PhD Scientific Days 2026

Poster Session 2.B - Molecular Medicine

Engineering a Fluorescent Biosensor for Detecting Endogenous Ligands of the Orphan Receptor GPR84

Name of the presenter

Kaszás, Diána

Institute/workplace of the presenter

SE Department of Phisiology

Authors

Kaszás Diána¹, Szekeres Alíz¹, Benoa Roux¹, Vágóné Kiss Klaudia¹, Puskás Attila¹, Enyedi Balázs¹
1: SE Department of Phisiology

Text of the abstract

Introduction
GPR84 is an inflammation-induced G protein-coupled receptor (GPCR) expressed in myeloid cells and upregulated in neutrophils and monocytes during inflammatory responses. Although initially proposed to function as a medium-chain fatty acid receptor, this has increasingly been questioned. Its responsiveness to diverse synthetic agonists and the proof of multiple ligand-binding sites suggests that GPR84 may recognize non-lipid endogenous ligands. Thus, the physiological ligands and activation dynamics of GPR84 remain poorly defined.
Aims
We aimed to develop genetically encoded fluorescent biosensors based on GPR84 for detection of receptor activation in vivo. By generating orthologue-specific variants for the human, mouse and zebrafish receptors, we sought to facilitate identification of endogenous GPR84 ligands and characterize GPR84 activation in inflammatory contexts.
Methods
Orthologue receptor activation by the synthetic agonist 6-OAU was first validated in Gqi5-assisted calcium measurements using Fluo-4 in human, mouse, and zebrafish GPR84-expressing Hek293 cells. GPR84-based fluorescent biosensors were then engineered by inserting cpEGFP into the intracellular regions of the receptor and by generating libraries through linker length variation and subsequent randomization of linker sequences, ICL2, and cpEGFP. Variants were screened in Hek293 cells by FACS-based enrichment followed by fluorescence microscopy.
Results
Calcium assays confirmed activation of human, mouse and zebrafish GPR84 orthologues by 6-OAU. A first-generation GPR84-GEM variant showed a ligand-induced fluorescence response with an EC50 of 1.17 ± 0.30 µM. Transfer of the optimized linker design to murine and zebrafish GPR84 yielded functional orthologue biosensors that also responded to 5 µM 6-OAU. Second-generation libraries are under screening, while ICL2-targeted designs showed impaired membrane localization and require further optimization.
Conclusion
Development of a functional GPR84-based fluorescent biosensor provides a tool for visualizing receptor activation with high spatial and temporal resolution. This approach supports in vivo studies using orthologue-specific sensors and identification of physiologically relevant GPR84 ligands.
Funding
NRDIO K 147351, ADVANCED 152292, 2025-1.2.1-HURIZONT-2025-00025 and Semmelweis Egyetem Predoktori Ösztöndíj