Tyrosine 220 in the 5th Transmembrane Domain of the Neuromedin B
Receptor Is Critical for the High Selectivity of the Peptoid Antagonist
PD168368*
Kenji
Tokita
,
Simon J.
Hocart§,
Tatsuro
Katsuno
,
Samuel A.
Mantey
,
David H.
Coy§, and
Robert T.
Jensen
¶
From the
Digestive Diseases Branch, NIDDK, National
Institutes of Health, Bethesda, Maryland 20892-1804 and the
§ Department of Medicine, Peptide Research Laboratories,
Tulane University Health Sciences Center,
New Orleans, Louisiana 70112
Received for publication, July 10, 2000, and in revised form, September 22, 2000
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ABSTRACT |
Peptoid antagonists are increasingly being
described for G protein-coupled receptors; however, little is known
about the molecular basis of their binding. Recently, the peptoid
PD168368 was found to be a potent selective neuromedin B receptor
(NMBR) antagonist. To investigate the molecular basis for its
selectivity for the NMBR over the closely related receptor for
gastrin-releasing peptide (GRPR), we used a chimeric receptor approach
and a site-directed mutagenesis approach. Mutated receptors were
transiently expressed in Balb 3T3. The extracellular domains of the
NMBR were not important for the selectivity of PD168368. However,
substitution of the 5th upper transmembrane domain (uTM5) of the NMBR
by the comparable GRPR domains decreased the affinity 16-fold. When the
reverse study was performed by substituting the uTM5 of NMBR into the GRPR, a 9-fold increase in affinity occurred. Each of the 4 amino acids
that differed between NMBR and GRPR in the uTM5 region were exchanged,
but only the substitution of Phe220 for Tyr in the
NMBR caused a decrease in affinity. When the reverse study was
performed to attempt to demonstrate a gain of affinity in the GRPR, the
substitution of Tyr219 for Phe caused an increase in
affinity. These results suggest that the hydroxyl group of
Tyr220 in uTM5 of NMBR plays a critical role for high
selectivity of PD168368 for NMBR over GRPR. Receptor and ligand
modeling suggests that the hydroxyl of the Tyr220 interacts
with nitrophenyl group of PD168368 likely primarily by hydrogen
bonding. This result shows the selectivity of the peptoid PD168368,
similar to that reported for numerous non-peptide analogues with other
G protein-coupled receptors, is primarily dependent on interaction with
transmembrane amino acids.
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INTRODUCTION |
Recently the "peptoid" approach was described for the design
of low molecular weight, nonpeptide ligands (peptoid), using the
chemical structure of mammalian neuropeptides as a starting point (1,
2). These peptoids may act as either agonists or antagonists at
neuropeptide receptors. To date, several classes of peptoid antagonists
for receptors of gastrointestinal
(GI)1
hormones/neurotransmitters have been described including for cholecystokinin (3-5), somatostatin (6), tachykinins (7-9), or
bombesin (10) receptors. They have been proven useful in helping to
examine the role of these receptors in mediating various physiological
and pathophysiological processes and they may be useful as therapeutic
agents in such conditions as panic attacks (1, 2, 5). Whereas there
have been a number of studies of the molecular basis of action of
nonpeptide antagonists for various GI hormone/neurotransmitter
receptors (11-15) within the heptahelical G protein-coupled receptors
(GPCRs), almost nothing is known about the molecular basis of action
for peptoid antagonists. For most small molecule ligands for GPCRs such
as nonpeptide antagonists, transmembrane regions play an important role
in determining the high affinity binding (16, 17). However, the
essential receptor domains for the high selectivity of peptoid binding
are still unclear.
Neuromedin B (NMB) and gastrin-releasing peptide (GRP), mammalian
homologues of the amphibian tetradecapeptide bombesin, are small
amidated peptides with structurally related carboxyl termini (18).
These peptides mediate a spectrum of biological activities by binding
to two structurally and pharmacologically distinct receptors, the NMB
receptor (NMBR) and GRP receptor (GRPR) (19, 20). These peptides have
important effects in the central nervous system including
thermoregulation (21), satiety (22), control of circadian rhythm (23),
and in peripheral tissues causing stimulation of gastrointestinal
hormone release (18, 24, 25), activation of macrophages (26), and
effects on development (27, 28). These peptides also have potent growth
effects causing proliferation of normal cells (18, 29) and various
tumor cell lines (18, 30). The NMBR and GRPR are members of the
bombesin receptor family within the GPCR superfamily and share ~50%
overall amino acid sequence identity (19, 20). Both the NMBR and GRPR are widely distributed in the central nervous system and alimentary tract (18). Although the potential physiological role of GRP and its
receptor has been a major focus of research (18, 20), the role of NMB
in physiological or pathophysiological processes has received much less
attention. Some studies suggest that NMB may play an important role in
a number of biological processes, including causing growth of some
tumor cells (31), a modulatory role in suppression of feeding behavior
or gastric emptying (32), control of the
hypothalamic-pituitary-adrenocortical axis and thyrotropin release
(33), sensory transmission in the spinal cord (34), excitation of
serotonin neurons in the dorsal raphe nucleus (35), control of
potassium secretion by the blood-brain barrier (36), and smooth muscle
contractility (37). However, which of these are physiological or the
principal roles of NMBR activation in pathological processes remains
unclear. The development of selective, high affinity NMBR agonists and
antagonists would enable a more precise definition of the role of NMB
in these processes.
In contrast to GRPR antagonists for which numerous classes of high
affinity antagonists have been described (38), the discovery process
for NMBR antagonists has been slower. None of the strategies successfully used previously yielded potent antagonists when applied to
the NMBR (38, 39). However, a peptoid based on a 3-amino acid template,
PD165929, was recently characterized as a high affinity NMBR antagonist
(10). In a previous study, we evaluated the pharmacology of a second
generation peptoid from this series, PD168368 (40, 41). The results
confirmed that PD168368 was a potent and selective antagonist for NMBR
over GRPR regardless of species origin, which could prove generally
useful in understanding the role of NMBR in physiological and
pathological processes.
