From the Departments of Medicine and
Immunology, Duke University Medical Center,
Durham, North Carolina 27710
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ABSTRACT |
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Formylated peptides (e.g.
n-formyl-Met-Leu-Phe (fMLP)) and platelet-activating factor (PAF)
mediate chemotactic and cytotoxic responses in leukocytes through
receptors coupled to G proteins that activate phospholipase C (PLC). In
RBL-2H3 cells, fMLP utilizes a pertussis toxin (ptx)-sensitive G
protein to activate PLC, whereas PAF utilizes a ptx-insensitive G
protein. Here we demonstrate that fMLP, but not PAF, enhanced
intracellular cAMP levels via a ptx-sensitive mechanism. Protein kinase
A (PKA) inhibition by H-89 enhanced inositol phosphate formation
stimulated by fMLP but not PAF. Furthermore, a membrane-permeable cAMP
analog 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) inhibited
phosphoinositide hydrolysis and secretion stimulated by fMLP but not
PAF. Both cpt-cAMP and fMLP stimulated PLC3
phosphorylation in intact RBL cells. The purified catalytic subunit of
PKA phosphorylated PLC
3 immunoprecipitated from RBL cell
lysate. Pretreatment of intact cells with cpt-cAMP and fMLP, but not
PAF, resulted in an inhibition of subsequent PLC
3
phosphorylation by PKA in vitro. These data demonstrate
that fMLP receptor, which couples to a ptx-sensitive G protein,
activates both PLC and cAMP production. The resulting PKA activation
phosphorylates PLC
3 and appears to block the ability of
G
to activate PLC. Thus, both fMLP and PAF generate
stimulatory signals for PLC
3, but only fMLP produces a
PKA-dependent inhibitory signal. This suggests a novel mechanism for
the bidirectional regulation of receptors which activate PLC by
ptx-sensitive G proteins.
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INTRODUCTION |
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Many extracellular signaling molecules including
neurotransmitters, hormones, and chemoattractants mediate their
biological responses via the activation of G protein-coupled receptors
through stimulation of adenylyl cyclase, phospholipase C
(PLC),1 and ion channels (1). Continuous
agonist stimulation leads to waning of the biological response by a
process termed desensitization (2). Receptor phosphorylation by G
protein-coupled receptor kinases as well as by second
messenger-activated kinases, such as protein kinase A (PKA) and protein
kinase C, are important in receptor desensitization (2, 3).
Additionally, chemoattractant responses are regulated at the level of
PLC (4). Chemoattractants such as formyl peptides (e.g.,
n-formyl-Met-Leu-Phe (fMLP)), the anaphylatoxin C5a, and
interleukin-8 activate PLC by releasing
subunits
(G
) of a pertussis toxin (ptx)-sensitive G protein,
likely Gi
2 (5, 6). The chemoattractant receptor for PAF
couples to both ptx-sensitive and -insensitive G proteins. The latter,
G
q, likely activates PLC
by a different mechanism (7,
8).
fMLP and PAF receptors have been shown to display differences in
susceptibility to desensitization (9). This laboratory has developed
methodology to study the regulation of chemoattractant receptors in the
leukocyte-like RBL-2H3 (RBL) cell line (10-13). Using this model, it
was found that a membrane permeable cAMP analog caused inhibition of
Ca2+ mobilization stimulated by fMLP but not PAF (4). This
difference could be potentially related to the distinct G protein usage
of these receptors. The present study characterizes this observation and demonstrates that fMLP causes an increase in cAMP production both
in neutrophils and transfected RBL cells and that the resulting PKA
activation leads to inhibition of a biological response, secretion. In
addition, the data show that PLC3 is a direct substrate
for phosphorylation by PKA and that fMLP receptor-stimulated
PLC
3 phosphorylation by PKA provides a previously
unrecognized mechanism for the counter regulation of cellular
activation.
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EXPERIMENTAL PROCEDURES |
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Materials--
[32P]Orthophosphate (8500-9120
Ci/mmol), myo-[2-3H(N)]inositol (24.4 Ci/mmol), [-32P]ATP (6000 Ci/mmol), and
[
-32P]GTP (6000 Ci/mmol) were purchased from NEN.
fMLP, PAF
(1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine), and the protein kinase A inhibitor H-89 were purchased from Calbiochem. Recombinant C5a and cpt-cAMP were purchased from Sigma.
Affinity-purified polyclonal antibody against PLC
3 was
obtained from Santa Cruz Biotechnology. Pertussis toxin and all tissue
culture reagents were purchased from Life Technologies, Inc. The
catalytic subunit of PKA was obtained from Promega. The
Radioimmunoassay kit for cAMP measurement was purchased from Amersham
Corp.
