From the Department of Physiology, University College London, London WC1E 6JJ, United Kingdom
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ABSTRACT |
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Activation of intact human neutrophils by fMLP stimulates phospholipase D (PLD) by an unknown signaling pathway. The small GTPase, ADP-ribosylation factor (ARF), and Rho proteins regulate the activity of PLD1 directly. Cell permeabilization with streptolysin O leads to loss of cytosolic proteins including ARF but not Rho proteins from the human neutrophils. PLD activation by fMLP is refractory in these cytosol-depleted cells. Readdition of myr-ARF1 but not non-myr-ARF1 restores fMLP-stimulated PLD activity. C3 toxin, which inactivates Rho proteins, reduces the ARF-reconstituted PLD activity, illustrating that although Rho alone does not stimulate PLD activity, it synergizes with ARF. To identify the signaling pathway to ARF and Rho activation by fMLP, we used pertussis toxin and wortmannin to examine the requirement for heterotrimeric G proteins of the Gi family and for phosphoinositide 3-kinase, respectively. PLD activity in both intact cells and the ARF-restored response in cytosol-depleted cells is inhibited by pertussis toxin, indicating a requirement for Gi2/Gi3 protein. In contrast, wortmannin inhibited only fMLP-stimulated PLD activity in intact neutrophils, but it has no effect on myr-ARF1-reconstituted activity. fMLP-stimulated translocation of ARF and Rho proteins to membranes is not inhibited by wortmannin. It is concluded that activation of Gi proteins is obligatory for ARF/Rho activation by fMLP, but activation of phosphoinositide 3-kinase is not required.
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INTRODUCTION |
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Phospholipase D (PLD)1
is an important signal-transducing enzyme in a wide variety of cells
and catalyzes the hydrolysis of phosphatidylcholine (PC) to produce the
potential second messenger phosphatidic acid (PA) (1-4). Studies in
neutrophils and HL60 cells have identified a requirement for both
cytosolic and membrane components for PLD activation when stimulated
with the nonhydrolyzable analog of GTP, GTPS. Through the use of
reconstitution studies utilizing HL60 membranes (5) or cytosol-depleted
cells (6), ARF1 and ARF3 have been identified as activators of
GTP
S-stimulated PLD activity. The requirement for Rho in PLD
activation was identified separately because RhoGDI, which extracts Rho
from membranes, inhibited neutrophil PLD (7). Subsequent studies
utilizing purified protein demonstrated that Rho proteins stimulated
PLD directly (8-10).
Although both ARF and Rho alone can stimulate PLD, other cytosolic
factors have been shown to augment these responses in human neutrophils. A 50-kDa factor has been shown to act synergistically with
both ARF (11, 12) and Rho in stimulating PLD (13). Similarly, both G
proteins also act synergistically with protein kinase C to activate
PLD. Both synergistic (14, 15) and additive (16, 17) responses have
been reported for the simultaneous addition of ARF and Rho. A single
PLD that is synergistically responsive to ARF/Rho/protein kinase C
has been cloned and is now referred to as hPLD1 (10).
The roles of ARF and Rho are well documented with regard to PLD
activation by GTPS, but their roles in G protein-coupled receptors
and receptor tyrosine kinases are not well defined and may be different
for distinct receptors. In the case of platelet-derived growth factor
and epidermal growth factor, PLD activity is reported to be downstream
of phospholipase C activation (18-20). However, in other cases,
including fMLP-stimulated neutrophils (21), protein kinase C plays, if
any, a minor role. Using Clostridium toxins, Rho proteins
have been implicated in the activation of PLD in human embryonic kidney
cells overexpressing the m3 muscarinic acetylcholine receptor (22), and
in Rat 1 fibroblasts stimulated with lyso-PA and endothelin (23).
Insulin-mediated PLD activation is dependent on ARF and Rho proteins,
and an involvement of PI 3-kinase has been implicated (24, 25).
