A new Kunitz-type snake toxin family associated with an original mode of interaction with the vasopressin 2 receptor.

BACKGROUND AND PURPOSE: Venomous animals express numerous Kunitz-type peptides. The mambaquaretin-1 (MQ1) peptide identified from the Dendroaspis angusticeps venom is the most selective antagonist of the arginine-vasopressin V2 receptor (V2R) and the only unique Kunitz-type peptide active on a GPCR. We aimed to exploit other mamba venoms to enlarge the V2R-Kunitz peptide family and gain insight into the MQ1 molecular mode of action. EXPERIMENTAL APPROACH: We used a bio-guided screening assay to identify novel MQs and placed them phylogenetically. MQs were produced by solid-phase peptide synthesis and characterized in vitro by binding and functional tests and in vivo by diuresis measurement in rats. KEY RESULTS: Eight additional MQs were identified with nanomolar affinities for the V2R, all antagonists. MQs form a new subgroup in the Kunitz family, close to the V2R non-active dendrotoxins and to two V2R-active cobra toxins. Sequence comparison between active and non-active V2R Kunitz peptides highlighted five positions, among which four are involved in V2R interaction and belong to the two large MQ1 loops. We finally determined that eight positions, part of these two loops, interact with the V2R. The variant MQ1-K39A showed a higher affinity for the hV2R, but not for the rat V2R. CONCLUSIONS AND IMPLICATIONS: A new function and mode of action is associated with the Kunitz peptides. The number of MQ1 residues involved in V2R binding is large and may explain its absolute selectivity. MQ1-K39A represents the first step in the improvement of the MQ1 design from a medicinal perspective.

Characterization of a functional V1B vasopressin receptor in the male rat kidney: evidence for cross talk between V1B and V2 receptor signaling pathways.

Although vasopressin V1B receptor (V1BR) mRNA has been detected in the kidney, the precise renal localization as well as pharmacological and physiological properties of this receptor remain unknown. Using the selective V1B agonist d[Leu4, Lys8]VP, either fluorescent or radioactive, we showed that V1BR is mainly present in principal cells of the inner medullary collecting duct (IMCD) in the male rat kidney. Protein and mRNA expression of V1BR were very low compared with the V2 receptor (V2R). On the microdissected IMCD, d[Leu4, Lys8]VP had no effect on cAMP production but induced a dose-dependent and saturable intracellular Ca2+ concentration increase mobilization with an EC50 value in the nanomolar range. This effect involved both intracellular Ca2+ mobilization and extracellular Ca2+ influx. The selective V1B antagonist SSR149415 strongly reduced the ability of vasopressin to increase intracellular Ca2+ concentration but also cAMP, suggesting a cooperation between V1BR and V2R in IMCD cells expressing both receptors. This cooperation arises from a cross talk between second messenger cascade involving PKC rather than receptor heterodimerization, as supported by potentiation of arginine vasopressin-stimulated cAMP production in human embryonic kidney-293 cells coexpressing the two receptor isoforms and negative results obtained by bioluminescence resonance energy transfer experiments. In vivo, only acute administration of high doses of V1B agonist triggered significant diuretic effects, in contrast with injection of selective V2 agonist. This study brings new data on the localization and signaling pathways of V1BR in the kidney, highlights a cross talk between V1BR and V2R in the IMCD, and suggests that V1BR may counterbalance in some pathophysiological conditions the antidiuretic effect triggered by V2R activation.NEW & NOTEWORTHY Although V1BR mRNA has been detected in the kidney, the precise renal localization as well as pharmacological and physiological properties of this receptor remain unknown. Using original pharmaceutical tools, this study brings new data on the localization and signaling pathways of V1BR, highlights a cross talk between V1BR and V2 receptor (V2R) in the inner medullary collecting duct, and suggests that V1BR may counterbalance in some pathophysiological conditions the antidiuretic effect triggered by V2R activation.

