Platelet-Activating Factor-Induced Chemokine Gene Expression Requires NF-κB Activation and Ca2+/calcineurin Signaling Pathways: Inhibition by Receptor Phosphorylation and β-Arrestin Recruitment

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Venkatesha, Rampura T.
Ahamed, Jasimuddin
Nuesch, Christopher
Zaidi, Asifa K.
Ali, Hydar

Previously, we reported that platelet-activating factor (PAF) stimulates higher G protein activation and a more robust Ca2+ mobilization in RBL-2H3 cells expressing earboxyl terminus deletion, phosphorylation-deficient mutant of PAF receptor (mPAFR) when compared with the wild-type receptor (PAFR). However, PAF did not provide sufficient signal for CC chemokine receptor ligand 2 (CCL2) production in cells expressing mPAFR. Based on these findings, we hypothesized that receptor phosphorylation provides a G protein-independent signal that synergizes with Ca2+ mobilization to induce CCL2 production. Here, we show that a mutant of PAFR (D289A), which does not couple to G proteins, was resistant to agonist-induced receptor phosphorylation. Unexpectedly, we found that when this mutant was coexpressed with mPAFR, it restored NF-κB activation and CCL2 production. PAF caused translocation of β-arrestin from the cytoplasm to the membrane in cells expressing PAFR but not a phosphorylation-deficient mutant in which all Ser/Thr residues were replaced with Ala (AST-PAFR). Interestingly, PAF induced significantly higher NF-κB and nuclear factor of activated T cells (NFAT)-luciferase activity as well as CCL2 production in cells expressing ΔST-PAFR than those expressing PAFR. Furthermore, a Ca2+/calcineurin inhibitor completely inhibited PAF-induced NFAT activation and CCL2 production but not NF-κB activation. These findings suggest that the carboxyl terminus of PAFR provides a G protein-independent signal for NF-κB activation, which synergizes with G protein-mediated Ca2+/calcineurin activation to induce CCL2 production. However, receptor phosphorylation and β-arrestin recruitment inhibit CCL2 production by blocking both NF-κB activation and Ca 2+/calcineurin-dependent signaling pathways

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