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

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.. T

An arginyl in the N-terminus of the V1a vasopressin receptor is part

of the conformational switch controlling activation by agonist

Stuart R Hawtin1,*, Victoria J Wesley1, John Simms1, Rosemary A Parslow1, Alice Miles1, Kim McEwan1, Mary Keen2and Mark Wheatley1

1

School of Biosciences and2Department 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 V1a vasopressin receptor (V1aR) 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 V1aR is critical

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]V1aR 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 Kito EC50, an indicator of efficacy, was increased for all substitutions Consequently, although [R46X]V1aR constructs have a lower affinity for agonist, once AVP has bound all 19 are more likely than the wildtype

V1aR to become activated Therefore, in the wildtype V1aR, Arg46 constrains the inactive conformation of the receptor

On binding AVP this constraint is alleviated, promoting the transition to active V1aR 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]oxymeso-tocin) 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: V1a, V1band V2(V1aR,

V1bR and V2R 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 V1aR 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 (V2R) and adrenocorticotropic hormone secretion (V1bR) This has been the stimulus for the development of a range of V1aR antagonists, initially peptides [7] and more recently non-peptides [8,9] Although agonists and antagonists exhibit competitive binding to the V1aR, 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 V1aR provides agonist-specific binding epitopes, as truncation of the distal segment of the V1aR 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: m.wheatley@bham.ac.uk

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)

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 V1aR could be functionally rescued

by a chimeric construct in which the N-terminus of the

OTR is replaced with the corresponding sequence of

the V1aR [10] The role of the N-terminus has been

addressed recently by alanine-scanning mutagenesis of the

N-terminus of the V1aR; 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 V1aR 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 V1aR 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(CH2)5Tyr(Me)2AVP] and linear

antagonist, phenylacetyl-D-Tyr(Me)2Arg6Tyr(NH2)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 SdaI were

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 Arg46fi

Xaa46 substitutions, and a unique Eco81I restriction site

(bold) The PCR cycling conditions were as follows:

denaturing, 94C (1 min); annealing, 60 C (2 min);

exten-sion, 72C (1 min) for 30 cycles followed by extension at

72C (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) CO2in air at

37C Cells were seeded at a density of approximately

5· 105cells 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 [Phe3-3,4,5-3H]AVP (68.5 CiÆ mmol)1; DuPont NEN, Stevenage, Herts, UK) or the

V1aR-selective peptide antagonist [Phe3-3,4,5-3H] d(CH2)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 theFIG.P program (Biosoft, Cambridge, UK) Individual IC50 values obtained for competing ligands were corrected for radioligand occu-pancy as described [19] using the radioligand affinity (Ki) experimentally determined for each individual construct AVP-induced inositol phosphate production

HEK 293T cells were seeded at a density of 2.5· 105cells per well in poly D-lysine coated 12 well plates and transfected after 24 h using TransfastTM (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-[2-3H]inositol (22.0 CiÆmmol)1; DuPont NEN) for 24 h Cells were washed twice with NaCl/Pi

and 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 Incuba-tions were terminated by adding 0.5 mL of 5% (w/v) perchloric acid containing 1 mM EDTA and 1 mgÆmL)1 phytic acid hydrolysate (final concentrations) Samples were neutralized with 1.2M KOH, 10 mM EDTA,

50 mM 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–InsP3) was eluted with 10 mL of 850 mM

NH4COOH containing 0.1M HCOOH as described [21] and quantified by scintillation counting

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 V1aR (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 V1aR The

wildtype and mutant receptors were expressed at the same

level of approximately 1–2 pmolÆmg)1protein 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(CH2)5Tyr(Me)2AVP] [18], the linear peptide

antagonist [phenylacetyl-D-Tyr(Me)2Arg6Tyr(NH2)9AVP]

