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Originally published In Press as doi:10.1074/jbc.M607639200 on September 21, 2006

J. Biol. Chem., Vol. 281, Issue 50, 38478-38488, December 15, 2006
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Charged Extracellular Residues, Conserved throughout a G-protein-coupled Receptor Family, Are Required for Ligand Binding, Receptor Activation, and Cell-surface Expression*

Stuart R. Hawtin{ddagger}12, John Simms{ddagger}1, Matthew Conner{ddagger}, Zoe Lawson{ddagger}, Rosemary A. Parslow{ddagger}, Julie Trim§, Andrew Sheppard§, and Mark Wheatley{ddagger}3

From the {ddagger}School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom and §Ferring Research Ltd., Southampton Science Park, 1 Venture Road, Southampton SO16 7NP, United Kingdom

Received for publication, August 10, 2006 , and in revised form, September 8, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
For G-protein-coupled receptors (GPCRs) in general, the roles of extracellular residues are not well defined compared with residues in transmembrane helices (TMs). Nevertheless, extracellular residues are important for various functions in both peptide-GPCRs and amine-GPCRs. In this study, the V1a vasopressin receptor was used to systematically investigate the role of extracellular charged residues that are highly conserved throughout a subfamily of peptide-GPCRs, using a combination of mutagenesis and molecular modeling. Of the 13 conserved charged residues identified in the extracellular loops (ECLs), Arg116 (ECL1), Arg125 (top of TMIII), and Asp204 (ECL2) are important for agonist binding and/or receptor activation. Molecular modeling revealed that Arg125 (and Lys125) stabilizes TMIII by interacting with lipid head groups. Charge reversal (Asp125) caused re-ordering of the lipids, altered helical packing, and increased solvent penetration of the TM bundle. Interestingly, a negative charge is excluded at this locus in peptide-GPCRs, whereas a positive charge is excluded in amine-GPCRs. This contrasting conserved charge may reflect differences in GPCR binding modes between peptides and amines, with amines needing to access a binding site crevice within the receptor TM bundle, whereas the binding site of peptide-GPCRs includes more extracellular domains. A conserved negative charge at residue 204 (ECL2), juxtaposed to the highly conserved disulfide bond, was essential for agonist binding and signaling. Asp204 (and Glu204) establishes TMIII contacts required for maintaining the beta-hairpin fold of ECL2, which if broken (Ala204 or Arg204) resulted in ECL2 unfolding and receptor dysfunction. This study provides mechanistic insight into the roles of conserved extracellular residues.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
G-protein-coupled receptors (GPCRs)4 exhibit a common tertiary structure comprising seven transmembrane helices (TMs) linked by extracellular loops (ECLs) and intracellular loops. The atomic detail of this general GPCR fold has been elucidated for bovine rhodopsin (bRho) using x-ray crystallography (1). This confirmed that the chromophore 11-cis-retinal is covalently linked to Lys296(7.43) in transmembrane helix VII (TMVII) via a protonated Schiff-base and projects into a binding pocket formed within the TM bundle where it interacts with amino acid side chains and water molecules (1, 2).5 Likewise, the binding pocket for small biogenic amine neurotransmitters such as acetylcholine and norepinephrine is buried deep within the TM bundle (3). Nevertheless, it is known from the bRho x-ray structure that the extracellular domains possess defined structure and are orientated to interact with each other and with the TM helices. Indeed ECL2 of bRho forms a beta-hairpin that plunges down into the helical bundle to form a plug over the chromophore. Furthermore, the orientation of ECL2 in the majority of GPCRs is restrained by a conserved disulfide bond between ECL2 and the top of TMIII (1, 2).

The neurohypophysial peptide hormones vasopressin (AVP) and oxytocin (OT) generate a wide range of physiological effects, including vasopressor and antidiuretic and uterotonic actions (4, 5). The effects of AVP/OT are mediated by a family of receptors (V1aR, V1bR, V2R, and OTR), which together with the vasotocin receptor (VTR), mesotocin receptor, and isotocin receptor from lower vertebrates constitute a subfamily of the rhodopsin/beta-adrenergic receptor class of GPCRs (family A). The V1aR, V1bR, and OTR couple to phospholipase C thereby generating inositol 1,4,5-trisphosphate and diacylglycerol as second messengers, whereas the V2R stimulates adenylyl cyclase. The V1aR is widely distributed and mediates nearly all of the actions of AVP with the exceptions of antidiuresis (V2R) and ACTH secretion (V1bR). Activation of the OTR stimulates contraction of the uterine myometrium during labor and causes lactation. In addition to the characteristic architecture of GPCRs, members of the neurohypophysial peptide hormone receptor family share certain sequence motifs and exhibit related pharmacologies (5-7). The hormone-binding site of these receptors includes residues in the TM bundle (8) and ECL1 (9-11). It has also been reported that the N termini of the V1aR and OTR are required for agonist binding (12, 13). In particular, two charged residues (Arg46 and Glu54) in the V1aR N terminus are required for high affinity agonist binding but not antagonist binding (14, 15). Likewise, Arg34 in the N terminus of the OTR is required for agonist binding (16).

For GPCRs in general, the roles of extracellular residues are not well understood compared with residues in the TM domain. Nevertheless, extracellular residues are important for binding both amine (17) and peptide (18) ligands and have been implicated in ligand receptor-subtype specificity (19), binding allosteric modulators (20), switching ligand agonist/antagonist properties (21), and human immunodeficiency virus co-receptor activity (22). The aim of this study was to use the V1aR to systematically investigate the function of extracellular charged residues that are highly conserved throughout a subfamily of peptide GPCRs. By using a combination of mutagenesis and molecular modeling, our results indicate that specific conserved charged residues in ECL1, ECL2, and ECL3 fulfill important roles in ligand binding, receptor activation, domain conformation, and cell-surface expression.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—AVP was purchased from Sigma. The cyclic peptide antagonist (CA) 1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-(O-methyl)tyrosine AVP (d(CH2)5Tyr(Me)2AVP), and linear peptide antagonist (LA) phenylacetyl (PhAc)-D-Tyr(Me)2Arg6Tyr(NH2)9AVP were from Bachem (St. Helens, UK). SR 49059 was a gift from Sanofi Recherche (Toulouse, France). Cell culture media, buffers, and supplements were purchased from Invitrogen. Restriction enzymes were obtained from MBI Fermentas (Sunderland, UK).

