The relaxin family peptide receptor 4 (RXFP4) and its ligand insulin-like peptide 5 (INSL5): signalling pathways and physiological roles
RXFP4 is recognised as the cognate GPCR for INSL5 although little is known about its signal transduction pathways and biological roles. My thesis investigates the signalling pathways activated by RXFP4 in a recombinant cell culture system and also identifies tissues and cell lines that endogenously express RXFP4, with subsequent evaluation of its endogenous signalling profile and possible physiological roles. In CHO cells stably expressing RXFP4, INSL5 inhibits cAMP and stimulates MAPK and AKT-S6RP pathways, and increased cell proliferation. INSL5 is expressed in discrete enteroendocrine cells in the colon. Stimulation of NCI-H716, an enteroendocrine L-cell line that endogenously expresses RXFP4, by INSL5 recapitulated many known signalling pathways previously observed in CHO-RXFP4. INSL5 also inhibits forskolin-stimulated GLP-1 secretion, suggesting a possible role for INSL5-RXFP4 in the control of GLP-1 secretion.
Development of improved methods for predictive modelling of G protein-coupled receptors
G protein-coupled receptors (GPCRs) are a superfamily of transmembrane proteins that play crucial roles in cell physiology. Being key drug targets in many diseases, drug discovery efforts harness structural information about these receptors to rationally design drugs in the field of structure-based drug design (SBDD). This thesis presents the development of computational tools to manage and assess large GPCR structural model datasets, applies these tools to define key features linked with the best performing models in SBDD and integrates this knowledge into a novel computational workflow that predicts new GPCR models, which may offer new opportunities in SBDD programs.
Supervisor: Helen Irving
Does the novel GC activity of IRAK3 affect its singalling?
A novel class of guanylate cyclases have recently been discovered that differ to classical guanylate cyclases. Guanylate cyclases are enzymes that catalyse the formation of cGMP, an important second messenger involved in countless biological processes. IRAK3 is an immune protein which functions to reduce the immune response, therefore acting as an anti-inflammatory protein. IRAK3 was shown to contain a guanylate cyclase centre, therefore it was hypothesised that IRAK3 may produce cGMP. The effect this novel guanylate cyclase centre has on the function of IRAK3 in the innate signalling cascade was investigated. This study demonstrates that the cGMP produced by the guanylate cyclase centre may function to modulate the function of IRAK3 and perhaps other proteins in its vicinity, therefore mediate the immune response.
Allosteric targeting of the dopamine D2 receptor
One-third of medicines target proteins called G protein-coupled receptors (GPCRs), which are found all over the body and are involved in numerous diseases and disorders. One example is the common mental disorder schizophrenia, in which there is an excessive activity of a chemical messenger within the brain. The chemical messenger causes its effect by interacting with a GPCR known as the dopamine receptor. Current medicines completely block this interaction but cause severe side-effects. We have identified a drug that only partially blocks the action of the chemical messenger. This thesis investigates the mechanism of action behind this novel action. Understanding this mechanism will potentially allow for the rational design of improved drugs for this important therapeutic target.
Characterisation of β-adrenoceptor responses in brite adipocyte and skeletal muscle metabolism
Skeletal muscle and adipose tissue are important targets in the treatment of metabolic diseases that include obesity and type 2 diabetes. This thesis has focused on the role of a group of cell surface receptors, the β-adrenoceptors, on the metabolic activity in these tissues. These receptors may represent a novel target in the treatment of metabolic diseases by raising energy usage and normalising the excess energy imbalance that drive these conditions.
Adenosine A2B receptor pharmacology and its role in pathophysiology
The adenosine A2B receptor is an important but poorly understood drug target. This thesis investigated new paradigms of adenosine A2B receptor signalling and behaviour and identified drugs that could selectively promote beneficial outcomes in prostate cancer cells and cardiac fibroblasts. This information helps to inform how targeting the adenosine A2B receptor may be used as a treatment for prostate cancer and cardiovascular disease.
Biased agonism at the adenosine family of G protein-coupled receptors
This thesis focused on investigating an important class of proteins that have been shown to play a role in a number of diseases, including heart disease, stroke and inflammation. Targeting these proteins with drugs is difficult, as severe side effects are commonly associated with treatment. In these studies, it was found that drugs without side effects could be developed. These drugs were able to do this by interacting with the proteins in a unique manner. The findings from this thesis suggest that targeting these proteins in a different way may be the key to avoiding side effects in future medicines.
Thesis under examination
Defining the Functional Expression of TRPV4 in Sensory Ganglia and the Gastrointestinal Tract
Transient Receptor Potential Vanilloid 4 (TRPV4) is a widely expressed sensory protein activated by diverse stimuli ranging from mechanical stress to inflammatory lipids and heat. Current research focuses predominantly on how TRPV4 expressed by nerve cells contributes to pain and inflammation. Using fine dissection techniques and imaging based methods, we identified TRPV4 expression by numerous non-neuronal cells, both surrounding pain sensing nerve cells, and in the gut. Thus, many processes considered as originating from nerve cells possibly initiate from non-neuronal cells. The findings of this thesis have major implications for drug discovery, and for our understanding of TRPV4-mediated cellular processes.
Structure and function studies on the calcitonin receptor, a Class B1 GPCR
This thesis examines four common natural variants of the calcitonin receptor (CTR), a GPCR involved in bone remodelling and calcium homeostasis, confirming that receptor splicing can change CTR function, and revealing that a common polymorphism can also change CTR signalling. Additionally, the CTR was observed to constitutively internalise in different cell systems. The use of different agonists, some of which biased, has started to explore the mechanistic basis of how ligands control CTR function, showing that different ligands distinctly activate the CTR through unique interactions with the receptor, and that different pathways are controlled by distinct portions of the receptor.