Physiological functions and mechanisms of action of the short chain fatty acid receptor FFAR2 dissected using allosteric and biased activating ligands

G protein-coupled receptors (GPCRs) are the most successfully exploited group of proteins for the development of medicines. A variety of GPCRs are activated by metabolic intermediates and play key roles in controlling blood and tissue levels of dietary energy sources, including glucose and fatty acids. This has promoted interest in targeting such receptors as potential treatments for ‘metabolic’ disorders including diabetes. GPCR-targetting medicines traditionally work by interacting with the same part of the receptor as the naturally-produced hormone and are called ‘orthosteric’ ligands. Those that activate the receptor are called agonists and those that block receptor function are called antagonists. GPCRs send signals into cells by activating G proteins and such effects are often limited in time when the GPCR subsequently interacts with an arrestin protein. Many if not all GPCRs can send signals into cells via multiple G proteins and, until recent years, it was broadly assumed that (orthosteric) agonist ligands simply mimicked the same pathways of activation and inactivation produced by the natural hormone. This is described as ‘canonical’ signalling. Recent times have shown this to be far too simplistic. Various orthosteric agonists can display ‘bias’ in that they cause activation of only subsets of the signals associated with the natural ligands, and some also fail to promote interactions with signal terminating arrestins. In addition, many GPCR-activating ligands bind at a site or sites other than where the natural ligands bind. These are termed ‘allosteric’. Because they bind elsewhere they frequently generate signals via distinct ‘non-canonical’ means. As a corollary of this they may act in an additive way, synergise with, or block in a non-competitive manner, actions of orthosteric ligands. This cannot be predicted a priori and must be tested directly. This is generally done initially by examining effects of co-added ligands in cells transfected to express a GPCR of interest at relatively high levels compared to those found in real tissues. Whilst very helpful this can only guide expectations, and such studies then need to be performed in native tissues to provide clearer understanding. For a GPCR called Free Fatty Acid Receptor 2 (FFAR2) three classes of allosteric activators have been described. To understand how they might work we recently obtained atomic level cryo-EM structures of each of these with the GPCR and, via mutagenesis and in silico molecular dynamics simulations, shown how they signal. Whilst two of the classes interact with FFAR2 is a broadly similar way, the third interacts in a completely different manner, and all of them work in ways never previously described for any other GPCR activator. We have also studied how each modulates orthosteric agonist function in transfected cells. We will now take such basic knowledge into tissues isolated from transgenic mouse lines we have developed that are optimised to study functions of FFAR2. This involved replacing mouse FFAR2 with variants of the human form and adding a peptide sequence into the receptor protein to allow easy visualisation of the GPCR and its immunoprecipitation for detailed studies on regulated post-translational modifications. As FFAR2 is associated with metabolic health we will focus on adipose tissue, pancreatic function and hormone release from entero-endocrine cells. These studies will provide insights into how to combine activators of FFAR2 to selectively optimise function across tissues and guide future design of novel medicines.