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Classically, G-protein-coupled receptors (GPCRs) are thought to activate G protein from the plasma membrane and are subsequently desensitized by β-arrestin (β-arr). However, some GPCRs continue to signal through G protein from internalized compartments, mediated by a GPCR–G protein–β-arr ‘megaplex’. Nevertheless, the molecular architecture of the megaplex remains unknown. Here, we present its cryo-electron microscopy structure, which shows simultaneous engagement of human G protein and bovine β-arr to the core and phosphorylated tail, respectively, of a single active human chimeric β2-adrenergic receptor with the C-terminal tail of the arginine vasopressin type 2 receptor (β2V2R). All three components adopt their canonical active conformations, suggesting that a single megaplex GPCR is capable of simultaneously activating G protein and β-arr. Our findings provide a structural basis for GPCR-mediated sustained internalized G protein signaling.

The vasopressin V2 receptor (V2R), a class A G protein-coupled receptor, is essential for regulating body water homeostasis. V2R antagonists have emerged as promising treatments for hyponatremia; however, the absence of structural information for antagonist-bound V2R hampers our understanding of antagonist recognition and the targeted design of V2R antagonists. In this study, we present two cryo-electron microscopy structures of inactive V2R bound to the clinically approved antagonists tolvaptan and conivaptan. Combined with functional analyses and molecular dynamic simulations, these structures reveal distinct binding poses: tolvaptan is deeply inserted within the binding pocket, whereas conivaptan is positioned at a shallower depth. Integrated analyses further define critical pharmacophoric features governing antagonist activity and unveil a TM7 helical conformation-dependent antagonism mechanism that is distinct from classical GPCR inactivation modes. Our findings deepen understanding of antagonist recognition and antagonism of V2R, providing a foundation for the development of V2R-targeted therapies. The vasopressin V2 receptor (V2R) is a key regulator of water balance, and its antagonists are promising therapeutics for hyponatremia. Here, the authors offer structural insights into antagonist recognition and antagonism of V2R.

Inhibitors of the arginine-vasopressin (AVP) V2 receptor (V2R) are key therapeutic compounds for treating hyponatremia or polycystic kidney diseases. Rational drug design based on experimental G protein-coupled receptor structures is a powerful avenue to develop better drugs. So far, the lack of inhibitor-bound V2R structures has impaired this strategy. Here we describe the cryo-electron microscopy structures of the V2R in complex with two selective inverse agonists, the non-peptide Tolvaptan (TVP) and the green mamba snake Mambaquaretin toxin (MQ1). Both ligands bind into the orthosteric binding site but with substantial differences. TVP binds deeper than MQ1, and directly contacts the toggle switch residue W2846.48 in the transmembrane domain 6. The Kunitz-fold toxin displays extensive contacts with extracellular and transmembrane residues. As anticipated from TVP and MQ1 pharmacological properties, both structures represent inactive V2R conformations. Their comparison with those of the active AVP-bound V2R reveals the molecular mechanisms modulating receptor activity. The mini-protein MQ1-bound V2R structure suggests a new pharmacology approach for treating water homeostasis and renal diseases.

Arrestins recognize different receptor phosphorylation patterns and convert this information to selective arrestin functions to expand the functional diversity of the G protein-coupled receptor (GPCR) superfamilies. However, the principles governing arrestin-phospho-receptor interactions, as well as the contribution of each single phospho-interaction to selective arrestin structural and functional states, are undefined. Here, we determined the crystal structures of arrestin2 in complex with four different phosphopeptides derived from the vasopressin receptor-2 (V2R) C-tail. A comparison of these four crystal structures with previously solved Arrestin2 structures demonstrated that a single phospho-interaction change results in measurable conformational changes at remote sites in the complex. This conformational bias introduced by specific phosphorylation patterns was further inspected by FRET and 1 H NMR spectrum analysis facilitated via genetic code expansion. Moreover, an interdependent phospho-binding mechanism of phospho-receptor-arrestin interactions between different phospho-interaction sites was unexpectedly revealed. Taken together, our results provide evidence showing that phospho-interaction changes at different arrestin sites can elicit changes in affinity and structural states at remote sites, which correlate with selective arrestin functions. The interaction between a GPCR, such as the vasopressin receptor-2 (V2R), and arrestin depends on the receptors’ phosphorylation pattern. Here authors use FRET and NMR to analyze the phosphorylation patterns of the V2R-arrestin complex and show that phospho-interactions are the key determinants of selective arrestin conformational states and correlated functions.

