Florey Department of Neuroscience and Mental Health - Theses
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ItemInvestigating ligand- and cell-specific signal transduction at relaxin family peptide receptor 1( 2021)Relaxin, a peptide hormone and the endogenous agonist of the relaxin family peptide receptor 1 (RXFP1), has shown substantial promise for the therapeutic treatment of cardiovascular disorders and fibrosis. RXFP1 has received considerable therapeutic interest as a drug target over the years, and a lot of effort has been put into the development of novel ligands targeting this receptor. However, we do not understand how novel RXFP1 agonists work because we do not understand RXFP1 signalling in enough detail, and do not fully understand which pathways are important for the therapeutic actions, where they are activated in the signal transduction cascade, and in what cell types. Furthermore, development of novel ligands raises the question of biased signalling, which is the ability of different ligands for the same receptor to preferentially activate certain signal transduction pathways relative to one another. It may be possible to utilise bias to create effective drugs that have fewer side effects, but this requires us to first understand which effectors and signal transduction pathways are important for the therapeutic versus harmful actions. Furthermore, recent research has highlighted the importance of measuring the temporal aspects of signalling in order to understand bias, as signalling and bias can change over time, and using end point assays can produce a misleading picture of efficacy and bias depending on which time point was chosen. Additionally, validating findings from recombinant cells in physiologically-relevant cells is also important to understand how ligands signal across cell types, and to distinguish biased signalling from cell-specific signalling. The development of sensitive, real-time assays that can be used across cell types will aid our understanding of the molecular mechanisms underlying the actions of relaxin and other ligands targeting this promising receptor. The general aim of this thesis was to apply and develop bioluminescence resonance energy transfer (BRET)-based methods in conjunction with human native and primary cells in order to examine real-time signalling and bias at RXFP1. First, the functionality and versatility of the CAMYEL (cAMP sensor using YFP-Epac-Rluc) real-time BRET-based cAMP biosensor was demonstrated for RXFP1 and the related GPCRs RXFP2, RXFP3, and RXFP4. CAMYEL was a sensitive alternative to end point assays, as it detected concentration-dependent changes in cAMP activity at all receptors in recombinant cell lines, was dynamic and reversible, detected kinetic differences between different ligands for the same receptor, and showed potencies comparable to those seen in end point assays. CAMYEL was cloned into a lentiviral vector, and lentivirus was used to transduce THP-1 cells, which endogenously express low levels of RXFP1. THP-1 CAMYEL cells showed robust cAMP activation after relaxin stimulation and will therefore streamline the process of screening novel RXFP1 ligands. The lentiviral vector will also allow for the transduction of many mammalian cell types for real-time analysis of cAMP activity at various GPCRs, including in primary cells. However, it appeared that the CAMYEL assay was unable to detect a delayed, Gi3-mediated phase of RXFP1 cAMP activity that has been demonstrated using other assays, suggesting that CAMYEL might not detect cAMP generated in specific compartments of the cell. Second, we developed, validated, and characterised a BRET-based biosensor for cGMP activity, known as CYGYEL (cyclic GMP sensor using YFP-PDE5-Rluc8), based on the Forster/fluorescence resonance energy transfer (FRET) biosensor cGES-DE5. CYGYEL was cloned into a lentiviral vector, enabling its use across different mammalian cell types. CYGYEL was initially characterised in HEK293T cells, where it was shown to be sensitive, dynamic, reversible, and also very selective for the detection of cGMP over cAMP. CYGYEL was then used to detect cGMP after transduction of human primary vascular cells, namely endothelial and smooth muscle cells. CYGYEL detected differences in cGMP signalling kinetics both between cell types, and also between ligands that increased cGMP production via soluble versus membrane guanylate cyclases. So far we have been unsuccessful at detecting GPCR-mediated cGMP using CYGYEL, but further work is required in this area. Regardless, CYGYEL still has many uses for drug discovery. Finally, we used a variety of BRET-based assays for G protein association, second messenger activity, and ERK1/2 activity, as well as physiologically-relevant primary cells, in order to understand the mechanisms of action underlying the beneficial actions of the relaxin peptide analogue B7-33. According to previous work, B7-33 appeared to show cell-specific signalling and biological responses, whereby it had weak activity in recombinant and cancer cells, but potent activity in fibroblasts and vascular cells, as well as