Receptor RXFP3 Research Articles

Engineering of Novel Analogues That Are More Receptor-Selective and Potent than the Native Hormone, Insulin-like Peptide 5 (INSL5).

Insulin-like peptide 5 (INSL5) targets the G protein-coupled receptor, relaxin family peptide receptor 4 (RXFP4), predominantly coexpressed in the colorectum. While INSL5 also binds to the related receptor RXFP3, it does not activate it. The INSL5/RXFP4 axis is a promising target for treating gastrointestinal disorders such as constipation. Despite its therapeutic potential, the clinical application of INSL5 has been hindered by synthesis complexities, necessitating the need for more accessible yet potent mimetics. In this study, we engineered an INSL5 analogue A13:B7-24-GG, featuring a simplified two-chain, two-disulfide scaffold with 32 amino acids, as opposed to the 45 amino acids found in native INSL5 (two-chain, three-disulfide), improving the synthesis yield by 19.5-fold. Additionally, A13:B7-24-GG demonstrates ∼4-fold higher potency (EC50 = 1.17 nM vs 4.57 nM) and ∼11 times greater selectivity than native INSL5, with significantly reduced RXFP3 binding affinity, positioning it as a promising new therapeutic candidate for the treatment of constipation.

Mechanisms of biased agonism by Gαi/o-biased stapled peptide agonists of the relaxin-3 receptor.

The neuropeptide relaxin-3 is composed of an A chain and a B chain held together by disulfide bonds, and it modulates functions such as anxiety and food intake by binding to and activating its cognate receptor RXFP3, mainly through the B chain. Biased ligands of RXFP3 would help to determine the molecular mechanisms underlying the activation of G proteins and β-arrestins downstream of RXFP3 that lead to such diverse functions. We showed that the i, i+4 stapled relaxin-3 B chains, 14s18 and d(1-7)14s18, were Gαi/o-biased agonists of RXFP3. These peptides did not induce recruitment of β-arrestin1/2 to RXFP3 by GPCR kinases (GRKs), in contrast to relaxin-3, which enabled the GRK2/3-mediated recruitment of β-arrestin1/2 to RXFP3. Relaxin-3 and the previously reported peptide 4 (an i, i+4 stapled relaxin-3 B chain) did not exhibit biased signaling. The staple linker of peptide 4 and parts of both the A chain and B chain of relaxin-3 interacted with extracellular loop 3 (ECL3) of RXFP3, moving it away from the binding pocket, suggesting that unbiased ligands promote a more open conformation of RXFP3. These findings highlight roles for the A chain and the N-terminal residues of the B chain of relaxin-3 in inducing conformational changes in RXFP3, which will help in designing selective biased ligands with improved therapeutic efficacy.

The RXFP3 receptor is functionally associated with cellular responses to oxidative stress and DNA damage.

DNA damage response (DDR) processes, often caused by oxidative stress, are important in aging and -related disorders. We recently showed that G protein-coupled receptor (GPCR) kinase interacting protein 2 (GIT2) plays a key role in both DNA damage and oxidative stress. Multiple tissue analyses in GIT2KO mice demonstrated that GIT2 expression affects the GPCR relaxin family peptide 3 receptor (RXFP3), and is thus a therapeutically-targetable system. RXFP3 and GIT2 play similar roles in metabolic aging processes. Gaining a detailed understanding of the RXFP3-GIT2 functional relationship could aid the development of novel anti-aging therapies. We determined the connection between RXFP3 and GIT2 by investigating the role of RXFP3 in oxidative stress and DDR. Analyzing the effects of oxidizing (H2O2) and DNA-damaging (camptothecin) stressors on the interacting partners of RXFP3 using Affinity Purification-Mass Spectrometry, we found multiple proteins linked to DDR and cell cycle control. RXFP3 expression increased in response to DNA damage, overexpression, and Relaxin 3-mediated stimulation of RXFP3 reduced phosphorylation of DNA damage marker H2AX, and repair protein BRCA1, moderating DNA damage. Our data suggests an RXFP3-GIT2 system that could regulate cellular degradation after DNA damage, and could be a novel mechanism for mitigating the rate of age-related damage accumulation.

