y1, Y2, and Y4 receptor amino acid sequences (Fig. 1). As seen in Fig. 1, the hhY2 amino acid sequence shows a surprisingly low overall identity of 31% with the Y1 receptor. The alignment scores for the TM domains are 41 and 43% identity with the Y1 and the Y4 receptors, respectively. When compared with other neuropeptide GPCR sequences, the amino acid transmembrane domains identities are very low, ranging from 26% (human bradykinin(2, 35) ) to 33% (human neurokinin 1 and PR4, a Y2-like Drosophila receptor(36, 37) ) with the notable exception of the orphan sequence MUSGIR score at 43%(38) . Using the human Y2 probe, Northern hybridizations reveal a unique band at 4.3 kb in human brain after a 3-day exposure (Fig. 2A). This is in good agreement with the 4.2-kb cDNA that we isolated by expression cloning and indicates that our cDNA clone is full length or nearly full length. The mRNA encoding the human Y2 receptor subtype is present in significant amounts in amygdala, corpus callosum, hippocampus, and subthalamic nucleus. A faint band is detectable in caudate nucleus, hypothalamus, and substantia nigra. No signal could be detected in thalamus. It should be noted that the Clontech brain MTN blot does not contain any mRNA from cortex or brain stem. No detectable signal was observed on Northern blots containing mRNA extracted from human peripheral tissues after an 8-day exposure (data not shown). Southern hybridizations to human genomic DNA followed by high stringency washes (Fig. 2B) suggest that the human genome contains a single Y2 receptor gene (single band with EcoRI, HindIII, BamHI, and PstI). The faint bands at 9 and 12 kb observed with BglII can be explained by the presence of two BglII restriction sites in the coding region of the Y2 sequence and are also consistent with a single Y2 receptor gene.
Figure 1: Alignment of the deduced amino acid sequence of the human hippocampal Y2 receptor (GenBank(TM) U36269) with the human Y1 and Y4 receptor sequences. The Pile Up algorithm (Genetics Computer Group) was used for the alignment with the human Y1 receptor (9, 10) and the human Y4 receptor (see accompanying manuscript(46) ). Putative transmembrane domains are boxed and numbered I-VII. Residues identical between the Y1, Y2, and Y4 receptors are shaded in gray.
Figure 2: Northern and Southern blot analysis. A, Northern blot of human mRNA (2 µg/lane) from six different regions of the brain hybridized at high stringency. A 3-day exposure is shown. B, Southern blot of human DNA (8 µg/lane) hybridized at high stringency. An 18-h exposure is shown (see ``Materials and Methods'' for details).
Characterization of the novel receptor cloned from pool 189
was accomplished using radioligand binding and functional assays. I-PYY (0.06 nM) bound specifically to membranes
from hhY2-transfected COS-7 cells (but not from mock-transfected cells)
at 30 °C. The association curve was biphasic, with approximately
55% of the specific binding following a rapid time course (k
= 1.28 ± 0.02
min
, t
= 0.5 min) and
45% following a slower time course (k
=
0.02 ± 0.00 min
, t
= 37 min). Equilibrium binding composed of both phases was
95% complete within 120 min and 100% complete within 240 min. The
biphasic time course suggests the possibility of multiple conformations
for the receptor/ligand complex. hY1-transfected COS-7 cell membranes,
when studied under the same conditions, yielded a monophasic
association curve with 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 hY2 receptors were conducted for 120 min.
I-PYY binding to the transiently expressed hhY2 receptor
was specific and saturable at
I-PYY concentrations
ranging from 0.5 pM to 3.0 nM. Binding data were fit
to a one-site model with an apparent pK
=
10.17 ± 0.05 (0.067 nM) and B
= 7.7 ± 0.7 pmol/mg membrane protein (n = 5). 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).
hhY2 bound human PP family members in I-PYY membrane binding assays in rank order (Table 1): PYY
NPY PP. pK
values were
in close range for parent compounds and their COOH-terminal fragments.
Noteworthy ligands include PYY
, a major form of
PYY-like immunoreactivity in human plasma(39) , and also
C2-NPY, a Cys
to Cys
disulfide-stabilized
derivative with an 8-amino octanoic linker replacing
NPY
(40) . NPY was rendered inactive by
hydrolysis of the carboxyl-terminal amide to NPY free acid.