To attempt to provide insight into the molecular basis of the
specificity of the peptoid PD168368 for the NMBR receptor, in the
present study we have investigated in detail the high affinity of
PD168368 for the NMBR, and its selectivity for the NMBR over the GRPR
using a chimeric and mutagenesis approach. To identify which domains of
the NMBR are important for high affinity binding of PD168368, we used a
chimeric receptor approach, which has proven useful in elucidating the
structural basis of GPCR interaction with ligands (42). A site-directed
mutagenesis approach was then used to identify critical amino acid(s)
within these domains. Here we report the peptoid antagonist PD168368's
selectivity for the NMBR over the GRPR depends primarily on an
interaction with amino acids in the 5th upper transmembrane region of
the NMBR. Detailed site-directed mutagenesis studies demonstrate that
Tyr220 in this region is a key amino acid for high affinity
PD168368 binding. Computer modeling of the receptor and analysis of the ligand support the conclusion that PD168368 interacts with the hydroxyl
group of Tyr220 principally through hydrogen bonding.
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EXPERIMENTAL PROCEDURES |
Materials--
pcDNA3 was from Invitrogen (Carlsbad, CA).
Oligonucleotides were from Midland Certified Reagent Company (Midland,
TX) and Life Technologies, Inc. SeamlessTM cloning kit and
QuikChangeTM site-directed mutagenesis kit were from Stratagene (La
Jolla, CA). Restriction endonucleases (HindIII,
XbaI, and EcoRI), fetal bovine serum (FBS),
penicillin-streptomycin, LipofectAMINETM reagent, LipofectAMINETM
Plus reagent, and trypsin-EDTA (0.05% trypsin, 0.53 mM
EDTA-4Na) were from Life Technologies, Inc. Dulbecco's modified
Eagle's medium (DMEM) and Dulbecco's phosphate-buffered saline were
from Biofluids, Inc. (Rockville, MD). Balb 3T3 cells were from American
Type Culture Collection (Rockville, MD). A 100 × 20-mm tissue
culture dish (Falcon® 3003) was from Becton Dickinson
(Plymouth, United Kingdom). Bombesin (Bn) was from Peninsula
Laboratories, Inc. (Belmont, CA). Na125I (2,200 Ci/mmol)
was from Amersham Pharmacia Biotech.
1,3,4,6-Tetrachloro-3
,6
-diphenylglycouril (IODO-GEN®) and dithiothreitol were from Pierce. Bovine
serum albumin fraction V and HEPES were from ICN Pharmaceutical Inc.
(Aurora, OH). Soybean trypsin inhibitor type I-S and bacitracin were
from Sigma. Nyosil M20 oil (specific gravity 1.0337) was from Nye
Lubricants Inc. (New Bedford, MA). PD168368 was a gift from Robert
Pinnock (Medicinal Chemistry Department, Pfizer Global Research,
Cambridge, United Kingdom). All other chemicals were of the highest
purity commercially available.
Construction of Chimeric and Mutant Receptors--
The cDNAs
of the mouse GRPR and rat NMBR were identical to those described
previously (42). The receptor extracellular domains or upper
transmembrane domains used to make chimeric receptors were those
identified using hydropathy plots for the GRPR and for the NMBR. The
amino acids in these regions of the two receptors were aligned using
the Wisconsin Package (Version 9.1; Genetics Computer Group, Madison,
WI) for comparisons. The cDNA of the wild-type mouse GRPR was
inserted between the HindIII site and XbaI site
of pcDNA3, and the wild-type rat NMBR was inserted into the
EcoR-I site of pcDNA3. Both the GRPR/NMBR
extracellular domain chimeras and upper transmembrane chimeras were
constructed using the SeamlessTM cloning kit. Mutant receptors were
made by using the QuikChangeTM site-directed mutagenesis kit,
following the manufacturer's instructions except that the annealing
temperature was 60 °C and the DpnI digestion was for
2 h. Nucleotide sequence analysis of the entire coding region was
performed using an automated DNA sequencer (ABI PrismTM 377 DNA
sequencer; Applied Biosystems Inc., Foster City, CA).
Cell Transfection--
Balb 3T3 cells were seeded in a 10-cm
diameter tissue culture dish at a density of 106 cells/dish
and grown overnight at 37 °C in DMEM supplemented with 10% (v/v)
FBS, 100 units/ml penicillin, and 100 mg/ml streptomycin. The following
morning cells were transfected with 5 µg of plasmid DNA by cationic
lipid-mediated method using 30 µl of LipofectAMINETM reagent and 20 µl of LipofectAMINETM Plus reagent in serum-free DMEM for 3 h
at 37 °C. At the end of the incubation period, the medium was
replaced with DMEM supplemented with 10% (v/v) FBS, 100 units/ml
penicillin, and 100 mg/ml streptomycin. Cells were maintained at
37 °C, with a 5% CO2 atmosphere and were used 48 h
later for binding assays.
Preparation of 125I-[Tyr4]Bn and
125I-[D-Tyr6,
-Ala11,Phe13,Nle14]Bn-(6-14)--
125I-[Tyr4]Bn
and
125I-[D-Tyr6,
-Ala11,Phe13,Nle14]Bn-(6-14)
at a specific activity of 2,200 Ci/mmol were prepared by a modification
of the methods described previously (39, 40, 43). Briefly, 0.8 µg of
IODO-GEN in chloroform was transferred to a vial, dried under a stream
of nitrogen and washed with 100 µl of KH2PO4
(pH 7.4). To this vial, 20 µl of 0.5 M
KH2PO4 (pH 7.4), 8 µg of peptide in 4 µl of
water, and 2 mCi (20 µl) of Na125I were added, mixed
gently and incubated at room temperature for 6 min. The incubation was
stopped by the addition of 100 µl of distilled water and in the case
of 125I-[Tyr4]Bn, 300 µl of 1.5 M dithiothreitol was also added to reduce the oxidized
methionines. The iodination mixture was re-incubated at 80 °C for 60 min. The reaction mixtures were applied to a Sep-Pak (Waters
Associates, Milford, MA), and free 125I was eluted with 5 ml of water followed by 5 ml of 0.1% (v/v) trifluoroacetic acid. The
radiolabeled peptides were eluted with 200 µl of sequential elutions
(×10) with 60% acetonitrile in 0.1% trifluoroacetic acid. The two or
three fractions with the highest radioactivity were combined and
purified on a reverse-phase, high performance liquid chromatography
with a µBondaPak column (0.46 × 25 cm). The column was eluted
with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid
(v/v) from 16-60% acetonitrile in 60 min, and 1-ml fractions were
collected and checked for radioactivity and receptor binding. The pH of
the pooled fractions were adjusted to 7 using 0.2 M Tris
(pH 9.5), and radioligands were stored in aliquots with 0.5% bovine
serum albumin at
20 °C.
