Unveiling a novel memory center in human brain: neurochemical identification of the nucleus incertus, a key pontine locus implicated in stress and neuropathology.

BACKGROUND: The nucleus incertus (NI) was originally described by Streeter in 1903, as a midline region in the floor of the fourth ventricle of the human brain with an 'unknown' function. More than a century later, the neuroanatomy of the NI has been described in lower vertebrates, but not in humans. Therefore, we examined the neurochemical anatomy of the human NI using markers, including the neuropeptide, relaxin-3 (RLN3), and began to explore the distribution of the NI-related RLN3 innervation of the hippocampus. METHODS: Histochemical staining of serial, coronal sections of control human postmortem pons was conducted to reveal the presence of the NI by detection of immunoreactivity (IR) for the neuronal markers, microtubule-associated protein-2 (MAP2), glutamic acid dehydrogenase (GAD)-65/67 and corticotrophin-releasing hormone receptor 1 (CRHR1), and RLN3, which is highly expressed in NI neurons in diverse species. RLN3 and vesicular GABA transporter 1 (vGAT1) mRNA were detected by fluorescent in situ hybridization. Pons sections containing the NI from an AD case were immunostained for phosphorylated-tau, to explore potential relevance to neurodegenerative diseases. Lastly, sections of the human hippocampus were stained to detect RLN3-IR and somatostatin (SST)-IR. RESULTS: In the dorsal, anterior-medial region of the human pons, neurons containing RLN3- and MAP2-IR, and RLN3/vGAT1 mRNA-positive neurons were observed in an anatomical pattern consistent with that of the NI in other species. GAD65/67- and CRHR1-immunopositive neurons were also detected within this area. Furthermore, RLN3- and AT8-IR were co-localized within NI neurons of an AD subject. Lastly, RLN3-IR was detected in neurons within the CA1, CA2, CA3 and DG areas of the hippocampus, in the absence of RLN3 mRNA. In the DG, RLN3- and SST-IR were co-localized in a small population of neurons. CONCLUSIONS: Aspects of the anatomy of the human NI are shared across species, including a population of stress-responsive, RLN3-expressing neurons and a RLN3 innervation of the hippocampus. Accumulation of phosphorylated-tau in the NI suggests its possible involvement in AD pathology. Further characterization of the neurochemistry of the human NI will increase our understanding of its functional role in health and disease.

Characterization of chicken Relaxin3 gene: mRNA expression and response to reproductive hormone treatment in ovarian granulosa cells, and single nucleotide polymorphisms associated with egg laying traits in hens.

As a protein structurally similar to insulin, relaxin3 (RLN3) plays a role in promoting arousal, suppressing depressive or anxious behaviors. Two studies revealed the increase of RLN3 expression during chicken follicle selection. In this study, by real-time quantitative PCR and luciferase assay, mRNA expression and single nucleotide polymorphisms (SNPs) of chicken RLN3 were investigated. The mRNA expression of chicken RLN3 was higher in the granulosa cell of hierarchal follicles (Post-GCs) than that of pre-hierarchal follicles (Pre-GCs). In Pre-GCs, the mRNA expression of chicken RLN3 was stimulated by FSH and progesterone; in Post-GCs, it was stimulated by higher concentration of estrogen and FSH, however, was inhibited by progesterone. Four SNPs including g.-655G > C, g-592G > A, g.-372T > A and g.-282G > C were identified in the critical promoter region from -1291 bp to -207 bp of chicken RLN3, among which g.-655G > C, and g-592G > A were associated with age at first laying and clutch size, respectively, in Zaozhuang Sunzhi chickens. At g.-655G > C and g-592G > A, allele C and allele A had higher transcriptional activity, respectively. These data suggest that RLN3 plays an important role in chicken follicle development and SNPs in its promoter region are potential DNA markers for improving egg production traits.

Acute Stress Affects the Relaxin/Insulin-Like Family Peptide Receptor 3 mRNA Expression in Brain of Pubertal Male Wistar Rats.

