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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March 2002
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Research Article|
February 22 2002
The death domain of Rab3 guanine nucleotide exchange protein in GDP/GTP exchange activity in living cells
Thierry COPPOLA;
Thierry COPPOLA
∗Institut de Biologie Cellulaire et de Morphologie, University of Lausanne, Rue du Bugnon 9, 1005 Lausanne, Switzerland
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Véronique PERRET-MENOUD;
Véronique PERRET-MENOUD
∗Institut de Biologie Cellulaire et de Morphologie, University of Lausanne, Rue du Bugnon 9, 1005 Lausanne, Switzerland
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Sonia GATTESCO;
Sonia GATTESCO
∗Institut de Biologie Cellulaire et de Morphologie, University of Lausanne, Rue du Bugnon 9, 1005 Lausanne, Switzerland
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Sarah MAGNIN;
Sarah MAGNIN
∗Institut de Biologie Cellulaire et de Morphologie, University of Lausanne, Rue du Bugnon 9, 1005 Lausanne, Switzerland
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Isabel POMBO;
Isabel POMBO
†Institut Pasteur, Unité d'Immuno-Allergie, Paris, France
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Ulrich BLANK;
Ulrich BLANK
†Institut Pasteur, Unité d'Immuno-Allergie, Paris, France
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Romano REGAZZI
Romano REGAZZI
1
∗Institut de Biologie Cellulaire et de Morphologie, University of Lausanne, Rue du Bugnon 9, 1005 Lausanne, Switzerland
1To whom correspondence should be addressed (e-mail Romano.Regazzi@ibcm.unil.ch).
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Publisher: Portland Press Ltd
Received:
August 09 2001
Revision Received:
November 13 2001
Accepted:
December 13 2001
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 362 (2): 273–279.
Article history
Received:
August 09 2001
Revision Received:
November 13 2001
Accepted:
December 13 2001
Citation
Thierry COPPOLA, Véronique PERRET-MENOUD, Sonia GATTESCO, Sarah MAGNIN, Isabel POMBO, Ulrich BLANK, Romano REGAZZI; The death domain of Rab3 guanine nucleotide exchange protein in GDP/GTP exchange activity in living cells. Biochem J 1 March 2002; 362 (2): 273–279. doi: https://doi.org/10.1042/bj3620273
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