title: Distinct roles of receptor phosphorylation, G protein usage, and mitogen-activated protein kinase activation on platelet activating factor-induced leukotriene C(4) generation and chemokine production.
abstract: Platelet activating factor (PAF) interacts with cell surface G protein-coupled receptors on leukocytes to induce degranulation, leukotriene C(4) (LTC(4)) generation, and chemokine CCL2 production. Using a basophilic leukemia RBL-2H3 cell line expressing wild-type PAF receptor (PAFR) and a phosphorylation-deficient mutant (mPAFR), we have previously demonstrated that receptor phosphorylation mediates desensitization of PAF-induced degranulation. Here, we sought to determine the role of receptor phosphorylation on PAF-induced LTC(4) generation and CCL2 production. We found that PAF caused a significantly enhanced LTC(4) generation in cells expressing mPAFR when compared with PAFR cells. In contrast, PAF-induced CCL2 production was greatly reduced in mPAFR cells. Pertussis toxin and U0126, which inhibit G(i) and p44/42 mitogen-activated protein kinase (ERK) activation, respectively, caused very little inhibition of PAF-induced CCL2 production (approximately 20% inhibition). In contrast, these inhibitors almost completely blocked both PAF-induced ERK phosphorylation and LTC(4) generation in PAFR cells. However, in mPAFR cells pertussis toxin only partially inhibited PAF-induced ERK phosphorylation. A Ca(2+)/calmodulin inhibitor had no effect on PAF-induced ERK phosphorylation in PAFR cells but completely blocked the response in mPAFR cells. These data demonstrate that receptor phosphorylation, which serves to desensitize PAF-induced LTC(4) generation, is required for chemokine CCL2 production. They also indicate a previously unrecognized selectivity in G protein usage and ERK activation for PAF-induced responses. Whereas PAF-induced CCL2 production is, in large part, mediated independently of G(i) activation or ERK phosphorylation, LTC(4) generation requires ERK phosphorylation, which is mediated by different G proteins depending on the phosphorylation status of the receptor.
authors.affiliation: Department of Pathology, University of Pennsylvania School of Dental Medicine, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Jasimuddin
authors.initials: J
authors.lastname: Ahamed
authors.forename: Hydar
authors.initials: H
authors.lastname: Ali
mesh_terms: D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D018925:Chemokines / Q000096:biosynthesis*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D049452:Green Fluorescent Proteins; D006801:Humans; D017997:Leukotriene C4 / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D020935:MAP Kinase Signaling System; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation; D010972:Platelet Activating Factor / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D013997:Time Factors; D014162:Transfection
chemical_list: D018925:Chemokines; D008164:Luminescent Proteins; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C039351:platelet activating factor receptor; D049452:Green Fluorescent Proteins; D017997:Leukotriene C4; D020928:Mitogen-Activated Protein Kinases; D002118:Calcium