Cell Culture and Assays--
RBL cells stably expressing
epitope-tagged fMLP and PAF receptors were used throughout this study
(4, 10, 11, 14). Cell culture, neutrophil purification, GTPase
activity, phosphoinositide hydrolysis, Ca2+ mobilization,
secretion, and in vivo PLC3 phosphorylation
were performed exactly as described by us previously (4, 9, 10). HL-60
cells were differentiated with 1.3% dimethyl sulfoxide for 5-6 days.
In vitro phosphorylation of PLC
3 was
performed essentially as described for PLC
2 (15).
Briefly, cells (5 × 106) were treated with various
agents or buffer for 5 min in the presence of
isobutylmethylxanthine (IBMX) (400 µM), lysed, and immunoprecipitated with anti-PLC
3 antibody. The immune
complex was washed with a buffer containing 40 mM Tris-HCl,
(pH 7.4), MgOAc (20 mM), ATP (20 µM) and
resuspended in the same buffer (50 µl) supplemented with 2 µCi of
[
32P-ATP]. Phosphorylation was started via the
addition of 1 µl of purified PKA. The reaction was stopped by adding
1 ml of ice-cold buffer, and the immune complex was washed three times.
The proteins were resolved on a 6% SDS-polyacrylamide gel
electrophoresis and visualized by autoradiography. For cAMP assay,
cells (0.5-1.0 × 106/ml) were preincubated for 10 min with 400 µM IBMX and stimulated with fMLP, C5a, or
PAF. The reactions were quenched, and cAMP measurements were carried
out as described in the cAMP kit manual.
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RESULTS |
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Differential Regulation of fMLP- and PAF-mediated Phosphoinositide Hydrolysis and Secretion by cAMP-- RBL cells were preincubated with or without a membrane-permeable cAMP analog, cpt-cAMP (1 mM; 5 min) and dose responses of fMLP- and PAF-stimulated phosphoinositide hydrolysis and degranulation were determined. As shown in Fig. 1A, cpt-cAMP caused a substantial inhibition of fMLP-stimulated generation of inositol phosphates. In contrast, PAF-mediated phosphoinositide hydrolysis was inhibited by only ~30% (Fig. 1B). Furthermore, cpt-cAMP substantially inhibited secretion stimulated by fMLP but had no effect on the response to PAF (Fig. 1, C and D). The half-maximal and maximal concentrations of cpt-cAMP for inhibition of fMLP-mediated responses were ~0.1 mM and ~1 mM, respectively (Fig. 1, E and F). Cpt-cAMP also caused a substantial inhibition of intracellular Ca2+ mobilization stimulated by fMLP but not PAF (4). To test whether cAMP also inhibited responses to other chemoattractant receptors that activate PLC via a ptx-sensitive G protein, its effect on C5a-stimulated Ca2+ mobilization in RBL cells was tested. In the absence of cpt-cAMP, stimulation with C5a (1 nM) resulted an increase of intracellular mobilization of 162 ± 5.6 nM (n = 4) over basal. In the presence of cpt-cAMP (1 mM, 5 min), this response was reduced to 27 ± 2.2 nM (83% inhibition).
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Effect of cAMP on fMLP- and PAF-mediated GTPase Activity-- To determine the effect of cpt-cAMP on G protein activation, RBL cells were treated with buffer or cpt-cAMP, then membranes were prepared, and the ability of fMLP and PAF to stimulate GTPase activity was measured. Both fMLP and PAF stimulated GTPase activity in a dose-dependent manner in membranes from buffer or cpt-cAMP-treated cells showing that cpt-cAMP pretreatment had no effect on this PAF- or fMLP-stimulated response (data not shown).
In Vivo and in Vitro Phosphorylation of
PLC3--
Using antibodies that specifically recognize
different PLC
isoforms, it was shown that of the known PLC
isoforms only PLC
3 is expressed in RBL cells (4). To
determine if other PLC
isoforms are expressed in RBL cells at levels
below the detection of antibodies, specific oligonucleotide primers for
different PLC
isoforms were used for reverse
transcriptase-polymerase chain reaction on RNA from RBL cells. Rat
brain RNA was used as a control. PLC
3 was the only
PLC
isoform detected in RBL cells (data not shown).