The current literature suggests that relaxin-3/relaxin/insulin-like family peptide receptor 3 (RLN-3/RXFP-3) system is involved in the pathophysiology of affective disorders because the results of anatomical and pharmacological studies have shown that the RLN-3 signaling pathway plays a role in modulating the stress response, anxiety, arousal, depression-like behavior, and neuroendocrine homeostasis. The risk of developing mental illnesses in adulthood is increased by exposure to stress in early periods of life. The available data indicate that puberty is especially characterized by the development of the neural system and emotionality and is a "stress-sensitive" period. The presented study assessed the short-term changes in the expression of RLN-3 and RXFP-3 mRNA in the stress-dependent brain regions in male pubertal Wistar rats that had been subjected to acute stress. Three stressors were applied from 42 to 44 postnatal days (first day: a single forced swim; second day: stress on an elevated platform that was repeated three times; third day: restraint stress three times). Anxiety (open field, elevated plus maze test) and anhedonic-like behavior (sucrose preference test) were estimated during these tests. The corticosterone (CORT) levels and blood morphology were estimated. We found that the RXFP-3 mRNA expression decreased in the brainstem, whereas it increased in the hypothalamus 72 h after acute stress. These molecular changes were accompanied by the increased levels of CORT and anxiety-like behavior detected in the open field test that had been conducted earlier, that is, 24 h after the stress procedure. These findings shed new light on the neurochemical changes that are involved in the compensatory response to adverse events in pubertal male rats and support other data that suggest a regulatory interplay between the RLN-3 pathway and the hypothalamus-pituitary-adrenal axis activity in the mechanisms of anxiety-like behavior.

Novel RXFP3 negative allosteric modulator RLX-33 reduces alcohol self-administration in rats.

There is much interest in identifying novel pharmacotherapeutic targets that improve clinical outcomes for the treatment of alcohol use disorder (AUD). One promising target for therapeutic intervention is the relaxin family peptide 3 (RXFP3) receptor, a cognate receptor for neuropeptide relaxin-3, which has previously been implicated in regulating alcohol drinking behavior. Recently, we developed the first small-molecule RXFP3-selective negative allosteric modulator (NAM) RLX-33. Therefore, the goal of the present work was to characterize the impact of this novel NAM on affective-related behaviors and alcohol self-administration in rats. First, the effects of RLX-33 were tested on alcohol and sucrose self-administration in Wistar and alcohol-preferring P rats to determine the dose-response profile and specificity for alcohol. Then, we assessed the effects of systemic RLX-33 injection in Wistar rats in a battery of behavioral assays (open-field test, elevated zero maze, acoustic startle response test, and prepulse inhibition) and tested for alcohol clearance. We found that the lowest effective dose (5 mg/kg) reduced alcohol self-administration in both male and female Wistar rats, while in alcohol-preferring P rats, this effect was restricted to males, and there were no effects on sucrose self-administration or general locomotor activity. The characterization of affective and metabolic effects in Wistar rats generally found few locomotor, affective, or alcohol clearance changes, particularly at the 5 mg/kg dose. Overall, these findings are promising and suggest that RXFP3 NAM has potential as a pharmacological target for treating AUD.

RLN3/RXFP3 Signaling in the PVN Inhibits Magnocellular Neurons via M-like Current Activation and Contributes to Binge Eating Behavior.

