Relaxin Family Peptide Receptor 3 Research Articles

The TRY‐motif in the Relaxin Family Peptide Receptor 3 (RXFP3) functions as a Molecular Switch Between DNA Damage Response and Cell Cycle Control

The majority of Rhodopsin-like Class A G protein-coupled receptors (GPCRs) contain the conserved Aspartate–Arginine–Tyrosine (DRY) motif in transmembrane helix 3 (TM3). This DRY-motif is responsible for the presence of an ionic lock, consisting of a salt bridge between the positively charged R3.50 in TM3 and the negatively charged glutamic acid (E6.30) on TM6 in typical rhodopsin-like GPCRs. While the R3.50 is 96% conserved across all class A GPCRs, D3.49 and Y3.51 are 66, and 67% conserved respectively. This variance in D3.49 and Y3.51 residues potential identifies a locus for evolutionary modification of GPCR signaling. The relaxin family peptide receptor 3 (RXFP3), a Class A GPCR, which plays a crucial role in the aging process via providing resilience to stress, possesses a natural variant of the DRY motif, where Aspartate is replaced by Threonine, creating a TRY motif. This variation enhances the ligand-independent activity of the RXFP3 receptor. We investigated how this natural variation may be implicated in its stress-resistance role through selective mutation of the TRY motif back to a canonical DRY sequence. Using an unbiased quantitative proteomic perturbation response approach, we found that protein network analysis revealed that DNA damage repair functions are supported by the presence of a TRY motif while reversion to a DRY sequence in the RXFP3 diverts signaling activity instead to cell-cycle/cellular senescence control. Thus it is likely that the RXFP3 has been naturally mutated to a TRY sequence to promote its anti-aging stress sensor role.

Hydrophobic interactions of relaxin family peptide receptor 3 with ligands identified using a NanoBiT-based binding assay

Relaxin family peptide receptor 3 (RXFP3) is a G protein-coupled receptor implicated in the regulation of food intake and stress response upon activation by the neuropeptide relaxin-3. In recent studies, interactions of RXFP3 with some natural or synthetic ligands have been investigated. In the present study, we identified the hydrophobic interactions of human RXFP3 with the chimeric agonist R3/I5 and the chimeric antagonist R3(ΔB23-27)R/I5 using a newly developed NanoBiT-based homogenous binding assay. We first demonstrated that the conserved large aliphatic B15Ile and B19Ile were important for the binding of the agonist and antagonist to RXFP3, because alanine replacement significantly decreased their receptor-binding potency. Thereafter, we demonstrated that the conserved large aliphatic Leu246 and Leu248 in extracellular loop 2 were important for RXFP3 binding to the agonist and antagonist, because alanine replacement significantly decreased the binding affinity of RXFP3 for both ligands. Finally, we deduced probable hydrophobic interactions based on the ability of RXFP3 mutants to distinguish the wild-type and mutant ligands: Leu246 of RXFP3 interacted with B15Ile of both ligands, while Leu248 of RXFP3 interacted with both B15Ile and B19Ile of the agonist and antagonist. The present results not only provided new insights into the interaction mechanism of RXFP3 with agonists and antagonists, but also demonstrated usefulness of the NanoBiT-based homogenous binding assay to study the interaction mechanism of certain receptors with their ligands.

Characterization of the relaxin family peptide receptor 3 system in the mouse bed nucleus of the stria terminalis

The bed nucleus of the stria terminalis (BNST) is a critical node involved in stress and reward-related behaviors. Relaxin family peptide receptor 3 (RXFP3) signaling in the BNST has been implicated in stress-induced alcohol seeking behavior. However, the neurochemical phenotype and connectivity of BNST RXFP3-expressing (RXFP3+) cells have yet to be elucidated. We interrogated the molecular signature and electrophysiological properties of BNST RXFP3+ neurons using a RXFP3-Cre reporter mouse line. BNST RXFP3+ cells are circumscribed to the dorsal BNST (dBNST) and are neurochemically heterogeneous, comprising a mix of inhibitory and excitatory neurons. Immunohistochemistry revealed that ~48% of BNST RXFP3+ neurons are GABAergic, and a quarter of these co-express the calcium-binding protein, calbindin. A subset of BNST RXFP3+ cells (~41%) co-express CaMKIIα, suggesting this subpopulation of BNST RXFP3+ neurons are excitatory. Corroborating this, RNAscope® revealed that ~35% of BNST RXFP3+ cells express vVGluT2 mRNA, indicating a subpopulation of RXFP3+ neurons are glutamatergic. RXFP3+ neurons show direct hyperpolarization to bath application of a selective RXFP3 agonist, RXFP3-A2, while around 50% of cells were depolarised by exogenous corticotrophin releasing factor. In behaviorally naive mice the majority of RXFP3+ neurons were Type II cells exhibiting Ih and T type calcium mediated currents. However, chronic swim stress caused persistent plasticity, decreasing the proportion of neurons that express these channels. These studies are the first to characterize the BNST RXFP3 system in mouse and lay the foundation for future functional studies appraising the role of the murine BNST RXFP3 system in more complex behaviors.

