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Activation and Regulation of Platelet-Activating Factor Receptor: Role of G_i and G_q in Receptor-Mediated Chemotactic, Cytotoxic, and Cross-Regulatory Signals¹

Stephan L. Brown, Venkatakrishna R. Jala, Sandeep K. Raghuwanshi^*, Mohd W. Nasser^*, Bodduluri Haribabu and Ricardo M. Richardson^2,*

^* Julius L. Chambers Biomedical/Biotechnology Research Institute and Department of Biology, North Carolina Central University, Durham, NC 27707; Department of Biochemistry, Meharry Medical College, Nashville, TN 37208; and James Graham Brown Cancer Center and Department of Microbiology and Immunology, University of Louisville Health Sciences Center, Louisville, KY 40202

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycerolphosphocholine; PAF) induces leukocyte accumulation and activation at sites of inflammation via the activation of a specific cell surface receptor (PAFR). PAFR couples to both pertussis toxin-sensitive and pertussis toxin-insensitive G proteins to activate leukocytes. To define the role(s) of G_i and G_q in PAF-induced leukocyte responses, two G-protein-linked receptors were generated by fusing G_i3 (PAFR-G_i3) or G_q (PAFR-G_q) at the C terminus of PAFR. Rat basophilic leukemia cell line (RBL-2H3) stably expressing wild-type PAFR, PAFR-G_i3, or PAFR-G_q was generated and characterized. All receptor variants bound PAF with similar affinities to mediate G-protein activation, intracellular Ca²⁺ mobilization, phosphoinositide (PI) hydrolysis, and secretion of -hexosaminidase. PAFR-G_i3 and PAFR-G_q mediated greater GTPase activity in isolated membranes than PAFR but lower PI hydrolysis and secretion in whole cells. PAFR and PAFR-G_i3, but not PAFR-G_q, mediated chemotaxis to PAF. All three receptors underwent phosphorylation and desensitization upon exposure to PAF but only PAFR translocated arrestin to the cell membrane and internalized. In RBL-2H3 cells coexpressing the PAFRs along with CXCR1, IL-8 (CXCL8) cross-desensitized Ca²⁺ mobilization to PAF by all the receptors but only PAFR-G_i3 activation cross-inhibited the response of CXCR1 to CXCL8. Altogether, the data indicate that G_i exclusively mediates chemotactic and cross-regulatory signals of the PAFR, but both G_i and G_q activate PI hydrolysis and exocytosis by this receptor. Because chemotaxis and cross-desensitization are exclusively mediated by G_i, the data suggest that differential activation of both G_i and G_q by PAFR likely mediate specific as well as redundant signaling pathways.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Leukocyte accumulation and activation at sites of inflammation and infection are mediated in part by chemoattractants released by invading microorganisms (i.e., N-formylated peptides, fMLP) or produced by the host (a cleavage product of the fifth component of complement, C5a; IL-8 or CXCL8; leukotriene B₄; and platelet-activating factor (PAF)³ (1, 2). Chemoattractants interact with specific cell surface G protein-coupled receptors (GPCRs) to activate effector systems such as phospholipase C (PLC), ion channels, and kinase cascades (1, 2, 3, 4). Like other members of the GPCR family, chemoattractant receptors become desensitized upon agonist exposure, resulting in a loss of cellular responsiveness (1, 5, 6). Chemoattractants induce receptor phosphorylation followed by arrestin recruitment to the cell membrane and internalization, which play an important role in down-regulating cellular responses (7, 8, 9, 10).

PAF is produced by a variety of cells (i.e., neutrophils, macrophages, and B cells), and may mediate physiological as well as pathological conditions such as allergic inflammation, anaphylaxis, and asthma (11, 12, 13). Although most chemoattractant receptors couple to a pertussis toxin (Ptx)-sensitive G protein, (predominantly G_i), to activate PLC, PAFR couples to both Ptx-sensitive and Ptx-insensitive G proteins to mediate cellular responses (14, 15, 16, 17, 18, 19). Reconstitution studies in COS-7 cells demonstrated that PAFR can couple to G_q, G₁₁, or G₁₆ to activate PLC and mediate phosphoinositide (PI) hydrolysis (16). However, in vascular endothelial cells, PAFR was shown to couple specifically to G_q to activate PLC3 (20). In RBL-2H3 cells that predominantly express PLC3, we have shown that PAFR-mediated intracellular Ca²⁺ mobilization was resistant to Ptx whereas secretion and PI hydrolysis were markedly attenuated (21, 22). Responses to PAFR were susceptible to cross-regulation by the peptide chemoattractant receptors formylpeptide receptor (FR), C5aR, and CXCR1 but not vice versa (23). However, a C terminus-deleted mutant of PAFR, deficient in receptor phosphorylation, induced greater G protein activation and cross-desensitized cellular responses to both FR and C5aR (23). In the present work, we sought to determine the precise role of G_i and G_q in PAFR-mediated cellular activation and regulation. For that purpose, PAFR was modified to express either G_i3 (PAFR-G_i3) or G_q (PAFR- G_q) at the C terminus and was stably expressed in the rat basophilic leukemia cell line (RBL-2H3). The ability of the fusion receptors to mediate cell motility, cytotoxicity, and to activate regulatory and cross-regulatory pathways was investigated. The data herein indicate that PAFR uses G_i exclusively to mediate cell motility and cross-regulatory signals, but both G_i and G_q, to mediate secretory activities. Thus, chemoattractant receptors may couple to different G proteins to activate redundant as well as specific pathways.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Materials

