From the Departments of Medicine and
¶ Immunology, Duke University Medical Center,
Durham, North Carolina 27710
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ABSTRACT |
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Interleukin-8 (IL-8) receptor A
(CXCR1) couples to a pertussis toxin-sensitive G protein to mediate
phospholipase C (PLC
) activation and cellular responses.
Responses to CXCR1 are attenuated by prior exposure of neutrophils to
either IL-8, a cleavage product of the fifth component of complement
(C5a) or n-formylated peptides (formylmethionylleucylphenylalanine, fMLP). To characterize the role of
receptor phosphorylation in the regulation of the CXCR1, a
phosphorylation-deficient mutant, M2CXCR1, was constructed. This
receptor, stably expressed in RBL-2H3 cells, coupled more efficiently
to G protein and stimulated enhanced phosphoinositide hydrolysis, cAMP
production, exocytosis, and phospholipase D activation, and was
resistant to IL-8-induced receptor internalization. The rate and total
amount of ligand stimulated actin polymerization remained unchanged,
but interestingly, chemotaxis was decreased by ~30% compared with
the wild type receptor. To study the role of receptor phosphorylation
in cross-desensitization of chemoattractant receptors, M2CXCR1
was coexpressed with cDNAs encoding receptors for
either fMLP (FR), C5a (C5aR), or platelet-activating factor (PAFR). Both C5aR and PAFR were cross-phosphorylated upon M2CXCR1 activation, resulting in attenuated guanosine
5'-3'-O-(thio)triphosphate (GTP
S) binding in membranes.
In contrast, FR and M2CXCR1 were resistant to cross-phosphorylation and
cross-inhibition of GTP
S binding by other receptors. Despite the
resistance of M2CXCR1 to cross-phosphorylation and receptor/G
protein uncoupling, its susceptibility to cross-desensitization of its
Ca2+ response by fMLP and C5a, was equivalent to CXCR1.
Regardless of the enhancement in certain receptor functions in M2CXCR1
compared with the wild type CXCR1, the mutated receptors mediated
equivalent PLC
3 phosphorylation and
cross-desensitization of Ca2+ mobilization by FR, C5aR, and
PAFR. The results herein indicate that phosphorylation of CXCR1
regulates some, but not all of the receptors functions. While receptor
phosphorylation inhibits G protein turnover, PLC activation,
Ca2+ mobilization and secretion, it is required for normal
chemotaxis and receptor internalization. Since phosphorylation of CXCR1
had no effect on its ability to induce phosphorylation of
PLC
3 or to mediate class-desensitization, these
activities may be mediated by independently regulated pathways.
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INTRODUCTION |
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IL-81 is a member of the structurally related family of cytokines, called chemokines, which mediate a number of biological activities including chemotaxis of leukocytes (1, 2). Cellular responses to IL-8 are initiated by specific cell surface receptors that couple to pertussis toxin-sensitive heterotrimeric G proteins (3). Two IL-8 receptor subtypes have been identified in human neutrophils, IL-8 receptor A or CXCR1 and IL-8 receptor B or CXCR2 (4, 5). CXCR1 is specific for IL-8, whereas CXCR2 also binds other chemokines including MGSA, GRO, and NAP2, to mediate cellular responses (3, 6). Both CXCR1 and CXCR2 undergo phosphorylation and desensitization upon agonist stimulation (7-9).