Binge-eating disorder is the most common eating disorder. Various neuropeptides play important roles in the regulation of feeding behavior, including relaxin-3 (RLN3), which stimulates food intake in rats through the activation of the relaxin-family peptide-3 receptor (RXFP3). Here we demonstrate that a likely mechanism underlying the orexigenic action of RLN3 is RXFP3-mediated inhibition of oxytocin- and arginine-vasopressin-synthesizing paraventricular nucleus (PVN) magnocellular neurosecretory cells. Moreover, we reveal that, in male and female rats, this action depends on M-like potassium conductance. Notably, higher intra- and peri-PVN RLN3 fiber densities were observed in females, which may constitute an anatomic substrate for observed sex differences in binge-eating disorder. Finally, in a model of binge-eating in female rats, RXFP3 blockade within the PVN prevented binge-eating behavior. These data demonstrate a direct RLN3/RXFP3 action in the PVN of male and female rats, identify the associated ionic mechanisms, and reveal that hypothalamic RLN3/RXFP3 signaling regulates binge-eating behavior.SIGNIFICANCE STATEMENT Binge-eating disorder is the most common eating disorder worldwide, affecting women twice as frequently as men. Various neuropeptides play important roles in the regulation of feeding behavior, including relaxin-3, which acts via the relaxin-family peptide-3 receptor (RXFP3). Using a model of binge-eating, we demonstrated that relaxin-3/RXFP3 signaling in the hypothalamic paraventricular nucleus (PVN) is necessary for the expression of binge-eating behavior in female rats. Moreover, we elucidated the neuronal mechanism of RLN3/RXFP3 signaling in PVN in male and female rats and characterized sex differences in the RLN3 innervation of the PVN. These findings increase our understanding of the brain circuits and neurotransmitters involved in binge-eating disorder pathology and identify RXFP3 as a therapeutic target for binge-like eating disorders.

Estrous Cycle Modulation of Feeding and Relaxin-3/Rxfp3 mRNA Expression: Implications for Estradiol Action.

INTRODUCTION: Food intake varies during the ovarian hormone/estrous cycle in humans and rodents, an effect mediated mainly by estradiol. A potential mediator of the central anorectic effects of estradiol is the neuropeptide relaxin-3 (RLN3) synthetized in the nucleus incertus (NI) and acting via the relaxin family peptide-3 receptor (RXFP3). METHODS: We investigated the relationship between RLN3/RXFP3 signaling and feeding behavior across the female rat estrous cycle. We used in situ hybridization to investigate expression patterns of Rln3 mRNA in NI and Rxfp3 mRNA in the hypothalamic paraventricular nucleus (PVN), lateral hypothalamic area (LHA), medial preoptic area (MPA), and bed nucleus of the stria terminalis (BNST), across the estrous cycle. We identified expression of estrogen receptors (ERs) in the NI using droplet digital PCR and assessed the electrophysiological responsiveness of NI neurons to estradiol in brain slices. RESULTS: Rln3 mRNA reached the lowest levels in the NI pars compacta during proestrus. Rxfp3 mRNA levels varied across the estrous cycle in a region-specific manner, with changes observed in the perifornical LHA, magnocellular PVN, dorsal BNST, and MPA, but not in the parvocellular PVN or lateral LHA. G protein-coupled estrogen receptor 1 (Gper1) mRNA was the most abundant ER transcript in the NI. Estradiol inhibited 33% of type 1 NI neurons, including RLN3-positive cells. CONCLUSION: These findings demonstrate that the RLN3/RXFP3 system is modulated by the estrous cycle, and although further studies are required to better elucidate the cellular and molecular mechanisms of estradiol signaling, current results implicate the involvement of the RLN3/RXFP3 system in food intake fluctuations observed across the estrous cycle in female rats.

Transcriptional up-regulation of relaxin-3 by Nur77 attenuates ß-adrenergic agonist-induced apoptosis in cardiomyocytes.

