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

Abstract

The non-activating allosteric modulator AZ1729, specific for free fatty acid receptor 2 (FFA2R), transfers the orthosteric FFA2R agonists propionate and the P2Y2R specific agonist ATP into activating ligands that trigger an assembly of the neutrophil superoxide generating NADPH-oxidase. The homologous priming effect on the propionate response and the heterologous receptor cross-talk sensitized ATP response mediated by AZ1729 are functional characteristics shared with Cmp58, another non-activating allosteric FFA2R modulator. In addition, AZ1729 also turned Cmp58 into a potent activator of the superoxide generating neutrophil NADPH-oxidase, and in agreement with the allosteric modulation concept, the effect was reciprocal in that Cmp58 turned AZ1729 into a potent activating allosteric agonist. The activation signals down-stream of FFA2R when stimulated by the two interdependent allosteric modulators were biased in that, unlike for orthosteric agonists, the two complementary modulators together triggered an activation of the NADPH-oxidase, but not any transient rise in the cytosolic concentration of free Ca2+. In addition, following AZ1729/Cmp58 activation, signaling by the desensitized FFA2Rs was functionally selective in that the orthosteric agonist propionate could still induce a transient rise in intracellular Ca2+. The novel neutrophil activation and receptor down-stream signaling pattern mediated by the two cross-sensitizing allosteric FFA2R modulators represents a new regulatory mechanism that controls receptor signaling.Significance StatementA novel activation mechanism of a G-protein coupled free fatty acid receptor (FFA2R), is synergistically triggered by two otherwise non-activating allosteric modulators in the absence of orthosteric agonists. The receptor down-stream signaling proved to be functionally selective (biased); a superoxide generating enzyme is assembled and activated without involvement of the Ca2+ signaling pathway. The novel activation mechanism and the receptor down-stream signaling pattern mediated by the two cross-sensitizing allosteric FFA2R modulators, represents a new regulatory mechanism for control of GPCR-signaling.

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

“…A tentative binding/signaling/activation-model, postulating three receptor binding sites in FFAR2 is presented. This model is based on data showing that the non-activating allosteric FFAR2 modulators AZ1729 and Cmp58 interdependently activate neutrophils to produce/release superoxide (O2 -), and that the signaling profile downstream of the receptor is biased (10) in that no transient rise in intracellular Ca 2+ is triggered during this activation of FFAR2. Neutrophil activation by orthosteric FFAR2 agonists is modulated in the same way by the non-activating compounds Cmp58 and AZ1729 but the enhanced response is achieved through binding of the two modulators to distinctly different allosteric receptor sites.…”

“…It is interesting to note, that the allosterically modulated FFAR2s were able to signal through different G-proteins containing either Gai or Gaq subunits ("induced-bias" (12,13)). Based on earlier findings, it is reasonable to assume that FFAR2 has multiple ligand binding sites allowing different allosteric modulators to distinctly affect the affinity/efficacy of orthosteric ligands; we recently showed that AZ1729 lacks direct activating effects in its own but turns the natural FFAR2 agonist propionate into a potent activator of the neutrophil NADPH-oxidase, and that neutrophils are activated by the non-activating modulator AZ1729 when combined with another non-activating allosteric modulator, Cmp58 (10). The novel activation/sensitization mediated by the two allosteric FFAR2 modulators was reciprocal, and represented a new regulatory mechanism that controls GPCR signaling.…”

“…Such an allosteric GPCR modulation is, according to the dogma for allosteric GPCR-modulation, solely affecting the signaling properties of agonists that specifically interact with the receptor that is allosterically modulated (6,7). This GPCR-modulation dogma has, however, been challenged and shown to be more complex than initially anticipated, when it was shown that allosteric modulators specific for Free Fatty Acid Receptor 2 (FFAR2) affect signaling mediated not only by orthosteric FFAR2 agonists but also by specific agonists for the ATP-receptor P2Y2R and for formyl peptide receptors (FPRs; (8)(9)(10).…”

“…The copyright holder for this this version posted October 27, 2020. ; https://doi.org/10.1101/2020.10.27.356923 doi: bioRxiv preprint 5 transient rise in the cytosolic concentration of free Ca 2+ ([Ca 2+ ]i); (10)). The results obtained suggested that the two allosteric FFAR2 modulators (Cmp58 and AZ1729) were recognized by FFAR2 through different receptor sites.…”

Functional and signaling characterization of the neutrophil FPR2 selective agonist Act-389949

“…The copyright holder for this preprint (which was this version posted March 8, 2019. ; https://doi.org/10.1101/571604 doi: bioRxiv preprint To further explore Act-389949 mediated GPCR cross-talk in human neutrophils, we determined the Act-389949 induced neutrophil response in the presence of an FFA2R (free fatty acid receptor 2) specific allosteric modulator [54]. This allosteric modulator (Cmp58) has been shown to prime neutrophils for the responses induced by low concentrations of FPR agonists, a priming achieved through a novel receptor cross-talk mechanism [54,55]. In accordance with published data, the NADPH-oxidase activity induced by low nanomolar concentrations of Act-389949 in neutrophils pre-incubated with the allosteric FFA2R modulator was also augmented compared to the response in naïve neutrophils (Fig 6C).…”