[Pro
] was also disruptive, as exemplified by
pK
values for
[Leu
,Pro
]NPY,
[Pro
]PYY, and PP. An increase in binding
affinity resulted when the COOH terminus of human PP was modified to
more closely resemble human NPY, as in
[Ile
,Gln
]PP. These data indicate a
strong interaction between hhY2 and COOH-terminal peptide residues of
PYY and NPY. Points of contrast between hhY2 and hY1 structure/affinity
profiles include 1) a relative insensitivity of hhY2 to
NH
-terminal peptide deletion and 2) a disruptive effect of
Pro
on hhY2 binding affinity. This binding profile
demonstrates that the newly isolated clone encodes a human Y2 receptor.
Native Y2 receptors are coupled to three second messenger pathways
in the human neuroblastoma cell line SMS-KAN: cAMP, intracellular
Ca mobilization, and K
-induced
Ca
influx through
-conotoxin-sensitive
channels(41) . Two of these pathways, cAMP and Ca
mobilization, were evaluated in hhY2 stably transfected 293
cells. Incubation of intact 293 cells with 10 µM forskolin
generated an average 12-fold increase in [cAMP] (n = 10). Simultaneous incubation with human PYY decreased the
forskolin-stimulated [cAMP] with an E
of 52 ± 5% and with high potency (pEC
= 9.47 ± 0.15, n = 10) in 293 cells
stably transfected with hY2 (Fig. 3A), but not in
untransfected cells. The inhibitory response was mimicked by NPY and
COOH-terminal fragments, whereas Pro
analogs were
relatively inactive (Table 1); pEC
values were in
close range of pK
values derived from
I-PYY binding to COS-7 cell membranes. These results are
in agreement with data from SMS-KAN cells and further establish cAMP
reduction as a common signaling pathway for the entire Y-type receptor
family (1, 39; see also Bard et al.(46) ).
Intracellular free [Ca
] was increased by 1
µM human PYY in 293 cells stably transfected with hY2
(
[Ca
]
= 50 ±
7 nM, n = 11), but not in untransfected cells.
The Ca
transient was detected within 30 s of PYY
application (Fig. 3B). In comparison, Y2 receptors
inhibited bradykinin and angiotensin II-induced Ca
flux in SMS-KAN cells(39) . These data reveal a
complexity in Ca
signaling that can be further
evaluated by studying hhY2 and species homologs in a variety of
transfected cells.
Figure 3:
Signaling events in intact 293 cells
stably transfected with hY2. A, inhibition of
forskolin-stimulated [cAMP]. Data shown are representative of
five or more independent experiments. B, stimulation of
intracellular free [Ca]. Human PYY (1
µM) was added at the time indicated by the arrow.
Data shown were selected from 11 recordings made in three independent
experiments.
This is the first report of a Y2 receptor clone
whose distribution and pharmacology support the potential for in
vivo activation by NPY and PYY or fragments. A previous report
described the cloning of a Y2-like receptor (PR4) from Drosophila and functional characterization in oocytes(37) . PR4 was
activated by mammalian peptides (PYY, NPY, C2-NPY,
[Pro]NPY, and PP) at concentrations between 0.03
and 3 µM with a Y2-like rank order, but functional
invertebrate ligands were not identified. As there have been no
published reports of an NPY analog in Drosophila,
classification of PR4 as Y2-like could reasonably be viewed as
tentative.
In summary, we have cloned the gene for a novel human
hippocampal Y-type receptor and generated a pharmacological profile
using NPY and related peptide family members. hhY2 closely resembles
pharmacologically defined Y2 receptors in a variety of cell and tissue
models(1) . Y2 receptors were first proposed to exist in rat
vas deferens, for example, based on the ability of both NPY and
NPY to block field-stimulated contraction through
presynaptic inhibition of norepinephrine release(42) . Y2
receptors in hippocampus exhibit higher binding affinity for
[Ile
,Gln
]PP than for PP and
[Leu
,Pro
]NPY (43) .
Hippocampal Y2 receptors are proposed to play a role in memory based on
behavioral effects induced by NPY and fragments(44) . Other
functions (e.g. analgesia and antisecretory effects) are
indicated by widespread distribution of Y2 receptors in dorsal root
ganglia, intestinal enterocytes, and
elsewhere(1, 2, 45) . As such, the hhY2
receptor is likely to be involved in a number of physiological
processes that can be further studied using selective agonists and
antagonists.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U36269[GenBank].