[³²P]Orthophosphate (8500–9120 Ci/mM), myo[2-[³H]inositol (24.4 Ci/mM), [³H]WEB 2086 (10.5 Ci/mM), [-³²P]GTP (6000 Ci/mM), and ¹²⁵I-labeled IL-8 were purchased from PerkinElmer. IL-8 (monocyte derived) was purchased from PeproTech. Geneticin (G418) and all tissue culture reagents were purchased from Invitrogen Life Technologies. Hemagglutinin (HA) mAb, protein G-agarose, and protease inhibitors were from Boehringer Mannheim. Indo-1 acetoxymethyl ester and pluronic acid were from Molecular Probes. PAF, GDP, GTP, and ATP were purchased from Sigma-Aldrich. ERK and phospho-ERK Abs were obtained from Cell Signaling. G_i3 and G_q Abs were purchased from Santa Cruz Biotechnology. pcDNA 3.1 containing human G_q and G_i3 were obtained from the Guthrie cDNA Resource Center. cDNA encoding the GFP-tagged arrestin-1 (arr) was a gift from Dr. M. G. Caron (Duke University, Durham, NC). All other reagents were obtained from commercial sources.

Construction of epitope-tagged PAFR and PAFR fusion receptors

Nucleotides encoding a 9-aa HA-epitope sequence (YPYDVPDYA) were inserted between the NH2-terminal initiator methionine and the second amino acid of human PAFR by PCR as previously described (21). To generate the fusion receptors, the HA-PAFR cDNA was altered by adding a HindIII site on the 5' end and replacing the stop codon with a triple alanine sequence followed by a KpnI restriction site at the 3' end. The open reading frame of the receptor cDNA was amplified by PCR using two specific primers: 5'-ACG TAC AAG CTT ATG TAC CCA TAC GAC GTC CCA GAC TAC GCT GAG CCA CAT GAC TCC TCC CAC ATG-3' forward and 5'-TCG ATC GGT ACC TGC TGC TGC TGC CGC ATT TTT GAG GGA ATT GCC AGG GAT CTG-3' reverse. The resulting PCR product as well as the pcDNA-G_q and pcDNA-G_i3 vectors which contain a HindIII-KpnI site at the 5' end of the G cDNAs were digested with HindIII and KpnI and then ligated. All receptor G protein fusion clones were sequenced to confirm the intended fusion constructs and lack of secondary mutations.

Cell culture and transfection

RBL-2H3 cells were maintained as monolayer cultures in DMEM supplemented with 15% FBS, 2 mM glutamine, penicillin (100 U/ml), and streptomycin (100 mg/ml) (19, 24). These cells (1 x 10⁷ cells) were transfected by electroporation with pcDNA3 containing the receptor cDNAs (20 µg). Geneticin-resistant cells were selected by subculturing the transfected cells in growth medium supplemented with geneticin (1 mg/ml) and cell surface expression of the receptors was monitored by FACS analysis as described previously (21). Double-transfectant RBL-2H3 cells expressing CXCR1 along with PAFR (PAFR-CXCR1) or the fusion receptors (PAFR-G_i3-CXCR1 or PAFR-G_q-CXCR1) were generated as described previously (23).

Radioligand-binding assays

RBL-2H3 cells were subcultured overnight in 24-well plates (0.5 x 10⁶ cells/well) in growth medium. Cells were placed on ice, washed three times with ice-cold PBS containing 10 mg/ml BSA, and incubated for 2–4 h in DMEM (250 µl) containing the radiolabeled WEB or PAF (0.1 nM). Reactions were stopped with 1 ml of ice-cold PBS, and cells were washed five times. Cells were then solubilized with radioimmunoprecipitation assay buffer (200 µl) and dried under vacuum, and bound radioactivity was counted. Nonspecific radioactivity bound was determined in the presence of 500 nM unlabeled ligand (21).

