Bioluminescence resonance energy transfer to investigate the cross talk between the formyl peptide receptors

Sadani Cooray1, Adrian Clark2, Rod Flower1, Mauro Perretti1. 1Centre for Biochemical Pharmacology, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, EC1M 6BQ, London, UK, 2Centre for Endocrinology, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, EC1M 6BQ, London, UK.

The human FPR receptor family comprises of 3 members (FPR1, FRP2/ALX and FPR3) which are highly promiscuous in that they are responsive to a variety of agonists leading to a number of different cellular events [Ye RD et al., Pharmacol Rev 2009]. Intriguingly, FPR2/ALX is the first GPCR known to be able to bind to both peptides and lipids [Perretti M et al., Nat Med 2002].

To investigate the versatility and potential ligand biased agonism of FPR2/ALX, we explored the possibility that FPRs may exist as dimeric structures and tested whether these interactions may be modulated by different ligands. We used a transfection approach with tagged receptor plasmids, and anti-tag antibodies for co-immunoprecipitation (Co-IP) experiments, in HEK293 cells. Bioluminescence Resonance Energy Transfer (BRET) was conducted using HEK 293 cells transfected with Renilla luciferase (pRluc) and Enhanced Yellow Fuorescence Protein (pEYFP) tagged FPRs. Readings were collected using the Polarstar Omega plate reader (BMG Labtech). The BRET ratio was defined as emission at 530nm (light emitted by EYFP)/emission at 480nm (light emitted by pRluc). Experiments were repeated three times with distinct cell preparations.

Confocal microscopy showed co-localisation between these receptors. Co-IP did not show an increase in the physical interaction between the FPR1 homodimers or the FPR1-FPR3 heterodimers. The FPR1-FPR2/ALX heterodimerisation, as determined by Co-IP, was not affected in the presence of a number of FPR1 or FPR2/ALX agonists that included: proteins such as human Annexin A1 (AnxA1; 100nM, Serum Amyloid protein A (SAA; 0.1μM), peptides such as peptide Ac2-26 (10μM), antiflammin 2 (AF2; 10μM) and formyl-Met-Leu-Phe (fMLP; 0.1μM), and smaller molecules such as Lipoxin A4 (LXA4; 0.1μM) and compound 43 (from Amgen; 1μM). The BRET signal obtained in HEK293 cells transfected with FPR1-pRluc and FPR1-pEYFP (or FPR3-pEYFP) constructs was not enhanced when cells were stimulated with 10 µM Ac2-26 for 10 min. However, upon treatment with this peptide, a significant enhancement in the BRET signal between FPR1 and FPR2/ALX was observed in cells expressing FPR2/ALX-pRluc and FPR1-pEYFP. The influence of ligand binding on FPR1 and FPR2/ALX was further evaluated using the other agonists and the selective antagonists for FPR1 (Cyclosporin H; 10 µM) or FPR2/ALX (WRW4; 10 µM). The BRET signal was significantly enhanced in the presence of the agonists, but not the antagonists. Monitoring the BRET analysis between FPR1 and FPR2/ALX using a real time protocol, up to 10 min post-stimulation, showed a signal enhancement when the different agonists were used.

Therefore, in contrast to the Co-IP experiments, BRET analyses showed ligand-dependent increase in signal, leading to the suggestion that the signal enhancement may be consequent to conformational changes within each receptor rather than consequent to an increase in the physical interaction between FPR1 and FPR2/ALX proteins.

Funded by the Wellcome Trust (programme grant 086867/Z/08/Z).