Article |
Address correspondence to Dr. Nicolas Vitale, Centre National de la Recherche Scientifique, Unité Propre de Recherche 2356, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France. Tel.: 33-388-45-67-12. Fax: 33-388-60-16-64. E-mail: vitalen{at}neurochem.u-strasbg.fr
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Abstract |
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Key Words: ARF6; exocytosis; PC12 cells; phospholipase D; secretory granule
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Introduction |
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ADP ribosylation factor (ARF)* GTPases play important roles in membrane trafficking events (Moss and Vaughan, 1998; Donaldson and Jackson, 2000). ARF6, the most structurally divergent member of the family (Chavrier and Goud, 1999), is found exclusively at the plasma and/or endosomal membranes in various cells types (Peters et al., 1995; Cavenagh et al., 1996). The protein has been proposed to play a role in cell motility (Song et al., 1998; Santy and Casanova, 2001), vesicle recycling at the plasma membrane (Radhakrishna and Donaldson, 1997; D'Souza-Schorey et al., 1998), Fc-mediated phagocytosis (Zhang et al., 1999a) and insulin-regulated secretion (Yang and Muekler, 1999), and Glut-4 translocation (Millar et al., 1999). Taken together, these findings suggest that ARF6 is an ubiquitous regulator of vesicle transport at the plasma membrane.
ARF6 is unique among the six mammalian ARFs in its ability to stimulate cortical actin rearrangements (Radhakrishna et al., 1999; Boshans et al., 2000). Because ARF6 also activates phospholipase D (PLD; Massenburg et al., 1994), a lipid-modifying enzyme recently described as a key factor for exocytosis in neurons and neuroendocrine cells (Humeau et al., 2001; Vitale et al., 2001), it would be attractive to hypothesize that ARF6 functions by coordinating reorganization of the actin cytoskeleton with the lipid modifications required for neuronal exocytosis. We demonstrate here that ARF6 is the sole member of the ARF family able to exert direct control on the exocytotic machinery. We show that ARF6 is in its inactive GDP-bound state when associated with secretory granules but then switches to its active GTP-bound state at the plasma membrane upon cell stimulation. Our results indicate that the guanine nucleotide exchange factor, ARF nucleotide binding site opener (ARNO), is a regulator of ARF6 functions in this setting. Finally, we show that ARF6 participates in the regulation of secretion, most likely through the direct activation of PLD1 at the site of exocytosis.
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Results |
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PLD1 is a molecular partner of ARF6 in the exocytotic machinery
ARF proteins including ARF6 are established activators of PLD. In neurons and neuroendocrine cells, we previously described that a plasma membraneassociated PLD1 plays an important function in a late postdocking step of exocytosis (Humeau et al., 2001; Vitale et al., 2001). In order to test whether PLD1 might be an effector of ARF6 in the exocytotic process, we generated a mutant of ARF6 unable to stimulate PLD activity, namely ARF6(N48I), based on analogy with a similar mutant generated for ARF1 by Jones et al. (1999). As illustrated in Fig. 8 A, GTPS binding to recombinant myrARF6(N48I) was indistinguishable from that of WT myrARF6. Dissociation of GDPßS from myrARF6(N48I) and myrARF6 was also very similar (unpublished data). ARNO stimulated GTP
S binding to myrARF6(N48I) and to myrARF6 to a similar extent (Fig. 8 A). Additionally, GIT1, a GAP protein for ARF6 (Vitale et al., 2000a), stimulated GTP hydrolysis on myrARF6(N48I) (Fig. 8 B). Together, these experiments indicate that the N48I mutation does not affect the activation/inactivation cycle of ARF6 and its regulation by endogenous accessory proteins. We next tested the ability of myrARF6(N48I) to stimulate known ARF effectors. In presence of GTP, cholera toxin ADP-ribosyltransferase activity was stimulated similarly by myrARF6(N48I) and myrARF6 (Fig. 8 C). However, as intended, the mutation N48I abolished almost completely the ability of ARF6 to stimulate PLD1 (Fig. 8 D), making ARF6(N48I) an ideal tool to test the involvement of the ARF6-PLD pathway in a cellular function.