Western blot analysis

RBL-2H3 cells (5 x 10⁶) expressing the receptors were washed three times with ice-cold PBS, lysed with radioimmunoprecipitation assay, and immunoprecipitated with HA Ab as described previously (25). The immunoprecipitates were resolved in 10% SDS-PAGE, transferred to a nitrocellulose membrane, and probed with Abs against either G_i3 or G_q. Detection was conducted with HRP-conjugated sheep anti-mouse Ab and ECL. For ERK activity, membranes were prepared and assayed for protein concentration as described previously (25). Membrane proteins (50 µg) were resolved in 10% SDS-PAGE, transferred to a nitrocellulose membrane, and probed with Abs against either ERK 1/2 or phospho-ERK 1/2 (26).

Calcium measurement

Cells (5 x 10⁶) were removed, washed with HEPES-buffered HBSS, and loaded with 1 mM Indo I-AM in the presence of 1 mM pluronic acid for 30 min at room temperature. Then the cells were washed and resuspended in 1.5 ml of buffer. Intracellular calcium increase in the presence of different ligands at the indicated doses was measured as described (27).

Phosphoinositide hydrolysis and secretion of -hexosaminidase

RBL-2H3 cells were subcultured overnight in 96-well culture plates (50,000 cells/well) in inositol-free medium supplemented with 10% dialyzed FBS and 1 µCi/ml [³H]inositol. Then cells were washed with HEPES-buffered HBSS containing 100 mM LiCl and 0.1% BSA and incubated in the same buffer with and without stimulants. Reactions were stopped by adding 200 ml of chloroform:methanol:4 N HCl (100:200:2). The generation of inositol phosphates was determined as reported (21, 25). For secretion of -hexosaminidase, cells were subcultured as above, treated with and without agonist, and -hexosaminidase release in cell lysates was assessed as previously reported (21, 27).

GTPase activity

Cells were treated with appropriate concentrations of stimulants and membranes were prepared as already described (21). GTPase activity, using 10 µg of membrane preparations, was conducted as described previously (21).

Chemotaxis

RBL-2H3 cells stably expressing PAFR and fusion receptor were incubated with calcein AM ionophore for 30 min (28). Cells were resuspended (1.0 x 10⁵/20 µl) in RPMI 1640 without phenol red. Different concentrations of PAF were loaded in a neuroprobe 96-well plate. The cells were added to the top of the filter and incubated for 3–4 h at 37°C. After incubation, the top of the filter was washed five times with medium and fluorescence intensity of the bottom well plate was measured in a PerkinElmer fluorescence microplate reader.

Phosphorylation of the epitope-tagged receptors

Receptor phosphorylation was performed as described previously (21, 26). Briefly, RBL-2H3 cells (5 x 10⁶) expressing the receptors were subcultured overnight in 60-mm tissue culture dishes. The following day, the cells were rinsed twice with 5 ml of phosphate-free DMEM and incubated in the same medium supplemented with [³²P]orthophosphate (150 µCi/dish) for 90 min to metabolically label the intracellular ATP pool. Then labeled cells were stimulated with or without PAF (100 nM) or PMA (100 nM) for 5 min at 37°C. The phosphorylated receptors were immunoprecipitated with the HA Ab, analyzed by SDS electrophoresis, and visualized by autoradiography.

Fluorescence microscopy studies

RBL cells stably expressing PAFR or PAFR-G_i3 or PAFR-G_q were transfected with arr-GFP by electroporation and grown for 24 h and replated on glass bottom dishes (thickness of glass is 0.17 mm) and allowed to adhere for 1 h at 37°C. The cells were washed with RPMI 1640 medium without phenol red and observed using oil immersion x60 objective lens. The fluorescence images were collected with the TE-FM Epi-Fluorescence system attached to the Nikon Inverted Microscope Eclipse TE300. All images were captured by the cool snap HQ digital B/W CCD (Roper Scientific) camera. A Lamda 10-2 optical filter changer (Sutter Instrument) was used to capture images of arr-GFP (GFP, FITC exciter (492 nm), and emitter GRNYFP (535 ± 15 nm); Chroma Technology). All the data obtained was analyzed on Metamorph 4.6r5 software (29). All images shown are representative of at least six separate cells from at least three separate experiments.

Receptor internalization

For flow cytometric analysis, cells were detached by versene treatment, washed with PBS, resuspended in the same medium (1–5 x 10⁶ cells) and treated with PAF (100 nM) for 60 min at 37°C. Cells were washed three times with ice-cold PBS and incubated with HA Ab (1 µg/ml) in a total volume of 400 µl of PBS for 60 min at 4°C. The cells were then washed and incubated with fluorescein (FITC)-anti-mouse IgG for another 60 min at 4°C. Cells were then washed and analyzed for cell surface expression of the receptor on a BD Biosciences FACScan cytometer (30).