A snake toxin as a theranostic agent for the type 2 vasopressin receptor.

Rationale: MQ1, a snake toxin which targets with high nanomolar affinity and absolute selectivity for the type 2 vasopressin receptor (V2R), is a drug candidate for renal diseases and a molecular probe for imaging cells or organs expressing V2R. Methods: MQ1's pharmacological properties were characterized and applied to a rat model of hyponatremia. Its PK/PD parameters were determined as well as its therapeutic index. Fluorescently and radioactively labeled MQ1 were chemically synthesized and associated with moderate loss of affinity. MQ1's dynamic biodistribution was monitored by positron emission tomography. Confocal imaging was used to observe the labeling of three cancer cell lines. Results: The inverse agonist property of MQ1 very efficiently prevented dDAVP-induced hyponatremia in rats with low nanomolar/kg doses and with a very large therapeutic index. PK (plasma MQ1 concentrations) and PD (diuresis) exhibited a parallel biphasic decrease. The dynamic biodistribution showed that MQ1 targets the kidneys and then exhibits a blood and kidney biphasic decrease. Whatever the approach used, we found a T1/2α between 0.9 and 3.8 h and a T1/2ß between 25 and 46 h and demonstrated that the kidneys were able to retain MQ1. Finally, the presence of functional V2R expressed at the membrane of cancer cells was, for the first time, demonstrated with a specific fluorescent ligand. Conclusion: As the most selective V2 binder, MQ1 is a new promising drug for aquaresis-related diseases and a molecular probe to visualize in vitro and in vivo V2R expressed physiologically or under pathological conditions.

Misfolding of vasopressin receptors: biased agonist pharmacochaperones as potential therapeutics.

Biased agonists and pharmacological chaperones have demonstrated their potential to harness G protein-coupled receptor signaling and trafficking, and have collectively opened new possibilities in G protein-coupled receptor drug discovery. Combining pharmacological chaperoning and biased agonism properties into a unique given molecule would be of high therapeutic interest in many human diseases resulting from G protein-coupled receptor mutation and misfolding. This strategy perfectly fits to congenital Nephrogenic Diabetes Insipidus which is a typical conformational disease. In most of the cases, it is associated to inactivating mutations of the renal arginine-vasopressin V2 receptor leading to misfolding and intracellular retention of the receptor, causing the inability of patients to concentrate their urine in response to the antidiuretic hormone. Cell-permeable pharmacological chaperones have been successfully challenged to restore plasma membrane localization of the receptor mutants and to rescue their function. Interestingly, different classes of pharmacological chaperones of the V2 receptor have proven their usefulness and efficacy, such as antagonists, agonists as well as biased agonists. These compounds, particularly small-molecule biased agonists which elicit the V2-induced Gs protein-dependent signaling pathway, but not V2-related arrestin-dependent cell responses, represent a potential therapeutic treatment of this X-linked genetic pathology.

Pharmacological Chaperones as Potential Therapeutic Strategies for Misfolded Mutant Vasopressin Receptors.

Pharmacological chaperones recently opened new possibilities in G protein-coupled receptor drug discovery. Even more interestingly, some unique ligands combine pharmacological chaperoning and biased agonism properties, boosting their therapeutic interest in many human diseases resulting from G protein-coupled receptor mutation and misfolding. These compounds displaying dual characteristics would constitute a perfect treatment for congenital Nephrogenic Diabetes Insipidus, a typical conformational disease. This X-linked genetic pathology is mostly associated with inactivating mutations of the renal arginine-vasopressin V2 receptor leading to misfolding and intracellular retention of the receptor, causing the inability of patients to concentrate their urine in response to the antidiuretic hormone. Cell-permeable pharmacological chaperones have been successfully challenged to restore plasma membrane localization of many V2 receptor mutants. In addition, different classes of specific ligands such as antagonists, agonists as well as biased agonists of the V2 receptor have proven their usefulness in rescuing mutant receptor function. This is particularly relevant for small-molecule biased agonists which only trigger Gs protein activation and cyclic adenosine monophosphate production, the V2-induced signaling pathway responsible for water reabsorption. In parallel, high-throughput screening assays based on receptor trafficking rescue approaches have been developed to discover novel V2 pharmacological chaperone molecules from different chemical libraries. These new hit compounds, which still need to be pharmacologically characterized and functionally tested in vivo, represent promising candidates for the treatment of congenital Nephrogenic Diabetes Insipidus.

