Distinct Roles of Receptor Phosphorylation, G Protein Usage, and Mitogen-activated Protein Kinase Activation on Platelet Activating Factor-induced Leukotriene C4 Generation and Chemokine Production

Platelet activating factor (PAF) interacts with cell surface G protein-coupled receptors on leukocytes to induce degranulation, leukotriene C4(LTC4) 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 LTC4 generation and CCL2 production. We found that PAF caused a significantly enhanced LTC4 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 Gi and p44/42 mitogen-activated protein kinase (ERK) activation, respectively, caused very little inhibition of PAF-induced CCL2 production (∼20% inhibition). In contrast, these inhibitors almost completely blocked both PAF-induced ERK phosphorylation and LTC4 generation in PAFR cells. However, in mPAFR cells pertussis toxin only partially inhibited PAF-induced ERK phosphorylation. A Ca2+/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 LTC4 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 Gi activation or ERK phosphorylation, LTC4 generation requires ERK phosphorylation, which is mediated by different G proteins depending on the phosphorylation status of the receptor. Platelet activating factor (PAF) interacts with cell surface G protein-coupled receptors on leukocytes to induce degranulation, leukotriene C4(LTC4) 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 LTC4 generation and CCL2 production. We found that PAF caused a significantly enhanced LTC4 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 Gi and p44/42 mitogen-activated protein kinase (ERK) activation, respectively, caused very little inhibition of PAF-induced CCL2 production (∼20% inhibition). In contrast, these inhibitors almost completely blocked both PAF-induced ERK phosphorylation and LTC4 generation in PAFR cells. However, in mPAFR cells pertussis toxin only partially inhibited PAF-induced ERK phosphorylation. A Ca2+/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 LTC4 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 Gi activation or ERK phosphorylation, LTC4 generation requires ERK phosphorylation, which is mediated by different G proteins depending on the phosphorylation status of the receptor. platelet-activating factor G protein-coupled receptor leukotriene C4 mitogen-activated protein extracellular signal-regulated kinase CC chemokine receptor ligand 2 (formerly known as MCP-1) PAF receptor mutant PAFR pertussis toxin cytosolic phospholipase A2 enzyme immunoassay enzyme-linked immunosorbent assay β-arrestin 2 green fluorescent protein Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3 phosphocholine, PAF)1 is an important mediator of inflammation that is released from mast cells, platelets, neutrophils, monocytes, and macrophages (1Venable M.E. Zimmerman G.A. McIntyre T.M. Prescott S.M. J. Lipid Res. 1993; 34: 691-702Abstract Full Text PDF PubMed Google Scholar, 2Braquet P. Touqui L. Shen T.Y. Vargaftig B.B. Pharmacol. Rev. 1987; 39: 97-145PubMed Google Scholar). PAF activates cell surface G protein-coupled receptors (GPCRs) to induce divergent biological functions (3Izumi T. Shimizu T. Biochim. Biophys. Acta. 1995; 1259: 317-333Crossref PubMed Scopus (208) Google Scholar). PAF is a potent leukocyte chemoattractant (4Haribabu B. Zhelev D.V. Pridgen B.C. Richardson R.M. Ali H. Snyderman R. J. Biol. Chem. 1999; 274: 37087-37092Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) that also induces degranulation (5Verghese M.W. Charles L. Jakoi L. Dillon S.B. Snyderman R. J. Immunol. 1987; 138: 4374-4380PubMed Google Scholar, 6Ali H. Richardson R.M. Tomhave E.D. DuBose R.A. Haribabu B. Snyderman R. J. Biol. Chem. 1994; 269: 24557-24563Abstract Full Text PDF PubMed Google Scholar), leukotriene C4(LTC4) generation (7Syrbu S.I. Waterman W.H. Molski T.F. Nagarkatti D. Hajjar J.J. Sha'afi R.I. J. Immunol. 1999; 162: 2334-2340PubMed Google Scholar, 8Myou S. Sano H. Fujimura M. Zhu X. Kurashima K. Kita T. Nakao S. Nonomura A. Shioya T. Kim K.P. Munoz N.M. Cho W. Leff A.R. Nat. Immunol. 