Activation of Mitogen-activated Protein Kinases by Formyl Peptide Receptors Is Regulated by the Cytoplasmic Tail*

Madhavi J. RaneDagger , John M. ArthurDagger §, Eric R. Prossnitz, and Kenneth R. McLeishDagger §parallel **

From the Departments of Dagger  Medicine and § Biochemistry and Molecular Biology, University of Louisville Health Science Center and the parallel  Veterans Affairs Medical Center, Louisville, Kentucky 40202 and the  Department of Cell Biology and Physiology, University of New Mexico, Health Sciences Center, Albuquerque, New Mexico 87131

    ABSTRACT
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Wild type formyl peptide receptors (FPRwt) and receptors deleted of the carboxyl-terminal 45 amino acids (FPRdel) were stably expressed in undifferentiated HL-60 promyelocytes. Expression of FPRwt reconstituted N-formylmethionyl-leucyl-phenylalanine (FMLP)-stimulated extracellular signal-regulated kinase (ERK) and p38 kinase activity. Expression of FPRdel resulted in a 2-5-fold increase in basal ERK and p38 kinase activity, whereas FMLP failed to stimulate either mitogen-activated protein kinase (MAPK). Pertussis toxin abolished FMLP stimulation of both MAPKs in FPRwt cells but had no effect on either basal or FMLP-stimulated MAPK activity in FPRdel cells. FMLP stimulated a concentration-dependent increase in guanosine 5'-3-O-(thio)triphosphate (GTPgamma S) binding in membranes from FPRwt but not FPRdel cells. GTPgamma S inhibited FMLP binding to FPRwt but not FPRdel membranes. Photoaffinity labeling with azidoanilide-[gamma -32P]GTP in the presence or absence of FMLP showed increased labeling only in FPRwt membranes. Immunoprecipitation of alpha i2 and alpha q/11 from solubilized, photolabeled membranes showed that FPRwt were coupled to alpha i2 but not to alpha q/11. FPRwt cells demonstrated calcium mobilization following stimulation with FMLP, whereas FPRdel cells showed no increase in intracellular calcium. We conclude that the carboxyl-terminal tail of FPRs is necessary for ligand-mediated activation of Gi proteins and MAPK cascades. Deletion of the carboxyl-terminal tail results in constitutive activation of ERK and p38 kinase through a Gi2-independent pathway.

    INTRODUCTION
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Chemoattractants, including formylated peptides, C5a, leukotriene B4, platelet-activating factor, and CXC chemokines (e.g. interleukin 8), are proinflammatory agents that recruit polymorphonuclear leukocytes (PMNs)1 to a site of infection or inflammation, stimulate respiratory burst activity, and induce release of lysosomal enzymes (1-3). Genes for chemoattractant receptors have been cloned and sequenced (1, 4), and all are members of the superfamily of G protein-coupled receptors (GPCRs) containing seven transmembrane domains with an extracellular amino-terminal domain and an intracellular carboxyl-terminal tail separated by three intracellular loops and three extracellular loops (5). Formyl peptide receptors (FPRs), as well as C5a and leukotriene B4 receptors, couple to pertussis toxin-sensitive Gi proteins (6-8), whereas platelet-activating factor receptors activate Go and/or Gq, in addition to Gi proteins (9, 10). Transient co-transfection of chemoattractant receptors and Galpha 16 permits activation of phospholipase C in Cos cells (11-13).

The domains of GPCRs that interact with G proteins have been examined in studies using mutant and chimeric receptors or synthetic peptides corresponding to specific receptor domains. These studies indicate that the third intracellular loop and the amino-terminal region of the carboxyl-terminal tail are essential for coupling of adrenergic and muscarinic receptors and rhodopsin to G proteins (14-17). Chemoattractant receptors differ structurally from other GPCRs in that they have a relatively short third intracellular loop (1), suggesting that chemoattractant receptors may interact with G proteins using domains different from those used by other GPCRs. Studies using site-directed replacement mutants of FPRs failed to demonstrate a role for the third cytoplasmic loop in G protein activation (18). Additionally, synthetic peptides consisting of the entire third cytoplasmic loop failed to inhibit G protein-dependent, high affinity ligand binding and physical coupling of formyl peptide receptor to G proteins (19). On the other hand, studies using synthetic peptides corresponding to the second intracellular loop and the proximal portion of the carboxyl-terminal tail of human FPRs disrupt the physical interaction with Gi proteins (19-21). Additionally, phosphorylation of the carboxyl-terminal tail during desensitization of FPRs results in uncoupling of the receptor from G proteins (22). These studies indicate that the second intracellular loop and carboxyl-terminal tail contribute to the physical interaction of FPRs with G proteins; however, the role of these domains in FPR activation of G proteins and effectors has not been examined.