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DISCUSSION |
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Chemotactic, microbiocidal, and cytotoxic effects of phagocytic leukocytes are stimulated by chemoattractants such as formylated peptides and PAF via the G protein-coupled receptor activation of PLC (16). The ability of fMLP to produce a transient increase in cAMP production in neutrophils is well documented (17-20); however, the physiological effects of this phenomenon were not known. A recent study did demonstrate that a PKA inhibitor enhanced superoxide production stimulated by fMLP in human neutrophils (21), but the mechanism of this effect was also unknown. It also remained to be determined whether PAF stimulated cAMP production in neutrophils and whether inhibition of PKA resulted in the regulation of PAF-mediated biological responses as well. The present work utilizing RBL cells stably expressing fMLP and PAF receptors demonstrated that fMLP, but not PAF, caused an increase in cAMP formation and that preincubation of cells with a membrane permeable cAMP analog resulted in inhibition of both phosphoinositide hydrolysis and exocytotic release of granules stimulated by fMLP. Furthermore, the PKA inhibitor H-89 enhanced fMLP-stimulated phosphoinositide hydrolysis. These data suggest that cAMP produced by fMLP provides a mechanism for counter regulation of an fMLP-stimulated biological response, secretion via the inhibition of PLC activation. This phenomenon appears to be specific for fMLP versus PAF, as the latter did not cause cAMP generation nor did exogenously added cAMP inhibit PAF-induced phosphoinositide hydrolysis or secretion. This difference in the regulation of fMLP- and PAF-mediated responses in transfected RBL cells is likely to be physiologically relevant as similar differences in the generation of cAMP and the regulation of cellular functions by cAMP were observed in the present study in human neutrophils and neutrophil-like HL-60 cells. These differences in the functional regulation of chemoattractant receptors are likely a consequence of G protein usage as suggested by the distinct ptx sensitivity of fMLP versus PAF receptors, with the former being sensitive and the latter is at least partially resistant (8, 10, 11).
In the studies reported here, fMLP stimulated a 40-50% increase in
cAMP over basal levels in neutrophils and HL-60 cells (Fig. 3A). These data are consistent with previous findings from
this and other laboratories (17-19). Furthermore, the ability of a PKA inhibitor to enhance fMLP-stimulated superoxide generation in neutrophils (21) suggests that the small fMLP-stimulated cAMP increase
is sufficient to counter regulate the response to fMLP. This contention
is supported by the finding that phosphodiesterase inhibitors which
cause ~50% increase of cellular cAMP also result in a substantial
inhibition of fMLP-stimulated superoxide release in neutrophils (22).
The mechanism by which fMLP causes an increase in cAMP is not known
(18, 23). Of the nine adenylyl cyclases identified, activation of types
I and III are inhibited by all three forms of Gi proteins
(24-26). In contrast, types II and IV are activated by Gs
and G of ptx-sensitive G proteins in a synergistic
manner (23). In guinea pig neutrophils, fMLP greatly potentiates cAMP
production stimulated by prostaglandin E1 receptor and this
enhancement is totally inhibited by ptx (27). In HEK 293 cells, which
endogenously expresses adenylyl cyclase type III, fMLP causes an
inhibition of cAMP production (28, 29). This inhibitory effect of fMLP
likely results from the interaction of Gi
with type III
adenylyl cyclase. However, in the same cell line transiently expressing
adenylyl cyclase type II, fMLP causes the stimulation of cAMP
production and this response is inhibited by ptx (30). This indicates
that fMLP can either stimulate or inhibit cAMP formation depending on
the subtype of adenylyl cyclase expressed. The observation that fMLP caused cAMP formation in RBL cells and that this response was completely inhibited by ptx suggest that G
directly interacts with adenylyl cyclases types II or IV to stimulate cAMP production. This contention is supported by the finding that PAF, which
utilizes a ptx insensitive mechanism to cause intracellular Ca2+ mobilization in RBL cells, HL-60 cells and
neutrophils, did not cause cAMP production in any of these cell types
(Figs. 2A and 3A).
The demonstration that cAMP did not cause phosphorylation of fMLP
receptor (11) and had no effect on fMLP-stimulated GTPase activity
indicates that its ability to block fMLP-stimulated inositol phosphate
generation and secretion is not mediated at the level of the receptor
or its coupling to G protein. Inhibition of membrane inositol
phospholipid resynthesis and thus a reduction in the availability of
substrate for PLC has been postulated as a mechanism by which cAMP
inhibits fMLP-stimulated generation of inositol phosphates in human
neutrophils (31). This mechanism is unlikely because cAMP did not
inhibit phosphoinositide hydrolysis stimulated by PAF in RBL cells, and
it had no effect on PAF-induced Ca2+ mobilization in human
neutrophils and HL-60 cells. The selective inhibition of fMLP response
by cAMP is therefore likely to be mediated via the modification of PLC.
