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J. Biol. Chem., Vol. 279, Issue 43, 44606-44612, October 22, 2004

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Platelet-activating Factor-induced Chemokine Gene Expression Requires NF-B Activation and Ca²⁺/Calcineurin Signaling Pathways

INHIBITION BY RECEPTOR PHOSPHORYLATION AND -ARRESTIN RECRUITMENT^*

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

From the Department of Pathology, University of Pennsylvania, School of Dental Medicine, Philadelphia, Pennsylvania 19104

Received for publication, July 16, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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-B activation and CCL2 production. PAF caused translocation of -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 (ST-PAFR). Interestingly, PAF induced significantly higher NF-B and nuclear factor of activated T cells (NFAT)-luciferase activity as well as CCL2 production in cells expressing ST-PAFR than those expressing PAFR. Furthermore, a Ca²⁺/calcineurin inhibitor completely inhibited PAF-induced NFAT activation and CCL2 production but not NF-B activation. These findings suggest that the carboxyl terminus of PAFR provides a G protein-independent signal for NF-B activation, which synergizes with G protein-mediated Ca²⁺/calcineurin activation to induce CCL2 production. However, receptor phosphorylation and -arrestin recruitment inhibit CCL2 production by blocking both NF-B activation and Ca²⁺/calcineurin-dependent signaling pathways.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Platelet-activating factor (PAF)¹ (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is an important mediator of inflammation that is released from mast cells, platelets, neutrophils, monocytes, and macrophages (1, 2). PAF activates cell surface G protein-coupled receptors (GPCRs) to induce divergent biological functions (3–6). PAF is a potent leukocyte chemoattractant (7), and it also induces degranulation (8, 9), leukotriene C₄ generation (10, 11), and chemokine gene expression in a wide variety of cells (12–17). Although PAF-induced chemokine production appears to depend on the activation of transcription factor NF-B, a crucial transcription factor regulating the expression of many proinflammatory cytokines and immunoregulatory molecules (12, 16, 18, 19), the early receptor-mediated signaling pathway that initiates this response has not been determined.

Receptor phosphorylation by G protein-coupled receptor kinase and the subsequent recruitment of -arrestin are essential for uncoupling the receptor from G proteins (20). -Arrestin also acts as an adapter molecule, leading to the formation of a scaffold in the cytoplasm of cells. This complex directly interacts with Src, Raf-1, extracellular signal-regulated kinase, c-Jun amino-terminal kinase-3, and a small GTP-binding protein, ADP-ribosylation factor 6 and the oncoprotein Mdm-2 to induce their activation (21–24). At a functional level, -arrestin signaling is required for GPCR-induced chemotaxis in lymphocytes, lymphoid enhancer factor transcriptional activity in transfected cell lines and development of allergic asthma in vivo (25–28). Recently, -arrestin has shown to inhibit NF-B activation in response to some but not all GPCRs (29, 30). Although -arrestin mediates agonist-induced internalization of PAFR (31–33), the role of this adapter molecule on PAFR signaling and biological responses has not been determined.

We have recently shown that PAF-induced chemokine CCL2 production in transfected RBL-2H3 cells requires G-protein-dependent Ca²⁺ mobilization and protein kinase C activation (31). We also reported the surprising observation that a carboxyl terminus deletion, phosphorylation-deficient mutant of PAFR (mPAFR), which couples to G protein more efficiently than the wild type receptor, did not provide sufficient signal for chemokine CCL2 production. Based on these findings, we hypothesized that PAF-induced CCL2 production requires the synergistic interaction of G protein-independent and G protein-dependent signals (31). In the present study, we show that G protein-independent signaling is mediated via the carboxyl terminus of the receptor without involving receptor phosphorylation. We also demonstrate that receptor phosphorylation and the subsequent -arrestin recruitment inhibits PAF-induced NF-B activation, Ca²⁺/calcineurin-mediated nuclear factor of activated T cells (NFAT) activation, and CCL2 production.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and cyclosporin A were purchased from Calbiochem. 12CA5 and anti-mouse IgG-RPE antibodies were obtained from Roche Applied Science and Southern Biotechnology Associates (Birmingham, AL), respectively. All tissue culture reagents were purchased from Invitrogen. The CCL2 sandwich ELISA kit was purchased from BioSource International (Camarillo, CA). pNF-B-Luc and pN-FAT-luc plasmid were purchased from Stratagene (La Jolla, CA). pRL Renilla control luciferase vector (pRL-SV40) and dual luciferase reporter assay system were obtained from Promega (Madison, WI). [³²P]Orthophosphate (8500–9120 Ci/mmol) was obtained from PerkinElmer Life Sciences.

