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

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 (GTPγS) binding in membranes from FPRwt but not FPRdel cells. GTPγS inhibited FMLP binding to FPRwt but not FPRdel membranes. Photoaffinity labeling with azidoanilide-[γ-32P]GTP in the presence or absence of FMLP showed increased labeling only in FPRwt membranes. Immunoprecipitation of αi2 and αq/11 from solubilized, photolabeled membranes showed that FPRwt were coupled to αi2 but not to αq/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.

Chemoattractants, including formylated peptides, C5a, leukotriene B 4 , 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)(2)(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 B 4 receptors, couple to pertussis toxin-sensitive G i proteins (6 -8), whereas platelet-activating factor receptors activate G o and/or G q , in addition to G i proteins (9,10). Transient co-transfection of chemoattractant receptors and G␣ 16 permits activation of phospholipase C in Cos cells (11)(12)(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 carboxylterminal tail of human FPRs disrupt the physical interaction with G i 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)(24)(25)(26)(27)(28)(29)(30)(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 differ-ent 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 G i protein-dependent activation of both ERK and p38 kinases.

EXPERIMENTAL PROCEDURES
Materials-FMLP, hygromycin, and geneticin (G418) were obtained from Sigma. GDP, GTP and GTP␥S were obtained from Boehringer Mannheim. [ 35 S]GTP␥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-G␣ i2 antibody were obtained from Chemicon (Temecula, CA). Polyclonal antisera against G␣ 12 and G␣ 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 14amino 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-G␣ 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 G␣ q produced in Sf9 cells by immunoblotting, whereas the antisera did not interact with bacterially expressed G␣ i1 , G␣ i2 , or G␣ 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 carboxylterminal 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% CO 2 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 g/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 g/ml hygromycin. Crude plasma membrane fractions were prepared by nitrogen cavitation as described previously (7).
Flow Cytometry Assay for Formyl Peptide Binding-Binding of Nformyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein to FPRs was performed as described previously (34). FPRwt-transfected HL-60 cells and FPRdeltransfected HL-60 cells were harvested by centrifugation, washed with Krebs-Ringer phosphate buffer, and resuspended in Krebs-Ringer phosphate buffer to 1 ϫ 10 6 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 M 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-Lysfluorescein occurred at 100 nM for FPRwt HL-60 cells and 1 M for FPRdel HL-60 cells. Quantum 24 fluorescein microbeads (Flow Cytometry Standards Corp., San Juan, PR) containing 4 ϫ 10 3 to 6.6 ϫ 10 4 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 ϫ 10 6 /ml cells were stimulated with FMLP for the indicated time and at the indicated concentration. Stimulation was terminated by a brief centrifugation (2500 ϫ g for 20 s), and the cell pellet was immediately lysed in 0.5 ml of ice-cold lysis buffer containing 50 mM ␤-glycerophosphate (pH 7.2), 100 M sodium vanadate, 1 mM EDTA, 1 mM dithiothreitol, 2 mM MgCl 2 , 0.5% Triton X-100, 5 g/ml leupeptin, and 0.09 units/ml aprotinin. The cell lysates were centrifuged for 15 min at 15,000 ϫ g at 4°C to remove cellular debris. The cleared lysates were applied to 0.5 ml DEAE-Sephacel (Amersham Pharmacia Biotech) minicolumns that had been pre-equilibrated in Buffer A containing 50 mM ␤-glycerophosphate (pH 7.2), 100 M 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-l aliquots of each eluate to 20 l of reaction mixture containing 50 mM ␤-glycerophosphate (pH 7.2), 0.1 mM sodium orthovanadate, 0.2 mM ATP, 20 mM MgCl 2 , 1 mM EGTA, 0.5 l [␥-32 P]ATP, and EGFR 662-681 synthetic peptide (Macromolecular Resources, Colorado State University, Ft. Collins, CO). Following incubation for 15 min at 30°C, reactions were terminated by spotting 30 l 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 ϫ 10 6 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 ϫ 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 ϫ g for 15 min at 4°C, cleared lysates were incubated with 5 l/sample of anti-p38 antisera and incubated for 1 h at 4°C. Protein A-Sepharose beads (15 l 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 ϫ g for 2 min, and the beads were washed once in lysis buffer and once in kinase buffer containing 25 mM Hepes, 25 mM ␤-glycerophosphate, 25 mM MgCl 2 , 2 mM dithiothreitol, and 0.1 mM sodium orthovanadate. The kinase assay was initiated by the addition of 40 l of kinase buffer containing 5 Ci [␥-32 P]ATP and 3 g 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 l of 5ϫ Laemmli SDS sample buffer. Samples were boiled and briefly centrifuged, and the products were resolved by 10% SDS-PAGE. The incorporation of 32 P was visualized by autoradiography and quantified on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Photoaffinity Labeling of Plasma Membrane G Proteins-[␥-32 P]GTP azidoanalide (AA-GTP) was synthesized as described previously (32). Photoaffinity labeling of G proteins from FPRwt and FPRdel HL-60 membranes (100 g/condition) with AA-GTP was performed as described previously (32). Briefly, plasma membranes were incubated in the presence or absence of 10 M FMLP in buffer containing 30 mM N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid, pH 7.5, 100 mM NaCl, 5 mM MgCl 2 , 5 g/ml soy trypsin inhibitor, 1 Ci of AA-GTP, and 2 M 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 l 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 G␣ i2 or G␣ q/11 at a dilution of 1:50. G protein antibody complexes were recovered by the addition of 25 l of protein A-Sepharose beads. Following incubation for 30 min, the beads were washed with cold phosphate-buffered saline and resuspended in 60 l 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).
GTP␥S Binding Assay-GTP␥S binding was performed as described previously (7). Briefly, assays were performed in a reaction mixture (100 l) containing 50 mM triethanolamine/HCl, pH 7. Receptor Binding Assay-FMLP binding assays were performed in a reaction mixture (100 l) containing 50 mM Tris, pH 7.5, 1 mM EDTA, 5 mM MgCl 2 , as described previously (7). Reactions were initiated by addition of 15-25 g 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-[ 3 H]Phe bound in the presence of excess ligand from the total N-formyl-Met-Leu-[ 3 H]Phe bound. Binding parameters were estimated using a nonlinear 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 ϫ sion wavelengths of 490 and 520 nm, respectively. The calcium ionophore ionomycin was used as a positive control.

