Florey Department of Neuroscience and Mental Health - Theses
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ItemProtein engineering of arginine vasopressin receptor V1A for structural biology( 2021)Peptide hormone arginine vasopressin (AVP) and its cognate G protein-coupled receptor (GPCR), V1A, belong to the vasopressin-oxytocin signalling system – a highly complex and widespread endocrine system in the human body. AVP activation of V1A causes a stimulatory contractile effect on vascular and uterine smooth muscle, while, in the brain, AVP and V1A play an important role in anxiety, aggression and social behaviours. Due to these physiological mechanisms, AVP and V1A have been of therapeutic interest for decades; initially for the treatment of peripheral conditions such as heart failure and menstrual cramps, while recently, V1A antagonists have shown promise for treating aspects of autism spectrum disorder (ASD). Despite this longstanding interest, there are currently no approved drugs selectively targeting V1A. Subtype-selectivity is important among the vasopressin-oxytocin receptor family, as these receptors share a high degree of sequence similarity and structural homology, yet have differing, and sometimes opposing, effects. Furthermore, as there are currently no experimental 3D structures of V1A, there is a lack of understanding on exactly how AVP binds to V1A at the molecular level, which limits the design and optimisation of new, V1A-selective drugs. GPCR structural biology is a rapidly developing field; however, the low-expression level and instability of many GPCRs, including V1A, can be problematic for purification and subsequent structural study. This thesis aimed to apply various protein engineering techniques to V1A, in order to facilitate the purification and subsequent biochemical study of this important, but long unfulfilled, potential therapeutic target. Chapter 2 covers the first GPCR-engineering venture, which was to introduce expression and stability augmenting missense mutations to V1A via a well-characterised method – directed evolution. This method, which uses E. coli display of receptor mutants, has successfully produced high-expressing, stabilised receptor mutants for the structural study of other neuropeptide receptors, including NTS1 and NK1. However, upon application of this method to V1A, stabilised V1A mutants were not produced. Instead, a V1A truncate was selected, which contained a small section of the V1A N terminus. The reason this protein was selected was unclear – but can primarily be attributed to the issues that arise when using E. coli for GPCR expression. It was evident that further engineering of V1A would require the use a different cell type, such as insect or mammalian cells. Chapter 3 explores the development of a mammalian cell-based directed evolution method. This new method was developed to overcome the expression issues encountered using E. coli, and required optimisation and innovation to accommodate the new cell type, including the use of lentivirus as the gene-delivery method. The method was developed, optimised and applied to V1A – producing V1A mutants that exhibited improved expression levels compared to wild-type V1A. Furthermore, several mutations were identified that appeared in multiple V1A mutants, indicating that they were contributing to this expression-boosting effect. Chapter 4 covers the purification of V1A using a combination of protein engineering approaches – including the introduction of the expression-boosting point mutations discovered in Chapter 3. Ultimately, functional V1A was able to be purified with the aid of these mutations, which provided a substantial increase in functional yield of purified receptor. The two main outcomes of this thesis are: the development of a mammalian cell-based directed evolution method that improves the functional yield of purified receptor – and the successful purification of functional V1A. Firstly, the new, mammalian cell-based selection method presents as a generic, rapid and viable means of improving the heterologous expression of low-expressing GPCRs – and potentially other complex membrane proteins with expression issues. Secondly, the substantial increase the yield of purified, functional V1A is a significant breakthrough in the biochemical study of V1A, as it enables the application of structural biology techniques, such as cryo-electron microscopy (cryo-EM), as well as ligand screening methods for new, V1A-selective ligands.