Annexin-1 (ANXA1), has been described as possessing anti-inflammatory activity in a range of models. The mechanism(s) by which ANXA1 exerts these biological effects is unclear, however recent evidence suggests that it may interact with G-protein-coupled receptors for formylated peptides, termed FPR and FPRL1/ALXR (Walther et al., 2000; Perretti et al., 200 2). In this study we determined whether ANXA1 or its bio-active peptides bound to either the FPR or ALXR receptor, and whether such interactions modulated the activation of the MAP kinase Erk 1/2, a common signalling event linked to this family of receptors.

HEK-293 cells were stably transfected with constructs encoding the human FPR and ALXR receptors. Transfected cells were used in binding studies, employing an iodinated N-terminal peptide of ANXA1, Ac2-26 ( Amersham). Unlabelled homoligand, human recombinant ANAX1 ( hr-ANXA1) and the ANXA1 nonapeptide, antiflammin-2 (AF-2; HDMNKVLDL, residues 246-254) were also tested as well as a selective agonist for human ALXR, W-peptide (WKYMVm). Activation of Erk in response to ANXA1/peptide exposure was assessed by western blotting using a phospho-specific antibody (detects both Erk 1 and 2) and normalised to total cellular Erk content (Cell Signalling Technology). All data are from n=3-4 distinct experiments.

Analysis of Ac2-26 binding using classical saturation binding assays proved problematic, as the specific binding curve broke down at concentrations above 12 nM ligand. Homoligand competition binding however, demonstrated that iodinated Ac2-26 competed at both the FPR and ALXR receptors with mean EC₅₀ values of 1.4 and 1.8 µM respectively. These values are comparable to earlier data (Perretti et al., 2002), however the Hill coefficients for these curves were less than 1. hr-ANXA1 displaced the tracer at the ALXR receptor with a calculated mean EC₅₀ of 0.15 µM (Hill coefficient 1.15), but not at the FPR receptor (EC₅₀> 1600 µM). Similar observations with ALXR transfected cells were made for AF-2 (mean EC₅₀ = 1.21 µM) and W-peptide (mean EC₅₀ = 0.29 µM). Erk activation at both the FPR and ALXR receptors was evident 5-15 min following stimulation with h r-ANXA1 (0.5 µM) or peptide Ac2-26
(10 µM); this effect was maintained for up to 3-6 hours, but was totally absent in mock-transfected cells. hr-ANXA1 and Ac2-26-induced Erk activation at both the FPR and ALXR receptors was inhibited by pertussis toxin ( 1 µg/ml for 2 h, Calbiochem).

These data would suggest that whilst ANXA1 and its derived peptides appear to have a greater binding specificity for the human ALXR receptor, these molecules can functionally act through both receptor types to activate Erk. ANXA1 appears to bind with a degree of positive co-operativity to the ALXR receptor.

Perretti M. et al. (2002). Nat Med, 8, 1296-1302
Walther A et al. (2000) Mol Cell, 5, 831-840

This work was supported by the Arthritis Research Campaign (P0583)