Generation of PAF Receptor Mutants—A hemagglutinin-tagged human PAFR cloned in pRK-5 was restriction-digested with HindIII and BamHI and inserted into pcDNA3.1 (+) vector. Mutants of PAFR (D289A and Y293A) were generated by site-directed mutagenesis using the QuikChange mutagenesis kit (Stratagene). A phosphorylation-deficient mutant of PAFR (ST-PAFR) was constructed by the PCR method using the 5' oligonucleotide (5'-TGC AAG CTT GCA ATG TAC CCA TAC GAC GTC CCA GAC TAC GCT GAG CCA CAT GAC TCC TCC CAC ATG GAC-3') and a 3' oligonucleotide complementary to the PAFR tail replacing all serine and threonine residues with alanine (5'-GCAT GGATCC CT AAT TTT TGA GGG CAT TGC CAG GGA TCT GGT TGA ATG GCA CAA CCA CTT CAG CGA CCG CAT CCG CGG CGG CCC GGG CGC ATT TCC GGG CAG CGC GCA TGG CGT AGA ACT TTT CGG CGA GGT GCT T-3'). Mutants were cloned into pcDNA3 vector as described (31) and confirmed by sequencing.

Cell Culture, Transfection, Receptor Phosphorylation, and Degranulation—RBL-2H3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum, glutamine (2 mM), penicillin (100 units/ml), and streptomycin (100 µg/ml) (9, 34). Mouse embryonic fibroblast cell lines (MEFs) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 µg/ml) (35). Transient transfection of RBL-2H3 cells and MEFs were performed as described previously (31, 36). Briefly, cells (2 x 10⁷) were mixed with appropriate ratios of cDNA constructs in a total volume of 200 µl of transfection medium. Cells were then electroporated with a single pulse using a Bio-Rad gene pulser. Cells were cultured in complete growth medium, and experiments were performed 16–18 h after transfection.

To determine cell surface receptor expression, cells (1 x 10⁶) were incubated with 12CA5 or isotope-matched antibody, followed by incubation for 1 h with a secondary antibody conjugated with phycoerythrin and analyzed on a FACStar^PLUS flow cytometer (BD Biosciences) (31, 36). Cell surface receptor expression was also determined by ELISA using 12CA5 antibody, as described previously (37). Receptor phosphorylation was determined as described previously (9). For degranulation, cells (5 x 10⁴ cells/well) were cultured overnight in a 96-well tissue culture plate. Cells were washed with HEPES-buffered saline and stimulated with PAF, and the extent of degranulation was determined by measuring the release of -hexosaminidase (9, 34).

Confocal Microscopy—Cells expressing hemagglutinin-tagged receptors and -arrestin 2/green fluorescent protein conjugate (-arr2-GFP) were plated on coverslips in 24-well plates. The cells were stimulated with 100 nM PAF for 5 min at 37 °C. The reaction was stopped by adding 3 volumes of cold phosphate-buffered saline, and cells were fixed with 4% paraformaldehyde solution for 30 min at room temperature. The cells were permeabilized by 0.1% saponin (Sigma) for 20 min at room temperature. To visualize receptor expression, cells were incubated with 12CA5 antibody followed by biotin-labeled anti-mouse IgG and streptavidin Cy5 (Jackson ImmunoResearch). Cells were observed using a laser-scanning confocal microscope (Bio-Rad Radiance 2100) with a x 100 lens. The GFP was excited using a 488-nm argon laser and detected at emission 515 ± 30 nm, and Cy5 was excited at 647 nm and detected at 660 long pass.

Assay of NF-B Luciferase and NFAT Luciferase Activity—RBL-2H3 and MEF cells were transiently co-transfected with 22.5 µg of PAFR constructs, 7.5 µg of pNF-B-Luc, or pNFAT-luc plasmid and 0.5 µg of pRL-SV40 Renilla plasmid by electroporation. The following day, cells were serum-starved for 2–4 h and stimulated with 10 nM PAF for another 6 h. Reactions were stopped by washing cells with ice-cold phosphate-buffered saline. Cells were then lysed with 100 µl of lysis buffer, and luciferase activity was determined using the dual luciferase reporter assay system (Promega). The firefly luciferase activities were normalized to Renilla luciferase activity.