in vivo. Our results across several cell types indicated that B7-33 is a biased agonist that favours signalling via Gi3/cGMP over Gs/cAMP, relative to relaxin which signals potently via both pathways. These findings are consistent with B7-33’s actions as a potent vasodilator and anti-fibrotic, which depend on cGMP rather than cAMP. Relatedly, we demonstrated that B7-33 shows transient cAMP activity relative to relaxin in all cell types tested, and that in a real-time cAMP assay involving ligand washout, the cAMP response from B7-33 dropped drastically relative to relaxin, suggesting that B7-33 dissociated from RXFP1 far more readily than relaxin. We thus hypothesised that the bias shown by B7-33 is related to kinetics, whereby the relaxin/RXFP1 complex catalyses more cycles of Gs activation due to the sustained duration of the active receptor conformation, relative to B7-33 which has a faster off-rate associated with its weaker activation of Gs. However, both agonists equally activate Gi3 suggesting that the relative rates of activation and deactivation of the different G proteins may also be important. Finally, it was also observed that ERK1/2 is activated by its upstream effectors in a cell type-specific manner. Specifically, whereas previous findings have shown that ERK1/2 is primarily downstream of Gi/o in native cells, our findings show that ERK1/2 is activated downstream of Gs in HEK-RXFP1 cells, which explains B7-33’s weak ERK1/2 activation in HEK-RXFP1 cells but potent activation in native cells. These findings have implications for the development of novel biased drugs targeting RXFP1, as it is believed that the negative actions of exogenously-administered relaxin, including for example its ability to promote tumour growth in mouse models in vivo, are related to its potent cAMP activity. Conversely, equi-molar doses of B7-33 do not promote tumour growth but do retain the beneficial actions of relaxin which occur via Gi. Thus, we could potentially aim to retain the kinetic bias to maintain potent cGMP signalling, while minimising cAMP activity, and at the same time aim to develop compounds that are more drug-like with longer half-lives.
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ItemStudies on the mechanism of binding and activation of relaxin family peptide receptors( 2020)The peptide hormone relaxin is involved in reproductive processes but has also been investigated for several decades as a treatment for a range of disease states such as scleroderma, acute heart failure, and fibrotic conditions. The receptor for relaxin, RXFP1, is an integral membrane protein belonging to the G protein coupled receptor (GPCR) family. RXFP1 is therefore a therapeutically tractable target for which a thorough understanding of its mechanism of binding and activation is required to develop better relaxin-like drugs. The aims of these studies are to investigate the mechanism by which relaxin binds and activates RXFP1 using a variety of molecular pharmacology approaches in a HEK293T cell model system recombinantly expressing RXFP1 in various forms. Specifically, a hypothesis was tested that a homodimer of RXFP1 might be the minimal functional unit required for receptor activation. GPCR dimers are postulated to interact via their transmembrane helices, so initial investigations aimed to disrupt RXFP1 homodimerisation by incorporation of peptides representing single transmembrane segments of RXFP1 as well as recombinant expression of RXFP1 transmembrane domains. There was no evidence that RXFP1 homodimerisation is required for receptor activation. Following this, the evidence for RXFP1 homodimerisation was re-evaluated in the development of two methods which utilise principles of Bioluminescence Resonance Energy Transfer (BRET). Firstly, split Nanoluciferase was used to tag cell surface localised RXFP1 receptors in combination with mCitrine-tagged RXFP1 and BRET was measured to assess relative receptor proximity. This indicated that RXFP1 is unlikely to be a stable homodimer, intracellularly localised receptors predominate, and there is no change in receptor:receptor proximity upon relaxin stimulation. Secondly, Nanoluciferase-tagged RXFP1 receptors were used in combination with fluorescently labelled relaxin and BRET was measured to track relaxin:RXFP1 binding interactions. This allowed sensitive, real time measurements of the relaxin:RXFP1 binding interactions, demonstrating a multi-step mechanism of relaxin binding in which the linker domain of RXFP1 is critical for high-affinity interactions. Furthermore, there was no evidence of negative co-operativity of relaxin binding, contrary to previous reports which were used as evidence of RXFP1 homodimerisation. Overall, these studies indicate that relaxin does not activate RXFP1 via a mechanism involving a receptor homodimer. Several molecular tools were developed which will be useful for future investigations into RXFP1 pharmacology. This work adds incremental detail to the understanding of how relaxin activates RXFP1, hopefully leading to the development of novel therapeutically useful relaxin-like molecules in future.