Whole Cell Radioligand Binding Assays--
Competitive binding
assays were performed 48 h after transfection as described
previously (43, 44). Briefly, disaggregated transiently transfected
cells were incubated for 1 h at room temperature in 250 µl of
binding buffer (pH 7.4) with the ligands
125I-[Tyr4]Bn (2,200 Ci/mmol) or
125I-[D-Tyr6,
-Ala11,Phe13,Nle14]Bn-(6-14)
(2,200 Ci/mmol) in the presence of the indicated concentration of
unlabeled peptides. The binding buffer contained 98 mM
NaCl, 6 mM KCl, 11.5 mM glucose, 5 mM fumarate, 5 mM glutamate, 5 mM pyruvate, 24.5 mM HEPES, 0.2% (v/v) essential amino acid
solution, 2.5 mM KH2PO4, 1 mM MgCl2, 0.5 mM CaCl2,
0.2% (w/v) bovine serum albumin, 0.05% (w/v) bacitracin, and 0.01%
(w/v) soybean trypsin inhibitor. The cell concentration was adjusted to
0.15-9 × 106 cells/ml to assure that no more than
20% of the total added radioactive ligand bound. Bound tracer was then
separated from unbound tracer by layering 100 µl of the binding
reaction on top of an oil phase (100 µl of Nyosil M20, Nye Lubricants
Inc., New Bedford, MA) in a 0.4-ml microcentrifuge tube (PGC
Scientific, Frederick, MD), and pelleting the cells through the oil by
centrifugation at 10,000 × g in a Microfuge ETM
(Beckman, Palo Alto, CA) for 3 min. The supernatant was aspirated, and
the pelleted cells were rinsed twice with distilled water. The amount
of radioactivity bound to the cells was measured in a Cobra II
counter (Packard, Downers, IL). Aliquots (100 µl) of the incubation
mixture were taken in duplicate to determine the total radioactivity.
Binding was expressed as the percentage of total radioactivity that was
associated with the cell pellet. All binding values represented
saturable binding (i.e. total binding minus nonsaturable
binding). Nonsaturable binding was <15% of the total binding in all
experiments. Each point was measured in duplicate, and each experiment
was replicated at least three times. Calculation of IC50
values was performed with a curve-fitting program, KaleidaGraph
graphing software (Synergy Software, Reading, PA).
Visualization of PD168368--
In an attempt to visualize the
hydrogen bonding potential and likely hydrogen bonding donor and
acceptor sites of PD168368, we generated a model for the ligand in the
molecular modeling suite Sybyl 6.6 (Tripos Inc., St. Louis, MO). The
model was initially generated in a reasonable low energy conformation
using Concord (45, 46), a program widely used to generate
three-dimensional structures. This model was then optimized by energy
minimization using the Sybyl 6.6 Tripos force field with atomic charges
calculated by the method of Gasteiger-Hückel (47-49). The
molecular surface was visualized as a Connolly dot surface. The
molecular volume was 558 Å2.
Binding Site Model--
The coordinates of the NMB transmembrane
receptor model (NMBR_Vriend) were retrieved from the GPCR data base
server on the World Wide Web (50). In a attempt to better define
the NMBR binding pocket of PD168368, the receptor was modeled using the
-carbon template from bacteriorhodopsin, a seven-transmembrane receptor characterized by electron cryomicroscopy (51), the WHAT IF
program (52), and Sybyl 6.6 (Tripos, Inc.). Following optimization of
the model receptor transmembrane bundle by extensive energy
minimization using the Kollman all force field (51) in Sybyl 6.6, putative solvent accessible binding sites were explored using the
SiteID module in Sybyl 6.6. The surface of the solvent cluster filling
the binding site was visualized using the Molcad module of Sybyl.
 |
RESULTS |
Wild-type NMBR and GRPR--
The Bn-related natural occurring
agonists Bn and GRP (Fig. 1, Table I) had
high affinities (IC50 values
of 0.5 and 2 nM, respectively) for the GRPR, and NMB (Fig.
1, Table I) for the NMBR (IC50 = 1.0 nM). Bn
had a 3-fold and GRP an 11-fold selectivity for the GRPR, whereas NMB
had 100-fold selectivity for the NMBR (Table I). PD168368 (Fig. 1) had
96-fold lower affinity for the NMBR (IC50 = 96 ± 8 nM) than NMB (Table I). However, PD168368 also had a very
low affinity for the GRPR (IC50 = 3,500 ± 110 nM) with the result that PD168368 had a 36-fold selectivity
for the NMBR over the GRPR (Table I).

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Fig. 1.
Structure of bombesin, gastrin-releasing
peptide (GRP-(14-27)), NMB, and the peptoid antagonist,
PD168368.
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Table I
Comparison of binding affinities of Bn-related naturally occurring
peptides and the peptoid antagonist, PD168368, for wild-type GRPR and
NMBR
Balb 3T3 cells were transfected with either wild-type GRPR or NMBR and
incubated with 50 pM 125I-[Tyr4]Bn alone
or with increasing concentrations of the indicated unlabeled
peptides/peptoid. The concentration causing half-maximal inhibition of
binding, IC50, was determined by using the curve-fitting
program KaleidaGraph. Values are mean ± S.E. from four different
experiments and in each experiment each value was determined in
duplicate.