Assay of Chemokine (CCL2) Production—RBL-2H3 cells (0.25 x 10⁶/well) were cultured in complete growth medium overnight. Cells were serum-starved for 2–4 h and stimulated with PAF to a final concentration of 10 nM for 6 h. Supernatants were collected and stored frozen at –80 °C until analysis. CCL2 levels were quantified using sandwich ELISA as described previously (31, 36).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We have previously shown that agonist-induced phosphorylation of PAFR does not require G protein activation (9). To determine the role of receptor phosphorylation and G protein-independent signaling on PAF-induced transcription factor NF-B activation and CCL2 production, we generated two previously characterized mutants of PAFR that do not couple to G proteins (38, 39). These mutants were constructed by making single substitutions in the putative internalization motif DPXXY in the seventh transmembrane domain of the receptor. Changing aspartate 289 into alanine (D289A) blocks agonist-induced -arrestin binding and receptor internalization (38). In contrast, replacing the distal tyrosine residue with alanine (Y293A) abolishes G protein coupling but does not interfere with its ability to interact with -arrestin or to undergo internalization in transfected COS cells (38, 39). We first sought to characterize the properties of D289A and Y293A in transiently transfected basophilic leukemia RBL-2H3, a cell line that we have extensively utilized as a model to study functional regulation of leukocyte chemoattractant receptors (9, 40, 41). RBL-2H3 cells transiently transfected with PAFR, and its D289A or Y293A mutants were labeled with [³²P]orthophosphate and exposed to a concentration of PAF (100 nM) that is 10 times higher than required for optimal receptor phosphorylation (9). We found that PAF caused robust phosphorylation of PAFR and Y293A but not D289A mutant (Fig. 1A). The resistance of D289A to undergo agonist-induced phosphorylation is not due to inefficient transfection or unequal protein loading as receptor expression for each transfectants was carefully monitored and protein concentration was adjusted to assure that equal numbers of receptor were used for each experiment.

View larger version (24K): [in this window] [in a new window]   FIG. 1. Receptor phosphorylation does not mediate PAF-induced NF-B activation and CCL2 production. A, transient transfectants were generated in RBL-2H3 cells expressing equal numbers of PAFR, D289A, or Y293A. Cells were labeled with [32P]orthophosphate and exposed to PAF (100 nM for 5 min), and receptor phosphorylation was determined. B, RBL-2H3 cells coexpressing PAFR, D289A, or Y293A with NF-B luciferase and Renilla luciferase plasmids were stimulated with PAF (10 nM) for 6 h, and NF-B luciferase activity was determined in cell lysate. Data presented are relative luciferase activity normalized to Renilla luciferase activity as relative luciferase units (RLU). C, cells expressing PAFR, D289A, or Y293A were stimulated with PAF (10 nM) for 6 h, and CCL2 production was determined by ELISA. The data in A are representative of three similar experiments, and the data presented in B and C are mean ± S.E. of three separate experiments performed in triplicate.

To determine the role of carboxyl terminus of PAFR and G protein-independent signaling on PAF-induced responses, we used RBL-2H3 cells stably expressing mPAFR (31) and transiently expressed D289A (Fig. 2A). D289A had no effect on PAF-induced degranulation (Fig. 2B) but significantly enhanced mPAFR-induced NF-B luciferase activity (Fig. 2C) and CCL2 production (Fig. 2D) and restored them to levels similar to those observed in cells expressing the wild-type receptor (see Fig. 1). These findings demonstrate that the carboxyl terminus of PAFR does not regulate degranulation but is required for CCL2 gene expression.

View larger version (18K): [in this window] [in a new window]   FIG. 2. D289A mutant of PAFR does not desensitize PAF-induced degranulation but enhances NF-B activation and CCL2 production. RBL-2H3 cells stably expressing mPAFR were transiently transfected with control vector pcDNA3.1 (mPAFR-Mock) or D289A (mPAFR-D289A) in the presence of NF-B luciferase and Renilla luciferase plasmids. A, cell surface receptor expression was measured by ELISA. B, cells were stimulated with PAF (10 nM) for 20 min, and supernatant was assayed for -hexosaminidase release. Cells were also stimulated with PAF (10 nM) for 6 h, and NF-B-luciferase activity was measured from the cell lysate (C), and CCL2 production was determined in supernatant by ELISA (D). The data presented are mean ± S.E. of four experiments performed in triplicate. *, p < 0.05 in mPAFR-mock versus mPAFR-D289A.

