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• W2072417697 abstract "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. 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. Phospholipase D (PLD) 1The abbreviations used are: PLD, phospholipase D; PC, phosphatidylcholine; PA, phosphatidic acid; GTPγS, 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.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) (1Singer W.D. Brown H.A. Sternweis P.C. Annu. Rev. Biochem. 1997; 66: 475-509Crossref PubMed Scopus (350) Google Scholar, 2Exton J.H. J. Biol. Chem. 1997; 272: 15579-15582Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 3Exton J.H. Physiol. Rev. 1997; 77: 303-320Crossref PubMed Scopus (388) Google Scholar, 4Cockcroft S. Prog. Lipid Res. 1997; 35: 345-370Crossref Scopus (56) Google Scholar). 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, GTPγS. Through the use of reconstitution studies utilizing HL60 membranes (5Brown H.A. Gutowski S. Moomaw C.R. Slaughter C. Sternweis P.C. Cell. 1993; 75: 1137-1144Abstract Full Text PDF PubMed Scopus (823) Google Scholar) or cytosol-depleted cells (6Cockcroft S. Thomas G.M.H. Fensome A. Geny B. Cunningham E. Gout I. Hiles I. Totty N.F. Troung O. Hsuan J.J. Science. 1994; 263: 523-526Crossref PubMed Scopus (586) Google Scholar), ARF1 and ARF3 have been identified as activators of GTPγS-stimulated PLD activity. The requirement for Rho in PLD activation was identified separately because RhoGDI, which extracts Rho from membranes, inhibited neutrophil PLD (7Bowman E.P. Uhlinger D.J. Lambeth J.D. J. Biol. Chem. 1993; 268: 21509-21512Abstract Full Text PDF PubMed Google Scholar). Subsequent studies utilizing purified protein demonstrated that Rho proteins stimulated PLD directly (8Malcolm K.C. Ross A.H. Qiu R.-G. Symons M. Exton J.H. J. Biol. Chem. 1994; 269: 25951-25954Abstract Full Text PDF PubMed Google Scholar, 9Siddiqi A.R. Smith J.L. Ross A.H. Qiu R.-G. Symons M. Exton J.H. J. Biol. Chem. 1995; 270: 8466-8473Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 10Hammond S.M. Jenco J.M. Nakashima S. Cadwallader K. Gu Q. Cook S. Nozawa Y. Prestwich G.D. Frohman M.A. Morris A.J. J. Biol. Chem. 1997; 272: 3860-3868Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar). 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 (11Lambeth J.D. Kwak J.-Y. Bowman E.P. Perry D. Uhlinger D.J. Lopez I. J. Biol. Chem. 1995; 270: 2431-2434Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 12Bourgoin S. Harbour D. Desmarais Y. Takai Y. Beaulieu A. J. Biol. Chem. 1995; 270: 3172-3178Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) and Rho in stimulating PLD (13Kwak J.-Y. Lopez I. Uhlinger D.J. Ryu S.H. Lambeth J.D. J. Biol. Chem. 1995; 270: 27093-27098Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Similarly, both G proteins also act synergistically with protein kinase Cα to activate PLD. Both synergistic (14Singer W.D. Brown H.A. Bokoch G.M. Sternweis P.C. J. Biol. Chem. 1995; 270: 14944-14950Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 15Kuribara H. Tago K. Yokozeki T. Sasaki T. Takai Y. Morii N. Narumiya S. Katada T. Kanaho Y. J. Biol. Chem. 1995; 270: 25667-25671Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) and additive (16Martin A. Brown F.D. Hodgkin M.N. Bradwell A.J. Cook S.J. Hart M. Wakelam M.J.O. J. Biol. Chem. 1996; 271: 17397-17403Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 17Abousalham A. Liossis C. O'Brien L. Brindley D.N. J. Biol. Chem. 1997; 272: 1069-1075Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) responses have been reported for the simultaneous addition of ARF and Rho. A single PLD that is synergistically responsive to ARF/Rho/protein kinase Cα has been cloned and is now referred to as hPLD1 (10Hammond S.M. Jenco J.M. Nakashima S. Cadwallader K. Gu Q. Cook S. Nozawa Y. Prestwich G.D. Frohman M.A. Morris A.J. J. Biol. Chem. 1997; 272: 3860-3868Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar). The roles of ARF and Rho are well documented with regard to PLD activation by GTPγS, 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 (18Yeo E.-J. Kazlauskas A. Exton J.H. J. Biol. Chem. 1994; 269: 27823-27826Abstract Full Text PDF PubMed Google Scholar, 19Yeo E.