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Discussion |
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The granule-associated ARF6 is activated by ARNO at the plasma membrane upon PC12 cell stimulation
We previously compared the distribution of ARF6 in subcellular fractions obtained from resting and stimulated chromaffin cells and found that ARF6 apparently dissociates from secretory granules and binds to the plasma membrane upon cell stimulation (Caumont et al., 1998). Similarly, we found here that overexpressed ARF6 proteins translocate from secretory granule to plasma membranecontaining fractions in stimulated PC12 cells. Immunofluorescence and ultrastructural analysis confirm the association of ARF6 with secretory granules and its relocalization at the plasma membrane upon cell stimulation. A reasonable explanation is that the plasma membrane location of ARF6 results from the docking of secretory granules to sites of exocytosis. The observation that ARF6 remains attached to the plasma membrane upon subcellular fractionation of stimulated cells may reflect the direct interaction of ARF6 with a plasma membranebound protein. The nucleotide exchange factor ARNO is an attractive candidate to be the plasma membrane-bound partner of ARF6 (Frank et al., 1998b). Indeed, ARNO colocalizes with ARF6 in stimulated PC12 cells. Furthermore, we show here that ARF6 is in its inactive GDP-bound state when associated with secretory granules, but becomes activated when recruited to the plasma membrane. Conversely, overexpression of ARNO increases GTP binding to ARF6 at the plasma membrane whereas a catalytically inactive ARNO mutant dramatically inhibits it. It is interesting to note that the constitutively GDP-bound ARF6(T27N) could also be found associated to the plasma membrane in stimulated cells, suggesting that GDP/GTP exchange is not required for the ARF6 association with plasma membrane. This observation is in line with the idea that granules bring ARF6 to the plasma membrane, making the protein transiently available for ARNO upon docking at the exocytotic sites.
GDP-bound ARFs interact weakly with membranes through hydrophobic interactions of the myristate and electrostatic interactions of cationic residues with anionic lipids (Antonny et al., 1997). Binding of GTP releases the NH2-terminal helix from the protein core allowing a stronger interaction with membranes through the NH2-terminal hydrophobic residues (Antonny et al., 1997). Using purified chromaffin granules, we previously described that ARF6 coimmunoprecipitates with trimeric G protein ß subunits (Galas et al., 1997), suggesting that ß subunits could serve as anchors for GDP-bound ARF6 in the secretory granule membrane. An appealing speculation is that the ARF6/ARNO interaction is in some way under the control of the granule-associated G protein ß
subunits. Thus, activation of ARF6 would require the docking of granules to appropriate ARNO-containing domains on the plasma membrane and concomitant changes in the interactions of ARF6 with ß
subunits. In favor of this idea, several recent reports describe ß
subunit regulation of the exocytotic fusion machinery, downstream of calcium entry, in secretory cells (Pinxteren et al., 1998; Gensse et al., 2000) and as well as in neuronal presynaptic terminals (Blackmer et al., 2001).
ARNO-induced ARF6 activation is critical for exocytosis in PC12 cells
We previously reported that a synthetic peptide, corresponding in sequence to the myristoylated NH2-terminal domain of ARF6 inhibited, in a dose-dependent manner, calcium-evoked catecholamine secretion from permeabilized chromaffin cells (Galas et al., 1997; Caumont et al., 1998). More recently, we described the presence of ARNO at the plasma membrane in chromaffin cells and demonstrated that overexpression of WT ARNO enhanced secretagogue-evoked secretion from PC12 cells whereas the catalytically inactive ARNO(E156K) mutant inhibited it (Caumont et al., 2000). These results suggested, but did not prove, that an ARNO-regulated ARF6 plays an important role in exocytosis. Indeed, ARNO is exchange factor for both ARF1 and ARF6, and both ARFs can be detected at the plasma membrane in chromaffin cells (Galas et al., 1997). Moreover, although ARF peptides have been described to block ARF activities in various cellular processes (Barr and Huttner, 1996; Le Stunff et al., 2000; Mukherjee et al., 2000), their specificity has also been questioned (Fensome et al., 1994). Using direct means, we demonstrate here for the first time that ARF6 is the sole member of the ARF family involved in the molecular pathway underlying calcium-regulated exocytosis. Although the involvement of ARF6 in peripheral vesicle trafficking has been reported previously in various cell types, this specific requirement for ARF6 in secretion is surprising and may be linked to its granular location and its specific activation at granule targeting sites.