Two mitogen-activated protein kinase (MAPK) cascades, the extracellular signal-regulated kinases (ERKs) and p38 kinases, are stimulated in PMNs by chemoattractants (23-31). ERKs are reported to participate in PMN adherence and respiratory burst activation (23, 29, 30), whereas p38 kinases participate in PMN adherence, chemotaxis, and respiratory burst activity (27, 28, 30). Nick et al. (28) reported that pertussis toxin inhibited ERK activation but not p38 kinase activation by FMLP in human PMNs. Additionally, chemoattractants stimulate different levels of MAPK activity. For example, interleukin 8 and platelet-activating factor stimulate a weaker ERK response than C5a and FMLP (25, 28). These findings suggest that specific domains of chemoattractant receptors regulate MAPK activity by stimulating different G protein-coupled pathways or by disparate rates of activation of the same pathway. The present study was designed to determine the role of the carboxyl-terminal tail in FPR-mediated activation of ERK and p38 MAPKs. A deletion mutant of the carboxyl-terminal tail of FPRs was constructed by site-directed mutagenesis, and both mutated and wild type receptors were stably expressed in undifferentiated HL-60 cells. Activation of MAPK cascades and G proteins by wild type and mutant receptors was examined. Our results indicate that the carboxyl-terminal tail of FPRs plays a significant role in control of basal activity and ligand-stimulated Gi protein-dependent activation of both ERK and p38 kinases.

    EXPERIMENTAL PROCEDURES
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Materials-- FMLP, hygromycin, and geneticin (G418) were obtained from Sigma. GDP, GTP and GTPgamma S were obtained from Boehringer Mannheim. [35S]GTPgamma S was obtained from NEN Life Science Products. Viral vector M13mp18 and site-directed mutagenesis kit were obtained from Bio-Rad. pCEP4 was obtained from Invitrogen (San Diego, CA). Oligonucleotides were obtained from DNA Technologies Inc. (Gaithersburg, MD). Goat anti-rabbit fluorescein isothiocyanate and monoclonal anti-Galpha i2 antibody were obtained from Chemicon (Temecula, CA). Polyclonal antisera against Galpha 12 and Galpha 13 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Pertussis toxin was from LIST Biological Laboratories (Campbell, CA). Fluo-3 was from Molecular Probes (Eugene, OR).

A polyclonal anti-p38 antisera was raised in rabbits using the 14-amino acid peptide CFVPPPLDQEEMES corresponding to the carboxyl terminus. The specificity of the p38 antisera was defined by immunoblotting of recombinant p38 from bacterial lysates. A polyclonal anti-Galpha q/11 antisera was raised in rabbits using the 10-amino acid peptide QLNLKEYNLV corresponding to the carboxyl terminus and was provided by Dr. Thomas W. Gettys (Medical University of South Carolina, Charleston, SC). The specificity of this antisera was determined by identification of Galpha q produced in Sf9 cells by immunoblotting, whereas the antisera did not interact with bacterially expressed Galpha i1, Galpha i2, or Galpha i3 (32).

Construction of Deletion Mutants-- The wild type formyl peptide receptor (FPRwt) gene was subcloned into the EcoRI site of the viral vector M13mp18 to isolate single-stranded DNA for site-directed mutagenesis. The site-directed mutagenesis procedure was based on the method of Kunkel et al. (33). A SnaBI site was created at amino acid number 301 between putative transmembrane region 7 and the carboxyl-terminal tail. The EcoRI/SnaBI fragment of the FPR deletion mutant (FPRdel) receptor was shuttled through pBluescript vector and was subcloned into the KpnI/HindIII site of the pCEP4 vector. The carboxyl-terminal 45 amino acids of FPR were deleted in the expressed mutant.

Cell Culture-- HL-60 cells, obtained from American Type Culture Collection (Manassas, VA), were grown in suspension culture in a humidified atmosphere with 8% CO2 at 37 °C in RPMI 1640 medium supplemented with 10% (v/v) horse serum, 1% (v/v) nonessential amino acids, 1 mM L-glutamine, 50 units/ml of penicillin, and 50 µg/ml streptomycin. HL-60 cells stably expressing FPRwt were established as described previously and maintained in medium containing geneticin (34). FPRdel was transfected into undifferentiated HL-60 cells by the calcium phosphate precipitation method (35). Stably transfected cells were selected by cultivation in medium supplemented with 200 µg/ml hygromycin. Crude plasma membrane fractions were prepared by nitrogen cavitation as described previously (7).

Flow Cytometry Assay for Formyl Peptide Binding-- Binding of N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein to FPRs was performed as described previously (34). FPRwt-transfected HL-60 cells and FPRdel-transfected HL-60 cells were harvested by centrifugation, washed with Krebs-Ringer phosphate buffer, and resuspended in Krebs-Ringer phosphate buffer to 1 × 106 cells/ml. To determine the concentration of N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein that produced saturation binding, HL-60 cells transfected with FPRwt, FPRdel, or vector alone were incubated with increasing concentrations of N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein at 4 °C for 20 min. Nonspecific binding was determined in the presence of 20 µM FMLP. The cells were analyzed on a Coulter Epics Elite II flow cytometer (Coulter, Hialeah, FL). Saturation of binding sites by N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein occurred at 100 nM for FPRwt HL-60 cells and 1 µM for FPRdel HL-60 cells. Quantum 24 fluorescein microbeads (Flow Cytometry Standards Corp., San Juan, PR) containing 4 × 103 to 6.6 × 104 molecules of equivalent soluble fluorochromes were used as standards to determine the average number of FPRs on the cell surface.

