Defining signal selection from the free fatty acid receptor FFA4; implications for physiological functions

G protein-coupled receptors (GPCRs) are a large family of proteins, located predominantly at the surface of cells, that act as sensors of changes in the external environment of cells and tissues of the body. Although members of the GPCR family are already the most successful class of drug targets, relatively few of them have yet been targeted in this way. We believe this is due to a lack of understanding of the basic underpinning biology of many GPCRs, and a paucity of pharmacological tool compounds and appropriate animal models to test their in vivo function in both normal physiology and in the context of disease. Although traditionally viewed as being designed to detect and instruct cells to respond to alterations in the concentration of various hormones and neurotransmitters, in recent times it has become clear that a substantial number of GPCRs are designed to respond directly to alterations in metabolites that are produced by digestion of foodstuffs and, in so doing, they alert organs of the body to co-ordinate responses to either a dearth or a surplus of nutrients. Given that many diseases that are currently rising to epidemic proportions, e.g. diabetes and obesity, develop from a 'Western lifestyle' of intake of a marked excess of highly calorific foods and a lack of exercise, then mimicking or blocking the effects of the GPCRs for such metabolic products may have beneficial effects. Indeed, for the receptor we plan to study in detail, which is called free fatty acid receptor 4 (FFA4), understanding of its ability when activated to regulate blood glucose levels, and to increase the effectiveness of the hormone insulin, has resulted in ongoing efforts by the pharmaceutical industry to assess if activation of FFA4 might be a novel approach to treat type II diabetes.
Like many other GPCRs, FFA4 is expressed by range of types of cells in the body, leading to suggestions that either activating or blocking this receptor might also have benefits in other conditions. It is clear that FFA4 can instruct cells to respond by controlling a number of different signalling pathways and in the proposed studies we plan to unravel this complexity and to understand which signals are predominant in different types of cells and tissues and how this determines the outcome of activating FFA4. We will do this in a number of ways. One of the most exciting and important will be to utilise cells and tissues from mice, that in our current studies on this receptor that BBSRC have funded, we have genetically engineered to express a form of FFA4 that can only interact with so called G proteins and not with arrestins. Arrestins are a key set of signalling proteins that interact strongly with activated FFA4. Studies that compare functions of FFA4 in cells and tissues from normal and arrestin-interaction deficient mice will provide this information, and this will be examined in tissues that range from different types of fat cells, to cells of the immune system. These have been selected because of the roles FFA4 has been suggested to play in areas that range from fat cell development and inflammation, to how resistance develops to drugs that are used to shrink cancers in chemotherapy.
In parallel with these large, overarching objectives we also plan to develop and utilise chemicals that block the actions of FFA4 that are much improved on those currently available, map in detail the expression pattern of FFA4 and employ cells we have developed from current BBSRC funding that have been 'genome-edited' to eliminate specific subsets of signalling pathways to allow us to assess if different types of molecule that activate FFA4 can selectively control the signalling potential of FFA4. By the end of the proposed studies FFA4 will no longer be a poorly studied GPCR and we will be in position to state with some confidence if regulating this receptor might be a means to treat 'Western lifestyle' diseases beyond diabetes.

Upon activation FFA4 can engage with a broad range of individual G protein subtypes and, following subsequent phosphorylation, interacts strongly with members of the arrestin family of signalling adaptors. FFA4 is expressed by cells as diverse as those of the entero-endocrine system, the immune system, airway epithelial cells and adipocytes. Although described as a 'Gq-coupled' receptor it is clear from combinations of chemical inhibitors, second messenger regulation measurements and signalling pathway knock-down studies that only subsets of FFA4 functions are actually transduced by 'Gq-coupled' mechanisms. In particular, previous studies suggest that FFA4-mediated blockade of the production of anti-inflammatory mediators from macrophages reflects engagement of the receptor with beta-arrestin-2 and that FFA4-induced release of the satiety hormone ghrelin requires activation of Gi-family G proteins. To fully understand the expression pattern of FFA4 and to address which signals are integral to different FFA4-mediated physiological end points we have generated transgenic 'knock-in' lines of mice that express either an epitope-tagged form of wild type FFA4 or a phosphorylation-deficient variant of the receptor in which agonist-induced interaction with arrestins is all but ablated. These animals will be used for both ex vivo and in vivo analysis of the key signals that are required to allow FFA4 to regulate white adipocyte differentiation, the browning of white fat, and the release from immune cells of both inflammatory mediators and a paracrine regulator of the efficacy of platinum-containing chemotherapeutics. In parallel, we will characterise and use novel series of FFA4 antagonists and employ both HEK293 cell lines that have been 'genome-edited' to eliminate signalling via either G protein subsets or arrestins and sensors of both G protein and arrestin activation to assess if either distinct agonist ligands differentially regulate FFA4 signalling pathways.

As noted in 'who will benefit from the research', there is a widespread interest in efforts to better understand the key biological functions and mechanisms of action of GPCRs. This is designed to improve confidence in the potential to target specific members of the GPCR superfamily in a therapeutic context as well as to provide greater basic knowledge of the roles of this important family of proteins. As highlighted in 'Case for Support', Caldan Therapeutics, co-founded by Milligan and an academic chemist based in Denmark, is assessing the potential for agonists of FFA4 to become novel medicines for the treatment of type II diabetes. In part, this 'spin-out' stemmed from and was underpinned by studies funded by BBSRC. Clearly Caldan Therapeutics is not the only company with an active programme in this therapeutic area that is considering FFA4 as a potential target. As such work resulting from this application is likely to be actively studied within the pharmaceutical industry. More broadly, because this application focuses on functions of FFA4 that are not directly related to glucose homeostasis then outcomes from the studies will be monitored carefully by the pharmaceutical industry for indications that ligands at FFA4 might find opportunities in a different set of therapeutic settings. Currently the view is that successful targeting of FFA4 in diabetes will require molecules with agonist action. However, as highlighted in the proposal and through the 'Open Innovation' platform established and co-ordinated by Astra-Zeneca we have been able to access a number of novel FFA4 receptor antagonists. At this stage these are poorly characterised, but once we have done so they will provide both important tool compounds for broader analysis of functions of FFA4 and may act to stimulate interest in areas such as limiting the development of resistance to the effectiveness of chemotherapeutic drugs that we have highlighted as a potential outcome from the studies. Once more the pharmaceutical industry will follow the outcome of these studies closely.
The project also has great potential in terms of staff training. The PDRA will benefit from opportunities to perform cutting edge research in a broad swathe of areas relevant to modern pharmacological and physiological studies. Moreover, for the part time technician the studies they will perform will provide links to the pharmaceutical industry because we will share outcomes of analysis of the potential FFA4 antagonists with Astra-Zeneca ahead of public disclosure. Moreover, the sets of collaborations we have established with internationally leading laboratories and teams to deliver key elements of the Programme of Work in which the PIs have less direct experience, will enhance team- and networking opportunities for the PDRA. This training will ensure the greatest range of subsequent career opportunities.