In this study we have examined the recruitment of ARF and Rho proteins to membranes by fMLP and the ability of these proteins to regulate receptor-controlled PLD in human neutrophils. We had reported previously that fMLP-dependent activation was compromised in differentiated HL60 cells depleted of their cytosolic contents (26). We report that both ARF and Rho proteins are regulated by the fMLP receptor via a pertussis toxin-sensitive heterotrimeric G protein. Although activation of PLD in intact neutrophils is inhibited by wortmannin, a relatively selective inhibitor of PI 3-kinase, wortmannin treatment is not inhibitory to the fMLP-stimulated recruitment of ARF and Rho to membranes. We conclude that the activation of PLD by the fMLP receptor is dependent on receptor-activated ARF and Rho proteins in human neutrophils coupled via Gi proteins and is not obligatorily dependent on PI 3-kinase activation.
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EXPERIMENTAL PROCEDURES |
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Materials-- Neutrophils were purified from blood from healthy volunteers or isolated from buffy coats that were obtained from the North London Blood Transfusion Center. Recombinant ARF1 and myr-ARF1 proteins were purified from Escherichia coli as described previously (6). Anti-RhoA antibodies were obtained from Santa Cruz Biotechnology Ltd. The anti-ARF antibodies used have been described previously (27). All other reagents were obtained as described previously (26).
Preparation of Neutrophils-- Neutrophils were prepared according to established procedures (28). 50 ml of anti-coagulated blood or a buffy coat pack was mixed with an equal volume of 2% dextran solution in phosphate-buffered saline, pH 7.2, to aggregate erythrocytes. After 20 min at room temperature, the leukocyte-rich upper layer was removed and layered onto 10 ml of Lymphoprep and centrifuged at 2,000 rpm for 20 min to separate neutrophils from other white cells. Contaminating erythrocytes were removed by hypotonic lysis.
Analysis of ARF and Rho Leakage from Permeabilized Neutrophils-- Neutrophils were permeabilized for varying lengths of time with 0.4 IU/ml streptolysin O. At the required time points, 1-ml aliquots were removed and centrifuged. The proteins from the supernatants (after precipitation with trichloroacetic acid) and cell pellets were resuspended in sample buffer and analyzed for ARF and RhoA proteins by Western blot analysis using appropriate antibodies.
Labeling of Neutrophils with [3H]Alkyllyso-PC-- Neutrophils were washed twice in HEPES buffer (20 mM HEPES, 137 mM NaCl, 3 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 1 mg/ml glucose, and 0.1 mg/ml bovine serum albumin, pH 7.2) and finally resuspended in 1.5 ml. The cells were incubated for 30 min at 37 °C with [3H]alkyllyso-PC (10 µCi). The cells were harvested by centrifugation to remove unincorporated label, and the cells were washed with either HEPES buffer (for intact cell experiments) or PIPES buffer (20 mM PIPES, 137 mM NaCl, 3 mM KCl, 1 mg/ml glucose, and 0.1 mg/ml bovine serum albumin, pH 6.8) for permeabilized cell experiments.
Assay for PLD Activity in Intact Neutrophils-- Neutrophils were suspended in HEPES buffer and pretreated with 5 µM cytochalasin B for 5 min. 50-µl aliquots were transferred to tubes containing 2% EtOH (1% final in the assay) in the presence or absence of fMLP (1 µM final). After a 10-min incubation at 37 °C, assays were quenched with 700 µl of CHCl3:MeOH (1:1). After phase separation with 350 µl of water, the chloroform phase was recovered. The chloroform phase was dried under vacuum and redissolved in 50 µl of chloroform. Samples were spotted onto Whatman LK6TLC silica plates. The plates were developed in chloroform:methanol:acetic acid:water (75:45:3:1), dried at room temperature, and the lipid spots localized with iodine vapors. The spots corresponding to PEt and PC were excised after iodine sublimation and put into scintillation vials. The lipids were extracted with 250 µl of methanol and counted for radioactivity after the addition of scintillation fluid. Results are expressed as the percentage of dpm incorporated into PC. Lipid standard containing PA and PEt which was prepared as described previously (29) was added for localization of lipids after separation by TLC.
