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. 1996 Aug 2;134(4):935–947. doi: 10.1083/jcb.134.4.935

Aluminum fluoride stimulates surface protrusions in cells overexpressing the ARF6 GTPase

PMCID: PMC2120964  PMID: 8769418

Abstract

To study the effector function of the ADP- ribosylation factor (ARF) 6 GTP-binding protein, we transfected HeLa cells with wild-type, epitope- tagged ARF6. Previously shown to indirectly activate the ARF1 GTPase, aluminum fluoride (AIF) treatment of ARF6-transfected cells resulted in a redistribution of both ARF6 and actin to discrete sites on the plasma membrane, which became increasingly protrusive over time. The effects of AIF were reversible, specific to cells transfected with wild-type ARF6, and resembled the cellular protrusions observed in cells expressing the GTPase defective mutant of ARF6. Importantly, the protrusions observed in cells transfected with ARF6 were distinct from the enhanced stress fibers and membrane ruffles observed in cells transfected with RhoA and Rac1, respectively. In cells forming protrusions, there was an apparent stimulation of macropinocytosis and membrane recycling within the protrusive structures. In contrast, no block in transferrin uptake or alteration of the distribution of clathrin AP-2 complexes was detected in these cells. The AIF-induced, ARF6- dependent formation of protrusive structures was blocked by cytochalasin D and inhibitors of the lipoxygenase pathway. These observations support a novel role for the ARF6 GTPase in modeling the plasma membrane and underlying cytoskeleton.