The relaxin family peptides have been shown to exert several beneficial effects on the heart, including anti-apoptosis, anti-fibrosis, and anti-hypertrophy activity. Understanding their regulation might provide new opportunities for therapeutic interventions, but the molecular mechanism(s) coordinating relaxin expression in the heart remain largely obscured. Previous work demonstrated a role for the orphan nuclear receptor Nur77 in regulating cardiomyocyte apoptosis. We therefore investigated Nur77 in the hopes of identifying novel relaxin regulators. Quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) data indicated that ectopic expression of orphan nuclear receptor Nur77 markedly increased the expression of latexin-3 (RLN3), but not relaxin-1 (RLN1), in neonatal rat ventricular cardiomyocytes (NRVMs). Furthermore, we found that the ß-adrenergic agonist isoproterenol (ISO) markedly stimulated RLN3 expression, and this stimulation was significantly attenuated in Nur77 knockdown cardiomyocytes and Nur77 knockout hearts. We showed that Nur77 significantly increased RLN3 promoter activity via specific binding to the RLN3 promoter, as demonstrated by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays. Furthermore, we found that Nur77 overexpression potently inhibited ISO-induced cardiomyocyte apoptosis, whereas this protective effect was significantly attenuated in RLN3 knockdown cardiomyocytes, suggesting that Nur77-induced RLN3 expression is an important mediator for the suppression of cardiomyocyte apoptosis. These findings show that Nur77 regulates RLN3 expression, therefore suppressing apoptosis in the heart, and suggest that activation of Nur77 may represent a useful therapeutic strategy for inhibition of cardiac fibrosis and heart failure.

Distinct but overlapping binding sites of agonist and antagonist at the relaxin family peptide 3 (RXFP3) receptor.

The relaxin-3 neuropeptide activates the relaxin family peptide 3 (RXFP3) receptor to modulate stress, appetite, and cognition. RXFP3 shows promise as a target for treating neurological disorders, but realization of its clinical potential requires development of smaller RXFP3-specific drugs that can penetrate the blood-brain barrier. Designing such drugs is challenging and requires structural knowledge of agonist- and antagonist-binding modes. Here, we used structure-activity data for relaxin-3 and a peptide RXFP3 antagonist termed R3 B1-22R to guide receptor mutagenesis and develop models of their binding modes. RXFP3 residues were alanine-substituted individually and in combination and tested in cell-based binding and functional assays to refine models of agonist and antagonist binding to active- and inactive-state homology models of RXFP3, respectively. These models suggested that both agonists and antagonists interact with RXFP3 via similar residues in their B-chain central helix. The models further suggested that the B-chain Trp27 inserts into the binding pocket of RXFP3 and interacts with Trp138 and Lys271, the latter through a salt bridge with the C-terminal carboxyl group of Trp27 in relaxin-3. R3 B1-22R, which does not contain Trp27, used a non-native Arg23 residue to form cation-π and salt-bridge interactions with Trp138 and Glu141 in RXFP3, explaining a key contribution of Arg23 to affinity. Overall, relaxin-3 and R3 B1-22R appear to share similar binding residues but may differ in binding modes, leading to active and inactive RXFP3 conformational states, respectively. These mechanistic insights may assist structure-based drug design of smaller relaxin-3 mimetics to manage neurological disorders.

Binding conformation and determinants of a single-chain peptide antagonist at the relaxin-3 receptor RXFP3.

The neuropeptide relaxin-3 and its receptor relaxin family peptide receptor-3 (RXFP3) play key roles in modulating behavior such as memory and learning, food intake, and reward seeking. A linear relaxin-3 antagonist (R3 B1-22R) based on a modified and truncated relaxin-3 B-chain was recently developed. R3 B1-22R is unstructured in solution; thus, the binding conformation and determinants of receptor binding are unclear. Here, we have designed, chemically synthesized, and pharmacologically characterized more than 60 analogues of R3 B1-22R to develop an extensive understanding of its structure-activity relationships. We show that the key driver for affinity is the nonnative C-terminal Arg23 Additional contributors to binding include amino acid residues that are important also for relaxin-3 binding, including Arg12, Ile15, and Ile19 Intriguingly, amino acid residues that are not exposed in native relaxin-3, including Phe14 and Ala17, also interact with RXFP3. We show that R3 B1-22R has a propensity to form a helical structure, and modifications that support a helical conformation are functionally well-tolerated, whereas helix breakers such as proline residues disrupt binding. These data suggest that the peptide adopts a helical conformation, like relaxin-3, upon binding to RXFP3, but that its smaller size allows it to penetrate deeper into the orthosteric binding site, creating more extensive contacts with the receptor.