Prior exposure of neutrophils to the chemoattractants fMLP, C5a, or
IL-8 resulted in attenuation of each others intracellular Ca2+ mobilization, a phenomenon called "class
desensitization" (10, 11). Activation of either FR or C5aR in
co-transfected RBL-2H3 cells, resulted in cross-phosphorylation and
cross-desensitization of CXCR1 (12). Likewise, C5aR underwent
cross-phosphorylation and cross-desensitization upon CXCR1 activation
(12). FR was, however, resistant to cross-phosphorylation, although
inositol 1,4,5-trisphosphate production and Ca2+
mobilization were attenuated by a first dose of either C5a or IL-8
(12). Taken together, these observations suggested that a shared
component(s) distal to receptor/G protein is(are) involved in
chemoattractant receptor class desensitization. Chemoattractant receptors have been shown to couple to both PLC2 and
PLC
3 to stimulate leukocyte responses (13, 14). Liu and
Simon (15) have recently shown in Cos-7 cells that protein kinase A
(PKA)-mediated phosphorylation of PLC
2 prevented its
activation by G
. Recent studies from this laboratory
have demonstrated that receptors for the chemoattractants PAF, fMLP,
and the CXC chemokine SDF1 induced phosphorylation of
PLC
3 via both protein kinase A- and C-activating
pathways (16-18). Thus, phosphorylation of PLC
, subsequent to
chemoattractant receptor activation, may play a regulatory role in
cross-desensitization.
To determine the role of receptor and PLC3
phosphorylation in CXCR1 regulation, a receptor mutant, M2CXCR1,
resistant to heterologous phosphorylation was transfected in RBL-2H3
cells. This mutant was more active than CXCR1 in stimulating G protein activation and some but not all subsequent responses. In this study, we
characterize M2CXCR1 regulation and cross-regulation of other
chemoattractant receptor and assess the role of phosphorylation in
receptor cross-desensitization. The data presented here suggest that
class-desensitization of CXCR1 is not regulated by receptor phosphorylation, suggesting a pathway distal to receptor/G protein activation for PLC regulation. Moreover, this IL-8-induced
phosphorylation of PLC
3 may play an important role in
class desensitization.
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EXPERIMENTAL PROCEDURES |
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Materials--
[32P]Ortophosphate (8500-9120
Ci/mmol), myo-[2-3H] inositol (24.4 Ci/mmol), [35S]GTPS (1300 Ci/mmol), and
[
-32]GTP (6000 Ci/mmol) were purchased from NEN Life
Science Products. 125I-labeled IL-8 was obtained from
Amersham Corp. IL-8 (monocyte-derived) was purchased from Genzyme. PAF
was from Calbiochem. Geniticin (G418) and all tissue culture reagent
were purchased from Life Technologies, Inc. Monoclonal 12CA5 antibody,
protein G-agarose, and protease inhibitors were purchased from
Boehringer Manheim. Polyclonal antibody against PLC
3 was
obtained from Santa Cruz Biotechnology. fMLP, Indo-1 acetoxymethyl
ester, and pluronic acid were purchased from Molecular Probes. C5a and
8-(4-chlorophenylthio)-cAMP, phorbol 12-myristate 13-acetate (PMA),
GDP, GTP, GTP
S, and ATP were purchased from Sigma. All other
reagents are from commercial sources.
Construction of Epitope-tagged CXCR1, M1CXCR1, and M2CXCR1-- Nucleotides encoding a nine-amino acid epitope sequence (YPYDVPDYA) was inserted between the N-terminal initiator methionine and the second amino acid of each cDNA by polymerase chain reaction as described previously (7, 19, 20). Alanine substitution of serine and threonine residues of the carboxyl terminus of CXCR1 to generate M2CXCR1 was carried out by polymerase chain reaction.
Cell Culture and Transfection-- RBL-2H3 cells were maintained as monolayer cultures in Earle's modified Eagle's medium supplemented with 15% fetal bovine serum, 2 mM glutamine, penicillin (100 units/ml), and streptomycin (100 mg/ml) (19). RBL-2H3 cells (1 × 107 cells) were transfected by electroporation with pcDNA3 containing the receptor cDNAs (20 µg) and geneticin-resistant cells were cloned into single cell by fluorescein-activated cell sorting analysis.