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Selected References

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  1. Adams A. E., Johnson D. I., Longnecker R. M., Sloat B. F., Pringle J. R. CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae. J Cell Biol. 1990 Jul;111(1):131–142. doi: 10.1083/jcb.111.1.131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bar-Sagi D., Feramisco J. R. Induction of membrane ruffling and fluid-phase pinocytosis in quiescent fibroblasts by ras proteins. Science. 1986 Sep 5;233(4768):1061–1068. doi: 10.1126/science.3090687. [DOI] [PubMed] [Google Scholar]
  3. Bonifacino J. S., Suzuki C. K., Lippincott-Schwartz J., Weissman A. M., Klausner R. D. Pre-Golgi degradation of newly synthesized T-cell antigen receptor chains: intrinsic sensitivity and the role of subunit assembly. J Cell Biol. 1989 Jul;109(1):73–83. doi: 10.1083/jcb.109.1.73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bretscher A. Microfilament structure and function in the cortical cytoskeleton. Annu Rev Cell Biol. 1991;7:337–374. doi: 10.1146/annurev.cb.07.110191.002005. [DOI] [PubMed] [Google Scholar]
  5. Brown H. A., Gutowski S., Moomaw C. R., Slaughter C., Sternweis P. C. ADP-ribosylation factor, a small GTP-dependent regulatory protein, stimulates phospholipase D activity. Cell. 1993 Dec 17;75(6):1137–1144. doi: 10.1016/0092-8674(93)90323-i. [DOI] [PubMed] [Google Scholar]
  6. Chabre M. Aluminofluoride and beryllofluoride complexes: a new phosphate analogs in enzymology. Trends Biochem Sci. 1990 Jan;15(1):6–10. doi: 10.1016/0968-0004(90)90117-t. [DOI] [PubMed] [Google Scholar]
  7. Chin D. J., Straubinger R. M., Acton S., Näthke I., Brodsky F. M. 100-kDa polypeptides in peripheral clathrin-coated vesicles are required for receptor-mediated endocytosis. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9289–9293. doi: 10.1073/pnas.86.23.9289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cockcroft S., Thomas G. M., Fensome A., Geny B., Cunningham E., Gout I., Hiles I., Totty N. F., Truong O., Hsuan J. J. Phospholipase D: a downstream effector of ARF in granulocytes. Science. 1994 Jan 28;263(5146):523–526. doi: 10.1126/science.8290961. [DOI] [PubMed] [Google Scholar]
  9. Colombo M. I., Inglese J., D'Souza-Schorey C., Beron W., Stahl P. D. Heterotrimeric G proteins interact with the small GTPase ARF. Possibilities for the regulation of vesicular traffic. J Biol Chem. 1995 Oct 13;270(41):24564–24571. doi: 10.1074/jbc.270.41.24564. [DOI] [PubMed] [Google Scholar]
  10. Condeelis J. Life at the leading edge: the formation of cell protrusions. Annu Rev Cell Biol. 1993;9:411–444. doi: 10.1146/annurev.cb.09.110193.002211. [DOI] [PubMed] [Google Scholar]
  11. D'Souza-Schorey C., Li G., Colombo M. I., Stahl P. D. A regulatory role for ARF6 in receptor-mediated endocytosis. Science. 1995 Feb 24;267(5201):1175–1178. doi: 10.1126/science.7855600. [DOI] [PubMed] [Google Scholar]
  12. Dascher C., Balch W. E. Dominant inhibitory mutants of ARF1 block endoplasmic reticulum to Golgi transport and trigger disassembly of the Golgi apparatus. J Biol Chem. 1994 Jan 14;269(2):1437–1448. [PubMed] [Google Scholar]
  13. Donaldson J. G., Cassel D., Kahn R. A., Klausner R. D. ADP-ribosylation factor, a small GTP-binding protein, is required for binding of the coatomer protein beta-COP to Golgi membranes. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6408–6412. doi: 10.1073/pnas.89.14.6408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Donaldson J. G., Finazzi D., Klausner R. D. Brefeldin A inhibits Golgi membrane-catalysed exchange of guanine nucleotide onto ARF protein. Nature. 1992 Nov 26;360(6402):350–352. doi: 10.1038/360350a0. [DOI] [PubMed] [Google Scholar]
  15. Donaldson J. G., Kahn R. A., Lippincott-Schwartz J., Klausner R. D. Binding of ARF and beta-COP to Golgi membranes: possible regulation by a trimeric G protein. Science. 1991 Nov 22;254(5035):1197–1199. doi: 10.1126/science.1957170. [DOI] [PubMed] [Google Scholar]
  16. Donaldson J. G., Klausner R. D. ARF: a key regulatory switch in membrane traffic and organelle structure. Curr Opin Cell Biol. 1994 Aug;6(4):527–532. doi: 10.1016/0955-0674(94)90072-8. [DOI] [PubMed] [Google Scholar]
  17. Finazzi D., Cassel D., Donaldson J. G., Klausner R. D. Aluminum fluoride acts on the reversibility of ARF1-dependent coat protein binding to Golgi membranes. J Biol Chem. 1994 May 6;269(18):13325–13330. [PubMed] [Google Scholar]