View this table: [in this window] [in a new window]   TABLE I Amino acid sequences of the carboxyl terminus of PAFR, a deletion mutant (mPAFR), and phosphorylation-deficient mutant (ST-PAFR), in which all Ser and Thr residues were replaced with Ala

View larger version (54K): [in this window] [in a new window]   FIG. 3. Phosphorylation-deficient mutant ST-PAFR does not interact with -arrestin. A, RBL-2H3 cells expressing equal numbers of PAFR and ST-PAFR, were labeled with [32P]orthophosphate exposed to PAF (100 nM for 5 min), and receptor phosphorylation was determined. B, RBL-2H3 cells expressing PAFR and ST-PAFR were transfected with -arrestin 2-GFP and stimulated with PAF (100 nM) for 5 min, fixed in paraformaldehyde, and permeabilized in saponin. The cells were incubated with 12CA5 antibody followed by biotin-labeled anti-mouse IgG and streptavidin-Cy5. The interaction between receptor and -arrestin 2-GFP was detected by confocal microscopy. The data shown are representative of three similar experiments.

View larger version (22K): [in this window] [in a new window]   FIG. 4. Receptor phosphorylation plays an inhibitory role on PAF-induced degranulation, NF-B activation, and CCL2 production. Transient transfectants were generated in RBL-2H3 cells expressing PAFR or ST-PAFR. A, cell surface receptor expression was determined by fluorescence-activated cell sorting analysis. Mock-transfected cells were used as a control. B, cells expressing PAFR or ST-PAFR were incubated in the absence and presence of PAF (10 nM for 20 min), and the release of -hexosaminidase was determined. Cells were incubated with or without PAF (10 nM) for 6 h, and NF-B luciferase activity was determined in cell lysate (C), and CCL2 production was determined in supernatants by ELISA (D). The data shown in A is representative of three similar experiments, and data in B–D are mean ± S.E. of three separate experiments performed in triplicate.

View larger version (16K): [in this window] [in a new window]   FIG. 5. Constitutively active mutant of -arrestin inhibits PAF-induced responses in RBL-2H3 cells expressing ST-PAFR. RBL-2H3 cells coexpressing ST-PAFR with GFP, arr-GFP, or R169E-arr-GFP were stimulated with PAF (10 nM) for 20 min, and -hexosaminidase release was determined (A). Cells were stimulated with PAF for 6 h, and NF-B luciferase activity (B) and CCL2 production (C) were determined as described under "Experimental Procedures." The data presented are mean ± S.E. of three separate experiments performed in triplicate. Percentage inhibitions are shown in parentheses.

View larger version (17K): [in this window] [in a new window]   FIG. 6. -Arrestin provides an inhibitory signal for PAF-induced NF-B activation. Mouse embryonic fibroblasts from knockout mice that lack both -arrestin 1 and -arrestin 2 (MEF 1/2KO) and its wild-type littermate (MEF-WT) were transiently co-transfected with PAFR and NF-B/Renilla luciferase constructs by electroporation. A, cell surface receptor expression was determined by flow cytometry. B, cells were also stimulated with PAF (10 nM) for 20 min, and the cell surface receptor expression was determined by flow cytometry. The data are expressed as percentage of receptors that are present on the cell surface in the absence of agonist stimulation (–PAF; 100%). C, cells were serum-starved for 4 h and stimulated with PAF (10 nM) for another 6 h. Cell lysates were used to determine NF-B luciferase activity. The data shown in A is representative of three similar experiments, and data in B and C are mean ± S.E. of three separate experiments performed in triplicate. *, p < 0.05 in wild-type versus -arrestin deficient cells.