ERK Assay-- An in vitro kinase assay for ERK activity was performed as described previously (30). Following a 5-min preincubation in Krebs-Ringer phosphate buffer containing 5 mM dextrose at 37 °C, 30 × 106/ml cells were stimulated with FMLP for the indicated time and at the indicated concentration. Stimulation was terminated by a brief centrifugation (2500 × g for 20 s), and the cell pellet was immediately lysed in 0.5 ml of ice-cold lysis buffer containing 50 mM beta -glycerophosphate (pH 7.2), 100 µM sodium vanadate, 1 mM EDTA, 1 mM dithiothreitol, 2 mM MgCl2, 0.5% Triton X-100, 5 µg/ml leupeptin, and 0.09 units/ml aprotinin. The cell lysates were centrifuged for 15 min at 15,000 × g at 4 °C to remove cellular debris. The cleared lysates were applied to 0.5 ml DEAE-Sephacel (Amersham Pharmacia Biotech) mini-columns that had been pre-equilibrated in Buffer A containing 50 mM beta -glycerophosphate (pH 7.2), 100 µM sodium vanadate, 1 mM EDTA, 1 mM dithiothreitol. The columns were then washed with 5 ml of Buffer A followed by elution of the enzyme-containing fraction with 1 ml buffer A containing 0.5 M NaCl. In vitro kinase reactions were performed by addition of 20-µl aliquots of each eluate to 20 µl of reaction mixture containing 50 mM beta -glycerophosphate (pH 7.2), 0.1 mM sodium orthovanadate, 0.2 mM ATP, 20 mM MgCl2, 1 mM EGTA, 0.5 µl [gamma -32P]ATP, and EGFR662-681 synthetic peptide (Macromolecular Resources, Colorado State University, Ft. Collins, CO). Following incubation for 15 min at 30 °C, reactions were terminated by spotting 30 µl from each assay onto P81 Whatman filter paper. Filters were washed three times in 150 mM phosphoric acid and once in acetone, dried, and counted by scintillation spectroscopy. Nonspecific binding was determined by assaying reaction mixture with elution buffer and subtracted from each result. Each assay was performed in triplicate, and the results were averaged.

p38 Kinase Assay-- p38 kinase activity was measured by an immune complex kinase assay using ATF-2 as substrate, as described previously (30). Briefly, 30 × 106 cells were preincubated for 5 min in Krebs-Ringer phosphate buffer containing 5 mM dextrose and then stimulated with FMLP for the indicated time and at the indicated concentration. The reactions were terminated by a 20 s centrifugation at 2500 × g followed by lysis with 0.5 ml of cold lysis buffer containing 20 mM Tris pH 7.5, 1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, 20 mM NaF, 0.2 mM sodium vanadate, 1 mM EDTA, 1 mM EGTA, and 5 mM phenylmethylsulfonyl fluoride. Following centrifugation at 15,000 × g for 15 min at 4 °C, cleared lysates were incubated with 5 µl/sample of anti-p38 antisera and incubated for 1 h at 4 °C. Protein A-Sepharose beads (15 µl of 1:1 slurry in lysis buffer) were added to the lysates and incubated for 1 h at 4 °C to precipitate the immune complexes. Samples were centrifuged at 15,000 × g for 2 min, and the beads were washed once in lysis buffer and once in kinase buffer containing 25 mM Hepes, 25 mM beta -glycerophosphate, 25 mM MgCl2, 2 mM dithiothreitol, and 0.1 mM sodium orthovanadate. The kinase assay was initiated by the addition of 40 µl of kinase buffer containing 5 µCi [gamma -32P]ATP and 3 µg of ATF-21-110 to the washed beads. Reactions were incubated for 15 min at 30 °C and then terminated by the addition of 15 µl of 5× Laemmli SDS sample buffer. Samples were boiled and briefly centrifuged, and the products were resolved by 10% SDS-PAGE. The incorporation of 32P was visualized by autoradiography and quantified on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Photoaffinity Labeling of Plasma Membrane G Proteins-- [gamma -32P]GTP azidoanalide (AA-GTP) was synthesized as described previously (32). Photoaffinity labeling of G proteins from FPRwt and FPRdel HL-60 membranes (100 µg/condition) with AA-GTP was performed as described previously (32). Briefly, plasma membranes were incubated in the presence or absence of 10 µM FMLP in buffer containing 30 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 5 µg/ml soy trypsin inhibitor, 1 µCi of AA-GTP, and 2 µM GDP at 30 °C for 10 min and then illuminated with ultraviolet light (302 nm) for 3 min. The samples were centrifuged at 10,000 rpm for 5 min and then resuspended in buffer containing 50 mM sodium phosphate, pH 7.4, 1 mM dithiothreitol, and 0.5% SDS. The samples were heated at 60 °C for 5 min and then solubilized in the same buffer containing 1.25% Nonidet P-40, 1.25% sodium deoxycholate, and 190 mM sodium chloride. The samples were centrifuged immediately, and the pellet was discarded. To determine the total G proteins labeled, 20 µl of the supernatant was separated by 10% SDS-PAGE followed by autoradiography and densitometry. To determine the AA-GTP binding to specific G proteins, the remaining supernatant was precleared with protein A-Sepharose beads for 15 min. Precleared solubilizate was incubated overnight with specific antisera for Galpha i2 or Galpha q/11 at a dilution of 1:50. G protein antibody complexes were recovered by the addition of 25 µl of protein A-Sepharose beads. Following incubation for 30 min, the beads were washed with cold phosphate-buffered saline and resuspended in 60 µl of Laemmli buffer. Labeled G protein subunits were separated by 10% SDS-PAGE under reducing conditions and identified by autoradiography. Relative densities of the G protein bands were determined with a Personal Densitometer SI (Molecular Dynamics).