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ItemUnderstanding the interactions of relaxin-3 with its receptor RXFP3( 2018)Relaxin-3 is a highly conserved neuropeptide that activates the Relaxin Family Peptide Receptor 3 (RXFP3). The relaxin-3/RXFP3 neuropeptide system is involved in the modulation of stress, appetite, mood, cognition, memory and learning, thereby presenting RXFP3 as an excellent putative target for the pharmacological treatment of a range of neurological disorders. To fully assess its clinical potential, it is imperative to develop small molecule agonist and antagonist of RXFP3 that can cross the blood-brain barrier. However, designing such drugs is challenging and requires structural details of relaxin-3/RXFP3 interactions to facilitate structure-based drug design (SBDD). Thus, the overarching aim of this project was to understand and identify how relaxin-3 and a single-chain peptide antagonist known as H3 B1-22R interact with RXFP3. It is established that ArgB26, ArgB16 and ArgB12 of relaxin-3 interacts with Glu141, Asp145 and Glu244 on RXFP3 respectively whereas TrpB27 interacts with Trp138 of RXFP3. However, the interactions for the hydrophobic binding residues on relaxin-3 and H3 B1-22R on RXFP3 remain elusive. Using a combination of site-directed mutagenesis, homology modelling and docking studies to predict RXFP3 residues that were interacting with H3 B1-22R and the hydrophobic binding residues of relaxin-3, refined models highlighted that relaxin-3 and H3 B1-22R had overlapping binding sites and used similar residues (Arg12, Ile15, Arg16 and Ile19) on the central B-chain to bind to extracellular loops (EL) 1 and 2 (EL2) of RXFP3. TrpB27 of relaxin-3 inserted into the binding pocket to interact not only with Trp138, but the C-terminal carboxyl also formed a salt-bridge with Lys271. H3 B1-22R which lacks the Trp27 has a non-native Arg23 to form cation-pi and salt-bridge interactions with Trp138 and Glu141. Overall, relaxin-3 and H3 B1-22R had overlapping binding sites but distinct binding modes, leading to the active and inactive conformational states of RXFP3 respectively. To further refine these H3 B1-22R/RXFP3 interactions, disulfide-trapping was employed but was unsuccessful. Structural details of ligand-receptor binding are fundamentally required for accurate SBDD. Therefore, a parallel approach was undertaken to understand the RXFP3 stability upon purification from the membrane and to engineer RXFP3 with fusion proteins and rationally-designed thermostabilising mutations. This study presented that RXFP3 could be expressed in HEK293F mammalian cells, solubilised in DDM/CHAPS/CHS and purified with StrepTactin. Stability of purified RXFP3 was then analysed in the thiol-specific 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) thermal shift assay. Subsequently, RXFP3 fused with the apocytochrome b562RIL (BRIL), T4 Lysozyme (T4L) and a novel monomeric ultra-stable green fluorescent protein (muGFP) were demonstrated to increase the apo-state RXFP3 stability by at least 6⁰C, 11⁰C and 13⁰C respectively, suggesting the potential for RXFP3 to be thermostabilised with protein engineering methods. Collectively, this thesis provided new insights into the elucidation of relaxin-3 and H3 B1-22R interactions and binding modes at RXFP3. Additionally, the ability to purify and engineer RXFP3 now paves a strong foundation to further study ligand-binding interactions in higher-resolution techniques such as nuclear magnetic resonance (NMR) or surface plasmon resonance (SPR).