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Expression and pharmacological characterization of the fusion receptors PAFR-G_i3 and PAFR-G_q in RBL-2H3 cells

To determine the role of G_i and G_q in PAFR-mediated leukocyte functions, two fusion receptors, PAFR-G_i3 (PAFR expressing G_i3 at the C terminus) and PAFR-G_q (PAFR expressing G_q at the C terminus), were constructed and stably expressed in RBL-2H3 cells. Expression of the receptors were confirmed by FACS analysis using the HA mAb, which specifically binds the 9-aa HA epitope-tag expressed at N-terminal of the receptor (Fig. 1A). Expression of the G linked at the C terminus (C-tail) of the receptors was confirmed by Western blotting. Cell lysates from PAFR-G_q, PAFR-G_i3, and PAFR were immunoprecipitated with HA and immunoblotted with anti-G_i3 (Fig. 1B, left panel) or anti-G_q (Fig. 1B, right panel) specific Abs. As shown in Fig. 1B (left panel, lane 2), anti- G_i3 Ab recognized a specific band at 80 kDa which corresponds to PAFR-G_i3 fusion receptor. The anti-G_q Ab also recognized a specific band slightly above 80 kDa (Fig. 1B, right panel, lane 1) which represents the PAFR-G_q fusion receptor.

View larger version (22K): [in this window] [in a new window]   FIGURE 1. Expression and pharmacological characterization of PAFR-Gi3, PAFR-Gq, and PAFR in RBL-2H3 cells. A, FACS analysis of surface expression of wild-type PAFR and fusion mutants PAFR-Gi3 and PAFR-Gq receptors expressed in RBL-2H3 cells after staining with the HA mAb. B, Cells (5 x 106) stably expressing PAFR-Gq, PAFR-Gi3, and PAFR were lysed and immunoprecipitated with HA Ab as described in Materials and Methods. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-Gi3 or anti-Gq. The results shown are representative of one of three experiments. C, Competition binding of [3H]WEB 2086 by different concentrations of WEB 2086. The results shown are representative of one of three experiments.

Functional characterization of PAFR-G_i3 and PAFR-G_q in RBL-2H3 cells

Upon activation by PAF, PAFR-G_i3, PAFR-G_q, and PAFR stimulated dose-dependent PI hydrolysis (Fig. 2A) and secretion of -hexosaminidase (Fig. 2B) as well as time-dependent activation of intracellular Ca²⁺ mobilization (Fig. 2C) and GTPase activity in membranes (Fig. 2D). PAFR-G_q and PAFR-G_i3 induced greater GTPase activity (0.17 pM of Pi/mg of protein) relative to PAFR (0.12 pM of Pi/mg of protein) but mediated lower PI hydrolysis and secretion, and less sustained intracellular Ca²⁺ mobilization.

View larger version (39K): [in this window] [in a new window]   FIGURE 2. Functional characterization of PAFR-Gi3, PAFR-Gq, and PAFR in RBL-2H3 cells. A, For the generation of inositol phosphates ([3H]IPs), cells were cultured overnight in the presence of [3H]inositol (1 µCi/ml). Cells were preincubated (10 min, 37°C) with a HEPES-buffered saline containing 10 mM LiCl in a total volume of 200 µl and stimulated with different concentrations of PAF for 10 min. Supernatant was used to determine the release of [3H]IPs. Data are represented as fold over basal. The experiment was repeated four times with similar results. B, For secretion, 10 µl of the supernatant for PI hydrolysis (A) was removed and -hexosaminidase released was measured. Data are represented as percentage of total -hexosaminidase in the cells. The experiment was repeated four times with similar results. C, For intracellular calcium mobilization, RBL cells (5 x 106) were loaded with indo-1 and PAF (100 nM) stimulated Ca2+ mobilization was measured. Representative tracings of four experiments are shown. D, For GTPase activity, membranes (10 µg of membrane proteins) were prepared as described in Materials and Methods and assayed for PAF-stimulated GTP hydrolysis. The data are presented as picomoles per milligram of protein. The data shown are the means of three different experiments performed in triplicate.

