Exploiting a novel molecular toolkit to explore cell type specific adenosine receptor pharmacology and regulation at endogenous levels of expression.

The process of cell to cell communication is a vital and integral part of all life and controls the inner workings of organisms allowing them to respond, adapt and survive. G protein-coupled receptors (GPCRs) are a large group of related proteins that are located on the surface of cells. These receptors can detect molecules outside of the cell and activate cell signalling pathways inside cells. It has become clear, in recent years, that GPCR pharmacology (the mechanisms by which drugs interact with them) is dependent on the local membrane environment within which the GPCR resides, and there is growing evidence that their function can be regulated by interactions with neighbouring proteins (including other GPCRs) within oligomeric complexes. This may provide novel opportunities to develop drugs which are targeted at GPCRs in a particular cell type or subcellular domain and provide a way to circumvent on-target side effects (i.e. where the drug binds to the right receptor but in the wrong location). Adenosine is an important endogenous nucleoside that is released locally and acts via four different GPCRs (A1AR, A2AAR, A2BAR, A3AR). Adenosine receptors have a widespread distribution and have been implicated in renal, respiratory and cardiovascular diseases, as well as cancer. Local generation of adenosine is markedly enhanced, and adenosine receptor expression changed, under metabolic stress e.g. during hypoxia, inflammation and ischaemia and this protects cells, tissues and organs from collateral damage during infection and inflammation. There is increasing evidence for the presence of several adenosine receptors in homodimer and heteromeric complexes. In our previous programme grant (MR/N020081/1), we showed that heteromeric complexes can be produced for all combinations of adenosine receptor. Others have also reported complexes between A2AAR and A2BAR. A2AAR-D2 dopamine receptor heteromers have been detected in striatum and have been proposed to be a viable target for Parkinson's disease. Finally, there is evidence for A1AR-beta1-adrenoceptor and A1AR-beta2-adrenoceptor heteromers which have altered ligand binding and signaling characteristics. To date, however, the majority of studies have used recombinantly overexpressed receptors in model cell lines to probe for oligomerisation using methods such as bioluminescence resonance energy transfer (BRET). However, such experiments can be confounded by overexpression which can increase the propensity of GPCRs to form dimers. There is therefore an urgent need to develop new approaches to monitor receptor expression levels and to understand the effect of oligomerisation on GPCR function in native cells at endogenous levels of receptor expression and their propensity to form stable and transient complexes. We have recently developed highly sensitive methods to quantify receptor expression levels, their pharmacology and protein-protein interactions that are particularly effective at the low levels of receptor expression found in native cells. These approaches include fluorescent ligand technologies, ligand-directed covalent labelling of endogenous GPCRs, CRISPR/Cas9 genome editing in combination with NanoBiT technologies and single molecule biophysical approaches in single living cells and more complex physiologically relevant ex vivo isolated blood vessels and heart preparations. The aim of this programme is study how receptor expression levels and receptor oligomerisation are regulated in native cells from the cardiovascular and immune systems, which endogenously express adenosine receptors that play key roles in health and disease. We aim to investigate these interactions in human endothelial cells, smooth muscle cells, fibroblasts, macrophages and stem cell-derived cardiomyocytes. We also aim to determine how these interactions are affected by hypoxia and inflammatory mediators.

The urgent need to understand how adenosine receptors are regulated and organised in their local cellular environment is illustrated by recent studies that show that G protein coupled receptor (GPCR) expression in a given cell type is highly heterogenous and that the receptor repertoire of an individual cell can change dramatically during disease progression. We have combined medicinal chemistry, molecular pharmacology and physiology to develop ground-breaking fluorescent ligand technologies to interrogate endogenous GPCR molecular pharmacology at the single cell level and in ex vivo cardiovascular tissues. These include development of novel suites of fluorescent ligands together with the application of super-resolution microscopy, fluorescence correlation spectroscopy, genome-editing, nanobodies, NanoBRET and ligand-directed covalent labelling of receptors with fluorescent tags. Initial proof of concept work with each of these approaches initially was in recombinant cells lines expressing the target receptor of interest. However, in recent years our focus has been to develop highly subtype selective fluorescent ligands (agonists and antagonists), ligand-directed covalent tagging strategies and to utilise CRISPR/Cas9 genome-editing and NanoBiT-conjugated nanobodies to tag receptors at endogenous levels of expression. In this new programme of work, we will apply these approaches to study adenosine receptor pharmacology, oligomerisation and the influence of protein-protein interactions on adenosine receptor signalling and location in different cell types and tissues. Furthermore, the use of pluripotent stem cell technologies, and the ability to differentiate cells into different phenotypes (from cardiomyocytes, fibroblasts and endothelial cell to neurones), provides a fantastic platform to use our new imaging technologies to investigate the impact of local cellular environment on GPCR signalling which should have wide-ranging implications for drug discovery.