Mutant Receptor Constructs—Mutation of the V1aR was made using a PCR approach as described previously (15). The mutant receptor constructs [D112A]V1aR, [R116A]V1aR, [R118A]V1aR, [D121A]V1aR, and [R125A]V1aR were engineered using the antisense oligonucleotides as follows: 5'-GGC-AAA-CAC-CTG-CAG-GTG-CTT-CAC-CAC-GCG-GCA-CAG-CCA-GTC-GGG-CCC-GCG-GAA-GCG-GTA-GGT-GAT-GGC-CCA-GC-3';5'-GGC-AAA-CAC CTG-CAG-GTG-CTT-CAC-CAC-GCG-GCA-CAG-CCA-GTC-GGG-CCC-GCG-GAA-GGC-GTA-GG-3'; 5'-GGC-AAA-CAC-CTG-CAG-GTG-CTT-CAC-CAC-GCG-GCA-CAG-CCA-GTC-GGG-CCC-GGC-GAA-GCG-G-3'; 5'-GGC-AAA-CAC-CTG-CAG-GTG-CTT-CAC-CAC-GCG-GCA-CAG-CCA-GGC-GGG-CCC-3'; and 5'-GGC-AAA-CAC-CTG-CAG-GTG-CTT-CAC-CAC-GGC-GCA-CAG-CCA-GTC-GGG-CCC-3', respectively. Each primer contained a unique SdaI restriction site (underlined) and base changes (shown in boldface) to generate each individual Ala substitution plus base changes to create a silent ApaI restriction site for diagnostic purposes (shown in italics). The same cloning strategy was employed to generate the mutant constructs [D112E]V1aR, [D112K]V1aR, [D112R]V1aR, [R116D]V1aR, [R116E]V1aR, [R116K]V1aR, [R125D]V1aR, and [R125K]V1aR using the antisense oligonucleotides as follows: 5'-GGC-AAA-CAC-CTG-CAG-GTG-CTT-CAC-CAC-GCG-GCA-CAG-CCA-GTC-GGG-CCC-GCG-GAA-GCG-GTA-GGT-GAT-CTC-CCA-GC-3'; 5'-GGC-AAA-CAC-CTG-CAG-GTG-CTT-CAC-CAC-GCG-GCA-CAG-CCA-GTC-GGG-CCC-GCG-GAA-GCG-GTA-GGT-GAT-TTT-CCA-GC-3'; 5'-GGC-AAA-CAC-CTG-CAG-GTG-CTT-CAC-CAC-GCG-GCA-CAG-CCA-GTC-GGG-CCC-GCG-GAA-GCG-GTA-GGT-GAT-GCG-CCA-GC-3'; 5'-GGC-AAA-CAC-CTG-CAG-GTG-CTT-CAC-CAC-GCG-GCA-CAG-CCA-GTC-GGG-CCC-GCG-GAA-GTC-GTA-GGT-G-3'; 5'-GGC-AAA-CAC-CTG-CAG-GTG-CTT-CAC-CAC-GCG-GCA-CAG-CCA-GTC-GGG-CCC-GCG-GAA-CTC-GTA-GGT-G-3'; 5'-GGC-AAA-CAC-CTG-CAG-GTG-CTT-CAC-CAC-GCG-GCA-CAG-CCA-GTC-GGG-CCC-GCG-GAA-TTT-GTA-GGT-G-3'; 5'-GGC-AAA-CAC-CTG-CAG-GTG-CTT-CAC-CAC-GTC-GCA-CAG-CCA-GTC-GGG-CCC-3'; and 5'-GGC-AAA-CAC-CTG-CAG-GTG-CTT-CAC-CAC-TTT-GCA-CAG-CCA-GTC-GGG-CCC-3', respectively. PCR products were subcloned into the HA epitope-tagged rat V1aR coding sequence in the mammalian expression vector pcDNA3.1 (Invitrogen) utilizing unique HindIII and SdaI restriction sites.

The mutations [E193A]V1aR, [E195A]V1aR, [K201A]V1aR, and [D204A]V1aR were made using antisense oligonucleotides as follows: 5'-GGC-GCG-GGT-ACC-CCA-GGG-CTG-GAT-GAA-GGT-AGC-CCA-GCA-GTC-TTG-GGT-TTT-AGT-GCC-ATT-GTT-CAC-CTC-GAT-TGC-GAT-CAC-AGA-G-3'; 5'-GGC-GCG-GGT-ACC-CCA-GGG-CTG-GAT-GAA-GGT-AGC-CCA-GCA-GTC-TTG-GGT-TTT-AGT-GCC-ATT-GTT-CAC-CGC-GAT-TTC-G-3'; 5'-GGC-GCG-GGT-ACC-CCA-GGG-CTG-GAT-GAA-CGT-TGC-CCA-GCA-GTC-TTG-GGT-TGC-AGT-GCC-ATT-G-3'; and 5'-GGC-GCG-GGT-ACC-CCA-GGG-CTG-GAT-GAA-GGT-AGC-CCA-GCA-GGC-TTG-GGT-TTT-AGT-GC-3', respectively. These primers contained the base changes (shown in boldface) to incorporate the Ala mutations and unique KpnI restriction site (underlined) used for subcloning. The [D204E]V1aR and [D204R]V1aR mutations were also engineered using this strategy using antisense oligonucleotides as follows: 5'-GGC-GCG-GGT-ACC-CCA-GGG-CTG-GAT-GAA-GGT-AGC-CCA-GCA-CTC-TTG-GGT-TTT-AGT-GC-3' and 5'-GGC-GCG-GGT-ACC-CCA-GGG-CTG-GAT-GAA-GGT-AGC-CCA-GCA-GCG-TTG-GGT-TTT-AGT-GC-3'. The construct [R216A]V1aR was made using sense oligonucleotide 5'-G-CCC-TGG-GGT-ACC-GCC-GCG-TAC-GTG-ACC-TGG-ATG-ACC-TCA-GGT-GTC-TTC-GTG-G-3'. This primer contained five base changes in the V1aR sequence (shown in boldface) that created the Ala mutation (shown in italics) unique silent Pfl23II and Eco81I restriction sites (for diagnostic purposes) and a KpnI restriction site (underlined) for subcloning into the V1aR.

The [E332A]V1aR mutation was made by PCR using both sense and antisense oligonucleotides. The sense primer was 5'-C-GAT-TCA-GCA-AAC-CCA-TCGATA-ACA-ATC-ACG-GCG-3'. This primer contained four base changes in the V1aR sequence (indicated in boldface) that created a unique functional ClaI restriction site (underlined) without altering the amino acid sequence and incorporated the Glu332 -> Ala mutation (shown in italics). A KpnI/EcoRI digest of this PCR fragment was subcloned into the V1aR. The constructs [D323A]V1aR and [D330A]V1aR were made by PCR with pcDNA3-[E332A]V1aR as template. Mutant antisense oligonucleotides were 5'-CGT-GAT-TGT-TAT-CGA-TGG-GTT-TTC-TGA-ATC-GGT-CCA-GAT-GAA-ATT-CTC-AGC-CCA-GAC-TGA-CC-3' and 5'-CGT-GAT-TGT-TAT-CGA-TGG-GTT-TTC-TGA-AGC-GGT-CCA-GAT-GAA-ATT-CTC-ATC-C-3' for [D323A]V1aR and [D330A]V1aR mutations, respectively. These primers contained base changes (shown in boldface) for the required Ala substitution and unique ClaI restriction site (underlined). The PCR products were subcloned into pcDNA3-[E332A]V1aR utilizing unique SdaI and ClaI restriction sites. All receptor constructs were confirmed by automated fluorescent sequencing (University of Birmingham, Birmingham, UK).

Cell Culture and Transfection—HEK 293T cells were routinely cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum in humidified 5% (v/v) CO2 in air at 37 °C. Cells were seeded at a density of ~5 x 105 cells/100-mm dish and transfected after 48 h using a calcium phosphate precipitation protocol with 10 µg of DNA/dish (16).

Radioligand Binding Assays—A washed cell membrane preparation of HEK 293T cells, transfected with the appropriate receptor construct, was prepared as described previously (23), and the protein concentration was determined using the BCA protein assay kit (Pierce) using bovine serum albumin as standard. Radioligand binding assays were performed as described previously (24) using either the natural agonist [Phe3-3,4,5-3H]AVP (0.5-1.5 nM), (64.2 Ci/mmol; PerkinElmer Life Sciences) or the V1aR-selective peptide antagonist [Phe3-3,4,5-3H]d(CH2)5Tyr(Me)2AVP (0.5-1.5 nM) (99 Ci/mmol; PerkinElmer Life Sciences) (25) as tracer ligand. Binding data were analyzed by nonlinear regression to fit theoretical Langmuir binding isotherms to the experimental data using PRISM Graphpad (Graphpad Software Inc., San Diego). Individual IC50 values obtained for competing ligands were corrected for radioligand occupancy as described (26) using the radioligand affinity (Kd) experimentally determined for each construct.