V1b vasopressin receptor trafficking and signaling: Role of arrestins, G proteins and Src kinase.

The signaling pathway of G protein-coupled receptors is strongly linked to their trafficking profile. Little is known about the molecular mechanisms involved in the vasopressin receptor V1b subtype (V1b R) trafficking and its impact on receptor signaling and regulation. For this purpose, we investigated the role of ß-arrestins in receptor desensitization, internalization and recycling and attempted to dissect the V1b R-mediated MAP kinase pathway. Using MEF cells Knocked-out for ß-arrestins 1 and 2, we demonstrated that both ß-arrestins 1 and 2 play a fundamental role in internalization and recycling of V1b R with a rapid and transient V1b R-ß-arrestin interaction in contrast to a slow and long-lasting ß-arrestin recruitment of the V2 vasopressin receptor subtype (V2 R). Using V1b R-V2 R chimeras and V1b R C-terminus truncations, we demonstrated the critical role of the V1b R C-terminus in its interaction with ß-arrestins thereby regulating the receptor internalization and recycling kinetics in a phosphorylation-independent manner. In parallel, V1b R MAP kinase activation was dependent on arrestins and Src-kinase but independent on G proteins. Interestingly, Src interacted with hV1b R at basal state and dissociated when receptor internalization occurred. Altogether, our data describe for the first time the trafficking profile and MAP kinase pathway of V1b R involving both arrestins and Src kinase family.

Green mamba peptide targets type-2 vasopressin receptor against polycystic kidney disease.

Polycystic kidney diseases (PKDs) are genetic disorders that can cause renal failure and death in children and adults. Lowering cAMP in cystic tissues through the inhibition of the type-2 vasopressin receptor (V2R) constitutes a validated strategy to reduce disease progression. We identified a peptide from green mamba venom that exhibits nanomolar affinity for the V2R without any activity on 155 other G-protein-coupled receptors or on 15 ionic channels. Mambaquaretin-1 is a full antagonist of the V2R activation pathways studied: cAMP production, beta-arrestin interaction, and MAP kinase activity. This peptide adopts the Kunitz fold known to mostly act on potassium channels and serine proteases. Mambaquaretin-1 interacts selectively with the V2R through its first loop, in the same manner that aprotinin inhibits trypsin. Injected in mice, mambaquaretin-1 increases in a dose-dependent manner urine outflow with concomitant reduction of urine osmolality, indicating a purely aquaretic effect associated with the in vivo blockade of V2R. CD1-pcy/pcy mice, a juvenile model of PKD, daily treated with 13 [Formula: see text]g of mambaquaretin-1 for 99 d, developed less abundant (by 33%) and smaller (by 47%) cysts than control mice. Neither tachyphylaxis nor apparent toxicity has been noted. Mambaquaretin-1 represents a promising therapeutic agent against PKDs.

Red fluorescent turn-on ligands for imaging and quantifying G protein-coupled receptors in living cells.

Classical fluorescence-based approaches to monitor ligand-protein interactions are generally hampered by the background signal of unbound ligand, which must be removed by tedious washing steps. To overcome this major limitation, we report here the first red fluorescent turn-on probes for a G protein-coupled receptor (oxytocin receptor) at the surface of living cells. The peptide ligand carbetocin was conjugated to one of the best solvatochromic (fluorogenic) dyes, Nile Red, which turns on emission when reaching the hydrophobic environment of the receptor. We showed that the incorporation of hydrophilic octa(ethylene glycol) linker between the pharmacophore and the dye minimized nonspecific interaction of the probe with serum proteins and lipid membranes, thus ensuring receptor-specific turn-on response. The new ligand was successfully applied for background-free imaging and quantification of oxytocin receptors in living cells.

