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Molecular Endocrinology 19(11):2871–2881
Copyright © 2005 by The Endocrine Society
doi: 10.1210/me.2005-0148
The N-Terminal Juxtamembrane Segment of the V1a
Vasopressin Receptor Provides Two Independent
Epitopes Required for High-Affinity Agonist Binding
and Signaling
Stuart R. Hawtin,* Victoria J. Wesley,* John Simms, Cymone C. H. Argent, Khalid Latif, and
Mark Wheatley
School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom
It is fundamentally important to define how agonist-receptor interaction differs from antagonistreceptor interaction. The V1a vasopressin receptor
(V1aR) is a member of the neurohypophysial hormone subfamily of G protein-coupled receptors.
Using alanine-scanning mutagenesis of the N-terminal juxtamembrane segment of the V1aR, we
now establish that Glu54 (1.35) is critical for arginine vasopressin binding. The mutant [E54A]V1aR
exhibited decreased arginine vasopressin affinity
(1700-fold) and disrupted signaling, but antagonist
binding was unaffected. Mutation of Glu54 had an
almost identical pharmacological effect as mutation of Arg46, raising the possibility that agonist
binding required a mutual interaction between
Glu54 and Arg46. The role of these two charged
residues was investigated by 1) substituting Glu54;
2) inserting additional Glu/Arg in transmembrane
helix (TM) 1; 3) repositioning the Glu/Arg in TM1;
and 4) characterizing the reciprocal mutant [R46E/
E54R]V1aR. We conclude that 1) the positive/negative charges need to be precisely positioned in
this N terminus/TM1 segment; and 2) Glu54 and
Arg46 function independently, providing two discrete epitopes required for high-affinity agonist
binding and signaling. This study explains why Glu
and Arg, part of an -R(X3)L/V(X3)E(X3)L- motif, are
conserved at these loci throughout this G proteincoupled receptor subfamily and provides molecular insight into key differences between agonist
and antagonist binding requirements. (Molecular
Endocrinology 19: 2871–2881, 2005)
G
bovine rhodopsin (bRho), where the Schiff base linkage of 11-cis retinal to Lys296 in TM7 positions the
chromophore within the membrane-embedded domain of the protein (1). Although the ligand-binding
pocket of ␤-adrenergic receptors and bRho is contained within the TM domain, the N-terminal domain of
these receptors is not functionally inert. The crystal
structure of bRho revealed that the N-terminal domain
is actually very structured (1) and, furthermore, point
mutations to Pro23 and Gln28 in this domain have been
linked to the degenerative disease, autosomal dominant retinitis pigmentosa (4). Likewise, polymorphisms
in the N terminus of the human ␤2-adrenergic receptor
at residue 16 (Gly or Arg) and residue 27 (Gln or Glu)
have also been reported to affect receptor function.
Glu27 exhibits decreased agonist-promoted downregulation, whereas Gly16 has enhanced agonist-induced down-regulation, which has been associated
with nocturnal asthma (5).
We established in a previous study (6) that the N
terminus of the V1a vasopressin receptor (V1aR) is functionally important. In particular, it was shown that Arg46
located within the N terminus is critical for high-affinity
vasopressin (AVP) binding but is not required for highaffinity antagonist binding (7). The neurohypophysial
peptide hormone AVP has a wide range of physiological
effects, including vasopressor and antidiuretic actions,
mediated by a family of specific AVP receptors classified
PROTEIN-COUPLED RECEPTORS (GPCRs) exhibit a common tertiary structure comprising
seven transmembrane helices (TMs) linked by extracellular and intracellular loops (1). On initial examination, it may appear that the N-terminal domain of
GPCRs is merely a peripheral structural element within
this established receptor architecture. However, the N
terminus is actually an important domain with respect
to GPCR function. The ligand-binding platform for
many peptide ligands is provided by the receptor TM
helices plus extracellular domains including the N terminus (2). Indeed, large glycohormones, such as LH,
will bind with high affinity to the isolated N terminus of
the receptor (3). In contrast, it is well established that
the binding pocket for small amine neurotransmitters,
such as norepinephrine, is buried deep within the TM
helical bundle (2). A similar situation is observed with
First Published Online June 30, 2005
* S.R.H. and V.J.W. contributed equally to the manuscript.
Abbreviations: AVP, [Arginine8]vasopressin; bRho, bovine
rhodopsin; GPCR, G-protein-coupled receptor; HEK, human
embryonic kidney; InsP, inositol phosphate; InsP3, inositol
trisphosphate; OTR, oxytocin receptor; TM, transmembrane
helix; V1aR, V1a vasopressin receptor; V1bR, V1b vasopressin
receptor; V2R, V2 vasopressin receptor; VPR, AVP receptor.
Molecular Endocrinology is published monthly by The
Endocrine Society (http://www.endo-society.org), the
foremost professional society serving the endocrine
community.
2871
2872 Mol Endocrinol, November 2005, 19(11):2871–2881
as the V1aR, the V1b vasopressin receptor (V1bR), and the
V2 vasopressin receptor (V2R) (8, 9). The V1aR is widely
distributed and mediates nearly all of the actions of AVP
with the notable exceptions of antidiuresis (V2R) and
ACTH secretion (V1bR). All three AVP receptor (VPR)
subtypes have been cloned from a range of different
species and possess the characteristic structural motifs
of the rhodopsin/␤-adrenergic receptor family (family A)
of GPCRs (1). The V1aR and the V1bR couple to phospholipase C, thereby generating inositol 1,4,5-trisphosphate and diacylglycerol as second messengers,
whereas the V2R couples to adenylyl cyclase. The peptide hormone oxytocin is structurally homologous to AVP
but fulfils discrete physiological functions including contraction of the uterus at parturition (10). The three VPR
subtypes, together with the oxytocin receptor (OTR) and
receptors for vasotocin, mesotocin, and isotocin from
lower vertebrates, constitute a subfamily of the rhodopsin/␤-adrenergic receptor class of GPCRs. The natural
agonists for all of these receptors are analogs of the
neurohypophysial peptide hormones AVP and oxytocin
(10). In addition to the characteristic architecture of
GPCRs, members of the neurohypophysial peptide hormone receptor family share certain sequence motifs and
exhibit related pharmacologies (10–12).
Defining at the molecular level how agonist-receptor
interaction differs from antagonist-receptor interaction is
fundamentally important to understanding hormonestimulated cell signaling by this subfamily of GPCRs.