-J. Exton J.H. J. Biol. Chem. 1995; 270: 3980-3988Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 20Lee Y.H. Kim H.S. Pai J.-K. Ryu S.H. Suh P. J. Biol. Chem. 1994; 269: 26842-26847Abstract Full Text PDF PubMed Google Scholar). However, in other cases, including fMLP-stimulated neutrophils (21Kanaho Y. Nishida A. Nozawa Y. J. Immunol. 1992; 149: 622-628PubMed Google Scholar), 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 (22Schmidt M. Rumenapp U. Bienek C. Keller J. von Eichel-Streiber C. Jakobs K.H. J. Biol. Chem. 1996; 271: 2422-2426Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), and in Rat 1 fibroblasts stimulated with lyso-PA and endothelin (23Malcolm K.C. Elliott C.M. Exton J.H. J. Biol. Chem. 1996; 271: 13135-13139Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Insulin-mediated PLD activation is dependent on ARF and Rho proteins, and an involvement of PI 3-kinase has been implicated (24Shome K. Vasudevan C. Romero G. Curr. Biol. 1997; 7: 387-396Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 25Karnam P. Standaert M.L. Galloway L. Farese R.V. J. Biol. Chem. 1997; 272: 6136-6140Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). 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 (26Stutchfield J. Cockcroft S. Biochem. J. 1993; 293: 649-655Crossref PubMed Scopus (122) Google Scholar). 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. 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 (6Cockcroft S. Thomas G.M.H. Fensome A. Geny B. Cunningham E. Gout I. Hiles I. Totty N.F. Troung O. Hsuan J.J. Science. 1994; 263: 523-526Crossref PubMed Scopus (586) Google Scholar). Anti-RhoA antibodies were obtained from Santa Cruz Biotechnology Ltd. The anti-ARF antibodies used have been described previously (27Whatmore J. Morgan C.P. Cunningham E. Collison K.S. Willison K.R. Cockcroft S. Biochem. J. 1996; 320: 785-794Crossref PubMed Scopus (66) Google Scholar). All other reagents were obtained as described previously (26Stutchfield J. Cockcroft S. Biochem. J. 1993; 293: 649-655Crossref PubMed Scopus (122) Google Scholar). Neutrophils were prepared according to established procedures (28Cockcroft S. Biochim. Biophys. Acta. 1984; 795: 37-46Crossref PubMed Scopus (100) Google Scholar). 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. 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. 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. Neutrophils were suspended in HEPES buffer and pretreated with 5 μmcytochalasin 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 (29Geny B. Cockcroft S. Biochem. J. 1992; 284: 531-538Crossref PubMed Scopus (83) Google Scholar) was added for localization of lipids after separation by TLC. Permeabilization of cells to deplete the cytosol and reconstitution of PLD activity were essentially carried out as described previously (6Cockcroft S. Thomas G.M.H. Fensome A. Geny B. Cunningham E. Gout I. Hiles I. Totty N.F. Troung O. Hsuan J.J. Science. 1994; 263: 523-526Crossref PubMed Scopus (586) Google Scholar, 30Geny B. Fensome A. Cockcroft S. Eur. J. Biochem. 1993; 215: 389-396Crossref PubMed Scopus (35) Google Scholar). 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. Intact labeled neutrophils were pretreated with 500 ng/ml pertussis toxin for 2 h at 37 °C as described previously (31Cockcroft S. Stutchfield J. FEBS Lett. 1989; 245: 25-29Crossref PubMed Scopus (84) Google Scholar). Cells were then processed as required. Cytosol-depleted neutrophils were incubated with 1 μg/ml C3 toxin in the presence of 0.5 mmNAD for 10 min at 37 °C. Intact cells were incubated at 37 °C for 10 min in the presence of 100 nmwortmannin. 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 μmcytochalasin 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 (27Whatmore J. Morgan C.P. Cunningham E. Collison K.S. Willison K.R. Cockcroft S. Biochem. J. 1996; 320: 785-794Crossref PubMed Scopus (66) Google Scholar). 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 mmMgCl2, and 1 μm Ca2+. 100 μm GTP, 1 μm fMLP, or 10 μmGTPγS was present as indicated. After incubation for 10 min at 37 °C, the samples were processed as described above for intact cells. 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.1 A 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. 