(Received for publication, December 20, 1996, and in revised form, February 13, 1997)
From the Departments of Medicine and
¶ Immunology, Duke University Medical Center,
Durham, North Carolina 27710
Platelet-activating factor (PAF) stimulates a
diverse array of cellular responses through receptors coupled to G
proteins that activate phospholipase C (PLC). Truncation of the
cytoplasmic tail of the receptor to remove phosphorylation sites
(mutant PAF receptor, mPAFR) results in enhancement of PAF-stimulated
responses. Here we demonstrate that PAF or phorbol 12-myristate
13-acetate (PMA) pretreatment inhibited wild type PAFR-induced
PLC-mediated responses by ~90%, whereas these responses to the
phosphorylation-deficient mPAFR were inhibited by ~50%, despite
normal G protein coupling, suggesting a distal inhibitory locus. PAF
and PMA, as well as a membrane permeable cyclic AMP analog, stimulated
phosphorylation of PLC3. A protein kinase C (PKC) inhibitor blocked
phosphorylation of PLC
3 stimulated by PAF and PMA but not by cAMP.
Activation of protein kinase A (PKA) by cAMP did not result in
inhibition of Ca2+ mobilization stimulated by PAF. In
contrast, cAMP did inhibit the response to formylpeptide
chemoattractant receptor. These data suggest that homologous
desensitization of PAF-mediated responses is regulated via
phosphorylation at two levels in the signaling pathway, one at the
receptor and the other at PLC
3 mediated by PKC but not by PKA.
Phosphorylation of PLC
3 by PKA could explain the inhibition of
formylpeptide chemoattractant receptor signaling by cAMP. As PAF and
formylpeptide chemoattractant receptors activate PLC via different G
proteins, phosphorylation of PLC
3 by PKC and PKA could provide
distinct regulatory control for classes of G protein-coupled
receptors.
Platelet-activating factor
(1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine,
PAF)1 plays important roles in inflammation
and has physiological effects on cardiovascular, reproductive, and
central nervous systems (1). PAF mediates its effects via the
activation of a seven transmembrane domain G protein-coupled receptor
(2, 3). One of the consequences of PAF receptor activation is the
phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol
4,5-bisphosphate leading to the activation of protein kinase C (PKC)
and mobilization of intracellular Ca2+ (4). Molecular
cloning has revealed three classes of PLC: PLC, PLC
, and PLC
,
and each of these occur in several isoforms (5, 6). All four PLC
isoforms are activated to varying extent by
subunit of a class of G
proteins known as Gq (7-10). PLC
2 and PLC
3 are also
activated by the
subunit of the Gi family of G
proteins (11-13). Tyrosine phosphorylation of PLC
is required for
its activation, but the mechanism by which PLC
is activated is
unknown (5). PAFR couples to both pertussis toxin (Ptx)-sensitive and
Ptx-insensitive G proteins in a variety of cells (4, 14, 15). In B
cells and platelets, PAF activates PLC
(16, 17), but the identity of
PLCs activated by PAF in phagocytic leukocytes is unknown.
In phagocytes, PAF-stimulated G protein activation and Ca2+
mobilization are desensitized by prior treatment of cells with PAF and
other chemoattractants such as the formylated peptides, C5a and
interleukin-8 (18). This laboratory has developed stably transfected
RBL-2H3 cells to study molecular mechanisms of leukocyte chemoattractant receptor regulation (15, 19-21). Using wild type and
phosphorylation-deficient PAFR expressed in this cell line, we now
describe the novel finding that homologous desensitization of
PAF-mediated phosphoinositide hydrolysis and Ca2+
mobilization is mediated via two processes; one at the level of
receptor phosphorylation and the other at the level of PLC activation
apparently by PKC-dependent phosphorylation of PLC3.
Bisindolylmaleimide or GF 109203X (BIM),
8-(4-chlorophenylthio)-adenosine 3-cyclic monophosphate (Cpt-cAMP) and
the tyrosine kinase inhibitor PP1 were purchased from Calbiochem.