This study explores the role of the juxtamembrane segment of the N terminus of the V1aR, at the interface of the
N terminus and TM1. We establish that Glu54 is required
for high-affinity agonist binding but is not required for
antagonist binding. The side-chain properties required
for this role at residue 54 are evaluated, and we demonstrate that although Glu54 and Arg46 are both required for
agonist binding and are in close proximity to each other,
they function independently. In addition, a role for Leu58
in V1aR function is identified.
RESULTS
Pharmacological Characterization of AlanylSubstituted V1aR Constructs
The N terminus of the V1aR fulfils an important role in
binding agonists, particularly Arg46, within this domain
(7). In addition to requiring the N terminus, it is known
that binding contacts for AVP are provided by residues
in the TM helical bundle of the V1aR (13). The aim of
this study was to investigate the role of the N terminus/
TM1 interface region of the V1aR in high-affinity agonist binding. Based on the crystal structure of bRho,
the membrane-embedded TM1 of the V1aR would be
predicted to start at Leu50 (1). To identify the contribution to agonist binding provided by individual residues in the distal segment of the N terminus, residues
between Arg46 and Leu50 inclusive (see Fig. 1) were
individually mutated to alanine to generate the constructs
Hawtin et al. • Agonist Binding to the V1aR
[R46A]V1aR, [N47A]V1aR, [E48A]V1aR, [E49A]V1aR, and
[L50A]V1aR. In addition, it was noted that Glu54 (1.35) [using
the nomenclature proposed by Ballesteros and Weinstein
(14)] is located one turn below Leu50 in TM1 and, furthermore, all members of the neurohypophysial peptide hormone receptor family cloned to date have a glutamyl at this
locus (Fig. 1). In addition, this conserved Glu (1.35) is
flanked by a Leu one turn above (1.31) and a Leu one turn
below (1.39) in all VPRs and OTRs cloned to date. The
hydrophobic nature of the two Leu side chains that sandwich Glu54 (1.35) would modulate the ionization properties
of the side chain carboxyl group and furthermore, could be
important for shielding the negative charge of the Glu. Consequently, the role of Glu54 (1.35) and Leu58 (1.39) were also
investigated by engineering the mutant constructs
[E54A]V1aR and [L58A]V1aR. Each mutant receptor construct was then expressed in human embryonic kidney
(HEK) 293T cells, and the pharmacological characteristics
were compared with wild-type V1aR. This characterization
was aided by the fact that four different classes of ligand are
available for probing changes in the ligand-binding profile of
V1aR constructs. In each case, competition radioligand
binding curves were determined using the natural agonist
AVP and three different structural classes of antagonist: 1)
cyclic peptide antagonist [d(CH2)5Tyr(Me)2AVP (15)] containing a 20-membered ring formed by a disulfide bond
between Cys1 and Cys6; 2) linear peptide antagonist ([phenylacetyl-D-Tyr(Me)2Arg6Tyr(NH2)9]AVP (16)); and 3), nonpeptide antagonist [SR 49059 (17)]. The dissociation constant (Kd) values are presented in Table 1, corrected for
radioligand occupancy. The wild-type and mutant constructs were all expressed at the same level of 1–2 pmol/mg
protein. The mutant constructs [N47A]V1aR, [E48A]V1aR,
[E49A]V1aR, and [L50A]V1aR exhibited a pharmacological
profile very similar to that of wild-type receptor, although the
Kd for AVP was slightly raised (2- to 3-fold) in each case
(Table 1). Substitution of Leu58 in the construct [L58A]V1aR
affected the affinity of all of the ligands investigated to a
similar extent, with the affinity of AVP decreasing 7-fold and
the affinity of the three different classes of antagonist decreasing 2- to 9-fold (Table 1). In marked contrast,
[E54A]V1aR displayed a profound decrease in affinity for
AVP with the Kd increasing 1700-fold compared with that of
the wild-type receptor. The affinity of each of the three
different classes of antagonist, however, was unaffected by
this mutation (Fig. 2A and Table 1). Consequently, the
[E54A]V1aR construct revealed that Glu 54 was critical for
high-affinity agonist binding, but this residue did not have a
role in binding antagonists of any class (peptide or nonpeptide, cyclic or linear).
The capability of each of the mutant receptor constructs to generate an intracellular signal in response to
the natural agonist AVP was also investigated. In each
case, AVP-induced accumulation of InsPs was assayed.
From the resulting dose-response curves, the EC50 and
maximum response (Emax) were determined for each
construct, and these are presented in Table 2. The EC50
value for [E48A]V1aR, [E49A]V1aR, [L50A]V1aR was
slightly higher than for the wild-type receptor in each
case (between 3- and 7-fold), reflecting the slight de-
Mol Endocrinol, November 2005, 19(11):2871–2881 2873
Hawtin et al. • Agonist Binding to the V1aR
Fig. 1. Comparison of the Sequence of the N Terminus of the Neurohypophysial Peptide Hormone Receptors Cloned from
Different Species
The sequences of the N terminus of the V1aR, OTR, V1bR, and V2R from different species have been aligned. The position of
the top of the first transmembrane helix (TM1) is indicated by a dashed line. The species of origin is shown by a single-letter code
preceding the receptor subtype: r, rat; m, mouse; v, vole; s, sheep; h, human; p, pig; b, cow; mky, rhesus monkey; d, dog. Also
shown is the N-terminal sequence of the vasotocin and isotocin receptors from teleost fish and an amphibian mesotocin receptor.
The conserved arginyl and glutamyl in all of the aligned sequences, equivalent to Arg46 and Glu54 in the rat V1aR, respectively,
is indicated by a box and is labeled.
crease in affinity of AVP at these constructs (Table 1). In
addition, the Emax values for [E48A]V1aR and [L50A]V1aR
were slightly higher than for wild-type V1aR (Table 2).