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Extracellular Chimeric Receptors--
To begin to explore the
molecular basis for this difference in affinity of PD168368 for NMBR
and GRPR, four chimeric receptors with the extracellular domains of
GRPR substituted for the comparable domains in NMBR (loss of affinity
chimeras, see Fig. 2) and four chimeras
with the extracellular domains of NMBR substituted into GRPR (gain of
affinity chimeras, see Fig. 3) were made.
The affinities of PD168368 for the NMBR chimeras in which the 1st, 2nd,
and 4th extracellular domain in the NMBR was substituted by the
comparable domain of GRPR were similar to wild-type NMBR (Fig. 2, Table
II). Substitution of the 3rd
extracellular domain in the NMBR by the comparable domain of the GRPR
decreased the affinity almost 2-fold from 96 ± 7.8 nM
to 180 ± 17 nM (Fig. 2, Table II). However, this represented only a small change compared with the 36-fold lower affinity PD168368 had for the wild type GRPR compared with the wild
type NMBR (Fig. 2, Tables I and II). To confirm these results, gain of
affinity chimeras were made by substituting in the GRPR, the comparable
extracellular domains of the NMBR (Fig. 3). The 3rd extracellular
domain substitution into the GRPR increased potency, causing a 3-fold
gain in affinity from 3,500 ± 110 nM to 1,100 ± 49 nM (Fig. 3, Table II). However, chimeras formed by the
insertions of the 1st, 2nd, or 4th extracellular loop of NMBR into GRPR
caused no change in affinity (Fig. 3, Table II).

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Fig. 2.
Importance of extracellular receptor domains
for determining PD168368 selectivity for NMBR over GRPR: loss of
affinity NMBR chimeras. The chimeric NMBRs were formed by
replacing each of the extracellular loops one at a time by the
comparable GRPR loop as shown in the diagram at the
top. The affinity was measured by competitive radioligand
displacement of 50 pM
125I-[Tyr4]Bn by PD168368 at the
concentrations shown. Each point on each dose-inhibition curve is the
mean from three separate experiments, and in each experiment each point
was measured in duplicate. e1-, e2-,
e3-, and e4-GRPR refer to substitution of this
extracellular loop of the GRPR for the comparable loop in the
NMBR.
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Fig. 3.
Importance of extracellular receptor domains
for determining PD168368 selectivity for NMBR over GRPR: gain of
affinity GRPR chimeras. The chimeric GRPR's were formed by
replacing each of the extracellular loops of GRPR by the comparable
loop of the NMBR one at a time as shown in the diagram. The
affinity was measured by competitive radioligand displacement of 50 pM 125I-[Tyr4]Bn by PD168368 at
the concentrations shown. Each point on the dose-inhibition curve is
the mean from three separate experiments, and in each experiment each
point was measured in duplicate. e1-, e2-,
e3-, and e4-NMBR refer to substitution of this
extracellular loop of the NMBR for the comparable loop in the
GRPR.
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Table II
Affinities of PD168368 for wild-type NMBR, loss of affinity NMBR, and
gain of affinity GRPR extracellular loop chimeric receptors and
wild-type GRPR
Affinities were calculated by competitive displacement of 50 pM 125I-[Tyr4]Bn by PD168368. Values
represent the mean ± S.E. from at least three independent
experiments.
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Upper Transmembrane Chimeric Receptors--
Because substitutions
of the extracellular domains caused only a small change in affinity of
PD168368, suggesting they were playing only a minor role in the
selectivity of PD168368 for the NMBR, we applied a similar chimeric
approach to the receptor's upper transmembrane regions, which are the
transmembranes areas most likely to interact with the ligand. The
affinities of potential loss of affinity NMBR chimeras in which 1st,
2nd, 3rd, 4th, and 7th upper transmembrane domain in the NMBR was
substituted by the comparable domain from GRPR showed no change in
affinity for PD168368 compared with the wild type NMBR (Fig.
4, Table
III). However, substitution of the 5th or
the 6th upper transmembrane domain of the NMBR by the comparable GRPR
domains decreased the affinity 16-fold (i.e. 96 ± 7.8 nM to 1,540 ± 150 nM) and 2-fold (96 ± 7.8 nM to 190 ± 9.4 nM), respectively
(Fig. 4, Table III). When the reverse study was performed to make
potential gain of affinity chimeras by substituting into the GRPR the
5th upper transmembrane domain of NMBR, a 9-fold increase in affinity
(3,500 ± 110 nM to 390 ± 36 nM)
occurred (Fig. 5, Table III). In
contrast, substitutions of the other upper transmembrane domains of
NMBR into the GRPR, including the upper 6th transmembrane region of NMBR, caused no gain of affinity (Fig. 5, Table III). A simultaneous substitution into the GRPR of the 3rd extracellular domain and the 5th
upper transmembrane domain of NMBR resulted in a 36-fold increase in
affinity for PD168368 and the resultant receptor had similar affinity
for PD168368 as the wild type NMBR (Fig. 5, Table III).

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Fig. 4.
Importance of the uTM regions in determining
selectivity of PD168368 for NMBR over GRPR: loss of affinity NMBR
transmembrane chimeras. The chimeric uTM NMBRs were formed by
replacing each of the transmembrane domains of NMBR by the comparable
domain of the GRPR one at a time as shown in the diagram at
the top of the figure. Affinities were measured by
competitive radioligand displacement of 50 pM
125I-[Tyr4]Bn or
125I-[D-Tyr6, -Ala11,Phe13,Nle14]Bn-(6-14)
by PD168368 at the concentrations shown. Each point on the
dose-inhibition curve is the mean from three separate experiments, and
in each experiment each point was measured in duplicate.
uTM1-, uTM2-, uTM3-, uTM4-,
uTM5-, uTM6-, and
uTM7-GRPR-NMBR refer to replacement by
this transmembrane domain of the NMBR by that from the GRPR.
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Table III
Affinities of PD168368 for wild-type NMBR, loss of affinity NMBR, and
gain of affinity GRPR upper transmembrane chimeric receptors and
wild-type GRPR
Affinities were calculated by competitive displacement of 50 pM 125I-[Tyr4]Bn or
125I-[D-Tyr6, -Ala11,Phe13,Nle14]Bn-(6-14)
(*) by PD168368. Values represent the mean ± S.E. from
at least three independent experiments.