Effect of Ptx in PAFR-G_i3- and PAFR-G_q-mediated cellular responses in RBL-2H3 cells

We next determined the effect of Ptx on PAF-mediated receptor activation. As shown in Fig. 3, Ptx had no effect on PAFR-G_i3- or PAFR-G_q-mediated PI hydrolysis (Fig. 3A), secretion of -hexosaminidase (Fig. 3B), and intracellular Ca²⁺ mobilization (data not shown). As expected, Ptx inhibited by 50% PAFR-induced PI hydrolysis and secretion of -hexosaminidase (Fig. 3). The lack of inhibitory effect of Ptx on PAFR-G_i3-mediated responses likely suggests that the G_i3 attached to the C-tail of the receptor is resistant to ADP ribosylation. PAFR-Gi2 expressed in RBL-2H3 cells that mediated intracellular Ca²⁺ mobilization and PI hydrolysis to PAF as well as PAFR-Gi3 was also resistant to Ptx pretreatment (data not shown).

View larger version (20K): [in this window] [in a new window]   FIGURE 3. Effect of pertussis toxin in PAFR-Gi3, PAFR-Gq, and PAFR-mediated PI hydrolysis and secretion of -hexosaminidase. A, RBL-2H3 cells were cultured overnight as described in the Fig. 1 legend with or without Ptx (100 ng/ml). Cells were stimulated with PAF (100 nM) for 10 min and supernatant was used to determine the release of [3H]IPs. The experiment was repeated three times with similar results. B, For secretion, 10 µl of the supernatant for PI hydrolysis (A) was removed and -hexosaminidase release was measured. Data are represented as fold over basal. The experiment was repeated three times with similar results. *, p < 0.05.

PAFR-G_i3- and PAFR-G_q-mediated chemotaxis

The fusion receptors were also analyzed for their ability to mediate chemotaxis to PAF. PAFR-G_i3 and PAFR, but not PAFR-G_q, induced dose-dependent chemotaxis to PAF, with maximum response at 1 nm (Fig. 4A). PAFR-G_i3 showed a 3-fold increase in chemotaxis as compared with wild-type PAFR. Ptx has no significant effect on PAFR-G_i3-mediated chemotaxis but totally inhibited response to PAFR (Fig. 4B).

View larger version (17K): [in this window] [in a new window]   FIGURE 4. PAFR-Gi3, PAFR-Gq, and PAFR mediated chemotaxis. A, RBL-2H3 cells were labeled with calcein AM ionophore for 30 min. Cells (1.0 x 105/20 µl) were incubated at 37°C with different concentrations of PAF. Chemotaxis was assessed in a 96-well microprobe chambers as described in Materials and Methods. The results are represented as chemotactic index (mean of the fluorescence intensity per well). The results are representative of one of four experiments performed in triplicate. B, Cells were pretreated with or without Ptx (100 ng/ml) overnight. Chemotaxis in the presence of 1 nm PAF was measured. The results are representative of three separate experiments. *, p < 0.05 and **, p < 0.01.

PAFR-G_i3- and PAFR-G_q-mediated ERK activation

In vascular smooth muscle, PAF was shown to mediate cell migration via a MAPK-dependent pathway (31). To assess the ability of the fusion receptors to activate MAPK, we measured ERK1/2 phosphorylation in response to PAF. As shown in Fig. 5A, PAFR, PAFR-G_i3, and PAFR-G_q induced time-dependent phosphorylation of ERK1/2 in response to PAF (100 nM) stimulation. Maximum response was obtained at 1 min. PAFR induced ERK1/2 phosphorylation to a greater extent (50%, Fig. 5B) relative to PAFR-G_i3 (30%, Fig. 5C) or PAFR-G_q (17%, Fig. 5D). PAFR-G_i3, however, showed a more sustained ERK1/2 phosphorylation as compared with PAFR (Fig. 5, B vs C).

View larger version (21K): [in this window] [in a new window]   FIGURE 5. PAFR-Gi3, PAFR-Gq, and PAFR-induced ERK 1/2 phosphorylation. RBL-2H3 cells expressing PAFR, PAFR-Gi3, PAFR-Gq, and PAFR were stimulated with PAF (100 nM) for 0–10 min. ERK 1/2 phosphorylation was determined by Western blotting using a phospho-ERK (pERK) specific Ab (A). The extents of ERK 1/2 phosphorylation (B–D) are expressed as percentage of total ERK 1/2 (tERK). Data shown are representative of three experiments.

Phosphorylation of PAFR-G_i3 and PAFR-G_q in RBL-2H3 cells

RBL-2H3 cells expressing PAFR-G_i3, PAFR-G_q, or PAFR were labeled with ³²P, treated with 100 nM PAF or PMA, and cell lysates were immunoprecipitated with HA Ab. As shown in Fig. 6A, PAFR (left panel; 42 kDa, monomer and 85 kDa, dimer; Refs. 19 and 32), PAFR-G_i3 (middle panel; 90 kDa), and PAFR-G_q (right panel; 95 kDa) were phosphorylated homologously by PAF (lanes 2) and heterologously by PMA (lanes 3) pretreatment. The specificity of the kinases was tested by measuring the effect of the protein kinase C inhibitor, staurosporine, on receptor phosphorylation. As previously shown (21), staurosporine (100 nM) had no effect on PAF-induced phosphorylation of PAFR (Fig. 6B, left panel, lanes 4 vs 3) but partially inhibited PMA-mediated phosphorylation (lanes 6 vs 5). In contrast, staurosporine totally inhibited PMA-mediated (lanes 6 vs 5), but partially PAF-mediated (lanes 4 vs 3), phosphorylation of PAFR-G_i3 (Fig. 6B, middle panel) and PAFR-G_q (Fig. 6B, right panel).