Discovery of a small molecule RXFP3/4 agonist that increases food intake in rats upon acute central administration.

The relaxin family peptide receptors have been implicated in numerous physiological processes including energy homeostasis, cardiac function, wound healing, and reproductive function. Two family members, RXFP3 and RXFP4, are class A GPCRs with endogenous peptide ligands (relaxin-3 and insulin-like peptide 5 (INSL5), respectively). Polymorphisms in relaxin-3 and RXFP3 have been associated with obesity, diabetes, and hypercholesterolemia. Moreover, central administration of relaxin-3 in rats has been shown to increase food intake, leading to body weight gain. Reported RXFP3 and RXFP4 ligands have been restricted to peptides (both endogenous and synthetic) as well as a low molecular weight positive allosteric modulator requiring a non-endogenous orthosteric ligand. Described here is the discovery of the first potent low molecular weight dual agonists of RXFP3/4. The scaffold identified is competitive with a chimeric relaxin-3/INSL5 peptide for RXFP3 binding, elicits similar downstream signaling as relaxin-3, and increases food intake in rats following acute central administration. This is the first report of small molecule RXFP3/4 agonism.

Effect of relaxin-3 on Kiss-1, gonadotropin-releasing hormone, and gonadotropin subunit gene expression.

PURPOSE: Relaxin-3 is a hypothalamic neuropeptide that belongs to the insulin superfamily. We examined whether relaxin-3 could affect hypothalamic Kiss-1, gonadotropin-releasing hormone (GnRH), and pituitary gonadotropin subunit gene expression. METHODS: Mouse hypothalamic cell models, mHypoA-50 (originated from the hypothalamic anteroventral periventricular region), mHypoA-55 (originated from arcuate nucleus), and GT1-7, and the mouse pituitary gonadotroph LßT2 were used. Expression of Kiss-1, GnRH, and luteinizing hormone (LH)/follicle-stimulating hormone (FSH) ß-subunits was determined after stimulation with relaxin-3. RESULTS: RXFP3, a principle relaxin-3 receptor, was expressed in these cell models. In mHypoA-50 cells, relaxin-3 did not exert a significant effect on Kiss-1 expression. In contrast, the Kiss-1 gene in mHypoA-55 was significantly increased by 1 nmol/L relaxin-3. These cells also express GnRH mRNA, and its expression was significantly stimulated by relaxin-3. In GT1-7 cells, relaxin-3 significantly upregulated Kiss-1 expression; however, GnRH mRNA expression in GT1-7 cells was not altered. In primary cultures of fetal rat neuronal cells, 100 nmol/L relaxin-3 significantly increased GnRH expression. In pituitary gonadotroph LßT2, both LHß- and FSHß-subunit were significantly increased by 1 nmol/L relaxin-3. CONCLUSIONS: Our findings suggest that relaxin-3 exerts its effect by modulating the expression of Kiss-1, GnRH, and gonadotropin subunits, all of which are part of the hypothalamic-pituitary-gonadal axis.

Cholesterol modulates the binding properties of human relaxin family peptide receptor 3 with its ligands.

Relaxin family peptide receptor 3 (RXFP3) is implicated in the regulation of food intake and stress response upon activation by its cognate agonist relaxin-3. As an A-class G protein-coupled receptor, RXFP3 is an integral plasma membrane protein with seven transmembrane domains, yet influence of the membrane lipids on its function remains unknown. In the present study, we disclosed that cholesterol, an essential membrane lipid for mammalian cells, modulated the binding properties of human RXFP3 with its ligands. We first demonstrated that depletion of cholesterol from host human embryonic kidney (HEK) 293T cells by methyl-ß-cyclodextrin altered ligand-binding properties of the overexpressed human RXFP3, such as increasing its binding potency with some antagonists and decreasing its binding affinity with a NanoLuc-conjugated R3/I5 tracer. Thereafter, we demonstrated that two B-chain residues, B5Tyr and B12Arg, were primarily responsible for the increased binding potency of these antagonists with human RXFP3 under the cholesterol depletion condition. Our results suggest that cell membrane cholesterol interacts with human RXFP3 and modulates its ligand-binding properties, providing new insights into the influence of membrane lipids on RXFP3 function.

