Vasopressin receptors: structure/function relationships and signal transduction in target cells

Vasopressin, a hypothalamic hormone, acts on its target tissues via three different G protein coupled receptors. The vasopressin V1a and V1b receptors, associated to Gq protein and phospholipase C, are responsible for vasoconstriction and regulation of the corticotroph axis respectively. The V2 vasopressin receptor is coupled to Gs protein and adenylyl cyclase and is responsible for water reabsorption in the renal collecting duct. Mutations of the V2 receptor are involved in diabetes insipidus and most of these mutations result in an endoplasmic reticulum (ER) retention of the mutated receptor. With the V1b receptor model, we have identified a proximal sequence of the C-terminal segment, which is crucial for ER export. Mutations in this short domain result in ER accumulation and degradation of the receptor. SSR 149415, a nonpeptide antagonist of V1bR, which is permeable to cell membrane, is able to rescue the mutant phenotype and acts as a pharmacological chaperone.     

Mapping the binding site of arginine vasopressin to V1a and V1b vasopressin receptors

Starting from the 2.8-A resolution x-ray structure of bovine rhodopsin, three-dimensional molecular models of the complexes between arginine vasopressin and two receptor subtypes (V1a, V1b) have been built. Amino acid sequence alignment and docking studies suggest that four key residues (1.35, 2.65, 4.61, and 5.35) fine tune the binding of vasopressin and related peptide agonists to both receptor subtypes. To validate these predictions, a series of single or double mutants were engineered at V1a and V1b receptor subtypes and tested for their binding and functional properties. Two negatively charged amino acids at positions 1.35 and 2.65 are key anchoring residues to the Arg8 residue of arginine vasopressin. Moreover, two amino acids (V(4.61) and P(5.35)) delineating a hydrophobic subsite at the human V1b receptor are responsible for the recognition of V1b selective peptide agonists. Last, one of the latter positions (5.35) is hypothesized to explain the pharmacological species differences between rat and human vasopressin receptors for a V1b peptide agonist. Altogether these refined three-dimensional models of V1a and V1b human receptors should enable the identification of further new selective V1a and V1b agonists as pharmacological but also therapeutic tools.     

Mechanisms of cell-surface rerouting of an endoplasmic reticulum-retained mutant of the vasopressin V1b/V3 receptor by a pharmacological chaperone

Cell-surface expression and biological functions of several intracellular-retained G protein-coupled receptors are restored by membrane-permeable ligands called pharmacological chaperones. We have previously demonstrated that a mutation of the hydrophobic motif 341FNX2LLX3L350 in the C terminus of the human pituitary vasopressin V3 receptor (MUT V3R) led to it being retained in the endoplasmic reticulum (ER). Here, we establish the precise role of this motif and investigate whether SSR149415, a non-peptide V3R antagonist, behaves as a pharmacological chaperone for the ER-retained MUT V3R. The absence of the mutated receptor in the plasma membrane is linked to its prolonged association with the molecular chaperone calnexin in the ER and to its intensive degradation by the ubiquitin-proteasomal machinery. However, this is not because of a lack of oligomerization, as demonstrated by the presence of MUT V3R homodimers in the ER. Treatment with SSR149415 restores expression of the mutated receptor on the cell surface and its correct maturation, resulting into the functional recovery of its signaling properties. SSR149415 acts by stabilizing a native-like conformation of the V3R, reducing its association with calnexin and, thus, favoring a secretory pathway rather than the proteasomal degradation pathway. In conclusion, the FN(X)2LL(X)3L sequence is an important motif for the V3R conformation, and the misfolding resulting from its mutation alters the receptor export but can be reverted by SSR149415.     

