Báo cáo khoa học: An arginyl in the N-terminus of the V1a vasopressin receptor is part of the conformational switch controlling activation by agonist docx

An arginyl in the N-terminus of the V
1a
vasopressin receptor is part
of the conformational switch controlling activation by agonist
Stuart R. Hawtin
1,
*, Victoria J. Wesley
1
, John Simms
1
, Rosemary A. Parslow
1
, Alice Miles
1
, Kim McEwan
1
,
Mary Keen
2
and Mark Wheatley
1
1
School of Biosciences and
2
Department of Pharmacology, Division of Neuroscience, The Medical School, University of Birmingham,
Edgbaston, Birmingham, UK
Defining how the agonist–receptor interaction differs from
that of the antagonist–receptor and understanding the
mechanisms of receptor activation are fundamental issues in
cell signalling. The V
1a

vasopressin receptor (V
1a
R) is a
member of a family of related G-protein coupled receptors
that are activated by neurohypophysial peptide hormones,
including vasopressin (AVP). It has recently been reported
that an arginyl in the distal N-terminus of the V
1a
Riscritical
for binding agonists but not antagonists. To determine
specific features required at this locus to support high affinity
agonist binding and second messenger generation, Arg46
was substituted by all other 19 encoded amino acids. Our
data establish that there is an absolute requirement for
arginyl, as none of the [R46X]V
1a
R mutant constructs sup-
ported high affinity agonist binding and all 19 had defective
signalling. In contrast, all of the mutant receptors possessed
wildtype binding for both peptide and nonpeptide antago-
nists. The ratio of K
i
to EC
50
, an indicator of efficacy, was
increased for all substitutions. Consequently, although
[R46X]V
1a
R constructs have a lower affinity for agonist,
once AVP has bound all 19 are more likely than the wildtype

V
1a
R to become activated. Therefore, in the wildtype V
1a
R,
Arg46 constrains the inactive conformation of the receptor.
On binding AVP this constraint is alleviated, promoting the
transition to active V
1a
R. Our findings explain why arginyl is
conserved at this locus throughout the evolutionary lineage
of the neurohypophysial peptide hormone receptor family of
G-protein coupled receptors.
Keywords: GPCR; vasopressin; ligand binding; cell signa-
ling; peptide hormone.
The neurohypophysial hormones are nonapeptides, ami-
dated at the C-terminus and with an intramolecular
disulphide bond between positions one and six, which
creates a 20-membered ring and a tripeptide tail. Peptides of
the vasopressin (AVP)-like family possess a positively
charged residue at position eight whereas those of the
oxytocin-like family have a neutral residue. Lower verte-
brates possess vasotocin (AVT; [Ile3]vasopressin) rather
than AVP, whereas isotocin ([Ser4,Ile8]oxytocin) and meso-
tocin ([Ile8]oxytocin) are the evolutionary precursors of
oxytocin [1]. The receptors that mediate the effects of these
hormones are G-protein coupled receptors (GPCRs) and
include the isotocin receptor, the mesotocin receptor, the
oxytocin receptor (OTR), the vasotocin receptor and three
subtypes of vasopressin receptor: V

1a
,V
1b
and V
2
(V
1a
R,
V
1b
RandV
2
R respectively). In addition to the characteristic
architecture of GPCRs [2], the neurohypophysial peptide
hormone receptors exhibit certain conserved sequence
motifs and share related pharmacologies (reviewed in
[3–6]). This has allowed these receptors to be classified as
a subfamily of GPCRs.
The V
1a
R mediates a plethora of responses to AVP in
addition to the well-characterized vasopressor effect [3].
Consequently, this receptor subtype is widely distributed
and generates nearly all of the physiological actions of AVP
with the notable exceptions of antidiuresis (V
2
R) and
adrenocorticotropic hormone secretion (V
1b
R). This has

been the stimulus for the development of a range of V
1a
R
antagonists, initially peptides [7] and more recently non-
peptides [8,9]. Although agonists and antagonists exhibit
competitive binding to the V
1a
R, only agonists promote the
active receptor conformation with subsequent second
messenger generation. Understanding how the agonist–
receptor interaction differs from that of the antagonist–
receptor and defining the mechanisms of receptor activation
are fundamental issues in cell signalling.
The N-terminus of the V
1a
R provides agonist-specific
binding epitopes, as truncation of the distal segment of
the V
1a
R N-terminus prevents high affinity AVP binding
but does not affect antagonist binding [10]. A similar
Correspondence to M. Wheatley, School of Biosciences, University of
Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
Fax: + 44 121 414 5925, Tel.: + 44 121 414 3981,
E-mail:
Abbreviations: AVP, [Arg8]vasopressin; AVT, [Arg8]vasotocin;
GPCR, G-protein coupled receptor; InsP, inositol phosphate; InsP
3
,
inositol trisphosphate; OTR, oxytocin receptor; [R46X]V