Whole Cell Vasopressin V1a Receptor Binding —HEK 293T cells were plated onto 12-well plates at a density of 2.5 x 105 cells/well in poly-D-lysine-coated 12-well plates and transfected after 24 h using TransfastTM (Promega Corp., Southampton, UK). After 36 h, each well received 0.5 ml of binding buffer (described above) containing 2% (w/v) BSA, 1-2 nM V1aR-selective peptide antagonist PhAc-D-Tyr(Me)2Arg6(3,4[3H]Pro)(3,5[3H]Tyr)9NH2-AVP (22 Ci/mmol; custom synthesis Phoenix Pharmaceuticals, INC. Belmont, CA) as tracer ligand in the presence (nonspecific) or absence (total) of 1 µM LA. Plates were incubated for 90 min at 37 °C, before removal of the medium by aspiration. After three rinses with ice-cold phosphate-buffered saline, 0.5 ml of 0.1 M NaOH was added to each well to extract radioactivity. After 15 min of incubation at 37 °C, the fluid from the plates was transferred to scintillation vials containing 10 ml of HiSafe3 scintillant mixture for counting. Cell-surface expression values were corrected for radioligand occupancy as described (26) using the radioligand affinity (Kd) experimentally determined for each construct.

Determination of Cell-surface Expression Using Enzyme-linked Immunosorbent Assay —All receptor constructs incorporated an HA epitope tag in the N terminus that enabled cell surface expression to be determined by enzyme-linked immunosorbent assay (27). Briefly, HEK 293T cells were seeded at a density of 1 x 105 cells/well in poly-D-lysine-coated 12-well plates and transfected after 24 h using TransfastTM (Promega Corp., Southampton, UK). After 36 h, cells were fixed with 3.7% (v/v) formaldehyde in TBS (20 mM Tris, pH 7.5, 150 mM NaCl) for 15 min at 37 °C and then washed three times with TBS. Nonspecific binding was blocked with 1% (w/v) BSA in TBS for 45 min. Anti-HA primary antibody (HA-7; Sigma) was diluted to 1:1000 in TBS containing 1% (w/v) BSA for 60 min at room temperature with occasional shaking, followed by three gentle washes with TBS. Cells were briefly re-blocked with 1% (w/v) BSA in TBS for 15 min, prior to incubation with secondary antibody (alkaline phosphatase-conjugated goat anti-mouse; Bio-Rad), and diluted to 1:3000 in 1% (w/v) BSA/TBS for 60 min with occasional shaking. Cells were washed three times with TBS before a colorimetric alkaline phosphate substrate (Bio-Rad) was added and incubated at 37 °C for 30 min. A 100-µl aliquot from each well was mixed with an equal volume of 0.4 M NaOH prior to measuring absorbance at 405 nm. Results were normalized against a wild-type control processed in parallel. Nontransfected cells were used to determine background. All experiments were performed in quadruplicate.

AVP-induced Inositol Phosphates Production—HEK 293T cells were seeded at a density of 2.5 x 105 cells/well in poly-D-lysine-coated 12-well plates and transfected after 24 h using TransfastTM (Promega). AVP-induced accumulation of inositol phosphates (InsPs) was assayed as described previously (12). Briefly, following pre-labeling of transfected cells with 2 µCi/ml myo-[2-3H]inositol (22.0 Ci/mmol; PerkinElmer Life Sciences) in inositol-free Dulbecco's modified Eagle's medium containing 1% (v/v) fetal calf serum, a mixed fraction containing mono-, bis-, and trisphosphates (InsP-InsP3) was collected following stimulation by AVP, at the concentrations indicated, in the presence of 10 mM LiCl.

Molecular Modeling of the V1aR—The V1aR sequence was aligned against the sequence corresponding to the crystal structure coordinates of bRho using ClustalW (28). The alignment was then used to generate homology models using MODELLER version 6.2 (29). A collection of 200 model structures was generated and ranked based on an objective function score 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 (MD) simulations of the receptor embedded in a hydrated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayer. MD simulations were carried out using the GROMOS96 force-field parameters, with minor modifications, as implemented in GROMACS (30). Partial charges for the heavy atoms of Lys and Arg side chains were determined using the 6-31G basis set as implemented in GAMESS US.


Figure 1
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  		FIGURE 1.
The extracellular face of neurohypophysial hormone receptors. A, sequence alignment of the extracellular loop regions of vasopressin and oxytocin receptors cloned from different species. The sequences of the extracellular loop regions (ECL1, ECL2, and ECL3) of the V1aR, OTR, V1bR, and V2R from different species have been aligned. The species of origin is indicated 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 sequence of the vasotocin and isotocin receptors from teleost fish and an amphibian mesotocin receptor. Conserved charged residues within these domains that were investigated in this study are boxed and numbered according to the rV1aR sequence. Sequences cited were obtained from Swiss Protein Database and GenEMBL. B, schematic diagram of the extracellular face of the rV1aR. Residues shown as white text on a black circle are the conserved charged residues investigated in this study.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Role of Charged Residues in the First Extracellular Loop (ECL1) of the V1aR—The sequences of the extracellular loops, plus the extracellular boundaries, of the neurohypophysial peptide hormone subfamily of GPCRs are aligned in Fig. 1A. Within ECL1 (including the extracellular regions of TMII and TMIII), there are five conserved charged residues as follows: Asp112, Arg116, Arg118, Asp121, and Arg125 (Fig. 1B, residue number is based on rV1aR). Residues Arg116, Asp121, and Arg125 are absolutely conserved across all VPR/OTRs cloned to date (Fig. 1A). Asp112 is also conserved with the exception of the human V2R (Lys) and the VTR (Glu). Arg118 is only conserved in V1aRs, although positively charged residues are present at this locus in all V2Rs (Fig. 1A).

To assess the importance of these conserved residues in V1aR function, each residue was substituted individually by Ala and then pharmacologically characterized using the natural agonist AVP and three different structural classes of antagonist as follows: (i) CA, [d(CH2)5Tyr(Me)2]AVP (25); (ii) LA ([PhAc-D-Tyr(Me)2Arg6Tyr(NH2)9]AVP (31)); and (iii) nonpeptide antagonist (SR 49059; (32)). The Kd values are presented in Table 1, corrected for radioligand occupancy. Mutating Arg116, Arg118, Asp121, or Arg125 to Ala had only a slight effect on the binding of the agonist AVP or the three different classes of antagonist (Table 1 and Fig. 2). [D112A]V1aR was also essentially wild type, although the Kd value for LA was slightly (5-fold) increased. Furthermore, the mutations [D112A]V1aR, [R118A]V1aR, and [D121A]V1aR had little effect on signaling, with EC50 values for AVP-stimulated inositol phosphate (InsP-InsP3) accumulations comparable with wild-type V1aR (Table 1). In contrast, [R116A]V1aR and [R125A]V1aR had a marked effect on signaling, increasing the EC50 value 70- and 16-fold, respectively, compared with the wild type (Fig. 3, A and B).