Biological characterization of rodent and human vasopressin V1b receptors using SSR-149415, a nonpeptide V1b receptor ligand

[(3)H]SSR-149415 is the first tritiated nonpeptide vasopressin V(1b) receptor (V(1b)R) antagonist ligand. It was used for studying rodent (mouse, rat, hamster) and human V(1b)R from native or recombinant origin. Moreover, a close comparison between the human and the mouse V(1b)R was performed using SSR-149415/[(3)H]SSR-149415 in binding and functional studies in vitro. [(3)H]SSR-149415 binding was time-dependent, reversible, and saturable. Scatchard plot analysis gave a single class of high-affinity binding sites with apparent equilibrium dissociation constant (K(d)) approximately 1 nM and maximum binding density (B(max)) values from 7,000 to 300,000 sites/cell according to the cell line. In competition experiments, [(3)H]SSR-149415 binding was stereospecific and dose-dependently displaced by reference peptide and nonpeptide arginine vasopressin (AVP)/OT ligands following a V(1b) rank order of affinity: SSR-149415 = AVP > dCha > dPen > dPal > dDavp > SSR-126768A > SR-49059 > SSR-149424 > OT > SR-121463B. Species differences between human, rat, mouse, and hamster V(1b)R were observed. Autoradiography studies with [(3)H]SSR-149415 on rat and human pituitary showed intense specific labeling confined to corticotroph cells and absence of labeling in the other tissues examined. SSR-149415 potently and stereospecifically antagonized the AVP-induced inositol phosphate production and intracellular Ca(2+) increase (EC(50) from 1.83 to 3.05 nM) in recombinant cell lines expressing either the mouse or the human V(1b)R. AVP (10(-7) M) exposure of AtT20 cells expressing mouse or human EGFP-tagged V(1b)R induced their rapid internalization. Preincubation with 10(-6) M SSR-149415 counteracted the internalization process. Moreover, recycling of internalized receptors was observed upon 10(-6) M SSR-149415 treatment. Thus SSR-149415/[(3)H]SSR-149415 are unique tools for studying animal and human V(1b)R.     

Potent and selective oxindole-based vasopressin 1b receptor antagonists with improved pharmacokinetic properties

Herein we report the discovery of a novel series of vasopressin 1b (V1b) receptor antagonists, starting from potent but metabolically labile oxindole SSR149415. Masking the proline N,N-dimethyl amide moiety as an oxazole and attaching a benzylic amine moiety to the northern phenyl ring resulted in potent and selective V1b receptor antagonists with improved metabolic stability and improved pharmacokinetic properties in rat. Compound 18c was found to be efficacious in a rat model of anti-depressant activity.     

An expanded V2 receptor retention signal

Following ligand-promoted internalization the human type 2 vasopressin receptor (hV2R) is not recycled to the cell surface after removal of the agonist. A retention motif consisting of a serine triplet present in the cytoplasmic tail was previously found to require for retention, but serine 357, and threonines 359, 360 located upstream were not examined. Evidence is now presented that substitution of these amino acids did not change V2 internalization although it reduced the levels of arginine vasopressin (AVP)-induced phosphorylation as compared to the wild type (WT). Contrary to the WT hV2R, these mutant receptors were recycled to the cell surface after a 2 h incubation in the absence of AVP identifying these changed residues as additional members of the retention motif of the hV2R.     

Central vasopressin V(1a) and V(1b) receptors modulate the cardiovascular response to air-jet stress in conscious rats.

This study investigates the contribution of central vasopressin receptors in the modulation of systolic arterial pressure (SAP) and heart rate (HR) response to air-jet stress in conscious Wistar rats equipped with a femoral arterial catheter and intracerebroventricular cannula using novel non-peptide and selective vasopressin V(1a) (SR49059) and V(1b) (SSR149415) antagonists. The effects of stress on SAP and HR were evaluated by measuring the maximal response to stress, the latency of the maximal response, the duration of the recovery period, and the increase in the low frequency (LF) short-term variability component. Stress induced a parallel and almost immediate increase in both SAP and HR, followed by enhanced LF SAP variability in the recovery period. Pretreatment of rats with V(1a) antagonist did not affect the maximal increase or the latency of SAP and HR response to acute stress, but shortened the recovery period of SAP and HR and prevented the increase in LF SAP. The V(1b) antagonist reduced the maximal increase in SAP without affecting HR and their latencies, shortened the recovery period of SAP and inhibited the increase in LF SAP variability. These results indicate that both central V(1a) and V(1b) receptors mediate cardiovascular changes induced by air-jet stress in conscious rats.     

