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BioRxiv : the Preprint Server For... Jul 2023The small size and flexibility of G protein-coupled receptors (GPCRs) have long posed a significant challenge to determining their structures for research and...
The small size and flexibility of G protein-coupled receptors (GPCRs) have long posed a significant challenge to determining their structures for research and therapeutic applications. Single particle cryogenic electron microscopy (cryoEM) is often out of reach due to the small size of the receptor without a signaling partner. Crystallization of GPCRs in lipidic cubic phase (LCP) often results in crystals that may be too small and difficult to analyze using X-ray microcrystallography at synchrotron sources or even serial femtosecond crystallography at X-ray free electron lasers. Here, we determine the previously unknown structure of the human vasopressin 1B receptor (V1BR) using microcrystal electron diffraction (MicroED). To achieve this, we grew V1BR microcrystals in LCP and transferred the material directly onto electron microscopy grids. The protein was labeled with a fluorescent dye prior to crystallization to locate the microcrystals using cryogenic fluorescence microscopy, and then the surrounding material was removed using a plasma-focused ion beam to thin the sample to a thickness amenable to MicroED. MicroED data from 14 crystalline lamellae were used to determine the 3.2 Å structure of the receptor in the crystallographic space group 1. These results demonstrate the use of MicroED to determine previously unknown GPCR structures that, despite significant effort, were not tractable by other methods.
PubMed: 37461729
DOI: 10.1101/2023.07.05.547888 -
International Journal of Molecular... Jul 2022Vasopressin type-2 receptor (V2R) is ectopically expressed and plays a pathogenic role in clear cell renal cell carcinoma (ccRCC) tumor cells. Here we examined how V2R...
Vasopressin type-2 receptor (V2R) is ectopically expressed and plays a pathogenic role in clear cell renal cell carcinoma (ccRCC) tumor cells. Here we examined how V2R signaling within human ccRCC tumor cells (Caki1 cells) stimulates stromal cancer-associated fibroblasts (CAFs). We found that cell culture conditioned media from Caki1 cells increased activation, migration, and proliferation of fibroblasts in vitro, which was inhibited by V2R gene silencing in Caki1 cells. Analysis of the conditioned media and mRNA of the V2R gene silenced and control Caki1 cells showed that V2R regulates the production of CAF-activating factors. Some of these factors were also found to be regulated by YAP in these Caki1 cells. YAP expression colocalized and correlated with V2R expression in ccRCC tumor tissue. V2R gene silencing or V2R antagonist significantly reduced YAP in Caki1 cells. Moreover, the V2R antagonist reduced YAP expression and myofibroblasts in mouse xenograft tumors. These results suggest that V2R plays an important role in secreting pro-fibrotic factors that stimulate fibroblast activation by a YAP-dependent mechanism in ccRCC tumors. Our results demonstrate a novel role for the V2R-YAP axis in the regulation of myofibroblasts in ccRCC and a potential therapeutic target.
Topics: Animals; Antidiuretic Hormone Receptor Antagonists; Cancer-Associated Fibroblasts; Carcinoma, Renal Cell; Cell Line, Tumor; Culture Media, Conditioned; Fibroblasts; Humans; Kidney; Kidney Neoplasms; Mice; Receptors, Vasopressin; Tumor Microenvironment; Vasopressins; Xenograft Model Antitumor Assays
PubMed: 35886951
DOI: 10.3390/ijms23147601 -
Frontiers in Neuroendocrinology Jul 2016Oxytocin (OT) and vasopressin (AVP) are important hypothalamic neuropeptides that regulate peripheral physiology, and have emerged as important modulators of brain... (Review)
Review
Oxytocin (OT) and vasopressin (AVP) are important hypothalamic neuropeptides that regulate peripheral physiology, and have emerged as important modulators of brain function, particularly in the social realm. OT structure and the genes that ultimately determine structure are highly conserved among diverse eutherian mammals, but recent discoveries have identified surprising variability in OT and peptide structure in New World monkeys (NWM), with five new OT variants identified to date. This review explores these new findings in light of comparative OT/AVP ligand evolution, documents coevolutionary changes in the oxytocin and vasopressin receptors (OTR and V1aR), and highlights the distribution of neuropeptidergic neurons and receptors in the primate brain. Finally, the behavioral consequences of OT and AVP in regulating NWM sociality are summarized, demonstrating important neuromodulatory effects of these compounds and OT ligand-specific influences in certain social domains.