GTPgamma S Binding Assay-- GTPgamma S binding was performed as described previously (7). Briefly, assays were performed in a reaction mixture (100 µl) containing 50 mM triethanolamine/HCl, pH 7.4, 1 mM dithiothreitol, 1 mM EDTA, 1 mM MgCl2, 150 mM NaCl, 1.0 µM GDP, and 0.02-0.04 µCi/tube of [35S]GTPgamma S. Reactions were initiated by the addition of 2-10 µg of membrane protein/tube. Each reaction was allowed to proceed at 30 °C for 20 min and terminated by rapid filtration through Whatman GF/C filters. Filters were counted by liquid scintillation spectrometry. Specific binding was calculated by subtracting the amount of [35S]GTPgamma S bound in the presence of 10 µM GTPgamma S from total [35S]GTPgamma S bound and expressed as fmol of GTPgamma S bound per mg of membrane protein.

Receptor Binding Assay-- FMLP binding assays were performed in a reaction mixture (100 µl) containing 50 mM Tris, pH 7.5, 1 mM EDTA, 5 mM MgCl2, as described previously (7). Reactions were initiated by addition of 15-25 µg of membrane protein and incubated for 30 min at 25 °C. Reactions were terminated by rapid filtration through Whatman GF/C filters, which were dried, placed in 4 ml of scintillation mixture, and counted in a liquid scintillation spectrometer. Specific binding was calculated by subtracting the amount of N-formyl-Met-Leu-[3H]Phe bound in the presence of excess ligand from the total N-formyl-Met-Leu-[3H]Phe bound. Binding parameters were estimated using a non-linear least squares curve fitting procedure (SCTFIT), as described previously (7).

Flow Cytometric Assay for Formyl Peptide Receptors-- Binding of N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein to FPRs was performed as described previously (34). Cells were harvested by centrifugation, washed in PBS, and resuspended at 1 × 106 cells/ml in PBS. Binding was performed in 1 ml with 10 nM N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein (Molecular Probes) for 15 min on ice. Specificity of binding sites was determined by addition of 10 µM fMet-Leu-Phe. The cells were analyzed by flow cytometry (Epics Profile II, Coulter). Quantum 24 fluorescein microbeads (Flow Cytometry Standards Corp., San Juan, PR) containing 4 × 103 to 6.6 × 104 molecules of equivalent soluble fluorochromes were used as standards to determine the average number of FPRs per cell. Data were stored and analyzed using WinList 3.0 (Verity Software House, Inc., Topsham, ME).

Calcium Mobilization-- HL-60 cells at 1 × 106 ml were incubated with Fluo-3 for 30 min at 37 °C followed by stimulation with 0.3 µM FMLP. The increase in fluorescence intensity attributed to the increase in intracellular calcium was monitored as a function of time with a confocal microscope (Meridian, Okemos, MI) using excitation and emission wavelengths of 490 and 520 nm, respectively. The calcium ionophore ionomycin was used as a positive control.

Immunoblot for G Proteins-- Proteins from undifferentiated HL-60, FPRwt HL-60, and FPRdel HL-60 membranes were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes, and immunoblotted with Galpha i2, Galpha q/11, Galpha 12, and Galpha 13 antisera. Bound antibody was detected by chemiluminesence (Renaissance, NEN Life Science Products).

    RESULTS
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Expression of FPRwt and FPRdel in HL-60 Cells-- Flow cytometry was used to confirm FPR expression in stably transfected, undifferentiated HL-60 cells. Saturation of binding sites occurred at 100 nM N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein in FPRwt HL-60 cells and 1 µM N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein in FPRdel HL-60 cells. Fig. 1 shows the histograms of one flow cytometric experiment representative of three separate experiments in which binding of N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein to vector-, FPRwt-, and FPRdel-transfected cells was measured. The specific binding of N-formyl-Nleu-Leu-Phe-Nleu-Tyr-Lys-fluorescein was used to calculate receptor numbers for both the cell types. FPRwt was expressed at an average of 15,000 receptors/cell, whereas FPRdel was expressed an average of 20,000 receptor/cell. Both FPRwt and FPRdel were expressed in 70-80% of transfected cells (results not shown). Vector-transfected undifferentiated HL-60 cells did not express receptors capable of specifically binding N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein. These results indicate that cells transfected with FPRwt and FPRdel represent a relatively homogeneous population and that deletion of the carboxyl-terminal tail does not result in an altered tertiary structure of FPRs that prevents either expression or ligand binding.