  18. Hall A. Small GTP-binding proteins and the regulation of the actin cytoskeleton. Annu Rev Cell Biol. 1994;10:31–54. doi: 10.1146/annurev.cb.10.110194.000335. [DOI] [PubMed] [Google Scholar]
  19. Hammond S. M., Altshuller Y. M., Sung T. C., Rudge S. A., Rose K., Engebrecht J., Morris A. J., Frohman M. A. Human ADP-ribosylation factor-activated phosphatidylcholine-specific phospholipase D defines a new and highly conserved gene family. J Biol Chem. 1995 Dec 15;270(50):29640–29643. doi: 10.1074/jbc.270.50.29640. [DOI] [PubMed] [Google Scholar]
  20. Hansen S. H., Sandvig K., van Deurs B. Molecules internalized by clathrin-independent endocytosis are delivered to endosomes containing transferrin receptors. J Cell Biol. 1993 Oct;123(1):89–97. doi: 10.1083/jcb.123.1.89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hawkins P. T., Eguinoa A., Qiu R. G., Stokoe D., Cooke F. T., Walters R., Wennström S., Claesson-Welsh L., Evans T., Symons M. PDGF stimulates an increase in GTP-Rac via activation of phosphoinositide 3-kinase. Curr Biol. 1995 Apr 1;5(4):393–403. doi: 10.1016/s0960-9822(95)00080-7. [DOI] [PubMed] [Google Scholar]
  22. Helms J. B., Rothman J. E. Inhibition by brefeldin A of a Golgi membrane enzyme that catalyses exchange of guanine nucleotide bound to ARF. Nature. 1992 Nov 26;360(6402):352–354. doi: 10.1038/360352a0. [DOI] [PubMed] [Google Scholar]
  23. Hewlett L. J., Prescott A. R., Watts C. The coated pit and macropinocytic pathways serve distinct endosome populations. J Cell Biol. 1994 Mar;124(5):689–703. doi: 10.1083/jcb.124.5.689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Higashijima T., Graziano M. P., Suga H., Kainosho M., Gilman A. G. 19F and 31P NMR spectroscopy of G protein alpha subunits. Mechanism of activation by Al3+ and F-. J Biol Chem. 1991 Feb 25;266(6):3396–3401. [PubMed] [Google Scholar]
  25. Janmey P. A. Phosphoinositides and calcium as regulators of cellular actin assembly and disassembly. Annu Rev Physiol. 1994;56:169–191. doi: 10.1146/annurev.ph.56.030194.001125. [DOI] [PubMed] [Google Scholar]
  26. Kahn R. A. Fluoride is not an activator of the smaller (20-25 kDa) GTP-binding proteins. J Biol Chem. 1991 Aug 25;266(24):15595–15597. [PubMed] [Google Scholar]
  27. Kahn R. A., Gilman A. G. The protein cofactor necessary for ADP-ribosylation of Gs by cholera toxin is itself a GTP binding protein. J Biol Chem. 1986 Jun 15;261(17):7906–7911. [PubMed] [Google Scholar]
  28. Kahn R. A., Kern F. G., Clark J., Gelmann E. P., Rulka C. Human ADP-ribosylation factors. A functionally conserved family of GTP-binding proteins. J Biol Chem. 1991 Feb 5;266(4):2606–2614. [PubMed] [Google Scholar]
  29. Kahn R. A., Yucel J. K., Malhotra V. ARF signaling: a potential role for phospholipase D in membrane traffic. Cell. 1993 Dec 17;75(6):1045–1048. doi: 10.1016/0092-8674(93)90314-g. [DOI] [PubMed] [Google Scholar]
  30. Kanner S. B., Reynolds A. B., Vines R. R., Parsons J. T. Monoclonal antibodies to individual tyrosine-phosphorylated protein substrates of oncogene-encoded tyrosine kinases. Proc Natl Acad Sci U S A. 1990 May;87(9):3328–3332. doi: 10.1073/pnas.87.9.3328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kozma R., Ahmed S., Best A., Lim L. The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts. Mol Cell Biol. 1995 Apr;15(4):1942–1952. doi: 10.1128/mcb.15.4.1942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ktistakis N. T., Linder M. E., Roth M. G. Action of brefeldin A blocked by activation of a pertussis-toxin-sensitive G protein. Nature. 1992 Mar 26;356(6367):344–346. doi: 10.1038/356344a0. [DOI] [PubMed] [Google Scholar]
  33. Lamaze C., Schmid S. L. The emergence of clathrin-independent pinocytic pathways. Curr Opin Cell Biol. 1995 Aug;7(4):573–580. doi: 10.1016/0955-0674(95)80015-8. [DOI] [PubMed] [Google Scholar]
  34. Liscovitch M., Cantley L. C. Signal transduction and membrane traffic: the PITP/phosphoinositide connection. Cell. 1995 Jun 2;81(5):659–662. doi: 10.1016/0092-8674(95)90525-1. [DOI] [PubMed] [Google Scholar]
  35. Massenburg D., Han J. S., Liyanage M., Patton W. A., Rhee S. G., Moss J., Vaughan M. Activation of rat brain phospholipase D by ADP-ribosylation factors 1,5, and 6: separation of ADP-ribosylation factor-dependent and oleate-dependent enzymes. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11718–11722. doi: 10.1073/pnas.91.24.11718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nobes C. D., Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell. 1995 Apr 7;81(1):53–62. doi: 10.1016/0092-8674(95)90370-4. [DOI] [PubMed] [Google Scholar]