Vasopressin receptors and pharmacological chaperones: from functional rescue to promising therapeutic strategies.

Conformational diseases result from protein misfolding and/or aggregation and constitute a major public health problem. Congenital Nephrogenic Diabetes Insipidus is a typical conformational disease. In most of the cases, it is associated to inactivating mutations of the renal arginine-vasopressin V2 receptor gene leading to misfolding and intracellular retention of the receptor, causing the inability of patients to concentrate their urine in response to the antidiuretic hormone. Cell-permeable pharmacological chaperones have been successfully challenged to restore plasma membrane localization of the receptor mutants and to rescue their function. Interestingly, different classes of specific ligands such as antagonists (vaptans), agonists as well as biased agonists of the V2 receptor have proven their usefulness as efficient pharmacochaperones. These compounds represent a potential therapeutic treatment of this X-linked genetic pathology.

Structural insights into biased G protein-coupled receptor signaling revealed by fluorescence spectroscopy.

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters, representing the largest group of therapeutic targets. Recent studies show that some GPCRs signal through both G protein and arrestin pathways in a ligand-specific manner. Ligands that direct signaling through a specific pathway are known as biased ligands. The arginine-vasopressin type 2 receptor (V2R), a prototypical peptide-activated GPCR, is an ideal model system to investigate the structural basis of biased signaling. Although the native hormone arginine-vasopressin leads to activation of both the stimulatory G protein (Gs) for the adenylyl cyclase and arrestin pathways, synthetic ligands exhibit highly biased signaling through either Gs alone or arrestin alone. We used purified V2R stabilized in neutral amphipols and developed fluorescence-based assays to investigate the structural basis of biased signaling for the V2R. Our studies demonstrate that the Gs-biased agonist stabilizes a conformation that is distinct from that stabilized by the arrestin-biased agonists. This study provides unique insights into the structural mechanisms of GPCR activation by biased ligands that may be relevant to the design of pathway-biased drugs.

Expression, purification and NMR characterization of the cyclic recombinant form of the third intracellular loop of the vasopressin type 2 receptor.

The vasopressin type 2 (V2R) receptor belongs to the class of G-protein coupled receptors. It is mainly expressed in the membrane of kidney tubules, where it is activated by the extracellular arginine vasopressin. In men, inactivating and activating mutations cause nephrogenic diabetes insipidus and the nephrogenic syndrome of inappropriate antidiuresis respectively. Like most GPCRs, V2R's third intracellular loop (V2R-i3) is involved in the binding and activation of its major effector, the GαS protein. We overexpressed the V2R224₋274 fragment corresponding to V2R-i3 as a fusion protein with thioredoxin A at the N-terminus and a hexahistidine tag between the two proteins. Recombinant V2R-i3 was designed to harbor N- and C-terminal cysteines, in order to introduce a disulfide bond between N- and C-terminal extremities and hence reproduce the hairpin fold presumably present in the full-length receptor. The fusion protein was produced as inclusion bodies in Escherichia coli and purified by nickel affinity chromatography under denaturing conditions. After a refolding step, thioredoxin and hexahistidine tags were specifically cleaved with the tobacco etch virus protease. The hydrolysis yield, initially very low, increased up to 80% thanks to optimization of buffers and refolding methods. The cleaved fragment, V2224₋274, devoid of any tag, was then eluted with low imidazole concentrations in a second nickel affinity chromatography in denaturing conditions. The final yield was sufficient to prepare a ¹5N-¹³C labeled NMR sample suitable for triple resonance experiments. We assigned all NMR resonances and confirmed the correct peptide sequence. As expected, the peptide forms a hairpin stabilized by a disulfide bond between its N- and C-terminal parts, thus mimicking its native structure in the full-length receptor. This study may provide a strategy for producing and studying the structure/function relationship of GPCR fragments.