The hydrophobic amino acid residues in the membrane-proximal C tail of the G protein-coupled vasopressin V2 receptor are necessary for transport-competent receptor folding

It is believed that the membrane-proximal C tail of the G protein-coupled receptors forms an additional alpha helix with amphipathic properties (helix 8). It was previously shown for the vasopressin V2 receptor (V2R) that a conserved dileucine motif (L(339), L(340)) in this putative helix 8 is necessary for endoplasmic reticulum (ER) to Golgi transfer of the receptor. Here, we demonstrate that the other hydrophobic residues forming the non-polar side of this helix (F(328), V(332) and L(336)) are also transport-relevant. In contrast, the multiple serine residues contributing to the more hydrophilic side (S(330), S(331), S(333), S(334), S(338)) do not influence receptor trafficking. In addition, we show unambiguously by the use of pharmacological chaperones that the hydrophobic residues of the putative helix 8 do not form a transport signal necessary for receptor sorting into ER to Golgi vesicles. Instead, they are necessary to establish a transport-competent folding state in the early secretory pathway.     

Analysis of interactions responsible for vasopressin binding to human neurohypophyseal hormone receptors-molecular dynamics study of the activated receptor-vasopressin-G(alpha) systems.

Vasopressin (CYFQNCPRG-NH(2), AVP) is a semicyclic endogenous peptide, which exerts a variety of biological effects in mammals. The main physiological roles of AVP are the regulation of water balance and the control of blood pressure and adrenocorticotropin hormone (ACTH) secretion, mediated via three different subtypes of vasopressin receptors: V1a, V1b and V2 receptors (V1aR, V1bR and V2R, respectively). They are the members of the class A, G-protein-coupled receptors (GPCRs). AVP also modulates several behavioral and social functions. In this study, the interactions responsible for AVP binding to vasopressin V1a and V2 receptors versus the closely related oxytocin ([I3,L8]AVP, OT) receptor (OTR) have been investigated. Three-dimensional models of the activated receptors were constructed using multiple sequence alignment, followed by homology modeling using the complex of activated rhodopsin with Gt(alpha) C-terminal peptide of transducin MII-Gt(338-350) prototype as a template. AVP was docked into the receptor-G(alpha) systems. The three lowest-energy pairs of receptor-AVP-G(alpha) (two complexes per each receptor) were selected. The 1-ns unconstrained molecular dynamics (MD) of complexes embedded into the fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipid bilayer was conducted in the AMBER 7.0 force field. Six relaxed receptor-AVP-G(alpha) models were obtained. The residues responsible for AVP binding to vasopressin receptors have been identified and a different mechanism of AVP binding to V2R than to V1aR has been proposed.     

Discovery of 4,5-diphenyl-1,2,4-triazole derivatives as a novel class of selective antagonists for the human V(1A) receptor

In the search for a novel class of selective antagonists for the human V(1A) receptor, high-throughput screening (HTS) of the Yamanouchi chemical library using CHO cells expressing the cloned human V(1A) (hV(1A)) receptor led to the discovery of 5-(4-biphenyl)-4-(2-methoxyphenyl)-3-methyl-1,2,4-triazole (3) which possessed the novel 4,5-diphenyl-1,2,4-triazole structure. Subsequent structure-activity relationships studies on a series of the 4,5-diphenyl-1,2,4-triazole derivatives related to 3 revealed that the 4,5-diphenyl-1,2,4-triazole structure played an essential role in exerting high affinity for the hV(1A) receptor and that introduction of a basic amine moiety to the methoxy part of the 4-phenyl ring was effective in the improvement of both affinity for the hV(1A) receptor and selectivity versus the hV(2) receptor. Compound 3 and the 2-(morphorino)ethoxy derivative (11b) were shown to be antagonists for the hV(1A) receptor, from their effects on AVP-induced [Ca(2+)](i) response in CHO cells expressing the hV(1A) receptor.     

