The N-Terminal Juxtamembrane Segment of the V1a Vasopressin Receptor Provides Two Independent Epitopes Required for High-Affinity Agonist Binding and Signaling

Stuart R. Hawtin1, Victoria J. Wesley1, John Simms, Cymone C. H. Argent, Khalid Latif and Mark Wheatley

School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom

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: m.wheatley{at}bham.ac.uk.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
It is fundamentally important to define how agonist-receptor interaction differs from antagonist-receptor 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 protein-coupled receptor subfamily and provides molecular insight into key differences between agonist and antagonist binding requirements.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
G 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 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 ß-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 ß2-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 down-regulation, 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 high-affinity 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 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/ß-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/ß-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, 11, 12).

Defining at the molecular level how agonist-receptor interaction differs from antagonist-receptor interaction is fundamentally important to understanding hormone-stimulated 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Pharmacological Characterization of Alanyl-Substituted 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. 1Go) were individually mutated to alanine to generate the constructs [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. 1Go). 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 1Go, 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 1Go). 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 1Go). 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. 2AGo and Table 1Go). 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).



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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.

 

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Table 1. Pharmacological Profile of Wild-Type and Alanyl-Substituted V1aRs

 


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Fig. 2. Pharmacological Characterization of Mutant V1aRs

HEK 293T cells were transiently transfected with either wild-type V1aR (open symbols; {circ}, {square}) or mutant receptor construct (solid symbols; {bullet}, {blacksquare}). 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 ({square}, {blacksquare}) or the antagonist d(CH2)5Tyr(Me)2AVP ({circ}, {bullet}) 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 µM). A theoretical Langmuir binding isotherm has been fitted to the experimental data as described in Materials and Methods.

 
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 2Go. 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 decrease in affinity of AVP at these constructs (Table 1Go). In addition, the Emax values for [E48A]V1aR and [L50A]V1aR were slightly higher than for wild-type V1aR (Table 2Go). Substitution of Leu58 slightly decreased AVP binding affinity but had a more pronounced effect on intracellular signaling. Consequently, [L58A]V1aR possessed a 7-fold lower affinity for AVP than wild-type V1aR (Table 1Go) but exhibited a 24-fold increase in the EC50 for AVP-induced InsPs accumulation compared with wild-type V1aR (Table 2Go). 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. 3AGo). 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 1Go). 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.


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Table 2. AVP-Induced Signaling by Wild-Type and Alanyl-Substituted V1aRs

 


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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 ({square}); [E54A]V1aR, ({circ}); [E54D]V1aR ({blacksquare}) or [E54R]V1aR ({bullet}). B, Wild-type V1aR ({square}); [L50A]V1aR, ({circ}); [L50P]V1aR ({blacksquare}). 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.

 
Specific Requirement for Glutamate at Residue 54 for High-Affinity Agonist Binding and Second Messenger Generation
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 3Go). 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 3Go).


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Table 3. Pharmacological Characterization of Glu54 and Leu50 Mutant Receptors

 
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. 3AGo). 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. 3AGo and Table 4Go). 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. 3AGo and Table 4Go).


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Table 4. AVP-Induced Signaling by Glu54 and Leu50 Mutant Receptors

 
Glu54 and Arg46 Are Both Required for High-Affinity 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, substitution 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 1Go). 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 2Go). 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 {alpha}-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. 4Go, A and B). Support for this extracellular extension of the TM1 helix is provided by secondary structure prediction, which also indicates that the {alpha}-helical conformation of TM1 continues up to Arg46 (Fig. 4CGo). It is well established that inserting a prolyl into an {alpha}-helical secondary structure distorts the helix. Therefore we engineered the construct [L50P]V1aR to disrupt any {alpha}-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. 2BGo and Table 3Go) and disrupted intracellular signaling approximately 250-fold (Fig. 3BGo and Table 4Go). 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 3Go).



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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.

 
The roles of Glu54 and Arg46 were investigated further. With reference to Fig. 5AGo, a series of mutant receptors was engineered to investigate the importance of the 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. 5BGo). Assessment of the signaling capability of these charged residue mutants revealed that second messenger generation was also compromised compared with wild-type V1aR (Fig. 6AGo). The dose-response curves for [L50E]V1aR, [L50R]V1aR, and [L50E/E54A]V1aR were all right shifted approximately 60-fold (Fig. 6BGo), whereas the mutant receptors [R46A/L50R]V1aR and [R46E/E54R]V1aR failed to respond to AVP challenge even at 10 µM AVP.