1a
R,
Arg fi mutant vasopressin V
1a
receptor at position 46;
V
1a
R, vasopressin V
1a
receptor; V
1b
R, vasopressin V
1b
receptor;
V
2
R, vasopressin V
2
receptor.
*Present address: Institute of Cell Signalling, Medical School,
Queen’s Medical Centre, Nottingham, UK.
(Received 13 May 2003, revised 25 September 2003,
accepted 3 October 2003)
Eur. J. Biochem. 270, 4681–4688 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03865.x
situation has been reported for other members of the
neurohypophysial hormone receptor family; for example,
the distal N-terminus is required for agonist binding to
the OTR [11,12] and also to the vasotocin receptor [13],
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 V
1a
R could be functionally rescued
by a chimeric construct in which the N-terminus of the
OTR is replaced with the corresponding sequence of
the V
1a
R [10]. The role of the N-terminus has been
addressed recently by alanine-scanning mutagenesis of the
N-terminus of the V
1a
R; this revealed that a single residue
(Arg46) located in the distal segment of this domain is
critical for high affinity agonist binding but not antag-
onist binding [14]. 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
[15]. Indeed, an arginyl is completely conserved at this
locus in all members of the neurohypophysial peptide
hormone receptor family cloned to date, suggesting that
this residue fulfils an important common role required
specifically for agonist binding throughout this subfamily
of GPCRs.
The aim of this study was to understand the structural
requirements at this key locus of the V
1a
R which endow
high affinity agonist binding, by systematically mutating the
critical arginyl to all the other 19 amino acids encoded in

mRNA. In addition, this study identifies Arg46 as part of
the conformational switch mechanism which controls
conversion of inactive V
1a
R to active receptor in response
to AVP.
Experimental procedures
Materials
AVP was purchased from Sigma. The cyclic antagonist,
1-(b-mercapto-b,b-cyclopentamethylenepropionic acid), 2-
(O-methyl)tyrosine AVP [d(CH
2
)
5
Tyr(Me)2AVP] and linear
antagonist, phenylacetyl-
D
-Tyr(Me)2Arg6Tyr(NH
2
)9AVP
were from Bachem (St Helens, UK). The nonpeptide
antagonist (SR 49059) was provided by Sanofi Recherche
(Toulouse, France). Cell culture media, buffers and supple-
ments were purchased from Gibco (Uxbridge, UK).
Restriction enzymes Eco81I, Pfl23II and SdaIwere
obtained from MBI fermentas (Sunderland, UK).
Mutant receptor constructs
Mutation of Arg46 to each of the 19 encoded amino acids
was made by a PCR approach. Mutant sense oligonucle-
otides (5¢-GGGGGCCTTAGGGGACGTAXXXAATGA

GGAGCTGG-3¢) contained the appropriate base change
(shown as XXX) for each of the corresponding Arg46 fi
Xaa46 substitutions, and a unique Eco81I restriction site
(bold). The PCR cycling conditions were as follows:
denaturing, 94 °C (1 min); annealing, 60 °C (2 min); exten-
sion, 72 °C (1 min) for 30 cycles followed by extension at
72 °C (7 min). All mutant PCR products were subcloned
into the receptor utilizing unique Eco81I and SdaI
restriction sites. All receptor constructs were confirmed
by automated fluorescent sequencing (Alta Bioscience,
Birmingham, UK).
Cell culture and transfection
HEK 293T cells were routinely cultured in Dulbecco’s
modified Eagles medium supplemented with 10% (v/v)
fetal bovine serum, penicillin (100 IUÆmL
)1
) and strepto-
mycin (100 lgÆmL
)1
) in humidified 5% (v/v) CO
2
in air at
37 °C. Cells were seeded at a density of approximately
5 · 10
5
cells per 100 mm dish and transfected after 48 h
using a calcium phosphate precipitation protocol with
10 lg DNA per dish.
Radioligand binding assays
A washed cell membrane preparation of HEK 293T cells,