Substitution of Leu58 slightly decreased AVP binding
affinity but had a more pronounced effect on intracellular
signaling. Consequently, [L58A]V1aR possessed a 7-fold
Table 1. Pharmacological Profile of Wild-Type and Alanyl-Substituted V1aRs
AVP
CA
LA
SR 49059
Cell Surface
Expression
(% wt)
1.0 ⫾ 0.1
1300 ⫾ 190
2.4 ⫾ 0.4
1.8 ⫾ 0.3
2.2 ⫾ 0.4
3.3 ⫾ 0.5
1700 ⫾ 270
6.9 ⫾ 1.7
0.7 ⫾ 0.3
0.8 ⫾ 0.1
0.4 ⫾ 0.1
1.1 ⫾ 0.6
1.2 ⫾ 0.2
0.8 ⫾ 0.4
1.0 ⫾ 0.5
6.2 ⫾ 1.5
0.5 ⫾ 0.1
0.3 ⫾ 0.1
1.1 ⫾ 0.2
1.6 ⫾ 0.6
2.3 ⫾ 0.2
1.7 ⫾ 0.6
0.3 ⫾ 0.1
0.9 ⫾ 0.4
1.9 ⫾ 0.3
1.9 ⫾ 0.4
1.5 ⫾ 0.5
2.3 ⫾ 0.2
1.8 ⫾ 0.5
2.0 ⫾ 0.9
1.5 ⫾ 0.5
9.2 ⫾ 2.0
100
106 ⫾ 7
101 ⫾ 8
102 ⫾ 6
101 ⫾ 8
111 ⫾ 5
96 ⫾ 6
96 ⫾ 2
Binding Affinities Kd (nM)
Receptor
Wild-type V1aR
R46Aa
N47Aa
E48A
E49A
L50A
E54A
L58A
IC50 values derived from competition binding experiments were determined by nonlinear regression after fitting Langmuir binding
isotherms. The affinity (Kd) and cell surface expression of receptor constructs were determined as described in Materials and
Methods. Data shown are the mean ⫾ SEM of three separate experiments performed in triplicate.
a
Data taken from Ref. 7. CA, Cyclic peptide antagonist; LA, linear peptide antagonist; SR 49059, nonpeptide antagonist.
2874 Mol Endocrinol, November 2005, 19(11):2871–2881
Hawtin et al. • Agonist Binding to the V1aR
Table 2. AVP-Induced Signaling by Wild-Type and AlanylSubstituted V1aRs
Receptor
Wild-type V1aR
R46Aa
N47Aa
E48A
E49A
L50A
E54A
L58A
Stimulation of InsP–InsP3
EC50 Values (nM)
Emax Values (fold)
0.4 ⫾ 0.1
26 ⫾ 3.0
0.6 ⫾ 0.1
1.5 ⫾ 0.1
2.0 ⫾ 0.1
2.8 ⫾ 0.3
25 ⫾ 8
9.4 ⫾ 3.1
5.5 ⫾ 1.8
4.2 ⫾ 0.2
9.6 ⫾ 2.3
8.1 ⫾ 0.1
6.8 ⫾ 0.5
8.6 ⫾ 0.5
3.6 ⫾ 0.7
8.0 ⫾ 2.8
EC50 and Emax values of AVP-induced accumulation of InsP–
InsP3 in cells expressing wild-type and mutant receptors.
Values shown are the mean ⫾ SEM of three separate experiments performed in triplicate.
a
Data taken from Ref. 7. Emax values are fold-stimulation over
basal.
Specific Requirement for Glutamate at Residue
54 for High-Affinity Agonist Binding and Second
Messenger Generation
Fig. 2. Pharmacological Characterization of Mutant V1aRs
HEK 293T cells were transiently transfected with either wildtype V1aR (open symbols; E, 䡺) or mutant receptor construct
(solid symbols; F, f). A, [E54A]V1aR; or B, [L50P]V1aR. Competition radioligand binding studies were then performed with a
membrane preparation of these HEK 293T cells using AVP (䡺,
f) or the antagonist d(CH2)5Tyr(Me)2AVP (E, F) as competing
ligand. Data are the mean ⫾ SEM of three separate experiments
each performed in triplicate. Values are expressed as percent
specific binding where nonspecific binding was defined by
d(CH2)5Tyr(Me)2AVP (1 ␮M). A theoretical Langmuir binding isotherm has been fitted to the experimental data as described in
Materials and Methods.
lower affinity for AVP than wild-type V1aR (Table 1) but
exhibited a 24-fold increase in the EC50 for AVP-induced
InsPs accumulation compared with wild-type V1aR (Table 2). The Emax value for [L58A]V1aR, however, was
comparable to that for wild-type receptor. Mutation of
Glu54 had a marked effect on the InsPs dose-response
curve with the curve right-shifted and the EC50 value
increased 63-fold compared with wild-type V1aR (Fig.
3A). Whole cell binding assays using [3H]antagonist as
tracer and HEK 293T cells expressing wild-type V1aR or
mutant V1aR constructs revealed that all of the receptor
constructs cited in this study exhibited the same cell
surface expression as wild-type V1aR (Table 1). Consequently, the disrupted intracellular signaling of
[E54A]V1aR and [L58A]V1aR compared with wild-type
V1aR was not due to these constructs failing to be trafficked efficiently to the plasma membrane.
To evaluate the properties of the Glu54 residue that
underlie its importance to V1aR function, we engineered the constructs [E54D]V1aR and [E54R]V1aR.
These mutant receptors probed the importance of the
charge of Glu54, by either preserving the negative
charge ([E54D]V1aR) or reversing the charge at this
locus ([E54R]V1aR). The affinity of AVP was reduced by
1700-fold and 2700-fold compared with wild-type
V1aR for [E54D]V1aR and [E54R]V1aR, respectively (Table 3). In contrast, the binding affinities of the three
different antagonists were unchanged compared with
wild-type V1aR, with the exception of linear peptide
antagonist binding to [E54R]V1aR, which was decreased 6-fold (Table 3).
Assessment of the intracellular signaling capability of
the Glu54 mutant constructs revealed that the nature of
the amino acid at this locus dictated second messenger
generation in response to AVP (Fig. 3A). Neither of the
substituted residues could support wild-type intracellular
signaling. Reversing the nature of the charge (construct
[E54R]V1aR) was very detrimental and raised the EC50 for
InsPs accumulation more than 600-fold compared with
wild type (Fig. 3A and Table 4). Even preserving the
negative charge, with the conservative substitution
[E54D]V1aR, greatly perturbed second messenger generation, resulting in a 250-fold increase in EC50 compared with wild type (Fig. 3A and Table 4).
