Platelet-activating factor-induced chemokine gene expression requires NF-kappaB activation and Ca2+/calcineurin signaling pathways. Inhibition by receptor phosphorylation and beta-arrestin recruitment.
Previously, we reported that platelet-activating factor (PAF) stimulates higher G protein activation and a more robust Ca2+ mobilization in RBL-2H3 cells expressing carboxyl 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-kappaB activation and CCL2 production. PAF caused translocation of beta-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 (DeltaST-PAFR). Interestingly, PAF induced significantly higher NF-kappaB and nuclear factor of activated T cells (NFAT)-luciferase activity as well as CCL2 production in cells expressing DeltaST-PAFR than those expressing PAFR. Furthermore, a Ca2+/calcineurin inhibitor completely inhibited PAF-induced NFAT activation and CCL2 production but not NF-kappaB activation. These findings suggest that the carboxyl terminus of PAFR provides a G protein-independent signal for NF-kappaB activation, which synergizes with G protein-mediated Ca2+/calcineurin activation to induce CCL2 production. However, receptor phosphorylation and beta-arrestin recruitment inhibit CCL2 production by blocking both NF-kappaB activation and Ca2+/calcineurin-dependent signaling pathways.
Department of Pathology, University of Pennsylvania, School of Dental Medicine, Philadelphia, Pennsylvania 19104, USA.
Rampura T
RT
Venkatesha
Jasimuddin
J
Ahamed
Christopher
C
Nuesch
Asifa K
AK
Zaidi
Hydar
H
Ali
D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D019703:Calcineurin / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D018932:Chemokine CCL2 / Q000378:metabolism; D018925:Chemokines / Q000378:metabolism*; D016572:Cyclosporine / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism*; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D010972:Platelet Activating Factor / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats; D015398:Signal Transduction; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D000071557:beta-Arrestins
D019390:Arrestins; D018932:Chemokine CCL2; D018925:Chemokines; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D016328:NF-kappa B; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010972:Platelet Activating Factor; D014157:Transcription Factors; D000071557:beta-Arrestins; D016572:Cyclosporine; D008156:Luciferases; D019703:Calcineurin; D002118:Calcium