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.

Overexpression of relaxin family peptide receptor 3 in Escherichia coli and characterization of its ligand binding properties

Abstract Relaxin family peptide receptor 3 (RXFP3) is an A-class G protein-coupled receptor. It is implicated in the regulation of food intake and stress response upon activation by the neuropeptide relaxin-3. So far, preparation of enough functional RXFP3 for structural analysis is still challenging due to its integral membrane protein nature. In the present study, we overexpressed an N-terminally secretory maltose-binding protein (sMBP)-fused shortened human RXFP3 in prokaryotic host Escherichia coli. A small fraction of the sMBP-RXFP3 fusion protein could be integrated into the E. coli cell membrane and this fraction retained ligand binding function although the measured binding parameters were different from those of sMBP-RXFP3 overexpressed in mammalian cells due to different cell membrane lipids of the different host cells. Our present work paved the way for production of functional RXFP3 using the low-cost E. coli expression system.

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-β-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.

GABAergic Neurons in the Rat Medial Septal Complex Express Relaxin-3 Receptor (RXFP3) mRNA.

The medial septum (MS) complex modulates hippocampal function and related behaviors. Septohippocampal projections promote and control different forms of hippocampal synchronization. Specifically, GABAergic and cholinergic projections targeting the hippocampal formation from the MS provide bursting discharges to promote theta rhythm, or tonic activity to promote gamma oscillations. In turn, the MS is targeted by ascending projections from the hypothalamus and brainstem. One of these projections arises from the nucleus incertus in the pontine tegmentum, which contains GABA neurons that co-express the neuropeptide relaxin-3 (Rln3). Both stimulation of the nucleus incertus and septal infusion of Rln3 receptor agonist peptides promotes hippocampal theta rhythm. The Gi/o-protein-coupled receptor, relaxin-family peptide receptor 3 (RXFP3), is the cognate receptor for Rln3 and identification of the transmitter phenotype of neurons expressing RXFP3 in the septohippocampal system can provide further insights into the role of Rln3 transmission in the promotion of septohippocampal theta rhythm. Therefore, we used RNAscope multiplex in situ hybridization to characterize the septal neurons expressing Rxfp3 mRNA in the rat. Our results demonstrate that Rxfp3 mRNA is abundantly expressed in vesicular GABA transporter (vGAT) mRNA- and parvalbumin (PV) mRNA-positive GABA neurons in MS, whereas ChAT mRNA-positive acetylcholine neurons lack Rxfp3 mRNA. Approximately 75% of Rxfp3 mRNA-positive neurons expressed vGAT mRNA (and 22% were PV mRNA-positive), while the remaining 25% expressed Rxfp3 mRNA only, consistent with a potential glutamatergic phenotype. Similar proportions were observed in the posterior septum. The occurrence of RXFP3 in PV-positive GABAergic neurons gives support to a role for the Rln3-RXFP3 system in septohippocampal theta rhythm.

Expression and localisation of RXFP3 in human spermatozoa and impact of INSL7 on sperm functions.

Insulin-like peptide 7 (INSL7) or relaxin-3 is a member of the insulin superfamily that is recently discovered. This hormone interacts with relaxin family peptide receptor 3 (RXFP3). Although recent studies of INSL7 have focused on its function in the brain as a neuropeptide, spermatozoa may be a candidate target of INSL7 due to its detection in testes and contains binding sites. Therefore, this study aims to analyse the expression and localisation of RXFP3 on human spermatozoa and to assess the effect of INSL7 on human sperm motility. We have incubated normal semen samples in different doses of INSL7. Sperm motility was analysed by Computer Assisted Sperm Analysis. Moreover, localisation and expression of RXFP3 were assessed in human spermatozoa by immunofluorescence and RT-PCR respectively. This study indicated that RXFP3 mainly localised in the post-acrosomal region of sperm head and neck. However, we did not observe expression of RXFP3 mRNA in human spermatozoa. This study showed that INSL7 alleviated the natural decline in sperm motility after a 4-hr incubation period. This was particularly observed in the 1.8pmol/L treated samples. These data suggested that most likely expression of RXFP3 arrested in spermiogenesis, but the RXFP3 peptide existed on the surface of mature spermatozoa.

