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• W2010454617 abstract "RalA and RalB constitute a family of highly similar Ras-related GTPases widely distributed in different tissues. Recently, active forms of Ral proteins have been shown to bind to the exocyst complex, implicating them in the regulation of cellular secretion. Since RalA is present on the plasma membrane in neuroendocrine chromaffin and PC12 cells, we investigated the potential role of RalA in calcium-regulated exocytotic secretion. We show here that endogenous RalA is activated during exocytosis. Expression of the constitutively active RalA (G23V) mutant enhances secretagogue-evoked secretion from PC12 cells. Conversely, expression of the constitutively inactive GDP-bound RalA (G26A) or silencing of the RalA gene by RNA interference led to a strong impairment of the exocytotic response. RalA was found to co-localize with phospholipase D1 (PLD1) at the plasma membrane in PC12 cells. We demonstrate that cell stimulation triggers a direct interaction between RalA and ARF6-activated PLD1. Moreover, reduction of endogenous RalA expression level interfered with the activation of PLD1 observed in secretagogue-stimulated cells. Finally, using various RalA mutants selectively impaired in their ability to activate downstream effectors, we show that PLD1 activation is essential for the activation of secretion by GTP-loaded RalA. Together, these results provide evidence that RalA is a positive regulator of calcium-evoked exocytosis of large dense core secretory granules and suggest that stimulation of PLD1 and consequent changes in plasma membrane phospholipid composition is the major function RalA undertakes in calcium-regulated exocytosis. RalA and RalB constitute a family of highly similar Ras-related GTPases widely distributed in different tissues. Recently, active forms of Ral proteins have been shown to bind to the exocyst complex, implicating them in the regulation of cellular secretion. Since RalA is present on the plasma membrane in neuroendocrine chromaffin and PC12 cells, we investigated the potential role of RalA in calcium-regulated exocytotic secretion. We show here that endogenous RalA is activated during exocytosis. Expression of the constitutively active RalA (G23V) mutant enhances secretagogue-evoked secretion from PC12 cells. Conversely, expression of the constitutively inactive GDP-bound RalA (G26A) or silencing of the RalA gene by RNA interference led to a strong impairment of the exocytotic response. RalA was found to co-localize with phospholipase D1 (PLD1) at the plasma membrane in PC12 cells. We demonstrate that cell stimulation triggers a direct interaction between RalA and ARF6-activated PLD1. Moreover, reduction of endogenous RalA expression level interfered with the activation of PLD1 observed in secretagogue-stimulated cells. Finally, using various RalA mutants selectively impaired in their ability to activate downstream effectors, we show that PLD1 activation is essential for the activation of secretion by GTP-loaded RalA. Together, these results provide evidence that RalA is a positive regulator of calcium-evoked exocytosis of large dense core secretory granules and suggest that stimulation of PLD1 and consequent changes in plasma membrane phospholipid composition is the major function RalA undertakes in calcium-regulated exocytosis. RalA and RalB constitute a family of proteins within the Ras branch of monomeric GTPases (1Chardin P. Tavitian A. Nucleic Acids Res. 1989; 17: 4380Crossref PubMed Scopus (61) Google Scholar). They are highly similar, sharing over 85% amino acid sequence identity, and display a widespread overlapping tissue distribution (2Bhullar R.P. Yang S. Mol. Cell Biochem. 