GTPγS Binding Assay for Melatonin Receptors in Mouse Brain Tissue.

The [35S]GTPγS assay is a method that measures the level of G protein activation by determining the binding of [35S]GTPγS, a non-hydrolyzable and radioactively labeled GTP analog, to Gα subunit of heterotrimeric G protein upon activation of G protein-coupled receptors (GPCR). The power of this assay lies in the fact that it measures an early event of GPCR signaling in cells expressing recombinant receptors and cells and tissues expressing endogenous receptors. The present protocol describes a sensitive method for studying G protein activation by melatonin receptors MT1 and MT2, in membranes prepared from mouse brain. Immunoprecipitation of [35S]GTPγS-labeled G proteins with Gα subunit specific antibodies (Gi, Gq, etc.) allows to determine the activation of specific G proteins. The assay can be easily applied to other tissues.

Apelin and Vasopressin: The Yin and Yang of Water Balance.

Apelin, a (neuro)vasoactive peptide, plays a prominent role in controlling body fluid homeostasis and cardiovascular functions. Experimental data performed in rodents have shown that apelin has an aquaretic effect via its central and renal actions. In the brain, apelin inhibits the phasic electrical activity of vasopressinergic neurons and the release of vasopressin from the posterior pituitary into the bloodstream and in the kidney, apelin regulates renal microcirculation and counteracts in the collecting duct, the antidiuretic effect of vasopressin occurring via the vasopressin receptor type 2. In humans and rodents, if plasma osmolality is increased by hypertonic saline infusion/water deprivation or decreased by water loading, plasma vasopressin and apelin are conversely regulated to maintain body fluid homeostasis. In patients with the syndrome of inappropriate antidiuresis, in which vasopressin hypersecretion leads to hyponatremia, the balance between apelin and vasopressin is significantly altered. In order to re-establish the correct balance, a metabolically stable apelin-17 analog, LIT01-196, was developed, to overcome the problem of the very short half-life (in the minute range) of apelin in vivo. In a rat experimental model of vasopressin-induced hyponatremia, subcutaneously (s.c.) administered LIT01-196 blocks the antidiuretic effect of vasopressin and the vasopressin-induced increase in urinary osmolality, and induces a progressive improvement in hyponatremia, suggesting that apelin receptor activation constitutes an original approach for hyponatremia treatment.

Brain ACE2 activation following brain aminopeptidase A blockade by firibastat in salt-dependent hypertension.

In the brain, aminopeptidase A (APA), a membrane-bound zinc metalloprotease, generates angiotensin III from angiotensin II. Brain angiotensin III exerts a tonic stimulatory effect on the control of blood pressure (BP) in hypertensive rats and increases vasopressin release. Blocking brain angiotensin III formation by the APA inhibitor prodrug RB150/firibastat normalizes arterial BP in hypertensive deoxycorticosterone acetate (DOCA)-salt rats without inducing angiotensin II accumulation. We therefore hypothesized that another metabolic pathway of brain angiotensin II, such as the conversion of angiotensin II into angiotensin 1-7 (Ang 1-7) by angiotensin-converting enzyme 2 (ACE2) might be activated following brain APA inhibition. We found that the intracerebroventricular (icv) administration of RB150/firibastat in conscious DOCA-salt rats both inhibited brain APA activity and induced an increase in brain ACE2 activity. Then, we showed that the decreases in BP and vasopressin release resulting from brain APA inhibition with RB150/firibastat were reduced if ACE2 was concomitantly inhibited by MLN4760, a potent ACE2 inhibitor, or if the Mas receptor (MasR) was blocked by A779, a MasR antagonist. Our findings suggest that in the brain, the increase in ACE2 activity resulting from APA inhibition by RB150/firibastat treatment, subsequently increasing Ang 1-7 and activating the MasR while blocking angiotensin III formation, contributes to the antihypertensive effect and the decrease in vasopressin release induced by RB150/firibastat. RB150/firibastat treatment constitutes an interesting therapeutic approach to improve BP control in hypertensive patients by inducing in the brain renin-angiotensin system, hyperactivity of the beneficial ACE2/Ang 1-7/MasR axis while decreasing that of the deleterious APA/Ang II/Ang III/ATI receptor axis.

Pharmacological evidence for transactivation within melatonin MT2 and serotonin 5-HT2C receptor heteromers in mouse brain.