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Fig. 5.
Importance of the uTM regions in determining
selectivity of PD168368 for NMBR over GRPR: gain of affinity GRPR
transmembrane chimeras. The chimeric uTM GRPRs were formed by
replacing each of the transmembrane domains of GRPR by the comparable
domain of the NMBR one at a time as shown in the diagrams at
the top of the figure. Affinities were measured by
competitive radioligand displacement of 50 pM
125I-[Tyr4]Bn by PD168368 at the
concentrations shown. Each point on the dose-inhibition curve is the
mean from three separate experiments, and in each experiment each point
was measured in duplicate. uTM1-, uTM2-,
uTM3-, uTM4-, uTM5-, uTM6-,
and uTM7-NMBR-GRPR refer to replacement by this
transmembrane domain of the GRPR by that from the NMBR.
Arrows indicate large changes in affinity of the GRPR.
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Point Mutants in the 5th Upper Transmembrane Domain--
To
identify which amino acid(s) in the 5th upper transmembrane domain of
the NMBR and GRPR were responsible for the selectivity of PD168368 for
the NMBR, the amino acid sequence of this region of the GRPR and NMBR
was compared (Fig. 6). The two receptors differed in 4 amino acids in the 5th transmembrane region (Fig. 6).
Specifically, the isoleucine in position 216 in the NMBR was replaced
by a serine in the GRPR, tyrosine 220 by phenylalanine, phenylalanine
221 by tyrosine, and leucine 222 in NMBR by valine in the comparable
position of GRPR (Fig. 6). To determine the importance of these four
amino acid differences, potential loss of affinity NMBR point mutations
were made by substituting into the NMBR the comparable amino acid from
the GRPR (Figs. 6 and 7) and four potential gain of affinity GRPR point
mutations were made by substituting into the GRPR the comparable amino
acid from the NMBR (Figs. 6 and 8). For the potential loss of affinity
NMBR point mutations, three of the substitutions (Ser216
for Ile, Tyr221 for Phe, and Val222 for Leu)
did not significantly alter the high
affinity for PD168368 compared with the wild type NMBR (Table IV, Fig.
7). In contrast, the substitution of
Phe220 for Tyr in the NMBR caused a 8.6-fold decrease in
affinity (96 ± 7.8 nM to 830 ± 63 nM) (Table IV, Fig. 7). When the reverse study was
performed to attempt to demonstrate a gain of affinity in the GRPR, the
substitution of Tyr219 for Phe caused 6.4-fold increase in
affinity (Table IV, Fig. 8). The other
three potential GRPR gain of affinity point mutants showed no gain in
affinity for PD168368 (Table IV, Fig. 8).

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Fig. 6.
Alignment of amino acid sequences in the 5th
upper transmembrane domain of NMBR and GRPR. Boxes
indicate divergent amino acids between these two receptors in the 5th
upper transmembrane region. Shown are the four NMBR and four GRPR point
mutants made to explore the importance of each of the four amino acid
differences for determining PD168368 selectivity.
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Table IV
Affinities of PD168368 for the wild-type, receptor uTM5 loss and gain
of affinity point mutants of NMBR and GRPR
Affinities were calculated by competitive displacement of 50 pM 125I-[Tyr4]Bn or
125I-[D-Tyr6, -Ala11,Phe13,Nle14]Bn-(6-14)
(*) by PD168368. Values represent the mean ± S.E. from
at least three independent experiments. The numbers refer to the
position in either the NMBR or GRPR of the amino acid substitutions as
shown in Fig. 6. [I216S]NMBR refers to replacement of the isoleucine
in position 216 of the NMBR by serine, which exists in the comparable
position in GRPR (see Fig. 6).
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Fig. 7.
Importance of specific amino
acids in the receptors 5th upper transmembrane region in
determining selectivity of PD168368 for the NMBR over the GRPR: loss of
affinity NMBR point mutations. The point mutants in the NMBR were
formed by replacing each of the amino acid(s) of the 5th upper
transmembrane region of NMBR by the comparable amino acid(s) of the
GRPR one at a time, as shown in Fig. 6. Affinities were measured by
competitive radioligand displacement of 50 pM
125I-[Tyr4]Bn or
125I-[D-Tyr6, -Ala11,Phe13,Nle14]Bn-(6-14)
by PD168368 at the concentrations shown. Each point on the
dose-inhibition curve is the mean from three separate experiments, and
in each experiment each point was measured in duplicate.
I216S refers to replacement of the isoleucine in position
216 of the NMBR by serine, which exists in the comparable position in
GRPR (see Fig. 6).
|
|

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Fig. 8.
Importance of specific amino
acids in the receptors 5th upper transmembrane region in
determining selectivity of PD168368 for the NMBR over the GRPR: gain of
affinity GRPR point mutations. The point mutants in the GRPR were
formed by replacing each of the amino acid(s) of the 5th upper
transmembrane region of GRPR by the comparable amino acid(s) of the
NMBR one at a time, as shown in Fig. 6. Affinities were measured by
competitive radioligand displacement of 50 pM
125I-[Tyr4]Bn by PD168368 at the
concentrations shown. Each point on the dose-inhibition curve is the
mean from three separate experiments, and in each experiment each point
was measured in duplicate. [S215I] refers to replacement
of the serine in position 215 of the GRPR by isoleucine, which exists
in the comparable position in NMBR (see Fig. 6). The arrows
indicate mutants showing a large change in affinity from the native
GRPR.
|
|
Visualization of PD168368--
To attempt to identify using the
computer modeling suite Sybyl 6.6 and calculating charges by the method
of Gasteiger-Hückel with the molecular energy minimized, the
hydrogen bonding potential of PD168368 was visualized (Fig.
9). This generated a Connolly surface of
the molecule, colored blue in hydrogen bond accepting regions and red in hydrogen bond donating regions (Fig. 9).
The molecular volume was 558 Å2. PD168368 had a large
hydrogen bond accepting region around the nitrophenyl group and smaller
hydrogen bond acceptor regions associated with the pyridyl group and
amide bonds (Fig. 9).

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|
Fig. 9.
