In Vitro Pharmacological Characterization of RXFP3 Allosterism: An Example of Probe Dependency

Recent findings suggest that the relaxin-3 neural network may represent a new ascending arousal pathway able to modulate a range of neural circuits including those affecting circadian rhythm and sleep/wake states, spatial and emotional memory, motivation and reward, the response to stress, and feeding and metabolism. Therefore, the relaxin-3 receptor (RXFP3) is a potential therapeutic target for the treatment of various CNS diseases. Here we describe a novel selective RXFP3 receptor positive allosteric modulator (PAM), 3-[3,5-Bis(trifluoromethyl)phenyl]-1-(3,4-dichlorobenzyl)-1-[2-(5-methoxy-1H-indol-3-yl)ethyl]urea (135PAM1). Calcium mobilization and cAMP accumulation assays in cell lines expressing the cloned human RXFP3 receptor show the compound does not directly activate RXFP3 receptor but increases functional responses to amidated relaxin-3 or R3/I5, a chimera of the INSL5 A chain and the Relaxin-3 B chain. 135PAM1 increases calcium mobilization in the presence of relaxin-3NH2 and R3/I5NH2 with pEC50 values of 6.54 (6.46 to 6.64) and 6.07 (5.94 to 6.20), respectively. In the cAMP accumulation assay, 135PAM1 inhibits the CRE response to forskolin with a pIC50 of 6.12 (5.98 to 6.27) in the presence of a probe (10 nM) concentration of relaxin-3NH2. 135PAM1 does not compete for binding with the orthosteric radioligand, [125I] R3I5 (amide), in membranes prepared from cells expressing the cloned human RXFP3 receptor. 135PAM1 is selective for RXFP3 over RXFP4, which also responds to relaxin-3. However, when using the free acid (native) form of relaxin-3 or R3/I5, 135PAM1 doesn't activate RXFP3 indicating that the compound's effect is probe dependent. Thus one can exchange the entire A-chain of the probe peptide while retaining PAM activity, but the state of the probe's c-terminus is crucial to allosteric activity of the PAM. These data demonstrate the existence of an allosteric site for modulation of this GPCR as well as the subtlety of changes in probe molecules that can affect allosteric modulation of RXFP3

Differential expression of mesotocin receptors in the uterus and ovary of the pregnant tammar wallaby

Mesotocin, an oxytocin-like peptide, is released in highest concentrations during parturition in macropodid marsupials. In late pregnant wallabies, uterine sensitivity to mesotocin increases markedly in the myometrium of the gravid uterus. This coincides with a significant increase in myometrial mesotocin receptor concentrations 3-4 days before term. To date, there is no information on mesotocin receptor gene expression in female wallaby reproductive tissues. This study aimed to examine mesotocin receptor gene expression in the uterus and ovaries of pregnant tammar wallabies, and to localise mesotocin receptors within the uterus. An RT-PCR strategy produced a consensus nucleotide sequence of 834 bp, which encoded 278 amino acids of transmembrane domains I to VI. This protein sequence has approximately 80% homology with the bovine and rat oxytocin receptor exon 2 region. Only one mesotocin receptor was detected in the tammar genome. The myometrium and mammary gland both expressed a 4.1 kb mesotocin receptor gene transcript. Myometrial mesotocin receptor gene expression increased on day 22 of the 26-day gestation and was significantly higher in the gravid than the non-gravid uterus in late pregnancy. This pattern of mesotocin receptor gene expression paralleled mesotocin receptor concentrations. Mesotocin binding sites were localised only to the myometrium, the highest densities being observed in the gravid uterus. Finally, this study showed high expression of mesotocin receptors in the corpus luteum. The pattern of luteal mesotocin receptor expression differed from the myometrium, with a decrease in mesotocin receptors occurring on the day of expected births

Engineering of chimeric peptides as antagonists for the G protein-coupled receptor, RXFP4

