Defining the functional roles of the enigmatic G protein-coupled receptor GPR35

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Despite suggestions that it may be a receptor for a metabolite of tryptophan (kynurenic acid), a lipid (a form of lysophosphatidic acid) or a chemokine (CXCL17) GPR35 remains an 'orphan' GPCR. However, expression patterns, genome wide association studies and activation by a previously used anti-asthma medicine, suggest important roles for GPR35 in inflammatory conditions of the gut and airways/lung. Here we aim to investigate the mechanisms of GPR35 signal transduction and regulation, and to employ pharmacological tool compounds in combination with genetically modified mice to dissect the modes of action and physiological role and therapeutic potential of GPR35. This will involve using approaches, including G protein biosensors and CRISPR/cas knock-out of G proteins and arrestins, to investigate the mechanistic basis of GPR35 signalling. In particular we will evaluate the prospect that currently available GPR35 ligands may show signalling bias by preferentially promoting coupling through G proteins or arrestins. Interestingly, mouse and human GPR35 show large differences in basic pharmacology, including that a pair of GPR35 'antagonists' have affinity only for the human receptor. We will exploit this difference to explore the in vivo functions of GPR35 by generating mutant mouse lines expressing human GPR35. In this way the action of human specific GPR35 chemical tools can be used to probe the function and potential clinical relevance of GPR35. We will also generate knock-in mice expressing a form of GPR35 which is G protein 'biased' and cannot be phosphorylated or engage beta-arrestins to define the in vivo modes of signal transduction of GPR35. Hence, by combining in vitro analysis using the 'gene edited' cells and sensors with ex vivo and in vivo analysis of the transgenic 'knock-in' lines, with particularly focus on lung and colon inflammatory disease models, we will provide unique insights into the function of GPR35 and indicate its therapeutic potential.

Who will benefit from this research?

The most immediate beneficiaries from the research will be academic researchers with interests both specifically related to GPR35 and, more generally, as described in the 'Case for Support' in 'Western lifestyle' inflammatory diseases. This is an area that is attracting enormous interest, with clear links between diet and health that extend to 'healthy aging' and possible intervention in disease or lifestyle via 'functional foods'. Although only recently becoming widely appreciated, many metabolites derived from food sources in the diet are now known to function as key homeostatic beacons and do so, at least in part by activating group of GPCRs expressed by cells and tissues that sense metabolic status. However, beyond these specific health-related aspects, there is vast interest in novel approaches to better understand GPCR function in general, and both the novel sensors we describe and the HEK293 cell lines lacking various G protein or arrestins and the results generated using them will be of great interest to virtually all of the vast number of researchers who work on GPCR-induced signalling. This includes stakeholders across the pharma/biotech sector as well as academic teams. Although the only current and ongoing clinical trials targeting GPR35 that we are aware of (from Patara Pharma (http://patarapharma.com/)) employ an undefined GPR35 agonist coded as 'PA101B' which is described as 'a GPR35 agonist immune modulator with mast cell stabilizing properties' and is being assessed to treat chronic cough and indolent systemic mastocytosis, there is also considerable interest in the mode of action of the anti-asthma medicine sodium cromoglycate, which displays modest potency as an agonist at GPR35. As many companies have 'respiratory disease' programmes, novel insights emerging from these studies are likely to attract attention also in this sphere. A number of companies (see e.g. Mackenzie et al., (2014) Mol. Pharmacol. 85, 91-104) have assisted us in the search for high potency agonist ligands of GPR35 and it is likely they would remain extremely interested in the outcomes and progress of these studies.

How will they benefit from this research?
As well as greater insight into specific roles of 'biased' signalling the research community will benefit from access to the many novel tools and reagents we have and will generate within this project. We will provide the transgenic mouse lines to appropriate interested partners as described within the 'Data management plan'. In a similar manner, if agreed to within MTAs developed by our Japanese collaborators at Tohoku University, we will also provide the gene edited 'knock out' cell lines as described, once key output publications have been achieved. The project has great potential in terms of staff training in that the PDRAs will benefit from opportunities to perform cutting edge research in a broad swathe of areas relevant to modern pharmacological and physiological studies and to enhance team working via the need to integrate work from two sites. They will also benefit greatly from the opportunities provided to travel and to work with our key collaborators in Germany to access to high end equipment and technologies (see Letters of Support from Evi Kostenis and Carsten Hoffmann). This training will ensure the greatest range of subsequent career opportunities.