From the Departments of 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 (GTP 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 G 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.
Materials--
FMLP, hygromycin, and geneticin (G418) were
obtained from Sigma. GDP, GTP and GTP Medicine and
§ Biochemistry and Molecular Biology,
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
Top
Abstract
Introduction
Procedures
Results
Discussion
References
S) binding in
membranes from FPRwt but not FPRdel cells. GTP
S inhibited FMLP
binding to FPRwt but not FPRdel membranes. Photoaffinity labeling with azidoanilide-[
-32P]GTP in the presence or absence of
FMLP showed increased labeling only in FPRwt membranes.
Immunoprecipitation of
i2 and
q/11 from
solubilized, photolabeled membranes showed that FPRwt were coupled to
i2 but not to
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
Top
Abstract
Introduction
Procedures
Results
Discussion
References
16 permits activation of phospholipase C
in Cos cells (11-13).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
S were obtained from Boehringer
Mannheim. [35S]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).
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 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 -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
-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
-glycerophosphate (pH 7.2), 0.1 mM sodium orthovanadate, 0.2 mM ATP, 20 mM MgCl2, 1 mM EGTA, 0.5 µl [
-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 -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
[
-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--
[-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 G
i2 or
G
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).
GTPS Binding Assay--
GTP
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]GTP
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]GTP
S bound in the
presence of 10 µM GTP
S from total
[35S]GTP
S bound and expressed as fmol of GTP
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 Gi2, G
q/11, G
12, and G
13 antisera. Bound antibody was
detected by chemiluminesence (Renaissance, NEN Life Science
Products).
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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.
|
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 × 107 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.
|
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 × 107 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.
|
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 GTPS binding and GTP
S inhibition of FMLP binding in plasma
membranes from FPRwt and FPRdel expressing cells. FPRwt membranes
exhibited a concentration-dependent increase in GTP
S
binding upon addition of FMLP (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.
|
|
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 × 107 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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DISCUSSION |
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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 GTPS 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 (25-28, 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 GTPS 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 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 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.
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ACKNOWLEDGEMENT |
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We acknowledge the excellent technical assistance of Suzanne Eades.
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FOOTNOTES |
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* 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
kinaseGTPS, guanosine 5'-3-O-(thio)triphosphateAA, azidoanalidePAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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