Reconstitution of ARF-regulated PLD in Cytosol-depleted Neutrophils-- Permeabilization of cells to deplete the cytosol and reconstitution of PLD activity were essentially carried out as described previously (6, 30). Briefly, labeled neutrophils were permeabilized with streptolysin O (0.4 IU/ml) for 10 min in the presence of 100 nM Ca2+ in 5 ml at 37 °C. The permeabilized cells were centrifuged at 4 °C and resuspended in cold PIPES buffer and aliquoted (50 µl) into tubes kept on ice containing appropriate reagents. The assays were carried out in the presence of 1 mM MgATP, 100 µM GTP (unless indicated otherwise), 2 mM MgCl2, 1 µM Ca2+, and 1% EtOH. ARF proteins and fMLP were added as indicated in the individual figure legends. After a 30-min incubation at 37 °C, the reactions were quenched as described above.
Pertussis Toxin Treatment-- Intact labeled neutrophils were pretreated with 500 ng/ml pertussis toxin for 2 h at 37 °C as described previously (31). Cells were then processed as required.
C3 Transferase Treatment-- Cytosol-depleted neutrophils were incubated with 1 µg/ml C3 toxin in the presence of 0.5 mM NAD for 10 min at 37 °C.
Wortmannin Treatment-- Intact cells were incubated at 37 °C for 10 min in the presence of 100 nM wortmannin.
Translocation of ARF and Rho to Membrane Compartments-- 1 ml of intact cells (107 cells/ml) was incubated in the presence or absence of 1 µM fMLP for 1 or 10 min at 37 °C. The cells were pretreated with 5 µM cytochalasin B as indicated. At the end of the incubation, the cells were sedimented at 4 °C; resuspended in buffer; and treated with diisopropyl fluorophosphate, a serine protease inhibitor, for 5 min at 4 °C. After centrifugation, the cells were resuspended in 1 ml, and a mixture of protease inhibitors was added (27). After sonication, the samples were centrifuged at 100,000 × g for 1 h at 4 °C to obtain the membrane fraction. The membrane fractions were resuspended in sample buffer, boiled, and run on SDS-PAGE. After transfer onto polyvinylidene difluoride, blots were probed with either anti-ARF or anti-Rho antibodies. Detection was by enhanced chemiluminescence.
For translocation of ARF to membranes in permeabilized cells, 1 ml of cells (107 cells/ml) was incubated with streptolysin O in the presence of 1 mM MgATP, 2 mM MgCl2, and 1 µM Ca2+. 100 µM GTP, 1 µM fMLP, or 10 µM GTP ![]() |
RESULTS |
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ARF Restores fMLP-dependent PLD Activity in Cytosol-depleted Human Neutrophils-- To examine a requirement for ARF proteins in fMLP-stimulated PLD activity, human neutrophils were permeabilized with streptolysin O for 10 min to deplete the cells of their freely diffusable cytosolic proteins. Fig. 1A illustrates that this protocol depletes the majority of the ARF proteins from the permeabilized cells, and they are recovered in the external medium. This loss is coincident with the inability of fMLP to stimulate PLD activity in cytosol-depleted cells (Ref. 26 and Fig. 1B). fMLP regains the ability to stimulate PLD activity provided that myr-ARF1 is also added to the permeabilized cells (Fig. 1B). It was noted that adding myr-ARF1 alone raised the basal activity of PLD, and this was dependent on the presence of GTP. In the reconstituted assay, the time course of PLD activation by fMLP reached a maximum at 30 min. Fig. 1, C and D, illustrates that reconstitution of PLD activity with myr-ARF1 and fMLP is concentration-dependent. For the remainder of the experiments, fMLP was used at 1 µM, and myr-ARF1 was used at 50 µg/ml.