Expression of FPRwt and FPRdel in HL
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 ϫ 10 Ϫ7 M FMLP. FMLP stimulated a 5.8 Ϯ 1.9-fold (mean Ϯ S.E.; n ϭ 3) increase in ERK activity in FPRwt HL-60 cells, whereas FMLP failed to stimulate an increase in ERK activity (1.3 Ϯ 0.1-fold; n ϭ 3) in FPRdel HL-60 cells (Fig.  3A). Basal activity in FPRdel cells was 3.1 Ϯ 1.4-fold (mean Ϯ S.E.; n ϭ 3) higher than in FPRwt. FMLP stimulated a 3.5 Ϯ 0.7-fold (mean Ϯ S.E.; n ϭ 6) increase in p38 kinase activity in FPRwt cells, whereas only a 1.3 Ϯ 0.1-fold (mean Ϯ S.E.; n ϭ 6) increase in p38 activity was stimulated in FPRdel cells (Fig.  3B). Basal activity in FPRdel HL-60 cells was 3.4 Ϯ 0.7 (mean Ϯ S.E., n ϭ 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 G i 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.
Role of the Carboxyl-terminal Tail in G Protein Activation-Because both pertussis toxin and FPRdel inhibited FMLP stim- ( Fig. 4A). On the other hand, GTP␥S binding was not increased by FMLP in FPRdel plasma membranes. Additionally, basal GTP␥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 GTP␥S reduced FMLP binding to FPRwt membranes in a concentration-dependent manner, whereas GTP␥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.
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 G i 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 Ϫ5 M FMLP. Autoradiography after SDS-PAGE separa-tion 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 G␣ i2 and G␣ q/11 (Fig. 5B). Immunoprecipitated G␣ 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 G␣ i2 was decreased in FPRdel membranes compared with FPRwt membranes (Fig. 5B). No increase in G␣ q/11 photolabeling was seen in either FPRwt or FPRdel membranes following addition of FMLP (data not shown). Immunoblotting of G␣ i2 in membranes from undifferentiated HL-60, FPRwt and FPRdel HL-60 membranes showed equal density of G␣ i2 and G␣ 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 ␣ 12 or ␣ 13 was detected, and neither of these ␣ subunits was detected by immunoblotting of HL-60 plasma membranes (data not shown).
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 G i proteinmediated 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 ϫ 10 Ϫ7 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 differ-

DISCUSSION
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 carboxylterminal 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 GTP␥S binding and GTP␥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 ␣i2 and ␣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)(26)(27)(28)42). To determine the role of the carboxylterminal tail of FPRs in receptor function, the ability of FMLP to stimulate ERK and p38 MAPK activation, calcium tran-sients, 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 carboxylterminal 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 concentrationdependent increase in GTP␥S binding. Second, basal GTP␥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 GTP␥S binding (6). Third, GTP␥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 G␣ subunits following AA-GTP photoaffinity labeling in the presence of FMLP confirmed that FPRwt couple to G proteins containing ␣ i2 but not to those containing G␣ q . FPRdel are not constitutively active for Gi proteins, because there was reduced basal photoaffinity labeling of immunoprecipitated ␣ 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 G i2 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 G␣ 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 ␣ 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 ␣ 12 or ␣ 13 was seen, and immunoblotting did not detect these subunits in HL-60 cells. We were unable to directly examine photoaffinity labeling of ␣ 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 ␣ 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.