Radioligand Binding Assays-- RBL-2H3 cells were subcultured overnight in 24-well plates (0.5 × 106 cells/well) in growth medium. Cells were then rinsed with Dulbecco's modified Eagle's medium supplemented with 20 mM Hepes, pH 7.4, and 10 mg/ml bovine serum albumin and incubated on ice for 2-4 h in the same medium (250 µl) containing 125I-IL-8. Reactions were stopped with 1 ml of ice-cold phosphate-buffered saline containing 10 mg/ml bovine serum albumin, and washed four times with the same buffer. Then cells were lysed with 0.1 N NaOH (250 µl), dried under vacuum, and bound radioactivity was evaluated by counting in a gamma counter. Nonspecific radioactivity bound was determined in the presence of 300 nM unlabeled IL-8. For internalization experiments, cells were incubated with 100 nM IL-8 for 0-60 min at 37 °C. After the incubation period, cells were washed three times with ice-cold phosphate-buffered saline and 125I-IL-8 binding was carried out as described above.
GTPase Activity and [35S]GTPS
binding--
Cells were treated with appropriate concentrations of
stimulants and membranes were prepared as already described (19). GTPase activity and [35S]GTP
S binding using 10-20
µg of membrane preparations were carried out as described previously
(7, 19-21).
Phosphoinositide Hydrolysis and Calcium Measurement-- RBL-2H3 cells were subcultured overnight in 96-well culture plates (50,000 cells/well) in inositol-free medium supplemented with 10% dialyzed fetal bovine serum and 1 µCi/ml [3H]inositol. The generation of inositol phosphates was determined as reported previously (19). For calcium mobilization, cells (3 × 106) were loaded with 1 µM Indo 1-acetoxymethyl ester in the presence of 1 µM pluronic acid for 30 min at room temperature. Then the cells were washed and resuspended in 1.5 ml of buffer. Intracellular calcium increase in the presence and absence of ligands was measured as described (7).
Phospholipase D Activation and cAMP Generation and Measurement-- For phospholipase D activation, phosphatidylethanol (PtdEtOH) formation was measured as described previously (11, 21). cAMP generation and measurement was carried out as described previously (16, 22) using the Amersham kit.
Actin Polymerization and Chemotaxis-- Actin polymerization assays were performed essentially as described previously (19). RBL-2H3 cells (50,000) were incubated at 37 °C with different concentration of IL-8. Chemotaxis was assessed in 48-well microchemotaxis chambers, using polyvinylpyrrolidone-free 5-µm pore size membranes. Migration was allowed to continue for 5 h at 37 °C in 5% CO2. The membrane was removed, the upper surface was washed with phosphate-buffered saline and scraped, fixed, and stained. The results are represented as chemotaxis index (mean number of cells per high power field for chemokine dilution/mean number of cells per high power field for medium) (23).
Receptors and PLC3 Phosphorylation and
Immunoprecipitation--
Phosphorylation of receptors was performed as
described previously (16). RBL-2H3 cells (2.5 × 106)
were subcultured overnight in 60-mm tissue culture dishes. The following day the cells were rinsed twice with 5 ml of phosphate-free Dulbecco's modified Eagle's medium and incubated in the same medium supplemented with [32P]orthophosphate (150 µCi/dish)
for 90 min to metabolically label the intracellular ATP pool. Then
labeled cells were stimulated with the indicated stimulants or vehicle
for 5-7 min at 37 °C. The reactions were stopped by placing the
cells on ice. The cells were washed twice with ice-cold
phosphate-buffered saline and harvested with cold detergent-containing
lysis buffer (1 ml/dish) supplemented with appropriate protease and
phosphatase inhibitors. The phosphorylated receptors were
immunoprecipitated with the 12CA5 antibody, analyzed by
SDS-electrophoresis, and visualized by autoradiography.
PLC
3 phosphorylation upon agonist stimulation was
determined as described above, using a rabbit polyclonal
PLC
3 specific antibody instead of 12CA5 as described
previously (16-18).
