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

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

G protein-coupled receptors (GPCRs) are cell surface proteins that play key roles in allowing cells to respond to external cues and signals. They are routinely considered to do so by activating one or more of a group of so called G proteins. Because of these key roles in controlling responses to many hormones and neurotransmitters a substantial number of GPCRs are the molecular targets for currently employed medicines and, in general, GPCRs are considered 'tractable' i.e. that molecules that activate or block their activation can be found and developed as potential new medicines. However, there are a number of GPCRs that are poorly characterised and the roles they play in the control of physiological functions are unknown or uncertain. In such cases the potential for them to be targets for new medicines in the longer term is often assessed partially by the effects produced in mice in which the gene encoding the GPCR has been eliminated (knocked-out) or, where such evidence is available, in studies in which variations in the sequence of the gene encoding the GPCR in humans is linked to the potential to develop a disease. GPR35 is such a GPCR. It is known to be present in the colon and variations in sequence of the protein have been linked to the potential development of inflammatory diseases of the lower gut including Ulcerative Colitis. There may also be a more general role for GPR35 in the regulation of inflammation. For example, some ligands that are able to activate GPR35 have been useful medicines for the treatment of airway/lung inflammatory disease asthma.
There are two major challenges to better understand if GPR35 could be a useful target for new medicines. Firstly, although there are two chemicals available that can block the function of the human form of GPR35, neither of them work at either the rat or mouse versions of GPR35. This means that it is very difficult to explore the function of GPR35 in rodent models of physiology and disease although such models are vital to provide support to build a case that this might be worth testing in human patients. Secondly, as the mechanisms of signal transduction by GPR35 are both unusual and poorly explored it is unclear which of the various signals generated by GPR35 might be most appropriate to mimic or block to treat disease.
In the proposed studies we plan to use a variety of highly innovative approaches to overcome these challenges. We have used a technique called 'gene-editing' to develop a range of cell lines in which only subsets of signals that can be generated by a GPCR are actually induced. These will allow us to assess if different activators of GPR35 are likely to cause different effects in physiological systems. The second key approach will involve the production of transgenic (i.e. genetically modified) mice. Overall, mice and humans have a very similar set of genes. However, in some circumstances ligands that can activate or block a human receptor do not have the same effect at the mouse receptor. This is the case for GPR35. As such, we plan to 'humanise' mice, by replacing the mouse gene for GPR35 with the equivalent gene from humans. This will produce mice in which the responses to GPR35 ligands will be akin to those we would anticipate if we activated or blocked GPR35 in humans and will provide a much clearer picture of how GPR35 ligands might affect the development of treatment of diseases in humans. Although the key objectives of the application are to develop approaches that will deepen our understanding of the roles of GPR35 and how it functions, the results obtained will greatly influence future decisions on whether this receptor might become a new target for the development of novel medicines.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description Although this grant is still in progress we have made key advances in each of three areas. Firstly we have developed assays based on so called SPASM sensors containing the receptor GPR35 and various G proteins that have allowed us to map the G protein selectivity of this receptor and to explain at the level of an individual amino acid why this receptor is able to couple highly selectivity to G13. Secondly, we have made substantial progress in defining the basis for interactions between this receptor and the adaptor proteins arrestin 2 and arrestin3. Thirdly we have regenerated a pair of unique transgenic mouse lines that will allow us to asses the physiological function of GPR35 in a precise manner.
Exploitation Route We are already actively collaborating with groups in the UK and in France to do
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology

 
Description This has led us to a collaboration with the Bio-pharmaceutical company Sosei-Heptares which although separate from this work focuses on a related receptor and has resulted in staff from Glasgow spending time at the company and vice versa.
First Year Of Impact 2019
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Title GPR35 SPASM sensors 
Description We have developed various sensors that are able to report on the ability of ligands to activate the receptor GPR35. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact The sensors described within the publication Mackenzie AE, Quon T, Lin LC, Hauser AS, Jenkins L, Inoue A, Tobin AB, Gloriam DE, Hudson BD, Milligan G. (2019) Receptor selectivity between the G proteins Ga12 and Ga13 is defined by a single leucine-to-isoleucine variation. FASEB J. 2019 Jan 2:fj201801956R. doi: 10.1096/fj.201801956R have been provided to the Californian company Precision IBD under MTA to assist ins their efforts to identify novel drug-like molecules active at GPR35 
 
Title Transgenic mice expressing GPR35 
Description Transgenic 'knock-in' mouse lines expressing, humanised, epitope-tagged and phosphorylation-resistant forms of GPR35 
Type Of Material Biological samples 
Year Produced 2019 
Provided To Others? Yes  
Impact to early 
 
Description GPR35 and lower gut inflammation. 
Organisation French National Institute of Agricultural Research
Country France 
Sector Academic/University 
PI Contribution We characterised GPR35 knock out mice and generate a set of transgenic GPR35 knock-in mouse lines
Collaborator Contribution The partners in France are experts in the gut microbiota and tryptophan metabolism an dare exploring the roles of GPR35 in this context
Impact No outcomes to dates. Animals have only recently been sent to the collaborators.
Start Year 2019
 
Description NMR studies on receptor-arrestin interactions 
Organisation Shandong University
Country China 
Sector Academic/University 
PI Contribution we have provide mutated plasmids, background information and direction of travel to this collaboration
Collaborator Contribution 19F NMR studies
Impact None to date
Start Year 2017
 
Description link to Astra-Zeneca 
Organisation AstraZeneca
Country United Kingdom 
Sector Private 
PI Contribution We have supported the successful application within AstraZeneca for a post-doctoral fellow on how short chain fatty acids regulate B-cell development and function. The ongoing interaction with AZ is also providing us with the opportunity to further develop novel ligands for the DREADD receptor in partnership with AZ by providing libraries of potential ligands for us to screen. This has been extremely fruitful and joint publication detailing this work will be submitted for publication in 2019.
Collaborator Contribution Astra-Zeneca developed the ideas around the internal post-doctoral programme e but wished to use animal models we developed within the BBSRC funded research programme. As this derived from a BBSRC funded IPA with Astra-Zeneca as Industrial partner we were delighted to assist.
Impact No outputs to date, although A Novel Allosteric Activator of Free Fatty Acid 2 Receptor Displays Unique Gi-functional Bias. Bolognini D, Moss CE, Nilsson K, Petersson AU, Donnelly I, Sergeev E, König GM, Kostenis E, Kurowska-Stolarska M, Miller A, Dekker N, Tobin AB, Milligan G. J Biol Chem. 2016 Sep 2;291(36):18915-31. is linked to this
Start Year 2016
 
Description patho-physiogical regulation of GPR35 
Organisation University of Cambridge
Department University of Cambridge, BBSRC IGF Drosophila Genomics Facility
Country United Kingdom 
Sector Academic/University 
PI Contribution generation of transgenic knock in mouse lines expressing various forms of the receptor GPR35
Collaborator Contribution The team at Cambridge have published very interesting studies on the interactions between GPR35 and the sodium potassium pump
Impact Too early
Start Year 2019
 
Description  
IP Reference  
Protection Protection not required
Year Protection Granted
Licensed Yes
Impact none to date