Residues 334 and 338 in transmembrane segment 8 of human equilibrative nucleoside transporter 1 are important determinants of inhibitor sensitivity, protein folding, and catalytic turnover

Equilibrative nucleoside transporters (ENTs) are important for the metabolic salvage of nucleosides and the cellular uptake of antineoplastic and antiviral nucleoside analogs. Human equilibrative nucleoside transporter 1 (hENT1) is inhibited by nanomolar concentrations of structurally diverse compounds, including dipyridamole, dilazep, nitrobenzylmercaptopurine ribonucleoside (NBMPR), draflazine, and soluflazine. Random mutagenesis and screening by functional complementation for inhibitor-resistant mutants in yeast revealed mutations at Phe-334 and Asn-338. Both residues are predicted to lie in transmembrane segment 8 (TM 8), which contains residues that are highly conserved in the ENT family. F334Y displayed increased V(max) values that were attributed to increased rates of catalytic turnover, and N338Q and N338C displayed altered membrane distributions that appeared to be because of protein folding defects. Mutations of Phe-334 or Asn-338 impaired interactions with dilazep and dipyridamole, whereas mutations of Asn-338 impaired interactions with draflazine and soluflazine. A helical wheel projection of TM 8 predicted that Phe-334 and Asn-338 lie in close proximity to other highly conserved and/or hydrophilic residues, suggesting that they form part of a structurally important region that influences interactions with inhibitors, protein folding, and rates of conformational change during the transport cycle.     

Investigation of mechanism of desmopressin binding in vasopressin V2 receptor versus vasopressin V1a and oxytocin receptors: molecular dynamics simulation of the agonist-bound state in the membrane-aqueous system

The vasopressin V2 receptor (V2R) belongs to the Class A G protein-coupled receptors (GPCRs). V2R is expressed in the renal collecting duct (CD), where it mediates the antidiuretic action of the neurohypophyseal hormone arginine vasopressin (CYFQNCPRG-NH2, AVP). Desmopressin ([1-deamino, 8-D]AVP, dDAVP) is strong selective V2R agonist with negligible pressor and uterotonic activity. In this paper, the interactions responsible for binding of dDAVP to vasopressin V2 receptor versus vasopressin V1a and oxytocin receptors has been examined. Three-dimensional activated models of the receptors were constructed using the multiple sequence alignment and the complex of activated rhodopsin with Gt(alpha) C-terminal peptide of transducin MII-Gt(alpha) (338-350) prototype (Slusarz, R.; Ciarkowski, J. Acta Biochim Pol 2004 51, 129-136) as a template. The 1-ns unconstrained molecular dynamics (MD) of receptor-dDAVP complexes immersed in the fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) membrane model was conducted in an Amber 7.0 force field. Highly conserved transmembrane residues have been proposed as being responsible for V2R activation and G protein coupling. Molecular mechanism of the dDAVP binding has been suggested. The internal water molecules involved in an intricate network of the hydrogen bonds inside the receptor cavity have been identified and their role in the stabilization of the agonist-bound state proposed.     

The N-terminal juxtamembrane segment of the V1a vasopressin receptor provides two independent epitopes required for high-affinity agonist binding and signaling

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.     

Antagonism of V1b receptors promotes maternal motivation to retrieve pups in the MPOA and impairs pup-directed behavior during maternal defense in the mpBNST of lactating rats

Recent studies using V1b receptor (V1bR) knockout mice or central pharmacological manipulations in lactating rats highlighted the influence of this receptor for maternal behavior. However, its role in specific brain sites known to be important for maternal behavior has not been investigated to date. In the present study, we reveal that V1bR mRNA (qPCR) and protein levels (Western blot) within either the medial preoptic area (MPOA) or the medial-posterior part of the bed nucleus of the stria terminalis (mpBNST) did not differ between virgin and lactating rats. Furthermore, we characterized the effects of V1bR blockade via bilateral injections of the receptor subtype-specific antagonist SSR149415 within the MPOA or the mpBNST on maternal behavior (maternal care under non-stress and stress conditions, maternal motivation to retrieve pups in a novel environment, maternal aggression) and anxiety-related behavior in lactating rats. Blocking V1bR within the MPOA increased pup retrieval, whereas within the mpBNST it decreased pup-directed behavior, specifically licking/grooming the pups, during the maternal defense test. In addition, immediately after termination of the maternal defense test, V1bR antagonism in both brain regions reduced nursing, particularly arched back nursing. Anxiety-related behavior was not affected by V1bR antagonism in either brain region. In conclusion our data indicate that V1bR antagonism significantly modulates different aspects of maternal behavior in a brain region-dependent manner.     