Association of G protein-coupled receptors into heterodimeric complexes has been reported for over 50 receptor pairs in vitro but functional in vivo validation remains a challenge. Our recent in vitro studies defined the functional fingerprint of heteromers composed of Gi -coupled melatonin MT2 receptors and Gq -coupled serotonin 5-HT2C receptors, in which melatonin transactivates phospholipase C (PLC) through 5-HT2C . Here, we identified this functional fingerprint in the mouse brain. Gq protein activation was probed by [35 S]GTPγS incorporation followed by Gq immunoprecipitation, and PLC activation by determining the inositol phosphate levels in brain lysates of animals previously treated with melatonin. Melatonin concentration-dependently activated Gq proteins and PLC in the hypothalamus and cerebellum but not in cortex. These effects were inhibited by the 5-HT2C receptor-specific inverse agonist SB-243213, and were absent in MT2 and 5-HT2C knockout mice, fully recapitulating previous in vitro data and indicating the involvement of MT2 /5-HT2C heteromers. The antidepressant agomelatine had a similar effect than melatonin when applied alone but blocked the melatonin-promoted Gq activation due to its 5-HT2C antagonistic component. Collectively, we provide strong functional evidence for the existence of MT2 /5-HT2C heteromeric complexes in mouse brain. These heteromers might participate in the in vivo effects of agomelatine.

LGR5 controls extracellular matrix production by stem cells in the developing intestine.

The Lgr5 receptor is a marker of intestinal stem cells (ISCs) that regulates Wnt/b-catenin signaling. In this study, phenotype analysis of knockin/knockout Lgr5-eGFP-IRES-Cre and Lgr5-DTReGFP embryos reveals that Lgr5 deficiency during Wnt-mediated cytodifferentiation results in amplification of ISCs and early differentiation into Paneth cells, which can be counteracted by in utero treatment with the Wnt inhibitor LGK974. Conditional ablation of Lgr5 postnatally, but not in adults, alters stem cell fate toward the Paneth lineage. Together, these in vivo studies suggest that Lgr5 is part of a feedback loop to adjust the Wnt tone in ISCs. Moreover, transcriptome analyses reveal that Lgr5 controls fetal ISC maturation associated with acquisition of a definitive stable epithelial phenotype, as well as the capacity of ISCs to generate their own extracellular matrix. Finally, using the ex vivo culture system, evidences are provided that Lgr5 antagonizes the Rspondin 2-Wnt-mediated response in ISCs in organoids, revealing a sophisticated regulatory process for Wnt signaling in ISCs.

Melatonin MT1 and MT2 receptor ERK signaling is differentially dependent on Gi/o and Gq/11 proteins.

G protein-coupled receptors (GPCRs) transmit extracellular signals into cells by activating G protein- and ß-arrestin-dependent pathways. Extracellular signal-regulated kinases (ERKs) play a central role in integrating these different linear inputs coming from a variety of GPCRs to regulate cellular functions. Here, we investigated human melatonin MT1 and MT2 receptors signaling through the ERK1/2 cascade by employing different biochemical techniques together with pharmacological inhibitors and siRNA molecules. We show that ERK1/2 activation by both receptors is exclusively G protein-dependent, without any participation of ß-arrestin1/2 in HEK293 cells. ERK1/2 activation by MT1 is only mediated though Gi/o proteins, while MT2 is dependent on the cooperative activation of Gi/o and Gq/11 proteins. In the absence of Gq/11 proteins, however, MT2 -induced ERK1/2 activation switches to a ß-arrestin1/2-dependent mode. The signaling cascade downstream of G proteins is the same for both receptors and involves activation of the PI3K/PKCζ/c-Raf/MEK/ERK cascade. The differential G protein dependency of MT1 - and MT2 -mediated ERK activation was confirmed at the level of EGR1 and FOS gene expression, two ERK1/2 target genes. Gi/o /Gq/11 cooperativity was also observed in Neuroscreen-1 cells expressing endogenous MT2 , whereas in the mouse retina, where MT2 is engaged into MT1 /MT2 heterodimers, ERK1/2 signaling is exclusively Gi/o -dependent. Collectively, our data reveal differential signaling modes of MT1 and MT2 in terms of ERK1/2 activation, with an unexpected Gi/o /Gq/11 cooperativity exclusively for MT2 . The plasticity of ERK activation by MT2 is highlighted by the switch to a ß-arrestin1/2-dependent mode in the absence of Gq/11 proteins and by the switch to a Gi/o mode when engaged into MT1 /MT2 heterodimers, revealing a new mechanism underlying tissue-specific responses to melatonin.

O-linked melatonin dimers as bivalent ligands targeting dimeric melatonin receptors.