1998; 179: 49-55Crossref PubMed Scopus (4) Google Scholar). Like most Ras family GTPases, Ral proteins have been implicated in the regulation of various cell biological processes, including oncogenic transformation, endocytosis, and actin-cytoskeleton dynamics (3Feig L.A. Trends Cell Biol. 2003; 13: 419-425Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Ral proteins have the potential to be activated by many different extracellular signals. One of the best known Ral-activating pathways is via Ral-specific guanine nucleotide exchange factors that become activated by binding GTP-bound Ras in response to many types of upstream signals, including almost all tyrosine kinase receptors and several G-protein-linked receptors (3Feig L.A. Trends Cell Biol. 2003; 13: 419-425Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 4Bos J.L. EMBO J. 1998; 17: 6776-6782Crossref PubMed Scopus (288) Google Scholar, 5Reuther G.W. Der C.J. Curr. Opin. Cell. Biol. 2000; 12: 157-165Crossref PubMed Scopus (348) Google Scholar). However, Ras-independent mechanisms of Ral activation occur as well. For instance, in platelets and in fibroblasts, elevation of intracellular calcium levels directly induces Ral activation without a contribution of Ras (6Hofer F. Berdeaux R. Martin G.S. Curr. Biol. 1998; 8: 839-842Abstract Full Text Full Text PDF PubMed Google Scholar). The small GTPase Rap has been also identified as a Ral activator under some conditions (7Wolthuis R.M. Franke B. van Triest M. Bauer B. Cool R.H. Camonis J.H. Akkerman J.W. Bos J.L. Mol. Cell. Biol. 1998; 18: 2486-2491Crossref PubMed Scopus (130) Google Scholar). Ral interacts with several protein effectors through two protein-protein interaction sites. The first binds phospholipase D1 (PLD1) 1The abbreviations used are: PLD, phospholipase D; siRNA, small interference RNA; shRNA, small hairpin RNA; GH, growth hormone. via an N-terminal 11-amino acid sequence. Ral weakly stimulates PLD1 activity but operates synergistically with small GTPases of the ARF family (8Luo J.Q. Liu X. Frankel P. Rotunda T. Ramos M. Flom J. Jiang H. Feig L.A. Morris A.J. Kahn R.A. Foster D.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3632-3637Crossref PubMed Scopus (119) Google Scholar, 9Kim J.H. Lee S.D. Han J.M. Lee T.G. Kim Y. Park J.B. Lambeth J.D. Suh P.G. Ryu S.H. FEBS Lett. 1998; 430: 231-235Crossref PubMed Scopus (89) Google Scholar). The second is an effector-binding loop, which mediates interaction with Ral-binding protein 1 (RalBP1, also known as RLIP76) and filamin. RalBP1 was the first Ral effector to be identified and was originally distinguished by its GTPase-activating protein domain, which has the potential to regulate Rac and Cdc42 GTPases negatively (10Cantor S.B. Urano T. Feig L.A. Mol. Cell. Biol. 1995; 15: 4578-4584Crossref PubMed Scopus (261) Google Scholar, 11Park S.H. Weiberg R.A. Oncogene. 1995; 11: 2349-2355PubMed Google Scholar, 12Jullien-Flores V. Dorseuil O. Romero F. Letourneur F. Saragosti S. Berger R. Tavitian A. Gacon G. Camonis J.H. J. Biol. Chem. 1995; 270: 22473-22477Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Filamin is an actin cross-linking protein that mediates filopodia formation (13Ohta Y. Suzuki N. Nakamura S. Hartwig J.H. Stossel T.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2122-2128Crossref PubMed Scopus (376) Google Scholar). More recently, Ral was found associated in a GTP-dependent manner with the mammalian exocyst (14Brymora A. Valova V.A. Larsen M.R. Roufogalis B.D. Robinson P.J. J. Biol. Chem. 2001; 276: 29792-29797Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 15Moskalenko S. Henry D.O. Rosse C. Mirey G. Camonis J.H. White M.A. Nat. Cell Biol. 2002; 4: 66-72Crossref PubMed Scopus (355) Google Scholar, 16Sugihara K. Asano S. Tanaka K. Iwamatsu A. Okawa K. Ohta Y. Nat. Cell Biol. 2002; 4: 73-78Crossref PubMed Scopus (204) Google Scholar), a multiprotein complex that functions in polarized cells in membrane delivery to specific domains of the plasma membrane (17Terbush D.R. Maurice T. Roth D. Novick P. EMBO J. 1996; 15: 6483-6494Crossref PubMed Scopus (681) Google Scholar, 18Hsu S.C. Ting A.E. Hazuka C.D. Davanger S. Kenny J.W. Kee Y. Scheller R.H. Neuron. 