Differential effects of relaxin-3 and a selective relaxin-3 receptor agonist on food and water intake and hypothalamic neuronal activity in rats

The neuropeptide relaxin-3 (RLN3) binds with high affinity to its cognate receptor, relaxin-family peptide receptor 3 (RXFP3), and with lower affinity to RXFP1, the cognate receptor for relaxin. Intracerebroventricular (icv) administration of RLN3 in rats strongly increases food and water intake and alters the activity of the hypothalamic-pituitary-adrenal (HPA) and gonadal (HPG) axes, but the relative involvement of RXFP3 and RXFP1 in these effects is not known. Therefore, the effects of icv administration of equimolar (1.1 nmol) amounts of RLN3 and the RXFP3-selective agonist RXFP3-A2 on food and water intake, plasma levels of corticosterone, testosterone, and oxytocin and c-fos mRNA expression in key hypothalamic regions in male rats were compared. Food intake was increased by both RLN3 and RXFP3-A2, but the orexigenic effects of RXFP3-A2 were significantly stronger than RLN3, 30 and 60min after injection. Water intake and plasma corticosterone and testosterone levels were significantly increased by RLN3, but not by RXFP3-A2. Conversely, RXFP3-A2 but not RLN3 decreased oxytocin plasma levels. RLN3, but not RXFP3-A2, increased c-fos mRNA levels in the parvocellular (PVNp) and magnocellular (PVNm) paraventricular and supraoptic (SON) hypothalamic nuclei, in the ventral medial preoptic area (MPAv), and in the organum vasculosum of the lamina terminalis (OVLT). A significant increase in c-fos mRNA expression was induced in the perifornical lateral hypothalamic area (LHApf) by RLN3 and RXFP3-A2. These results suggest that RXFP1 is involved in the RLN3 stimulation of water intake and activation of the HPA and HPG axes. The reduced food intake stimulation by RLN3 compared to RXFP3-A2 may relate to activation of both orexigenic and anorexigenic circuits by RLN3.

Central amygdala relaxin-3/relaxin family peptide receptor 3 signalling modulates alcohol seeking in rats.

Alcohol use disorders are a leading cause of preventable deaths worldwide, and stress is a major trigger of relapse. The neuropeptide relaxin-3 and its cognate receptor, relaxin family peptide receptor 3 (RXFP3), modulate stress-induced relapse to alcohol seeking in rats, and while the bed nucleus of the stria terminalis has been implicated in this regard, the central nucleus of the amygdala (CeA) also receives a relaxin-3 innervation and CeA neurons densely express RXFP3 mRNA. Moreover, the CeA is consistently implicated in both stress and addictive disorders. Yohimbine precipitates relapse-like behaviour in rodents, although exactly how yohimbine induces relapse is unknown, possibly by increasing stress levels and inducing heightened cue reactivity. In the current study, we examined the effects of yohimbine (1mg·kg-1 , i.p.) on anxiety-like behaviour in alcohol-experienced rats. Furthermore, we assessed CeA neuronal activation following yohimbine-induced reinstatement of alcohol seeking and the role of the relaxin-3/RXFP3 signalling within the CeA in yohimbine-induced reinstatement to alcohol seeking. Low-dose yohimbine was anxiogenic in rats with a history of alcohol use. Furthermore, yohimbine-induced reinstatement of alcohol seeking increased Fos activation in CeA corticotrophin-releasing factor, dynorphin and GABA neurons compared with naïve and vehicle controls. Bilateral intra-CeA injections of the selective RXFP3 antagonist, R3(B1-22)R, attenuated yohimbine-induced reinstatement of alcohol seeking. Collectively, these data suggest that the CeA is a node where yohimbine acts to induce reinstatement of alcohol seeking and implicate the relaxin-3/RXFP3 system within the CeA in this process.

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 33N-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.