PLD1 is a major downstream effector of ARF6 in the exocytotic process
One of the proposed functions of ARF6 is to mediate actin rearrangements (Chavrier and Goud, 1999). Activation of ARF6 can also lead to significant changes in the lipid composition of specific membrane domains, as the protein is known to directly activate two lipid-modifying enzymes, PLD and phosphatidylinositol-4-phosphate 5-kinase (Honda et al., 1999). Thus, activation of ARF6 at the exocytotic sites may provide a mechanism whereby secretory cells can engineer a localized remodeling of the actin cytoskeleton with phospholipid modifications, so that the fusion machinery can negotiate the exocytotic event. Using rhodamine-conjugated phalloidin to visualize actin filaments, we have investigated whether ARF6 mutants affect the depolymerization of cortical actin observed in stimulated cells. However, we could not correlate the strong inhibition of secretion induced by ARF6(T27N) to a stabilization of the cortical actin barrier (unpublished data). Accordingly, we found that the secretagogue-dependent movement of secretory granules to the cell periphery was similar in control and in ARF6(T27N)-expressing cells. Thus, ARF6 is probably not involved in the partial depolymerization of the cortical actin cytoskeleton that enables recruitment of the reserve pool of secretory granules to the plasma membrane. This finding is in line with the idea that ARF6 is activated after docking of secretory granules to the sites of exocytosis. Whether ARF6 mediates some other subtle modifications of the actin cytoskeleton functionally required in late stages of the exocytotic machinery cannot be excluded and will be an interesting future issue.
We recently demonstrated that PLD1 constitutes a critical factor for regulated exocytosis in neuroendocrine cells (Vitale et al., 2001) and neurons (Humeau et al., 2001), operating subsequent to the cytoskeletal-mediated recruitment of secretory granules to exocytotic sites (Vitale et al., 2001). To directly probe the idea that ARF6 is the upstream activator of the plasma membrane-bound PLD1 in the exocytotic pathway, we generated a novel ARF6(N48I) mutant that specifically lost its ability to stimulate PLD1, based on analogy with a similar ARF1 mutant defective in PLD activation but still able to activate phosphatidylinositol-4-phosphate 5-kinase (Jones et al., 1999; Skippen et al., 2002). Expression of ARF6(N48I) inhibited secretagogue-evoked GH secretion to an extent similar to the constitutively inactive ARF6(T27N), suggesting that the stimulation of PLD1 is the major function ARF6 undertakes in regulated exocytosis. Accordingly, ARF6 interacts directly with PLD1 at the plasma membrane in stimulated cells. Our previous observation that the introduction of specific anti-ARNO antibodies into permeabilized chromaffin cells inhibited in a similar manner catecholamine secretion and PLD activation (Caumont et al., 2000) further corroborates the ARNOARF6PLD1 cascade in the exocytotic process.