View larger version (15K): [in this window] [in a new window]   FIG. 7. Cyclosporin A completely inhibits PAF-induced NFAT activation and CCL2 production but only partially inhibits NF-B activation. RBL-2H3 cells transiently expressing PAFR, mPAFR, and ST-PAFR were treated with and without cyclosporin A (100 nM) for 30 min and stimulated with PAF (10 nM) for 6 h. A, PAF-induced NFAT-luciferase activity; B, NF-B-luciferase activity measured from cell lysate; C, CCL2 production measured by ELISA. The data presented are mean ± S.E. of three experiments performed in triplicate.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have previously shown that PAF stimulates higher G protein activation and a more robust Ca²⁺ mobilization in RBL-2H3 cells expressing carboxyl terminus deletion, phosphorylation-deficient mutant, mPAFR when compared with the wild-type receptor (PAFR) (31). However, PAF did not provide sufficient signal for 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. PAFR mutant D289A, containing a single substitution in the putative internalization motif DPXXY in its seventh transmembrane domain does not interact with -arrestin but signals via a G protein-independent pathway (38, 39). We found that D289A was resistant to agonist-induced receptor phosphorylation. Furthermore, PAF did not induce NF-B activation or CCL2 production in cells expressing D289A. Our expectation was that when D289A is coexpressed with mPAFR, it would have little or no effect on PAF-induced CCL2 gene expression. Surprisingly, we found that D289A restored PAF-induced NF-B activation and CCL2 production. These findings suggest that the carboxyl terminus of PAFR, but not receptor phosphorylation, mediates G protein-independent signal for NF-B activation, which synergizes with Ca²⁺ mobilization to induce CCL2 gene expression.

The role of NF-B activation on chemokine CCL2 production is well established (19, 50, 51). However, Satonaka et al. (48), recently demonstrated that CCL2 gene expression in vascular myocytes requires Ca²⁺/calmodulin-mediated calcineurin activation. The ability of D289A to restore PAF-induced CCL2 production in mPAFR cells provided us with an important tool to delineate the roles of distinct signaling pathways on PAF-induced responses. An interesting finding of the present study was that while PAF caused significantly lower NF-B activation in cells expressing mPAFR when compared with PAFR, the reverse was the case for NFAT activation (see Fig. 7). Despite the fact that PAF stimulated a robust calcineurin-mediated NFAT activation in mPAFR cells, a G protein-independent signal for NF-B activation by D289A was required to restore CCL2 production. These findings suggest that the carboxyl terminus of PAFR provides a G protein-independent signaling for NF-B activation, which synergizes with mPAFR-induced Ca²⁺/calcineurin-mediated signaling pathway to induce CCL2 gene expression. This contention is supported by the finding that a Ca²⁺/calcineurin inhibitor completely blocked PAF-induced NFAT activation and CCL2 production but only partially inhibited NF-B activation.

An interesting finding of the present study was that although the carboxyl terminus of PAFR is required for PAF-induced CCL2 gene expression, phosphorylation of the receptor at Ser/Thr residues within this domain and the subsequent -arrestin recruitment inhibits this response. This contention is supported by the following observations. First, PAFR but not ST-PAFR interacts with -arrestin in response to PAF. Second, PAF was more active in stimulating CCL2 gene expression in RBL-2H3 cells expressing ST-PAFR when compared with PAFR. Third, a constitutively active mutant of -arrestin (R169E) caused a substantial inhibition of PAF-induced CCL2 gene expression. Fourth, PAF-induced NF-B activation was significantly enhanced in -arrestin knockout MEFs when compared with wild-type MEFs.

The mechanism by which receptor phosphorylation and -arrestin recruitment inhibit PAF-induced CCL2 gene expression is not known. Recently, it has been shown that interaction of -arrestin with IB or its upstream kinases inhibit NF-B activation stimulated by some but not all GPCRs (29, 30). In the present study, we have shown that receptor phosphorylation and -arrestin recruitment inhibits PAF-induced NF-B activation. We further demonstrated that PAF-induced NF-B activation is enhanced in -arrestin knockout MEFs when compared with wild-type cells. These findings are consistent with the idea that -arrestin modifies IB or its upstream kinases to inhibit PAF-induced CCL2 production in RBL-2H3 cells. We also showed that PAF-induced NFAT activation was enhanced in cells expressing ST-PAFR when compared with PAFR and that this Ca²⁺/calcineurin-mediated response is essential for CCL2 production. This finding suggests that the ability of receptor phosphorylation and -arrestin recruitment to inhibit CCL2 production involves the modification of at least two components in the PAFR signaling pathway. One involves the inhibition of NF-B, and the other involves modulation of Ca²⁺/calcineurin-mediated signaling pathway.