1996; 17: 1209-1219Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 19Lipschutz J.H. Mostov K.E. Curr. Biol. 2002; 12: R212-R214Abstract Full Text Full Text PDF PubMed Google Scholar). Several evidence support the idea that Ral proteins are intimately linked to vesicular trafficking events at the plasma membrane. First, RalBP1 regulates recycling of epidermal growth factor and insulin receptors by interacting with epsin homology domain proteins involved in endocytosis (20Yamaguchi A. Urano T. Goi T. Feig L.A. J. Biol. Chem. 1997; 272: 31230-31234Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 21Ikeda M. Ishida O. Hinoi T. Kishida S. Kikuchi A. J. Biol. Chem. 1998; 273: 814-821Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 22Santolini E. Salcini A.E. Kay B.K. Yamabhai M. Di Fiore P.P. Exp. Cell Res. 1999; 253: 186-209Crossref PubMed Scopus (115) Google Scholar). RalBP1 also binds to the AP2 adaptor complex (23Jullien-Flores V. Mahe Y. Mirey G. Leprince C. Meunier-Bisceuil B. Sorkin A. Camonis J.H. J. Cell Sci. 2000; 113: 2837-2844Crossref PubMed Google Scholar), which plays a key role in clathin-mediated endocytosis. Second, Ral has been shown to participate in receptor-mediated endocytosis through a process that involves PLD (24Shen Y. Xu L. Foster D.A. Mol. Cell. Biol. 2001; 21: 595-602Crossref PubMed Scopus (186) Google Scholar). Third, activated Ral proteins have been implicated in the targeting of Golgi-derived vesicles to the basolateral membrane in epithelial cells (15Moskalenko S. Henry D.O. Rosse C. Mirey G. Camonis J.H. White M.A. Nat. Cell Biol. 2002; 4: 66-72Crossref PubMed Scopus (355) Google Scholar, 25Shipitsin M. Feig L.A. Mol. Cell. Biol. 2004; 24: 5746-5756Crossref PubMed Scopus (113) Google Scholar). This possibility arose from the discovery that two subunits of the exocyst complex are downstream binding partners of active RalA and RalB (14Brymora A. Valova V.A. Larsen M.R. Roufogalis B.D. Robinson P.J. J. Biol. Chem. 2001; 276: 29792-29797Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 15Moskalenko S. Henry D.O. Rosse C. Mirey G. Camonis J.H. White M.A. Nat. Cell Biol. 2002; 4: 66-72Crossref PubMed Scopus (355) Google Scholar, 16Sugihara K. Asano S. Tanaka K. Iwamatsu A. Okawa K. Ohta Y. Nat. Cell Biol. 2002; 4: 73-78Crossref PubMed Scopus (204) Google Scholar, 26Moskalenko S. Tong C. Rosse C. Camonis J. White M.A. J. Biol. Chem. 2003; 278: 51743-51748Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). In budding yeast, the exocyst directs targeting of secretory vesicles to sites of rapid membrane growth (17Terbush D.R. Maurice T. Roth D. Novick P. EMBO J. 1996; 15: 6483-6494Crossref PubMed Scopus (681) Google Scholar). Consistent with a function in membrane addition, the analogous mammalian complex has been found at the tight junctions of epithelial cells, where it has been implicated in basolateral secretion (15Moskalenko S. Henry D.O. Rosse C. Mirey G. Camonis J.H. White M.A. Nat. Cell Biol. 2002; 4: 66-72Crossref PubMed Scopus (355) Google Scholar, 27Grindstaff K.K. Yeaman C. Anandasabapathy N. Hsu S.C. Rodriguez-Boulan E. Scheller R.H. Nelson W.J. Cell. 1998; 93: 731-740Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar) and in axonal growth cones of developing neurons (28Hazuka C.D. Foletti D.L. Hsu S.C. Kee Y. Hopf F.W. Scheller R.H. J. Neurosci. 1999; 19: 1324-1334Crossref PubMed Google Scholar). Thus, by promoting assembly of the exocyst complex (26Moskalenko S. Tong C. Rosse C. Camonis J. White M.A. J. Biol. Chem. 2003; 278: 51743-51748Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), Ral proteins have been proposed to regulate exocyst-mediated vesicle delivery to appropriate fusion sites at the plasma membrane. Release of hormones and neurotransmitters occurs through a specialized form of exocytosis that is tightly regulated in time and space by extracellular signals and cytosolic calcium levels. The presence of Ral proteins at high levels on synaptic vesicles (29Ngsee J.K. Elferink L.A. Scheller R.H. J. Biol. Chem. 1991; 266: 2675-2680Abstract Full Text PDF PubMed Google Scholar, 30Bielinski D.F. Pyun H.Y. Linko-Stentz K. Macara I.G. Fine R.E. Biochim. Biophys. Acta. 1993; 1151: 246-256Crossref PubMed Scopus (60) Google Scholar) and secretory granules in platelets (31Mark B.L. Jilkina O. Bhullar R.P. Biochim. Biophys. Res. Commun. 