How does PLD1 relate to our current understanding of the protein machinery responsible for regulated exocytosis? PLD1 catalyzes the hydrolysis of phosphatidylcholine to produce membrane-localized phosphatidic acid (PA). An exciting speculation relates to the changes in the lipid bilayer properties created by the activation of PLD. PA is a cone-shaped lipid which promotes membrane curvature and the formation of hemifusion intermediates required for the fusion of two membranes (Monck and Fernandez, 1994). Thus, the predicted effect of a local elevation of PA due to the activation of PLD1 at exocytotic sites would be to promote membrane bending, particularly in the presence of calcium, thereby facilitating membrane breakdown and subsequent formation of the exocytotic fusion pore. Accordingly, data obtained by amperometry on isolated chromaffin cells suggest that PLD1 affects the kinetics of the initial fusion event and/or the rate of the fusion pore opening (Vitale et al., 2001). Similarly, electrophysiological analysis suggest that the role played by PLD1 in neurotransmission is related to the fusogenic status of the presynaptic release sites (Humeau et al., 2001). Hence, the ARF6PLD1 complex may provide to the protein scaffold pulling membrane together the lipid counterparts required to open and/or expand the fusion pore.
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Materials and methods |
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The ARF and ARNO constructs, provided by J.E. Casanova (University of Virginia Health System, Charlottesville, VA), were described previously (Frank et al., 1998a; Morinaga et al., 2001). pXS-ARF6(N48I) and pXS-ARF6(N48I-Q67L) were generated by site-directed mutagenesis with a Quickchange mutagenesis kit from Stratagene (Vitale et al., 2000a).
Recombinant myrARF6 and myrARF6(N48I) were expressed and purified as described previously (Vitale et al., 2000b). Recombinant ARNO was expressed as described (Pacheco-Rodriguez et al., 1998).
Culture, transfection, and assay of GH release from PC12 cells
PC12 cells were grown in DME supplemented with glucose (4,500 mg/L) and containing 30 mM NaHCO3, 5% fetal bovine serum, 10% horse serum and 100 U/ml penicillin/streptomycin. Mammalian expression vectors were introduced into PC12 cells together with the GH plasmid pXGH5 (6-well dishes, 80% confluent, 0.4 µg/well of each plasmid) using GenePorter (Gene Therapy Systems) according to the manufacturer's instruction. After 5 h of incubation at 37°C, 2 ml of culture medium containing fetal bovine serum, horse serum, and antibiotics was added.
GH release experiments were performed 48 h after transfection. PC12 cells were washed twice with Locke's solution and then incubated for 10 min with calcium-free Locke's solution (basal release) or stimulated with an elevated K+ solution (Locke's containing 59 mM KCl and 85 mM NaCl). The supernatant was collected and the cells harvested by scraping in 10 mM phosphate buffered saline. The amounts of GH secreted into the medium and retained in the cells were measured using a radioimmunoassay kit (Nichols Institute). The amount of GH secretion is expressed as a percentage of total GH present in the cells before stimulation.
Distribution and nucleotide status of ARF6 in PC12 cell subcellular fractions
PC12 cells grown in 100-mm plates were transfected with various plasmids (for each 10 µg per plate) using GenePorter. 36 h after transfection, cells were incubated in 2 ml/plate phosphate- and serum-free DME medium, supplemented with 25 mM Hepes, pH 7.2, 2 mM pyruvate and 175 µci of [32P]orthophosphate for 24 h. PC12 cells were washed twice with Locke's solution and then incubated for 15 min with Locke's solution (basal release) or stimulated with an elevated K+ solution. Medium was removed and cells immediately scrapped in 1 ml of sucrose 0.32 M (20 mM Tris, pH 8.0). Cells were broken in a Dounce homogenizer and centrifuged at 800 g for 15 min. The supernatant was further centrifuged at 20,000 g for 20 min. The resulting supernatant was further centrifuged for 60 min at 100,000 g to obtain the cytosol (supernatant) and microsomes (pellet enriched in endosomes). The 20,000 g pellet containing the crude membrane fraction was resuspended in sucrose 0.32 M (20 mM Tris, pH 8.0), layered on a cushion sucrose density gradient (sucrose 11.6 M, 20 mM Tris, pH 8.0) and centrifuged for 90 min at 100,000 g to separate the plasma membrane from secretory granules (Caumont et al., 2000). The upper fractions containing SNAP-25 (plasma membrane marker) and the pellet containing GH and dopamineß-hydroxylase (secretory granule markers) were collected and resuspended in buffer A (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM MgCl2, 1% Triton X-100, 0.05% cholate, 0.005% SDS, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 1 mM NaF, 1 mM vanadate, 0.32 M sucrose). To immunoprecipitate ARF6 proteins, 5 µl anti-HA antiserum and 10 µl of protein A Sepharose beads were added to 500 µl of cytosol, plasma membrane, and secretory granule fractions. Immunoprecipitation was performed for 3 h at 4°C, followed by extensive washing of the protein A beads with ice cold buffer B (50 mM Hepes, pH 7.4, 0.5 M NaCl, 5 mM MgCl2, 0.1% Triton X-100, 0.05% cholate, 0.005% SDS). Elution was carried out in Laemmli buffer for samples subjected to SDS-PAGE and in 40 µl of 2 M formic acid (heated at 70°C for 3 min) for samples analyzed by thin layer chromatography (Vitale et al., 2000b).