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Fig. 1.   Flow cytometric analysis of N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein binding by vector-, FPRdel-, and FPRwt-transfected HL-60 cells. Vector- (A), FPRwt- (B), and FPRdel (C)-transfected HL-60 cells were incubated with 100, 100, and 1000 nM N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein, respectively, on ice for 20 min and analyzed by flow cytometry, as described under "Experimental Procedures." The results are from a single experiment representative of three separate experiments.

Reconstitution of FMLP-Stimulated MAPK Activity in FPRwt-transfected Undifferentiated HL-60 Cells-- FPRs expressed by HL-60 granulocytes following differentiation by dimethyl sulfoxide stimulate ligand-dependent activation of both ERKs and p38 kinases (30). To determine that expression of FPRwt in undifferentiated HL-60 cells reconstitutes the signal transduction pathways necessary for MAPK activation, ERK and p38 kinase activities were assayed following FMLP stimulation of FPRwt HL-60 cells. Similar to results in differentiated HL-60 granulocytes (30), maximal stimulation of ERK (mean ± S.E., 5.4 ± 0.3-fold; n = 3) and p38 (3.2 ± 0.4-fold; n = 3) activity occurred 1 min after addition of 3 × 10-7 M FMLP (Fig. 2). Neither ERK nor p38 kinase activity was stimulated by FMLP in vector-transfected, undifferentiated HL-60 cells (data not shown). Thus, signaling components necessary for FMLP stimulation of MAPK cascades are present in undifferentiated FPRwt HL-60 cells.


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Fig. 2.   Time course of ERK and p38 activity stimulated by FMLP in FPRwt HL-60. FPR HL-60 cells (20 × 106/ml) were stimulated for the indicated times with 3 × 10-7 M FMLP at 37 °C and assayed for MAPK activity. Panel A presents the results of ERK activity partially purified from cell lysates by DEAE-Sephacel chromatography. ERK was assayed for kinase activity in an in vitro kinase reaction using EGFR662-681 synthetic peptide as substrate. Peptide incorporated 32P was quantified by scintillation counting. Results are presented as mean ± S.E. of the fold increase over basal ERK activity for three separate experiments. Panel B presents results of p38 kinase activity separated from cell lysates by immunoprecipitation. Kinase activity was determined by [gamma -32P]ATP phosphorylation of ATF-2 by immunoprecipitated p38 for each time point. The data represent the mean ± S.E. of three separate experiments as fold increase in 32P incorporation over unstimulated cells determined by phosphorimaging.

Role of the Carboxyl-terminal Tail in MAPK Activation-- To examine the role of the carboxyl-terminal tail in MAPK activation, FMLP-stimulated ERK and p38 kinase activities were measured in FPRwt HL-60 cells and FPRdel HL-60 cells 1 min after addition of 3 × 10-7 M FMLP. FMLP stimulated a 5.8 ± 1.9-fold (mean ± S.E.; n = 3) increase in ERK activity in FPRwt HL-60 cells, whereas FMLP failed to stimulate an increase in ERK activity (1.3 ± 0.1-fold; n = 3) in FPRdel HL-60 cells (Fig. 3A). Basal activity in FPRdel cells was 3.1 ± 1.4-fold (mean ± S.E.; n = 3) higher than in FPRwt. FMLP stimulated a 3.5 ± 0.7-fold (mean ± S.E.; n = 6) increase in p38 kinase activity in FPRwt cells, whereas only a 1.3 ± 0.1-fold (mean ± S.E.; n = 6) increase in p38 activity was stimulated in FPRdel cells (Fig. 3B). Basal activity in FPRdel HL-60 cells was 3.4 ± 0.7 (mean ± S.E., n = 6)-fold higher than in FPRwt HL-60 cells. To determine whether FPRwt and FPRdel are coupled to ERK and p38 kinases by pertussis toxin-sensitive G proteins, cells were pretreated with 100 ng/ml pertussis toxin for 24 h. This time and concentration has been shown previously to inhibit Gi protein activation by FMLP in HL-60 cells (6). Pertussis toxin suppressed FMLP-stimulated ERK and p38 activities in FPRwt HL-60 cells by 94 and 88%, respectively, indicating that pertussis toxin-sensitive G proteins couple FPRwt to both MAPK cascades (Fig. 3, A and B). Basal ERK and p38 activities were not significantly altered by pertussis toxin pretreatment in cells expressing either FPRwt or FPRdel. Thus, deletion of the carboxyl-terminal tail uncouples FPRs from ligand-stimulated MAPK activation and results in increased basal MAPK activity.