A series of dimeric melatonin analogues 3a-e obtained by connecting two melatonin molecules through the methoxy oxygen atoms with spacers spanning 16-24 atoms and the agomelatine dimer 7 were synthesized and characterized in 2-[125-I]-iodomelatonin binding assays, bioluminescence resonance energy transfer (BRET) experiments, and in functional cAMP and ß-arrestin recruitment assays at MT1 and MT2 receptors. The binding affinity of 3a-e generally increased with increasing linker length. Bivalent ligands 3a-e increased BRET signals of MT1 dimers up to 3-fold compared to the monomeric control ligand indicating the simultaneous binding of the two pharmacophores to dimeric receptors. Bivalent ligands 3c and 7 exhibited important changes in functional properties on the Gi/cAMP pathway but not on the ß-arrestin pathway compared to their monomeric counterparts. Interestingly, 3c (20 atoms spacer) shows inverse agonistic properties at MT2 on the Gi/cAMP pathway. In conclusion, these findings indicate that O-linked melatonin dimers are promising tools to develop signaling pathway-based bivalent melatonin receptor ligands.

Orphan GPR61, GPR62 and GPR135 receptors and the melatonin MT2 receptor reciprocally modulate their signaling functions.

Understanding the function of orphan G protein-coupled receptors (GPCRs), whose cognate ligand is unknown, is of major importance as GPCRs are privileged drug targets for many diseases. Recent phylogenetic studies classified three orphan receptors, GPR61, GPR62 and GPR135 among the melatonin receptor subfamily, but their capacity to bind melatonin and their biochemical functions are not well characterized yet. We show here that GPR61, GPR62 and GPR135 do not bind [3H]-melatonin nor 2-[125I]iodomelatonin and do not respond to melatonin in several signaling assays. In contrast, the three receptors show extensive spontaneous ligand-independent activities on the cAMP, inositol phosphate and ß-arrestin pathways with distinct pathway-specific profiles. Spontaneous ß-arrestin recruitment internalizes all three GPRs in the endosomal compartment. Co-expression of the melatonin binding MT2 receptor with GPR61, GPR62 or GPR135 has several consequences such as (i) the formation of receptor heteromers, (ii) the inhibition of melatonin-induced ß-arrestin2 recruitment to MT2 and (iii) the decrease of elevated cAMP levels upon melatonin stimulation in cells expressing spontaneously active GPR61 and GPR62. Collectively, these data show that GPR61, GPR62 and GPR135 are unable to bind melatonin, but show a reciprocal regulatory interaction with MT2 receptors.

Design and validation of the first cell-impermeant melatonin receptor agonist.

BACKGROUND AND PURPOSE: The paradigm that GPCRs are able to prolong or initiate cellular signalling through intracellular receptors recently emerged. Melatonin binds to G protein-coupled MT1 and MT2 receptors. In contrast to most other hormones targeting GPCRs, melatonin and its synthetic analogues are amphiphilic molecules easily penetrating into cells, but the existence of intracellular receptors is still unclear mainly due to a lack of appropriate tools. EXPERIMENTAL APPROACH: We therefore designed and synthesized a series of hydrophilic melatonin receptor ligands coupled to the Cy3 cyanin fluorophore to reliably monitor its inability to penetrate cells. Two compounds, one lipophilic and one hydrophilic, were then functionally characterized in terms of their affinity for human and murine melatonin receptors expressed in HEK293 cells and their signalling efficacy. KEY RESULTS: Among the different ligands, ICOA-13 showed the desired properties as it was cell-impermeant and bound to human and mouse MT1 and MT2 receptors. ICOA-13 showed differential activities on melatonin receptors ranging from partial to full agonistic properties for the Gi /cAMP and ERK pathway and ß-arrestin 2 recruitment. Notably, ICOA-13 enabled us to discriminate between Gi /cAMP signalling of the MT1 receptor initiated at the cell surface and neuronal mitochondria. CONCLUSIONS AND IMPLICATIONS: We report here the first cell-impermeant melatonin receptor agonist, ICOA-13, which allows us to discriminate between signalling events initiated at the cell surface and intracellular compartments. Detection of mitochondrial MT1 receptors may have an important impact on the development of novel melatonin receptor ligands relevant for neurodegenerative diseases, such as Huntington disease.

Development of original metabolically stable apelin-17 analogs with diuretic and cardiovascular effects.