Variant amino acids in the extracellular loops of murine and human vasopressin V2 receptors account for differences in cell surface expression and ligand affinity

Cloning and sequencing of the murine chromosomal region XB harboring the murine vasopressin V(2) receptor (mV(2)R) gene and comparison with the orthologous human Xq28 region harboring the human vasopressin V(2) receptor (hV(2)R) revealed conservation of the genomic organization and a high degree of sequence identity in the V(2)R coding regions. Despite an identity of 87% of the amino acid sequences, both receptors show marked functional differences upon stable expression in Chinese hamster ovary cells: the mV(2)R displayed a 5-fold higher affinity for [(3)H]AVP than the human ortholog; similar differences were found for the AVP-mediated activation of adenylyl cyclase. Saturation binding experiments with transiently transfected intact COS.M6 cells showed that the mV(2)R was 3- to 5-fold less abundantly expressed at the cell surface than the hV(2)R. Laser scanning microscopy of fusion proteins consisting of the V(2)Rs and green fluorescent protein (GFP) (mV(2)R/GFP, hV(2)R/GFP) demonstrated that the hV(2)R/GFP was efficiently transported to the plasma membrane, whereas the mV(2)R/GFP was localized mainly within the endoplasmic reticulum. Chimeric hV(2)Rs, in which the first and/or second extracellular loop(s) were replaced by the corresponding loop(s) of the mV(2)R, revealed that the second extracellular loop accounts for the differences in ligand binding, but the first extracellular loop accounts for the reduced cell surface expression. The exchange of lysine 100 by aspartate in the first extracellular loop of hV(2)R was sufficient to reduce cell surface expression, which was accompanied by intracellular retention as observed in laser scanning microscopy analysis. Conversely, the exchange of aspartate 100 by lysine in the mV(2)R increased the cell surface expression and resulted in predominant plasma membrane localization. Thus, a single amino acid difference in the first extracellular loop between mV(2)R and hV(2)R determines the efficiency of cell surface expression.     

Pancreatic vasopressin V1b receptors: characterization in In-R1-G9 cells and localization in human pancreas

Vasopressin (AVP) receptors present in In-R1-G9 cells, a hamster glucagon-secreting alpha-pancreatic cell line, were characterized using SSR-149415, a selective nonpeptide V1b receptor antagonist, and reference AVP compounds. Binding experiments, using [3H]AVP as a ligand, identified a single population of high-affinity binding sites. SSR-149415 competitively inhibited this binding and exhibited nanomolar and stereospecific affinity for these sites. The affinity of various AVP/oxytocin ligands confirmed a V1b binding profile. In functional studies, AVP was a potent stimulant in inducing intracellular Ca2+ increase, glucagon secretion, and cell proliferation. These effects were fully antagonized by SSR-149415 with a nanomolar potency, whereas its diasteroisomer as well as two selective V1a and V2 receptor antagonists were much less potent. Additionally, the order of potency of AVP agonists and antagonists was in agreement with V1b-mediated effects. By RT-PCR, we confirmed the presence of V1b receptor mRNA in both In-R1-G9 cells and in human pancreas. The distribution pattern of V1b receptors investigated in human pancreas by immunohistochemistry showed strong labeling in islets of Langerhans, and colocalization studies indicated that this receptor was expressed in alpha-glucagon, beta-insulin, and somatostatin pancreatic cells. Thus, in In-R1-G9 cells, AVP mediates intracellular Ca2+ increase, glucagon secretion, and cell proliferation by activating V1b receptors, and these effects are potently antagonized by SSR-149415. Moreover, the presence of V1b receptors also found in human Langerhans islets could suggest hormonal control of AVP in human pancreas.     