2001; 2: 145-149Crossref PubMed Scopus (54) Google Scholar), and chemokine gene expression in a wide variety of cells (9Kravchenko V.V. Pan Z. Han J. Herbert J.M. Ulevitch R.J. Ye R.D. J. Biol. Chem. 1995; 270: 14928-14934Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 10Maruoka S. Hashimoto S. Gon Y. Takeshita I. Horie T. Am. J. Respir. Crit. Care Med. 2000; 161: 922-929Crossref PubMed Scopus (45) Google Scholar, 11Roth M. Nauck M. Yousefi S. Tamm M. Blaser K. Perruchoud A.P. Simon H.U. J. Exp. Med. 1996; 184: 191-201Crossref PubMed Scopus (74) Google Scholar, 12Nasu K. Narahara H. Matsui N. Kawano Y. Tanaka Y. Miyakawa I. Mol. Hum. Reprod. 1999; 5: 548-553Crossref PubMed Scopus (44) Google Scholar, 13Jocks T. Freudenberg J. Zahner G. Stahl R.A. Nephrol. Dial. Transplant. 1998; 13: 37-43Crossref PubMed Scopus (11) Google Scholar). Although much has been learned regarding the signaling pathways involved in PAF-induced chemotaxis and degranulation (4Haribabu B. Zhelev D.V. Pridgen B.C. Richardson R.M. Ali H. Snyderman R. J. Biol. Chem. 1999; 274: 37087-37092Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 6Ali H. Richardson R.M. Tomhave E.D. DuBose R.A. Haribabu B. Snyderman R. J. Biol. Chem. 1994; 269: 24557-24563Abstract Full Text PDF PubMed Google Scholar, 14Richardson R.M. Haribabu B. Ali H. Snyderman R. J. Biol. Chem. 1996; 271: 28717-28724Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), very little information is available on the mechanism by which PAF stimulates LTC4 generation and chemokine production. Receptor phosphorylation by G protein-coupled receptor kinase (GRK) and the subsequent recruitment of β-arrestin are essential for uncoupling the receptor from G proteins (15Lefkowitz R.J. Inglese J. Koch W.J. Pitcher J. Attramadal H. Caron M.G. Cold Spring Harbor Symp. Quant. Biol. 1992; 57: 127-133Crossref PubMed Google Scholar). Thus, phosphorylation-deficient mutants of chemoattractant receptors expressed in basophilic leukemia RBL-2H3 cells respond to the ligand for enhanced G protein activation, a more sustained Ca2+ mobilization, and a greater extent of degranulation when compared with cells expressing wild-type receptors (14Richardson R.M. Haribabu B. Ali H. Snyderman R. J. Biol. Chem. 1996; 271: 28717-28724Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 16Richardson R.M. DuBose R.A. Ali H. Tomhave E.D. Haribabu B. Snyderman R. Biochemistry. 1995; 34: 14193-14201Crossref PubMed Scopus (89) Google Scholar, 17Ahamed J. Haribabu B. Ali H. J. Immunol. 2001; 167: 3559-3563Crossref PubMed Scopus (31) Google Scholar). Receptor phosphorylation and β-arrestin recruitment have recently been shown to mediate MAP kinase activation for many GPCRs (18Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (712) Google Scholar, 19Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar, 20Luttrell L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1286) Google Scholar, 21Miller W.E. Lefkowitz R.J. Curr. Opin. Cell Biol. 2001; 13: 139-145Crossref PubMed Scopus (283) Google Scholar). However, the chemoattractants formylpeptide and the complement component C3a stimulate ERK phosphorylation via pathways that do not require receptor phosphorylation or β-arrestin recruitment (17Ahamed J. Haribabu B. Ali H. J. Immunol. 2001; 167: 3559-3563Crossref PubMed Scopus (31) Google Scholar, 22Gilbert T.L. Bennett T.A. Maestas D.C. Cimino D.F. Prossnitz E.R. Biochemistry. 2001; 40: 3467-3475Crossref PubMed Scopus (49) Google Scholar). We have recently shown that C3a receptor phosphorylation by the G protein-coupled receptor kinase provides a stimulatory signal that synergizes with ERK activation to induce chemokine CCL2 (also known as monocytes chemoattractant protein-1 or MCP-1) production (17Ahamed J. Haribabu B. Ali H. J. Immunol. 