transfected with the appropriate receptor construct, was
prepared as previously described [16] and the protein
concentration determined using the BCA protein assay kit
(Pierce Chemical Co., Tattenhall, Cheshire, UK) with
bovine serum albumin as the standard. Radioligand binding
assays were performed as described previously [17] using
either the natural agonist [Phe3-3,4,5-
3
H]AVP (68.5 CiÆ
mmol
)1
; DuPont NEN, Stevenage, Herts, UK) or the
V
1a
R-selective peptide antagonist [Phe3-3,4,5-
3
H] d(CH
2
)
5
-
Tyr(Me)2AVP (99 CiÆmmol
)1
; DuPont NEN) [18] as the
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,
Cambridge, UK). Individual IC
50
values obtained for
competing ligands were corrected for radioligand occu-
pancy as described [19] using the radioligand affinity (K
i
)
experimentally determined for each individual construct.
AVP-induced inositol phosphate production
HEK 293T cells were seeded at a density of 2.5 · 10
5
cells
per well in poly
D
-lysine coated 12 well plates and
transfected after 24 h using Transfast
TM
(Promega).
The assay for AVP-induced accumulation of inositol
phosphates was based on that described previously [20].
Essentially, at 16 h post-transfection, medium was
replaced with inositol-free Dulbecco’s modified Eagles
medium containing 1% (v/v) fetal bovine serum and
2 lCiÆmL
)1
myo-[2-
3
H]inositol (22.0 CiÆmmol
)1

; DuPont
NEN) for 24 h. Cells were washed twice with NaCl/P
i
and incubated in inositol-free medium containing 10 m
M
LiCl for 30 min, after which AVP was added at the
concentrations indicated for a further 30 min. Incuba-
tions were terminated by adding 0.5 mL of 5% (w/v)
perchloric acid containing 1 m
M
EDTA and 1 mgÆmL
)1
phytic acid hydrolysate (final concentrations). Samples
were neutralized with 1.2
M
KOH, 10 m
M
EDTA,
50 m
M
Hepes on ice for 1 h, insoluble material was
sedimented at 12 000 g for 5 min and supernatants were
loaded onto 0.8 mL AG1-X8 (formate form; Bio-Rad
Laboratories, Hemel Hempstead, UK). A mixed inositol
fraction containing mono-, bis- and tris-phosphates
(InsP–InsP
3
) was eluted with 10 mL of 850 m
M
NH

4
COOH containing 0.1
M
HCOOH as described [21]
and quantified by scintillation counting.
4682 S. R. Hawtin et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Results
The effect of systematic substitution of Arg46
on agonist binding
The structural requirements of the residue at position 46 in
the N-terminus of the V
1a
R (Fig. 1) were investigated, with
respect to supporting high affinity agonist binding, by site-
directed mutagenesis. The wildtype Arg46 was systematic-
ally substituted by all the other 19 encoded amino acids.
These receptor constructs were then characterized pharma-
cologically by radioligand binding assay after expression
in HEK 293T cells and compared to wildtype V
1a
R. The
wildtype and mutant receptors were expressed at the same
level of approximately 1–2 pmolÆmg
)1
protein. Four differ-
ent classes of ligand were available to probe the ligand
binding site: (a) peptide agonist; (b) cyclic peptide antagonist
possessing a disulphide bond, 20-membered ring and short
peptide tail; (c) linear peptide antagonist and (d) nonpeptide
antagonist. The binding affinities of the cyclic peptide

antagonist [d(CH
2
)
5
Tyr(Me)2AVP] [18], the linear peptide
antagonist [phenylacetyl-
D
-Tyr(Me)2Arg6Tyr(NH
2
)9AVP]
[22] and the nonpeptide antagonist (SR 49059) [23] for all
the 19 mutant receptors engineered, were comparable to
wildtype V
1a
R (Table 1). This was important, as it allowed
accurate quantification of the pharmacological characteris-
tics of each mutant construct by radioligand binding studies
using [
3
H]d(CH
2
)
5
Tyr(Me)2AVP as the tracer ligand.
Furthermore, normal antagonist binding established that
substitution of the arginine had not resulted in a distorted or
misfolded receptor protein. In contrast to the three classes of
antagonist, the binding of AVP was dramatically affected
by the substitution of Arg46, with the affinity of AVP for all
the 19 mutant receptor constructs decreasing between 700-

fold and 3000-fold compared to the wildtype (Table 1). As
an example, competition binding curves for one of the 19
engineered mutant receptors are presented in Fig. 2A,
which shows AVP and the nonpeptide antagonist SR 49059
binding to wildtype V
1a
R and the mutant construct
[R46A]V
1a
R. The decrease in agonist affinity observed
when Arg46 was mutated was apparent in both the presence
and absence of 5¢-guanylylimidodiphosphate (Fig. 2A).
Consequently, the decrease in agonist affinity did not
merely reflect uncoupling of the receptor–G-protein com-
plex. No residue other than the wildtype arginine could
support high affinity agonist binding. The inability to bind
agonist was universal and was observed regardless of the
physico-chemical characteristic of the residue side-chain, be
it positively charged, negatively charged, polar, aromatic or
aliphatic (Table 1). Addition of guanidinium ion (up to
10 m
M
) to the [R46A]V
1a
R construct did not rescue agonist
binding (data not shown).
The effect of systematic substitution of Arg46
on intracellular signalling
The stimulation of accumulated InsPs by increasing
concentrations of AVP, was assayed for each of the 19