An energy-minimized structure for
PD168368. PD168368 is shown as a ball and stick model (hydrogen,
cyan; carbon, white; nitrogen, blue;
oxygen, red). It is surrounded by a Connolly
molecular surface that has been colored to indicate hydrogen bond
acceptor (blue) and donor (red) regions.
Hydrophobic regions are colored gray.
|
|
Binding Site Model--
To attempt to better define the binding
pocket of PD168368 to NMBR, the NMBR (Fig.
10) was analyzed using modeling
programs. The NMBR was analyzed using the
-carbon template from the
bacteriorhodopsin receptor derived from electron microscopy (51), the
WHAT IF program (52), and Sybyl 6.6. This indicated a possible binding site near the extracellular surface, bounded by the extracellular surface and Pro120, Gln123, Leu124,
and Val127 of helix 3; Trp168,
Ser171, and Glu178 of helix 4;
Ile216 and Tyr220 of helix 5;
Trp279, Asn282, His283, and
Tyr286 of helix 6; and Ser313 of helix 7, with
a volume of >700 Å2 (Fig. 10). Amino acid side chains
facing the interior of the transmembrane bundle within 3.5 Å of the
solvent cluster were highlighted to indicate the putative binding site
of PD168368 (Fig. 10). The critical Tyr220 residue of the
5th transmembrane domain of NMBR was found to face the interior of the
putative binding pocket formed by transmembrane domains 3-7 (Fig.
10).

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Fig. 10.
The NMB transmembrane receptor putative
binding site surrounding the critical residue Tyr220.
Helices 1-7 are colored light cyan,
orange, red, purple, blue,
green, and yellow, respectively. The putative
binding site, established by computer modeling using SiteID, is
depicted as a gray opaque space-filling model. Residues
surrounding the binding site, including Tyr220
(green), are shown as stick models. Amino acid side chains
not facing the center of the transmembrane region are omitted for
clarity.
|
|
 |
DISCUSSION |
There are now more than 30 GPCRs mediating the action of GI
hormones or neurotransmitters such as bradykinin, GRP, or
cholecystokinin (CCK) (53). In most cases their roles in physiological
processes or in pathological conditions are still unclear because only
recently have high affinity antagonists been developed for some of
these receptors. The antagonists for these receptors generally fall into one of three types: peptide antagonists, nonpeptide antagonists, or peptoid antagonists, which have features of both peptides and nonpeptides (1, 2). In general the molecular basis of action of these
antagonists is poorly understood, and it is unclear whether it is
similar to the well studied adrenergic or muscarinic cholinergic receptor antagonists (54-56). Whereas there have been a number of
studies of the molecular basis of action of nonpeptide antagonists for
various GI hormone receptors (11-15), there are only a few studies of
peptide antagonists (12, 57, 58) and almost none for peptoid
antagonists (59). In this study we examined the molecular basis of
action of the novel peptoid receptor antagonist PD168368, which is
reported to be selective for the NMBR in the bombesin family of
receptors (10, 40, 41).
A number of our results support the conclusion that the NMBR receptor
extracellular domains do not play a prominent role in determining the
selectivity of PD168368 for NMBR over the structurally closely related
receptor, the GRPR. First, when extracellular domains of the GRPR were
substituted into the NMBR to assess loss of affinity for PD168368, only
the substitution of the 3rd extracellular domain in the NMBR by the
comparable domain of the GRPR altered affinity and the decrease in
affinity was less than 2-fold. Second, when the reverse study was
performed by substituting into the GRPR the extracellular domain of the
NMBR to produce gain of affinity chimeras, only the substitution of the
3rd extracellular domain in the GRPR by the comparable domain of the
NMBR altered affinity causing an almost 3-fold increase for PD168368.
The contribution of the 3rd extracellular domain for high selectivity
of PD168368 was small compared with the almost 40-fold higher affinity
PD168368 had for the native NMBR over GRPR. These results therefore
suggest that differences in the 3rd extracellular domain of these two receptors play only a small role in the selectivity of PD168368. This
result has both similarities and differences from studies on the
interaction of peptide and nonpeptide agonists and antagonists with
other GPCRs. Mutagenesis and biophysical analyses of several GPCRs
indicate that the receptor extracellular domain can be an important
receptor region for determining high affinity ligand binding (16). Only
a few studies have explored whether peptide antagonists interacting
with receptor extracellular domains. Such an interaction is important
for determining high affinity of the peptide antagonist JMV179 for the
human CCK-A receptor (12); however, for other peptide antagonists such
as the interaction of
D-Arg-[Hyp3,D-Phe7]bradykinin
(NPC567) with the B2 bradykinin receptor (57) and BQ-123
with the endothelin A (ET-A) receptor (13), the receptor extracellular
domains are not important for high affinity interaction. Numerous
studies show that with most nonpeptide antagonists such as the
interaction of losartan with the AT1b angiotensin II
receptor, L365,260 with the CCK-B/gastrin receptor, or BMS-182874 with
the ET-A receptor, the extracellular region does not contain important determinants for high affinity interaction (11, 13, 60). However,
interaction of nonpeptide substance P antagonist CP96345 with the 3rd
extracellular domain of the neurokinin 1 (NK1) receptor is important
for determining selectivity for this receptor (61). For a number of
peptide GI receptor neurotransmitter/hormones that function as agonists
high affinity receptor binding has been shown to dependent on
interaction with receptor extracellular domain, including CCK-8 with
the CCK-A or CCK-B receptor (62), bradykinin with B2
bradykinin receptor (63), and substance P with the NK1 receptor (64).
On the other hand, with many nonpeptide agonists such as
- and
-adrenergic agents, nucleosides, eicosanoids, muscarinic cholinergic
agents, and lipid moieties, the extracellular receptor domains are not
important generally in determining receptor selectivity and high
affinity interaction (17). However, with small molecular nonpeptide
agonists for the G protein-coupled Ca2+-sensing receptor;
Ca2+ and Gd3+ interaction with the large
NH2-terminal extracellular segment of the receptor is
important for high affinity interaction (65).