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RESULTS |
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Characterization of M2CXCR1 in RBL-2H3 Cells-- M2CXCR1 was previously expressed in RBL-2H3 cells and it was demonstrated that the mutant bound IL-8 with a dissociation constant (Kd) of 2.8 ± 0.7 nM and a Bmax of 7792 ± 284 receptors/cell. This was similar to that of wild type CXCR1 expressed in RBL-2H3 cells (Kd, 2.3 ± 0.3 nM; Bmax, 8532 ± 152 receptors/cell) or the native receptors in neutrophils (~1-2 nM) (3, 7), indicating that mutation of the four amino acid residues which comprise the M2 cluster (7) did not affect ligand binding. However, M2CXCR1-mediated inositol phosphates formation and secretion were ~5- and ~10-fold, respectively, higher than the wild type receptor (7). IL-8-induced GTPase activity in membranes, and cAMP production and phospholipase D activity in intact cells were also greater in cells expressing M2CXCR1 than CXCR1 (data not shown). IL-8-induced actin polymerization were similar for both M2CXCR1 and CXCR1-expressing cells (Fig. 1A). However, cells expressing M2CXCR1 showed a ~30% decrease in maximal chemotaxis compared with cells expressing CXCR1 (Fig. 1B). The EC50 (~0.1 nM), however, remained unchanged.
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Co-expression and Cross-phosphorylation of M2CXCR1-- The ability of M2CXCR1 to cross-phosphorylate and cross-desensitize chemoattractant responses was determined. Cells expressing CXCR1 and cells coexpressing M2CXCR1 and receptors for either fMLP (M2CXCR1-FR) or PAF (M2CXCR1-PAFR) were stimulated with either IL-8 (100 nM), fMLP (1 µM), PAF (100 nM), or PMA (100 nM). As shown in Fig. 2, CXCR1 (lane 2, ~70 kDa), FR (lane 6, ~65 kDa), and PAFR (lane 11, ~45 kDa) were homologously phosphorylated by their ligands. CXCR1 and PAFR were also phosphorylated by PMA (lanes 3 and 10, respectively) but not FR (lane 7) and M2CXCR1 (lanes 7 and 10). PAFR was cross-phosphorylated by M2CXCR1 activation (lane 9). Both M2CXCR1 (lanes 6 and 11) and FR (lane 5) were resistant to cross-phosphorylation. Some homologous phosphorylation of M2CXCR1 was detected with longer exposure of the autoradiogram (7) (data not shown).
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Cross-desensitization of M2CXCR1-- Ca2+ mobilization was measured to determine the relationship between cross-phosphorylation and cross-desensitization of receptor-mediated cellular responses. Ca2+ mobilization in response to an EC100 dose of either fMLP (100 nM; M2CXCR1-FR cells), PAF (10 nM; M2CXCR1-PAFR cells) or IL-8 (10 nM; M2CXCR1-FR and M2CXCR1-PAFR cells) was homologously desensitized by a first dose of the same ligand (data not shown). As shown in Fig. 3, the dose-response of either fMLP (panel A) or PAF (panel C) induced Ca2+ mobilization in M2CXCR1-FR and M2CXCR1-PAFR cells, respectively, was cross-desensitized by pretreatment of the cells with a first dose of IL-8 (10 nM). IL-8-induced Ca2+ mobilization in cells expressing M2CXCR1 was also cross-desensitized by pretreatment of the cells with a first dose of either fMLP (panel B) or C5a (data not shown) but not PAF (panel D). The extent of FR mediated cross-desensitization of M2CXCR1 (~60%) was equivalent to that of CXCR1 (57%) (12).
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Phosphorylation of PLC3 by CXCR1 and
M2CXCR1--
Peptide chemoattractants have been shown to couple to
PLC
to mediate cellular signals (13, 14). Both Western blot analysis and reverse transcriptase-polymerase chain reaction indicated that of
the four known PLC
isozymes only PLC
3 is expressed in RBL-2H3 cells (16, 18). In addition, both PAF and fMLP have recently
been shown to mediate PLC
3 phosphorylation in RBL-2H3 cells (16, 17). The ability of CXCR1 and the M2CXCR1 to mediate PLC
3 phosphorylation was determined.