mutant constructs and the dose–response characteristics
compared to the wildtype V
1a
R. None of the 19 encoded
amino acids could substitute the Arg46 and retain the
wildtype V
1a
R second messenger generation. Dose–
response curves for AVP-stimulated accumulation of
InsP–InsP
3
by the wildtype V
1a
R and a representative
mutant construct ([R46A]V
1a
R) are presented in Fig. 2B.
The degree of perturbation of second messenger generation
was dictated by the identity of the residue present at position
46, with the EC
50
for AVP-induced InsP–InsP
3
accumula-
tion increasing from between 10-fold to 600-fold compared
to the wildtype V
1a
R (Fig. 3B). The acidic residues at
position 46 in [R46E]V
1a

R and [R46D]V
1a
Rwerethemost
detrimental to signalling. This effect was due predominantly
to the negative charge, as removal of this charge in the
constructs [R46Q]V
1a
R and [R46N]V
1a
RincreasedtheE
max
andresultedinEC
50
values that were 28 fold and 14 fold
lower than their respective acids (Table 1). Histidine was the
least disruptive substitution, as the EC
50
of [R46H]V
1a
R
was only 10 fold higher than wildtype V
1a
R, although the
E
max
was depressed. Lysine could not replace arginine
because [R46K]V
1a
R was no better than [R46P]V
1a

R,
[R46V]V
1a
Ror[R46A]V
1a
R at signalling. [R46M]V
1a
R,
[R46C]V
1a
R and the hydroxyl-containing [R46S]V
1a
Rand
[R46T]V
1a
R were all similarly impaired with EC
50
values
increased approximately 100 fold over the wildtype. The
addition of hydroxyl to the aromatic ring of phenylalanine
had a neutral effect as the sensitivity of [R46F]V
1a
Rand
[R46Y]V
1a
R to AVP was almost identical. The residues Trp
and Gly represent the two extremes of side-chain size,
however, both [R46W]V
1a
Rand[R46G]V

1a
R exhibited a
similar increase in the EC
50
of AVP-induced InsP produc-
tion of  150 fold. A similar perturbation was observed with
the branched aliphatic substitutions in [R46L]V
1a
Rand
[R46I]V
1a
R. The wildtype V
1a
R and the 19 mutant V
1a
R
constructs are arranged in rank order with respect to
binding in Fig. 3A and with respect to second messenger
Fig. 1. Schematic diagram of the V
1a
R. The V
1a
R is illustrated as seven
a-helical transmembrane domains traversing the lipid bilayer. The
enlarged circle shows the amino acid sequence of the distal N-terminus
of the V
1a
R and indicates the position of Arg46 that was subjected to
systematic investigation in this study. Glycosylation sites which have
been shown to be modified by oligosaccharide [29] are indicated by

branched structures.
Ó FEBS 2003 The N-terminal switch of the V
1a
R(Eur. J. Biochem. 270) 4683
Table 1. Pharmacological profile of [R46X]V
1a
Rs. Mutant V
1a
Rs were expressed in HEK 293T cells and characterized pharmacologically. Dis-
sociation constants (K
i
)werecalculatedfromIC
50
values after correcting for the radioligand occupancy as described in Experimental procedures.
EC
50
and E
max
values relate to AVP-induced accumulation of InsP–InsP
3
in cells expressing wildtype (WT) or mutant receptors. Values shown are
the mean ± SEM of three separate experiments performed in triplicate. Data for R46A, R46K, R46L and R46E are taken from [14]. CA, cyclic
peptide antagonist; LA, linear peptide antagonist; SR 49059, nonpeptide antagonist.
4684 S. R. Hawtin et al. (Eur. J. Biochem. 270) Ó FEBS 2003
generation in Fig. 3B. Collectively, these results established
that Arg46 has a critical role in agonist activation of the
V
1a
R.
Discussion

