A Novel Allosteric Activator of Free Fatty Acid 2 Receptor Displays Unique Gi-functional Bias

Abstract

The short chain fatty acid receptor FFA2 is able to stimulate signaling via both Gi- and Gq/G11-promoted pathways. These pathways are believed to control distinct physiological end points but FFA2 receptor ligands appropriate to test this hypothesis have been lacking. Herein, we characterize AZ1729, a novel FFA2 regulator that acts as a direct allosteric agonist and as a positive allosteric modulator, increasing the activity of the endogenously produced short chain fatty acid propionate in Gi-mediated pathways, but not at those transduced by Gq/G11. Using AZ1729 in combination with direct inhibitors of Gi and Gq/G11 family G proteins demonstrated that although both arms contribute to propionate-mediated regulation of phospho-ERK1/2 MAP kinase signaling in FFA2-expressing 293 cells, the Gq/G11-mediated pathway is predominant. We extend these studies by employing AZ1729 to dissect physiological FFA2 signaling pathways. The capacity of AZ1729 to act at FFA2 receptors to inhibit β-adrenoreceptor agonist-promoted lipolysis in primary mouse adipocytes and to promote chemotaxis of isolated human neutrophils confirmed these as FFA2 processes mediated by Gi signaling, whereas, in concert with blockade by the Gq/G11 inhibitor FR900359, the inability of AZ1729 to mimic or regulate propionate-mediated release of GLP-1 from mouse colonic preparations defined this physiological response as an end point transduced via activation of Gq/G11.

Interdependent Allosteric FFA2R Modulators Synergistically Induce Functional Selective Activation and Desensitization in Neutrophils

“…No direct neutrophil activating effect was induced by AZ1729 (structure shown in Fig 1A), as shown by a lack of a change in [Ca 2+ ] i (Fig 1C). This is in agreement with the initial description of AZ1729 as a positive specific allosteric FFA2R modulator (Bolognini et al, 2016), as well as with the results obtained earlier with another allosteric FFA2R modulator, Cmp58 (Fig 1B and D; (Lind, 2019a; Martensson et al, 2018)). Further, AZ1729 positively modulated the response induced by propionate, an orthosteric FFA2R agonist that triggered a concentration-dependent transient increase in [Ca 2+ ] i in neutrophils (Fig 2A); the modulating effect was evident with propionate concentrations that were unable to induce an increase in [Ca 2+ ] i when added alone (Fig 2A).…”

“…Fura 2-AM was from Molecular Probes/Thermo Fisher Scientific (Waltham, MA, USA), and horseradish peroxidase (HRP) was obtained from Boehringer Mannheim (Mannheim, Germany). The allosteric FFA2R modulator AZ1729 (Bolognini et al, 2016) was a generous gift from AstraZeneca (Mölndal, Sweden) and the phenylacetamide compound (S)-2-(4-chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl)butanamide (PA;Cmp58 (Wang et al, 2010)) was obtained from Calbiochem-Merck Millipore (Billerica, USA) The Gαq inhibitor YM-254890 was purchased from Wako Chemicals (Neuss, Germany). The FFA2R antagonist CATPB ((S)-3-(2-(3-chlorophenyl)acetamido)-4-(4-(trifluoromethyl)-phenyl) butanoic acid) synthesized as described previously (Due-Hansen et al, 2015; Hudson et al, 2013; Hudson et al, 2012), was a generous gift from Trond Ulven (Odense university, Denmark).…”

“…A novel allosteric FFA2R ligand, AZ1729, was recently described and shown to be both a positive allosteric FFA2R modulator that increased the activity induced by conventional orthosteric agonists and a direct activating allosteric agonist (Bolognini et al, 2016). It is also interesting to note, that the allosterically modulated FFA2Rs were able to signal through different G-proteins containing the G α i and G α q subunits, respectively (“induced-bias” (Bolognini et al, 2016; Kenakin, 2015).…”

Host–microbiota interactions: epigenomic regulation

“…Recently, the microbial-derived short chain fatty acids (SFCAs) that are produced by commensal bacteria, such as Clostridia and Bifidobacteria , from fermentation of carbohydrates and fiber have emerged as central players mediating crosstalk between the microbiota and host [18,19]. In particular, propionate, acetate and butyrate, the three most abundant SCFAs in the intestinal lumen, have received increasing attention in the field due to their potential beneficial impact on host physiology including reduced inflammation and enhanced epithelial barrier function, although these effects have varied between studies [3,20–24].…”

Receptor selectivity between the G proteins Gα 12 and Gα 13 is defined by a single leucine‐to‐isoleucine variation

“…For example, although free fatty acid receptor 4 promotes secretion of the incretin glucagon‐like peptide 1 by activation of pertussis toxin–insensitive G proteins of the Gα q/11 family (14), regulation of release of the satiety hormone ghrelin requires activation of one or more pertussis toxin‐sensitive Gα i ‐family G protein (15). Equally, although free fatty acid receptor 2 acts to counter lipolysis in mouse white adipose tissue via a pertussis toxin–sensitive mechanism (16), effects of this receptor on secretion of glucagon‐like peptide 1 are instead mediated by Gα q/11 ‐family G proteins (16). Although such examples are clearly defined, there is often less understanding of the importance of selective interactions of a receptor with different members from within one of the G protein families and little insight into the molecular basis of such selectivity, and there are currently no comparative structures of a single GPCR in complex with 2 different G proteins.…”