  37. Peppelenbosch M. P., Qiu R. G., de Vries-Smits A. M., Tertoolen L. G., de Laat S. W., McCormick F., Hall A., Symons M. H., Bos J. L. Rac mediates growth factor-induced arachidonic acid release. Cell. 1995 Jun 16;81(6):849–856. doi: 10.1016/0092-8674(95)90005-5. [DOI] [PubMed] [Google Scholar]
  38. Peppelenbosch M. P., Tertoolen L. G., Hage W. J., de Laat S. W. Epidermal growth factor-induced actin remodeling is regulated by 5-lipoxygenase and cyclooxygenase products. Cell. 1993 Aug 13;74(3):565–575. doi: 10.1016/0092-8674(93)80057-l. [DOI] [PubMed] [Google Scholar]
  39. Peters P. J., Hsu V. W., Ooi C. E., Finazzi D., Teal S. B., Oorschot V., Donaldson J. G., Klausner R. D. Overexpression of wild-type and mutant ARF1 and ARF6: distinct perturbations of nonoverlapping membrane compartments. J Cell Biol. 1995 Mar;128(6):1003–1017. doi: 10.1083/jcb.128.6.1003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Racoosin E. L., Swanson J. A. M-CSF-induced macropinocytosis increases solute endocytosis but not receptor-mediated endocytosis in mouse macrophages. J Cell Sci. 1992 Aug;102(Pt 4):867–880. doi: 10.1242/jcs.102.4.867. [DOI] [PubMed] [Google Scholar]
  41. Randazzo P. A., Yang Y. C., Rulka C., Kahn R. A. Activation of ADP-ribosylation factor by Golgi membranes. Evidence for a brefeldin A- and protease-sensitive activating factor on Golgi membranes. J Biol Chem. 1993 May 5;268(13):9555–9563. [PubMed] [Google Scholar]
  42. Ridley A. J., Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 1992 Aug 7;70(3):389–399. doi: 10.1016/0092-8674(92)90163-7. [DOI] [PubMed] [Google Scholar]
  43. Ridley A. J., Paterson H. F., Johnston C. L., Diekmann D., Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell. 1992 Aug 7;70(3):401–410. doi: 10.1016/0092-8674(92)90164-8. [DOI] [PubMed] [Google Scholar]
  44. Robinson M. S., Kreis T. E. Recruitment of coat proteins onto Golgi membranes in intact and permeabilized cells: effects of brefeldin A and G protein activators. Cell. 1992 Apr 3;69(1):129–138. doi: 10.1016/0092-8674(92)90124-u. [DOI] [PubMed] [Google Scholar]
  45. Rothman J. E. Mechanisms of intracellular protein transport. Nature. 1994 Nov 3;372(6501):55–63. doi: 10.1038/372055a0. [DOI] [PubMed] [Google Scholar]
  46. Sandvig K., van Deurs B. Endocytosis without clathrin. Trends Cell Biol. 1994 Aug;4(8):275–277. doi: 10.1016/0962-8924(94)90211-9. [DOI] [PubMed] [Google Scholar]
  47. Steinman R. M., Mellman I. S., Muller W. A., Cohn Z. A. Endocytosis and the recycling of plasma membrane. J Cell Biol. 1983 Jan;96(1):1–27. doi: 10.1083/jcb.96.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Stossel T. P. On the crawling of animal cells. Science. 1993 May 21;260(5111):1086–1094. doi: 10.1126/science.8493552. [DOI] [PubMed] [Google Scholar]
  49. Swanson J. A., Watts C. Macropinocytosis. Trends Cell Biol. 1995 Nov;5(11):424–428. doi: 10.1016/s0962-8924(00)89101-1. [DOI] [PubMed] [Google Scholar]
  50. Takai Y., Sasaki T., Tanaka K., Nakanishi H. Rho as a regulator of the cytoskeleton. Trends Biochem Sci. 1995 Jun;20(6):227–231. doi: 10.1016/s0968-0004(00)89022-2. [DOI] [PubMed] [Google Scholar]
  51. Takebe Y., Seiki M., Fujisawa J., Hoy P., Yokota K., Arai K., Yoshida M., Arai N. SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol Cell Biol. 1988 Jan;8(1):466–472. doi: 10.1128/mcb.8.1.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Teal S. B., Hsu V. W., Peters P. J., Klausner R. D., Donaldson J. G. An activating mutation in ARF1 stabilizes coatomer binding to Golgi membranes. J Biol Chem. 1994 Feb 4;269(5):3135–3138. [PubMed] [Google Scholar]
  53. Traub L. M., Ostrom J. A., Kornfeld S. Biochemical dissection of AP-1 recruitment onto Golgi membranes. J Cell Biol. 1993 Nov;123(3):561–573. doi: 10.1083/jcb.123.3.561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Tsuchiya M., Price S. R., Tsai S. C., Moss J., Vaughan M. Molecular identification of ADP-ribosylation factor mRNAs and their expression in mammalian cells. J Biol Chem. 1991 Feb 15;266(5):2772–2777. [PubMed] [Google Scholar]
  55. Watts C., Marsh M. Endocytosis: what goes in and how? J Cell Sci. 1992 Sep;103(Pt 1):1–8. doi: 10.1242/jcs.103.1.1a. [DOI] [PubMed] [Google Scholar]
  56. van Deurs B., Petersen O. W., Olsnes S., Sandvig K. The ways of endocytosis. Int Rev Cytol. 1989;117:131–177. doi: 10.1016/s0074-7696(08)61336-4. [DOI] [PubMed] [Google Scholar]

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