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  		TABLE 1
Pharmacological profile of mutant V1aRs Mutant V1aRs were expressed in HEK 293T cells and characterized pharmacologically. Dissociation constants (Kd) were calculated from IC50 values and corrected for radioligand occupancy as described under "Experimental Procedures." Data shown are the mean ± S.E. (n = 3) of three replicates. SR 49059 indicates nonpeptide antagonist. EC50 and Emax values of AVP-induced accumulation of InsP-InsP3 in cells expressing wild-type (WT) and mutant receptors are shown. Values shown are the mean ± S.E. of three separate experiments performed in triplicate. NS indicates no stimulation. *, data were taken from Ref. 34. Basal values (mean ± S.E.) were 1217 ± 252, 1263 ± 224, 1135 ± 201, 1080 ± 158, 1214 ± 207, 1073 ± 223, 984 ± 198, 956 ± 214, 1075 ± 248, 1256 ± 258, 1208 ± 174, 1007 ± 125, 1283 ± 202, 1321 ± 230, 1299 ± 188, 1116 ± 207, 1302 ± 191, 1350 ± 260, 956 ± 239, 1150 ± 176, 1045 ± 115, 1263 ± 239, 1371 ± 206, 1298 ± 117, 1228 ± 223, 1008 ± 182, and 1251 ± 248 dpm for wild type, [D112A]V1aR, [D112E]V1aR, [D112K]V1aR, [D112R]V1aR, [D116A]V1aR, [D116D]V1aR, [D116E]V1aR, [D116K]V1aR, [R118A]V1aR, [D121A]V1aR, [R125A]V1aR, [R125D]V1aR, [R125K]V1aR, [E193A]V1aR, [E195A]V1aR, [D204A]V1aR, [D204E]V1aR, [D204R]V1aR, [K201A]V1aR, [R216A]V1aR, [R46D]V1aR, [R46D/D204R]V1aR, [R125D/D204R]V1aR, [D323A]V1aR, [D330A]V1aR, and [E332A]V1aR respectively. None of the mutant receptors displayed constitutive activity. Cell-surface expression was determined, mean ± S.E. (n = 3), in parallel experiments as a percentage relative to wild-type V1aR using an ELISA-based assay, or as pmol/mg protein (Bmax) using whole cell 3H-labeled LA binding where possible, as described under "Experimental Procedures."

 
A Positive Charge Is Required at Residue 116 in ECL1—The charge requirements of residue 116 were investigated further by engineering [R116D]V1aR, [R116E]V1aR (incorporating a negative charge), and [R116K]V1aR (maintaining a positive charge). A negative charge was not tolerated at this position, as the affinity of AVP decreased 1600- and 730-fold for [R116D]V1aR and [R116E]V1aR, respectively, compared with wild-type V1aR (Fig. 2A). In contrast, the binding affinities of the three different antagonists to [R116D]V1aR and [R116E]V1aR were relatively unchanged, although a small decrease (~6-fold) was observed for CA (Table 1). Incorporating a negative charge at this locus also perturbed receptor activation, increasing the EC50 value for [R116D]V1aR and [R116E]V1aR by 53- and 23-fold, respectively (Fig. 3A). In contrast, maintaining a positive charge at this position ([R116K]V1aR) resulted in a receptor that exhibited essentially wild-type binding (Fig. 2A and Table 1) and signaling (Fig. 3A).

A Positive Charge Is Required at Residue 125 in ECL1—The charge requirements of residue 125 were investigated further. Retaining a positive charge ([R125K]V1aR) resulted in a wild-type receptor profile (Table 1). In contrast, introduction of a negative charge at this locus ([R125D]V1aR) ablated specific binding of the radio-tracers (agonist and antagonist) and impaired signaling, with a marked decrease in AVP potency compared with wild-type V1aR (Fig. 3B). Molecular modeling of the V1aR indicated that Arg125 orientates into the lipid bilayer (Fig. 4A), with the side-chain methylene groups interacting with the lipid hydrocarbon tails and the guanidinium group interacting with the lipid phosphate head groups and solvent. These contacts are preserved in [R125K]V1aR, consistent with the wild-type characteristics of this mutant receptor. In contrast, molecular dynamics of [R125D]V1aR revealed a re-ordering of the phospholipids in this region resulting from mutual repulsion between the negatively charged lipid phosphate head group and the carboxyl of the Asp side chain. This re-ordering of the lipids increased solvent accessibility at the extracellular end of TMIII and TMIV (Fig. 4A).

Species-specific and Receptor Subtype-specific Differences at Position 112 in ECL1—An Asp is highly conserved at residue 112 throughout this family of GPCRs, with the exception of the VTR and the human V2R that possess Glu and Lys, respectively (Fig. 1). Pharmacological differences arising from this sequence variation were assessed. Conservative substitution ([D112E]V1aR) resulted in wild-type binding and intracellular signaling, with only a small change in affinity for the CA antagonist (3-fold). Reversing the charge in [D112K]V1aR also slightly decreased the affinity of CA (5-fold) and reduced the affinity of the linear antagonist LA 8-fold (Table 1) but was otherwise wild type (Fig. 3C). However, in marked contrast to [D112K]V1aR, the construct [D112R]V1aR exhibited low affinity for AVP (Fig. 2B) and perturbed signaling (Fig. 3C). These effects were not because of a nonspecific disruption of the receptor tertiary fold as the affinity of the three classes of antagonist was unchanged (Table 1).

Asp112 is located at the membrane/solvent interface at the extracellular end of TMII. Molecular modeling shows that when residue 112 is Glu or Lys, they occupy a similar position to Asp112, consistent with the near wild-type profile observed with these constructs. However, the increased side-chain length of Arg112 compared with Lys112 positions the positively charged guanidinium moiety of Arg112 3.0 Å from the carboxyl group of Glu54(1.35) (top of TMI), resulting in a charge-charge interaction between these two residues (Fig. 4B). A comparable interaction between the amine of Lys112 and Glu54 is far less likely as the functional groups are further apart (4.8 Å). Furthermore, the guanidinium of Arg has higher partial charges on its heavy atoms compared with the amine of Lys, which increases the potential of the Arg guanidinium to establish ionic interactions compared with the amine of Lys. In addition, the planar nature of the guanidinium group may aid directive interactions.

Role of Charged Residues in the 2nd Extracellular Loop (ECL2) of the V1aR—The ECL2 domain (including the extracellular borders of TMIV and TMV) of the V1aR contains five charged residues Glu193, Glu195, Lys201, Asp204, and Arg216 (Fig. 1B). Sequence analysis of ECL2 revealed the following: (i) charged residues are well conserved at loci corresponding to Glu195, Asp204, and Arg216 throughout the vertebrate VPR/OTR family, whereas Glu193 and Lys201 are found only in the V1aR subtype; (ii) Asp204 is absolutely conserved with the single exception of the chick VTR, which has a Glu (33); (iii) a negative charge (usually a Glu but an Asp in V2Rs) is conserved at residue 195 with the exception of the human V2R, which has Asn; and (iv) a positive charge (Arg/Lys) is conserved at position 216 but is replaced by a Pro in the sheep V1aR (Fig. 1A).

To assess the functional importance of these conserved charged residues, each residue was mutated individually to Ala, and the pharmacological characteristics were compared with wild-type V1aR (Table 1). With the exception of [D204A]V1aR, all the mutant constructs exhibited binding and signaling characteristics similar to wild type (Table 1). In marked contrast, [D204A]V1aR possessed a marked decrease in AVP affinity (2300-fold; Fig. 2C and Table 1) and impaired signaling (Fig. 3D). The affinity of [D204A]V1aR for the cyclic and nonpeptide antagonists remained unchanged (Table 1), indicating that the receptor protein was folded appropriately; nevertheless, the Kd value for LA was increased 20-fold relative to wild type (Table 1). The charge requirements at position 204 were investigated. Retaining a negative charge ([D204E]V1aR) resulted in wild-type ligand binding and signaling (Figs. 2C and 3D and Table 1), whereas reversing the charge ([D204R]V1aR) markedly decreased both AVP affinity (Fig. 2C) and signaling (Fig. 3D) and to a lesser extent LA and CA affinity (24- and 6-fold, respectively; Table 1). The binding of the nonpeptide antagonist to [D204R]V1aR was wild type.