In summary, we have shown that PAF-induced CCL2 gene expression requires the carboxyl terminus of PAFR and involves a complex interaction of G protein-dependent and -independent signaling pathways. However, phosphorylation of Ser/Thr residues within this region blocks gene expression via the modification of multiple signaling pathways. It is noteworthy that PAFR is not the only GPCR that requires its carboxyl terminus for the induction of NF-B activation and chemokine production. Schwarz et al. (52) showed that Kaposi's sarcoma-associated herpesvirus stimulates NF-B activation and CCL2 production and that deletion of the terminal five amino acids on the carboxyl terminus of its GPCR resulted in substantial inhibition of these responses. Most interestingly, Hernandez et al. recently showed that WHIM syndrome, an immunodeficiency disease characterized by neutropenia, is associated with deletion of the carboxyl terminus of the G protein-coupled chemokine receptor 4 (53). Therefore, the mechanism described in the present study for PAF-induced chemokine production is likely to be a general mechanism by which functions of different G protein-coupled receptors are regulated.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant HL-63372 and a American Heart Association Grant-in-Aid 0256361U. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: The Scripps Research Institute, 10550 Torrey Pines Rd., La Jolla, CA 92037.

^¶ To whom correspondence should be addressed: Dept. of Pathology, University of Pennsylvania School of Dental Medicine, 240 S. 40th St. (346 Levy Bldg.), Philadelphia, PA 19104-6002. Tel.: 215-573-1993; Fax: 215-573-2050; E-mail: ali{at}path.dental.upenn.edu.

¹ The abbreviations used are: PAF, platelet-activating factor; PAFR, PAF receptor; mPAFR, mutant of PAF receptor; GPCR, G protein-coupled receptor; CCL2, CC chemokine receptor ligand 2 (formerly known as MCP-1); GFP, green fluorescent protein; arr2-GFP, -arrestin 2-green fluorescent protein conjugate; NFAT, nuclear factor of activated T cells; ELISA, enzyme-linked immunosorbent assay; MEF, mouse embryo fibroblast.

