Identification of diverse functional domains in FPR2/ALX receptor activation by selective agonists

Stefania Bena, Vincenzo Brancaleone, Roderick J. Flower, Mauro Perretti. William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London, EC1M 6BQ, United Kingdom.

Inflammation is a protective response of the organism against injurious stimuli, affording their containment and removal to then initiate the healing process. When run un-controlled, however, inflammation is also key to tissue damage and injury. This project focuses on one effector of endogenous anti-inflammation, the G protein coupled receptor (GPCR) belonging to the formyl peptide (FPR) receptor family, termed FPR2/ALX. FPR2/ALX conveys the homeostatic signals of annexin A1 (AnxA1), a glucocorticoid inducible protein, and the short-lived lipid lipoxin A4. This receptor binds a variety of ligands including the pro-inflammatory mediator serum amyloid A (SAA) and the synthetic small molecule compound 43 (C43). We aimed, here, to identify the differential functional domains of FPR2/ALX ligands and define their impact on AnxA1 receptor-mediated responses. We also studied FPR2/ALX receptor heterologous desensitization.

HEK-293 cells transfected with naïve FPR2/ALX were loaded with FURA-2 (2 µM) and calcium fluxes determined as readout upon stimulation with AnxA1, SAA and C43 (concentrations ranging from 10nM to 1μM), with results being reported as % of the Ionomycin (1 µM) response, taken as 100%. To compare the ligand-binding sites activated by AnxA1, SAA and C43, calcium mobilization was also studied in HEK-293 cells transfected with chimaeric receptors, formed by distinct domains of FPR1 and FPR2/ALX, and kindly provided by Dr JM Wang (NIH) [1]. In selected experiment Western blotting analysis was used to monitor post-receptor ERK 1/2 activation (rabbit anti-phospho-p44/42 MAP kinase, and anti-total-p44/42, clone 137F5; Cell Signalling, Cambridge, USA).

AnxA1 elicited ∼40% calcium mobilization in cells with intact FPR2/ALX, optimal at 10 nM, and required the N-terminus (40% calcium mobilization) and the II and the III extracellular loops (60% of calcium mobilization) of FPR2/ALX. SAA (0.1 µM) afforded ∼50% of the Ionomycin response with naïve FPR2/ALX and chimaeric cells with the I and the II extracellular loops. No responses were observed in clones with the other loops (p<0.001). FPR2/ALX responsiveness to C43 (1-3 µM), which led to ∼85% of Ionomycin response, required only the I extracellular loop for full agonism. The same receptor domains were also required/sufficient to induce ERK 1/2 activation (10 min incubation time for all ligands). In desentization experiments, FPR2/ALX activation by AnxA1 was significantly desensitized (p<0.001) after 5 min pre-treatment with AnxA1, SAA and C43, i.e. either in homologous or heterologous ways. With the AnxA1 treatment, the desensitization of the FPR2/ALX receptor required the II and the III extracellular loops (40% of calcium mobilization, p<0.01) whereas the N-terminal domain was dispensed.

We have used HEK-293 cells transfected with whole or chimaeric FPR2/ALX isoforms to identify the domains responsible for AnxA1 activation, in relation to other agonists, and receptor desensitization.

1. Le, Y., R. D. Ye, W. Gong, J. Li, P. Iribarren, and J. M. Wang. 2005. Identification of functional domains in the formyl peptide receptor-like 1 for agonist-induced cell chemotaxis. Febs J 272:769-778.

Supported by the Wellcome Trust (programme 086867/Z/08)