Glu54 and Arg46 Are Both Required for HighAffinity Agonist Binding but Function
Independently
The effects on binding and intracellular signaling of
substituting Glu54 are almost identical to those observed by substituting Arg46. In both cases, substitu-
Mol Endocrinol, November 2005, 19(11):2871–2881 2875
Hawtin et al. • Agonist Binding to the V1aR
Table 4. AVP-Induced Signaling by Glu54 and Leu50
Mutant Receptors
Receptor
Wild-type V1aR
E54A
E54D
E54R
L50A
L50P
Stimulation of InsP–InsP3
EC50 values (nM)
Emax values (fold)
0.4 ⫾ 0.1
25 ⫾ 8
101 ⫾ 40
248 ⫾ 28
2.8 ⫾ 0.3
94 ⫾ 17
5.5 ⫾ 1.8
3.6 ⫾ 0.7
3.9 ⫾ 0.2
3.2 ⫾ 0.5
8.6 ⫾ 0.5
4.1 ⫾ 0.1
EC50 and Emax values of AVP-induced accumulation of InsP–
InsP3 in cells expressing wild-type and mutant receptors.
Values shown are the mean ⫾ SEM of three separate experiments performed in triplicate. Emax values are fold-stimulation over basal.
Fig. 3. Intracellular Signaling by Wild-Type and Mutant
V1aRs
AVP-induced accumulation of InsP–InsP3 in HEK 293T
cells transfected with A, wild-type V1aR (䡺); [E54A]V1aR, (E);
[E54D]V1aR (f) or [E54R]V1aR (F). B, Wild-type V1aR (䡺);
[L50A]V1aR, (E); [L50P]V1aR (f). Data are the mean ⫾ SEM of
three separate experiments, each performed in triplicate. Values are expressed as maximum stimulation induced by AVP
at the concentrations stated.
tion by alanyl ([E54A]V1aR and [R46A]V1aR) resulted in
a marked decrease in agonist affinity but did not affect
the binding of any class of antagonist (Table 1). Likewise, the dose-response curves for AVP-induced accumulation of InsPs by [E54A]V1aR and [R46A]V1aR
was equally right shifted compared with wild type, with
EC50 values increasing by approximately 65-fold in
each case (Table 2). These observations raise the possibility of mutual charge-charge interaction between
Glu54 and Arg46. Furthermore, molecular modeling of
the V1aR, based on the crystal structure of bRho,
revealed that the ␣-helical conformation of TM1 continues into the distal N terminus of the V1aR. This
positions Glu54 and Arg46 just two turns apart on the
same face of this helix (Fig. 4, A and B). Support for
this extracellular extension of the TM1 helix is provided
by secondary structure prediction, which also indicates that the ␣-helical conformation of TM1 continues up to Arg46 (Fig. 4C). It is well established that
inserting a prolyl into an ␣-helical secondary structure
distorts the helix. Therefore we engineered the construct [L50P]V1aR to disrupt any ␣-helical conformation of the sequence between Glu54 and Arg46 by
inserting a prolyl between these two residues. Introducing the mutation [L50P]V1aR did not affect cell
surface expression compared with wild-type V1aR but
selectively decreased AVP binding 550-fold (Fig. 2B
and Table 3) and disrupted intracellular signaling approximately 250-fold (Fig. 3B and Table 4). This is in
marked contrast to the effects of simply removing the
Leu50 side chain in the mutant [L50A]V1aR, which had
only very slight effects on receptor binding and signaling (Table 3).
The roles of Glu54 and Arg46 were investigated further.
With reference to Fig. 5A, a series of mutant receptors
was engineered to investigate the importance of the
Table 3. Pharmacological Characterization of Glu54 and Leu50 Mutant Receptors
AVP
CA
LA
SR 49059
Cell Surface
Expression
(% wt)
1.0 ⫾ 0.1
1700 ⫾ 270
1700 ⫾ 510
2700 ⫾ 700
3.3 ⫾ 0.5
550 ⫾ 90
0.7 ⫾ 0.3
1.0 ⫾ 0.5
0.8 ⫾ 0.4
0.4 ⫾ 0.1
0.8 ⫾ 0.4
0.9 ⫾ 0.1
0.5 ⫾ 0.1
0.3 ⫾ 0.1
0.6 ⫾ 0.1
2.8 ⫾ 0.1
1.7 ⫾ 0.6
0.4 ⫾ 0.1
1.9 ⫾ 0.3
1.5 ⫾ 0.5
6.0 ⫾ 1.4
1.5 ⫾ 0.7
1.8 ⫾ 0.9
1.3 ⫾ 0.7
100
96 ⫾ 6
109 ⫾ 16
97 ⫾ 1
111 ⫾ 5
105 ⫾ 6
Binding Affinities Kd (nM)
Receptor
Wild-type V1aR
E54A
E54D
E54R
L50A
L50P
The affinity (Kd) and cell surface expression of receptor constructs were determined as described in Materials and Methods. Data
shown are the mean ⫾ SEM of three separate experiments performed in triplicate. CA, Cyclic peptide antagonist; LA, linear peptide
antagonist; SR 49059, nonpeptide antagonist.
2876 Mol Endocrinol, November 2005, 19(11):2871–2881
Hawtin et al. • Agonist Binding to the V1aR
L50R]V1aR and [R46E/E54R]V1aR failed to respond to
AVP challenge even at 10 ␮M AVP.
Having established that both Arg46 and Glu54 are
required for high-affinity agonist binding, this raised
the possibility that a small peptide corresponding to
this segment of the V1aR could disrupt the normal
contacts of these side chains and thereby act as an
antagonist. The peptide N-acetyl-RNEELAKLE-amide
was synthesized, corresponding to the sequence of
the juxtamembrane segment between Arg46 and
Glu54. However, this peptide mimetic did not compete
with [3H]AVP binding to the wild-type V1aR or affect
intracellular signaling, even at 10 ␮M (data not shown).
Fig. 4. Molecular Model of the TM1 Helix of the V1aR Revealing the Alignment of Arg46, Leu50, and Glu54 on One Face
of the Helix
A molecular module of the entire V1aR embedded in a
hydrated lipid bilayer was constructed based on the crystal
structure of bRho, as described in Materials and Methods.