View larger version (75K): [in this window] [in a new window]   FIGURE 6. PAF and PMA-mediated phosphorylation of PAFR-Gi3, PAFR-Gq, and PAFR. A, 32P-RBL-2H3 cells (5 x 106/60 mm plate) expressing PAFR-Gi3, PAFR-Gq, and PAFR were incubated for 5 min with (lanes 2 and 3) or without (lane 1) stimulants. Cells were lysed, immunoprecipitated with HA-specific Ab and analyzed by SDS-PAGE and autoradiography. The results are from one of three representative experiments. B, 32P-labeled cells were preincubated for 3 min with (lanes 2, 4, and 6) or without (lanes 1, 3, and 5) staurosporine (100 nM) and stimulated with either PAF (lanes 3 and 4) or PMA (lanes 5 and 6) for 5 min. Cells were lysed, immunoprecipitated with HA Ab and analyzed as described above. The results are from one of two representative experiments.

Desensitization of PAFR-G_i3 and PAFR-G_q in RBL-2H3 cells

Intracellular Ca²⁺ mobilization in intact cells was measured to determine the susceptibility of the fusion receptors to desensitization. Cells were first exposed to PAF (1–100 nM) and rechallenged 3 min later with a second dose of 1 µM. As shown in Fig. 7, A–C, exposure of the cells to a first dose of PAF homologously desensitized response to a second dose, with an EC₅₀ of 3–4 nM. Pretreatment with PMA (100 nM) also desensitized PAFR-, PAFR-G_i3-, and PAFR-G_q-mediated Ca²⁺ mobilization to PAF (Fig. 7D).

View larger version (26K): [in this window] [in a new window]   FIGURE 7. Desensitization of PAFR-Gi3, PAFR-Gq, and PAFR-mediated intracellular calcium mobilization and GTPase activity. RBL-2H3 cells (5 x 106 cells) expressing (A) PAFR, (B) PAFR-Gi3, or (C) PAFR-Gq were loaded with the calcium indicator Indo-1 and exposed to a first dose of PAF (1, 5, 10, and 100 nM). Cells were rechallenged 3 min later with a second dose of PAF (1 µM) as indicated. Traces are representative of three experiments. D, Cells were pretreated with (doted traces) or without (solid traces) PMA (100 nM) for 3 min and rechallenged with PAF (100 nM). E, For GTPase activity, the cells were treated with PAF (100 nM) or PMA (100 nM) for 5 min. Membranes were prepared and assayed for agonist-stimulated GTP hydrolysis. The data are presented as percentage of control which is the net maximal stimulation obtained with untreated cells. Data shown are representative of three experiments performed in triplicate. *, p < 0.05 and **, p < 0.01.

PAFR-G_i3 and PAFR-G_q mediated CXCR1 cross-desensitization in RBL-2H3 cells

Activation of the CXCL8 receptor CXCR1 was shown to cross-desensitize cellular responses to PAFR but not vice versa (23). To further determine the relationship between G protein usage and cross-desensitization, RBL-2H3 cells stably coexpressing CXCR1 along with PAFR (PAFR-CXCR1), PAFR-G_i3 (PAFR-G_i3-CXCR1), or PAFR-G_q (PAFR-G_q-CXCR1) were generated. CXCL8- and PAF-mediated cross-desensitization of Ca²⁺ mobilization was measured. As shown in Table I, pretreatment of PAFR-CXCR1 and PAFR-G_i3-CXCR1 cells with a first dose of CXCL8 (10 nM) cross-desensitized PAF-induced Ca²⁺ mobilization by 33 ± 7% and 42 ± 4%, respectively. PAFR-G_q (PAFR-G_q-CXCR1 cells) was resistant to cross-desensitization by CXCL8 (4 ± 3%). CXCR1-mediated Ca²⁺ mobilization was resistant to cross-desensitization by PAFR-G_q (PAFR-G_q-CXCR1 cells, –5 ± 9%) and PAFR (PAFR-CXCR1 cells, 6 ± 5%) but not PAFR-G_i3 (PAFR-G_i3-CXCR1 cells, 51 ± 11%).