Identification of amino acid residues that direct differential ligand selectivity of mammalian and nonmammalian V1a type receptors for arginine vasopressin and vasotocin. Insights into molecular coevolution of V1a type receptors and their ligands

Arginine vasotocin (VT) is the ortholog in all nonmammalian vertebrates of arginine vasopressin (AVP) in mammals. We have previously cloned an amphibian V1atype vasotocin receptor (VT1R) that exhibited higher sensitivity for VT than AVP, while the mammalian V1a type receptor (V1aR) responded better to AVP than VT. In the present study, we identified the amino acid residues that confer differential ligand selectivity for AVP and VT between rat V1aR and bullfrog VT1R (bfVT1R). A chimeric rat V1aR having transmembrane domain (TMD) VI to the carboxyl-terminal tail (C-tail) of bfVT1R showed a reverse ligand preference for AVP and VT, whereas a chimeric VT1R with TMD VI to the C-tail of rat V1aR showed a great increase in sensitivity for AVP. A single mutation (Ile(315(6.53)) to Thr) in TMD VI of V1aR increased the sensitivity for VT, while a single mutation (Phe(313(6.51)) to Tyr or Pro(334(7.33)) to Thr) reduced sensitivity toward AVP. Interestingly the triple mutation (Phe(313(6.51)) to Tyr, Ile(6.53) to Thr, and Pro(7.33) to Thr) of V1aR increased sensitivity to VT but greatly reduced sensitivity to AVP, behaving like bfVT1R. Further, like V1aR, a double mutant (Tyr(306(6.51)) to Phe and Thr(327(7.33)) to Pro) of bfVT1R showed an increased sensitivity to AVP. These results suggest that Phe/Tyr(6.51), Ile/Thr(6.53), and Pro/Thr(7.33) are responsible for the differential ligand selectivity between rat V1aR and bfVT1R. This information regarding the molecular interaction of VT/AVP with their receptors may have important implications for the development of novel AVP analogs.     

CFTR: a cysteine at position 338 in TM6 senses a positive electrostatic potential in the pore

We investigated the accessibility to protons and thiol-directed reagents of a cysteine substituted at position 338 in transmembrane segment 6 (TM6) of CFTR to test the hypothesis that T338 resides in the pore. Xenopus oocytes expressing T338C CFTR exhibited pH-dependent changes in gCl and I-V shape that were specific to the substituted cysteine. The apparent pKa of T338C CFTR was more acidic than that expected for a cysteine or similar simple thiols in aqueous solution. The pKa was shifted toward alkaline values when a nearby positive charge (R334) was substituted with neutral or negatively charged residues, consistent with the predicted influence of the positive charge of R334, and perhaps other residues, on the titration of a cysteine at 338. The relative rates of chemical modification of T338C CFTR by MTSET+ and MTSES- were also altered by the charge at 334. These observations support a model for CFTR that places T338 within the anion conduction path. The apparent pKa of a cysteine substituted at 338 and the relative rates of reaction of charged thiol-directed reagents provide a crude measure of a positive electrostatic potential that may be due to R334 and other residues near this position in the pore.     

Structural implication for receptor oligomerization from functional reconstitution studies of mutant V2 vasopressin receptors

Previous studies have established that G-protein-coupled receptors (GPCRs) are composed of independent folding domains. Based on this findings we attempted to rescue the function of clinically relevant missense mutations (R137H, S167L, and R181C) within the N-terminal domain of the V2 vasopressin receptor (V2-R), by coexpressing mutated full-length (Y280C) and C-terminally truncated (E242X) receptor constructs in COS-7 cells. Coimmunoprecipitation and enzyme-linked immunosorbent assay studies demonstrated a specific association of E242X with full-length V2-Rs even in the presence of missense mutations. Systematic analysis of the structural requirements for the observed receptor/fragment association showed that N-terminal fragments containing at least transmembrane regions 1-3 interact with the full-length V2-R. Despite this specific interaction, no functional reconstitution was achieved for mutant V2-Rs following coexpression with E242X and Y280C. However, functional activity of R137H and R181C upon coexpression with E242X was regained by mutational disruption of the extracellular disulfide bond, which is highly conserved among GPCRs. Our data with the V2-R are consistent with a structural model in which class I GPCRs form contact oligomers by lateral interaction rather than by a domain-swapping mechanism.