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Myristoylation of ARF Is Essential for fMLP- but Not
GTPS-dependent PLD Activity--
ARF proteins are
myristoylated at their NH2 terminus, and this lipid
modification is thought to be important for efficient guanine
nucleotide exchange catalyzed by the ARF exchange factors (32-34).
Both myristoylated (myr-ARF1) and nonmyristoylated (ARF1) ARF proteins
were examined for their ability to restore fMLP-dependent PLD activity in cytosol-depleted neutrophils. Fig.
2 illustrates that myristoylation is
essential for the restoration of fMLP-dependent PLD
activity and also for the response observed with GTP alone. Consistent
with our own observation (6) and those of others (5, 35),
myristoylation is not required for GTP
S-dependent stimulation of PLD (Fig. 2). However, a 100-fold higher concentration of nonmyristoylated rARF1 is required for maximal stimulation compared
with fully myristoylated ARF1 for GTP
S-stimulated PLD activity (6).
Therefore, concentrations of recombinant ARF proteins used to examine
the requirement for myristoylation take this into account; for
nonmyristoylated ARF, a concentration of 750 µg/ml is used compared
with 50 µg/ml for myr-ARF1. (Effective myr-ARF1 used is approximately
5 µg/ml (500 nM) because of the 10% efficiency of
myristoylation in E. coli determined by mass spectroscopy
analysis.) These concentrations of recombinant myr-ARF1 and
non-myr-ARF1 reflect the equivalent loading of GTP
S when measured
in vitro. Fig. 2 also illustrates that the level of PLD stimulated by these proteins is similar in magnitude when GTP
S is
the activator.
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Pertussis Toxin Inhibits Myr-ARF1-restored
fMLP-dependent PLD Activity--
The fMLP receptor is
coupled to the pertussis toxin-sensitive heterotrimeric G proteins,
Gi2 and Gi3 (36), and subunits are the
direct regulators of phospholipase C
2 and PI 3-kinase (
isoform) (37, 38). To address the question of whether activation of the PLD activity by the fMLP receptor in the reconstituted assay
also requires a prior activation of Gi proteins, we
examined the influence of pertussis toxin pretreatment. Initially we
confirmed that, as reported previously (39, 40), pertussis toxin
pretreatment led to inhibition of the fMLP-stimulated PLD activity in
intact cells (Fig. 3A).
Pertussis toxin pretreatment also inhibits fMLP-stimulated PLD activity
when the agonist and the permeabilizing agent streptolysin O are added
simultaneously, conditions in which the cytosolic proteins are still
present (Fig. 3B). To establish a requirement for
Gi proteins in the regulation of PLD activity by myr-ARF1, we examined the effect of pertussis toxin pretreatment on the myr-ARF1-restored fMLP-dependent PLD activity in
cytosol-depleted neutrophils (Fig. 3C). Although the
stimulation of PLD activity observed in the combined presence of GTP
and myr-ARF1 was not inhibited significantly by pertussis toxin
treatment, the fMLP-stimulated activity was inhibited (Fig.
3C). These results confirm that regulation of myr-ARF1 by
the fMLP receptor is indirect and that one intervening component in the
pathway leading to myr-ARF1 activation has to include the
heterotrimeric Gi proteins.
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Rho Proteins Participate in PLD Activation by the fMLP
Receptor--
Previous studies in a variety of cells have indicated
that not only ARF but also Rho can activate PLD activity (1-4). It has
been reported that in HL60 cells, endogenous Rho is unlikely to play a
physiological role in PLD activation (16). However, these experiments
were performed using GTPS as an activator. To examine the
contribution of Rho proteins in the system analyzed here, we first
established whether Rho proteins leaked out of permeabilized cells.