Receptors which mediate the effects of the neurohypophy-
sial peptide hormone family are structurally and pharma-
cologically related and as such form a subclass of GPCRs
(reviewed in [3–6]). It has been shown recently that the
N-terminus of the V
1a
R provides agonist-specific binding
epitopes. Consequently, truncation of the V
1a
R N-terminus
prevented high affinity AVP binding but did not affect
antagonist binding [10]. A single arginyl is conserved in the
distal N-terminus of all V
1a
RsandOTRsclonedtodate,
suggesting functional importance (Fig. 1). This has now
been confirmed experimentally, as substitution of this
residue by alanine in the rat V
1a
R and the human OTR
Fig. 2. Pharmacological characterization of [R46A]V
1a
R. (A) Compe-
tition radioligand binding studies with agonist AVP in the absence
(h,j) or presence (n,m)of10
)4
M
5¢-guanylylimidodiphosphate, or
nonpeptide antagonist SR 49059 (s,d) were performed using a
membrane preparation of HEK 293T cells transiently transfected with

wildtype V
1a
R (open symbols) or [R46A]V
1a
R (filled symbols). 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(CH
2
)
5
Tyr(Me)2AVP (10 l
M
). A
theoretical Langmuir binding isotherm has been fitted to the experi-
mental data as described in Experimental procedures. (B) AVP-
induced accumulation of mono-, bis- and tris-phosphates (InsP–InsP
3
)
in HEK 293T cells transfected with wildtype V
1a
R(h)or[R46A]V
1a
R
mutant (j). Data are the mean ± SEM of three separate experiments
each performed in triplicate. Values are expressed as percent maximum
stimulation induced by AVP at the stated concentrations.
Fig. 3. Effect of substitution of Arg46 of the V
1a
R by all other encoded

amino acids. Wildtype V
1a
R(wt)and[R46X]V
1a
R constructs were
expressed in HEK 293T cells and characterized pharmacologically. (A)
Rank order of [R46X]V
1a
R constructs with respect to the binding
affinity (pK
i
) of AVP and (B) rank order of [R46X]V
1a
Rconstructs
with respect to pEC
50
value for AVP-induced accumulation of mono-,
bis- and tris-phosphates (InsP–InsP
3
). Data shown are the mean ±
SEM of three separate experiments each performed in triplicate. Basal
values (mean ± SEM) were 1108 ± 248, 956 ± 206, 958 ± 245,
1240 ± 207, 1064 ± 166, 996 ± 175, 968 ± 139, 1076 ± 213,
1024 ± 197, 1184 ± 117, 1228 ± 230, 1045 ± 205, 1252 ± 241,
1073 ± 186, 978 ± 169, 1037 ± 233, 1214 ± 206, 1008 ± 185,
1163 ± 202 and 1236 ± 242 d.p.m. for wildtype V
1a
R, R46H, R46Q,
R46N, R46P, R46V, R46F, R46Y, R46A, R46K, R46C, R46S,
R46M, R46G, R46I, R46T, R46W, R46L, R46D and R46E mutant

receptors, respectively. Mock-transfected cells did not bind ligand or
exhibit AVP-induced accumulation of inositol phosphates.
Ó FEBS 2003 The N-terminal switch of the V
1a
R(Eur. J. Biochem. 270) 4685
profoundly and selectively disrupted agonist binding and
signalling [14,15].
Having established the functional necessity of Arg46 in
the V
1a
R it was important to identify the features of the
arginyl side-chain that supported high affinity agonist
binding. A comprehensive approach was undertaken in
which each of the 19 alternative amino acids encoded by
mRNA were substituted at position 46 and the biological
characteristics of the mutant receptors assessed. None of the
other amino acids could replace Arg46 whilst still main-
taining the wildtype pharmacological characteristics with
respect to either agonist binding (Fig. 3A) or second
messenger generation (Fig. 3B). This was particularly
noticeable for AVP binding, where the affinity (K
i
)was
almost uniformly impaired irrespective of the nature of the
substitution (Fig. 3A). In contrast, second messenger gen-
eration was more sensitive to the amino acid at residue 46,
with the EC
50
value for AVP-induced InsP production
varying between 10 fold higher than the wildtype to 600-fold

higher (Fig. 3B). Lys46 was not an effective substitute for
Arg46. A major feature of the arginyl side-chain is the
guanidinium moiety. High concentrations of guanidinium
ion (10 m
M
) however, could not endow the [R46A]V
1a
R
construct with wildtype pharmacology. Therefore free
guanidinium could not be co-ordinated within the mutant
receptor in the appropriate orientation to recover agonist
binding. Although Lys46 is an inadequate substitution,
charge is nevertheless a significant aspect of the Arg at this
locus in the wildtype V
1a
R because reversal of the charge in
[R46E]V
1a
R and [R46D]V
1a
R resulted in the least respon-
sive receptors of the 20 studied (Fig. 3B). In addition,
removal of this negative charge with [R46Q]V
1a
Rand
[R46N]V
1a
R increased the responsiveness to AVP by
14-fold and 30-fold respectively (Table 1, Fig. 3B). While
differences in the observed E