For clarity, only the TM1 helix is shown with the position of
the side chains of Arg46, Leu50, and Glu54 indicated. A, TM1
viewed from above and B, TM1 viewed from within the plane
of the membrane. Residues 46, 50, and 54 are positioned on
the same face of the helix, and each of these residues is
separated by one turn of the helix. C, Secondary structure
prediction of the V1aR N terminus/TM1 helix juxtamembrane
segment. Secondary structure was predicted as described in
Materials and Methods. H, Helix; C, random coil.
relative positions of the Glu and Arg within the N terminus/TM1 helix juxtamembrane segment: 1) an additional
negative charge was inserted between Glu54 and Arg46
([L50E]V1aR); 2) an additional positive charge was inserted between Glu54 and Arg46 ([L50R]V1aR); 3) the
glutamyl was moved one turn higher in the helix so as to
be adjacent to Arg46 ([L50E/E54A]V1aR); 4) the arginyl
was moved one turn lower in the helix so as to be
adjacent to Glu54 ([R46A/L50R]V1aR); 5) the positions of
the glutamyl and arginyl were reversed ([R46E/
E54R]V1aR). Each mutant receptor construct was expressed in HEK 293T cells and pharmacologically characterized with respect to 1) binding the four different
classes of ligand, and 2) AVP-induced second messenger generation. Agonist binding affinity was dramatically
decreased compared with wild-type V1aR for all of the
mutants investigated, with the Kd for AVP increasing
between 1000- to 1900-fold. Antagonist binding, however, was only slightly affected in each case with the
largest change (2- to 5-fold for all classes of antagonist)
being observed when the charges were reversed ([R46E/
E54R]V1aR) (Fig. 5B). Assessment of the signaling capability of these charged residue mutants revealed that
second messenger generation was also compromised
compared with wild-type V1aR (Fig. 6A). The dose-response curves for [L50E]V1aR, [L50R]V1aR, and [L50E/
E54A]V1aR were all right shifted approximately 60-fold
(Fig. 6B), whereas the mutant receptors [R46A/
DISCUSSION
Defining how agonist-receptor interaction differs from
antagonist-receptor interaction at the molecular level
is of fundamental importance. For the V1aR, it has
been demonstrated that the N-terminal domain is required for high-affinity agonist binding but not antagonist binding (6). A similar situation has been reported
for other members of the neurohypophysial hormone
receptor family. For example, the N terminus is required for agonist binding to the OTR (18, 19) and also
to the vasotocin receptor (20), suggesting a common
role for the N-terminal domain in agonist binding
throughout this GPCR subclass. This is supported by
the observation that disruption of AVP binding to a
truncated V1aR could be functionally rescued by a
chimeric construct in which the N terminus of the OTR
replaced the corresponding sequence of the V1aR (6).
Further investigation identified Arg46 within the N terminus of the V1aR as critical for high-affinity agonist
binding but not for binding any class of antagonist (7).
The corresponding residue in the OTR is also an arginyl (Arg34) and furthermore, this arginyl is required for
high-affinity agonist binding to the OTR (21). Indeed,
an arginyl is absolutely conserved at this locus in all
members of the vertebrate neurohypophysial peptide
hormone receptor family cloned to date, suggesting
that this residue fulfils an important common role required specifically for agonist binding throughout the
neurohypophysial hormone receptor family.
In the current study, we have used site-directed mutagenesis to probe the function of the juxtamembrane
segment at the interface of the N terminus and TM1 of
the V1aR. Alanine scanning mutagenesis revealed that
none of the residues between Arg46 and Leu50 at the top
of TM1 were required for high-affinity ligand binding. In
contrast, Glu54, located one turn lower in TM1 than
Leu50, was critical for high-affinity binding of AVP, and
intracellular signaling, but was not required for the binding of any of the three classes of antagonist. The loss of
agonist binding observed when Glu54 was substituted
was not due to aberrant assembly of the receptor, or to
local distortion of the mature protein, as the binding of
both peptide and nonpeptide antagonists was largely
Hawtin et al. • Agonist Binding to the V1aR
Mol Endocrinol, November 2005, 19(11):2871–2881 2877
Fig. 5. Pharmacological Characterization of Mutant V1aRs Possessing Altered Glu and Arg Residues within the N Terminus/TM1
Helix Juxtamembrane Segment
A, Schematic representation of the relative position of arginyl and glutamyl residues within this segment in wild-type and mutant
V1aR constructs. In the ␣-helical conformation, residues 46, 50, and 54 stack on the same face of the helix. The top, middle, and
bottom circles represent the positions of arginyl (black R in a white circle) and glutamyl (white E in a black circle) when present
at residue 46, residue 50, and residue 54, respectively. B, Pharmacological characterization of wild-type and mutant V1aR
constructs. Dissociation constants (Kd) were calculated from IC50 values and corrected for radioligand occupancy as described
in Materials and Methods. Data shown are the mean ⫾ SEM of three separate experiments, each performed in triplicate. CA, Cyclic
peptide antagonist; LA, linear peptide antagonist; SR 49059, nonpeptide antagonist.
unaffected. Moreover, this preservation of antagonist
binding provided us with the means of accurately characterizing changes in agonist binding by using radioligand binding studies with [3H]antagonist as tracer.
Whole-cell radioligand binding studies with [3H]antagonist showed that the cell surface expressions of wildtype V1aR and [E54A]V1aR were comparable. Consequently, the disrupted signaling by [E54A]V1aR was not
due to defective trafficking of the construct to the plasma
membrane. A glutamyl is present at the corresponding
locus to Glu54 in all of the neurohypophysial peptide
hormone receptors cloned to date (Fig. 1), implying that
its functional importance is also conserved in other
members of this GPCR subfamily. The Leu residues that
flank the conserved Glu are also conserved in all VPRs
and OTRs (Fig. 1). Moreover, the sequence motif -R(X3)L/
V(X3)E(X3)L- in the N-terminal/TM1 juxtamembrane domain of the V1aR is conserved throughout the vertebrate
neurohypophysial peptide hormone GPCR family, consistent with functional importance. Leu50 (1.31) could
nevertheless be substituted by Ala with only marginal
effects on the properties of the receptor. Substitution of
Leu58 (1.39) by Ala reduced the affinity of all classes of
ligand by 2- to 9-fold and right shifted the dose-response
curve for InsPs production. As the aliphatic side chain of
Ala is considerably shorter than that of Leu, it has only
limited capability to establish hydrophobic interactions.
Consequently, a localized loosening of hydrophobic interactions between TM1 and neighboring helices within
the TM helical bundle probably underlies the observed
changes in the pharmacological profile of [L58A]V1aR
compared with wild-type V1aR.