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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 {alpha}-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.

 


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Fig. 6. Intracellular Signaling by Mutant V1aRs

A, AVP-induced accumulation of InsP–InsP3 in HEK 293T cells transfected with wild-type V1aR ({square}), [L50E]V1aR ({circ}), [L50R]V1aR ({blacksquare}), or [L50E/E54A]V1aR ({bullet}). 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 µM. Emax values are fold-stimulation over basal.

 
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 µM (data not shown).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 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 wild-type 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. 1Go), 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. 1Go). 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. 1Go). 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. 1Go). 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 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 1–4GoGoGoGo). 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 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 wild-type 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 high-affinity conformation for AVP. Molecular modeling of the V1aR indicates that the {alpha}-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. 4Go, A and B). Indeed, secondary structure prediction for this region of the V1aR also suggests an {alpha}-helical conformation extending from TM1 up to Arg46 (Fig. 4CGo). Such an extension of {alpha}-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 {alpha}-helical fold, which extends TM5 and TM6 by up to three turns out of the membrane (24). It is well documented that an {alpha}-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 {alpha}-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 {alpha}-helical conformation, residues Arg46, Leu50, and Glu54 stack on the same face of the helix (Fig. 4Go, A and B). The effect of altering the distribution, and nature, of the charge on this face was investigated systematically (Fig. 5AGo). 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 µM).

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/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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Material
AVP was purchased from Sigma Chemical Co. (St. Louis, MO). The cyclic antagonist 1-(ß-mercapto-ß,ß-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).

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-GAG-CTG-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 Glu54->Arg 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-GTA-CGC-AAT-GCG-GAG-C-3', 5'-G-GGG-GCC-TTA-GGG-GAC-GTA-CGC-AAT-GAG-GCG-CTG-G-3', 5'-G-GGG-GCC-TTA-GGG-GAC-GTA-CGC-AAT-GAG-GAG-GCG-GCC-AAA-CTG-G-3', 5'-G-GGG-GCC-TTA-GGG-GAC-GTA-CGC-AAT-GAG-GAG-GAG-GCC-AAA-CTG-G-3', 5'-G-GGG-GCC-TTA-GGG-GAC-GTA-CGC-AAT-GAG-GAG-CCG-GCC-AAA-CTG-G-3', 5'-G-GGG-GCC-TTA-GGG-GAC-GTA-CGC-AAT-GAG-GAG-CGG-GCC-AAA-CTG-G-3', 5'-G-GGG-GCC-TTA-GGG-GAC-GTA-CGC-AAT-GAG-GAG-TGG-GCC-AAA-CTG-G-3', 5'-G-GGG-GCC-TTA-GGG-GAC-GTA-CGC-AAT-GAG-GAG-GAG-GCC-AAA-CTG-GCA-ATC-GC-3', 5'-G-GGG-GCC-TTA-GGG-GAC-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'-GGG-CTG-GCT-AAG-CTG-GAA-ATC-GCT-GTG-GCG-GCA-GTG-3', and 5'-G-GGG-GCC-TTA-GGG-GAC-GTA-GAG-AAT-GAG-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-TGC-TAG-CAC-AGC-GAT-ATC-CAG-CTT-AGC-C-3' and 5'-AAT-CAC-TGC-TAG-CAC-AGC-GAT-CGC-CAG-CTT-AGC-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 x 105 cells per 100-mm dish and transfected after 48 h using a calcium phosphate precipitation protocol with 10 µg 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. Radioligand binding assays were performed as previously described (29) using either the natural agonist [Phe3-3,4,5-3H]AVP, (64.2 Ci/mmol; PerkinElmer, Beaconsfield, UK) or the V1aR-selective peptide antagonist [Phe3-3,4,5-3H]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 wild-type and mutant receptors was determined for each construct individually using whole-cell binding assays as described previously (31).

AVP-induced Inositol Phosphate Production
HEK 293T cells were seeded at a density of 2.5 x 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 µCi/ml myo-[2-3H]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 x 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).

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 µmol scale using N{alpha}-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
 
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.


    FOOTNOTES
 
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.

First Published Online June 30, 2005

1 S.R.H. and V.J.W. contributed equally to the manuscript. Back

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.

Received for publication April 11, 2005. Accepted for publication June 17, 2005.


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