Intranasal administration of a stapled relaxin-3 mimetic has anxiolytic- and antidepressant-like activity in rats.

Background and PurposeDepression and anxiety are common causes of disability, and innovative tools and potential pharmacological targets are actively sought for prevention and treatment. Therapeutic strategies targeting the relaxin‐3 peptide or its primary endogenous receptor, RXFP3, for the treatment of major depression and anxiety disorders have been limited by a lack of compounds with drug‐like properties. We proposed that a hydrocarbon‐stapled mimetic of relaxin‐3, when administered intranasally, might be uniquely applicable to the treatment of these disorders.Experimental ApproachWe designed a series of hydrocarbon‐stapled relaxin‐3 mimetics and identified the most potent compound using in vitro receptor binding and activation assays. Further, we assessed the effect of intranasal delivery of relaxin‐3 and the lead stapled mimetic in rat models of anxiety and depression.Key ResultsWe developed an i,i+7 stapled relaxin‐3 mimetic that manifested a stabilized α‐helical structure, proteolytic resistance, and confirmed agonist activity in receptor binding and activation in vitro assays. The stapled peptide agonist enhanced food intake after intracerebral infusion in rats, confirming in vivo activity. We showed that intranasal delivery of the lead i,i+7 stapled peptide or relaxin‐3 had orexigenic effects in rats, indicating a potential clinically translatable route of delivery. Further, intranasal administration of the lead i,i+7 stapled peptide exerted anxiolytic and antidepressant‐like activity in anxiety‐ and depression‐related behaviour paradigms.Conclusions and ImplicationsOur preclinical findings demonstrate that targeting the relaxin‐3/RXFP3 receptor system via intranasal delivery of an i,i+7 stapled relaxin‐3 mimetic may represent an effective treatment approach for depression, anxiety, and related neuropsychiatric disorders.

The human RXFP3 receptor regulates the conserved DNA damage response process

The human RXFP3 receptor regulates the conserved DNA damage response process

Functionality of an absolutely conserved glycine residue in the chimeric relaxin family peptide R3/I5.

The insulin superfamily is a group of homologous proteins that are further divided into the insulin family and relaxin family according to their distinct receptors. All insulin superfamily members contain three absolutely conserved disulfide linkages and a nonchiral Gly residue immediately following the first B-chain cysteine. The functionality of this conserved Gly residue in the insulin family has been studied by replacing it with natural L-amino acids or the corresponding unnatural D-amino acids. However, such analysis has not been conducted on relaxin family members. In the present study, we conducted chiral mutagenesis on the conserved B11Gly of the chimeric relaxin family peptide R3/I5, which is an efficient agonist for receptor RXFP3 and RXFP4. Similar to the effects on insulin family foldability, L-Ala or L-Ser substitution completely abolished the in vitro refolding of a recombinant R3/I5 precursor; whereas, D-Ala or D-Ser substitution had no detrimental effect on refolding of a semi-synthetic R3/I5 precursor, suggesting that the conserved Gly residue controls the foldability of relaxin family members. In contrast to the effect on insulin family activity, D-Ala or D-Ser replacement had no detrimental effect on the binding and activation potencies of the mature R3/I5 towards both RXFP3 and RXFP4, suggesting that the conserved Gly residue is irrelevant to the relaxin family's activity. The present study revealed functionality of the conserved B-chain Gly residue for a relaxin family peptide for the first time, providing an overview of its contribution to foldability and activity of the insulin superfamily.

The RXFP3‐GIT2 signaling system represents a potential multidimensional therapeutic target in age‐related disorders