Figure 2
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  		FIGURE 2.
Pharmacological characterization of ECL mutant receptors. Radioligand binding studies with AVP as competing ligand were performed using a membrane preparation of HEK 293T cells transiently transfected as follows. A, wild-type (Arg116) V1aR ({circ}); [R116A]V1aR (•); [R116E]V1aR ({blacktriangledown}); [R116D]V1aR ({blacktriangleup}); and [R116K]V1aR ({diamondsuit}). B, wild-type (Asp112) V1aR ({circ}); [D112A]V1aR (•); [D112E]V1aR ({blacktriangledown}); [D112K]V1aR ({diamondsuit}); and [D112R]V1aR ({blacksquare}). C, wild-type (Asp204) V1aR ({circ}); [D204A]V1aR (•); [D204E]V1aR ({blacktriangledown}); and [D204R]V1aR({blacksquare}). Data are the mean ± S.E. of three separate experiments each performed in triplicate using [3H]AVP (0.5-1.5 nM) or 3H-labeled CA (0.5-1.5 nM) as tracer. 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 under "Experimental Procedures."

 
Investigating a Possible Interaction between Asp204 and Arg125 or Arg46—Mutagenesis of Asp204 or Arg125 generated similar effects. Consequently, substitution by Ala ([D204A]V1aR and [R125A]V1aR) decreased the potency of AVP signaling by ~20-fold compared with wild-type V1aR (Fig. 3, D and B, respectively), and reversing the charge ([D204R]V1aR and [R125D]V1aR) increased the EC50 100-fold compared with wild-type V1aR. Although Arg125 (top of TMIII) and Asp204 (ECL2) are in different domains, they are located at opposite ends of the highly conserved disulfide bond and therefore in close proximity and spatially constrained. It was possible that a mutual charge interaction existed between these two residues, which was required for receptor activation. However, the double-reciprocal mutant [R125D/D204R]V1aR did not bind 3H-labeled tracer ligands, did not signal when challenged with AVP (>10 µM), and was poorly expressed (Table 1). Consequently, these data do not support a mutual interaction between Arg125 and Asp204.

We have established previously that a single residue (Arg46) located within the distal N terminus of the V1aR is critical for binding AVP but not peptide or nonpeptide antagonists (14) and that reversing the charge at this locus ([R46D]V1aR or [R46E]V1aR) impaired receptor function (34) in a similar manner to that observed for [D204R]V1aR in this study. Given that high affinity agonist binding required both Arg46 and Asp204, it was feasible that a direct intra-molecular ionic interaction between Arg46 and Asp204 may contribute to high affinity agonist binding and receptor activation. However, the double-reciprocal mutant [R46D/D204R]V1aR bound AVP with very low affinity (Kd = 2500 nM), a similar affinity to [R46D]V1aR or [D204R]V1aR (Table 1), and the signaling capability of [R46D/D204R]V1aR was also severely compromised. The overall tertiary fold of the receptor was nevertheless good as the nonpeptide antagonist bound with wild-type affinity, and the peptide antagonists CA and LA also bound with high affinity, albeit less than wild type (Table 1). Cell-surface expression of [R46D/D204R]V1aR was only ~20% of wild type. These data do not provide evidence for a direct interaction between Arg46 and Asp204.

Molecular modeling indicated that Asp204 lies at the center of a pocket defined by residues Lys128(3.29) (TMIII), Gln131(3.32) (TMIII), Trp206 (ECL2), Phe283(6.51) (TMVI), and Gln287(6.55) (TMVI). Asp204 forms a salt bridge with Lys128(3.29) and hydrogen bonds with Gln131(3.32), both in TMIII (Fig. 4C). These interactions with Lys128(3.29) and Gln131(3.32) are preserved in the conservative substitution [D204E]V1aR (not shown), consistent with the wild-type pharmacological profile (Table 1). Removal of the negative charge at this locus ([D204A]V1aR) resulted in a decrease in both AVP affinity and signaling potency (Fig. 3D and Table 1). MD simulation of [D204A]V1aR revealed that removal of the negative charge breaks the wild-type contacts between ECL2 and TMIII (Fig. 4D). This leads to a partial unfolding of the beta-hairpin within ECL2 (Fig. 4E). In addition, the side chain of Lys128 rotates away from its wild-type position and orientates toward TMVI (Fig. 4D). A similar perturbation was observed with the construct [D204R]V1aR, again leading to re-organization of ECL2. However, the introduction of an Arg at residue 204 also created an alternative hydrogen bonding network involving new interactions between Arg204 in ECL2 and residues in TMII (Gln104(2.57) and Gln108(2.61)) and TMVII (Ala272(7.42) and Ser273(7.43)) (Fig. 4F).


Figure 3
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  		FIGURE 3.
Intracellular signaling by ECL mutant receptors. AVP-induced accumulation of mono-, bis-, and trisphosphates in HEK 293T cells transiently transfected as follows. A, wild-type (Arg116) V1aR ({circ}); [R116A]V1aR (•); [R116E]V1aR ({blacktriangledown}); [R116D]V1aR ({blacktriangleup}); and [R116K]V1aR ({diamondsuit}). B, wild-type (Arg125) V1aR ({circ}); [R125A]V1aR (•); [R125D]V1aR ({blacktriangleup}); and [R125K]V1aR ({diamondsuit}). C, wild-type (Asp112) V1aR ({circ}); [D112A]V1aR (•); [D112E]V1aR ({blacktriangledown}); [D112K]V1aR ({diamondsuit}; and [D112R]V1aR ({blacksquare}). D, wild-type (Asp204) V1aR ({circ}); [D204A]V1aR (•); [D204E]V1aR ({blacktriangledown}); and [D204R]V1aR ({blacksquare}). Data are the mean ± S.E. of three separate experiments each performed in triplicate. Values are stimulation induced by AVP at the stated concentrations expressed as percent maximum.

 
Role of Charged Residues in ECL3 of the V1aR—ECL3 (including the extracellular borders of TMVI and TMVII) of the V1aR contains three conserved charged residues Asp323, Asp330, and Glu332 (Fig. 1B). Analysis of sequence alignments of ECL3 of the vertebrate VPR/OTR family revealed the following: (i) an Asp is completely conserved at the locus corresponding to Asp323; (ii) a negative charge (usually an Asp) is conserved at residue 330 in V1aRs, V1bRs, and VTRs but is replaced by Pro in OTRs and V2Rs; and (iii) a Glu is conserved at the locus corresponding to Glu332 with the exception of V1bRs that possess a Thr (Fig. 1A). To determine the functional importance of these residues, each residue was mutated individually to Ala. Pharmacological characterization established that [D323A]V1aR, [D330A]V1aR, and [E332A]V1aR were almost identical to wild-type V1aR with respect to binding all four classes of ligand (Table 1) and intracellular signaling (Table 1), indicating that the conserved charged residues in ECL3 had little or no role in these functions. However, it was noteworthy that [D323A]V1aR exhibited reduced cell-surface expression (~50%) compared with wild-type V1aR (Table 1).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The aim of this study was to use the V1aR to systematically investigate the function of extracellular charged residues that are highly conserved throughout a subfamily of peptide GPCRs. Within the ECL domains of the V1aR, the charged residues were subdivided into the following two groups: (i) those that are conserved in all members of the subfamily, and (ii) those that are conserved within a specific subtype. Thirteen conserved charged residues were identified in the ECL domains and associated TM boundaries, with five in ECL1, five in ECL2, and three in ECL3. Ala substitution within ECL1 had little effect on ligand binding. However, [R116A]V1aR and [R125A]V1aR exhibited impaired intracellular signaling (70- and 16-fold, respectively) indicating a role in receptor activation. Although [R125A]V1aR was expressed at 56% of wild type, this was unlikely to be responsible for the impaired signaling of [R125A]V1aR, as [D323A]V1aR was expressed at 52% of wild type but retained essentially wild-type signaling capability (Table 1). A positive charge is essential at residue 116, as retaining a positive charge ([R116K]V1aR) preserved wild-type signaling, and reversing the charge ([R116D]V1aR and [R116E]V1aR) not only compromised signaling but also profoundly decreased agonist affinity. This loss of AVP binding was agonist-specific and not because of aberrant assembly of the receptor as the binding of antagonists (peptide and nonpeptide) was unaffected. Consequently, Arg116 is required to stabilize the active R* conformation of the V1aR and is absolutely conserved throughout the vertebrate neurohypophysial hormone subfamily of GPCRs cloned to date (Fig. 1A).