26Stutchfield J. Cockcroft S. Biochem. J. 1993; 293: 649-655Crossref PubMed Scopus (122) Google Scholar and Fig. 1 B). fMLP regains the ability to stimulate PLD activity provided that myr-ARF1 is also added to the permeabilized cells (Fig. 1 B). 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. 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 (32Franco M. Chardin P. Chabre M. Paris S. J. Biol. Chem. 1995; 270: 1337-1341Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 33Randazzo P.A. Terui T. Sturch S. Fales H.M. Ferrige A.G. Kahn R.A. J. Biol. Chem. 1995; 270: 14809-14815Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 34Chardin P. Paris S. Antonny B. Robineau S. Beraud-Dufour S. Jackson C.L. Chabre M. Nature. 1996; 384: 481-484Crossref PubMed Scopus (409) Google Scholar). 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 (6Cockcroft S. Thomas G.M.H. Fensome A. Geny B. Cunningham E. Gout I. Hiles I. Totty N.F. Troung O. Hsuan J.J. Science. 1994; 263: 523-526Crossref PubMed Scopus (586) Google Scholar) and those of others (5Brown H.A. Gutowski S. Moomaw C.R. Slaughter C. Sternweis P.C. Cell. 1993; 75: 1137-1144Abstract Full Text PDF PubMed Scopus (823) Google Scholar, 35Massenburg D. Han J. Liyanage M. Patton W.A. Rhee S.G. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11718-11722Crossref PubMed Scopus (245) Google Scholar), myristoylation is not required for GTPγS-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 GTPγS-stimulated PLD activity (6Cockcroft S. Thomas G.M.H. Fensome A. Geny B. Cunningham E. Gout I. Hiles I. Totty N.F. Troung O. Hsuan J.J. Science. 1994; 263: 523-526Crossref PubMed Scopus (586) Google Scholar). 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 GTPγS when measuredin vitro. Fig. 2 also illustrates that the level of PLD stimulated by these proteins is similar in magnitude when GTPγS is the activator. The fMLP receptor is coupled to the pertussis toxin-sensitive heterotrimeric G proteins, Gi2 and Gi3 (36Gierschik P. Sidiropoulos D. Jakobs K.H. J. Biol. Chem. 1989; 264: 21470-21473Abstract Full Text PDF PubMed Google Scholar), and βγ subunits are the direct regulators of phospholipase Cβ2 and PI 3-kinase (γ isoform) (37Camps M. Carozzi A. Schnabel P. Scheer A. Parker P.J. Gierschik P. Nature. 1992; 360: 684-686Crossref PubMed Scopus (515) Google Scholar, 38Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nurnberg B. Gierschik P. Seedorf S. Hsuan J.J. Waterfield M.D. Wetzker R. Science. 1995; 269: 690-693Crossref PubMed Scopus (641) Google Scholar). 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 (39Pai J.-K. Siegel M.I. Egan R.W. Billah M.M. J. Biol. Chem. 1988; 263: 12472-12477Abstract Full Text PDF PubMed Google Scholar, 40Xie M. Jacobs L.S. Dubyak G.R. J. Clin. Invest. 1991; 88: 45-54Crossref PubMed Scopus (55) Google Scholar), pertussis toxin pretreatment led to inhibition of the fMLP-stimulated PLD activity in intact cells (Fig. 3 A). 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. 3 B). 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. 3 C). 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.3 C). 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. Previous studies in a variety of cells have indicated that not only ARF but also Rho can activate PLD activity (1Singer W.D. Brown H.A. Sternweis P.C. Annu. Rev. Biochem. 1997; 66: 475-509Crossref PubMed Scopus (350) Google Scholar, 2Exton J.H. J. Biol. Chem. 1997; 272: 15579-15582Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 3Exton J.H. Physiol. Rev. 1997; 77: 303-320Crossref PubMed Scopus (388) Google Scholar, 4Cockcroft S. Prog. Lipid Res. 1997; 35: 345-370Crossref Scopus (56) Google Scholar). It has been reported that in HL60 cells, endogenous Rho is unlikely to play a physiological role in PLD activation (16Martin A. Brown F.D. Hodgkin M.N. Bradwell A.J. Cook S.J. Hart M. Wakelam M.J.O. J. Biol. Chem. 1996; 271: 17397-17403Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). However, these experiments were performed using GTPγS 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. 