Pathological aspects of the hyper‐complex aging process potently control the generation of neurodegenerative diseases, such as Alzheimer's or Parkinson's disease. Pathological aging and neurodegeneration are share many functional commonalities, i.e. progressive loss of cell stress resilience, oxidative DNA damage and metabolic dysfunction. This loss of stress resistance, results in the accumulation of damage, leading to systemic dysfunction and eventual cell death.It is currently conceptualized that complex physiological process, such as aging, are likely controlled through hierarchical coordination of focused signaling systems by ‘hub’ or ‘keystone’ proteins. These master‐regulator proteins have the ability to coordinate responses to dynamic molecular events through coherent organization of protein‐protein interactions with multiple distinct partners, bridging multiple signaling pathways and coordinating multisystem processes, such as aging. G protein‐coupled receptor kinase interacting protein 2 (GIT2) has been identified as a keystone protein in neurometabolic aging: GIT2 functionality is strongly associated with oxidative stress, DNA damage, metabolic dysfunction, immunesenescence and lifespan alterations. As a G protein‐coupled receptor (GPCR) interacting protein, it is however possible to control this protein through a proxy modulator. It has long been established that GPCR activity influencing the expression levels of their interacting/signaling proteins – thus creating a self‐reinforcing signaling system. Transcriptomic analysis of the GIT2 knockout mouse tissues, showed that one GPCR was consistently downregulated in the absence of GIT2, namely Relaxin Family Peptide 3 receptor (RXFP3). The RXFP3 receptor demonstrates multiple functional synergies with GIT2, i.e. glucose metabolism regulation and responsiveness to oxidative stress and stress resilience. Using an unbiased approach to generate comprehensive in cellula activity signatures as well as investigation of functional RXFP3‐interactomes we found strong evidence for RXFP3 roles in DNA damage repair, oxidative stress management, cell cycle arrest control, and glucose metabolism functionalities. These data support the existence of a tight functional synergy between RXFP3 and GIT2 – potentially via direct signaling association. We confirmed this functional interaction using co‐immunoprecipitation, microscopy, and bioluminescence resonance energy transfer (BRET). As RXFP3 expression alteration should be able to affect GIT2 expression –thus reinforcing this signaling unit, we also also demonstrated expression co‐regulation of both partners. Lastly, we investigated RXFP3 expression in a mouse model for age‐associated neurodegeneration (Granulin KO mice) – as with GIT2 expression in aging we found RXFP3 increases in expression with age and neurodegeneration. We hypothesize that age‐related stresses reinforce a tight functional association between RXFP3 and GIT2, thus forging an important therapeutic target system that regulates cellular degradation and controls disease etiology.Support or Funding InformationFWO‐Odysseus and FWO Travel GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Un nuevo agente en los mecanismos de la adicción al alcohol y la ingesta: el núcleo incertus y el neuropéptido relaxina-3

Alcohol intake is facilitated by its relationship with eating behavior and both processes are highly influenced by situations of stress and anxiety. The dysregulation of these processes can reach pathological situations such as anorexia, bulimia or obesity. The neurobiological elements which underlie this control are not completely clarified. The nucleus incertus (NI) in the pontine tegmentum is a common element in the food intake and alcoholism. NI is characterized by using the neuropeptide relaxin-3 (RLN3) as transmitter and its receptor RXFP3. In the present review, we will analyze the participation of the NI-RLN3-RXFP3 system in these behaviors under conditions of anxiety or stress in animal models. The activation of NI has a positive effect on intake (orexigenic) and generates a wide response in the amygdala modulating anxiety states. The activity of RLN3-RXFP3 in the amygdala could affect alcohol addiction since the application of the RXFP3 antagonist in extended amygdala attenuates the relapse to alcohol induced by stress. The neuroanatomical data indicate that the NI-RLN3-RXFP3 system acts on the feeding behavior and alcohol intake by means of projections parallel to the canonical mesolimbic pathways. Thus, data in animal models indicate that the NI-RLN3-RXFP3 system should be taken into account as a target in the future treatment of disorders of eating and alcohol addictive behaviors.

Development of a Single-Chain Peptide Agonist of the Relaxin-3 Receptor Using Hydrocarbon Stapling.