A highly conserved rearrangement of residue contacts functions as a common step in the activation pathways of diverse G-protein-coupled receptors. Structural convergence in GPCRs A comprehensive structural analysis of 27 class A G-protein-coupled receptors (GPCRs) reveals that, despite the extensive diversity in the activation pathways between receptors, the pathways converge near the G-protein-coupling region. The convergence is mediated by a highly conserved structural rearrangement of residue contacts between transmembrane helices. These findings may explain how the activation steps initiated by diverse ligands enable GPCRs to bind a common repertoire of G proteins, and will have implications for the modelling and engineering of GPCRs for structure-based drug discovery. Class A G-protein-coupled receptors (GPCRs) are a large family of membrane proteins that mediate a wide variety of physiological functions, including vision, neurotransmission and immune responses 1 , 2 , 3 , 4 . They are the targets of nearly one-third of all prescribed medicinal drugs 5 such as beta blockers and antipsychotics. GPCR activation is facilitated by extracellular ligands and leads to the recruitment of intracellular G proteins 3 , 6 . Structural rearrangements of residue contacts in the transmembrane domain serve as ‘activation pathways’ that connect the ligand-binding pocket to the G-protein-coupling region within the receptor. In order to investigate the similarities in activation pathways across class A GPCRs, we analysed 27 GPCRs from diverse subgroups for which structures of active, inactive or both states were available. Here we show that, despite the diversity in activation pathways between receptors, the pathways converge near the G-protein-coupling region. This convergence is mediated by a highly conserved structural rearrangement of residue contacts between transmembrane helices 3, 6 and 7 that releases G-protein-contacting residues. The convergence of activation pathways may explain how the activation steps initiated by diverse ligands enable GPCRs to bind a common repertoire of G proteins.

RationaleArginine vasopressin (AVP) has dose- and sex-specific effects on social behavior, and variation in social responses is related to variation in the V1a receptor gene in animals. Whether such complexity also characterizes AVP effects on anxiety in humans, or whether V1a genotype is related to anxiety and/or AVP’s ability to affect it, remains to be determined.ObjectiveTo test if AVP has dose-dependent effects on anxiety in men and/or women and if a particular allele within the RS3 promoter region of the V1a receptor gene is associated with anxiety and/or AVP effects on anxiety.MethodMen and women self-administered 20 IU or 40 IU intranasal arginine vasopressin (AVP) and placebo in a double-blind, within-subjects design, and State (SA) and Trait (TA) anxiety were measured 60 min later. PCR was used to identify allelic variation within the RS3 region of the V1a receptor gene.ResultsAVP decreased SA in men across both doses, whereas only the lower dose had the same effect, across sexes, in individuals who carry at least one copy of a previously identified “risk” allele in the RS3 promoter of the V1a receptor gene. Additionally, after placebo, women who carried a copy of the allele displayed lower TA than women who did not, and AVP acutely increased TA scores in those women.ConclusionsExogenous AVP has modest sex- and dose-dependent effects on anxiety/affect in humans. Further, allelic variation in the V1a promoter appears associated with responsiveness to AVP’s effects and, at least in women, to stable levels of anxiety/affect.

Loss-of-function mutations in the type 2 vasopressin receptor (V2R) are a major cause of congenital nephrogenic diabetes insipidus (cNDI). In the context of partial cNDI, the response to desmopressin (dDAVP) is partially, but not entirely, diminished. For those with the partial cNDI, restoration of V2R function would offer a prospective therapeutic approach. In this study, we revealed that OPC-51803 (OPC5) and its structurally related V2R agonists could functionally restore V2R mutants causing partial cNDI by inducing prolonged signal activation. The OPC5-related agonists exhibited functional selectivity by inducing signaling through the G s -cAMP pathway while not recruiting β-arrestin1/2. We found that six cNDI-related V2R partial mutants (V88 2.53 M, Y128 3.41 S, L161 4.47 P, T273 6.37 M, S329 8.47 R and S333 8.51 del) displayed varying degrees of plasma membrane expression levels and exhibited moderately impaired signaling function. Several OPC5-related agonists induced higher cAMP responses than AVP at V2R mutants after prolonged agonist stimulation, suggesting their potential effectiveness in compensating impaired V2R-mediated function. Furthermore, docking analysis revealed that the differential interaction of agonists with L312 7.40 caused altered coordination of TM7, potentially contributing to the functional selectivity of signaling. These findings suggest that nonpeptide V2R agonists could hold promise as potential drug candidates for addressing partial cNDI.