max
values may reflect differ-
ences in transfection efficiencies, it is also possible that a
number of different active conformations have been gener-
ated, some better able to activate the G-protein Gq than
others.
Trp and Gly represent the two extremes of side-chain size,
with accessible surface areas of 210 A
˚
and 33 A
˚
respectively
[24], but [R46W]V
1a
Rand[R46G]V
1a
R had very similar
EC
50
values for InsP generation (Table 1). Therefore,
residue 46 is not spatially restricted within the receptor
architecture. Generation of an intracellular signal requires
the receptor to adopt an active conformation in addition to
binding the agonist. Ground-state (R) and active (R*)
conformations of GPCRs exist in equilibrium. Agonists
have a higher affinity for the active receptor (R*) which
stabilizes this conformation and subsequently establishes
productive R*–G-protein coupling (reviewed in [25]). All of
the Arg46 substitutions in this study produced a marked
reduction in the ability of AVP to both bind to the receptor

(Fig. 3A) and to initiate a second messenger response
(Fig. 3B). It might be supposed that the impaired signalling
simply reflected a reduced ability of the mutant receptors to
assume an active conformation. Such a situation would
clearly lead to a reduction in signalling response and would
also produce a decrease in agonist affinity, as agonists have
a low affinity for the ground state of the receptor. However,
close inspection of the data reveals that this is not the case.
Different amino acid substitutions affect binding affinity
and functional EC
50
values to markedly different extents.
For example, substitution by His and Trp in [R46H]V
1a
R
and [R46W]V
1a
R resulted in a decrease in affinity of
approximately 700-fold compared to the wildtype V
1a
R, but
the increase in EC
50
was 10-fold and 600-fold respectively
(Table 1). Indeed, there is no correlation between the K
i
and
EC
50
values for the mutant receptors (Fig. 4).

The ratio of EC
50
to K
i
is an indicator of efficacy, i.e. the
likelihood that a receptor will become activated and initiate
a functional response once an agonist has bound [26]. This
parameter (expressed as pEC
50
) pK
i
) was increased for all
19 substitutions compared to the wildtype Arg46, with the
precise value depending on the identity of the substituent
amino acid (Fig. 5). These data establish that the mutant
receptors are much less likely than the wildtype to bind
AVP, but once AVP has bound all 19 mutant receptors are
Fig. 4. Lack of correlation between
P
EC
50
values and
P
K
i
values for
[R46X]V
1a
R constructs. Each square represents one of the 19 different
[R46X]V

1a
R mutant constructs; m,wildtypeV
1a
R.
Fig. 5. Agonist-induced activation of the V
1a
R is dictated by the residue
at position 46. Wildtype V
1a
R(wt)and[R46X]V
1a
Rconstructswere
expressed in HEK 293T cells and characterized pharmacologically. In
each case the binding affinity (pK
i
)ofAVPandthepEC
50
value for
AVP-induced accumulation of mono-, bis- and tris-phosphates (InsP–
InsP
3
) was determined from three separate experiments each per-
formed in triplicate. The pEC
50
) pK
i
value was calculated and the
[R46X] substitutions presented in rank order.
4686 S. R. Hawtin et al. (Eur. J. Biochem. 270) Ó FEBS 2003
more likely than the wildtype V

1a
R to become activated.
This implies that arginyl at this locus is a constraining
residue which contributes to maintaining the conformational
switch of the V
1a
R in the off-state. The unique ability of
arginyl to fulfil this constraining function in the N-terminus
of the V
1a
R is similar to the unique constraining role of
alanyl previously reported for Ala293 in the distal i3-loop
of the a
1b
-adrenergic receptor [27]. Although substitution of
Arg46 facilitated agonist-induced transition of the V
1a
Rto
the active state, signalling was nevertheless still dependent
on agonist because an increase in basal signalling was not
observed. Furthermore, the marked decrease in agonist
affinity with the [R46X]V
1a
R constructs implies that Arg46
has an additional function facilitating high affinity agonist
binding. Consequently, mutation of Arg46 had the dual
effect of (a) decreasing agonist affinity and (b) promoting
the agonist-induced active conformation due to the loss of a
stabilizing constraint on the ground state of the receptor.
Therefore, the agonist had a lower affinity than the wildtype