Structure-activity studies of the insulin superfamily member, relaxin-3, have shown that its G protein-coupled receptor (RXFP3) binding site is contained within its central B-chain α-helix and this helical structure is essential for receptor activation. We sought to develop a single B-chain mimetic that retained agonist activity. This was achieved by use of solid phase peptide synthesis together with on-resin ruthenium-catalyzed ring closure metathesis of a pair of judiciously placed i,i+4 α-methyl, α-alkenyl amino acids. The resulting hydrocarbon stapled peptide was shown by solution NMR spectroscopy to mimic the native helical conformation of relaxin-3 and to possess potent RXFP3 receptor binding and activation. Alternative stapling procedures were unsuccessful, highlighting the critical need to carefully consider both the peptide sequence and stapling methodology for optimal outcomes. Our result is the first successful minimization of an insulin-like peptide to a single-chain α-helical peptide agonist which will facilitate study of the function of relaxin-3.

Sex‐specific effects of relaxin‐3 on food intake and body weight gain

This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc.

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 (Asp2.50 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.

Antagonism of RXFP-3 receptor in the lateral hypothalamus increases alcohol self-administration in rats

Event Abstract Back to Event Antagonism of RXFP-3 receptor in the lateral hypothalamus increases alcohol self-administration in rats Christina J. Perry1, 2*, Hanna E. Kastman1, 2, Sarah S. Ch'Ng1, 2, Leigh C. Walker1, 2, Craig M. Smith1, 2, Andrew L. Gundlach1, 2 and Andrew J. Lawrence1, 2 1 The Florey Institute of Neuroscience and Mental Health, Behavioural Neuroscience, Australia 2 The University of Melbourne, Florey Department of Neuroscience and Mental Health, Australia Alcohol is the most widely abused drug worldwide, and alcohol abuse is a significant contributor to the global burden of disease. The search for novel and effective therapeutic targets to treat alcoholism is an ongoing task, especially given the high rates of relapse observed following alcohol rehabilitation. RXFP-3, the cognate receptor for the neuropeptide relaxin-3, is a potential therapeutic target. Recently we showed that RXFP3 is necessary for alcohol consumption and seeking, since central antagonism of this receptor reduced alcohol self-administration and relapse-like behaviour in alcohol preferring rats. Relaxin-3 is predominantly expressed in the brainstem, in neurons that project rostrally to the septum, hippocampus and lateral hypothalamus (LH). Of these, the lateral hypothalamus in particular is frequently implicated in alcohol consumption and relapse to alcohol-seeking. We sought, therefore, to examine the effect of RXFP3 antagonism in the LH. Alcohol-preferring P rats were trained to lever press for 10% ethanol for a minimum of 30 days. The selective antagonist R3(B1-22)R was then infused bilaterally into the LH prior to a standard self-administration session. In contrast to the effect of a central (icv) or intra-BNST infusion, rats showed increased alcohol consumption following administration of R3(B1-22)R compared to vehicle. To better understand this unexpected result we subsequently sought to characterise RXFP-3 positive neurons in the hypothalamus using a transgenic reporter mouse expressing GFP protein on RXFP-3 neurons. RXFP-3 receptors were expressed on orexin negative neurons that project to the ventral tegmental area (VTA). These findings have implications for understanding how relaxin 3 acts centrally to regulate alcohol-seeking and alcohol consumption, and on-going studies will elucidate the mechanism behind this finding, which somewhat parallels observations with the opioid receptor antagonist naltrexone. Keywords: Hypothalamus, Neuron, RXFP3, Alcohol consumption, alcohol seeking behaviour Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: YIC01: Young Investigator Colloquium 1 Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Perry CJ, Kastman HE, Ch'Ng SS, Walker LC, Smith CM, Gundlach AL and Lawrence AJ (2016). Antagonism of RXFP-3 receptor in the lateral hypothalamus increases alcohol self-administration in rats. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00053 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Aug 2016; Published Online: 11 Aug 2016. * Correspondence: Dr. Christina J Perry, The Florey Institute of Neuroscience and Mental Health, Behavioural Neuroscience, Melbourne, Victoria, Australia, christina.perry@mq.edu.au Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Christina J Perry Hanna E Kastman Sarah S Ch'Ng Leigh C Walker Craig M Smith Andrew L Gundlach Andrew J Lawrence Google Christina J Perry Hanna E Kastman Sarah S Ch'Ng Leigh C Walker Craig M Smith Andrew L Gundlach Andrew J Lawrence Google Scholar Christina J Perry Hanna E Kastman Sarah S Ch'Ng Leigh C Walker Craig M Smith Andrew L Gundlach Andrew J Lawrence PubMed Christina J Perry Hanna E Kastman Sarah S Ch'Ng Leigh C Walker Craig M Smith Andrew L Gundlach Andrew J Lawrence Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Role of relaxin-3/RXFP3 system in stress-induced binge-like eating in female rats

