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. 2002 Mar 1;362(Pt 2):273–279. doi: 10.1042/0264-6021:3620273

The death domain of Rab3 guanine nucleotide exchange protein in GDP/GTP exchange activity in living cells.

Thierry Coppola 1, Véronique Perret-Menoud 1, Sonia Gattesco 1, Sarah Magnin 1, Isabel Pombo 1, Ulrich Blank 1, Romano Regazzi 1
PMCID: PMC1222386  PMID: 11853534

Abstract

Rab3 GTPases regulate exocytosis of neurons, endocrine and exocrine cells. In the present paper, we report a system to measure the guanine nucleotide status of Rab3 proteins in living cells. The assay is based on the ability of the Rab3 interacting molecule RIM to extract selectively the GTP-bound form of Rab3. Using this system, we found that approx. 20% of wild-type Rab3A, -B, -C or -D transfected in the insulin-secreting cell line HIT-T15 is in the GTP-bound conformation. The pool of activated Rab3 is decreased under conditions that stimulate exocytosis or by co-expression of the Rab3 GTPase-activating protein. In contrast, co-expression of Mss4 or Rab3-GEP (guanine nucleotide exchange protein) increases by approx. 3-fold the GTP-bound pool of Rab3 isoforms. Rab3-GEP is very similar to MADD, a death domain-containing protein that associates with the type 1 tumour necrosis factor receptor. We observed that the death domain of Rab3-GEP is involved in intramolecular interactions and that deletions or mutations that affect this domain of the protein impair the nucleotide exchange activity towards Rab3. We propose that the death domain of Rab3-GEP acts as a molecular switch and co-ordinates multiple functions of the protein by exchanging its binding partners.