Serum relaxin-3 hormone relationship to male delayed puberty.

Puberty is the transitional period between childhood and adulthood, a process encompassing morphological, physiological and behavioural development to attain full reproductive capability. This study aimed to assess serum relaxin-3 hormone relationship with male delayed puberty. Sixty males were investigated as two equal groups: males with delayed puberty and healthy matched males as controls. They were subjected to history taking, clinical examination and estimation of serum FSH, LH, testosterone, relaxin-3 hormonal levels. The results showed that the secondary sexual characters in the patients group were at Tanner stages 1-2 and in the healthy controls at Tanner stages 3-5. The mean BMI in the patients group was significantly increased, whereas the mean levels of the span, testicular volume, serum LH, FSH, testosterone as well as relaxin-3 hormonal levels were significantly decreased compared with the healthy controls. Serum relaxin-3 levels showed significant positive correlation with the age, testis volume, span, Tanner stages, serum testosterone, FSH, LH hormones. In addition, serum relaxin-3 levels showed significant negative correlation with BMI. It is concluded that serum level of relaxin-3 hormone is an important mediator in the pathophysiological process of normal puberty being significantly decreased in males with delayed puberty.

Alterations of relaxin and its receptor system components in experimental diabetic cardiomyopathy rats.

High glucose induces apoptosis of cardiomyocytes and fibrosis of cardiac fibroblasts, contributing to diabetic cardiomyopathy. In this work, we explore the production of relaxin alterations and the significance of their receptor system components in the hearts of experimental diabetic cardiomyopathy rats. We measured rat relaxin-1 (equivalent to human relaxin-2), relaxin-3, RXFP1 and RXFP3 mRNA expression in the hearts of experimental diabetic cardiomyopathy rats. Neonatal rat ventricular myocytes (NRVMs) and cardiac fibroblasts were treated with 5.5 mmol/l normal glucose (NG) and 33 mmol/l high glucose (HG) for 0, 6, 12, 24, 48 and 72 h. Rat relaxin-1, relaxin-3, RXFP1 and RXFP3 mRNA expression were determined by real-time PCR. In the present study, we offer the first evidence that Relaxin-1 mRNA significantly increased and Relaxin-3 mRNA expression decreased at 4 and 8 weeks after STZ in the hearts of diabetic rats. In addition, significant down regulation of the mRNA expression of RXFP1 and RXFP3 was observed at 4 w after STZ; however, the mRNA expression levels of RXFP1 and RXFP3 were increased at 8 weeks after STZ. Apoptotic NRVMs induced by high glucose generate a decreased level of relaxin-1 and RXFP1. In HG-administered cardiac fibroblasts, Relaxin-1 mRNA was significantly increased and relaxin-3 mRNA was significantly decreased. Additionally, the mRNA expression of RXFP1 was decreased, and the mRNA expression of RXFP3 was increased. This results showed that an important role of relaxin-2, relaxin-3 and their receptors system in the regulation of diabetic cardiomyopathy.

A novel BRET-based binding assay for interaction studies of relaxin family peptide receptor 3 with its ligands.