Binge eating is frequently stimulated by stress. The neuropeptide relaxin-3 (RLN3) and its native receptor RXFP3 are implicated in stress and appetitive behaviors. We investigated the dynamics of the central RLN3/RXFP3 system in a newly established model of stress-induced binge eating. Female Sprague–Dawley rats were subjected to unpredictable intermittent 1-h access to 10% sucrose. When sucrose intake stabilized, rats were assessed for consistency of higher or lower sucrose intake in response to three unpredictable episodes of foot-shock stress; and assigned as binge-like eating prone (BEP) or binge-like eating resistant (BER). BEP rats displayed elevated consumption of sucrose under non-stressful conditions (30% > BER) and an additional marked increase in sucrose intake (60% > BER) in response to stress. Conversely, sucrose intake in BER rats was unaltered by stress. Chow intake was similar in both phenotypes on ‘non-stress’ days, but was significantly reduced by stress in BER, but not BEP, rats. After stress, BEP, but not BER, rats displayed a significant increase in RLN3 mRNA levels in the nucleus incertus. In addition, in response to stress, BEP, but not BER, rats had increased RXFP3 mRNA levels in the paraventricular and supraoptic nuclei of the hypothalamus. Intracerebroventricular administration of a selective RXFP3 antagonist, R3(B1-22)R, blocked the stress-induced increase in sucrose intake in BEP rats and had no effect on sucrose intake in BER rats. These results provide important evidence for a role of the central RLN3/RXFP3 system in the regulation of stress-induced binge eating in rats, and have therapeutic implications for eating disorders.

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.

Synthesis and pharmacological characterization of a europium-labelled single-chain antagonist for binding studies of the relaxin-3 receptor RXFP3.

Relaxin-3 and its endogenous receptor RXFP3 are involved in fundamental neurological signalling pathways, such as learning and memory, stress, feeding and addictive behaviour. Consequently, this signalling system has emerged as an attractive drug target. Development of leads targeting RXFP3 relies on assays for screening and ligand optimization. Here, we present the synthesis and in vitro characterization of a fluorescent europium-labelled antagonist of RXFP3. This ligand represents a cheap and safe but powerful tool for future mechanistic and cell-based receptor-ligand interaction studies of the RXFP3 receptor.

International Union of Basic and Clinical Pharmacology. XCV. Recent advances in the understanding of the pharmacology and biological roles of relaxin family peptide receptors 1-4, the receptors for relaxin family peptides.

Relaxin, insulin-like peptide 3 (INSL3), relaxin-3, and INSL5 are the cognate ligands for the relaxin family peptide (RXFP) receptors 1-4, respectively. RXFP1 activates pleiotropic signaling pathways including the signalosome protein complex that facilitates high-sensitivity signaling; coupling to Gα(s), Gα(i), and Gα(o) proteins; interaction with glucocorticoid receptors; and the formation of hetero-oligomers with distinctive pharmacological properties. In addition to relaxin-related ligands, RXFP1 is activated by Clq-tumor necrosis factor-related protein 8 and by small-molecular-weight agonists, such as ML290 [2-isopropoxy-N-(2-(3-(trifluoromethylsulfonyl)phenylcarbamoyl)phenyl)benzamide], that act allosterically. RXFP2 activates only the Gα(s)- and Gα(o)-coupled pathways. Relaxin-3 is primarily a neuropeptide, and its cognate receptor RXFP3 is a target for the treatment of depression, anxiety, and autism. A variety of peptide agonists, antagonists, biased agonists, and an allosteric modulator target RXFP3. Both RXFP3 and the related RXFP4 couple to Gα(i)/Gα(o) proteins. INSL5 has the properties of an incretin; it is secreted from the gut and is orexigenic. The expression of RXFP4 in gut, adipose tissue, and β-islets together with compromised glucose tolerance in INSL5 or RXFP4 knockout mice suggests a metabolic role. This review focuses on the many advances in our understanding of RXFP receptors in the last 5 years, their signal transduction mechanisms, the development of novel compounds that target RXFP1-4, the challenges facing the field, and current prospects for new therapeutics.