Internalization PAFR-G_i3 and PAFR-G_q in RBL-2H3 cells

PAF (100 nM) induced internalization of PAFR but not PAFR-G_i3 or PAFR-G_q (Fig. 8). PAFR was shown to internalize via a arr-dependent mechanism. To determine whether the fusion receptors activate arr, we transiently expressed a GFP-tagged arr-1 in RBL cells expressing PAFR, PAFR-G_i3, or PAFR-G_q. Fluorescence microscopy was used to study the time course of PAF-mediated arrestin translocation and internalization. Upon exposure to PAF (100 nM), PAFR but not PAFR-G_i3 or PAFR-G_q induced translocation of arr-1 to the cell membrane (Fig. 9).

View larger version (30K): [in this window] [in a new window]   FIGURE 8. PAF-induced internalization of PAFR-Gi3, PAFR-Gq, and PAFR in RBL-2H3 cells. RBL-2H3 cells (1 x 106 cells) expressing (A) PAFR, (B) PAFR-Gi3, or (C) PAFR-Gq were treated with (doted lines) or without (solid lines) PAF (100 nM) for 60 min. Cells were stained with FITC-labeled HA Ab and analyzed by FACS for cell surface expression of the receptors. The results are from one of four representative experiments.

View larger version (49K): [in this window] [in a new window]   FIGURE 9. PAFR-Gi3, PAFR-Gq, and PAFR-mediated arr-1 translocation. RBL-2H3 cells coexpressing GFP-tagged arr-1 and PAFR, PAFR-Gi3, or PAFR-Gq were treated with PAF (100 nM). Images were collected at different times with a TE-FM fluorescence system. Shown are representative confocal microscopic images of four different experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

The data herein demonstrate that PAFR couples efficiently to both G_i and G_q to activate similar as well as different pathways. This contention is supported by the following observations. First, the fusion receptors and PAFR bind PAF to mediate PI hydrolysis, secretion, intracellular Ca²⁺ mobilization, ERK1/2 phosphorylation, and GTPase activity (Figs. 2, 3, and 5). In contrast, PAFR and PAFR-G_i3, but not PAFR-G_q, mediated chemotaxis in response to PAF (Fig. 4). In addition, PAFR-G_i3, but not PAFR or PAFR-G_q, cross-regulated Ca²⁺ response to CXCR1 (Table I).

Previous studies with G-linked receptors have shown that the fusion does not alter the properties of either the G protein linked to the C terminus or the receptor (36, 37, 38, 39). The data herein also support these findings. The pharmacological properties of the fusion receptors (K_d: 1.48 ± 0.9 nM and 2.91 ± 0.3 nM for PAFR-G_i3 and PAFR-G_q, respectively) expressed in RBL-2H3 cells were similar to that of the wild-type PAFR (K_d: 3.15 ± 1.3 nM). PAFR-G_i3 and PAFR-G_q, however, mediated lower PI hydrolysis and exocytosis relative to PAFR (Figs. 2 and 3). Three possible explanations could account for this observation. First, maximal responses to PAFR are additive and as the wild-type receptor couples to both G_i and G_q, they may act synergistically to induce PI hydrolysis and secretion. Second, upon PAFR activation, both G and G are released to mediate leukocyte responses. In contrast, the PAFR-G protein fusion receptors could not release G into the cytosol to activate PLC. And, third, the fusion of the G with the receptor promotes its deactivation and reassociation with G, thereby, inhibiting receptor signaling. Supporting this contention is that both PAFR-G_i3 and PAFR-G_q mediated greater GTPase activity relative to PAFR (Fig. 2D). The ₂AR-G_s fusion receptor was also shown to induce greater G protein activation but was less efficient in stimulating adenylate cyclase activity relative to wild-type ₂AR (34).

PAFR-mediated PI hydrolysis and secretion are partially attenuated by Ptx (Fig. 3; Refs. 19 , 21 , 22). In the studies reported here, Ptx had no effect on PAFR-G_i3- and PAFR-G_q-mediated responses. Ptx was shown by others to inhibit cellular responses to the chemoattractant fusion receptor FR-G_i3 (39, 40). An explanation for the resistance of the PAFR-G_i3 to Ptx inhibition in our studies could be that the folding of the C terminus of the PAFR-G_i3 is different from that of the FR-G_i. Several GPCRs are palmitoylated on cysteine residues proximal to the cytosolic end of their seventh transmembrane to create a fourth intracellular loop (41). PAFR C-tail, but not FR, possesses several cysteine residues which are potential targets for palmitoylation (16, 42, 43, 44). Thus, it is possible that acylation of the PAFR-G_i3 C terminus upon activation anchors the G_i3-linked tail in the cellular membrane and hinders the access of the toxin to its cysteine target.