	ACKNOWLEDGMENTS

 
We thank Dr. Bruce Shenker and Ali Zekavat (University of Pennsylvania School of Dental Medicine, FACS Core Facility) for assistance with fluorescence-activated cell sorting analysis. We also thank Dr. Marc Caron (Duke University) and Dr. Jeffrey Benovic (Thomas Jefferson University) for providing the -arrestin plasmids. We are also grateful to Drs. Trudy Kohut and Robert Lefkowitz (Duke University) for providing mouse embryonic cell lines.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Venable, M. E., Zimmerman, G. A., McIntyre, T. M., and Prescott, S. M. (1993) J. Lipid Res. 34, 691–702[Medline] [Order article via Infotrieve]
2. Braquet, P., Touqui, L., Shen, T. Y., and Vargaftig, B. B. (1987) Pharmacol. Rev. 39, 97–145[Medline] [Order article via Infotrieve]
3. Izumi, T., and Shimizu, T. (1995) Biochim. Biophys. Acta 1259, 317–333[Medline] [Order article via Infotrieve]
4. Hikiji, H., Ishii, S., Shindou, H., Takato, T., and Shimizu, T. (2004) J. Clin. Invest. 114, 85–93[CrossRef][Medline] [Order article via Infotrieve]
5. Ishii, S., Nagase, T., Shindou, H., Takizawa, H., Ouchi, Y., and Shimizu, T. (2004) J. Immunol. 172, 7095–7102[Abstract/Free Full Text]
6. Rijneveld, A. W., Weijer, S., Florquin, S., Speelman, P., Shimizu, T., Ishii, S., and van der Poll, T. (2004) J. Infect. Dis. 189, 711–716[CrossRef][Medline] [Order article via Infotrieve]
7. Haribabu, B., Zhelev, D. V., Pridgen, B. C., Richardson, R. M., Ali, H., and Snyderman, R. (1999) J. Biol. Chem. 274, 37087–37092[Abstract/Free Full Text]
8. Verghese, M. W., Charles, L., Jakoi, L., Dillon, S. B., and Snyderman, R. (1987) J. Immunol. 138, 4374–4380[Abstract]
9. Ali, H., Richardson, R. M., Tomhave, E. D., DuBose, R. A., Haribabu, B., and Snyderman, R. (1994) J. Biol. Chem. 269, 24557–24563[Abstract/Free Full Text]
10. Syrbu, S. I., Waterman, W. H., Molski, T. F., Nagarkatti, D., Hajjar, J. J., and Sha'afi, R. I. (1999) J. Immunol. 162, 2334–2340[Abstract/Free Full Text]
11. Myou, 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., and Leff, A. R. (2001) Nat. Immunol. 2, 145–149[CrossRef][Medline] [Order article via Infotrieve]
12. Kravchenko, V. V., Pan, Z., Han, J., Herbert, J. M., Ulevitch, R. J., and Ye, R. D. (1995) J. Biol. Chem. 270, 14928–14934[Abstract/Free Full Text]
13. Maruoka, S., Hashimoto, S., Gon, Y., Takeshita, I., and Horie, T. (2000) Am. J. Respir. Crit. Care Med. 161, 922–929[Abstract/Free Full Text]
14. Roth, M., Nauck, M., Yousefi, S., Tamm, M., Blaser, K., Perruchoud, A. P., and Simon, H. U. (1996) J. Exp. Med. 184, 191–201[Abstract/Free Full Text]
15. Nasu, K., Narahara, H., Matsui, N., Kawano, Y., Tanaka, Y., and Miyakawa, I. (1999) Mol. Hum. Reprod. 5, 548–553[Abstract/Free Full Text]
16. Jocks, T., Freudenberg, J., Zahner, G., and Stahl, R. A. (1998) Nephrol. Dial. Transplant. 13, 37–43[Abstract/Free Full Text]
17. Beaudeux, J. L., Said, T., Ninio, E., Ganne, F., Soria, J., Delattre, J., Soria, C., Legrand, A., and Peynet, J. (2004) Clin. Chim. Acta 344, 163–171[CrossRef][Medline] [Order article via Infotrieve]
18. Ye, R. D., Kravchenko, V. V., Pan, Z., and Feng, L. (1996) Adv. Exp. Med. Biol. 416, 143–151[Medline] [Order article via Infotrieve]
19. Ueda, A., Ishigatsubo, Y., Okubo, T., and Yoshimura, T. (1997) J. Biol. Chem. 272, 31092–31099[Abstract/Free Full Text]
20. Lefkowitz, R. J., Inglese, J., Koch, W. J., Pitcher, J., Attramadal, H., and Caron, M. G. (1992) Cold Spring Harbor Symp. Quant. Biol. 57, 127–133[Medline] [Order article via Infotrieve]
21. Luttrell, 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., and Lefkowitz, R. J. (1999) Science 283, 655–661[Abstract/Free Full Text]
22. Miller, W. E., and Lefkowitz, R. J. (2001) Curr. Opin. Cell Biol. 13, 139–145[CrossRef][Medline] [Order article via Infotrieve]
23. Claing, A., Chen, W., Miller, W. E., Vitale, N., Moss, J., Premont, R. T., and Lefkowitz, R. J. (2001) J. Biol. Chem. 276, 42509–42513[Abstract/Free Full Text]
24. Wang, P., Gao, H., Ni, Y., Wang, B., Wu, Y., Ji, L., Qin, L., Ma, L., and Pei, G. (2003) J. Biol. Chem. 278, 6363–6370[Abstract/Free Full Text]