1996; 225: 40-46Crossref PubMed Scopus (33) Google Scholar) has led to the speculation that Ral may play a role in calcium-regulated exocytosis. Hence, a role for Ral in a regulatory aspect of neurotransmitter release has been identified through the analysis of neuronal tissue from transgenic mice expressing a dominant inhibitory form of RalA (32Polzin A. Shipitsin M. Goi T. Feig L.A. Turner T.J. Mol. Cell. Biol. 2002; 22: 1714-1722Crossref PubMed Scopus (67) Google Scholar). Release of glutamate from isolated synaptic endings after depolarization was found normal in the transgenic mice, but protein kinase C-mediated enhancement of glutamate secretion was suppressed. This suggested the participation of Ral in some forms of synaptic plasticity linked to the recruitment of synaptic vesicles but not in neuronal exocytosis per se. In neuroendocrine PC12 cells, Ral proteins have been implicated in exocytosis by regulating the assembly of exocyst complexes (15Moskalenko S. Henry D.O. Rosse C. Mirey G. Camonis J.H. White M.A. Nat. Cell Biol. 2002; 4: 66-72Crossref PubMed Scopus (355) Google Scholar). However, a recent report by Wang et al. (33Wang L. Li G. Sugita S. J. Biol. Chem. 2004; 279: 19875-19881Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) indicates that RalA plays its exocyst-mediated function in GTP-dependent but not in calcium-dependent exocytosis. Thus, the participation and precise function of Ral proteins in calcium-regulated secretion remains unclear. In chromaffin cells, we previously reported the presence of RalA on the plasma membrane, whereas RalB resided on some intracellular vesicles that were distinct from catecholamine-containing secretory granules (34Gasman S. Chasserot-Golaz S. Popoff M.R. Aunis D. Bader M.F. J. Cell Sci. 1999; 112: 4763-4771Crossref PubMed Google Scholar). This observation led us to further explore the role of RalA in the calcium-regulated exocytotic process. In the present paper, we show that RalA is a positive regulator of dense core granule exocytosis. Moreover, we obtained a series of results indicating that ARF6-regulated PLD1, present on the plasma membrane and implicated in the late stages of exocytosis (35Vitale N. Caumont A.S. Chasserot-Golaz S. Du G. Wu S. Sciorra V.A. Morris A.J. Frohman M.A. Bader M.F. EMBO J. 2001; 20: 2424-2434Crossref PubMed Scopus (203) Google Scholar, 36Vitale N. Chasserot-Golaz S. Bailly Y. Morinaga N. Frohman M.A. Bader M.F. J. Cell Biol. 2002; 159: 79-89Crossref PubMed Scopus (103) Google Scholar), participates in the downstream pathway by which RalA promotes calcium-regulated exocytosis. Plasmids and Small Interference RNA (siRNA)—RalA wild type and variants were as previously described (15Moskalenko S. Henry D.O. Rosse C. Mirey G. Camonis J.H. White M.A. Nat. Cell Biol. 2002; 4: 66-72Crossref PubMed Scopus (355) Google Scholar) and were expressed in PC12 cells using pRK5. The sequence encoding the Ral-binding domain of RalBP1/RLIP76 (amino acids 403-499) was inserted as a BamHI-EcoRI fragment into pRK5-Myc. Recombinant HA-ARF6 proteins were expressed in PC12 cells as described (36Vitale N. Chasserot-Golaz S. Bailly Y. Morinaga N. Frohman M.A. Bader M.F. J. Cell Biol. 2002; 159: 79-89Crossref PubMed Scopus (103) Google Scholar). The PLD1-nonresponsive HA-ARF6(N48I) mutant was generated by site-directed mutagenesis. Its nucleotide exchange activity, activation by ARNO, and inactivation by an ARF6-GAP; intracellular distribution when expressed in PC12 cells; and in vitro effect on PLD1 activity were characterized and previously described (36Vitale N. Chasserot-Golaz S. Bailly Y. Morinaga N. Frohman M.A. Bader M.F. J. Cell Biol. 2002; 159: 79-89Crossref PubMed Scopus (103) Google Scholar). Rat RalA cDNA fragments encoding the 19-nucleotide siRNA sequence (AAGGCAGGTTTCTGTAGAA) derived from the target transcript and separated from its reverse 19-nucleotide complement by a short spacer were annealed and cloned in front of the H1-RNA promoter from the pSuper vector (37Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3971) Google Scholar). The specificity of the sequence was verified by BLAST search against the gene data bank. For mammalian expression vectors encoding both small hairpin