Neutrophils were permeabilized with streptolysin O for various lengths
of time, and the supernatants and the cell pellets were analyzed for
Rho proteins. The majority of RhoA remains cell-associated in the
permeabilized cells even after 30 min. The amount of Rho released in
the supernatant was low compared with that retained in the cells (Fig.
4). Under these conditions, the majority
of the ARF proteins was found to leak out of the permeabilized cells
(Fig. 1A). Because Rho proteins are found mainly to be
cytosolic when cells are disrupted by homogenization (27), this would
suggest that RhoA proteins do not behave as freely diffusable proteins
under conditions in which the cellular architecture is maintained, as
is the case in permeabilized cells. In addition to RhoA, Rac proteins
were also retained in the permeabilized cells (data not shown).
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ARF and Rho Are Translocated to the Membrane Fraction on fMLP Stimulation-- ARF and RhoA are found predominantly in the postnuclear supernatant where they are present in a GDP-bound state. Nucleotide exchange and therefore activation results in the stable interaction of ARF and Rho with membranes (41, 42). The data presented indicate that ARF and Rho are required for fMLP stimulation of PLD in a reconstituted system. To verify that ARF and Rho proteins are activated in intact cells, the translocation of ARF and Rho proteins to the membranes was examined upon stimulation with fMLP. Intact neutrophils were pretreated with cytochalasin B and incubated in the presence or absence of fMLP for 1 and 10 min. At the end of the incubation, the cells were recovered and the membrane fractions prepared. The samples were run on SDS-PAGE, blotted to polyvinylidene difluoride, and probed with the appropriate antibodies. Both ARF and Rho translocated to the membrane fraction within a minute of stimulation. (Maximal activation of PLD activity by fMLP in intact cells occurs at 1 min (28, 43).) ARF proteins remained membrane-associated even after 10 min, but the association of Rho was diminished at 10 min (see Fig. 6A).
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Potential Role of PI 3-Kinase in PLD Activation-- Although we have established that ARF and Rho proteins are involved in the stimulation of PLD via the fMLP receptor, it remains to be ascertained how the monomeric GTPases are activated by the fMLP receptor. Activation of Gi proteins is clearly an intermediary step as observed by the inhibition with pertussis toxin treatment. A potential candidate that could couple Gi proteins to ARF and Rho activation is PIP3, the product of PI 3-kinase activation. Wortmannin, a direct inhibitor of PI 3-kinase (48, 49), has been reported to block the activation of fMLP-stimulated PLD activity in intact human neutrophils (48, 50). PIP3 could potentially recruit the ARF and Rho exchange factors to the plasma membrane via their PH (pleckstrin homology) domains (34, 52-55). The ARF exchange factors, ARNO, cytohesin, and GRP1, all contain a PH domain that can bind phosphoinositides and a SEC7 domain that is responsible for the exchange of GDP for GTP (34, 52-54). The guanine nucleotide exchange factors for Rho proteins all contain a Dbl homology domain responsible for GDP-GTP exchange and also a PH domain (55). To test for this possibility, we examined whether wortmannin could inhibit the translocation of ARF and Rho proteins in fMLP-stimulated intact cells. Contrary to our expectations, wortmannin did not block the translocation of ARF proteins to membranes and marginally inhibited RhoA translocation (Fig. 6A). We observed consistently that wortmannin alone increased the amount of Rho proteins associated in the membranes slightly.