Because the extracellular domains play only a minor role in the
selectivity of PD168368 for the NMBR over the GRPR, we applied a
similar chimeric approach to the receptor's upper transmembrane region, which is also likely to interact with small ligands (16, 17).
Our results support the conclusion that amino acids in the 5th upper
transmembrane region play a major role, and in the 6th upper
transmembrane region a minor role in determining the high selectivity
of PD168368 for the NMBR over the GRPR. In contrast, the amino acids in
the other five upper transmembrane regions did not play a significant
role in this ligand's selectivity. The importance of the 5th upper
transmembrane region for PD168368 selectivity is consistent with the
interaction of some small ligands (
- and
-adrenergic agents,
nucleosides, eicosanoids, or muscarinic cholinergic agents) (16, 17)
but not others (Ca2+, glutamate, or
-aminobutyric acid)
with their GPCRs, which provide evidence that upper transmembrane
domains can be important regions for determining the affinity of some
small molecular ligands to interact with receptors (17). High affinity
receptor binding of some peptide agonists have been shown to primarily
depend on interactions with the receptor upper transmembrane domains.
This includes interaction of various peptide agonists with angiotensin II receptor (66), the endothelin-A receptor (ETA-R) (15), neuropeptide Y1 receptor (67), and the
thyrotropin-releasing hormone receptor (68). However, with peptide
agonists for the B2 bradykinin receptor (63), the
corticotropin-releasing factor receptor (69), or µ- and
-opioid
receptors (70), the transmembrane regions are not important. In similar
fashion, several nonpeptide agonists have been shown to primarily
interact with GPCR upper transmembrane regions including nonpeptide
agonists for adrenergic receptors (54, 55), muscarinic cholinergic
receptors (56, 71, 72), or eicosanoid (73, 74). High affinity receptor binding of some peptide antagonists also have been shown to depend primarily on interaction with the upper transmembrane domains of
receptors such as the interaction of NPC567 and Hoe140 with the
B2 bradykinin receptor (57, 58) or BQ-123 with the
ETA-R (13), whereas with other GPCRs such as interaction of
CCK-JMV179 with the CCK-A receptor (12), the upper transmembrane
regions are not important. A number of studies show that for most
nonpeptide antagonists the interaction with the GPCRs' transmembrane
domains are the primary determinants of high affinity receptor
interaction (13, 14, 60, 75-77). For example, with the
AT1b angiotensin II receptor, the nonpeptide antagonist
losartan requires specific amino acid residues within the 3rd to 7th
transmembrane regions for high affinity binding (60). Similarly the
high affinity binding of the nonpeptide antagonist L365,260 with
CCK-B/gastrin receptor (75), BMS-182874 with ETA-R (13),
SB209670 and Ro 46-2005 with ETB-R (14), or CP96345 and
L-161,664 with NK1 receptor (76, 77) are all dependent
primarily on interaction with receptor upper transmembrane regions. Our
results show that the peptoid antagonist PD168368 primarily resembles
nonpeptide receptor antagonist's interaction with GPCRs in that its
high affinity and selectivity for the NMBR depends primarily on
interactions with the receptor upper transmembrane areas.
For a few other ligands, the 5th transmembrane region is also a
critical region to interact with a GPCR as we found in the present
study for PD168368. For example, the critical binding site for the
8-amino acid peptide agonist angiotensin II is in the 5th transmembrane
region of AT1a angiotensin II receptor (66). The high
affinity interaction of the nonpeptide agonists acetylcholine and
carbachol depends on interaction with the 5th transmembrane region of
the m3 muscarinic receptor (72). Furthermore, high affinity receptor
binding and selectivity for several nonpeptide antagonists depend on
interact with specific amino acids in the 5th transmembrane region,
including the binding of the nonpeptide corticotropin releasing factor
receptor 1 antagonist NBI27914 (69) to the human corticotropin
releasing factor receptor 1 and the binding of the NK1 receptor
antagonist L161,664 to the NK1 receptor (77).
To determine which amino acids in the 5th upper transmembrane region of
the NMBR account for the PD168368's selectivity, we performed a
comparative alignment of the amino acids in this region between the
NMBR and the GRPR. Within the 5th upper transmembrane domain, there
were four amino acid residues that differ between NMBR and GRPR (Fig.
6). These differences included a Ser for Ile change at position 216 of
the NMBR and Phe, Tyr, and Val at positions 219-221 of the GRPR for
Tyr, Phe, and Leu at positions 220-222 of the NMBR. Our results
support the conclusion that the tyrosine residue in position 220 of
NMBR instead of a phenylalanine in this position in the GRPR is the key
amino acid residue in determining the selectivity of the antagonist
PD168368 caused by the differences in the receptors' 5th transmembrane
region. In a previous study (42), we showed that the 5th transmembrane
domain of NMBR was critical also for high affinity NMB binding.
However, the isoleucine in position 216 was the key amino acid for high
affinity interaction of NMB with the NMBR over the GRPR. These results
demonstrate that the key receptor determinants for the selectivity of
the agonist NMB and peptoid antagonist PD168368 for the NMBR over the
GRPR are in very close proximity in the NMBR. This result is consistent
with the strategy used to design PD168368, which depended strongly on
incorporating critical structural features for determining high
affinity NMB binding to NMBR into the PD168368 (10). However, despite
these considerations the peptoid antagonist PD168368 and the peptide
agonist NMB show a significant difference in their determinants of high
affinity interaction with the NMBR. Specifically, the key amino acid in
the 5th transmembrane domain (Tyr220) for high affinity
NMBR interaction of PD168368 is different from the key amino acid for
the high affinity interaction of NMB (Ile216) with the
NMBR, supporting the conclusion that significantly different molecular
interactions are likely responsible for their high affinities for the
NMBR.