RNAs (shRNAs) and growth hormone (GH), a cassette containing the H1-RNA promoter and the silencing sequence was amplified by PCR as described previously (38Waselle L. Coppola T. Fukuda M. Iezzi M. El-Amraoui A. Petit C. Regazzi R. Mol. Biol. Cell. 2003; 10: 4103-4113Crossref Google Scholar) and subcloned within the HindIII and XbaI sites of pXGH5 (Nichols Institute, San Juan Capistrano, CA). The empty vector with no siRNA sequence was named pGHsuper. To estimate the silencing effect, the RalA-shRNA plasmid was electroporated (260 V, 1050 microfarads, for 17 ms) in 107 PC12 cells, and 72 h post-transfection, cells were used for Western blot and immunofluorescence experiments. The transfection efficiency under these conditions was measured by counting GH-positive cells and was found to be 53% (432 of 815) for pGHsuper and 55% (541 of 983) for RalA-shRNA. Chromaffin Cell Culture and Subcellular Fractionation—Chromaffin cells were isolated from fresh bovine adrenal glands by retrograde perfusion with collagenase, purified on self-generating Percoll gradients, and maintained in culture as previously described (39Bader M.F. Thiersé D. Aunis D. Ahnert-Hilger G. Gratzl M. J. Biol. Chem. 1986; 261: 5777-5783Abstract Full Text PDF PubMed Google Scholar). For immunocytochemistry, cells were cultured on fibronectin-coated glass coverslips at a density of 2.5 × 105 cells/12-mm plate. In some experiments, cells were homogenized in 10 mm imidazole, pH 7.4, containing protease inhibitors and then centrifuged at 200,000 × g for 45 min (40Chasserot-Golaz S. Vitale N. Sagot I. Delouche B. Dirrig S. Pradel L.A. Henry J.P. Aunis D. Bader M.F. J. Cell Biol. 1996; 133: 1217-1236Crossref PubMed Scopus (102) Google Scholar). The supernatant was saved (cytosol), and the pellet was resuspended in 10 mm imidazole, pH 7.4, 75 mm KCl, 2 mm MgCl2, 1 mm NaN3, and 0.5% Triton X-100 containing protease inhibitors. After centrifugation at 200,000 × g for 45 min, the supernatant (Triton X-100-soluble fraction) and the pellet (Triton X-100-insoluble fraction) were saved and solubilized in sample buffer for gel electrophoresis (40Chasserot-Golaz S. Vitale N. Sagot I. Delouche B. Dirrig S. Pradel L.A. Henry J.P. Aunis D. Bader M.F. J. Cell Biol. 1996; 133: 1217-1236Crossref PubMed Scopus (102) Google Scholar). Culture, Transfection, and Growth Hormone (GH) Release from PC12 Cells—PC12 cells were grown in Dulbecco's modified Eagle's medium supplemented with glucose (4500 mg/liter) and containing 30 mm NaHCO3, 5% fetal bovine serum, 10% horse serum, and 100 units/ml penicillin/streptomycin. Mammalian expression vectors were introduced into PC12 cells together with the GH plasmid pXGH5 (6-well dishes, 80% confluent, 4 μg/well of each plasmid) using GenePorter (Gene Therapy Systems) according to the manufacturer's instructions. 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 were 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. GH release experiments were performed on at least three different cell cultures. In the figures that are representative of a typical experiment, data are given as the mean of triplicate determinations performed on the same cell preparation ± S.E. Assay for PLD Activity—Transfected 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). Medium was replaced with 100 μl of ice-cold Tris (50 mm, pH 8.0), and cells were broken by three freeze and thaw cycles. Samples were collected, mixed with an equal amount of the Amplex Red reaction buffer (Amplex Red Phospholipase D assay kit, Molecular Probes, Inc., Eugene, OR), and the PLD activity was estimated after a 1-h incubation at 37 °C with a Mithras (Berthold) fluorimeter. A standard curve was performed with purified PLD from Streptomyces chromofuscus (Sigma). In Fig. 4D, data are given as the mean of six determinations performed on the same cell preparation ± S.E. Similar results were obtained on four different cell preparations. Antibodies and Immunofluorescence—The following antibodies were used: monoclonal anti-RalA and rabbit polyclonal anti-RalB (Transduction Laboratories, Lexington, KY); polyclonal anti-PLD1 raised in rabbits against the N-terminal domain of PLD1 (QCB, BIOSOURCE International, France); monoclonal anti-SNAP-25 antibodies (Sternberger Monoclonals Inc., Lutherville, MD); monoclonal anti-HA antibodies (Babco, Richmond, CA); monoclonal anti-Myc antibodies (Novocostra Laboratories); polyclonal anti-GH (Dr. A. F. Parlow, NIDDK, National Institutes of Health, Bethesda, MD); Alexa-488-anti-mouse and Alexa-555-anti-rabbit (Molecular Probes). Rabbit polyclonal anti-chromogranin A antibodies were prepared in our laboratory (41Ehrhart M. Grube D. Bader M.F. Aunis D. Gratzl M. J. Histochem. Cytochem. 