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Fig. 3.   Effect of pertussis toxin on FMLP-stimulated ERK and p38 activities. Panel A, FPRwt and FPRdel HL-60 cells were incubated for 24 h in the presence or absence of 100 ng/ml pertussis toxin (PTx). Cells were stimulated with 3 × 10-7 M FMLP for 1 min at 37 °C (open bars) or left unstimulated (solid bars), and ERK activity was measured in an in vitro kinase assay by 32P phosphorylation of EGFR662-681 in partially purified cell lysates. The results are expressed as mean ± S.E. cpm of ERK activity for three separate experiments. Panel B, FPRwt and FPRdel HL-60 cells were incubated for 24 h in the presence or absence of 100 ng/ml pertussis toxin (PTx). Cells were stimulated with 3 × 10-7 M FMLP for 1 min at 37 °C (open bars) or left unstimulated (solid bars), and p38 activity was measured. Results are expressed as mean ± S.E. arbitrary densitometric units from 4-6 separate experiments. Panel C, autoradiogram representative of experiments from which data were analyzed for panel B, depicting the increase in basal p38 kinase activity in membranes from FRPdel HL-60 cells.

Role of the Carboxyl-terminal Tail in G Protein Activation-- Because both pertussis toxin and FPRdel inhibited FMLP stimulation of MAPKs, the contribution of the carboxyl-terminal tail of FPRs to the interaction with G proteins was examined by FMLP-stimulated GTPgamma S binding and GTPgamma S inhibition of FMLP binding in plasma membranes from FPRwt and FPRdel expressing cells. FPRwt membranes exhibited a concentration-dependent increase in GTPgamma S binding upon addition of FMLP (Fig. 4A). On the other hand, GTPgamma S binding was not increased by FMLP in FPRdel plasma membranes. Additionally, basal GTPgamma S binding was reduced in FPRdel membranes, compared with FPRwt membranes, suggesting reduced affinity of FPRdel for ligand. Guanine nucleotides inhibit ligand binding to formyl peptide receptors by reducing receptor affinity (36-38). Addition of GTPgamma S reduced FMLP binding to FPRwt membranes in a concentration-dependent manner, whereas GTPgamma S had no effect on FMLP binding in FPRdel membranes (Fig. 4B). These data indicate that the absence of the carboxyl-terminal tail results in uncoupling of FPRs from G proteins.


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Fig. 4.   FMLP-stimulated GTPgamma S binding and GTPgamma S inhibition of FMLP binding to FPRwt and FPRdel HL-60 membranes. Panel A, FPRwt (solid circles) and FPRdel (open circles) HL-60 membranes were incubated for 20 min in the presence of increasing concentrations of FMLP. Results are expressed as mean ± S.E. percentage increase in GTPgamma S bound from 3 separate experiments. Panel B, FPRwt (solid circles) and FPRdel (open circles) HL-60 membranes were incubated for 20 min in the presence of increasing concentrations of GTPgamma S. Results are expressed as mean ± S.E. percentage inhibition of FMLP bound from three separate experiments.

The increased, pertussis toxin-independent basal ERK and p38 kinase activities in FPRdel HL-60 cells suggested that these mutant receptors were constitutively active for pathways independent of Gi proteins. To examine possible activation of other G proteins, FPRwt and FPRdel plasma membranes were photoaffinity labeled with AA-GTP before and after stimulation with 10-5 M FMLP. Autoradiography after SDS-PAGE separation of stimulated FPRwt plasma membrane proteins demonstrated increased labeling of a band at 42-43 kDa (Fig. 5A). On the other hand, basal AA-GTP labeling was reduced in FPRdel membranes, and no increase in AA-GTP labeling of FPRdel membranes was seen following FMLP stimulation (Fig. 5A). The identity of the G proteins that demonstrated increased labeling was examined by immunoprecipitation of solubilized photolabeled membranes with antibodies to Galpha i2 and Galpha q/11 (Fig. 5B). Immunoprecipitated Galpha i2 showed increased photolabeling in FMLP-stimulated FPRwt membranes, whereas no increase in FMLP-stimulated labeling occurred in FPRdel membranes (Fig. 5B). Basal photolabeling of Galpha i2 was decreased in FPRdel membranes compared with FPRwt membranes (Fig. 5B). No increase in Galpha q/11 photolabeling was seen in either FPRwt or FPRdel membranes following addition of FMLP (data not shown). Immunoblotting of Galpha i2 in membranes from undifferentiated HL-60, FPRwt and FPRdel HL-60 membranes showed equal density of Galpha i2 and Galpha q/11 subunits (data not shown), demonstrating that decreased photolabeling of G proteins in FPRdel HL-60 membranes was not due to decreased amounts of G proteins present. No basal or FMLP-stimulated photolabeling of alpha 12 or alpha 13 was detected, and neither of these alpha  subunits was detected by immunoblotting of HL-60 plasma membranes (data not shown).


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Fig. 5.   AA-GTP photolabeling of FPRwt- and FPRdel-containing HL-60 Membranes. Panel A, autoradiogram of AA-GTP-photolabeled G proteins in solubilized membranes from FPRwt and FPRdel HL-60 cells. Photolabeling of intact membranes was performed in the presence and absence of 1 × 10-5 M FMLP prior to solubilization. Results are representative of three separate experiments. Panel B, autoradiogram of AA-GTP photolabeling of Galpha i2 immunoprecipitated from solubilized membranes. Photolabeling of intact membranes was performed in the presence and absence of 1 × 10-5 M FMLP in FPRwt and FPRdel membranes prior to solubilization. Results are representative of three separate experiments.