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Selected References

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  1. Brondyk W. H., McKiernan C. J., Burstein E. S., Macara I. G. Mutants of Rab3A analogous to oncogenic Ras mutants. Sensitivity to Rab3A-GTPase activating protein and Rab3A-guanine nucleotide releasing factor. J Biol Chem. 1993 May 5;268(13):9410–9415. [PubMed] [Google Scholar]
  2. Brown T. L., Howe P. H. MADD is highly homologous to a Rab3 guanine-nucleotide exchange protein (Rab3-GEP) Curr Biol. 1998 Mar 12;8(6):R191–R191. [PubMed] [Google Scholar]
  3. Burton J. L., Burns M. E., Gatti E., Augustine G. J., De Camilli P. Specific interactions of Mss4 with members of the Rab GTPase subfamily. EMBO J. 1994 Dec 1;13(23):5547–5558. doi: 10.1002/j.1460-2075.1994.tb06892.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Collins R. N., Brennwald P., Garrett M., Lauring A., Novick P. Interactions of nucleotide release factor Dss4p with Sec4p in the post-Golgi secretory pathway of yeast. J Biol Chem. 1997 Jul 18;272(29):18281–18289. doi: 10.1074/jbc.272.29.18281. [DOI] [PubMed] [Google Scholar]
  5. Feinstein E., Kimchi A., Wallach D., Boldin M., Varfolomeev E. The death domain: a module shared by proteins with diverse cellular functions. Trends Biochem Sci. 1995 Sep;20(9):342–344. doi: 10.1016/s0968-0004(00)89070-2. [DOI] [PubMed] [Google Scholar]
  6. Fukui K., Sasaki T., Imazumi K., Matsuura Y., Nakanishi H., Takai Y. Isolation and characterization of a GTPase activating protein specific for the Rab3 subfamily of small G proteins. J Biol Chem. 1997 Feb 21;272(8):4655–4658. doi: 10.1074/jbc.272.8.4655. [DOI] [PubMed] [Google Scholar]
  7. Geppert M., Südhof T. C. RAB3 and synaptotagmin: the yin and yang of synaptic membrane fusion. Annu Rev Neurosci. 1998;21:75–95. doi: 10.1146/annurev.neuro.21.1.75. [DOI] [PubMed] [Google Scholar]
  8. Guan K. L., Dixon J. E. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991 Feb 1;192(2):262–267. doi: 10.1016/0003-2697(91)90534-z. [DOI] [PubMed] [Google Scholar]
  9. Iezzi M., Escher G., Meda P., Charollais A., Baldini G., Darchen F., Wollheim C. B., Regazzi R. Subcellular distribution and function of Rab3A, B, C, and D isoforms in insulin-secreting cells. Mol Endocrinol. 1999 Feb;13(2):202–212. doi: 10.1210/mend.13.2.0228. [DOI] [PubMed] [Google Scholar]
  10. Iwasaki K., Staunton J., Saifee O., Nonet M., Thomas J. H. aex-3 encodes a novel regulator of presynaptic activity in C. elegans. Neuron. 1997 Apr;18(4):613–622. doi: 10.1016/s0896-6273(00)80302-5. [DOI] [PubMed] [Google Scholar]
  11. Iwasaki K., Toyonaga R. The Rab3 GDP/GTP exchange factor homolog AEX-3 has a dual function in synaptic transmission. EMBO J. 2000 Sep 1;19(17):4806–4816. doi: 10.1093/emboj/19.17.4806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Martinez O., Goud B. Rab proteins. Biochim Biophys Acta. 1998 Aug 14;1404(1-2):101–112. doi: 10.1016/s0167-4889(98)00050-0. [DOI] [PubMed] [Google Scholar]
  13. Nonet M. L., Staunton J. E., Kilgard M. P., Fergestad T., Hartwieg E., Horvitz H. R., Jorgensen E. M., Meyer B. J. Caenorhabditis elegans rab-3 mutant synapses exhibit impaired function and are partially depleted of vesicles. J Neurosci. 1997 Nov 1;17(21):8061–8073. doi: 10.1523/JNEUROSCI.17-21-08061.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Nuoffer C., Wu S. K., Dascher C., Balch W. E. Mss4 does not function as an exchange factor for Rab in endoplasmic reticulum to Golgi transport. Mol Biol Cell. 1997 Jul;8(7):1305–1316. doi: 10.1091/mbc.8.7.1305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ohnishi H., Samuelson L. C., Yule D. I., Ernst S. A., Williams J. A. Overexpression of Rab3D enhances regulated amylase secretion from pancreatic acini of transgenic mice. J Clin Invest. 1997 Dec 15;100(12):3044–3052. doi: 10.1172/JCI119859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Oishi H., Sasaki T., Nagano F., Ikeda W., Ohya T., Wada M., Ide N., Nakanishi H., Takai Y. Localization of the Rab3 small G protein regulators in nerve terminals and their involvement in Ca2+-dependent exocytosis. J Biol Chem. 1998 Dec 18;273(51):34580–34585. doi: 10.1074/jbc.273.51.34580. [DOI] [PubMed] [Google Scholar]
  17. Pfeffer S. R., Dirac-Svejstrup A. B., Soldati T. Rab GDP dissociation inhibitor: putting rab GTPases in the right place. J Biol Chem. 1995 Jul 21;270(29):17057–17059. doi: 10.1074/jbc.270.29.17057. [DOI] [PubMed] [Google Scholar]
  18. Regazzi R., Ravazzola M., Iezzi M., Lang J., Zahraoui A., Andereggen E., Morel P., Takai Y., Wollheim C. B. Expression, localization and functional role of small GTPases of the Rab3 family in insulin-secreting cells. J Cell Sci. 1996 Sep;109(Pt 9):2265–2273. doi: 10.1242/jcs.109.9.2265. [DOI] [PubMed] [Google Scholar]
  19. Schievella A. R., Chen J. H., Graham J. R., Lin L. L. MADD, a novel death domain protein that interacts with the type 1 tumor necrosis factor receptor and activates mitogen-activated protein kinase. J Biol Chem. 1997 May 2;272(18):12069–12075. doi: 10.1074/jbc.272.18.12069. [DOI] [PubMed] [Google Scholar]
  20. Stahl B., von Mollard G. F., Walch-Solimena C., Jahn R. GTP cleavage by the small GTP-binding protein Rab3A is associated with exocytosis of synaptic vesicles induced by alpha-latrotoxin. J Biol Chem. 1994 Oct 7;269(40):24770–24776. [PubMed] [Google Scholar]
  21. 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]
  22. Tartaglia L. A., Ayres T. M., Wong G. H., Goeddel D. V. A novel domain within the 55 kd TNF receptor signals cell death. Cell. 1993 Sep 10;74(5):845–853. doi: 10.1016/0092-8674(93)90464-2. [DOI] [PubMed] [Google Scholar]
  23. Wada M., Nakanishi H., Satoh A., Hirano H., Obaishi H., Matsuura Y., Takai Y. Isolation and characterization of a GDP/GTP exchange protein specific for the Rab3 subfamily small G proteins. J Biol Chem. 1997 Feb 14;272(7):3875–3878. doi: 10.1074/jbc.272.7.3875. [DOI] [PubMed] [Google Scholar]
  24. Wang Y., Okamoto M., Schmitz F., Hofmann K., Südhof T. C. Rim is a putative Rab3 effector in regulating synaptic-vesicle fusion. Nature. 1997 Aug 7;388(6642):593–598. doi: 10.1038/41580. [DOI] [PubMed] [Google Scholar]
  25. Zhang Y., Zhou L., Miller C. A. A splicing variant of a death domain protein that is regulated by a mitogen-activated kinase is a substrate for c-Jun N-terminal kinase in the human central nervous system. Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2586–2591. doi: 10.1073/pnas.95.5.2586. [DOI] [PMC free article] [PubMed] [Google Scholar]

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