An interesting finding of this study is that PAFR-G_i3 mediated 3-fold greater chemotaxis relative to PAFR (Fig. 4). Furthermore, the PAFR-G_q, which stimulated greater GTPase activity than PAFR, did not mediate chemotaxis in response to PAF (Figs. 2 and 4). These results support previously published data suggesting that chemoattractant receptor-mediated cell motility requires G released predominantly from G_i (22, 44). Thus, the increase in chemotactic activity by the PAFR-G_i3 fusion receptor likely indicates greater G release from the G_i3-linked receptor relative to PAFR (Fig. 4). PAFR was also shown to mediate chemotaxis via a MAPK-dependent pathway stimulated by G (31). Indeed, PAFR (50%) and PAFR-G_i3 (30%) induced greater ERK 1/2 phosphorylation than PAFR-G_q (5%). Interestingly, PAFR-G_i3, which induced greater chemotaxis, mediated a more sustained ERK 1/2 phosphorylation relative PAFR (Fig. 5). These data support the role of MAPK in PAF-mediated chemotaxis, and indicate that the increase in chemotactic response to PAFR-G_i3 is likely due to the prolonged activation of the MAPK cascade.

A question addressed in this study was the role of G protein usage in PAFR regulation. Upon activation by PAF, PAFR was shown to undergo receptor phosphorylation and desensitization via two mechanisms: a G protein-coupled receptor kinase-dependent and a protein kinase C-dependent pathway (21, 45). As shown in Fig. 6, PAFR-G_i3 and PAFR-G_q underwent homologous and heterologous phosphorylation by both PAF and PMA. Furthermore, PAF and PMA pretreatment attenuated receptor-mediated GTPase activity in membrane and intracellular Ca²⁺ mobilization in intact cells (Fig. 7). These results suggest that the G linked at the C-tail of the receptors did not perturb the access of the kinases to the phosphorylatable serine and threonine residues. FR-G_i was also shown to desensitize and internalize as well as the wild-type FR upon activation by fMLP (39, 40). In contrast to FR-G_i, however, PAFR-G_q and PAFR-G_i3 were resistant to internalization by PAF (Fig. 8). PAFR was previously shown to internalize in a phosphorylation/arr-dependent fashion (21, 46). Deletion of the C terminus of PAFR, which prevented receptor phosphorylation and arrestin recruitment, inhibited receptor internalization (23). Because PAFR-G_q and PAFR-G_i3 undergo phosphorylation as well as PAFR, but failed to recruit arrestin (Figs. 6 and 9), this likely suggests that the presence of the G subunits linked at the C-tail of the receptors prevented their association with arr thereby inhibiting receptor internalization.

In summary, the data herein suggest that PAFR uses G released from both G_i and G_q to activate similar as well as specific cellular responses. PAF-induced chemotactic and cross-regulatory signals are mediated exclusively via G_i coupling whereas cytotoxicity related functions are mediated by both G_i and G_q. PAFR-Gi3 mediated greater chemotaxis but was resistant to internalization. Thus, the data also suggest that receptor internalization is not required for PAFR-mediated leukocyte motility. Kelley et al. (47) recently demonstrated that GPCR could temporally activate different PLC isoforms to mediate acute and sustained responses. PAFR was shown to activate both PLC₂ and PLC₃ (48, 49). Does PAFR temporally couple to different G protein and PLC isoforms to mediate chemotactic vs cytotoxic functions of leukocyte? Future studies using the fusion receptors described in this work will address this question.

	Acknowledgments

 
We are very grateful to Dr. Charles N. Serhan (Harvard Medical School) for the assistance provided to Dr. S. L. Brown during his PhD training. We also thank Dr. Marc Caron (Duke University) for providing the arr plasmids, and Drs. Ralph Snyderman (Duke University) and Ifeanyi J. Arinze (Meharry Medical College) for critical reading of the manuscript.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
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.

¹ This work was supported by National Institutes of Health Grants AI-38910 (to R.M.R.) and AI-52381 (to B.H.).

² Address correspondence and reprint requests to Dr. Ricardo M. Richardson, Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, 1801 Fayetteville Street, Durham, NC 27707. E-mail address: mrrichardson{at}nccu.edu

³ Abbreviations used in this paper: PAF, platelet-activating factor; GPCR, G protein-coupled receptor; PLC, phospholipase C; Ptx, pertussis toxin; PI, phosphoinositide; FR, formylpeptide receptor; HA, hemagglutinin; arr, arrestin; IP, inositol phosphate.

Received for publication February 6, 2006. Accepted for publication June 2, 2006.

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