Distribution, physiology and pharmacology of relaxin-3/RXFP3 systems in brain.

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.

Partial agonist activity of R3(BΔ23–27)R/I5 at RXFP3 – Investigation of in vivo and in vitro pharmacology

Relaxin family peptide receptor 3 (RXFP3) is a G-protein coupled receptor mainly expressed in the brain and involved in appetite regulation. Previous studies in lean Wistar rats during the light phase have shown that the chimeric peptide R3(BΔ23–27)R/I5 suppresses food intake stimulated by an RXFP3 agonist, but has no effect on food intake when administered alone. We wanted to further investigate if R3(BΔ23–27)R/I5 on its own is able to antagonize the basal tone of the relaxin-3/RXFP3 system and therefore characterized the pharmacology of R3(BΔ23–27)R/I5 in vivo and in vitro. R3(BΔ23–27)R/I5 was intracerebroventricularly (ICV) injected in diet induced obese (DIO) Wistar rats and food intake was automatically measured during the dark phase when feeding drive is high. In our hands, R3(BΔ23–27)R/I5 alone did not have a significant effect on food intake during 24h following administration. Consistent with previous results, relaxin-3 stimulated food intake in satiated lean rats. R3(BΔ23–27)R/I5 was characterized in vitro using [35S]-GTPγS binding and cAMP assays, both assessing Gαi-protein mediated signalling, and dynamic mass redistribution (DMR) assays capturing the integrated cell response. R3(BΔ23–27)R/I5 showed partial agonist activity in all three functional assays. Thus, since R3(BΔ23–27)R/I5 displays partial RXFP3 agonist properties in vitro, further in vivo studies including additional tool compounds are needed to address if antagonizing relaxin-3/RXFP3 basal tone is a therapeutically relevant mechanism to regulate food intake and body weight.

Signalling profiles of H3 relaxin, H2 relaxin and R3(BΔ23–27)R/I5 acting at the relaxin family peptide receptor 3 (RXFP3)

Relaxin family peptide receptor 3 (RXFP3) is expressed in brain areas important for processing sensory information and feeding, suggesting that it may be a target for anti-anxiety and anti-obesity drugs. We examined the effects of H3 relaxin, the biased agonist H2 relaxin and the antagonist, R3(BΔ23-27)R/I5, on RXFP3 signalling to establish their suitability as tools to assess the physiological roles of RXFP3. The signalling profile of the RXFP3 ligands was determined using reporter gene assays, multiplexed signalling assays and direct examination of receptor-G protein and receptor-β-arrestin interactions using BRET. H2 relaxin activated p38MAPK and ERK1/2 with lower efficacy than H3 relaxin, but had similar efficacy for JNK1/2 phosphorylation. H2 or H3 relaxin activation of p38MAPK, JNK1/2 or ERK1/2 involved Pertussis toxin-sensitive G-proteins. R3(BΔ23-27)R/I5 blocked H3 relaxin AP-1 reporter gene activation, but not H2 relaxin AP-1 activation or H3 relaxin NF-κB activation. R3(BΔ23-27)R/I5 activated the SRE reporter, but did not inhibit either H2 or H3 relaxin SRE activation. R3(BΔ23-27)R/I5 blocked H3 relaxin-stimulated p38MAPK and ERK1/2 phosphorylation, but was a weak partial agonist for p38MAPK and ERK1/2 signalling. p38MAPK activation by R3(BΔ23-27)R/I5 was G protein-independent. H3 relaxin-activated RXFP3 interacts with Gαi2 , Gαi3 , Gαo A and Gαo B whereas H2 relaxin or R3(BΔ23-27)R/I5 induce interactions only with Gαi2 or Gαo B . Only H3 relaxin promoted RXFP3/β-arrestin interactions that were blocked by R3(BΔ23-27)R/I5. Understanding signalling profile of drugs acting at RXFP3 is essential for development of therapies targeting this receptor.