1 A). Because Rho proteins are found mainly to be cytosolic when cells are disrupted by homogenization (27Whatmore J. Morgan C.P. Cunningham E. Collison K.S. Willison K.R. Cockcroft S. Biochem. J. 1996; 320: 785-794Crossref PubMed Scopus (66) Google Scholar), 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). Despite the retention of Rho proteins, the stimulation of PLD activity by fMLP is impaired in the cytosol-depleted cells (Fig. 1 B), which would suggest that Rho proteins do not play a major role in fMLP-stimulated PLD activation. However, RhoA has been shown to be a poor activator of PLD activity by itself; but when it is present together with ARF, a synergistic activation is observed (10Hammond S.M. Jenco J.M. Nakashima S. Cadwallader K. Gu Q. Cook S. Nozawa Y. Prestwich G.D. Frohman M.A. Morris A.J. J. Biol. Chem. 1997; 272: 3860-3868Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar). Thus, it was still possible that RhoA could be a contributory factor in the myr-ARF1-reconstituted response stimulated by fMLP in the permeabilized cells. C3 transferase ADP-ribosylates Rho proteins, thereby inactivating them. To investigate a role for Rho proteins, permeabilized cells were treated with C3 transferase. The myr-ARF1-restored fMLP-dependent PLD activity was partially reduced when Rho proteins were inactivated (Fig. 5). This experiment uncovers the Rho component to the PLD response observed in the presence of myr-ARF. Thus ARF and RhoA proteins act synergistically to regulate fMLP-stimulated PLD activity. ARF can stimulate PLD activity in the absence of Rho (C3-treated cells), whereas Rho requires the presence of ARF. 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 (41Regazzi R. Ullrich S. Khan R.A. Wollheim C.B. Biochem. J. 1991; 275: 639-644Crossref PubMed Scopus (35) Google Scholar, 42Walker M.W. Bobak D.A. Tsai S. Moss J. Vaughan M. J. Biol. Chem. 1992; 267: 3230-3235Abstract Full Text PDF PubMed Google Scholar). 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 (28Cockcroft S. Biochim. Biophys. Acta. 1984; 795: 37-46Crossref PubMed Scopus (100) Google Scholar, 43Billah M.M. Eckel S. Mullmann T.J. Egan R.W. Siegel M.I. J. Biol. Chem. 1989; 264: 17069-17077Abstract Full Text PDF PubMed Google Scholar).) ARF proteins remained membrane-associated even after 10 min, but the association of Rho was diminished at 10 min (see Fig.6 A). PLD activation by fMLP in intact neutrophils is enhanced greatly by pretreatment with cytochalasin B (43Billah M.M. Eckel S. Mullmann T.J. Egan R.W. Siegel M.I. J. Biol. Chem. 1989; 264: 17069-17077Abstract Full Text PDF PubMed Google Scholar). Several other neutrophil responses, including degranulation, respiratory burst, phospholipase A2 activation, and protein kinase C translocation, are also potentiated greatly by cytochalasin B (44Bennett J.P. Cockcroft S. Gomperts B.D. Biochim. Biophys. Acta. 1980; 601: 584-591Crossref PubMed Scopus (43) Google Scholar, 45Horn W. Karnovsky M.L. Biochem. Biophys. Res. Commun. 1986; 139: 1169-1175Crossref PubMed Scopus (23) Google Scholar, 46Rider L.G. Niedel J.E. J. Biol. Chem. 1987; 262: 5603-5608Abstract Full Text PDF PubMed Google Scholar, 47Meade C.J. Turner G.A. Bateman P.E. Biochem. J. 1986; 238: 425-436Crossref PubMed Scopus (51) Google Scholar). The priming of human neutrophils by cytochalasin B can also be mimicked by other physiological agonists, e.g. low concentrations of C5a, fMLP, or tumor necrosis factor, and is therefore of physiological relevance. To examine whether translocation of ARF and Rho was dependent on priming, the cells were stimulated with fMLP for 1 min with or without cytochalasin B pretreatment. The translocation of ARF was entirely dependent on cytochalasin B pretreatment, whereas some RhoA translocation could be observed in its absence. Cytochalasin B enhanced RhoA translocation (Fig. 6 B). These data indicate that similar to the activation of PLD by fMLP, the translocation of the two regulators, ARF and RhoA, is more efficient in primed cells. Fig. 6 C illustrates that in permeabilized cells, translocation of ARF can be observed without cytochalasin B treatment. Both fMLP a" @default.
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• W2072417697 title "ADP-ribosylation Factor and Rho Proteins Mediate fMLP-dependent Activation of Phospholipase D in Human Neutrophils" @default.
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