Lipoxin A4 (LXA4) is a potent negative modulator of the inflammatory response. The antiinflammatory activities of LXA4, such as inhibition of agonist-induced polymorphonuclear cell (PMN) chemotaxis and upregulation of β-2 integrins, require the expression of a G-protein-coupled, high-affinity LXA4 receptor (LXA4R). We now report that stimulation of PMN with proinflammatory agonist N-formyl peptides (FMLP), calcium ionophore A23187, or phorbol mirystate acetate (PMA) is followed by marked downregulation of LXA4 binding (B(max) decrease of ~45%) and decreased activation of phospholipases A2 (PLA2) and D (PLD). Elucidation of the mechanisms underlying these effects was addressed by structure-function analyses of the intracellular domains of LXA4R. Mutant molecule, S236/S237 → A/G (LXA4R(pk)) and Y302 → F (LXA4R(tk)) were obtained by site-directed mutagenesis to yield receptors lacking the putative targets for serine/threonine kinase- or tyrosine kinase-dependent phosphorylation. Expression of wild-type and mutated LXA4R sequences in CHO and HL-60 cells was used to examine LXA4 ligand-receptor interactions and signal transduction events. Results indicated that cells expressing LXA4R(pk) or LXA4R(tk) displayed sustained activation of PLA2 and PLD in contrast to the transient ones obtained with LXA4R(wt) (peak activation at 2-3 min). Moreover, inhibition of LXA4-dependent PLA2 activity by PMA in LXA4R(wt) transfected CHO cells was not observed in cells expressing LXA4R(pk). Phosphopeptide immunoblotting revealed that the functional differences between wild-type and mutant LXA4 receptors are accompanied by distinct changes in the receptor protein phosphorylation pattern. Further characterization of these and related LXA4R intracellular domains will help to better understand specific events that regulate the antiinflammatory activities of LXA4.
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