Apelin, a (neuro)vasoactive peptide, plays a prominent role in controlling cardiovascular functions and water balance. Because the in vivo apelin half-life is in the minute range, we aimed to identify metabolically stable apelin-17 (K17F) analogs. We generated P92 by classic chemical substitutions and LIT01-196 by original addition of a fluorocarbon chain to the N terminus of K17F. Both analogs were much more stable in plasma (half-life >24 h for LIT01-196) than K17F (4.6 min). Analogs displayed a subnanomolar affinity for the apelin receptor and behaved as full agonists with regard to cAMP production, ERK phosphorylation, and apelin receptor internalization. Ex vivo, these compounds induced vasorelaxation of rat aortas and glomerular arterioles, respectively, precontracted with norepinephrine and angiotensin II, and increased cardiac contractility. In vivo, after intracerebroventricular administration in water-deprived mice, P92 and LIT01-196 were 6 and 160 times, respectively, more efficient at inhibiting systemic vasopressin release than K17F. Administered intravenously (nmol/kg range) in normotensive rats, these analogs potently increased urine output and induced a profound and sustained decrease in arterial blood pressure. In summary, these new compounds, which favor diuresis and improve cardiac contractility while reducing vascular resistances, represent promising candidates for the treatment of heart failure and water retention/hyponatremic disorders.-Gerbier, R., Alvear-Perez, R., Margathe, J.-F., Flahault, A., Couvineau, P., Gao, J., De Mota, N., Dabire, H., Li, B., Ceraudo, E., Hus-Citharel, A., Esteoulle, L., Bisoo, C., Hibert, M., Berdeaux, A., Iturrioz, X., Bonnet, D., Llorens-Cortes, C. Development of original metabolically stable apelin-17 analogs with diuretic and cardiovascular effects.

New structural insights into the apelin receptor: identification of key residues for apelin binding.

Apelin is the endogenous ligand of the orphan 7-transmembrane domain GPCR APJ, now named the apelin receptor (ApelinR). Apelin plays a prominent role in body fluid and cardiovascular homeostasis. To better understand the structural organization of the ApelinR, we built 3 homology 3-dimensional (3D) models of the human ApelinR using the validated cholecystokinin receptor-1 3D model or the X-ray structures of the ß2-adrenergic and CXCR4 receptors as templates. Docking of the pyroglutamyl form of apelin 13 (pE13F) into these models revealed the conservation at the bottom of the binding site of a hydrophobic cavity in which the C-terminal Phe of pE13F was embedded. In contrast, at the top of the binding site, depending on the model, different interactions were visualized between acidic residues of the ApelinR and the basic residues of pE13F. Using site-directed mutagenesis, we showed that Asp 92, Glu 172, and Asp 282 of rat ApelinR are key residues in apelin binding by interacting with Lys 8, Arg 2, and Arg 4 of pE13F, respectively. These residues are only seen in the CXCR4-based ApelinR 3D model, further validating this model. These findings bring new insights into the structural organization of the ApelinR and the mode of apelin binding.

By interacting with the C-terminal Phe of apelin, Phe255 and Trp259 in helix VI of the apelin receptor are critical for internalization.

Apelin is the endogenous ligand of the orphan seven-transmembrane domain (TM) G protein-coupled receptor APJ. Apelin is involved in the regulation of body fluid homeostasis and cardiovascular functions. We previously showed the importance of the C-terminal Phe of apelin 17 (K17F) in the hypotensive activity of this peptide. Here, we show either by deleting the Phe residue (K16P) or by substituting it by an Ala (K17A), that it plays a crucial role in apelin receptor internalization but not in apelin binding or in Gα(i)-protein coupling. Then we built a homology three-dimensional model of the human apelin receptor using the cholecystokinin receptor-1 model as a template, and we subsequently docked K17F into the binding site. We visualized a hydrophobic cavity at the bottom of the binding pocket in which the C-terminal Phe of K17F was embedded by Trp(152) in TMIV and Trp(259) and Phe(255) in TMVI. Using molecular modeling and site-directed mutagenesis studies, we further showed that Phe(255) and Trp(259) are key residues in triggering receptor internalization without playing a role in apelin binding or in Gα(i)-protein coupling. These findings bring new insights into apelin receptor activation and show that Phe(255) and Trp(259), by interacting with the C-terminal Phe of the pyroglutamyl form of apelin 13 (pE13F) or K17F, are crucial for apelin receptor internalization.

Identification and pharmacological properties of E339-3D6, the first nonpeptidic apelin receptor agonist.

Apelin plays a prominent role in body fluid and cardiovascular homeostasis. To explore further upstream the role played by this peptide, nonpeptidic agonists and antagonists of the apelin receptor are required. To identify such compounds that do not exist to date, we used an original fluorescence resonance energy transfer-based assay to screen a G-protein-coupled receptor-focused library of fluorescent compounds on the human EGFP-tagged apelin receptor. This led to isolated E339-3D6 that displayed a 90 nM affinity and behaved as a partial agonist with regard to cAMP production and as a full agonist with regard to apelin receptor internalization. Finally, E339-3D6 induced vasorelaxation of rat aorta precontracted with noradrenaline and potently inhibited systemic vasopressin release in water-deprived mice when intracerebroventricularly injected. This compound represents the first nonpeptidic agonist of the apelin receptor, the optimization of which will allow development of a new generation of vasodilator and aquaretic agents.