Affinity purified polyclonal antibodies against PLC
1, PLC
2, and
PLC
3 were obtained from Santa Cruz Biotechnology. ECL Western blot analysis kit was purchased from Amersham Corp. Mouse monoclonal IgE and
antigen (dinitrophenylated BSA) were generously provided by Drs. Juan
Rivera and Henry Metzger (National Institutes of Health). All other
materials were obtained from sources previously described (15).
RBL-2H3 cells stably expressing ~3 × 104 receptors for PAFR and mPAFR were used (21). The cells were maintained in Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum, glutamine (2 mM), penicillin (100 units/ml), and streptomycin (100 mg/ml) (19). For phosphoinositide hydrolysis, cells (5 × 104 cells/well) were subcultured overnight in 96-well tissue culture plate with 1 µCi/ml of [3H]inositol in an inositol-free medium supplemented with 10% dialyzed fetal bovine serum. Cells were washed with HEPES-buffered saline containing 10 mM LiCl and 0.1% BSA and stimulated, and the generation of total [3H]inositol phosphates was determined. For Ca2+ mobilization, cells (3 × 106) were loaded with 1 µM indo-1/AM in the presence of 1 µM pluoronic acid for 30 min at room temperature. Cells were washed and resuspended in 1.5 ml HEPES-buffered saline, and intracellular Ca2+ mobilization was determined as described previously (19).
Western Blotting and Immunoprecipitation of PLCFor
Western blotting, lysate (150 µg protein) from RBL-2H3 cells, and for
comparison, lysates from bovine brain, COS cells, and HL-60 cells were
resolved on a 6% SDS-PAGE gel. The proteins were transferred to
nitrocellulose membrane, blocked with 3% milk, and incubated with 1.5 µg/ml of different anti-PLC antibodies. The immunoreactive
proteins were visualized using ECL Western blotting detection
system. Phosphorylation of PLC
3 was performed essentially as
described for the epitope-tagged chemoattractant receptors (19).
Briefly, RBL-2H3 cells (3 × 106) were subcultured
overnight in 60-mm tissue culture dishes. The following day, cells were
washed twice with 5 ml of phosphate-free Dulbecco's modified Eagle's
medium and incubated in the same medium plus
[32P]orthophosphate (150 µCi/dish) for 90 min. The
labeled cells were stimulated, and PLC
3 was immunoprecipitated from
cell lysate with anti-PLC
3 antibody. The proteins were resolved on a
6% SDS-PAGE gel and visualized by autoradiography.
The mechanism of PAF receptor desensitization was studied
using the rat basophilic leukemia (RBL-2H3) cells expressing wild type
PAFR and a mPAFR in which the cytoplasmic tail was truncated to delete
potential phosphorylation sites (21). In contrast to PAFR, mPAFR was
resistant to both ligand and PKC-induced receptor phosphorylation and
desensitization of G protein coupling, as measured by
[35S]GTPS binding to membranes (21, 22). However, when
indo-1-loaded cells expressing mPAFR were stimulated with PAF (10 nM), washed, and restimulated with PAF (10 nM),
the peak Ca2+ response was still homologously desensitized
by ~50% (Table I). PMA also inhibited mPAFR-mediated
Ca2+ mobilization by ~50%, suggesting a locus of
inhibition distal to G protein activation. In cells expressing wild
type PAFR, both PAF and PMA inhibited PAF-induced Ca2+
mobilization by >90%. PMA also caused substantial inhibition of
mPAF-induced generation of inositol phosphates and completely blocked
the response to PAF (Fig. 1). To further test the role of PKC on PAFR desensitization, cells expressing mPAFR and PAFR were
treated with PMA (100 nM) overnight to deplete PKC or
incubated with the PKC-inhibitor bisindolylmaleimide for 10 min to
block the activity of the enzyme. PAF-stimulated generation of inositol phosphates was then determined. Under both of these conditions, PAF-induced responses were enhanced 2-3-fold in cells expressing mPAFR
(Fig. 1). Overnight PMA treatment did not enhance the response to PAF
in cells expressing PAFR, but bisindolylmaleimide did potentiate the
response to PAF. Because PMA-induced PKC depletion involves activation
of the enzyme, it was possible that the differential effect of PMA on
mPAFR and PAFR cells resulted from PKC-induced phosphorylation and
down-regulation of PAFR but not mPAFR. Indeed, treatment of cells with
PMA (100 nM, overnight) resulted in ~50% decrease of the
wild type PAFR expression but had no effect on mPAFR as measured by the
PAFR antagonist [3H]WEB 2086 binding to intact cells
(data not shown). These data suggest that homologous desensitization of
PAF-mediated cellular responses is mediated by two processes. One
involves receptor phosphorylation, presumably by PKC and a G
protein-coupled receptor kinase, which uncouple the receptor from G
proteins (21). In addition, a new mechanism was identified as a
consequence of PKC-induced modification of a component distal to G
protein activation but proximal to the generation of inositol
phosphates.
