Y. Kang et al., Mutations of serine 236-237 and tyrosine 302 residues in the human lipoxinA(4) receptor intracellular domains result in sustained signaling, BIOCHEM, 39(44), 2000, pp. 13551-13557
Lipoxin A(4) (LXA(4)) is a potent negative modulator of the inflammatory re
sponse. The antiinflammatory activities of LXA(4), such as inhibition of ag
onist-induced polymorphonuclear cell (PMN) chemotaxis and upregulation of b
eta -2 integrins, require the expression of a G-protein-coupled, high-affin
ity LXA(4) receptor (LXA(4)R). We now report that stimulation of PMN with p
roinflammatory agonist N-formyl peptides (FMLP), calcium ionophore A(23187)
. or phorbol mirystate acetate (PMA) is followed by marked downregulation o
f LXA(4) binding (B-max decrease of similar to 45%) and decreased activatio
n of phospholipases A(2) (PLA(2)) and D (PLD). Elucidation of the mechanism
s underlying these effects was addressed by structure-function analyses of
the intracellular domains of LXA(4)R. Mutant molecule, S236/5237 -> A/G (LX
A(4)R(pk)) and Y302 -> F (LXA(4)R(tk)) were obtained by site-directed mutag
enesis to yield receptors lacking the putative targets fur serine/threonine
kinase- or tyrosine kinase-dependent phosphorylation. Expression of wild-t
ype and mutated LXA(4)R sequences in CHO and HL-60 cells was used to examin
e LXA(4) ligand-receptor interactions and signal transduction events. Resul
ts indicated that cells expressing LXA(4)R(pk) or LXA(4)R(tk) displayed sus
tained activation of PLA(2) and PLD in contrast to the transient ones obtai
ned with LXA(4)R(wt) (peak activation at 2-3 min). Moreover, inhibition of
LXA(4)-dependent PLA2 activity by PMA in LXA(4)R(wt) transfected CHO cells
was not observed in cells expressing LXA(4)R(pk). Phosphopeptide immunoblot
ting revealed that the functional differences between wild-type and mutant
LXA(4) receptors are accompanied by distinct changes in the receptor protei
n phosphorylation pattern. Further characterization of these and related LX
A(4)R intracellular domains will help to better understand specific events
that regulate the antiinflammatory activities of LXA(4).