Multiple ligand recognition sites in free fatty acid receptor 2 (FFAR2) direct distinct neutrophil activation patterns

AbstractNon-activating positive allosteric modulators specific for free fatty acid receptor 2 (FFAR2) increased the activity induced by orthosteric agonists to trigger a rise in intracellular Ca2+ ([Ca2+]i) and activate the O2− producing neutrophil NADPH-oxidase. In addition, two allosteric modulators (Cmp58 and AZ1729) recognized by different receptor domains on FFAR2, cooperatively triggered activation without any rise in [Ca2+]i. To gain insights into FFAR2 modulation and signaling, we set out to identify structurally diverse allosteric FFAR2 modulators. Initially, we identified two molecules that directly activate neutrophils and these were classified as an allosteric FFAR2 agonists and an orthosteric agonist, respectively. Based on the sensitizing effect on the neutrophil response to propionate, ten non-direct-activating molecules were classified as allosteric FFAR2 modulators. One of these synergistically activated neutrophils when combined with AZ1729, but not when combined with Cmp58. The remaining nine compounds synergistically induced the same type of biased neutrophil signaling but only when combined with Cmp58. The activation signals down-stream of FFAR2 when stimulated by two allosteric modulators with different binding sites were in most cases biased in that two complementary modulators together triggered an activation of the NADPH-oxidase, but no increase in [Ca2+]i. The neutrophil activation pattern achieved when two functionally “AZ1729- or “Cmp58-like” allosteric FFAR2 modulators were combined, supporting a model for activation in which FFAR2 has two different sites that selectively bind allosteric modulators. The novel neutrophil activation patterns and receptor down-stream signaling mediated by two cross-sensitizing allosteric modulators represent a new regulatory mechanism that controls FFAR2 receptor function.