Topics: Animals; Behavior, Animal; Brain; Oxytocin; Primates; Receptors, Oxytocin; Receptors, Vasopressin; Social Behavior; Vasopressins
PubMed: 27020799
DOI: 10.1016/j.yfrne.2016.03.004 -
Open Biology Jan 2018Recent observations suggest that atypical chemokine receptor (ACKR)3 and chemokine (C-X-C motif) receptor (CXCR)4 regulate human vascular smooth muscle function through...
Recent observations suggest that atypical chemokine receptor (ACKR)3 and chemokine (C-X-C motif) receptor (CXCR)4 regulate human vascular smooth muscle function through hetero-oligomerization with α-adrenoceptors. Here, we show that ACKR3 also regulates arginine vasopressin receptor (AVPR)1A. We observed that ACKR3 agonists inhibit arginine vasopressin (aVP)-induced inositol trisphosphate (IP) production in human vascular smooth muscle cells (hVSMCs) and antagonize aVP-mediated constriction of isolated arteries. Proximity ligation assays, co-immunoprecipitation and bioluminescence resonance energy transfer experiments suggested that recombinant and endogenous ACKR3 and AVPR1A interact on the cell surface. Interference with ACKR3 : AVPR1A heteromerization using siRNA and peptide analogues of transmembrane domains of ACKR3 abolished aVP-induced IP production. aVP stimulation resulted in β-arrestin 2 recruitment to AVPR1A and ACKR3. While ACKR3 activation failed to cross-recruit β-arrestin 2 to AVPR1A, the presence of ACKR3 reduced the efficacy of aVP-induced β-arrestin 2 recruitment to AVPR1A. AVPR1A and ACKR3 co-internalized upon agonist stimulation in hVSMC. These data suggest that AVPR1A : ACKR3 heteromers are constitutively expressed in hVSMC, provide insights into molecular events at the heteromeric receptor complex, and offer a mechanistic basis for interactions between the innate immune and vasoactive neurohormonal systems. Our findings suggest that ACKR3 is a regulator of vascular smooth muscle function and a possible drug target in diseases associated with impaired vascular reactivity.
Topics: Animals; Arginine Vasopressin; Cells, Cultured; HEK293 Cells; Humans; Inositol Phosphates; Male; Muscle, Smooth, Vascular; Protein Multimerization; Rats; Rats, Sprague-Dawley; Receptors, CXCR; Receptors, Vasopressin; beta-Arrestins
PubMed: 29386406
DOI: 10.1098/rsob.170207 -
FEBS Letters Jul 2021Although class A seven-transmembrane helix (7TM) receptor hetero-oligomers have been proposed, information on the assembly and function of such higher-order...
Although class A seven-transmembrane helix (7TM) receptor hetero-oligomers have been proposed, information on the assembly and function of such higher-order hetero-oligomers is not available. Utilizing bioluminescence resonance energy transfer (BRET), bimolecular luminescence/fluorescence complementation (BiLC/BiFC), and BiLC/BiFC BRET in HEK293T cells, we provide evidence that chemokine (C-X-C motif) receptor 4, atypical chemokine receptor 3, α -adrenoceptor, and arginine vasopressin receptor 1A form hetero-oligomers composed of 2-4 different protomers. We show that hetero-oligomerization per se and ligand binding to individual protomers regulate agonist-induced coupling to the signaling transducers of interacting receptor partners. Our findings support the concept that receptor hetero-oligomers form supramolecular machineries with molecular signaling properties distinct from the individual protomers. These findings provide a mechanism for the phenomenon of context-dependent receptor function.