32P-Labeled cells expressing CXCR1 or M2CXCR1 were
stimulated with either IL-8 (100 nM), PMA (100 nM) and 8-(4-chlorophenylthio)-cAMP (1 mM). As
shown in Fig. 4, upon IL-8 stimulation,
both the mutant (lane 6) and the wild type receptor
(lane 2) mediated phosphorylation of PLC
3 to
a similar extent, 1.81- and 1.77-fold over basal, respectively
(panel B). IL-8-induced phosphorylation of
PLC
3 is similar to that of 8-(4-chlorophenylthio)-cAMP
(panel A, lanes 4 and 8, and
panel B), but less than phosphorylation induced by PMA
(panel A, lanes 3 and 7, and
panel B).
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DISCUSSION |
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Phagocytic leukocytes respond to inflammatory mediators such as
IL-8, fMLP, and C5a by migrating to sites of inflammation where they
may exert their cytotoxic activities (25). Despite the presence of two
receptors for IL-8 in neutrophils (CXCR1 and CXCR2), certain cellular
responses to IL-8 are lower in magnitude compared with fMLP and C5a.
For example, IL-8 is a weaker stimulator of exocytosis and respiratory
burst (26). IL-8 has also been shown to be less effective than fMLP and
C5a in stimulating phosphoinositide hydrolysis and mitogen-activated
protein kinase activation in neutrophils (27). The results presented
here indicate that responses to IL-8 are modulated by specific
phosphorylation sites in the cytoplasmic tail of the receptor as well
as a site downstream of G protein activation. A phosphorylation
deficient receptor mutant (M2CXCR1) more effectively activated G
protein and was more resistant to agonist-mediated desensitization and
internalization. Despite the ability of this mutant to up-regulate
phosphoinositide hydrolysis, cAMP production, and phospholipase D
activation and secretion, actin polymerization was not affected, and
chemotaxis in response to IL-8 was actually diminished compared with
wild type CXCR1 (Fig. 1). These results may distinguish events which are important for leukocyte recruitment from those for cytotoxic functions. Cytoskeletal rearrangement and chemotaxis which are early
events in inflammation occur via pathways which require lower doses of
agonist to reach maximum responses. Cytotoxic activation such as
exocytosis of lysosomal enzymes and superoxide production require
higher doses of ligand (25). The molecular basis for these observations
may be provided by the findings described here. Prevention of receptor
phosphorylation which resulted in enhanced G protein activation at all
ligand doses was associated with enhanced cytotoxic activity yet
diminished chemotaxis and unchanged cytoskeletal actin assembly. This
can be inferred to indicate that cytotoxic activity requires higher G
protein turnover than chemotaxis. The finding that chemotaxis to IL-8
decreased in M2CXCR1 cells may be interpreted in two ways. First, that
receptor desensitization and/or internalization may be required for
normal gradient detection. However, this contention is not supported by
the recent findings that chemotaxis may be regulated via receptor
phosphorylation-independent pathways (28, 29). Second, since maximal
cell migration occurs at doses of agonist lower than those required for
Ca2+ mobilization or cAMP production (25), it is possible
that the pathway(s) regulating chemotaxis is/are sensitive to second
messenger levels and that the up-regulation of phospholipase C
activation mediated by M2CXCR1 plays a negative regulatory role in
sensing chemical gradients. Supporting this contention is that
truncation of the carboxyl tail of the monocyte chemoattractant protein
1 (MCP1) receptor (CCR2B) which enhanced receptor mediated
Ca2+ mobilization and cAMP production, diminished
chemotaxis in response to MCP1 (29). In addition, chemotaxis of
leukocytes was enhanced in PLC2 deficient mice in which
phosphoinositide hydrolysis, Ca2+ mobilization and
superoxide production was decreased (30).
Understanding molecular events underlying cross-desensitization of
receptors was facilitated by the availability of M2CXCR1. Phosphorylation of unoccupied receptors by second
messenger-dependent kinases activated via different
receptors appear to account for cross-desensitization at the level of
R/G protein coupling (12). Our previous studies with FR have revealed
that receptor cross-desensitization can also occur independently of
receptor phosphorylation and G protein uncoupling. A question addressed
in the present study is the role of CXCR1 phosphorylation in the
cross-desensitization of cellular responses. M2CXCR1, like FR (12, 21),
is resistant to cross-phosphorylation and cross-desensitization of
receptor mediated [35S]GTPS binding in membranes.
