# Free fatty acid receptor 2 allosterism is defined by cellular context

## TL;DR

This study shows how the FFA2R receptor's response to allosteric drugs depends on the cell type, with implications for drug development.

## Contribution

The paper reveals that FFA2R allosterism is cell-context dependent, with distinct signaling outcomes in different cell models.

## Key findings

- FFA2R activates multiple G proteins and recruits β-arrestin in HEK293 cells.
- Allosteric ligands Cmp58 and AZ1729 act as pathway-selective ago-PAMs, modulating G protein activity in the presence of propionate.
- FFA2R in neutrophils and HL60 cells shows a conserved Ca²⁺ response and ROS generation when allosterically modulated.

## Abstract

Allosteric modulators offer a way to fine-tune GPCR signaling in the presence of endogenous ligands. The short-chain fatty acid receptor FFA2R (GPR43) recognizes propionate and allosteric ligands such as Cmp58 and AZ1729. We characterized FFA2R signaling and allosteric modulation using multiple cell models including HEK293, HL60 cells and primary human neutrophils.

FFA2R activation was assessed using complementary assays in HEK293 and HL60 cells as well as primary human neutrophils. G protein activation and β-arrestin recruitment were profiled using ebBRET biosensors. Ca2+ mobilization was measured with Fura-2, and reactive oxygen species (ROS) generation was quantified by isoluminol chemiluminescence. Pharmacological tools included the FFA2R antagonist CATPB, the Gαq inhibitor YM-254,890, and pertussis toxin (PTX).

Propionate activated all tested G proteins except Gα12 in HEK293 cells and recruited both β-arrestin1 and β-arrestin2. The allosteric ligands Cmp58 and AZ1729 behaved as pathway-selective ago-PAMs. Alone they engaged a limited subset of G proteins with minimal β-arrestin recruitment, whereas in the presence of propionate they selectively potentiated Gαi1 while attenuating Gαq/11 and Gαi2/3. Fura-2 measurements coupled to YM-254,890 treatment established that FFA2R couples to Gαq-dependent Ca²⁺ mobilization in HEK293 cells; Cmp58, but not AZ1729, enhanced Ca²⁺ responses at submaximal propionate concentrations. In primary neutrophils, propionate elicited Ca²⁺ transients but did not trigger NADPH oxidase–dependent ROS on its own. Either Cmp58 or AZ1729 enabled propionate-driven ROS, and their combination produced robust ROS. Transient FFA2R expression in HL60 cells reconstituted this neutrophil-like functional profile, including allosteric activation of ROS.

Across systems, Ca²⁺ mobilization emerged as a conserved, receptor-proximal output of FFA2R, while allosteric modulation by Cmp58 and AZ1729 promoted Gαi/o-biased signaling that enabled ROS generation. These data define pathway-selective allosterism at FFA2R and highlight Ca²⁺ mobilization and ROS as informative readouts for therapeutic strategies that exploit allosteric control.

The online version contains supplementary material available at 10.1186/s12964-026-02730-5.