Effects of Carboxyl-terminal Tail on Calcium Mobilization-- Ligand stimulation of FPRs also results in an increase in cytosolic calcium concentration in HL-60 cells via a Gi protein-mediated stimulation of phospholipase C and subsequent generation of inositol 1,4,5-trisphosphate (39). To determine whether the carboxyl-terminal tail of FPRs also contributes to signal transduction pathways leading to calcium mobilization, intracellular calcium concentrations were determined in FPRwt- and FPRdel-expressing HL-60 cells before and after addition of 3 × 10-7 M FMLP. Calcium concentrations were determined by loading cells with Fluo-3 prior to stimulation with FMLP. Cells expressing FPRwt receptor responded to FMLP with a rapid increase in intracellular calcium concentration (Fig. 6). On the other hand, FMLP stimulation of cells expressing FPRdel showed no calcium mobilization. No differences in basal calcium levels were observed (fluorescence intensity (mean ± S.E.), 76 ± 16 versus 68 ±18; n = 43 and n = 38, FPRwt versus FPRdel, respectively). Both cell types demonstrated a similar increase in calcium concentration following addition of ionomycin (data not shown).


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Fig. 6.   Intracellular calcium response of FPRwt- and FPRdel-containing HL-60 cells to FMLP. FPRwt HL-60 cells (A) and FPRdel HL-60 cells (B) were incubated with Fluo-3 for 30 min followed by FMLP stimulation with 3 × 10-7 M (arrow) as described under "Experimental Procedures," and the increase in intracellular calcium was monitored as a function of time by a confocal microscopy.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Defining the structural basis for divergent PMN functional response and signal transduction pathway activation to chemoattractants provides the opportunity to develop strategies for pharmacologic regulation of these responses. The present study was designed to determine the role of the carboxyl-terminal tail of FPRs in the activation of MAPK cascades. Our data show that undifferentiated HL-60 cells provide a useful model in which to examine structure-function relationships of chemoattractant receptor activation of MAPKs. Stable expression of wild type FPRs permitted FMLP to stimulate a 3-5-fold increase in ERK and p38 kinase activities. The time course, concentration response, and pertussis toxin sensitivity were all similar to those seen in differentiated HL-60 cells and human PMNs (26, 28-30). Additionally, FMLP stimulation of GTPgamma S binding and GTPgamma S inhibition of FMLP binding to membranes from FPR HL-60 cells was similar to that seen in PMNs and HL-60 granulocytes. Our findings are consistent with a previous report showing that stable expression of FPRs in undifferentiated HL-60 cells resulted in the ability of FMLP to stimulate pertussis toxin-sensitive calcium mobilization (34). Thus, stable expression of FPRs in undifferentiated HL-60 cells permits examination of chemoattractant receptor activation of G proteins and MAPK cascades.

Various receptors use different domains to couple to G proteins, and specific rules or consensus sequences that determine G protein coupling do not exist. In PMNs and HL-60 cells, FPRs couple to G proteins containing alpha i2 and alpha i3 (21, 40, 41). Physical interaction of FPRs with Gi proteins is interrupted by synthetic peptides derived from the carboxyl-terminal tail but not those derived from the third intracellular loop (19-21). Synthetic peptides from the second intracellular loop have been reported to inhibit and to have no effect on FPR-G protein coupling (19, 20). To determine the role of the carboxyl-terminal tail in FPR activation of MAPKs, we constructed a mutant FPR in which carboxyl-terminal amino acids 301-346 were deleted and stably expressed this mutant receptor in undifferentiated HL-60 cells.

Recently, our laboratory has reported that FMLP stimulation of HL-60 granulocytes, a model for PMNs (39), activates ERK and p38 kinases (30), similar to results obtained in human PMNs (25-28, 42). To determine the role of the carboxyl-terminal tail of FPRs in receptor function, the ability of FMLP to stimulate ERK and p38 MAPK activation, calcium transients, and G protein activation was compared between undifferentiated HL-60 cells stably transfected with FPRwt and FPRdel. Deletion of the carboxyl-terminal tail prevented FMLP-stimulated ERK and p38 kinase activities and blocked FMLP-stimulated calcium transients. The observed differences in MAPK activation and calcium responses are unlikely to be due to differences in expression of FPRwt and FPRdel. Expression of both receptor types by transfected HL-60 cells was similar, as determined by fluorescein-labeled formyl peptide binding. It remains possible that the deletion mutation results in changes in the tertiary structure that impair ligand binding. However, the ability of the fluorescent formyl peptide to specifically bind to FPRDEL HL-60 cells suggests that any conformational change does not significantly affect ligand binding. Taken together, our data and the previously published results with synthetic peptides strongly suggest that the carboxyl-terminal tail of FPRs is required for G protein-coupled activation of MAPKs and phospholipase C-dependent calcium mobilization. Our data suggest that the mechanism of inhibition of MAPK activation and calcium mobilization in FPRdel HL-60 cells is uncoupling of the receptor-G protein interaction. This conclusion is based on several findings. First, FMLP failed to stimulate guanine nucleotide exchange by G proteins in FPRdel membranes, as shown by the absence of a concentration-dependent increase in GTPgamma S binding. Second, basal GTPgamma S binding was significantly reduced in FPRdel membranes, compared with FPRwt membranes. We have previously shown that uncoupling of formyl peptide receptors from G proteins by pretreatment with pertussis toxin results in a reduction of basal GTPgamma S binding (6). Third, GTPgamma S failed to inhibit FMLP binding in FPRdel membranes. Guanine nucleotides reduce G protein-coupled receptor affinity for ligand, resulting in a reduced level of ligand binding (36-38). Fourth, photoaffinity labeling of G proteins in FPRwt and FPRdel membranes showed that FMLP failed to stimulate increased labeling of G proteins in FPRdel membranes, and basal labeling was reduced compared with FPRwt membranes. Fifth, the concentration of fluoresceinated formyl peptide required to saturate binding sites was greater in FPRdel HL-60 cells. Because the number of receptors was similar in the two groups of cells, this finding suggests a reduced affinity for ligand of FPRdel. Finally, pertussis toxin pretreatment did not alter either stimulated or basal ERK and p38 activities in FPRdel HL-60 cells.