2001; 167: 3559-3563Crossref PubMed Scopus (31) Google Scholar). Unlike the C3a receptor, which couples to Gi, the PAF receptor interacts with the Gi and Gq family of G proteins to induce distinct biological responses (4Haribabu B. Zhelev D.V. Pridgen B.C. Richardson R.M. Ali H. Snyderman R. J. Biol. Chem. 1999; 274: 37087-37092Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 23Ali H. Richardson R.M. Haribabu B. Snyderman R. J. Biol. Chem. 1999; 274: 6027-6030Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). In the present study, we sought to determine the roles of receptor phosphorylation, G protein usage, and ERK phosphorylation on PAF-induced LTC4 generation and CCL2 production. For this purpose, RBL-2H3 cells expressing the wild-type PAF receptor (PAFR) and a phosphorylation-deficient mutant of PAFR (mPAFR) were used (14Richardson R.M. Haribabu B. Ali H. Snyderman R. J. Biol. Chem. 1996; 271: 28717-28724Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Here, we demonstrate that receptor phosphorylation, which serves to desensitize PAF-induced LTC4 generation, is required for chemokine CCL2 production. Furthermore, CCL2 production is, in large part, mediated independently of Gi activation or ERK phosphorylation. In contrast, LTC4 generation is dependent on ERK phosphorylation, which is mediated via different mechanisms depending on the phosphorylation status of the receptor. PAF, fluphenazine, Ro-31-8220, and U0126 were purchased from Calbiochem. [3H]PAF (1-O-hexadecyl-[acetyl-3H(N)]) (499.5 GBq/mmol) was obtained from PerkinElmer Life Sciences. Rabbit anti-p44/42 MAP kinase and anti-phospho-p44/42 MAP kinase antibodies were obtained from New England Biolabs and antibodies were obtained from and Pertussis toxin and were purchased from and were from A assay was purchased from LTC4 and were purchased from A CCL2 was purchased from was obtained from RBL-2H3 cells expressing PAFR and mPAFR were used in this H. Richardson R.M. Tomhave E.D. DuBose R.A. Haribabu B. Snyderman R. J. Biol. Chem. 1994; 269: 24557-24563Abstract Full Text PDF PubMed Google Scholar, 14Richardson R.M. Haribabu B. Ali H. Snyderman R. J. Biol. Chem. 1996; 271: 28717-28724Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). cells were in with and H. Richardson R.M. Tomhave E.D. DuBose R.A. Haribabu B. Snyderman R. J. Biol. Chem. 1994; 269: 24557-24563Abstract Full Text PDF PubMed Google Scholar, H. Richardson R.M. Tomhave E.D. Snyderman R. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). of PAFR and mPAFR were used for by For with of in the of for and (17Ahamed J. Haribabu B. Ali H. J. Immunol. 2001; 167: 3559-3563Crossref PubMed Scopus (31) Google Scholar) were were in and were Cell surface receptor expression was by cells with or an by and on a For Ca2+ mobilization, cells were with in the of for were in of and was as previously H. Richardson R.M. Tomhave E.D. DuBose R.A. Haribabu B. Snyderman R. J. Biol. Chem. 1994; 269: 24557-24563Abstract Full Text PDF PubMed Google Scholar). For degranulation, cells were in a were with with PAF, and the extent of degranulation was by the of H. Richardson R.M. Tomhave E.D. DuBose R.A. Haribabu B. Snyderman R. J. Biol. Chem. 1994; 269: 24557-24563Abstract Full Text PDF PubMed Google Scholar, H. Richardson R.M. Tomhave E.D. Snyderman R. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). were to the of receptors present in PAFR and mPAFR cells as by R.A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). RBL-2H3 cells expressing PAFR and mPAFR were in were with and in of the or with were for were with and with of was by of the cell were on the of cell by the of cells in RBL-2H3 cells were with PAF for 2 was by of Cell were in of in a and for of the cell was a assay as in the RBL-2H3 cells expressing PAFR or mPAFR were in were with PAF for and were and CCL2 (17Ahamed J. Haribabu B. Ali H. J. Immunol. 2001; 167: 3559-3563Crossref PubMed Scopus (31) Google Scholar) and LTC4 were and respectively, as in the receptors with were on cells were with PAF for was by of and the cells were and with for cell surface receptor cells were with the by the were a with a was by a and was and (17Ahamed J. Haribabu B. Ali H. J. Immunol. 