Insulin-like peptide 5 (INSL5) is a very important pharma target for treating human conditions such as anorexia and diabetes. However, INSL5 with two chains and three disulfide bridges is an extremely difficult peptide to assemble by chemical or recombinant means. In a recent study, we were able to engineer a simplified INSL5 analogue 13 which is a relaxin family peptide receptor 4 (RXFP4)-specific agonist. To date, however, no RXFP4-specific antagonist (peptide or small molecule) has been reported in the literature. The focus of this study was to utilize the non-specific RXFP3/RXFP4 antagonist ΔR3/I5 as a template to rationally design an RXFP4 specific antagonist. Unexpectedly, we demonstrated that ΔR3/I5 exhibited partial agonism at RXFP4 when expressed in CHO cells which is associated with only partial antagonism of INSL5 analogue activation. In an attempt to improve RXFP4 specificity and antagonist activity we designed and chemically synthesized a series of analogues of ΔR3/I5. While all the chimeric analogues still demonstrated partial agonism at RXFP4, one peptide (Analogue 17) exhibited significantly improved RXFP4 specificity. Importantly, analogue 17 has a simplified structure which is more amenable to chemical synthesis. Therefore, analogue 17 is an ideal template for further development into a specific high affinity RXFP4 antagonist which will be an important tool to probe the physiological role of RXFP4/INSL5 axis

C-Terminus of the B-Chain of Relaxin-3 Is Important for Receptor Activity

Human relaxin-3 is a neuropeptide that is structurally similar to human insulin with two chains (A and B) connected by three disulfide bonds. It is expressed primarily in the brain and has modulatory roles in stress and anxiety, feeding and metabolism, and arousal and behavioural activation. Structure-activity relationship studies have shown that relaxin-3 interacts with its cognate receptor RXFP3 primarily through its B-chain and that its A-chain does not have any functional role. In this study, we have investigated the effect of modification of the B-chain C-terminus on the binding and activity of the peptide. We have chemically synthesised and characterized H3 relaxin as C-termini acid (both A and B chains having free C-termini; native form) and amide forms (both chains' C-termini were amidated). We have confirmed that the acid form of the peptide is more potent than its amide form at both RXFP3 and RXFP4 receptors. We further investigated the effects of amidation at the C-terminus of individual chains. We report here for the first time that amidation at the C-terminus of the B-chain of H3 relaxin leads to significant drop in the binding and activity of the peptide at RXFP3/RXFP4 receptors. However, modification of the A-chain C-terminus does not have any effect on the activity. We have confirmed using circular dichroism spectroscopy that there is no secondary structural change between the acid and amide form of the peptide, and it is likely that it is the local C-terminal carboxyl group orientation that is crucial for interacting with the receptors

Characterisation of a cell-free synthesised G-protein coupled receptor

G-protein coupled receptors are the largest family of integral membrane proteins found within the human genome. They function as receptors and modulators to a wide range of ligands and responses which are crucial for human health. GPCR study, specifically the investigation of structure and interaction to cognate ligands, is of high priority. Limitations for structural study can be traced in part, to obtaining suitable quantities of recombinant protein. We sought to address the limitations of traditional recombinant technologies by utilising an Escherichia coli based cell-free protein synthesis (CFPS) approach for production of a thermostable neurotensin receptor 1 (en2NTS1). Initial results were promising, with a high amount (up to 2 mg/mL) of en2NTS1 produced, that had attained correct secondary structure. Meanwhile, concurrent experiments indicated that CFPS produced en2NTS1 showed non-competitive binding to the peptide ligand neurotensin8-13 when compared to E. coli produced en2NTS1. 1H-13C HMQC SOFAST NMR spectra were indicative of disrupted tertiary structure for CFPS produced 13CH3-methionine labelled en2NTS1. The results obtained, indicate CFPS produced en2NTS1 is not forming a discrete tertiary structure and that further development of the CFPS technique needs to be carried out