Basal ERK and p38 kinase activities were significantly higher in FPRdel HL-60 cells, than in FPRwt HL-60 cells. This finding suggests that removal of the carboxyl-terminal tail of FPR may result in a constitutively active receptor. The differences in basal MAPK activity was not due to selection of a specific clone of FPRdel cells with high MAPK activity, because transfected HL-60 cells were selected by antibiotic resistance. Immunoprecipitation of specific Galpha subunits following AA-GTP photoaffinity labeling in the presence of FMLP confirmed that FPRwt couple to G proteins containing alpha i2 but not to those containing Galpha q. FPRdel are not constitutively active for Gi proteins, because there was reduced basal photoaffinity labeling of immunoprecipitated alpha i2 in membranes from FPRdel HL-60 cells, and pertussis toxin did not alter this labeling. The increased basal MAPK activity in FPRdel cells was not the result of uncoupling of FPR from Gi2 proteins, because pertussis toxin pretreatment of FPRwt cells failed to reproduce the increase in basal ERK or p38 kinase activity. Okamoto et al. (43) showed that removal of a portion of the carboxyl-terminal tail of endothelin B receptors resulted in uncoupling from Gi proteins, but their ability to activate Gq was not affected. Co-transfection of FPRs and Galpha 16 into COS cells results in FMLP-stimulated phospholipase C activation (11). Therefore, we considered the possibility that FPRdel constitutively activate other G proteins. Immunoprecipitation of alpha q/11 subunits failed to demonstrate increased photoaffinity labeling in FPRdel membranes or following FMLP stimulation of FPRwt and FPRdel membranes. No basal or stimulated labeling of alpha 12 or alpha 13 was seen, and immunoblotting did not detect these subunits in HL-60 cells. We were unable to directly examine photoaffinity labeling of alpha 16, because an immunoprecipitating antibody was unavailable. However, the absence of increase basal or FMLP-stimulated photolabeling in solubilized membranes from FPRdel HL-60 cells suggests that this pathway was not activated. Our results suggest that the carboxyl-terminal tail acts to inhibit activation of an alternative signal transduction pathway when FPRs are uncoupled from alpha i2-containing G proteins. Elimination of the carboxyl-terminal tail not only uncouples FPRs from Gi proteins but alters the FPR by either unmasking of an active site upon removal of stearic hindrance of the carboxyl-terminal tail or a conformational change in the remaining receptor. The mutant receptor is then constitutively active for an alternative pathway resulting in ERK and p38 MAPK activation. The failure of FPRdel membranes to show increased basal photolabeling with AA-GTP suggests that the alternative pathway is G protein-independent; however, the components of this pathway remain to be determined.

    ACKNOWLEDGEMENT

We acknowledge the excellent technical assistance of Suzanne Eades.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant AI36357 (to E. R. P.), an American Heart Association grant-in-aid (to E. R. P.), the Department of Veterans Affairs (to K. R. M.), the American Heart Association, Kentucky Affiliate (to K. R. M. and M. J. R.), and the Jewish Hospital of Louisville Foundation (to K. R. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: Kidney Disease Program, 615 S. Floyd St., University of Louisville, Louisville, KY 40202-1718. Tel.: (502) 852-5757; Fax: 502-852-7643; E-mail: kmcleish{at}e-mail.kdp-baptist.louisville.edu.

The abbreviations used are: PMN, polymorphonuclear leukocyte; FMLP, N-formylmethionyl-leucyl-phenylalanineFPR, formyl peptide receptorFPRwt, wild type FPRFPRdel, FPR lacking the carboxyl-terminal tailGPCR, G protein-coupled receptorMAPK, mitogen-activated protein kinaseERK, extracellular signal-regulated kinaseGTPgamma S, guanosine 5'-3-O-(thio)triphosphateAA, azidoanalidePAGE, polyacrylamide gel electrophoresis.
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Abstract
Introduction
Procedures
Results
Discussion
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