Using reverse transcriptase-polymerase chain reaction (this study) and
Western blotting with PLC isoform-specific antibodies (4) it was
shown that PLC
3 is the only known PLC
isozyme
expressed in RBL cells. Furthermore, both fMLP and cpt-cAMP caused
phosphorylation of PLC
3 in this cell line. In addition, purified catalytic subunit of PKA phosphorylated PLC
3
immunoprecipitated from an RBL cell lysate. The observation that
preincubation of cells with cpt-cAMP blocked subsequent in
vitro PLC
3 phosphorylation by PKA suggests that
PLC
3 is a direct substrate for PKA. The ability of
cpt-cAMP to inhibit fMLP-induced phosphoinositide hydrolysis and
secretion is likely mediated via the phosphorylation of
PLC
3 by PKA. Importantly, the finding that fMLP
stimulated the formation of cAMP and that pretreatment of cells with
fMLP resulted in a partial inhibition of PKA-stimulated
PLC
3 phosphorylation in vitro indicate that
fMLP-stimulated PLC
3 phosphorylation is mediated, at
least in part, by PKA. The ability of the PKA inhibitor H-89 to enhance
fMLP-stimulated inositol phosphates generation suggests that counter
regulation of fMLP-stimulated biological responses is likely mediated
via the PKA-induced phosphorylation of PLC
3. This form
of inhibition appears to be specific for fMLP versus PAF,
which did not stimulate cAMP formation and did not block PLC
3 phosphorylation by PKA in vitro.
Furthermore, the PKA inhibitor H-89 had no effect on the
ptx-insensitive component of PAF-mediated generation of inositol
phosphates.
The data presented herein revealed that fMLP activated both
PLC3 and cAMP production via a ptx-sensitive pathway.
Interestingly, PKA activated by this mechanism phosphorylated
PLC
3 and blocked the subsequent activation of PLC by
fMLP. It is likely that fMLP activates PLC
3 through the
release of G
(8, 10, 11). Thus the PKA-mediated phosphorylation
of PLC
3 can be hypothesized to selectively block
activation by G
as opposed to G
q, which is activated by PAF. This suggests a novel selective counter regulatory pathway for certain G protein-coupled receptors such as
those for chemoattractants that activate a ptx-sensitive signaling pathway. Other receptors such as the
2-adrenergic, dopamine
D2 and adenosine A1 receptors also activate
both adenylyl cyclase and PLC (23). Given that PLC
3 is
expressed abundantly in many cells and tissues such as platelets,
leukocytes, brain, testes, and lung (32-34), phosphorylation of
PLC
3 by PKA may be a novel mechanism for the counter
regulation of some but not other G protein-coupled receptors.
Receptors that couple to Gs and cause an elevation of
intracellular cAMP levels are known to inhibit PLC-mediated responses to other receptors that couple to ptx-sensitive G protein (15). The
data herein suggest that the mechanism for this phenomenon may be
through the phosphorylation of PLC3 by PKA at a site
which blocks G
-mediated activation. PLC
other than
PLC
3 may also be regulated by PKA. For example,
phosphorylation of PLC
2 by PKA has been suggested as a
mechanism by which cAMP inhibits fMLP-stimulated phosphoinositide
hydrolysis in differentiated HL-60 cells (15). It is, however,
important to note that the neutrophil-like HL-60 cells express both
PLC
2 and PLC
3 (4). Furthermore, cAMP
causes phosphorylation of PLC
3 in the human monocyte-like U937 cells and the murine macrophage-like J774.1 cells
(4). The finding that fMLP-stimulated phosphoinositide hydrolysis and
Ca2+ mobilization were not completely blocked in
neutrophils isolated from mice deficient in PLC
2
suggests that both PLC
2 and PLC
3 are
activated by fMLP (35). Unlike PLC
2, which is expressed only in certain cells of hematopoietic origin, PLC
3 is
expressed in many cell types and tissues (32, 33). Therefore,
cross-talk between the adenylyl cyclase and PLC pathways is likely to
be mediated via the phosphorylation of PLC
2,
PLC
3, or both depending on the cell type on which the
receptors are expressed.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL-54166 (to H. A.), AI-38910 (to R. M. R), 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.
¶ Supported by a fellowship from the Italian Association for Cancer Research. Current address: Instituto di Ricerche Farmacologiche "Mario Negri," Milan, Italy.
§ To whom correspondence should be addressed: Dept. of Medicine, Duke University Medical Center, 201C MSRB, Box 3680, Durham, NC 27710. Tel.: 919-681-6756; Fax: 919-684-4434; E-mail: ali00001{at}mc.duke.edu.
1 The abbreviations used: PLC, phospholipase C; fMLP, n-formyl-Met-Leu-Phe; PAF, platelet-activating factor; G protein, GTP-regulatory protein; RBL, rat basophilic leukemia; cpt-cAMP, 8-(4-chlorophenylthio)-adenosine 3':cyclic monophosphate; PKA, protein kinase A; ptx, pertussis toxin; IBMX, isobutylmethylxanthine.
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REFERENCES |
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