[Pharmacological chaperones: a potential therapeutic treatment for conformational diseases]. / Chaperons pharmacologiques : un espoir thérapeutique pour les pathologies conformationnelles.

Many genetic and neurodegenerative diseases in humans result from protein misfolding and/or aggregation. These diseases are named conformational diseases. As a result, the misfolded non functional proteins are rejected and misrouted by the cellular quality control system, and cannot play their endogenous physiological roles. Specific compounds (ligands, substrates or inhibitors) known as pharmacological chaperones are able to bind and stabilize these misfolded proteins. Their interaction allows the target proteins to escape the quality control system and to be functionally rescued. These pharmacochaperones may possess different intrinsic activity: they can be antagonists (inhibitors), agonists (activators) or allosteric modulators of the target receptors, ionic channels or enzymes. Pharmacological chaperones have obviously a therapeutic potential to treat rare diseases like cystic fibrosis, retinitis pigmentosa, nephrogenic diabetes insipidus, Fabry disease, Gaucher disease, but also for cancers and more frequent and highly invalidant neurodegenerative disorders such as Alzheimer's disease or Parkinson's disease.

Leukotriene BLT2 receptor monomers activate the G(i2) GTP-binding protein more efficiently than dimers.

Accumulating evidence indicates that G protein-coupled receptors can assemble as dimers/oligomers but the role of this phenomenon in G protein coupling and signaling is not yet clear. We have used the purified leukotriene B(4) receptor BLT2 as a model to investigate the capacity of receptor monomers and dimers to activate the adenylyl cyclase inhibitory G(i2) protein. For this, we overexpressed the recombinant receptor as inclusion bodies in the Escherichia coli prokaryotic system, using a human alpha(5) integrin as a fusion partner. This strategy allowed the BLT2 as well as several other G protein-coupled receptors from different families to be produced and purified in large amounts. The BLT2 receptor was then successfully refolded to its native state, as measured by high-affinity LTB(4) binding in the presence of the purified G protein G alpha(i2). The receptor dimer, in which the two protomers displayed a well defined parallel orientation as assessed by fluorescence resonance energy transfer, was then separated from the monomer. Using two methods of receptor-catalyzed guanosine 5'-3-O-(thio)triphosphate binding assay, we clearly demonstrated that monomeric BLT2 stimulates the purified G alpha(i2) beta(1) gamma(2) protein more efficiently than the dimer. These data suggest that assembly of two BLT2 protomers into a dimer results in the reduced ability to signal.

The constitutively active V2 receptor mutants conferring NSIAD are weakly sensitive to agonist and antagonist regulation.

Patients having the nephrogenic syndrome of inappropriate antidiuresis present either the R137C or R137L V2 mutated receptor. While the clinical features have been characterized, the molecular mechanisms of functioning of these two mutants remain elusive. In the present study, we compare the pharmacological properties of R137C and R137L mutants with the wild-type and the V2 D136A receptor, the latter being reported as a highly constitutively active receptor. We have performed binding studies, second messenger measurements and BRET experiments in order to evaluate the affinities of the ligands, their agonist and antagonist properties and the ability of the receptors to recruit beta-arrestins, respectively. The R137C and R137L receptors exhibit small constitutive activities regarding the G(s) protein activation. In addition, these two mutants induce a constitutive beta-arrestin recruitment. Of interest, they also exhibit weak sensitivities to agonist and to inverse agonist in term of G(s) protein coupling and beta-arrestin recruitment. The small constitutive activities of the mutants and the weak regulation of their functioning by agonist suggest a poor ability of the antidiuretic function to be adapted to the external stimuli, giving to the environmental factors an importance which can explain some of the phenotypic variability in patients having NSIAD.