Arg125 is located close to the extracellular end of TMIII, immediately adjacent to the conserved disulfide bond, where it interacts with lipids. This Arg-lipid interaction has been referred to as "snorkeling" (35). The absolute conservation of this Arg throughout the neurohypophysial peptide hormone receptor family (Fig. 1A) implies functional importance. This is supported by a report that the naturally occurring mutation R113W in the human V2R (which corresponds to Arg125 in the V1aR) causes the receptor dysfunction responsible for nephrogenic diabetes insipidus in some patients (36). Furthermore, an alignment of 717 sequences of family A GPCRs, which bind peptide ligands, revealed that a positively charged residue is conserved at this position in 85% of receptors and that Asp and Glu are excluded (see the GPCR data base). This is indicative of a generic role for this residue in signaling by peptide-GPCRs, a notion supported by mutagenesis studies on the CXCR2 and angiotensin II type 1 receptors (37, 38). It is now well established that relative movement between TMIII, TMVI, and TMVII is central to the R -> R* transition of GPCRs (39). The location of Arg125 at the extracellular extremity of TMIII may allow it to act as a structural support for TMIII during receptor activation. Reversing the charge at this locus in [R125D]V1aR was very detrimental because of charge-charge repulsion between the side-chain carboxyl and the membrane lipid phosphate head groups. This repulsion resulted in re-ordering of the surrounding phospholipids, increased solvent accessibility at the extracellular end of TMIII/TMIV, and altered local conformation that could have ramifications along the length of TMIII. These conformational changes observed in silico would explain why Asp/Glu are excluded from this locus in peptide-binding GPCRs. In marked contrast, GPCRs for biogenic amines actually favor a negatively charged residue at the position corresponding to Arg125. Analysis of an alignment of 371 sequences of amine-GPCRs from different species revealed that ~70% have Glu/Asp at this locus (GPCR data base) with the exclusion of Arg/Lys. Exceptions to this trend are the H3 histamine receptor and trace amine receptors that do possess a positive charge. It is possible that the charge difference at this single locus between peptide-GPCRs and amine-GPCRs may reflect differences in the binding mode between these ligands. Biogenic amines access a binding site enclosed within the TM bundle, whereas peptides bind to extracellular domains plus TM helices. If Glu/Asp at the top of TMIII in amine-GPCRs (corresponding to Arg125 in V1aR) increases solvent penetration into the TM bundle (analogous to the mutant [R125D]V1aR), it may facilitate ligand access to the binding site. Support for such a mechanism is perhaps provided by [3H]propylbenzylcholine mustard ([3H]PrBCM) labeling of the M1 mAChR. In addition to alkylating Asp105 in TMIII (the "classical" amine counter-ion), [3H]PrBCM also labeled Asp99 (corresponding to Arg125 in the V1aR) (40). Furthermore, mutation of this Asp99 to Asn moderately decreased the affinity of a range of ligands and strongly decreased both alkylation by [3H]PrBCM and agonist-induced second messenger generation (41).


Figure 4
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  		FIGURE 4.
Molecular modeling of wild-type and mutant receptors. A, the overlapped positions of solvent molecules are presented for wild-type V1aR (yellow) and [R125D]V1aR (blue) simulations. It can be seen that the mutation [R125D]V1aR increased the solvent-accessible surface as a result of re-ordering of the phospholipids surrounding the extracellular end of TMIII. B, the mutant [D112R]V1aR enabled inappropriate hydrogen bonds (dotted green lines) to be formed between this residue and Glu54 in TMI. C, in the wild-type V1aR, Asp204 in ECL2 hydrogen bonds (dotted green lines) with Lys128 and Gln131 in TMIII. These contacts are broken in the mutant [D204A]V1aR (D) which results in partial unfolding of the beta-hairpin structure of ECL2. E, the partially unfolded ECL2 beta-hairpin structure of [D204A]V1aR (blue) is revealed when superimposed onto that of wild-type V1aR (yellow). F, the mutant [D204R]V1aR inappropriately hydrogen bonds (dotted green) with residues in TMVII and TMII. See text for details.

 
Asp112 in the V1aR is conserved throughout the neurohypophysial hormone receptor family with the exception of the human V2R and the VTR that possess Lys and Glu, respectively. It has been reported that this locus is important for binding some V2R-selective agonists (42). The substitutions [D112A]V1aR, [D112E]V1aR, and [D112K]V1aR had little effect on receptor function; introducing Arg112, however, impaired agonist binding and signaling but had little effect on any antagonist binding. Although Arg and Lys are superficially similar, [D112K]V1aR and [D112R]V1aR exhibited very different affinity for AVP. Molecular modeling revealed that Arg112 formed a stable ionic interaction with Glu54(1.35) at the top of TMI, which was absent with the shorter side chain of Lys112 (and also absent in wild type and [D112E]V1aR). Glu54(1.35) has recently been identified as a key residue for high affinity agonist binding and signaling (15); consequently, an inappropriate interaction with Arg112 may prevent Glu54(1.35) from adopting an optimal conformation for AVP binding and signaling.

ECL2 is usually the longest ECL in GPCRs and in bRho forms a beta-hairpin that projects into the binding crevice allowing the beta4-strand to contact retinal (1, 2). There is also evidence that this ECL2 fold is not restricted to bRho and occurs in other GPCRs (43). Of the five conserved charged residues substituted by Ala in ECL2 of the V1aR, only [D204A]V1aR had a marked effect on receptor function, with a profound decrease in AVP affinity, decreased LA affinity, and impaired signaling potency. Molecular modeling revealed that Asp204 provides interactions between ECL2 and TMIII by hydrogen bonding with Gln131(3.32) and forming a salt bridge with Lys128(3.29). Substitution by Ala in [D204A]V1aR disrupted this contact network resulting in partial unfolding of ECL2 and re-arrangement of the Lys128(3.29) side chain. Substitution of Lys128(3.29) or Gln131(3.32) by Ala also disrupted AVP binding and signaling (8), consistent with the proposed role for Asp204. The reduction in affinity of both AVP and LA was similar following either removal of the negative charge ([D204A]V1aR) or charge reversal ([D204R]V1aR) (Table 1). Although [D204R]V1aR and [D204A]V1aR had the same affinity for AVP, the decrease in potency of AVP-stimulated InsP-InsP3 production was greater with Arg204 than with Ala204 (Table 1), suggesting that Arg204 stabilized the receptor ground state. Molecular modeling indicated that for both of these constructs the interactions between ECL2 and TMIII were disrupted in a similar manner leading to re-organization of ECL2. However, the introduction of the longer side chain of Arg204 also created new interactions between ECL2-TMII (Gln104(2.57) and Gln108(2.61)) and ECL2-TMVII (Ala272(7.42) and Ser273(7.43)), which reduced the R -> R* transition. Interestingly, Ser273(7.43) corresponds to the retinal attachment site in bRho, and this locus has been implicated in activation of other GPCRs (44, 45). In addition, Ala substitution of both of the TMII contacts, Gln104(2.57) and Gln108(2.61), has been reported previously to perturb both ligand binding and intracellular signaling (8). Consequently, the inappropriate new contacts established by Arg204 are with residues required for receptor activation and explain the perturbed pharmacological profile observed with [D204R]V1aR. In contrast, the conservative substitution [D204E]V1aR, which occurs naturally in a chick VTR (33), maintains the normal ECL2-TMIII contacts and exhibits wild-type characteristics. Our investigations establish the importance of an acidic residue at position 204 and provide an explanation for the absolute conservation of Asp(Glu) at this locus throughout a subfamily of GPCRs. In addition, our study also provides a feasible mechanism for the naturally occurring "loss-of-function" mutation D191G in the human V2R (corresponding to Asp204 in the V1aR) that has been identified as a cause of nephrogenic diabetes insipidus in some families (46). Asp204 is juxtaposed to the disulfide bridge (Cys205), conserved in the majority of GPCRs, and is therefore under positional restraint. Interestingly, the residue corresponding to Asp204 has been reported to be functionally important in other GPCRs. For example, Met195 of the cholecystokinin-A receptor is required for interaction with cholecystokinin (47), and the mutation I185A affected CXCR4 co-receptor activity for some human immunodeficiency virus strains (48).