Topics: Bacterial Proteins; Chemokine CXCL12; Fluorescence Resonance Energy Transfer; Gene Expression; Genes, Reporter; HEK293 Cells; Humans; Kinetics; Luciferases; Luminescent Proteins; Plasmids; Protein Binding; Protein Conformation; Protein Interaction Domains and Motifs; Protein Multimerization; Receptors, Adrenergic, alpha-1; Receptors, CXCR; Receptors, CXCR4; Receptors, Vasopressin; Recombinant Fusion Proteins
PubMed: 34032285
DOI: 10.1002/1873-3468.14135 -
Genes, Brain, and Behavior Feb 2017We examined the extent to which the arginine vasopressin receptor 1a (AVPR1a) and dopamine receptor D4 (DRD4) were related to sensitive maternal behavior directly or...
We examined the extent to which the arginine vasopressin receptor 1a (AVPR1a) and dopamine receptor D4 (DRD4) were related to sensitive maternal behavior directly or indirectly via maternal social cognition. Participants were 207 (105 European-American and 102 African-American) mothers and their children (52% females). Sensitive maternal behavior was rated and aggregated across a series of tasks when infants were 6 months, 1 year and 2 years old. At 6 months, mothers were interviewed about their empathy, attributions about infant behavior and beliefs about crying to assess their parenting-related social cognition. Mothers with long alleles for AVPR1a and DRD4 engaged in more mother-oriented social cognition (i.e. negative attributions and beliefs about their infants' crying, β = 0.13, P < 0.05 and β = 0.16, P < 0.05, respectively), which in turn predicted less sensitive maternal behavior (β = -0.23, P < 0.01). Both indirect effects were statistically significant independent of one another and covariates [95% confidence interval (CI): -0.22, -0.03 and β = -0.03 for AVPR; 95% CI: -0.20, -0.03 and β = -0.04 for DRD4]. There were no significant direct effects of AVPR1a or DRD4 on maternal sensitivity (β = 0.02, P = .73 and β = -0.10, P = .57, respectively). The results did not vary for African-American and European-American mothers (Δχ = 18.76, Δdf = 16, P = 0.28). Results support the view that one mechanism by which maternal genes are associated with parental behavior is via social cognition.
Topics: Adult; Alleles; Arginine Vasopressin; Child, Preschool; Cognition; Female; Genetic Testing; Humans; Infant; Infant Behavior; Infant, Newborn; Male; Maternal Behavior; Mother-Child Relations; Polymorphism, Single Nucleotide; Receptors, Dopamine D4; Receptors, Vasopressin; Social Behavior; Stress, Psychological
PubMed: 27581946
DOI: 10.1111/gbb.12326 -
Nature Communications May 2021In contrast to male rats, aggression in virgin female rats has been rarely studied. Here, we established a rat model of enhanced aggression in females using a...
In contrast to male rats, aggression in virgin female rats has been rarely studied. Here, we established a rat model of enhanced aggression in females using a combination of social isolation and aggression-training to specifically investigate the involvement of the oxytocin (OXT) and arginine vasopressin (AVP) systems within the lateral septum (LS). Using neuropharmacological, optogenetic, chemogenetic as well as microdialysis approaches, we revealed that enhanced OXT release within the ventral LS (vLS), combined with reduced AVP release within the dorsal LS (dLS), is required for aggression in female rats. Accordingly, increased activity of putative OXT receptor-positive neurons in the vLS, and decreased activity of putative AVP receptor-positive neurons in the dLS, are likely to underly aggression in female rats. Finally, in vitro activation of OXT receptors in the vLS increased tonic GABAergic inhibition of dLS neurons. Overall, our data suggest a model showing that septal release of OXT and AVP differentially affects aggression in females by modulating the inhibitory tone within LS sub-networks.
Topics: Aggression; Animals; Arginine Vasopressin; Female; Microdialysis; Neurons; Oxytocin; Rats, Wistar; Receptors, Oxytocin; Receptors, Vasopressin; Septal Nuclei; Social Isolation; Rats
PubMed: 34006875
DOI: 10.1038/s41467-021-23064-5 -
Journal of Neuroendocrinology Jul 2020Oxytocin (OT) and arginine vasopressin (AVP), as well as synthetic ligands targeting their receptors (OTR, V1aR), are used in a wide variety of research contexts,...