2001; 167: 3559-3563Crossref PubMed Scopus (31) Google Scholar). RBL-2H3 cells expressing PAFR or mPAFR were with PAF in and the was different by the of a were with an of and to for were in and a was with in and with an for p44/42 MAP was by enhanced was and with an that with p44/42 MAP kinase (17Ahamed J. Haribabu B. Ali H. J. Immunol. 2001; 167: 3559-3563Crossref PubMed Scopus (31) Google Scholar, H. J. J. Immunol. 2000; PubMed Scopus Google Scholar). We have previously shown that PAF stimulates degranulation in RBL-2H3 cells expressing PAFR with an of H. Richardson R.M. Tomhave E.D. DuBose R.A. Haribabu B. Snyderman R. J. Biol. Chem. 1994; 269: 24557-24563Abstract Full Text PDF PubMed Google Scholar). In the present study, we these cells with different of PAF and and chemokine CCL2 production. for degranulation, PAF both LTC4 generation and CCL2 production with an of A and However, were in the of these responses. For LTC4 generation was In contrast, CCL2 production was not 2 a and for to We have previously shown that receptor phosphorylation to the desensitization of PAF-induced degranulation in leukocytes (14Richardson R.M. Haribabu B. Ali H. Snyderman R. J. Biol. Chem. 1996; 271: 28717-28724Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). determine the role of receptor phosphorylation on PAF-induced LTC4 generation and CCL2 RBL-2H3 cells expressing wild-type PAFR and phosphorylation-deficient mutant mPAFR were used (14Richardson R.M. Haribabu B. Ali H. Snyderman R. J. Biol. Chem. 1996; 271: 28717-28724Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). PAF an in PAFR and mPAFR cells PAF-induced LTC4 generation requires activation of T. K. K. T. M. H. Shimizu T. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). the of PAF to stimulate in PAFR and mPAFR cells was shown in PAF caused in PAFR and mPAFR cells. PAF also the generation of PAFR and mPAFR cells to 2 of PAFR cells with PAF for in CCL2 production and 2 this PAF not CCL2 production in mPAFR cells the that this receptor for Ca2+ mobilization, and LTC4 generation that the of CCL2 production in mPAFR cells a of production is of these cells with PAF for to to induce chemokine were to the of receptors present in the cells used in the RBL-2H3 cells expressed In contrast, mPAFR cells expressed receptors is that the of PAF to stimulate CCL2 production in mPAFR cells the expression of receptor We were previously in in RBL-2H3 cells expressing of For this we a to PAFR and mPAFR of receptor expression the is shown in A. Using this we the of PAF on LTC4 generation, degranulation, and CCL2 production. PAF significantly enhanced LTC4 generation and degranulation in mPAFR cells when compared with PAFR cells and In contrast, the of PAF to induce CCL2 production in mPAFR cells was that in PAFR cells These data that receptor phosphorylation, which PAF-induced degranulation and LTC4 generation, provides a stimulatory signal for CCL2 production. receptor phosphorylation is with the of β-arrestin from the to the Ferguson S.S. J. Caron M.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, W. Luttrell L.M. Pierce K.L. T.M. Lefkowitz R.J. S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). determine β-arrestin recruitment with PAF-induced were in RBL-2H3 cells PAFR or mPAFR and the β-arrestin fluorescent protein shown in PAF caused the of from the to the in PAFR cells. In contrast, PAF not induce this response in mPAFR cells PAFR couples to Gi in RBL-2H3 cells to induce chemotaxis (4Haribabu B. Zhelev D.V. Pridgen B.C. Richardson R.M. Ali H. Snyderman R. J. Biol. Chem. 1999; 274: 37087-37092Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). PAF also stimulates ERK phosphorylation in cells via a G protein T. Luttrell L.M. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). In contrast, PAF-induced degranulation requires both Gi and activation of phospholipase in the activation of protein kinase and the of Ca2+ (4Haribabu B. Zhelev D.V. Pridgen B.C. Richardson R.M. Ali H. Snyderman R. J. Biol. Chem. 1999; 274: 37087-37092Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 6Ali H. Richardson R.M. Tomhave E.D. DuBose R.A. Haribabu B. Snyderman R. J. Biol. Chem. 1994; 269: 24557-24563Abstract Full Text PDF PubMed Google Scholar). We the role of signaling G proteins on PAF-induced CCL2 production and LTC4 generation in PAFR cells. were with or pertussis toxin and effect on PAF-induced responses was shown in inhibited PAF-induced CCL2 production by In contrast, blocked LTC4 generation by determine the role of phospholipase we the of the inhibitors of protein kinase and Ca2+/calmodulin on PAF-induced responses. and almost completely blocked PAF-induced CCL2 production and A and the role of p44/42 MAP kinase activation on PAF-induced the effect of U0126 was MAP kinase inhibitor blocked CCL2 production by but inhibited LTC4 generation by in PAFR cells of these inhibitors were also on PAF-induced LTC4 generation in mPAFR cells. blocked PAF-induced LTC4 generation in mPAFR cells by is in to the in PAFR cells inhibited PAF-induced response by However, as in PAFR cells, Ro-31-8220, fluphenazine, or U0126 almost completely blocked PAF-induced LTC4 generation in mPAFR cells shown and PAF-induced LTC4 generation in PAFR and mPAFR cells to mediated by different G the role of different G protein usage on PAF-induced ERK phosphorylation, the of on PAFR and mPAFR responses were shown in caused inhibition of PAF-induced ERK phosphorylation in PAFR cells inhibition). In contrast, was much in this response in mPAFR cells However, caused almost inhibition of PAF-induced ERK phosphorylation in both cell A and had no effect on ERK phosphorylation mediated by PAF in PAFR cells but inhibited the response in mPAFR cells by U0126 blocked ERK phosphorylation in response to PAF in both cell A and PAF an important role in and S. Shimizu T. Lipid Res. 2000; 39: PubMed Scopus Google Scholar, G. G. G. Rev. 2000; PubMed Scopus Google Scholar). PAF stimulates chemotaxis and degranulation in leukocytes H. Richardson R.M. Tomhave E.D. DuBose R.A. Haribabu B. Snyderman R. J. Biol. Chem. 1994; 269: 24557-24563Abstract Full Text PDF PubMed Google Scholar, B. Snyderman R. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar). also LTC4 generation and chemokine production in a variety of cell (7Syrbu S.I. Waterman W.H. Molski T.F. Nagarkatti D. Hajjar J.J. Sha'afi R.I. J. Immunol. 1999; 162: 2334-2340PubMed Google Scholar, V.V. Pan Z. Han J. Herbert J.M. 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We have previously RBL-2H3 cells expressing PAFR and a mutant receptor (mPAFR) and demonstrated that receptor phosphorylation an important role in the desensitization of PAF-induced degranulation (14Richardson R.M. Haribabu B. Ali H. Snyderman R. J. Biol. Chem. 1996; 271: 28717-28724Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). of the present was to determine the role of receptor phosphorylation on PAF-induced LTC4 generation and chemokine CCL2 production. Here, we demonstrate that receptor phosphorylation PAF-induced LTC4 generation but provides a stimulatory signal for chemokine CCL2 production. We also distinct in both G protein usage and ERK phosphorylation on LTC4 generation and CCL2 production. We recently that complement component C3a stimulates CCL2 production via a that requires receptor phosphorylation (17Ahamed J. Haribabu B. Ali H. J. Immunol. 2001; 167: 3559-3563Crossref PubMed Scopus (31) Google Scholar). Furthermore, and M. J. Immunol. 2001; 167: PubMed Scopus Google Scholar) that stimulates chemokine gene expression via the activation of a However, of the in the of the receptor, which and in a in chemokine production. These that receptor phosphorylation provides a mechanism for chemokine gene receptor signal that mediates chemokine production is not A and of that the of receptors with the β-arrestin to the of a in the of cells. interacts with kinase and a factor to induce activation L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1286) Google Scholar, 21Miller W.E. Lefkowitz R.J. Curr. Opin. Cell Biol. 2001; 13: 139-145Crossref PubMed Scopus (283) Google Scholar, A. W. Miller W.E. N. J. Lefkowitz R.