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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ItemToward the structural characterisation of the relaxin receptor, RXFP1( 2021)Relaxin is a peptide hormone that is involved in several physiological processes such as pregnancy, collagen breakdown, fibrosis inhibition and vasodilation. It has been investigated for the use of several disease states such as scleroderma, fibrosis, cancer and most recently acute heart failure. Relaxin’s cognate receptor is the relaxin family peptide receptor 1 (RXFP1), an integral membrane protein belonging to the G protein-coupled receptor (GPCR) family with a complex, multistep activation mechanism which is still not well understood. Given the physiological roles of relaxin, RXFP1 is a promising target for the treatment of abovementioned conditions. However, there is currently a lack of a detailed mechanism in which relaxin mediated activation of RXFP1 occurs and this makes the design of relaxin-like compounds such as long active peptide mimetics, small molecules or biologics targeting RXFP1, or understanding and optimizing existing compounds that act at RXFP1 difficult. The lack of a detailed mechanism of RXFP1 activation can be attributed to the lack of full-length RXFP1 structures. While there are proposed models of this activation mechanism, these models were derived from studies on isolated domains of RXFP1 and thus it cannot be assumed that the findings are similar to that of a full-length RXFP1. Thus, the aim of this thesis was to work toward active and inactive state structures of full-length RXFP1 using cryo-electron microscopy (EM). By solving active and inactive state structures, we can overlay these structures to determine key conformational changes and key residues that interact with relaxin to determine a complete mode of relaxin mediated activation of RXFP1. However, these studies are hampered by the limitations of cryo-EM to study inactive state GPCRs and the low recombinant expression of WT RXFP1 which makes producing sufficient amounts of purified RXFP1 for these studies very difficult. In this thesis we optimised the expression and purification of RXFP1 for the purposes of cryo-EM studies. We also developed and optimised a novel tool, monomeric ultra-stable GFP (muGFP) as an intracellular loop 3 (ICL3) fusion partner to overcome the limitations of inactive state cryo-EM studies. We applied this to a thermostabilised variant of the alpha1A-adrenoceptor and demonstrated its utility for cryo-EM studies before applying it to RXFP1. Next, we applied an established workflow for the production of active state GPCR-G protein complexes in insect cells for cryo-EM studies to WT RXFP1 for the active state studies of the receptor. We also experimented with the expression and formation of an RXFP1-G protein complex in a mammalian expression systems. However, we were unable to proceed to cryo-EM studies of either inactive or active state RXFP1 due to inability to produce sufficient quantities of protein To overcome the limitation of poor protein yield, we developed a novel mammalian cell-based method of directed evolution. Existing methods of GPCR directed evolution are primarily E. coli based, and as RXFP1 is unable to be expressed in E. coli due to requiring post-translational modification, a mammalian system was required. We applied this novel method to RXFP1 and were able to evolve mutant #35, which demonstrated an ~9x increase in recombinant RXFP1 expression. Additionally, we also identified 2 mutants that demonstrated interesting pharmacological changes from WT. This includes a mutant that demonstrated an increase in basal signalling, and another mutant that demonstrates a decreased pEC50 for relaxin, that is a higher concentration of relaxin is required to produce an equivalent response in WT. By evolving high expressing mutant #35, we could potentially overcome the bottleneck of insufficient purified protein yield for cryo-EM studies. By applying mutant #35 to the workflows developed in this thesis, we can potentially enable downstream cryo-EM studies of RXFP1 through the ability to produce ~9x more protein than WT. Through enabling these studies, we may be able to elucidate the mechanism in which relaxin triggers RXFP1 activation in a full-length receptor. Understanding this mechanism in atomic resolution detail through cryo-EM studies could then facilitate rational drug design of novel relaxin-like mimetics for the treatment of acute heart failure or fibrosis or antagonists for the treatment of certain cancers.
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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.