1986; 34: 1673-1682Crossref PubMed Scopus (93) Google Scholar). For immunocytochemistry, chromaffin or PC12 cells on coated glass coverslips were fixed and immunostained as described previously (35Vitale N. Caumont A.S. Chasserot-Golaz S. Du G. Wu S. Sciorra V.A. Morris A.J. Frohman M.A. Bader M.F. EMBO J. 2001; 20: 2424-2434Crossref PubMed Scopus (203) Google Scholar, 40Chasserot-Golaz S. Vitale N. Sagot I. Delouche B. Dirrig S. Pradel L.A. Henry J.P. Aunis D. Bader M.F. J. Cell Biol. 1996; 133: 1217-1236Crossref PubMed Scopus (102) Google Scholar). Stained cells were visualized with a Zeiss confocal microscope LSM 510. Using the Zeiss CLSM instrument software 3.2, the amount of RalA labeling was measured and expressed as the average fluorescence intensity multiplied by the corresponding surface area and divided by the total surface of each cell. This allows a quantitative cell-to-cell comparison of the RalA immunoreactivity detected in cells. Immunoprecipitation—PC12 cells transfected with pXS-HA-ARF6 or pXS-HA-ARF6(N48I) were maintained in Locke's solution or stimulated with 59 mm K+. Cells were then lysed, and ARF6 proteins were immunoprecipitated with anti-HA antibodies and protein A-Sepharose beads as described (36Vitale N. Chasserot-Golaz S. Bailly Y. Morinaga N. Frohman M.A. Bader M.F. J. Cell Biol. 2002; 159: 79-89Crossref PubMed Scopus (103) Google Scholar). GTP-bound RalA was specifically pulled down using the Myc-RBD (Ral-binding domain) of Ral-binding protein 1 (RalBP1). Therefore, PC12 cells expressing Myc-RBD were maintained under resting conditions or stimulated for various periods of time with 59 mm K+. Cells were then lysed in ice-cold lysis buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 20 mm MgCl2, 5 mm EDTA, 1% Nonidet P-40, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, and mammalian protease inhibitors) (Sigma). Lysates were clarified by centrifugation and Myc-tagged proteins were immunoprecipitated from the supernatant of each sample using agarose-coupled mouse anti-Myc IgG. Precipitated proteins were resolved on 12% polyacrylamide-SDS gels and immunoblotted with anti-Myc (1:500) and anti-RalA (1:1000) antibodies. Blots were processed using the “Western-Light Plus” chemiluminescent detection system (Tropix, Bedford, MA). Intracellular Distribution of RalA in Resting and Stimulated Chromaffin and PC12 Cells—We investigated the intracellular distribution of native RalA in cultured chromaffin cells by immunofluorescence and confocal microscopy. As illustrated in Fig. 1, RalA was restricted to the cell periphery. Double labeling with antibodies against the plasma membrane marker SNAP25 or against the chromaffin granule protein chromogranin A (CGA) indicated its association with the plasma membrane but not with the membrane of secretory granules (Fig. 1A). Endogenous RalA was similarly present at the plasma membrane in the chromaffin cell tumor derivatives, PC12 cells (see Fig. 6A). Next, we compared the distribution of RalA in resting and secretagogue-stimulated cells. Stimulation of chromaffin cells with nicotine (Fig. 1B) or stimulation of PC12 cells with a depolarizing concentration of potassium (59 mm K+) (Fig. 6A) reduced the level of peripheral RalA immunoreactivity by ∼85% without apparently increasing it in the cytosol or in other intracellular compartments. One possible explanation for this observation is that the monoclonal antibody recognized an epitope that was masked in stimulated cells due to the formation of a RalA-protein complex.Fig. 6RalA interacts with