In a class of their own-RXFP1 and RXFP2 are unique members of the LGR family

The leucine-rich repeat-containing G protein-coupled receptors (LGRs) family consists of three groups: types A, B, and C and all contain a large extracellular domain (ECD) made up of the structural motif - the leucine-rich repeat (LRR). In the LGRs, the ECD binds the hormone or ligand, usually through the LRRs, that ultimately results in activation and signaling. Structures are available for the ECD of type A and B LGRs, but not the type C LGRs. This review discusses the structural features of LRR proteins, and describes the known structures of the type A and B LGRs and predictions that can be made for the type C LGRs. The mechanism of activation of the LGRs is discussed with a focus on the role of the low-density lipoprotein class A (LDLa) module, a unique feature of the type C LGRs. While the LDLa module is essential for activation of the type C LGRs, the molecular mechanism for this process is unknown. Experimental data for the potential interactions of the type C LGR ligands with the LRR domain, the transmembrane domain, and the LDLa module are summarized

Using the novel HiBiT tag to label cell surface relaxin receptors for BRET proximity analysis

Relaxin family peptide 1 (RXFP1) is the receptor for relaxin a peptide hormone with important therapeutic potential. Like many G protein-coupled receptors (GPCRs), RXFP1 has been reported to form homodimers. Given the complex activation mechanism of RXFP1 by relaxin, we wondered whether homodimerization may be explicitly required for receptor activation, and therefore sought to determine if there is any relaxin-dependent change in RXFP1 proximity at the cell surface. Bioluminescence resonance energy transfer (BRET) between recombinantly tagged receptors is often used in GPCR proximity studies. RXFP1 targets poorly to the cell surface when overexpressed in cell lines, with the majority of the receptor proteins sequestered within the cell. Thus, any relaxin-induced changes in RXFP1 proximity at the cell surface may be obscured by BRET signal originating from intracellular compartments. We therefore, utilized the newly developed split luciferase system called HiBiT to specifically label the extracellular terminus of cell surface RXFP1 receptors in combination with mCitrine-tagged receptors, using the GABAB heterodimer as a positive control. This demonstrated that the BRET signal detected from RXFP1-RXFP1 proximity at the cell surface does not appear to be due to stable physical interactions. The fact that there is also no relaxin-mediated change in RXFP1-RXFP1 proximity at the cell surface further supports these conclusions. This work provides a basis by which cell surface GPCR proximity and expression levels can be specifically studied using a facile and homogeneous labeling technique such as HiBiT

Colokinetic effect of an insulin-like peptide 5-related agonist of the RXFP4 receptor

BACKGROUND: Insulin-like peptide 5 (INSL5) is a hormone stored in colonic enteroendocrine cells that also contain the unrelated hormones, GLP-1 and PYY. It acts at the relaxin family peptide 4, RXFP4, receptor. RXFP4 is expressed by enteric neurons in the colon, and it has been speculated that INSL5, through its action on enteric neurons, might be involved in the control of colonic contractions. Similar to insulin and relaxin, INSL5 consists of A and B peptide chains linked by three disulfide bonds, two between the chains and one intrinsic to the A chain. Because of its complex structure, it is difficult to synthesize and to prepare peptide analogues to investigate its roles. We have recently developed a potent simplified peptide analogue, INSL5-A13 (INSL5 analogue 13). METHODS: In the present work, we have investigated the actions of INSL5-A13 in mice. We investigated the ability of INSL5-A13 to increase the speed of emptying of a bead from the colon, after expulsion had been slowed by the peripherally restricted opioid agonist, loperamide (1 mg/kg). KEY RESULTS: INSL5-A13 was a full agonist at the mouse RXFP4 expressed in HEK cells, with an EC50 of ~9 nmol/L. INSL5-A13 caused an acceleration of colorectal bead propulsion in mice constipated by loperamide in the dose range 0.2 to 60 µg/kg, with an EC50 of ~6 µg/kg in vivo. It also accelerated bead propulsion in untreated mice. Bead expulsion was not accelerated in RXFP4-/- mice. CONCLUSION AND INFERENCES: Our data suggest that RXFP4 agonists could be useful in the treatment of constipation