[22] and the nonpeptide antagonist (SR 49059) [23] for all

the 19 mutant receptors engineered, were comparable to

wildtype V1aR (Table 1) This was important, as it allowed

accurate quantification of the pharmacological

characteris-tics of each mutant construct by radioligand binding studies

using [3H]d(CH2)5Tyr(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 3000-700-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 V1aR and the mutant construct [R46A]V1aR 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 mM) to the [R46A]V1aR 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 V1aR None of the 19 encoded amino acids could substitute the Arg46 and retain the wildtype V1aR second messenger generation Dose– response curves for AVP-stimulated accumulation of InsP–InsP3 by the wildtype V1aR and a representative mutant construct ([R46A]V1aR) 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 EC50for AVP-induced InsP–InsP3 accumula-tion increasing from between 10-fold to 600-fold compared

to the wildtype V1aR (Fig 3B) The acidic residues at position 46 in [R46E]V1aR and [R46D]V1aR were the most detrimental to signalling This effect was due predominantly

to the negative charge, as removal of this charge in the constructs [R46Q]V1aR and [R46N]V1aR increased the Emax and resulted in EC50values that were 28 fold and 14 fold lower than their respective acids (Table 1) Histidine was the least disruptive substitution, as the EC50 of [R46H]V1aR was only 10 fold higher than wildtype V1aR, although the

Emax was depressed Lysine could not replace arginine because [R46K]V1aR was no better than [R46P]V1aR, [R46V]V1aR or [R46A]V1aR at signalling [R46M]V1aR, [R46C]V1aR and the hydroxyl-containing [R46S]V1aR and [R46T]V1aR were all similarly impaired with EC50values 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]V1aR and [R46Y]V1aR to AVP was almost identical The residues Trp and Gly represent the two extremes of side-chain size, however, both [R46W]V1aR and [R46G]V1aR exhibited a similar increase in the EC50of AVP-induced InsP produc-tion of 150 fold A similar perturbation was observed with the branched aliphatic substitutions in [R46L]V1aR and [R46I]V1aR The wildtype V1aR and the 19 mutant V1aR 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.

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 ) were calculated from IC 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.

generation in Fig 3B Collectively, these results established

that Arg46 has a critical role in agonist activation of the

V1aR

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 V1aR provides agonist-specific binding epitopes Consequently, truncation of the V1aR 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 V1aRs and OTRs cloned to date, suggesting functional importance (Fig 1) This has now been confirmed experimentally, as substitution of this residue by alanine in the rat V 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) of 10)4M 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 Experiexperi-mental 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 R constructs 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.

profoundly and selectively disrupted agonist binding and

signalling [14,15]

Having established the functional necessity of Arg46 in

the V1aR 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 (Ki) 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 EC50 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 mM) however, could not endow the [R46A]V1aR

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 V1aR because reversal of the charge in

[R46E]V1aR and [R46D]V1aR resulted in the least

respon-sive receptors of the 20 studied (Fig 3B) In addition,

removal of this negative charge with [R46Q]V1aR and

[R46N]V1aR increased the responsiveness to AVP by

14-fold and 30-fold respectively (Table 1, Fig 3B) While

differences in the observed Emaxvalues 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]V1aR and [R46G]V1aR had very similar

EC50 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 EC50 values to markedly different extents For example, substitution by His and Trp in [R46H]V1aR and [R46W]V1aR resulted in a decrease in affinity of approximately 700-fold compared to the wildtype V1aR, but the increase in EC50was 10-fold and 600-fold respectively (Table 1) Indeed, there is no correlation between the Kiand

EC50values for the mutant receptors (Fig 4)

The ratio of EC50to Kiis 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 pEC50) pKi) 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, wildtype V 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 R constructs were expressed in HEK 293T cells and characterized pharmacologically In each case the binding affinity (pK i ) of AVP and the pEC 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.

more likely than the wildtype V1aR to become activated.

This implies that arginyl at this locus is a constraining

residue which contributes to maintaining the conformational

switch of the V1aR in the off-state The unique ability of

arginyl to fulfil this constraining function in the N-terminus

of the V1aR is similar to the unique constraining role of

alanyl previously reported for Ala293 in the distal i3-loop

of the a1b-adrenergic receptor [27] Although substitution of

Arg46 facilitated agonist-induced transition of the V1aR to

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]V1aR 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

V1aR 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, dual-role residues were

also identified in transmembrane helix VII of the M1

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 V1aR is required for high

affinity agonist binding and signalling It is one of the

constraining residues which maintain the V1aR in the

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.

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