Relaxin‐3 innervation of the intergeniculate leaflet of the rat thalamus – neuronal tract‐tracing and in vitro electrophysiological studies

Behavioural state is controlled by a range of neural systems that are sensitive to internal and external stimuli. The relaxin-3 and relaxin family peptide receptor 3 (RXFP3) system has emerged as a putative ascending arousal network with putative involvement in regulation of stress responses, neuroendocrine control, feeding and metabolism, circadian activity and cognition. Relaxin-3/γ-aminobutyric acid neuron populations have been identified in the nucleus incertus, pontine raphe nucleus, periaqueductal grey (PAG) and an area dorsal to the substantia nigra. Relaxin-3-positive fibres/terminals densely innervate arousal-related structures in the brainstem, hypothalamus and limbic forebrain, but the functional significance of the heterogeneous relaxin-3 neuron distribution and its inputs to specific brain areas are unclear. Therefore, in this study, we used neuronal tract-tracing and immunofluorescence staining to explore the source of the dense relaxin-3 innervation of the intergeniculate leaflet (IGL) of the thalamus, a component of the neural circadian timing system. Confocal microscopy analysis revealed that relaxin-3-positive neurons retrogradely labelled from the IGL were predominantly present in the PAG and these neurons expressed corticotropin-releasing factor receptor-like immunoreactivity. Subsequently, whole-cell patch-clamp recordings revealed heterogeneous effects of RXFP3 activation in the IGL by the RXFP3 agonist, relaxin-3 B-chain/insulin-like peptide-5 A-chain (R3/I5). Identified, neuropeptide Y-positive IGL neurons, known to influence suprachiasmatic nucleus activity, were excited by R3/I5, whereas neurons of unidentified neurotransmitter content were either depolarized or displayed a decrease in action potential firing and/or membrane potential hyperpolarization. Our data identify a PAG to IGL relaxin-3/RXFP3 pathway that might convey stress-related information to key elements of the circadian system and influence behavioural state rhythmicity.

Cell-free protein synthesis as a tool to study RXFP3-relaxin-3 protein interactions.

With the discovery of the relaxin family peptide receptors there is interest in obtaining a clearer understanding of the structure of these proteins and the molecular mechanism of receptor-ligand interaction. As G-protein coupled receptors, obtaining milligram quantities for structural investigations is hampered by the inherent instability of these integral membrane proteins. In the current context, understanding of GPCR structural biology has increased dramatically with crystal structures of several inactive and now active forms solved. In addition, the first nuclear magnetic resonance structure of a GPCR was obtained which is of crucial importance to studying these receptors in a more "biologically relevant" setting. However despite this expansion in the field, most structures have been solved on modified systems so as to increase stability and are not necessarily representative of the native receptors. In relation to the relaxin family peptide receptors, we chose to investigate relaxin-family peptide receptor-3 expressed by cell-free protein synthesis. In contrast to in-vivo expression, cell-free was capable of producing large amounts of native receptor which makes it amenable to demanding structural studies.

H2 Relaxin Is a Biased Ligand Relative to H3 Relaxin at the Relaxin Family Peptide Receptor 3 (RXFP3)

Relaxin family peptide 3 receptors (RXFP3) are activated by H3-relaxin to inhibit forskolin-stimulated cAMP accumulation and stimulate extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. In this study, we sought to identify novel signaling pathways coupled to RXFP3 and to investigate whether other members of the relaxin peptide family activated these pathways. Two patterns of signaling were observed in RXFP3-expressing Chinese hamster ovary (CHO)-K1 and human embryonic kidney (HEK)-293 cells (CHO-RXFP3 and HEK-RXFP3) and murine septal neuron SN56 cell lines: 1) strong inhibition of forskolin-stimulated cAMP accumulation, ERK1/2 activation and nuclear factor (NF)-kappaB reporter gene activation in cells stimulated with H3 relaxin, with weaker activity observed for H2 relaxin, porcine relaxin, or insulin-like peptide (INSL) 3 and 2) strong stimulation of activator protein (AP)-1 reporter genes by H2 relaxin, with weaker activation observed with H3 or porcine relaxin. Two distinct ligand binding sites were identified on RXFP3-expressing cells using two different radioligands. (125)I-INSL5 A-chain/relaxin-3 B-chain chimera bound with high affinity to the RXFP3-expressing cells with competition by H3 relaxin or a H3 relaxin B-chain dimeric peptide, consistent with previous reports. Binding studies with (125)I-H2 relaxin revealed a distinct binding site with potent competition observed with H2 relaxin, H3 relaxin, or INSL3 and weaker competition with porcine relaxin. Thus H3 relaxin potently activates all signaling pathways coupled to RXFP3, whereas H2 relaxin is an AP-1-biased ligand relative to H3 relaxin.