Biased agonist pharmacochaperones of the AVP V2 receptor may treat congenital nephrogenic diabetes insipidus.

X-linked congenital nephrogenic diabetes insipidus (cNDI) results from inactivating mutations of the human arginine vasopressin (AVP) V2 receptor (hV(2)R). Most of these mutations lead to intracellular retention of the hV(2)R, preventing its interaction with AVP and thereby limiting water reabsorption and concentration of urine. Because the majority of cNDI-hV(2)Rs exhibit protein misfolding, molecular chaperones hold promise as therapeutic agents; therefore, we sought to identify pharmacochaperones for hV(2)R that also acted as agonists. Here, we describe high-affinity nonpeptide compounds that promoted maturation and membrane rescue of L44P, A294P, and R337X cNDI mutants and restored a functional AVP-dependent cAMP signal. Contrary to pharmacochaperone antagonists, these compounds directly activated a cAMP signal upon binding to several cNDI mutants. In addition, these molecules displayed original functionally selective properties (biased agonism) toward the hV(2)R, being unable to recruit arrestin, trigger receptor internalization, or stimulate mitogen-activated protein kinases. These characteristics make these hV(2)R agonist pharmacochaperones promising therapeutic candidates for cNDI.

Structure of the third intracellular loop of the vasopressin V2 receptor and conformational changes upon binding to gC1qR.

The V2 vasopressin receptor is a G-protein-coupled receptor that regulates the renal antidiuretic response. Its third intracellular loop is involved in the coupling not only with the GalphaS protein but also with gC1qR, a potential chaperone of G-protein-coupled receptors. In this report, we describe the NMR solution structure of the V2 i3 loop under a cyclized form (i3_cyc) and characterize its interaction with gC1qR. i3_cyc formed a left-twisted alpha-helical hairpin structure. The building of a model of the entire V2 receptor including the i3_cyc NMR structure clarified the side-chain orientation of charged residues, in agreement with literature mutagenesis reports. In the model, the i3 loop formed a rigid helical column, protruding deep inside the cytoplasm, as does the i3 loop in the recently elucidated structure of squid rhodopsin. However, its higher packing angle resulted in a different structural motif at the intracellular interface, which may be important for the specific recognition of GalphaS. Moreover, we could estimate the apparent K(d) of the i3_cyc/gC1qR complex by anisotropy fluorescence. Using a shorter and more soluble version of i3_cyc, which encompassed the putative site of gC1qR binding, we showed by NMR saturation transfer difference spectroscopy that the binding surface corresponded to the central arginine cluster. Binding to gC1qR induced the folding of the otherwise disordered short peptide into a spiral-like path formed by a succession of I and IV turns. Our simulations suggested that this folding would rigidify the arginine cluster in the entire i3 loop and would alter the conformation of the cytosolic extensions of TM V and TM VI helices. In agreement with this conformational rearrangement, we observed that binding of gC1qR to the full-length receptor modifies the intrinsic tryptophan fluorescence binding curves of V2 to an antagonist.

Differential coupling of the vasopressin V1b receptor through compartmentalization within the plasma membrane.

We show here that the rat vasopressin V(1b) receptor simultaneously activates both the G(q/11)-inositol phosphate (IP) and G(s)-cAMP pathways when transiently expressed in Chinese hamster ovary, human embryonic kidney (HEK) 293, and COS-7 cells and stimulated with arginine-vasopressin. Higher concentrations of the hormone, however, were needed to trigger the cAMP pathway. The nonmammalian analog arginine-vasotocin and the selective V(1b) agonist d[Cha(4)]vasopressin also activated the cAMP and IP pathways, although d[Cha(4)]-vasopressin elicited the two responses with equivalent potencies. We determined that the V(1b) receptor is present as a homodimer at the plasma membrane. Treatment of V(1b)-transfected HEK-293 cells with methyl-beta-cyclodextrin, a drug known to dissociate cholesterol-rich domains of the plasma membrane, shifted the EC(50) of the vasopressin-induced cAMP accumulation to lower concentrations and, remarkably, increased the hormone efficacy related to the activation of this second messenger system. In parallel, the vasopressin-mediated activation of the IP pathway was slightly reduced without modification of its EC(50). These results suggest that, as with many other G protein-coupled receptors, when transfected in heterologous cell systems, the V(1b) receptor forms dimers that signal differentially through the G(q/11) and G(s) proteins depending on the nature of the ligand as well as on its localization within specialized compartments of the plasma membrane. The present study thus illustrates how signal transduction associated with the activation of a G protein-coupled receptor can be versatile and highly dependent on both the cell context and the chemical nature of the extracellular signaling messenger.