Relaxin family peptide receptor 3 (RXFP3) is an A-class G protein-coupled receptor that is implicated in the regulation of food intake and stress response upon activation by its cognate agonist relaxin-3. To study its interaction with various ligands, we developed a novel bioluminescence resonance energy transfer (BRET)-based binding assay using the brightest NanoLuc as an energy donor and a newly developed cyan-excitable orange fluorescent protein (CyOFP) as an energy acceptor. An engineered CyOFP without intrinsic cysteine residues but with an introduced cysteine at the C-terminus was overexpressed in Escherichia coli and chemically conjugated to the A-chain N-terminus of an easily labeled chimeric R3/I5 peptide via an intermolecular disulfide linkage. After the CyOFP-conjugated R3/I5 bound to a shortened human RXFP3 (removal of 33 N-terminal residues) fused with the NanoLuc reporter at the N-terminus, high BRET signals were detected. Saturation binding and real-time binding assays demonstrated that this BRET pair retained high binding affinity with fast association/dissociation. Using this BRET pair, binding potencies of various ligands with RXFP3 were conveniently measured through competition binding assays. Thus, the novel BRET-based binding assay facilitates interaction studies of RXFP3 with various ligands. The engineered CyOFP without intrinsic cysteine residues may also be applied to other BRET-based binding assays in future studies.

Nucleus incertus corticotrophin-releasing factor 1 receptor signalling regulates alcohol seeking in rats.

Alcoholism is a chronic relapsing disorder, and stress is a key precipitant of relapse. The nucleus incertus (NI) is highly responsive to corticotrophin-releasing factor (CRF) and psychological stressors, receives a CRF innervation and expresses CRF1 and CRF2 receptor mRNA. Furthermore, the ascending NI relaxin-3 system is implicated in alcohol seeking in rats. Therefore, in alcohol-preferring rats, we examined the effect of bilateral injections into the NI of the CRF1 receptor antagonist, CP376395 or the CRF2 receptor antagonist, astressin-2B on yohimbine-induced reinstatement of alcohol seeking. Using quantitative PCR analysis of NI micropunches, we assessed the effects of chronic alcohol consumption on gene expression profiles for components of the relaxin-3 and CRF systems. Bilateral intra-NI injections of CP376395 (500 ng/0.25 µl) attenuated yohimbine-induced reinstatement of alcohol seeking. In contrast, intra-NI injections of astressin-2B (200 ng/0.25 µl) had no significant effect. In line with these data, CRF1 , but not CRF2 , receptor mRNA was upregulated in the NI following chronic ethanol intake. Relaxin family peptide 3 receptor mRNA was also increased in the NI following chronic ethanol. Our quantitative PCR analysis also identified CRF mRNA within the rat NI, and the existence of a newly identified population of CRF-containing neurons was subsequently confirmed by detection of CRF immunoreactivity in rat and mouse NI. These data suggest that NI neurons contribute to reinstatement of alcohol seeking, via an involvement of CRF1 signalling. Furthermore, chronic ethanol intake leads to neuroadaptive changes in CRF and relaxin-3 systems within rat NI.

Quantitative measurement of cell membrane receptor internalization by the nanoluciferase reporter: Using the G protein-coupled receptor RXFP3 as a model.

Nanoluciferase (NanoLuc) is a newly developed small luciferase reporter with the brightest bioluminescence to date. In the present work, we developed NanoLuc as a sensitive bioluminescent reporter to measure quantitatively the internalization of cell membrane receptors, based on the pH dependence of the reporter activity. The G protein-coupled receptor RXFP3, the cognate receptor of relaxin-3/INSL7, was used as a model receptor. We first generated stable HEK293T cells that inducibly coexpressed a C-terminally NanoLuc-tagged human RXFP3 and a C-terminally enhanced green fluorescent protein (EGFP)-tagged human RXFP3. The C-terminal EGFP-tag and NanoLuc-tag had no detrimental effects on the ligand-binding potency and intracellular trafficking of RXFP3. Based on the fluorescence of the tagged EGFP reporter, the ligand-induced RXFP3 internalization was visualized directly under a fluorescence microscope. Based on the bioluminescence of the tagged NanoLuc reporter, the ligand-induced RXFP3 internalization was measured quantitatively by a convenient bioluminescent assay. Coexpression of an EGFP-tagged inactive [E141R]RXFP3 had no detrimental effect on the ligand-binding potency and ligand-induced internalization of the NanoLuc-tagged wild-type RXFP3, suggesting that the mutant RXFP3 and wild-type RXFP3 worked independently. The present bioluminescent internalization assay could be extended to other G protein-coupled receptors and other cell membrane receptors to study ligand-receptor and receptor-receptor interactions.