The presence of a Glu at position 1.35 is absolutely
conserved throughout the neurohypophysial peptide
hormone receptor family cloned to date (Fig. 1). The side
chain of aspartyl possesses almost identical charge
characteristics to glutamyl but is one methylene shorter.
Nevertheless, we found that Asp cannot substitute for
Glu at position 1.35, not even partially. This indicates that
the presence of the negative charge at residue 54 is
insufficient per se to support high-affinity agonist binding; the negative charge must be correctly positioned by
the extra length provided by the glutamyl side chain.
Furthermore, this observation provides the rationale for
the absolute conservation of Glu (1.35) throughout this
receptor family (Fig. 1). Although mutation of the residue
at this locus in the cholecystokinin B receptor (R57A) also
decreased agonist binding (22), the importance of residue 1.35 for agonist binding/signaling is not a general
feature of family A GPCRs.
The ratio of EC50 to Kd is an indicator of efficacy, i.e.
the likelihood that a receptor will become activated and
2878 Mol Endocrinol, November 2005, 19(11):2871–2881
Fig. 6. Intracellular Signaling by Mutant V1aRs
A, AVP-induced accumulation of InsP–InsP3 in HEK 293T
cells transfected with wild-type V1aR (䡺), [L50E]V1aR (E),
[L50R]V1aR (f), or [L50E/E54A]V1aR (F). Data are the mean ⫾
SEM of three separate experiments each performed in triplicate. Values are expressed as maximum stimulation induced
by AVP at the concentrations stated. B, EC50 and Emax values
of AVP-induced accumulation of InsP–InsP3 in cells expressing wild-type and mutant receptors. Values shown are the
mean ⫾ SEM of three separate experiments performed in
triplicate. NR, No response detectable at 10 ␮M. Emax values
are fold-stimulation over basal.
initiate a functional response once an agonist has bound.
In the current study, this parameter was increased compared with the wild-type V1aR after mutation of Glu54 or
Arg46 (Tables 1–4). These data indicate that these mutant
receptors are much less likely than wild type to bind AVP,
but once AVP has bound, the mutant receptors are more
likely than wild-type V1aR to become activated. This implies that Glu54 and Arg46 are constraining residues that
contribute to maintaining the conformational switch of
the V1aR in the off state. Consequently, mutation of these
residues had the dual effect of 1) decreasing agonist
affinity and 2) promoting the agonist-induced active conformation resulting from loss of stabilizing constraints on
the ground state of the receptor.
It is noteworthy that the pharmacological effects of
mutating Glu54 or Arg46 are very similar, with substitution
of either residue profoundly decreasing agonist affinity
Hawtin et al. • Agonist Binding to the V1aR
and intracellular signaling but not affecting antagonist
binding (peptide, nonpeptide, cyclic, or linear). Furthermore, the structural requirements at both loci are very
specific. The construct [E54D]V1aR did not display wildtype pharmacology and, likewise, none of the 19 encoded amino acids could replace arginyl at position 46,
including lysyl (23). These observations raised the possibility that Glu54 and Arg46 form a mutual ionic interaction, which is required for the V1aR to adopt a highaffinity conformation for AVP. Molecular modeling of the
V1aR indicates that the ␣-helical structure of TM1 in the
receptor extends up to Arg46, which positions Glu54 and
Arg46 just two turns apart on the same face of the helix
(Fig. 4, A and B). Indeed, secondary structure prediction
for this region of the V1aR also suggests an ␣-helical
conformation extending from TM1 up to Arg46 (Fig. 4C).
Such an extension of ␣-helix out of the lipid bilayer is not
unusual. Spin-labeling studies have shown that the third
intracellular loop of bRho, immediately juxtaposed to the
membrane, is capable of adopting an ␣-helical fold,
which extends TM5 and TM6 by up to three turns out of
the membrane (24). It is well documented that an ␣-helix
can be made to kink by the introduction of a prolyl (25).
Introducing a prolyl between Glu54 and Arg46 in the construct [L50P]V1aR had a similar effect on the receptor
pharmacology as substituting Glu54 or Arg46, consistent
with disruption of an ␣-helix containing these two residues. The effect was not due to substituting Leu50 because the pharmacological profile of [L50A]V1aR was
similar to the wild-type receptor. In this ␣-helical conformation, residues Arg46, Leu50, and Glu54 stack on the
same face of the helix (Fig. 4, A and B). The effect of
altering the distribution, and nature, of the charge on this
face was investigated systematically (Fig. 5A). Insertion
of an additional charge (positive or negative) at position
50 selectively disrupted agonist binding and perturbed
intracellular signaling. These mutations (L50E and L50R)
position a charge directly between Glu54 and Arg46,
which would have the dual effect of attracting one of the
side chains and repelling the other. In addition, the close
proximity of a counter ion would reduce the effect of the
local charge. This study also establishes that the negative and positive charges, provided by Glu54 and Arg46,
respectively, need to be precisely positioned. Moving
either the Glu or the Arg one turn ([L50E/E54A]V1aR,
[R46A/L50R]V1aR, respectively) selectively disrupted agonist binding and cell signaling. Repositioning the Arg
seemed particularly disruptive with respect to cell signaling, as [R46A/L50R]V1aR failed to signal even with a
high concentration of AVP (10 ␮M).
If Arg46 and Glu54 interact in the wild-type V1aR, then
a construct possessing a double mutation in which these
two residues are reversed would be expected to preserve this mutual interaction and therefore bind agonist
with high affinity and signal effectively. Such a rationale
was applied to demonstrate an interaction between
Asn87 (2.50) and Asp318 (7.49) in TM2 and TM7, respectively, of the GnRH receptor (26). However, mutual interaction between Glu54 and Arg46 was excluded when a
V1aR construct with reciprocal mutation ([R46E/
Hawtin et al. • Agonist Binding to the V1aR
E54R]V1aR) failed to signal and had very low affinity for
AVP. The overall fold of this construct was not significantly different from wild-type V1aR, however, because
[R46E/E54R]V1aR exhibited only a slight reduction in
binding affinity for a range of antagonists compared with
wild-type V1aR and had similar cell surface expression.
Consequently, Glu54 and Arg46 are both required for
high-affinity agonist binding but are not prerequisites for
high-affinity antagonist binding. Although these two residues are important functionally, a peptide mimetic encompassing this segment of the receptor was unable to
perturb AVP binding to wild-type V1aR or affect signaling.