V
1a
R but once the agonist bound, the receptor was more
likely to signal. This dual role suggests that binding of the
agonist releases the Arg46-mediated constraint on the
ground state of the receptor, thereby promoting agonist-
induced activation. Interestingly, Ôdual-roleÕ residues were
also identified in transmembrane helix VII of the M
1
muscarinic acetylcholine receptor (mAChR) recently. In this
mAChR however, these residues stabilize the ground state
of the receptor but also subsequently stabilize the G-protein
interaction of the active receptor [28].
In summary, Arg46 of the V
1a
R is required for high
affinity agonist binding and signalling. It is one of the
constraining residues which maintain the V
1a
Rinthe
inactive conformation and as such is part of the receptor
activation switch. There is an absolute requirement for
arginyl at position 46 for these functions.
Acknowledgements
We are grateful to Dr Claudine Serradeil-Le Gal (Sanofi Recherche,
France) for providing a sample of SR 49059. This work was supported
by a grant to M.W. from the Biotechnology and Biological Sciences
Research Council.
References
1. Acher, R., Chauvet, J. & Chauvet, M.T. (1995) Man and the

chimera. Selective versus neutral oxytocin evolution. Adv. Exp.
Med. Biol. 395, 615–627.
2. Baldwin, J.C. (1993) The probable arrangement of the helices in
G-protein-coupled receptors. EMBO J. 12, 1693–1703.
3. Howl, J. & Wheatley, M. (1995) Molecular pharmacology of V
1a
vasopressin receptors. General Pharmacol. 26, 1143–1152.
4. Hibert, M., Hoflack, J., Trumpp-Kallmeyer, S., Mouillac, B.,
Chini,B.,Mahe,E.,Cotte,N.,Jard,S.,Manning,M.&Barberis,C.
(1999) Functional architecture of vasopressin/oxytocin receptors.
J. Recept. Signal Transduct. Res. 19, 589–596.
5. Gimpl, G. & Fahrenholz, F. (2001) The oxytocin receptor system:
Structure, function and regulation. Physiol. Rev. 81, 629–683.
6. Thibonnier, M., Coles, P., Thibonnier, A. & Shoham, M. (2001)
The basic and clinical pharmacology of non-peptide vasopressin
receptor antagonists. Annu. Rev. Pharmacol. Toxicol. 41, 175–202.
7. Manning, M., Bankowski, K. & Sawyer, W.H. (1987) Selective
agonists and antagonists of vasopressin. In Vasopressin (Gash, D.M.
& Boer, G.J., eds), pp. 335–368. Plenum Press, New York, NY,
USA.
8. Pettibone, D.J. & Freidinger, R.M. (1997) Discovery and devel-
opment of non-peptide antagonists of peptide hormone receptors.
Biochem. Soc. Trans. 25, 1051–1057.
9. Freidinger, R.M. & Pettibone, D.J. (1997) Small molecule ligands
for oxytocin and vasopressin receptors. Med. Res. Rev. 17, 1–16.
10. Hawtin,S.R.,Wesley,V.J.,Parslow,R.A.,Patel,S.&Wheatley,M.
(2000) Critical role of a subdomain of the N-terminus of the V
1a
vasopressin receptor for binding agonists; functional rescue by the
oxytocin N-terminus. Biochemistry 39, 13524–13533.

11. Postina, R., Kojro, E. & Fahrenholz, F. (1996) Separate agonist
and peptide antagonist binding sites of the oxytocin receptor
defined by their transfer into the V
2
vasopressin receptor. J. Biol.
Chem. 271, 31593–31601.
12. Hawtin, S.R., Howard, H.C. & Wheatley, M. (2001) Identification
of an extracellular segment of the oxytocin receptor providing
agonist-specific binding epitopes. Biochem. J. 354, 465–472.
13. Hausmann, H., Richters, A., Kreienkamp, H J., Meyerhof, W.,
Mattes, H., Lederis, K., Zwiers, H. & Richter, D. (1996) Muta-
tional analysis and molecular modeling of the nonapeptide hor-
mone binding domains of the [Arg
8
]vasotocin receptor. Proc. Natl
Acad. Sci. USA 93, 6907–6912.
14. Hawtin,S.R.,Wesley,V.J.,Parslow,R.A.,Patel,S.&Wheatley,M.
(2002) A single residue (Arg
46
) located within the N-terminus of
the V
1a
vasopressin receptor is critical for binding vasopressin
but not peptide or non-peptide antagonists. Mol. Endocrinol. 16,
600–609.
15. Wesley, V.J., Hawtin, S.R., Howard, H.C. & Wheatley, M. (2002)
Agonist-specific, high affinity binding epitopes are contributed by
an arginine in the N-terminus of the human oxytocin receptor.
Biochemistry 41, 5086–5092.
16.Wheatley,M.,Howl,J.,Yarwood,N.J.,Davies,A.R.L.&

