title: The death domain of Rab3 guanine nucleotide exchange protein in GDP/GTP exchange activity in living cells.
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.
authors.affiliation: Institut de Biologie Cellulaire et de Morphologie, University of Lausanne, Rue du Bugnon 9, 1005 Lausanne, Switzerland.
authors.forename: Thierry
authors.initials: T
authors.lastname: Coppola
authors.forename: Véronique
authors.initials: V
authors.lastname: Perret-Menoud
authors.forename: Sonia
authors.initials: S
authors.lastname: Gattesco
authors.forename: Sarah
authors.initials: S
authors.lastname: Magnin
authors.forename: Isabel
authors.initials: I
authors.lastname: Pombo
authors.forename: Ulrich
authors.initials: U
authors.lastname: Blank
authors.forename: Romano
authors.initials: R
authors.lastname: Regazzi
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D020033:Protein Isoforms / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D020692:rab3 GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D020662:Guanine Nucleotide Exchange Factors; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D020033:Protein Isoforms; C092115:Rab3ip protein, rat; D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020692:rab3 GTP-Binding Proteins