Immunoblotting, immunofluorescence, and confocal microscopy
One-dimensional SDS gel electrophoresis was performed on 10% acrylamide gels in Tris-Glycine buffer. The proteins were transferred to nitrocellulose sheets at a constant current of 120 mA for 1 h. Blots were developed using secondary antibodies coupled to horseradish peroxidase (Amersham Biosciences) and the immunoreactive bands detected using the ECL system (Amersham Biosciences).
For immunocytochemistry, PC12 cells grown on poly-D-lysinecoated glass coverslips were maintained in Locke's solution or stimulated with elevated K+. The cells were then fixed for 20 min in 4% paraformaldehyde in 0.12 M sodium/phosphate, pH 7.0, and for a further 10 min in fixative containing 0.1% Triton X-100. Immunostaining was performed as described previously (Vitale et al., 2001) and stained cells were visualized using a Zeiss confocal microscope LSM 510.
Preembedding immunoelectron microscopy
PC12 cells transfected with pXS-ARF6 were stimulated with 59 mM K+ and fixed for 45 min with 3% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M sodium/phosphate, pH 7.2. Immunostaining was performed using anti-HA antibodies according to the immunogold silver labeling procedure described by Yi et al. (2001).
Molecular characterization of myrARF6(N48I)
Time-course GTPS binding assays were performed with 0.5 µg of myrARF6 or myrARF6(N48I) essentially as described (Vitale et al., 1997). In the presence or absence of GIT1, provided by R.T. Premont (Duke University Medical Center, Durham, NC), GTPase activity of 0.5 µg of myrARF6 or myrARF6(N48I) was measured for 10 min at 30°C as described previously (Vitale et al., 2000b). ARF stimulation of cholera toxin-catalyzed ADP-ribosylagmatine formation was performed with 0.5 µg of myrARF6 or myrARF6(N48I) as described (Vitale et al., 1997). PLD activity assays were carried out using the in vitro head-group release assay for a 30-min time period as previously described (Zhang et al., 1999b). Recombinant WT and mutant ARF6 were loaded using 50 µM GTP
S as previously described (Frohman et al., 2000) and were used at 0.5 µM (1 µg/ 100 µl reaction). Assay blanks were subtracted. The source of PLD1 protein was a tet-regulated stable CHO cell line that had been induced for 24 h in a 35-mm dish before harvesting and sonication. The sonicated lysate was centrifuged for 30 min at 4°C and 20,000 g to pellet the PLD1, and the supernatant containing the Rho and ARF small GTP proteins removed. 100 µl of PBS containing protease inhibitors was added to the pellet which was then sonicated briefly to evenly resuspend it. 1 µl of the resulting PLD1 source was used for each assay sample.
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Footnotes |
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Acknowledgments |
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This work was supported by Association de la Recherche sur le Cancer ARC (Grant 5802 to M.F. Bader), and by grants from the National Institutes of Health to M.A. Frohman. We acknowledge the confocal microscopy facilities of IFR 37.
Submitted: 6 March 2002
Revised: 3 September 2002
Accepted: 6 September 2002
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References |
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