Parslow, R.A. (1997) Preparation of a membrane fraction for
receptor studies and solubilisation of receptor proteins with
retention of biological activity. Methods Mol. Biol. 73, 305–332.
17. Howl, J., Langel, U
¨
., Hawtin, S.R., Valkna, A., Yarwood, N.J.,
Saar, K. & Wheatley, M. (1997) Chimeric strategies for the
rational design of bioactive analogs of small peptide hormones.
FASEB J. 11, 582–590.
18. Kruszynski, M., Lammek, B., Manning, M., Seto, J., Haldar, J. &
Sawyer,W.H.(1980)1-(b-mercapto-b,b-cyclopentamethylene-
propionic acid), 2-(O-methyl) tyrosine arginine vasopressin and
1-(b-mercapto-b,b-cyclopentamethylenepropionic acid) arginine
vasopressin, two highly potent antagonists of the vasopressor
response to arginine vasopressin. J. Med. Chem. 23, 364–368.
19. Cheng, Y. & Prusoff, W.H. (1973) Relationship between the
inhibition constsant (K
i
) and concentration of inhibitor which
causes 50 % inhibition (IC
50
) of an enzyme reaction. Biochem.
Pharmacol. 22, 3099–3108.
20. Howl, J., Rudge, S.A., Lavis, R.A., Davies, A.R.L., Parslow, R.A.,
Hughes, P.J., Kirk, C.J., Michell, R.H. & Wheatley, M. (1995)
Rat testicular myoid cells express vasopressin receptors: receptor
structure, signal transduction and developmental regulation.
Endocrinology 136, 2206–2213.
21. Hawtin, S.R., Tobin, A., Patel, S. & Wheatley, M. (2001) Palmi-
toylation of theV

1a
vasopressinreceptor reveals different conforma-
tional requirements for signaling, agonist-induced receptor phos-
phorylation and sequestration. J. Biol. Chem. 276, 38139–38146.
22. Schmidt, A., Audigier, S., Barberis, C., Jard, S., Manning, M.,
Kolodziejczyk, A.S. & Sawyer, W.H. (1991) A radioiodinated
linear vasopressin antagonist. FEBS Lett. 282, 77–81.
23. Serradeil-Le Gal, C., Wagnon, J., Garcia, C., Lacour, C.,
Guiraudou, P., Christophe, B., Villanova, G., Nisato, D.,
Maffrand, J.P. & Le Fur, G.P. (1993) Biochemical and pharma-
cological properties of SR 49059, a new, potent, non-peptide
Ó FEBS 2003 The N-terminal switch of the V
1a
R(Eur. J. Biochem. 270) 4687
antagonist of rat and human vasopressin V
1a
receptors. J. Clin.
Invest. 92, 224–231.
24. Richardson, J.S. & Richardson, D.C. (1989) Principles and pat-
terns of protein conformation. In Prediction of Protein Structure
and the Principles of Protein Conformation (Fasman, G.D., ed.),
pp. 1–98. Plenum Press, New York, NY, USA.
25. Gether, U. & Kobilka, B.K. (1998) G-protein-coupled receptors:
II. Mechanism of agonist activation. J. Biol. Chem. 273, 17979–
17982.
26. Christopoulos, A. & El-Fakahany, E.E. (1999) Qualitative and
quantitative assessment of relative agonist efficacy. Biochem.
Pharmacol. 58, 735–748.
27. Kjelsberg,M.A.,Cotecchia,S.,Ostrowski,J.,Caron,M.G.&
Lefkowitz, R.J. (1992) Constitutive activation of the alpha

1B-adrenergic receptor by all amino acid substitutions at a single
site. J. Biol. Chem. 267, 1430–1433.
28. Lu, Z L., Saldanha, J.W. & Hulme, E.C. (2001) Transmembrane
domains 4 and 7 of the M
1
muscarinic acetylcholine receptor are
critical for ligand binding and the receptor activation switch.
J. Biol. Chem. 276, 34098–34104.
29. Hawtin, S.R., Davies, A.R.L., Matthews, G. & Wheatley, M.
(2001) Identification of the glycosylation sites utilized on the V
1a
vasopressin receptor and assessment of their role in receptor sig-
nalling and expression. Biochem. J. 357, 73–81.
4688 S. R. Hawtin et al. (Eur. J. Biochem. 270) Ó FEBS 2003