(Received for publication, July 11, 1995; and in revised form, July 28, 1995)
From the
Neuropeptide Y (NPY) is a 36-amino acid polypeptide that is
widely distributed in the central nervous system and periphery.
Pharmacological studies have suggested that there are at least three
receptor subtypes, Y1, Y2, and Y3. Cloning of the Y1 subtype has been
reported previously. Here we report the isolation by expression cloning
of a cDNA encoding a human NPY receptor displaying a pharmacology
typical of a Y2 receptor. COS-7 cells transfected with the cDNA express
high affinity binding sites for NPY, peptide YY, and
NPY, whereas
[Leu
,Pro
]NPY binds with lower
affinity. The receptor is 381 amino acids in length and has seven
putative transmembrane regions typical of G-protein-coupled receptors.
Comparison of the amino acid sequence of this Y2 receptor to that of
the human Y1 receptor indicates that the two receptors are 31%
identical at the amino acid level. Northern blot analyses reveal a
single 4-kilobase mRNA species and indicate that the messenger RNA is
present in many areas of the central nervous system. NPY induced
calcium mobilization and inhibited forskolin-stimulated cAMP
accumulation in Chinese hamster ovary cells that stably express the Y2
receptor cDNA, indicating that the recombinant Y2 receptor is
functionally coupled to second messenger systems.
Neuropeptide Y (NPY) ()is a 36-amino acid peptide
amide that is widely distributed in the central and peripheral nervous
systems. It belongs to a family of homologous peptides including the
gut peptide YY (PYY) and pancreatic polypeptide. NPY has been highly
conserved throughout evolution and is therefore thought to be an
important hormone/neurotransmitter. Centrally its effects include blood
pressure regulation, memory enhancement, anxiolysis/sedation, and
increased food intake, and in the periphery it affects vascular and
other smooth muscle activity, intestinal electrolyte secretion, and
urinary sodium excretion(1, 2) .
Heterogeneity
among NPY receptors has been observed. Based on the rank order of
potency of NPY and related peptides to displace I-NPY
binding, NPY receptors have been classified into at least three
receptor subtypes, Y1, Y2, and Y3(3) . Both the Y1 and Y2
subtypes have high affinity for NPY and PYY. The Y1 subtype has a high
affinity for the NPY analog
[Leu
,Pro
]NPY and low affinity for
C-terminal fragments of NPY such as NPY
. In
contrast, the Y2 receptor subtype has high affinity for
NPY
and low affinity for
[Leu
,Pro
]NPY. The Y3 subtype has a
low affinity for PYY. The existence of a fourth receptor, Y1a or Y4,
that is important in the feeding response has been hypothesized (2) .
The NPY Y1 receptor has been cloned from rat
forebrain(4) , human fetal brain(5) , bovine
hypothalamus, ()and murine genomic DNA(6) . It
belongs to the superfamily of G-protein-coupled receptors and appears
to couple to more than one second messenger
systems(5, 7) . To date the cloning of a Y2 receptor
has not been reported. Attempts to isolate a Y2 cDNA using homology to
the Y1 cDNA have not been successful. Here we report the isolation by
expression cloning (8) of a cDNA encoding a human NPY receptor
displaying a pharmacology typical of a Y2 receptor. The clone has been
functionally expressed in CHO cells where its activation causes
mobilization of calcium and inhibition of forskolin-stimulated cAMP
accumulation.
The CHO cells stably expressing the NPY receptor
(CHO-hY2) in a 24-well tissue culture plate (5 10
cells/well) were incubated in 250 µl of binding buffer C
(Dulbecco's modified Eagle's medium containing 0.1% BSA)
with 100 pM
I-PYY, with or without competitor,
at room temperature for 1 h. The cells were then washed twice with 0.5
ml of ice-cold buffer C, suspended in 0.5 M NaOH, transferred
to a counting tube, and counted in a
counter. The competition
binding and saturation binding data were analyzed by logistic.fit and
hyperbola.fit programs of Sigma Plot, respectively.
Neuropeptide Y is a peptide hormone with diverse and
important physiological effects in the central nervous system and
periphery that are mediated through multiple receptor subtypes. In
order to gain insight into the molecular mechanisms associated with
these effects, we undertook the cloning of a human NPY Y2 receptor. A
plasmid cDNA expression library was prepared from poly(A) RNA isolated from SMS-KAN cells, a human neuroblastoma cell line
that expresses high levels of Y2 receptor(14) . Plasmid library
DNA was transfected into COS-7 cells that are null for NPY binding; the
cells were assayed for NPY receptor expression by
I-PYY
binding. Detection was by autoradiography. A single pool (pool 398) was
identified that had increased levels of
I-PYY binding.
This pool was subdivided and the process was repeated until a pure cDNA
clone (pNPYR-398) was isolated.