The functional importance of Asp204 for agonist binding and signaling by V1aR is a property shared by Arg125 (this study) and also Arg46 in the N terminus (14). It was therefore possible that a charge-charge interaction between Arg125-Asp204 or Arg46-Asp204 was required for high affinity agonist binding. Interaction between these two charge pairs was theoretically possible as Arg125-Asp204 is located at opposite ends of the same disulfide bond, and in bRho the N terminus has been shown to make multiple contacts with ECL2 (1, 2). However, the double-reciprocal mutants [R125D/D204R]V1aR and [R46D/D204R]V1aR were both severely compromised; therefore, our data do not support a direct interaction of Asp204 with either Arg125 or Arg46.

Although ECL3 charged residues are important for peptide ligands binding to some GPCRs (49-51), substitution of the three conserved charged residues in ECL3 of the V1aR did not affect either ligand binding or activation of the receptor. However, the mutant [D323A]V1aR did exhibit decreased cell-surface expression (~50% of wild type). It is noteworthy that Asp323 is the only ECL3 charged residue absolutely conserved throughout the vertebrate neurohypophysial hormone receptors cloned to date, suggesting that it may fulfill an important role in maintaining cell-surface expression that is common to all members of this family. Our data do not support the suggestion (52) that ECL3 acidic residues might be implicated in binding AVP and vasotocin.

In conclusion, we have shown that key charged residues located throughout the extracellular face of the V1aR are required for normal receptor function, identifying Arg116 (ECL1), Arg125 (top of TMIII), and Asp204 (ECL2) as important for high affinity agonist binding and/or receptor activation and Asp323 (ECL3) as important for cell-surface expression. Consistent with their fundamental role in receptor function, these charged residues are highly conserved throughout the neurohypophysial hormone receptor subfamily of GPCRs.


   FOOTNOTES
 
* This work was supported by grants (to M. W.) from the Wellcome Trust, the Biotechnology and Biological Sciences Research Council, and Ferring Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors should be considered as joint first authors. Back

2 Present address: Novartis Pharma AG, WSJ-386.9.59, CH-4002 Basel, Switzerland. Back

3 To whom correspondence should be addressed: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK. Tel.: 44-121-4143981; Fax: 44-121-414-5925; E-mail: m.wheatley{at}bham.ac.uk.

4 The abbreviations used are: GPCR, G-protein-coupled receptor; AVP, [Arg8]vasopressin; bRho, bovine rhodopsin; CA, cyclic peptide antagonist; ECL, extracellular loop; HA, hemagglutinin; InsP, inositol phosphate; InsP3, inositol trisphosphate; LA, linear peptide antagonist; OT, oxytocin; OTR, oxytocin receptor; PhAc, phenylacetyl; TM, transmembrane helix; V1aR, V1a vasopressin receptor; V1bR, V1b vasopressin receptor; V2R, V2 vasopressin receptor; VTR, vasotocin receptor; BSA, bovine serum albumin; PrBCM, propylbenzylcholine mustard. Back