Oxytocin (OT) and arginine vasopressin (AVP), as well as synthetic ligands targeting their receptors (OTR, V1aR), are used in a wide variety of research contexts, although their pharmacological properties are determined in only a few species. Syrian hamsters (Mesocricetus auratus) have a long history of use as a behavioural and biomedical model for the study of OT and AVP and, more recently, hamsters have been used to investigate behavioural consequences of OT-mediated activation of V1aR. We aimed to determine the binding affinities of OT, AVP and the selective V1aR antagonist, Manning compound, for OTR and V1aR in hamster brains. We performed saturation binding assays to determine the Kd values for the selective OTR and V1aR radioligands, [ I]ornithine vasotocin analogue and [ I]linear vasopressin antagonist. We then performed competition binding assays to determine K values for OT, AVP and Manning compound at both the OTR and V1aR. We found that OT and AVP each had the highest affinity for their canonical receptors (OT-OTR Ki = 4.28 [95% confidence interval (CI) = 2.9-6.3] nmol L ; AVP-V1ar Ki = 4.70 [95% CI = 1.5-14.1] nmol L ) and had the lowest affinity for their non-canonical ligands (OT-V1aR = 495.2 [95% CI = 198.5-1276] nmol L ; AVP-OTR Ki = 36.1 [95% CI = 12.4-97.0] nmol L ). Manning compound had the highest affinity for the V1aR (MC-V1aR Ki = 6.87 [95% CI = 4.0-11.9] nmol L ; MC-OTR Ki = 213.8 [95% CI = 117.3-392.7] nmol L ), although Manning compound was not as selective for the V1aR in hamsters as has been reported for the receptor in rats. When comparing these data with previously published work, we found that the promiscuity of the V1aR in hamsters with respect to OT and AVP binding is more similar to the promiscuity of the human V1aR than to the rat V1aR receptor. Moreover, the selectivity of OT at hamster receptors is more similar to the selectivity of OT at human receptors than the selectivity of OT at rat receptors. These data highlight the importance of determining the pharmacological properties of behaviourally relevant compounds in diverse model species.
Topics: Animals; Arginine Vasopressin; Binding, Competitive; Brain; Cricetinae; Humans; Male; Mesocricetus; Oxytocin; Protein Binding; Rats; Receptors, G-Protein-Coupled; Receptors, Oxytocin; Receptors, Vasopressin
PubMed: 32662552
DOI: 10.1111/jne.12882 -
International Journal of Molecular... Aug 2021Oxytocin (OXT) is a neuropeptide involved in a plethora of behavioral and physiological processes. However, there is a prominent lack of 3D cell culture models that...
Oxytocin (OXT) is a neuropeptide involved in a plethora of behavioral and physiological processes. However, there is a prominent lack of 3D cell culture models that investigate the effects of OXT on a cellular/molecular level. In this study, we established a hypothalamic neuronal spheroid model to investigate the cellular response in a more realistic 3D setting. Our data indicate that the formation of spheroids itself does not alter the basic characteristics of the cell line and that markers of cellular morphology and connectivity are stably expressed. We found that both OXT and arginine vasopressin (AVP) treatment increase spheroid size (surface area and volume), as well as individual nucleus size, which serves as an indicator for cellular proliferation. The cellular response to both OXT and AVP seems mainly to be mediated by the AVP receptor 1a (V1aR); however, the OXT receptor (OXTR) contributes significantly to the observed proliferative effect. When we blocked the OXTR pharmacologically or knocked down the OXTR by siRNA, the OXT- or AVP-induced cellular proliferation decreased. In summary, we established a 3D cell culture model of the neuronal response to OXT and AVP and found that spheroids react to the treatment via their respective receptors but also via cross-talk between the two receptor types.
Topics: Animals; Arginine Vasopressin; Cell Line; Cell Proliferation; Hypothalamus; Oxytocin; Rats; Receptors, Oxytocin; Receptors, Vasopressin; Spheroids, Cellular
PubMed: 34445168
DOI: 10.3390/ijms22168464 -
The New England Journal of Medicine Apr 2015
Review
Topics: Animals; Aquaporins; Arginine Vasopressin; Humans; Kidney Tubules; Receptors, Vasopressin; Sodium-Potassium-Chloride Symporters; Water-Electrolyte Balance
PubMed: 25830425
DOI: 10.1056/NEJMra1404726