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). These that β-arrestin involved in PAF-induced CCL2 production. that PAF caused the recruitment of β-arrestin in cells expressing PAFR but not mPAFR is with this is, important to that the phosphorylation of PAFR and the of the receptor with β-arrestin do not require G protein activation Z. M. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, which had no effect on PAFR phosphorylation (4Haribabu B. Zhelev D.V. Pridgen B.C. Richardson R.M. Ali H. Snyderman R. J. Biol. Chem. 1999; 274: 37087-37092Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), caused a inhibition of PAF-induced ERK phosphorylation but had very little effect on CCL2 production. In U0126, which completely blocked PAF-induced ERK phosphorylation, not a inhibition of CCL2 production. These that β-arrestin mediates PAF-induced CCL2 via the activation of a that is of G protein activation and ERK phosphorylation. We have shown in the present that when PAFR and mPAFR were expressed in RBL-2H3 cells, PAF was to induce CCL2 production in mPAFR cells but and M. J. Immunol. 2001; 167: PubMed Scopus Google Scholar) also a for a wild-type and a mutant for a and These that receptor phosphorylation not a signal for chemokine production. is by the that the inhibition of responses as Ca2+ and activation to the inhibition of PAF-induced CCL2 production in PAFR cells. We have previously shown that chemokine production requires the of receptor and the G (17Ahamed J. Haribabu B. Ali H. J. Immunol. 2001; 167: 3559-3563Crossref PubMed Scopus (31) Google Scholar). In to the with the G signal for the C3a receptor ERK phosphorylation (17Ahamed J. Haribabu B. Ali H. J. Immunol. 2001; 167: 3559-3563Crossref PubMed Scopus (31) Google Scholar). These that chemokine production is mediated via and distinct pathways that in the G protein usage of the receptor. in the present that the of cells with U0126 or a Ca2+/calmodulin inhibitor to a inhibition of PAF-induced LTC4 generation is with the roles of ERK phosphorylation and Ca2+ on LTC4 generation (7Syrbu S.I. Waterman W.H. Molski T.F. Nagarkatti D. Hajjar J.J. Sha'afi R.I. J. Immunol. 1999; 162: 2334-2340PubMed Google Scholar, T. K. K. T. M. H. Shimizu T. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, K. J. Immunol. 1999; 162: Google Scholar, J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). Although PAF-induced ERK phosphorylation has been in the mechanism of activation has not been T. Luttrell L.M. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, Z. T. Y. K. Shimizu T. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, I. K. Shimizu T. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, I. M. K. Shimizu T. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). data indicate that PAF-induced ERK phosphorylation in RBL-2H3 cells is mediated by different depending on the phosphorylation status of the receptor. For PAF-induced ERK phosphorylation in PAFR cells requires activation of a G In contrast, this response in mPAFR cells both as as G We have previously shown that PAF-induced Ca2+ on the activation of H. Richardson R.M. Tomhave E.D. DuBose R.A. Haribabu B. Snyderman R. J. Biol. Chem. 1994; 269: 24557-24563Abstract Full Text PDF PubMed Google Scholar). that the Ca2+/calmodulin inhibitor blocked ERK phosphorylation in mPAFR but not PAFR cells the that receptor phosphorylation the G of PAF-induced ERK phosphorylation. In we have previously shown that receptor phosphorylation mediates the desensitization of PAF-induced degranulation (14Richardson R.M. Haribabu B. Ali H. Snyderman R. J. 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Full Text Full Text PDF PubMed Scopus Google Scholar). receptor phosphorylation is to have a greater on functions previously We and Ali Cell of of and S. of of for with cell and We also Caron for the β-arrestin