The vasopressin 1b receptor (Avpr1b) is critical for social memory and social aggression in rodents, yet little is known about its specific roles in these behaviors. Some clues to Avpr1b function can be gained from its profile of expression in the brain, which is largely limited to the pyramidal neurons of the CA2 region of the hippocampus, and from experiments showing that inactivation of the gene or antagonism of the receptor leads to a reduction in social aggression. Here we show that partial replacement of the Avpr1b through lentiviral delivery into the dorsal CA2 region restored the probability of socially motivated attack behavior in total Avpr1b knockout mice, without altering anxiety-like behaviors. To further explore the role of the Avpr1b in this hippocampal region, we examined the effects of Avpr1b agonists on pyramidal neurons in mouse and rat hippocampal slices. We found that selective Avpr1b agonists induced significant potentiation of excitatory synaptic responses in CA2, but not in CA1 or in slices from Avpr1b knockout mice. In a way that is mechanistically very similar to synaptic potentiation induced by oxytocin, Avpr1b agonist-induced potentiation of CA2 synapses relies on NMDA (N-methyl-D-aspartic acid) receptor activation, calcium and calcium/calmodulin-dependent protein kinase II activity, but not on cAMP-dependent protein kinase activity or presynaptic mechanisms. Our data indicate that the hippocampal CA2 is important for attacking in response to a male intruder and that the Avpr1b, likely through its role in regulating CA2 synaptic plasticity, is a necessary mediator.

Jet-lag symptoms arise from temporal misalignment between the internal circadian clock and external solar time. We found that circadian rhythms of behavior (locomotor activity), clock gene expression, and body temperature immediately reentrained to phase-shifted light-dark cycles in mice lacking vasopressin receptors V1a and V1b (V1a⁻ / ⁻ V1b⁻ / ⁻). Nevertheless, the behavior of V1a⁻ / ⁻ V1b⁻ / ⁻ mice was still coupled to the internal clock, which oscillated normally under standard conditions. Experiments with suprachiasmatic nucleus (SCN) slices in culture suggested that interneuronal communication mediated by V1a and V1b confers on the SCN an intrinsic resistance to external perturbation. Pharmacological blockade of V1a and V1b in the SCN of wild-type mice resulted in accelerated recovery from jet lag, which highlights the potential of vasopressin signaling as a therapeutic target for management of circadian rhythm misalignment, such as jet lag and shift work.

Ensuring the proper amount of water inside the body is essential for survival. One of the key factors in the maintenance of body water balance is water reabsorption in the collecting ducts of the kidney, a process that is regulated by aquaporin-2 (AQP2). AQP2 is a channel that is exclusively selective for water molecules and impermeable to ions or other small molecules. Impairments of AQP2 result in various water balance disorders, including nephrogenic diabetes insipidus (NDI), which is a disease characterized by a massive loss of water through the kidney and consequent severe dehydration. Dysregulation of AQP2 is also a cause of water retention with hyponatremia in heart failure, hepatic cirrhosis, and syndrome of inappropriate antidiuretic hormone secretion (SIADH). Antidiuretic hormone vasopressin is an upstream regulator of AQP2. Its binding to the vasopressin V2 receptor promotes AQP2 targeting to the apical membrane and thus enables water reabsorption. Tolvaptan, a vasopressin V2 receptor antagonist, is effective and widely used for water retention with hyponatremia. However, there are no studies showing improvement in hard outcomes or long-term prognosis. A possible reason is that vasopressin receptors have many downstream effects other than AQP2 function. It is expected that the development of drugs that directly target AQP2 may result in increased treatment specificity and effectiveness for water balance disorders. This review summarizes recent progress in studies of AQP2 and drug development challenges for water balance disorders.