The multifunctional protein GC1q-R interacts specifically with the i3 loop arginine cluster of the vasopressin V2 receptor.

In this study, we identified the multifunctional protein GC1q-R as a novel vasopressin V(2) receptor (V(2)R) interacting protein. For this purpose, we have developed a proteomic approach combining pull-down assays using a cyclic peptide mimicking the third intracellular loop of V(2)R as a bait and mass spectrometry analyses of proteins isolated from either rat or human kidney tissues or the HEK 293 cell line. Co-immunoprecipitation of GC1q-R with the c-Myc-tagged h-V(2)R expressed in a HEK cell line confirmed the existence of a specific interaction between GC1q-R and the V(2) receptor. Then, construction of a mutant receptor in i3 loop allowed us to identify the i3 loop arginine cluster of the vasopressin V(2) receptor as the interacting determinant for GC1q-R interaction. Using purified receptor as a bait and recombinant (74-282) GC1q-R, we demonstrated a direct and specific interaction between these two proteins via the arginine cluster.

Design and synthesis of cyclic and linear peptide-agarose tools for baiting interacting protein partners of GPCRs.

A ligation strategy for the synthesis of cyclic and linear peptides covalently linked to agarose beads designed as baits to identify new interacting partners of intracellular loops of the V2 vasopressin receptor, a member of the G-protein-coupled receptor family, is reported. The peptide-resin conjugates were subsequently shown to interact specifically with a fraction of proteins present in cellular lysates.

A cyclic peptide mimicking the third intracellular loop of the V2 vasopressin receptor inhibits signaling through its interaction with receptor dimer and G protein.

In this study, we investigated the mechanism by which a peptide mimicking the third cytoplasmic loop of the vasopressin V2 receptor inhibits signaling. This loop was synthesized as a cyclic peptide (i3 cyc) that adopted defined secondary structure in solution. We found that i3 cyc inhibited the adenylyl cyclase activity induced by vasopressin or a nonhydrolyzable analog of GTP, guanosine 5'-O-(3-thio)triphosphate. This peptide also affected the specific binding of [3H]AVP by converting vasopressin binding sites from a high to a low affinity state without any effect on the global maximal binding capacity. The inhibitory actions of i3 cyc could also be observed in the presence of maximally uncoupling concentration of guanosine 5'-O-(3-thio)triphosphate, indicating a direct effect on the receptor itself and not exclusively on the interaction between the Gs protein and the V2 receptor (V2-R). Bioluminescence resonance energy-transfer experiments confirmed this assumption, because i3 cyc induced a significant inhibition of the bioluminescence resonance energy-transfer signal between the Renilla reniformis luciferase and the enhanced yellow fluorescent protein fused V2-R. This suggests that the proper arrangement of the dimer could be an important prerequisite for triggering Gs protein activation. In addition to its effect on the receptor itself, the peptide exerted some of its actions at the G protein level, because it could also inhibit guanosine 5'-O-(3-thio)triphosphate-stimulated AC activity. Taken together, the data demonstrate that a peptide mimicking V2-R third intracellular loop affects both the dimeric structural organization of the receptor and has direct inhibitory action on Gs.