A negatively charged transmembrane aspartate residue controls activation of the relaxin-3 receptor RXFP3.

Relaxin-3 is an insulin/relaxin superfamily neuropeptide involved in the regulation of food intake and stress response via activation of its cognate receptor RXFP3, an A-class G protein-coupled receptor (GPCR). In recent studies, a highly conserved ExxxD motif essential for binding of relaxin-3 has been identified at extracellular end of the second transmembrane domain (TMD2) of RXFP3. For most of the A-class GPCRs, a highly conserved negatively charged Asp residue (Asp(2.50) using Ballesteros-Weinstein numbering and Asp128 in human RXFP3) is present at the middle of TMD2. To elucidate function of the conserved transmembrane Asp128, in the present work we replaced it with other residues and the resultant RXFP3 mutants all retained quite high ligand-binding potency, but their activation and agonist-induced internalization were abolished or drastically decreased. Thus, the negatively charged transmembrane Asp128 controlled transduction of agonist-binding information from the extracellular region to the intracellular region through maintaining RXFP3 in a metastable state for efficient conformational change induced by binding of an agonist.

Identification of hydrophobic interactions between relaxin-3 and its receptor RXFP3: implication for a conformational change in the B-chain C-terminus during receptor binding.

Relaxin-3 is an insulin/relaxin superfamily neuropeptide implicated in the regulation of food intake and stress response via activation of the G protein-coupled receptor RXFP3. Their electrostatic interactions have been recently identified, and involves three positively charged B-chain residues (B12Arg, B16Arg, and B26Arg) of relaxin-3 and two negatively charged residues (Glu141 and Asp145) in a highly conserved ExxxD motif at the extracellular end of the second transmembrane domain of RXFP3. To investigate their hydrophobic interactions, in the present work we deleted the highly conserved B-chain C-terminal B27Trp residue of relaxin-3, and mutated four highly conserved aromatic residues (Phe137, Trp138, Phe146, and Trp148) around the ExxxD motif of RXFP3. The resultant [∆B27W]relaxin-3 exhibited approximately tenfold lower binding potency and ~1000-fold lower activation potency towards wild-type RXFP3, confirming its importance for relaxin-3 function. Although the RXFP3 mutants could be normally trafficked to cell membrane, they had quite different activities. [F137A]RXFP3 could normally distinguish wild-type relaxin-3 and [∆B27W]relaxin-3 in binding and activation assays, whereas [W138A]RXFP3 lost most of this capability, suggesting that the Trp138 residue of RXFP3 forms hydrophobic interactions with the B27Trp residue of relaxin-3. The hydrophobic Trp138 residue and the formerly identified negatively charged Glu141 and Asp145 residues in the highly conserved WxxExxxD motif may thus form a functional surface that is important for interaction with relaxin-3. We hypothesize that the relaxin-3 B-chain C-terminus changes from the original folding-back conformation to an extended conformation during binding with RXFP3, to allow its B27Trp and B26Arg residues to interact with the Trp138 and Glu141 residues of RXFP3, respectively.

Orthosteric, Allosteric and Biased Signalling at the Relaxin-3 Receptor RXFP3.

Relaxin-3 is a neuropeptide that has roles in stress, memory and appetite regulation. The peptide acts on its cognate receptor RXFP3 to induce coupling to inhibitory G proteins to inhibit adenylyl cyclase and activate MAP-kinases such as ERK1/2, p38MAPK and JNK. Other relaxin family peptides can activate the receptor to produce alternative patterns of signalling and there is an allosteric modulator 135PAM1 that displays probe-selectivity. There are now a variety of selective peptide agonists and antagonists that will assist in the determination of the physiological roles of the relaxin-RXFP3 system and its potential as a drug target.