Signals Generated by Neutrophil Receptors for Danger Molecules Transactivate Allosterically Modulated FFA2R: Distinct response patterns are mediated by modulators recognized by different allosteric receptor sites

Abstract

Positive allosteric modulators for free fatty acid receptor 2 (FFA2R/GPR43), that affect receptor function through binding to two distinct allosteric binding sites, were used to determine the correlation between the responses induced in neutrophils by two distinct activation modes; FFA2R was activated either by the orthosteric agonist propionate or by a receptor transactivation mechanism that activated FFA2R from the cytosolic side of the neutrophil plasma membrane by signals generated by the neutrophil PAFR (receptor for platelet activating factor), P2Y2R (receptor for ATP), FPR1 (receptor for fMLF) and FPR2 (receptor for WKYMVM). We show that the transactivation signals that activate FFA2R in the absence of any orthosteric agonist were generated downstream of the signaling G protein that couple to PAFR and P2Y2R. This transactivation of allosterically modulated FFA2Rs, by signals generated by PAFR/P2Y2R, represents a novel mechanism by which a G protein coupled receptor can be activated. Weak correlations were obtained when the FFA2R activity was induced by the transactivation signals generated by PAFRs and P2Y2Rs were compared with the FFA2R activity induced by the orthosteric agonist propionate. Comparison of the responses for each allosteric modulator revealed that the ratio values, calculated from the peak values of the ATP and propionate responses, varied from 0.2 to 1. Depending on the allosteric modulator, the response induced by the two different mechanisms (orthosteric activation and receptor transactivation, respectively), was equal or the propionate response was more pronounced. Importantly, we conclude that FFA2R activation from outside (orthosteric activation) and inside (receptor cross-talk/transactivation) can be selectively affected by an allosteric FFA2R modulator.The allosterically modulated FFA2R is transactivated by signals generated by other GPCRs.The PAF and ATP receptors transactivate FFA2R from the cytosolic side of the membrane.The mechanisms that regulates activation of FFA2R from outside and inside differ.

G protein coupled pattern recognition receptors expressed in neutrophils: Recognition, activation/modulation, signaling and receptor regulated functions

“…FPR agonists is positively modulated (Figure 8). 145,159 The orthosteric agonist propionate is not able to replace the allosteric modu- Notably, the allosteric FFA2R modulators, have off target effects also on the response induced non-activating concentrations of FPR agonists. This suggests that not only the Gαq-coupled PAFR/P2Y 2 R but also receptors such as the FPRs, that couple to Gαi containing G proteins have the capacity to transactivate the allosterically modulated FFA2R from inside the plasma membrane.…”

“…Accordingly, FFA2R specific PAMs turns orthosteric FFA2R agonist into potent neutrophil activating agonists, but in addition the response induced also by ATP, PAF, and FPR agonists is positively modulated (Figure 8). 145,159 The orthosteric agonist propionate is not able to replace the allosteric modulator in this receptor transactivation process. The mechanism by which the agonist‐occupied PAFRs/P2Y 2 Rs activates allosterically modulated FFA2R, involves not yet identified intracellular receptor transactivation signals generated by PAFR/P2Y 2 R. The signaling is initiated down‐stream of the Gαq containing G protein coupled to PAFR/P2Y 2 R, and the signals generated transactivate FFA2Rs from the cytosolic side of the plasma membrane.…”