Addition of a Carboxy-Terminal Green Fluorescent Protein Does Not Alter the Binding and Signaling Properties of Relaxin Family Peptide Receptor 3

The relaxin family peptide receptor 3 (RXFP3) is the cognate receptor for the neuropeptide relaxin-3. RXFP3 was tagged at the carboxy-terminus with a variant of the green fluorescent protein (GFP(2)) for use in receptor localization studies. RXFP3-GFP(2) was examined to ensure it retained binding and signaling properties similar to untagged RXFP3. Competition for [(125)I]INSL5/H3 relaxin chimera binding to RXFP3 and RXFP3-GFP(2) indicated that the carboxy-terminal tag did not affect receptor binding or receptor internalization. RXFP3-GFP(2) activated ERK1/2 with a similar potency to RXFP3 when transiently expressed in CHO-K1 or HEK293T cells, suggesting that the GFP(2) tag did not affect receptor function. This study demonstrated that addition of a carboxy-terminal fusion protein to RXFP3 did not alter the binding or signaling properties of RXFP3, making RXFP3-GFP(2) a useful tool for future receptor localization and trafficking studies.

AP‐1 is activated by relaxin family peptides in cells expressing the relaxin family peptide receptor 3 (RXFP3)

Relaxin‐3 is a neuropeptide involved in appetite regulation and stress behaviour in rats. RXFP3 is the cognate receptor for relaxin‐3 that is coupled to cAMP inhibition and ERK1/2 activation in CHO‐K1 and HEK293 cell lines recombinantly expressing the receptor. Competition for [125I]‐INSL5/H3 relaxin chimera binding is observed with H3 relaxin and the B‐chain of H3 relaxin, but not with any other relaxin family peptides. We identified that H3 relaxin but also H2 relaxin and porcine relaxin activate transcription from AP‐1 elements in CHO and HEK cells transiently expressing human RXFP3 receptors, measured using a reporter gene. H2 relaxin was more potent at activating transcription than either H3 relaxin or porcine relaxin. Experiments with inhibitors of p38 MAPK (RWJ67657, 10 μM), MEK1/2 (PD98059, 20μM) and JNK (SP600125, 10μ M) showed that AP‐1 activation involved p38 MAPK in CHO‐K1 cells stimulated with H3 relaxin, H2 relaxin or porcine relaxin. In HEK293 cells, AP‐1 activation in response to H3 relaxin or porcine relaxin involved p38 MAPK, MEK1/2 and JNK, but, when stimulated with H2 relaxin, it involved only a MEK1/2 pathway. We propose that different relaxin family peptides interact at different sites on RXFP3, to activate distinct signalling pathways.This research was supported by National Health and Medical Research Council Project Grants 436713 and 454375

Contrasting pharmacological profiles for functional responses and binding in cells expressing the relaxin family peptide receptor 3 (RXFP3)

Relaxin-3 is a neuropeptide involved in appetite regulation and stress behaviours in rats (Tanaka et al. 2005, McGowan et al. 2005), that is the cognate ligand for the Gi-coupled RXFP3 receptor. In CHO-K1 cells expressing human RXFP3 (CHO-RXFP3), [125I]-INSL5/H3 relaxin chimera (100pM) bound to a single binding site, with competition observed with H3 relaxin (pIC50: 8.42 ± 0.25) but not H2 relaxin, porcine relaxin or INSL3 (n=6). However, in the same cells, inhibition of forskolin (30μM, 3 min) stimulated cAMP accumulation was observed not only with H3 relaxin (pEC50: 9.01 ± 0.27), but also with H2 relaxin (pEC50: 7.14 ± 0.59), porcine relaxin (pEC50: 7.41 ± 0.67) and INSL3 (pEC50: 7.45 ± 0.43) (n=10). We also established that RXFP3 activates ERK1/2 when stimulated with H3 relaxin (6.57 ± 0.78 fold/basal) and weakly with H2 relaxin (2.13 ± 0.74 fold/basal) but not with porcine relaxin or INSL3. The activation of ERK1/2 closely reflects the binding site characterized with [125I]-INSL5/H3 relaxin chimera, which is distinct from the site that leads to cAMP inhibition. Further characterization of RXFP3 signalling using reporter genes, revealed that H2 relaxin and porcine relaxin (all 0.1μM, 24h) could activate AP1 reporter genes, whereas INSL3 activated NFκB reporter genes (n=6). We propose that different relaxin family peptides interact with different binding sites on RXFP3, to activate distinct signalling pathways.