Pathophysiological regulation of lung function by the free fatty acid receptor FFA4.

Increased prevalence of inflammatory airway diseases including asthma and chronic obstructive pulmonary disease (COPD) together with inadequate disease control by current frontline treatments means that there is a need to define therapeutic targets for these conditions. Here, we investigate a member of the G protein-coupled receptor family, FFA4, that responds to free circulating fatty acids including dietary omega-3 fatty acids found in fish oils. We show that FFA4, although usually associated with metabolic responses linked with food intake, is expressed in the lung where it is coupled to Gq/11 signaling. Activation of FFA4 by drug-like agonists produced relaxation of murine airway smooth muscle mediated at least in part by the release of the prostaglandin E2 (PGE2) that subsequently acts on EP2 prostanoid receptors. In normal mice, activation of FFA4 resulted in a decrease in lung resistance. In acute and chronic ozone models of pollution-mediated inflammation and house dust mite and cigarette smoke-induced inflammatory disease, FFA4 agonists acted to reduce airway resistance, a response that was absent in mice lacking expression of FFA4. The expression profile of FFA4 in human lung was similar to that observed in mice, and the response to FFA4/FFA1 agonists similarly mediated human airway smooth muscle relaxation ex vivo. Our study provides evidence that pharmacological targeting of lung FFA4, and possibly combined activation of FFA4 and FFA1, has in vivo efficacy and might have therapeutic value in the treatment of bronchoconstriction associated with inflammatory airway diseases such as asthma and COPD.

FFA4/GPR120: Pharmacology and Therapeutic Opportunities.

Free Fatty Acid receptor 4 (FFA4), also known as GPR120, is a G-protein-coupled receptor (GPCR) responsive to long-chain fatty acids that is attracting considerable attention as a potential novel therapeutic target for the treatment of type 2 diabetes mellitus (T2DM). Although no clinical studies have yet been initiated to assess efficacy in this indication, a significant number of primary publications and patents have highlighted the ability of agonists with potency at FFA4 to improve glucose disposition and enhance insulin sensitivity in animal models. However, the distribution pattern of the receptor suggests that targeting FFA4 may also be useful in other conditions, ranging from cancer to lung function. Here, we discuss and contextualise the basis for these ideas and the results to support these conclusions.

Structural insight into allosteric modulation of protease-activated receptor 2.

Protease-activated receptors (PARs) are a family of G-protein-coupled receptors (GPCRs) that are irreversibly activated by proteolytic cleavage of the N terminus, which unmasks a tethered peptide ligand that binds and activates the transmembrane receptor domain, eliciting a cellular cascade in response to inflammatory signals and other stimuli. PARs are implicated in a wide range of diseases, such as cancer and inflammation. PARs have been the subject of major pharmaceutical research efforts but the discovery of small-molecule antagonists that effectively bind them has proved challenging. The only marketed drug targeting a PAR is vorapaxar, a selective antagonist of PAR1 used to prevent thrombosis. The structure of PAR1 in complex with vorapaxar has been reported previously. Despite sequence homology across the PAR isoforms, discovery of PAR2 antagonists has been less successful, although GB88 has been described as a weak antagonist. Here we report crystal structures of PAR2 in complex with two distinct antagonists and a blocking antibody. The antagonist AZ8838 binds in a fully occluded pocket near the extracellular surface. Functional and binding studies reveal that AZ8838 exhibits slow binding kinetics, which is an attractive feature for a PAR2 antagonist competing against a tethered ligand. Antagonist AZ3451 binds to a remote allosteric site outside the helical bundle. We propose that antagonist binding prevents structural rearrangements required for receptor activation and signalling. We also show that a blocking antibody antigen-binding fragment binds to the extracellular surface of PAR2, preventing access of the tethered ligand to the peptide-binding site. These structures provide a basis for the development of selective PAR2 antagonists for a range of therapeutic uses.

Distinct Phosphorylation Clusters Determine the Signaling Outcome of Free Fatty Acid Receptor 4/G Protein-Coupled Receptor 120.

It is established that long-chain free fatty acids includingω-3 fatty acids mediate an array of biologic responses through members of the free fatty acid (FFA) receptor family, which includes FFA4. However, the signaling mechanisms and modes of regulation of this receptor class remain unclear. Here, we employed mass spectrometry to determine that phosphorylation of mouse (m)FFAR4 occurs at five serine and threonine residues clustered in two separable regions of the C-terminal tail, designated cluster 1 (Thr(347), Thr(349), and Ser(350)) and cluster 2 (Ser(357)and Ser(361)). Mutation of these phosphoacceptor sites to alanine completely prevented phosphorylation of mFFA4 but did not limit receptor coupling to extracellular signal regulated protein kinase 1 and 2 (ERK1/2) activation. Rather, an inhibitor of Gq/11proteins completely prevented receptor signaling to ERK1/2. By contrast, the recruitment of arrestin 3, receptor internalization, and activation of Akt were regulated by mFFA4 phosphorylation. The analysis of mFFA4 phosphorylation-dependent signaling was extended further by selective mutations of the phosphoacceptor sites. Mutations within cluster 2 did not affect agonist activation of Akt but instead significantly compromised receptor internalization and arrestin 3 recruitment. Distinctly, mutation of the phosphoacceptor sites within cluster 1 had no effect on receptor internalization and had a less extensive effect on arrestin 3 recruitment but significantly uncoupled the receptor from Akt activation. These unique observations define differential effects on signaling mediated by phosphorylation at distinct locations. This hallmark feature supports the possibility that the signaling outcome of mFFA4 activation can be determined by the pattern of phosphorylation (phosphorylation barcode) at the C terminus of the receptor.