Binding of I-NPY and
I-PYY to COS-7 cells transfected with pNPYR-398 was
saturable and high affinity, with K
values of 0.58
and 0.27 nM, respectively. Specific binding to COS-7 cells
transfected with vector (pcDNA1) was negligible. Scatchard analysis
suggests that
I-NPY bound to a single class of binding
sites in COS-7 cells transfected with pNPYR-398 cDNA (data not shown).
To characterize the pharmacology of the NPY receptor encoded by
pNPYR-398 cDNA, I-NPY (100 pM) binding to COS-7
cells transfected with the recombinant plasmid was competed with
various NPY analogs (Fig. 1). PYY, NPY, and NPY
were the most effective competitors of
I-NPY
binding, followed by the Y2-specific agonist NPY
(Table 1). The Y1-specific agonist
[Leu
,Pro
]NPY inhibited
I-NPY binding only at high concentration with an
IC
of 0.5 µM.
[D-
Trp]NPY, a competitive antagonist of
NPY in rat hypothalamus(15) , inhibited
I-NPY
binding with an IC
of 2.9 µM. The rank order
of potency of NPY and related peptides to COS-7 cells expressing the
NPY receptor cDNA is PYY
NPY
NPY
>
NPY
> NPY
[Leu
,Pro
]NPY >
[D-Trp
]NPY. To further confirm the
characteristics of pNPYR-398 NPY receptor, the potency of the NPY
analogs to this receptor in competing for
I-PYY to COS-7
cells transfected with pNPYR-398 was studied. The results were similar
to those described for
I-NPY binding (Table 1).
These data are consistent with the previously reported pharmacology for
the Y2 subtype of NPY receptors(3) .
Figure 1:
Pharmacological
characterization of a human NPY receptor expressed in COS-7 cells.
Displacement of specific binding of I-NPY to membranes
from COS-7 cells transfected with pNPYR-398. Binding was determined in
the presence of the indicated concentrations of PYY (
), NPY
(
), NPY
(
), NPY
(
), [Leu
,Pro
]NPY
(
), and [D-
Trp]NPY
(
).
Fig. 2shows the nucleotide sequence of the pNPYR-398 cDNA and the deduced amino acid sequence for the encoded NPY Y2 receptor. The nucleotide sequence surrounding the initiation codon agrees well with Kozak's consensus sequences(16) . The receptor is 381 amino acids in length and has a molecular mass of approximately 42 kDa. Hydrophobicity analyses determined according to the method of Kyte and Doolittle (17) suggest the existence of seven transmembrane-spanning regions. There is a single putative N-linked glycosylation site in the N-terminal region. There is an aspartic acid in the putative second transmembrane region that is conserved in many G-protein-coupled receptors and frequently involved with signal transduction(18) . In the C-terminal tail there is a cysteine residue that might be involved in palmitolyation (19) as well as several serine residues that might be involved in regulatory phosphorylation. A long polyadenylation signal was found at the 3` end of the clone, suggesting that the cDNA clone encompasses most of the mRNA species observed in the Northern blot (Fig. 3).
Figure 2: Nucleic acid and amino acid sequence of a human Y2 receptor. The nucleotide sequence of clone pNPYR-398 and the deduced amino acid sequence for the encoded human NPY receptor, subtype 2 (Y2). The deduced amino acid sequence is shown under the nucleic acid sequence. The putative transmembrane regions I-VII are underlined; the borders of the regions were assigned based on hydrophobicity profile and comparison to the Y1 sequence. The asterisk (*) indicates a potential N-glycosylation site; the ``+'' indicates a potential palmitolyation site.
Figure 3:
Northern hybridization. Northern blot
analysis of human brain sections probed with a 1.9-kb fragment of
pNPYR-398 cDNA that contained the entire open reading frame of the Y2
receptor. A, lane1, amygdala; lane2, caudate nucleus; lane3, corpus
callosum; lane4, hippocampus; lane5, hypothalamus; lane 6, substantia nigra; lane7, subthalamic nucleus, lane 8,
thalamus. B, lane1, cerebellum; lane2, cortex; lane 3, medulla; lane4, spinal cord; lane 5,
occipital pole; lane 6, frontal lobe; lane7, temporal lobe; lane8, putamen.
Molecular size markers are indicated in kb. The blots were also probed
with P-labeled actin cDNA as a
standard.
Comparison of the amino acid sequence of this NPY receptor to that of the human Y1 receptor (5) reveals significant differences. Overall, the two receptors are 31% identical at the amino acid level. Their sequences show the greatest similarity in the first intracellular loop and the second and sixth transmembrane regions, being 63%, 61%, and 50% identical, respectively. On the nucleotide level, the coding regions of the two NPY receptors were 47% identical. This relatively low homology to the Y1 receptor may explain why cloning with Y1 probes has not been fruitful. A search of the GenBank(TM) data base revealed that the Y2 nucleic acid sequence showed some similarity to the NK-2R neurokinin A receptor and two putative opioid receptors(20, 21, 22) .