This study establishes that Glu54 and Arg46 operate independently, fulfilling two different roles that support
high-affinity agonist-receptor interaction and effective intracellular signaling. A feasible mechanism for the importance of Glu54 is suggested by molecular modeling of
AVP docked to the V1aR (27). This indicated that Glu54
was one of several residues delimiting the ligand-binding
cavity; therefore it is plausible that Glu54 may interact
directly with AVP. In contrast, Arg46 is positioned where
it can interact with other extracellular structures in the
receptor, which apparently allows Arg46 to constrain the
ground state of the receptor and form part of the conformational switch controlling activation by agonist (23).
In conclusion, we have established that a glutamyl is
specifically required at position 54 in the V1aR to support
high-affinity agonist binding and intracellular signaling,
but this residue does not contribute to antagonist binding. In addition, Leu58 also has a role in AVP-induced
signaling. The pharmacological importance of Glu54 reported in this study is analogous to the agonist-specific
requirement for an arginyl at residue 46 (23). Although
Glu54 and Arg46 are in close proximity in the N terminus/
TM1 juxtamembrane segment of the receptor, they function independently to fulfil different roles required for
high-affinity agonist binding and signaling. This explains
the very high degree of conservation of Glu and Arg at
these loci throughout the entire family of neurohypophysial peptide hormone receptors. In a wider context, it is
possible that the importance of the N-terminal/TM1 domain to receptor function may not be restricted to the
neurohypophysial peptide hormone family of GPCRs.
MATERIALS AND METHODS
Material
AVP was purchased from Sigma Chemical Co. (St. Louis,
MO). The cyclic antagonist 1-(␤-mercapto-␤,␤-cyclopentamethylenepropionic acid), 2-(O-methyl)tyrosine AVP (d(CH2)5
Tyr(Me)2AVP), and linear antagonist phenylacetyl-D-Tyr(Me)2
Arg6Tyr(NH2)9AVP were from Bachem U.K. (St. Helens, UK).
SR 49059 was obtained from Sanofi Recherche (Toulouse,
France). Cell culture media, buffers, and supplements were
purchased from GIBCO (Uxbridge, UK). Restriction enzymes
NheI, Eco81I and SdaI were obtained from MBI Fermentas
(Sunderland, UK), and BlpI was obtained from New England
BioLabs, Inc. (Hitchen, UK).
Mol Endocrinol, November 2005, 19(11):2871–2881 2879
Mutant Receptor Constructs
Mutation of the V1aR was made using a PCR approach as
described previously (28). The mutant receptor construct
[E54R]V1aR was made using the sense oligonucleotide:
5⬘-G-GGG-GCC-TTA-GGG-GAC-GTA-CGC-AAT-GAG-GAGCTG-GCT-AAG-CTG-AGA-ATC-GCT-GTG-CTA-GC-3⬘ and using R46A-V1aR construct as template (7). This primer contained a
unique Eco81I restriction site (italics and underlined) and five base
changes (shown in bold) to generate the appropriate base changes
for the Glu543Arg substitution and two new unique BlpI and NheI
restriction sites (underlined) respectively. This PCR product was
subcloned into the V1aR coding sequence in the mammalian expression plasmid pcDNA3 (Invitrogen) utilizing the previously engineered Eco8I1 (7) and SdaI restriction sites.
Mutant receptor constructs [E48A]V1aR, [E49A]V1aR,
[L50A]V1aR, [L50E]V1aR, [L50P]V1aR, [L50R]V1aR, [L50W]V1aR,
[L50E/E54A]V1aR and [R46A/L50R]V1aR were made using
sense oligonucleotides: 5⬘-G-GGG-GCC-TTA-GGG-GAC-GTACGC-AAT-GCG-GAG-C-3⬘, 5⬘-G-GGG-GCC-TTA-GGG-GACGTA-CGC-AAT-GAG-GCG-CTG-G-3⬘,
5⬘-G-GGG-GCC-TTAGGG-GAC-GTA-CGC-AAT-GAG-GAG-GCG-GCC-AAA-CTG-G3⬘, 5⬘-G-GGG-GCC-TTA-GGG-GAC-GTA-CGC-AAT-GAG-GAGGAG-GCC-AAA-CTG-G-3⬘, 5⬘-G-GGG-GCC-TTA-GGG-GACGTA-CGC-AAT-GAG-GAG-CCG-GCC-AAA-CTG-G-3⬘, 5⬘G-GGG-GCC-TTA-GGG-GAC-GTA-CGC-AAT-GAG-GAGCGG-GCC-AAA-CTG-G-3⬘, 5⬘-G-GGG-GCC-TTA-GGGGAC-GTA-CGC-AAT-GAG-GAG-TGG-GCC-AAA-CTG-G-3⬘, 5⬘G-GGG-GCC-TTA-GGG-GAC-GTA-CGC-AAT-GAG-GAG-GAGGCC-AAA-CTG-GCA-ATC-GC-3⬘, 5⬘-G-GGG-GCC-TTA-GGGGAC-GTA-GCC-AAT-GAG-GAG-CGG-GCC-AAA-CTG-G-3⬘
respectively and using [R46A]V1aR-pcDNA3 as template.
Each primer contained the unique Eco81I restriction site (underlined) and appropriate bases changes (shown in bold) to
introduce specific mutation(s) at the desired location within
the V1aR coding sequence. Each PCR product was subcloned into the pcDNA3-V1aR utilizing Eco81I and SdaI restriction sites.
The mutant receptor constructs [L58A]V1aR and [R46E/
E54R]V1aR were made using sense oligonucleotides: 5⬘-GGGCTG-GCT-AAG-CTG-GAA-ATC-GCT-GTG-GCG-GCA-GTG3⬘, and 5⬘-G-GGG-GCC-TTA-GGG-GAC-GTA-GAG-AATGAG-GAG-CTG-G-3⬘ respectively, using [E54R]V1aR as template. The [L58A] and [R46A/E54R] PCR products were subcloned into the V1aR-pcDNA3 utilizing either BlpI or Eco81I
(underlined in each primer, respectively) and SdaI restriction
sites. The mutant constructs [E54D]V1aR and [E54A]V1aR were
made using antisense oligonucleotides 5⬘-AAT-CAC-TGCTAG-CAC-AGC-GAT-ATC-CAG-CTT-AGC-C-3⬘ and 5⬘AAT-CAC-TGC-TAG-CAC-AGC-GAT-CGC-CAG-CTTAGC-C-3⬘, respectively. PCR products were subcloned into
V1aR-pcDNA3 utilizing unique HindIII and NheI (underlined)
restriction sites. All receptor constructs were confirmed by
automated fluorescent sequencing (University of Birmingham, UK).