To check that wortmannin was effective in the inhibition of PLD activity in intact cells in our hands, we confirmed that wortmannin inhibited the fMLP-stimulated PLD activation in intact cells as reported previously (48). However, only a partial inhibition by wortmannin was observed in acutely permeabilized cells examined under conditions in which cytosolic proteins are still present during the time of stimulation (data not shown). We next examined the reconstitution of PLD activity by myr-ARF1 in the cytosol-depleted neutrophils, and this was not inhibited by wortmannin (data not shown). The lack of involvement of PI 3-kinase in the activation of fMLP-stimulated PLD in the cytosol-depleted cells is also in line with our recent observations (56). We have shown that fMLP is unable to stimulate PI 3-kinase activation in cytosol-depleted cells because of the loss of PI 3-kinase (PMA Translocated ARF and RhoA to Membranes-- To demonstrate further the lack of involvement of PI 3-kinase in the recruitment of ARF and RhoA to membranes, we took advantage of the observation that PMA stimulation of PLD activity (1-4) is not inhibited by wortmannin (57). The ability of PMA to stimulate PLD activity could be caused by the activation of protein kinase C isozymes. Alternatively, PMA could cause translocation of ARF proteins to membranes as reported previously for rat basophilic leukemic mast cells (58). In human neutrophils, we observe that PMA recruits both ARF and Rho to membranes (Fig. 7A) under conditions in which no PI 3-kinase activation occurs.
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DISCUSSION |
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Activation of many G protein-coupled receptors and receptor tyrosine kinases in intact cells leads to stimulation of PLD activity, but the intervening components that lead to PLD activation remain to be delineated. Mammalian PLD has three known members, hPLD1a, hPLD1b, and PLD2; hPLD1a and hPLD1b differ by lacking a 38-amino acid insert, and both are regulated by ARF and Rho proteins. PLD2 is constitutively active. In HL60 cells, a cell line that is related to neutrophils, the predominant isoform that is expressed is hPLD1 (59).
ARF and Rho Proteins Are Involved in the Coupling of the fMLP
Receptor to PLD via a Heterotrimeric G Protein in Human
Neutrophils--
In this study, we present results demonstrating that
ARF and Rho proteins are involved in PLD regulation in fMLP-stimulated human neutrophils. Extended permeabilization depleted the cells of ARF
proteins but not RhoA, and this compromised the ability of fMLP to
stimulate PLD activity (Fig. 3). Myr-ARF1 (but not non-myr-ARF1)
restores fMLP-dependent PLD activity in these
cytosol-depleted cells (Fig. 1A). This result was surprising
initially because we had shown previously that nonmyristoylated ARF
proteins were equally competent at activating PLD when GTPS was used
as a stimulus. Higher concentrations of nonmyristoylated ARF are
required to elicit a response similar to that of myristoylated protein
in GTP
S-dependent PLD activation (5, 6). Myristoylation
is clearly required for efficient nucleotide exchange at physiological concentrations of Mg2+ when the receptor-driven system is
used (32, 34, 60). When GTP
S is used, the exchange factors must be
bypassed, and the limited exchange that occurs on nonmyristoylated ARF
could be the result of basal turnover. The recently described guanine
nucleotide exchange factor for ARF (ARNO) has been shown to promote
exchange rapidly on myristoylated proteins but not nonmyristoylated ARF (34, 60).
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ACKNOWLEDGEMENT |
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We thank Dr. Anne Ridley for the gift of C3 transferase.
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FOOTNOTES |
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* This work was supported by the Medical Research Council and the Wellcome Trust.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 Physiology,
Rockefeller Bldg., University College London, University St., London
WC1E 6JJ, U. K. Tel.: 44-71-209-6084/6259; Fax: 44-71-387-6368; E-mail: ucgbsxc{at}ucl.ac.uk.
1
The abbreviations used are: PLD, phospholipase
D; PC, phosphatidylcholine; PA, phosphatidic acid; GTPS, guanosine
5'-O-3-(thio)triphosphate; ARF, ADP-ribosylation factor; PI
3-kinase, phosphoinositide 3-kinase; fMLP,
formylmethionylleucylphenylalanine; PI 3-kinase, phosphoinositide 3-kinase; PIPES, 1,4-piperazinediethanesulfonic acid; PEt,
phosphatidylethanol; PAGE, polyacrylamide gel electrophoresis;
PIP3, phosphoinositide trisphosphate; PMA, phorbol
12-myristate 13-acetate.
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REFERENCES |
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