ARF6-activated PLD1 in stimulated PC12 cells. A, PC12 cells were transfected with the GFP-PLD1 expression plasmid alone or in combination with wild-type RalA. 48 h after transfection, cells were incubated for 10 min in Locke's solution (R) or stimulated with Locke containing 59 mm K+ (S). Cells were then fixed, and endogenous or overexpressed RalA was visualized by immunocytochemistry using anti-RalA antibodies and Alexa-555-conjugated secondary antibodies. In the merge images, the yellow-orange staining reveals areas where GFP-PLD1 and RalA co-localize. Masks represent the region of co-localization obtained by selecting the double-labeled pixels. Bars, 5 μm. B, PC12 cells expressing ARF6-HA or ARF6(N48I)-HA were incubated for 10 min in either Locke's solution (R) or in 59 mm K+ (S). Cells were scraped, and HA-tagged ARF6 proteins were immunoprecipitated. Samples were analyzed by electrophoresis and Western blotting using anti-ARF6, anti-PLD1, and anti-RalA antibodies. Similar observations were obtained from two different cell preparations.View Large Image Figure ViewerDownload (PPT) Subcellular fractionation experiments were performed on chromaffin cell homogenates to separate the cytosol, the Triton X-100-soluble fraction representing the membrane-bound compartment, and the Triton X-100-insoluble fraction representing the cytoskeleton and some detergent-resistant lipid microdomains. RalA was primarily detected in the Triton X-100-soluble fraction (Fig. 1C), suggesting that its presence in the cell periphery was most likely due to a binding to the plasma membrane rather than to an association with the actin filaments concentrated in the subplasmalemmal region. The distribution of RalA in the fractions prepared from nicotine-stimulated cells remained largely unchanged except for a slight increase detected in the Triton X-100-insoluble fraction (Fig. 1C). Taken together, these results suggest that RalA resides at the plasma membrane in resting cells. Since the protein was hardly detectable in stimulated cells, we concluded that RalA might be engaged in a putative complex with a regulator/effector protein formed in response to secretagogue-evoked stimulation. Secretagogue-evoked Stimulation Activates RalA in PC12 Cells—To probe the role of RalA in the control of dense core granule exocytosis, we first investigated whether RalA might be activated in response to a secretagogue that triggers exocytosis. Therefore, PC12 cells were maintained under resting conditions or stimulated for various periods of time with 59 mmK+, and RalA activation was assessed using the Ral-binding domain of RalBP1 (Myc-RBD) as a bait to trap the endogenous GTPase in its GTP-bound form (Fig. 2). We found that the amount of GTP-bound RalA is relatively low in resting cells. However, 2-10 min of stimulation with 59 mm K+ increased the level of cellular RalA-GTP by 3-10-fold, respectively (Fig. 2, A and B). Importantly, RalA-GTP rapidly decreased to basal levels as cells returned to the resting condition, revealing a tight coupling between membrane depolarization and RalA activation. Since the immediate consequence of membrane depolarization is calcium influx and secretion, these findings indicate that RalA activation is triggered by a rise in cytosolic calcium and thereby accompanies the exocytotic process. RalA Regulates Exocytosis of Large Dense Core Secretory Granules in PC12 Cells—To establish that RalA plays a role in exocytosis, we first examined the effect of expressing the Ral-binding domain of RalBP1 (Myc-RBD) in PC12 cells using growth hormone as a reporter for exocytosis (35Vitale N. Caumont A.S. Chasserot-Golaz S. Du G. Wu S. Sciorra V.A. Morris A.J. Frohman M.A. Bader M.F. EMBO J. 2001; 20: 2424-2434Crossref PubMed Scopus (203) Google Scholar). This protein fragment specifically binds to activated Ral-GTP and thereby interferes through competitive association with the ability of endogenous Ral proteins to interact with and stimulate effector molecules. As shown in Fig. 3, expression of Myc-RBD did not affect cell viability or GH" @default.
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