In several studies, a tyrosine residue in the GPCRs plays a critical
role in ligand-receptor interaction (13, 66, 68, 73, 77). The tyrosine
of the 3rd transmembrane domain of the thyrotropin-releasing hormone
receptor binds the pyroglutamyl moiety of thyrotropin-releasing hormone
through hydrogen bonding (68). The tyrosine of the 6th
transmembrane domain of the m2 muscarinic receptor is a critical amino
acid for the interaction with nonpeptide agonists (acetylcholine,
carbachol, oxotremorine M, and pilocarpine) (71), likely by a hydrogen
bonding mechanism. The tyrosine hydroxyl moiety
characteristically interacts with ligands by functioning as a hydrogen
bond donor or as a strong locus of cation-
binding (73). The
cation-
binding occurs through the side-chains of aromatic amino
acids such as phenylalanine, tyrosine, or tryptophan (78). In our study
the substitution of phenylalanine for tyrosine in the NMBR resulted in
a marked decrease in affinity for PD168368. Therefore, it might be
argued that the interaction of the tyrosine hydroxyl with PD168368
through hydrogen bonding is likely more important than cation-
binding. The calculated
(
G°) value of 0.5 kcal/mol
for the peptoid antagonist PD168368 obtained from the difference in
affinities by replacing Tyr220 of the NMBR by the
comparable Phe219 of the GRPR is consistent with the
elimination of a hydrogen bond (79). However, tyrosine is predicted to
have a higher cation-
binding potential than phenylalanine secondary
to the negative electrostatic potential of the oxygen (80). Therefore,
whether the tyrosine hydroxyl is involved in increasing negative
electrostatic potential due to cation-
binding site interaction or
to hydrogen bonding cannot be resolved by our studies.
To further explore the putative binding site for PD168368,
three-dimensional modeling of the NMBR based on the structure of bacteriorhodopsin (51) was employed. In this model, the critical Tyr220 residue on the 5th transmembrane domain of the NMBR
was found to face the interior of a large binding pocket formed
primarily by transmembrane domains 3-7 (Fig. 10). Examination of a
minimum energy conformation of the ligand showed that it is dominated by a large hydrogen bond accepting region around the nitrophenyl group,
and smaller acceptor regions associated with the pyridyl moiety and
amide bonds (Fig. 9). The ligand also has smaller hydrogen bond donor
regions associated with the Trp moiety and the amide linkages. The rest
of the molecule consists of uncharged hydrocarbon structures that
prefer a lipid or hydrophobic environment (Fig. 9). Thus, it is
feasible for the ligand PD168368 to be able to enter the
intratransmembrane binding pocket to interact with Tyr220
while simultaneously being capable of interacting with extracellular loops. If this interaction is primarily dependent on hydrogen bonding
as our data suggest, it is most likely that the hydroxyl of the
Tyr220 interacts with nitrophenyl group of PD168368 or
perhaps one of the other hydrogen acceptor groups on PD168368. This
proposal is supported by the fact that, in PD168368, the largest
hydrogen bond accepting group is the nitro group on the phenyl ring
(Fig. 9). This is also the most accessible of the hydrogen bond
acceptors in the ligand. The other hydrogen bond acceptors, the pyridyl group, and the backbone carbonyls are much less accessible. Our receptor model indicates that Pro120, Gln123,
Leu124, and Val127 of TM helix 3;
Trp168, Ser171, and Glu178 of TM
helix 4; Ile216 and Tyr220 of TM helix 5;
Trp279, Asn282, His283, and
Tyr286 of TM helix 6; and Ser313 of TM helix 7 are all facing the putative binding pocket and could interact with a
small ligand such as PD168368 (Figs. 9 and 10). Pro120,
Gln123, Leu124, and Val127 of TM
helix 3; Trp168, Ser171, and Glu178
of TM helix 4; Trp279, Asn282,
His283, and Tyr286 of TM helix 6; and
Ser313 of TM helix 7 in the NMBR are the same as the
comparable amino acids in the GRPR and therefore are unlikely to be
important in the selectivity of PD168368 for the NMBR over the GRPR. Of
the other two amino acids, Ile216 and Tyr220,
our mutagenesis studies show only Tyr220 is important in
determining the selectivity of PD168368 for the NMBR over the GRPR.
Therefore, we conclude that Tyr220 is the important amino
acid in determining the selectivity of PD168368 of the transmembrane
amino acids facing the binding pocket. Our studies suggest that amino
acids within the 3rd extracellular domain have a small effect on
determining the affinity of PD168368. Although the 3rd extracellular
domain of the NMBR is in close proximity to the putative binding pocket
of PD168368 (Fig. 10) in the present study because of its relative
small effect on PD168368 affinity, we did not investigate in detail
which amino acids within this domain are responsible for the slight
differences in affinity. We therefore cannot speculate on the type of
ligand receptor interaction responsible for this small effect of the
3rd extracellular domain.
In conclusion, our receptor chimeric studies showed that the 5th
transmembrane domain of the NMBR was a responsible region for the high
affinity and selectivity of the peptoid antagonist PD168368 for the
NMBR over the GRPR. Our mutagenesis studies show Tyr220 in
the 5th upper transmembrane domain of the NMBR was the key amino acid
interacting with this antagonist. To our knowledge, this is the first
study that reveals the molecular basis of action between a peptoid
antagonist and receptor. The described data from these studies allowed
us to obtain a more detailed picture of the receptor-ligand interaction
and propose a model that could account for important receptor-ligand
interactions. The availability of this model could be of use not only
for studying the molecular basis of the interaction of natural
agonists, but also for peptoid antagonists or peptide antagonists for
this important family of receptors.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence and reprint requests should be
addressed: Digestive Diseases Branch, NIDDK, National Institutes of Health, Bldg. 10, Rm. 9C-103, 10 Center Dr., MSC 1804, Bethesda, MD
20892-1804. Tel.: 301-496-4201; Fax: 301-402-0600; E-mail: robertj@bdg10.niddk.nih.gov.
Published, JBC Papers in Press, September 29, 2000, DOI 10.1074/jbc.M006059200
 |
ABBREVIATIONS |
The abbreviations used are:
GI, gastrointestinal;
NMB, neuromedin B;
NMBR, neuromedin B receptor;
GRP, gastrin-releasing peptide;
GRPR, gastrin-releasing peptide receptor;
GPCR, G protein-coupled receptor;
FBS, fetal bovine serum;
DMEM, Dulbecco's modified Eagle's medium;
Bn, bombesin;
CCK, cholecystokinin;
ET, endothelin;
NK1, neurokinin 1.
 |
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