Cell Culture and Transfection
HEK 293T cells were routinely cultured in DMEM supplemented with 10% (vol/vol) fetal calf serum (FCS) in humidified
5% (vol/vol) CO2 in air at 37 C. Cells were seeded at a density
of approximately 5 ⫻ 105 cells per 100-mm dish and transfected after 48 h using a calcium phosphate precipitation
protocol with 10 ␮g DNA/dish (21, 28).
Radioligand Binding Assays
A washed cell membrane preparation of HEK 293T cells,
transfected with the appropriate receptor construct, was prepared as previously described (21), and the protein concentration was determined using the BCA protein assay kit
(Pierce Chemical Co., Rockford, IL) with BSA as standard.
2880
Mol Endocrinol, November 2005, 19(11):2871–2881
Radioligand binding assays were performed as previously
described (29) using either the natural agonist [Phe3-3,4,53
H]AVP, (64.2 Ci/mmol; PerkinElmer, Beaconsfield, UK)
or the V1aR-selective peptide antagonist [Phe3-3,4,53
H]d(CH2)5Tyr(Me)2AVP (99 Ci/mmol; DuPont NEN, UK)
(15) as tracer ligand. Binding data were analyzed by nonlinear regression to fit theoretical Langmuir binding isotherms to
the experimental data using the Fig. P program (Biosoft, Milltown, NJ). Individual IC50 values obtained for competing ligands
were corrected for radioligand occupancy as described elsewhere (30) using the radioligand affinity (Kd) experimentally determined for each construct. Cell-surface expression of wildtype and mutant receptors was determined for each construct
individually using whole-cell binding assays as described previously (31).
Hawtin et al. • Agonist Binding to the V1aR
Synthesis of a Peptide Mimetic Corresponding
to the Functionally Important Segment of the N
Terminus/TM1 Interface
The peptide N-acetyl-RNEELAKLE-amide was synthesized
on a 10 ␮mol scale using N␣-9-fluoremylmethoxycarbonyl
(Fmoc)-protected amino acids with conventional solid-phase
methodology by Alta Bioscience (University of Birmingham,
UK). The structure of the peptide was confirmed by mass
spectrometry using a Bruker Biflex IV MALDI-TOF instrument
[Bruker BioSpin Ltd. (Coventry, UK)], and a matrix of gentisic
acid [1 mg/ml in methanol-chloroform (1:1)], which revealed
that the synthetic peptide had the expected molecular weight
(MH⫹ ⫽ 1143).
Acknowledgments
AVP-induced Inositol Phosphate Production
HEK 293T cells were seeded at a density of 2.5 ⫻ 105 cells
per well in poly D-lysine-coated 12-well plates and transfected after 24 h using Transfast (Promega, Southampton,
UK). The assay for AVP-induced accumulation of InsPs was
based on that described previously (32, 33). Essentially, 16 h
after transfection, medium was replaced with inositol-free
DMEM containing 1% (vol/vol) FCS and 2 ␮Ci/ml myo-[23
H]inositol (22.0 Ci/mmol; PerkinElmer) for 24 h. Cells were
washed twice with PBS, then incubated in inositol-free medium containing 10 mM LiCl for 30 min, after which AVP was
added at the concentrations indicated for a further 30 min.
Incubations were terminated by adding 0.5 ml of 5% (vol/vol)
perchloric acid containing 1 mM EDTA and 1 mg/ml phytic
acid hydrolysate. Samples were neutralized with 1.2 M KOH,
10 mM EDTA, 50 mM HEPES on ice for 1 h, insoluble material
sedimented at 12,000 ⫻ g for 5 min and supernatants loaded
onto Bio-Rad AG1-X8 columns (formate form) in 10 ml of
water. After the elution of inositol and glycerophosphoinositol
(10 ml of 25 mM NH4COOH containing 0.1 M HCOOH), a
mixed inositol fraction containing mono-, bis-, and trisphosphates (InsP–InsP3) was eluted with 10 ml of 850 mM
NH4COOH containing 0.1 M HCOOH, mixed with UltimaFlo
AF scintillation cocktail (PerkinElmer), and radioactivity was
quantified by liquid scintillation spectroscopy. EC50 values
were determined by nonlinear regression after fitting of logistic sigmoidal curves to the experimental data.
Secondary Structure Prediction
The sequence of the V1aR was submitted to the hidden
Markov model-based protein structure prediction, SAM-T02
(34), which utilizes an hidden Markov model engine to search
for homologous proteins from which a sequence alignment is
produced and a structure prediction obtained.
Receptor Modeling
The V1aR sequence was aligned against the recently reported
crystal structure coordinates of bRho using CLUSTALW (35).
The alignment was then used to generate homology models
using MODELLER version 6.2 (36). A collection of 200 model
structures was generated and ranked based on an objective
function provided by MODELLER version 6.2. From this ensemble, a single structure was selected for further analysis.
Further refinement of the homology model was achieved
through molecular dynamics simulations of the receptor embedded in a hydrated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayer. Molecular dynamics simulations were carried
out using the GROMOS96 force-field parameters, with minor
modifications, as implemented in GROMACS (37).
We thank Mrs. Rosemary A. Parslow for excellent technical
assistance, Professor Ülo Langel (Stockholm University,
Sweden) for useful discussions on the peptide mimetic, and
Dr. Claudine Serradeil-Le Gal (Sanofi Recherche, France) for
providing a sample of SR 49059.
Received April 11, 2005. Accepted June 17, 2005.
Address all correspondence and requests for reprints to:
Dr. Mark Wheatley, School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom. E-mail: [email protected].
This work was supported by grants from the Wellcome
Trust and the Biotechnology and Biological Sciences Research Council (to M.W.).
Current address for S.R.H.: Institute of Cell Signalling,
School of Biomedical Sciences, Queen’s Medical Centre,
University of Nottingham, Nottingham NG7 2UH, United
Kingdom.
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