|
Although most G protein-coupled receptors activate PLC, angiotensin
receptors in smooth muscle cells and PAF receptors in B cells and
platelets cause tyrosine phosphorylation and activation of PLC
(16,
17, 23). The molecular mechanism by which G protein-coupled receptors
activate PLC
has yet to be determined. PMA, which causes serine
phosphorylation of PLC
(24), leads to reduction of both tyrosine
phosphorylation of PLC
and generation of inositol phosphates
stimulated by cross-linking of cell surface IgE receptors with antigen
(25, 26). This suggests that if PAF activates PLC
, a similar
modification of PLC
by PKC could be involved in its homologous
desensitization. However, the tyrosine kinase inhibitor PP1 (27), which
blocked antigen (IgE)-stimulated Ca2+ mobilization by
>70%, presumably by inhibiting tyrosine phosphorylation of PLC
,
had no effect on PAF-mediated responses (Table II).
Elevation of intracellular cAMP has also been shown to cause serine
phosphorylation of PLC
and inhibition of antigen-stimulated
responses (24). As shown in Table II, the membrane permeable cAMP
analog 8-(4-chlorophenylthio)-adenosine 3
-cylic monophosphate
(cpt-cAMP) substantially inhibited IgE-mediated Ca2+
mobilization but had no effect on the PAF-mediated response. These data
suggest that unlike B cells and platelets (16, 17) PAF does not
activate PLC
in RBL-2H3 cells and that PKA does not down-regulate
PAF-mediated responses.
|
We therefore sought to determine whether a PLC isoform is activated
by PAF in RBL-2H3 cells. To identify the PLC
isoforms expressed in
RBL-2H3 cells, their lysates and lysates from bovine brain, COS and
HL-60 cells were separated by SDS-PAGE, and the presence of PLC
1,
PLC
2, and PLC
3 was determined by Western blotting using PLC
isoform-specific antibodies. All cells tested, including RBL-2H3 cells,
expressed PLC
3 in different amounts (Fig. 2).
Although PLC
1 and PLC
2 were expressed at high levels in brain and
HL-60 cells, respectively, neither could be detected in RBL-2H3 cells
(Fig. 2).
Studies with purified G proteins and PLC revealed that PLC1 and
PLC
3 are equally responsive to activation by G
q (28). It was therefore possible that in RBL-2H3 cells, which do not express
PLC
1, PAF activates PLC
3 and that PKC-induced modification of
this enzyme accounts for the distal component of PAFR homologous desensitization. To test this hypothesis, cells were labeled with [32P]orthophosphate and stimulated with PAF, and cell
lysate was immunoprecipitated with PLC
3-specific antibody. As shown
in Fig. 3 (A and B), PAF caused
phosphorylation of PLC
3 in a dose- and time-dependent
manner. PLC
3 phosphorylation was detectable at physiologic PAF
concentration of 0.3 nM and was maximum by 10 nM. PAF-induced PLC
3 phosphorylation was rapid, reached
a maximum by 15 s, and remained elevated for 10 min. To determine
whether there was a correlation between PKC-mediated PLC
3
phosphorylation and inhibition of PAF-stimulated responses, cells were
treated with or without the PKC inhibitor bisindolylmaleimide,
stimulated with PAF (10 nM) and PMA (100 nM),
and PLC
3 phosphorylation was determined. As shown in Fig.