The Y2 receptor is
believed to be a common presynaptic receptor (2) and to be the
predominant NPY receptor in the brain(23) . To study the
distribution of the mRNA corresponding to the Y2 receptor encoded by
pNPYR-398, Northern blots of poly(A) RNA from several
parts of the brain were hybridized with a 1.9-kb fragment of the Y2
cDNA that contained the entire receptor open reading frame. A single
4-kb mRNA transcript was detected in the amygdala, caudate nucleus,
corpus callosum, hippocampus, hypothalamus, and subthalamic nucleus (Fig. 3A), cortex, medulla, occipital pole, and frontal
lobe (Fig. 3B). No hybridization was detected in RNA
from peripheral tissues (data not shown). This suggests that either
another form of the Y2 receptor is found in the periphery or that the
Y2 mRNA transcript is present at levels undetectable by Northern
analysis. In situ hybridization studies are in progress to
address this question.
Southern blot analysis to human genomic DNA (Fig. 4) suggests that there is a single Y2 receptor gene. Since the Y2 receptor is known to be expressed in the periphery (1) these data support the hypothesis that either there is another form of the Y2 receptor in the periphery that is very different from the described here or the Y2 mRNA is present at very low levels.
Figure 4: Southern hybridization. Southern blot of human genomic DNA probed with the 1.9-kb fragment of Y2 receptor cDNA described in Fig. 3and washed under high stringency conditions. Each lane has approximately 12 µg of DNA. Molecular size markers are indicated in kb.
The truncated fragment of the pNPYR-398 clone containing the Y2 open
reading frame described above was subcloned into pcDNA3 and transfected
into CHO cells for stable expression. Competition binding experiments
demonstrated a pharmacology identical to that seen in the COS-7 cells (Table 1). In neuroblastoma cells, the Y2 receptor appears to
couple to multiple second messenger systems including the inhibition of
forskolin-stimulated cAMP accumulation and calcium
mobilization(24, 25) . To examine the functional
coupling of the recombinant Y2 receptor to second messenger systems,
the effect of NPY, [Leu,Pro
]NPY,
and NPY
on cAMP and calcium mobilization was
studied in the CHO-hY2 cells. Activation with 100 nM NPY,
[Leu
,Pro
]NPY, and
NPY
inhibited forskolin (10 µM)
stimulated cAMP accumulation by 72%, 7%, and 71%, respectively (Fig. 5A). When the cells were pretreated with
pertussis toxin (100 ng/ml), the peptides did not inhibit the
accumulation of forskolin-stimulated cAMP, suggesting that the Y2
receptor couples to G
. In addition, NPY did not
inhibit the accumulation of forskolin-stimulated cAMP in CHO cells
transfected with the pcDNA3 vector (data not shown).
Figure 5:
Functional coupling of the recombinant Y2
receptor to second messenger systems. A, inhibition of
forskolin (10 µM)-stimulated cAMP accumulation by NPY
analogs in CHO-hY2 cells: lane 1, vehicle; lane 2,
forskolin (10 µM); lane 3, NPY; lane 4,
[Leu,Pro
]NPY; lane 5,
NPY
, (lanes 3-5, peptides at 1
µM); lane 6, forskolin plus NPY; lane 7,
forskolin plus [Leu
,Pro
]NPY; lane 8, forskolin plus NPY
(lanes
6-8, forskolin at 10 µM and peptides at 100
nM). The cAMP values are expressed as a percentage of the cAMP
level observed in the presence of forskolin (100%). B. Calcium
mobilization by NPY analogs in CHO-hY2 cells: lane1,
NPY
(1 µM); lane2,
NPY
(1 µM); lane3, MCP-1 (5 µM); lane 4, galanin (5
µM). Three independent assays were performed for each
ligand tested. The data represent an average of the two best
experiments.
Activation of
the CHO-hY2 cells with the NPY analogs NPY (1
µM) and NPY
(1 µM)
resulted in a 13-14-fold increase in intracellular calcium (Fig. 5B). Neither MCP-1 (5 µM) or galanin
(5 µM) elicited a change in intracellular calcium levels.
Although calcium mobilization is frequently associated with
G-protein-coupled receptors linked to phospholipase C(26) ,
this is not necessarily the case(27) . The specific G-protein
mediating the increase of intracellular calcium described here is not
known at this time.
In summary, while NPY appears to be an important mediator of a wide variety of physiological functions, a clear understanding of how these functions are mediated has been hampered by the lack of cloned NPY receptors. The present cloning and pharmacological characterization of pNPYR-398 that encodes a human NPY Y2 receptor should facilitate the elucidation of the structure, function, regulation, and diversity of neuropeptide Y receptors as well as the identification of Y2 receptor-specific antagonists.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U32500[GenBank].