25. Chen, W., Hu, L. A., Semenov, M. V., Yanagawa, S., Kikuchi, A., Lefkowitz, R. J., and Miller, W. E. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 14889–14894[Abstract/Free Full Text]
26. Fong, A. M., Premont, R. T., Richardson, R. M., Yu, Y. R., Lefkowitz, R. J., and Patel, D. D. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7478–7483[Abstract/Free Full Text]
27. Ohsawa, M., Mizoguchi, H., Narita, M., Nagase, H., Dun, N. J., and Tseng, L. F. (2003) Neurosci. Lett. 346, 13–16[CrossRef][Medline] [Order article via Infotrieve]
28. Walker, J. K., Fong, A. M., Lawson, B. L., Savov, J. D., Patel, D. D., Schwartz, D. A., and Lefkowitz, R. J. (2003) J. Clin. Invest. 112, 566–574[CrossRef][Medline] [Order article via Infotrieve]
29. Gao, H., Sun, Y., Wu, Y., Luan, B., Wang, Y., Qu, B., and Pei, G. (2004) Mol. Cell 14, 303–317[CrossRef][Medline] [Order article via Infotrieve]
30. Witherow, D. S., Garrison, T. R., Miller, W. E., and Lefkowitz, R. J. (2004) Proc. Natl. Acad. Sci. U. S. A.
31. Ahamed, J., and Ali, H. (2002) J. Biol. Chem. 277, 22685–22691[Abstract/Free Full Text]
32. Lukashova, V., Asselin, C., Krolewski, J. J., Rola-Pleszczynski, M., and Stankova, J. (2001) J. Biol. Chem. 276, 24113–24121[Abstract/Free Full Text]
33. Perron, A., Chen, Z. G., Gingras, D., Dupre, D. J., Stankova, J., and Rola-Pleszczynski, M. (2003) J. Biol. Chem. 278, 27956–27965[Abstract/Free Full Text]
34. Ali, H., Richardson, R. M., Tomhave, E. D., Didsbury, J. R., and Snyderman, R. (1993) J. Biol. Chem. 268, 24247–24254[Abstract/Free Full Text]
35. Kohout, T. A., Lin, F. S., Perry, S. J., Conner, D. A., and Lefkowitz, R. J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 1601–1606[Abstract/Free Full Text]
36. Ahamed, J., Haribabu, B., and Ali, H. (2001) J. Immunol. 167, 3559–3563[Abstract/Free Full Text]
37. Sromek, S. M., and Harden, T. K. (1998) Mol. Pharmacol. 54, 485–494[Abstract/Free Full Text]
38. Le Gouill, C., Parent, J. L., Rola-Pleszczynski, M., and Stankova, J. (1997) J. Biol. Chem. 272, 21289–21295[Abstract/Free Full Text]
39. Chen, Z., Dupre, D. J., Le Gouill, C., Rola-Pleszczynski, M., and Stankova, J. (2002) J. Biol. Chem. 277, 7356–7362[Abstract/Free Full Text]
40. Ali, H., Fisher, I., Haribabu, B., Richardson, R. M., and Snyderman, R. (1997) J. Biol. Chem. 272, 11706–11709[Abstract/Free Full Text]
41. Ali, H., Sozzani, S., Fisher, I., Barr, A. J., Richardson, R. M., Haribabu, B., and Snyderman, R. (1998) J. Biol. Chem. 273, 11012–11016[Abstract/Free Full Text]
42. Richardson, R. M., Haribabu, B., Ali, H., and Snyderman, R. (1996) J. Biol. Chem. 271, 28717–28724[Abstract/Free Full Text]
43. Celver, J. P., Lowe, J., Kovoor, A., Gurevich, V. V., and Chavkin, C. (2001) J. Biol. Chem. 276, 4894–4900[Abstract/Free Full Text]
44. Kovoor, A., Celver, J., Abdryashitov, R. I., Chavkin, C., and Gurevich, V. V. (1999) J. Biol. Chem. 274, 6831–6834[Abstract/Free Full Text]
45. Kim, Y. M., and Benovic, J. L. (2002) J. Biol. Chem. 277, 30760–30768[Abstract/Free Full Text]
46. Rao, A., Luo, C., and Hogan, P. G. (1997) Annu. Rev. Immunol. 15, 707–747[CrossRef][Medline] [Order article via Infotrieve]
47. Henning, S. W., and Cantrell, D. A. (1998) Curr. Opin. Immunol. 10, 322–329[CrossRef][Medline] [Order article via Infotrieve]
48. Satonaka, H., Suzuki, E., Nishimatsu, H., Oba, S., Takeda, R., Goto, A., Omata, M., Fujita, T., Nagai, R., and Hirata, Y. (2004) Circ. Res. 94, 693–700[Abstract/Free Full Text]
49. Boss, V., Abbott, K. L., Wang, X. F., Pavlath, G. K., and Murphy, T. J. (1998) J. Biol. Chem. 273, 19664–19671[Abstract/Free Full Text]
50. Oda, T., Ueda, A., Shimizu, N., Handa, H., and Kasahara, T. (2002) J. Immunol. 169, 3329–3335[Abstract/Free Full Text]
51. Goebeler, M., Gillitzer, R., Kilian, K., Utzel, K., Brocker, E. B., Rapp, U. R., and Ludwig, S. (2001) Blood 97, 46–55[Abstract/Free Full Text]
52. Schwarz, M., and Murphy, P. M. (2001) J. Immunol. 167, 505–513[Abstract/Free Full Text]
53. Hernandez, P. A., Gorlin, R. J., Lukens, J. N., Taniuchi, S., Bohinjec, J., Francois, F., Klotman, M. E., and Diaz, G. A. (2003) Nat. Genet. 34, 70–74[CrossRef][Medline] [Order article via Infotrieve]

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