Despite this, intracellular Ca2+ mobilization mediated by
M2CXCR1 in response to IL-8 was cross-desensitized by pretreatment of
the cells with either fMLP or C5a (12). These results clearly indicate
cross-desensitization of M2CXCR1 at the level of a downstream effector
which is independent of receptor phosphorylation or R/G protein
uncoupling. This contention is supported by the observation that both
PMA and 8-(4-chlorophenylthio)-cAMP which, like fMLP, IL-8, and C5a,
caused phosphorylation of PLC
3 (Fig. 4), desensitized
Ca2+ mobilization in response to FR, C5aR, CXCR1, and
M2CXCR1 in both neutrophils and transfected RBL-2H3 cells (16, 17).
Prossnitz (31) using phosphorylation deficient mutants of FR have
recently reported that homologous desensitization of fMLP-mediated
Ca2+ mobilization is solely mediated by receptor
phosphorylation. These results are in contrast to the ones presented in
this work and may well reflect differences between homologous and
cross-desensitization. Homologous desensitization requires
phosphorylation of the agonist-occupied form of the receptor by a
receptor specific kinase and accessory proteins such as arrestin (32).
Cross-desensitization, in contrast, is agonist-independent. It occurs
via pathway(s) triggered by a different, but related, receptor and
involve modification of downstream effector(s) and, perhaps,
different accessory proteins.
Another question addressed in this study is the role of second
messenger production in receptor cross-regulation. M2CXCR1 induced
greater G protein turnover, phosphoinositide hydrolysis and cAMP
production than CXCR1. However, M2CXCR1 activation by IL-8 did not
result in greater cross-phosphorylation or cross-desensitization of
Ca2+ mobilization in response to either FR, PAFR, or C5aR
(22-30%) (Fig. 3, data not shown) compared with CXCR1 (20-30%) (12,
21). These results suggest that cross-desensitization of
receptor-mediated Ca2+ mobilization occurs via pathways
which are independent of receptor phosphorylation or rate of second
messenger production measured in the present work. Supporting that
contention is that phosphorylation of PLC3, which is
thought to be one of the target effector for class-desensitization (16,
17), is mediated by both CXCR1 and M2CXCR1 to the same extent (Fig.
4).
This work provides evidence for independent mechanisms for CXCR1
receptor mediated chemotactic versus cytotoxic functions of
phagocytic leukocytes. Whereas secretion was enhanced by removal of
specific phosphorylation sites in the cytoplasmic tail of the receptor,
chemotaxis and receptor internalization were inhibited by loss of
receptor phosphorylation. In contrast, IL-8 mediated actin
polymerization, PLC3 phosphorylation, receptor
cross-phosphorylation and cross-desensitization of Ca2+
response were not affected. Moreover, the lack of receptor
phosphorylation did not affect cross-desensitization of or by M2CXR1 at
the level of Ca2+ mobilization. These data further
underscore the presence of downstream effector(s) of receptor
class-desensitization which appear to be regulated independently of
receptor phosphorylation and enhanced G protein turnover.
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ACKNOWLEDGEMENT |
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We are grateful to Dr. Michael C. Grimm for helpful advice and assistance in setting up the chemotaxis assay.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AI-38910 (to R. M. R.), HL-54166 (to H. A.), and DE-03738 (to R. S.).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: Dept. of Medicine, Duke University Medical Center, Box 3680, Durham, NC 27710. Tel.: 919-684-5332; Fax: 919-684-4390; E-mail: richa021{at}mc.duke.edu.
1
The abbreviations used are: IL-8, interleukin-8;
fMLP, formylmethionylleucylphenylalanine; FR, fMLP receptor; C5a,
peptide from the fifth component of complement; C5aR, C5a receptor;
PMA, phorbol 12-myristate 13-acetate; GTPS, guanosine
5'-3'-O-(thio)triphosphate; G protein, GTP-regulatory
protein; PAF, platelet-activating factor; PAFR, PAF receptor; PLC,
phopholipase C.
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REFERENCES |
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