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.

The role of relaxin-3 and its receptor RXFP3 in defense of elevated body weight in diet-induced obesity

Incidence of overweight and obesity has dramatically increased during the past three decades. Treatment of this serious clinical problem is hindered by the fact that once obesity has developed, the elevated body weight is defended against weight-decreasing treatment strategies by mechanisms that are not yet fully understood. This review focuses on the neuronal mechanisms that contribute to the maintenance of obesity after it develops in the DIO rat model. Among the neuronal factors regulating energy intake, orexigenic neuropeptide relaxin-3 and its cognate receptor RXFP3 may play an important role in the defense of elevated body weight in DIO. The levels of expression of relaxin-3 mRNA in the brainstem nucleus incertus (NI) were significantly increased in the ad libitum feeding state in DIO rats compared to DR rats. However, the effects of relaxin-3 in the DIO ad libitum -fed rats may be compensated by a significant decrease in the levels of expression of RXFP3 mRNA in the food intake-regulating brain regions of DIO rats including the paraventricular hypothalamic nucleus (PVN), central amygdala (CeA), NI, and nucleus of the solitary tract (NTS). Remarkably, the DIO rats showed an immediate rebound in food intake at refeeding and regained all body weight lost during starvation. This significant increase in food intake during refeeding was accompanied by an increase in the levels of expression of RXFP3 in the parvocellular PVN, CeA, NI, and NTS in the DIO rats to the levels of the DR rats. Moreover, the expression of RXFP3 in the paraventricular thalamic nucleus was significantly higher in the refed DIO rats compared to the DR counterparts. A constitutive increase in the expression of relaxin-3 accompanied by a relative increase in the expression of RXFP3 in food intake-regulating brain regions during refeeding after food deprivation may contribute to the mechanisms of defense of elevated body weight in the DIO phenotype.

Relaxin-3 receptor (Rxfp3) gene knockout mice display reduced running wheel activity: Implications for role of relaxin-3/RXFP3 signalling in sustained arousal

Anatomical and pharmacological evidence suggests the neuropeptide, relaxin-3, is the preferred endogenous ligand for the relaxin family peptide-3 receptor (RXFP3) and suggests a number of putative stress- and arousal-related roles for RXFP3 signalling. However, in vitro and in vivo evidence demonstrates exogenous relaxin-3 can activate other relaxin peptide family receptors, and the role of relaxin-3/RXFP3 signalling in specific brain circuits and associated behaviours in mice is not well described. In this study, we characterised the behaviour of cohorts of male and female Rxfp3 gene knockout (KO) mice (C57/B6JRXFP3TM1/DGen), relative to wild-type (WT) littermates to determine if this receptor KO strain has a similar phenotype to its ligand KO equivalent. Rxfp3 KO mice displayed similar performance to WT littermates in several acute behavioural paradigms designed to gauge motor coordination (rotarod test), spatial memory (Y-maze), depressive-like behaviour (repeat forced-swim test) and sensorimotor gating (prepulse inhibition of acoustic startle). Notably however, male and female Rxfp3 KO mice displayed robust and consistent (dark phase) hypoactivity on voluntary home-cage running wheels (∼20-60% less activity/h), and a small but significant decrease in anxiety-like behavioural traits in the elevated plus maze and light/dark box paradigms. Importantly, this phenotype is near identical to that observed in two independent lines of relaxin-3 KO mice, suggesting these phenotypes are due to the elimination of ligand or receptor and RXFP3-linked signalling. Furthermore, this behavioural characterisation of Rxfp3 KO mice identifies them as a useful experimental model for studying RXFP3-linked signalling and assessing the selectivity and/or potential off-target actions of RXFP3 agonists and antagonists, which could lead to an improved understanding of dysfunctional arousal in mental health disorders, including depression, anxiety, insomnia and neurodegenerative diseases.