5 Residues in the TMs are referred to by residue number and the nomenclature of Ballosteros and Weinstein (53). Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Claudine Serradeil-Le Gal (Sanofi Recherche, France) for providing a sample of SR 49059.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Palczewski, K., Kumasaka, T., Hori, T., Behnke, C. A., Motoshima, H., Fox, B. A., Le Trong, I., Teller, D. C., Okada, T., Stenkamp, R. E., Yamamoto, M., and Miyano, M. (2000) Science 289, 739-745[Abstract/Free Full Text]
  2. Li, J., Edwards, P. C., Burghammer, M., Villa, C., and Schertler, G. F. X. (2004) J. Mol. Biol. 343, 1409-1438[CrossRef][Medline] [Order article via Infotrieve]
  3. Lu, Z. L., Saldanha, J. W., and Hulme, E. C. (2002) Trends Pharmacol. Sci. 23, 140-146[CrossRef][Medline] [Order article via Infotrieve]
  4. Thibonnier, M., Coles, P., Thibonnier, A., and Shoham, M. (2001) Annu. Rev. Pharmacol. Toxicol. 41, 175-202
  5. Gimpl, G., and Fahrenholz, F. (2001) Physiol. Rev. 81, 629-683[Abstract/Free Full Text]
  6. Howl, J., and Wheatley, M. (1995) Gen. Pharmacol. 26, 1143-1152[Medline] [Order article via Infotrieve]
  7. Hibert, M., Hoflack, J., Trumpp-Kallmeyer, S., Mouillac, B., Chini, B., Mahe, E., Cotte, N., Jard, S., Manning, M., and Barberis, C. (1999) J. Recept. Signal. Transduct. Res. 19, 589-596[Medline] [Order article via Infotrieve]
  8. Mouillac, B., Chini, B., Balestre, M.-N., Elands, J., Trump-Kallmeyer, S., Hoflack, J., Hibert, M., Jard, S., and Barberis, C. (1995) J. Biol. Chem. 270, 25771-25777[Abstract/Free Full Text]
  9. Howl, J., and Wheatley, M. (1996) Biochem. J. 317, 577-582
  10. Kojro, E., Eich, P., Gimpl, G., and Fahrenholz, F. (1993) Biochemistry 32, 13537-13544[CrossRef][Medline] [Order article via Infotrieve]
  11. Chini, B., Mouillac, B., Ala, Y., Balestre, M.-N., Trump-Kallmeyer, S., Hoflack, J., Elands, J., Hibert, M., Manning, M., Jard, S., and Barberis, C. (1995) EMBO J. 14, 2176-2182[Medline] [Order article via Infotrieve]
  12. Hawtin, S. R., Wesley, V. J., Parslow, R. A., Patel, S., and Wheatley, M. (2000) Biochemistry 39, 13524-13533[CrossRef][Medline] [Order article via Infotrieve]
  13. Hawtin, S. R., Howard, H. C., and Wheatley, M. (2001) Biochem. J. 354, 465-472[CrossRef][Medline] [Order article via Infotrieve]
  14. Hawtin, S. R., Wesley, V. J., Parslow, R. A., Simms, J., Miles, A., McEwan, K., and Wheatley, M. (2002) Mol. Endocrinol. 16, 600-609[Abstract/Free Full Text]
  15. Hawtin, S. R., Wesley, V. J., Simms, J., Argent, C. C. H., Latif, K., and Wheatley, M. (2005) Mol. Endocrinol. 19, 2871-2881[Abstract/Free Full Text]
  16. Wesley, V. J., Hawtin, S. R., Howard, H. C., and Wheatley, M. (2002) Biochemistry 41, 5086-5092[CrossRef][Medline] [Order article via Infotrieve]
  17. Shi, L., and Javitch, J. A. (2002) Annu. Rev. Pharmacol. Toxicol. 42, 437-467
  18. Ding, X. Q., Pinon, D. I., Furse, K. E., Lybrand, T. P., and Miller, L. J. (2002) Mol. Pharmacol. 61, 1041-1052[Abstract/Free Full Text]
  19. Zhao, M. M., Hwa, J., and Perez, D. M. (1996) Mol. Pharmacol. 50, 1118-1126[Abstract]
  20. Gnagey, A. L., Seidenberg, M., and Ellis, J. (1999) Mol. Pharmacol. 56, 1245-1253[Abstract/Free Full Text]
  21. Ott, T. R., Troskie, B. E., Roeske, R. W., Illing, N., Flanagan, C. A., and Millar, R. P. (2002) Mol. Endocrinol. 10, 1079-1088
  22. Brelot, A., Heveker, N., Montes, M., and Alizon, M. (2000) J. Biol. Chem. 275, 23736-23744[Abstract/Free Full Text]
  23. Hawtin, S. R., Tobin, A., Patel, S., and Wheatley, M. (2001) J. Biol. Chem. 276, 38139-38146[Abstract/Free Full Text]
  24. Howl, J., Langel, Ü., Hawtin, S. R., Valkna, A., Yarwood, N. J., Saar, K., and Wheatley, M. (1997) FASEB J. 11, 582-590[Abstract]
  25. Kruszynski, M., Lammek, B., Manning, M., Seto, J., Haldar, J., and Sawyer, W. H. (1980) J. Med. Chem. 23, 364-368[CrossRef][Medline] [Order article via Infotrieve]
  26. Cheng, Y., and Prusoff, W. H. (1973) Biochem. Pharmacol. 22, 3099-3108[CrossRef][Medline] [Order article via Infotrieve]
  27. Conner, A. C., Hay, D. L., Simms, J., Howitt, S. G., Schindler, M., Smith, D. M., Wheatley, M., and Poyner, D. R. (2005) Mol. Pharmacol. 67, 20-31[Abstract/Free Full Text]
  28. Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-4680[Abstract/Free Full Text]
  29. Sali, A., and Blundell, T. L. (1993) J. Mol. Biol. 234, 779-815[CrossRef][Medline] [Order article via Infotrieve]
  30. Lindahl, E., Hess, B., and van der Spoel, D. (2001) J. Mol. Med. 7, 306-317[CrossRef]
  31. Schmidt, A., Audigier, S., Barberis, C., Jard, S., Manning, M., Kolodziejczyk, A. S., and Sawyer, W. H. (1991) FEBS Lett. 282, 77-81[CrossRef][Medline] [Order article via Infotrieve]
  32. Serradeil-Le Gal, C., Wagnon, J., Garcia, C., Lacour, C., Guiraudou, P., Christophe, B., Villanova, G., Nisato, D., Maffrand, J. P., and Le Fur, G. P. (1993) J. Clin. Investig. 92, 224-231[Medline] [Order article via Infotrieve]
  33. Tan, F.-L., Lolait, S. J., Brownstein, M. J., Saito, N., MacLeod, V., Baeyens, D. A., Mayeux, P. R., Jones, S. M., and Cornett, L. E. (2000) Biol. Reprod. 62, 8-15[Abstract/Free Full Text]
  34. Hawtin, S. R., Wesley, V. J., Simms, J., Parslow, R. A., Simms, J., Miles, A., McEwan, K., Keen, M., and Wheatley, M. (2003) Eur. J. Biochem. 270, 4681-4688[Medline] [Order article via Infotrieve]
  35. Strandberg, E., and Killian, J. A. (2003) FEBS Lett. 544, 69-73[CrossRef][Medline] [Order article via Infotrieve]
  36. Birnbaumer, M., Gilbert, S., and Rosenthal, W. (1994) Mol. Endocrinol. 8, 886-894[Abstract/Free Full Text]
  37. Katancik, J. A., Sharma, A., and de Nardin, E. (2000) Cytokine 12, 1480-1488[CrossRef][Medline] [Order article via Infotrieve]
  38. Monnot, C., Bihoreau, C., Conchon, S., Curnow, K. M., Corvol, P., and Clauser, E. (1996) J. Biol. Chem. 271, 1507-1513[Abstract/Free Full Text]
  39. Schwartz, T. W., Frimurer, T. M., Holst, B., Rosenkilde, M. M., and Elling, C. E. (2006) Annu. Rev. Pharmacol. Toxicol. 46, 481-519
  40. Kurtenbach, E., Curtis, C. A. M., Pedder, E. K., Aitken, A., Harris, A. C. M., and Hulme, E. C. (1990) J. Biol. Chem. 265, 13702-13708[Abstract/Free Full Text]
  41. Fraser, C. M., Wang, C.-D., Robinson, D. A., Gocayne, J. D., and Venter, J. C. (1989) J. Biol. Chem. 36, 840-847
  42. Cotte, N., Balestre, M. N., Phalipou, S., Hibert, M., Manning, M., Barberis, C., and Mouillac, B. (1998) J. Biol. Chem. 273, 29462-29468[Abstract/Free Full Text]
  43. Shi, L., and Javitch, J. A (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 440-445[Abstract/Free Full Text]
  44. Marie, J., Richard, E., Pruneau, D., Paquet, J.-L., Siatka, C., Larguier, R., Ponce, C., Vassault, P., Groblewski, T., Maigret, B., and Bonnafous, J.-C. (2001) J. Biol. Chem. 276, 41100-41111[Abstract/Free Full Text]
  45. Lu, Z.-L., Saldanha, J. W., and Hulme, E. C. (2001) J. Biol. Chem. 276, 34098-34104[Abstract/Free Full Text]
  46. Arthus, M.-F., Lonergan, M., Crumley, M. J., Naumova, A. K., Morin, D., De Marco, L. A., Kaplan, B. S., Robertson, G. L., Sasaki, S., Morgan, K., Bichet, D. G., and Fujiwara, T. M. (2000) J. Am. Soc. Nephrol. 11, 1044-1054[Abstract/Free Full Text]
  47. Gigoux, V., Escrieut, C., Silvente-Poirot, S., Maigret, B., Gouilleux, L., Fehrentz, J. A., Gully, D., Moroder, L., Vaysse, N., and Fourmy, D. (1998) J. Biol. Chem. 273, 14380-14386[Abstract/Free Full Text]
  48. Brelot, A., Heveker, N., Adema, K., Hosie, M. J., Willett, B., and Alizon, M. (1999) J. Virol. 73, 2576-2586[Abstract/Free Full Text]
  49. Gigoux, V., Escrieut, C., Fehrentz, J. A., Poirot, S., Maigret, B., Moroder, L., Gully, D., Martinez, J., Vaysse, N., and Fourmy, D. (1999) J. Biol. Chem. 274, 20457-20464[Abstract/Free Full Text]
  50. Fromme, B. J., Katz, A. A., Roeske, R. W., Millar, R. P., and Flanagan, C. A. (2001) Mol. Pharmacol. 60, 1280-1287[Abstract/Free Full Text]
  51. Feng, Y.-H., Noda, K., Saad, Y., Liu, X.-P., Husain, A., and Karnik, S. S. (1995) J. Biol. Chem. 270, 12846-12850[Abstract/Free Full Text]
  52. Hausmann, H., Richters, A., Kreienkamp, H.-J., Meyerhof, W., Mattes, H., Lederis, K., Zwiers, H., and Richter, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6907-6912[Abstract/Free Full Text]
  53. Ballesteros, J. A., and Weinstein, H. (1995) Methods Neurosci. 25, 366-428[CrossRef]

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M. Conner, S. R. Hawtin, J. Simms, D. Wootten, Z. Lawson, A. C. Conner, R. A. Parslow, and M. Wheatley
Systematic Analysis of the Entire Second Extracellular Loop of the V1a Vasopressin Receptor: KEY RESIDUES, CONSERVED THROUGHOUT A G-PROTEIN-COUPLED RECEPTOR FAMILY, IDENTIFIED
J. Biol. Chem., June 15, 2007; 282(24): 17405 - 17412.
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