Florey Department of Neuroscience and Mental Health - Theses
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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).