3C, PAF- and PMA-induced PLC
3 phosphorylation was almost
completely inhibited by bisindolylmaleimide. Cpt-cAMP also
phosphorylated PLC
3 presumably through activation of PKA, and this
phosphorylation was not inhibited by bisindolylmaleimide. PLC
3
phosphorylation was also tested in two other cell lines that
endogenously express PAF receptors. These were the human monocyte-like
U937 cells and the murine macrophage-like J774.1 cells. In both cases,
PAF, PMA, and cpt-cAMP stimulated phosphorylation of PLC
3 (Fig.
3D).
The effect of cAMP elevation on PLC activation was determined using the
RBL-2H3 cells coexpressing PAF and formylpeptide receptors. In such
cells, cpt-cAMP did not inhibit Ca2+ mobilization to PAF,
although it did inhibit the response of the formylpeptide receptor
(Table II). Furthermore, in cells expressing mPAFR, cpt-cAMP (1 mM, 5 min) had no effect on PAF-stimulated generation of
[3H]inositol phosphates, whereas PMA (100 nM,
5 min) blocked this response by ~50%. Preincubation of cells with
cpt-cAMP had no effect on PMA-mediated inhibition of PAF-stimulated
response (data not shown). These data support the hypothesis that
PLC3 is likely activated by PAFR and that PLC
3 phosphorylation by
PKC, but not by PKA, is responsible for one component of PAFR
homologous desensitization.
PLC1 is phosphorylated by PKC (29), and PLC
2 is phosphorylated by
PKA (30). The present study revealed that both PKC and PKA
phosphorylate of PLC
3 in vivo (Fig. 3C). More
importantly, phosphorylation of PLC
3 by the different protein
kinases appears to be associated with distinct functional consequences.
Although phosphorylation of PLC
3 by PKC resulted in the inhibition
of responses stimulated by PAF, its phosphorylation by PKA was not associated with such inhibition. On the other hand, PKA activation by
cpt-cAMP inhibited formylpeptide receptor-mediated responses. In
RBL-2H3 cells PAF receptors activate PLC predominantly via a
Ptx-insensitive G protein, whereas formylpeptide-mediated response is
completely blocked by Ptx (15, 19). Therefore, differential inhibition
of responses to PAF and formylpeptide by PKC and PKA is likely due to
the different G protein subunits these receptors utilize to activate
PLC. It was demonstrated in vitro that G
q and
G
released from Ptx-sensitive G protein interact with distinct regions of PLC
3 to activate the enzyme (28). This led Smrcka and
Sternweis to propose that different receptors that utilize Gq or Gi could activate PLC
3 by producing
G
q or G
(28). The data presented herein support
this hypothesis and provide a molecular basis for the differential
regulation of G
q- and G
-mediated activation of PLC
3. It
is likely that PKC and PKA phosphorylate PLC
3 on distinct sites that
block its activation by G
q and G
, respectively.
Similar to RBL-2H3 cells, many human cell lines and tissues express
PLC3 but not PLC
1 or PLC
2 (9, 11, 28, 31). Given the duality
of PLC
3 activation by both G
q and G
and its
ubiquitous tissue distribution, phosphorylation of PLC
3 by PKC and
PKA may be general mechanisms by which functions of different G
protein/PLC-coupled receptors are regulated.
We thank Drs. Juan Rivera and Henry Metzger (National Institutes of Health) for providing IgE and dinitrophenylated bovine serum albumin.