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The death domain of Rab3 guanine nucleotide exchange protein in GDP/GTP exchange activity in living cells
Journal Article Research Support, Non-U.S. Gov't --- Old month value: Mar 1
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.
Amino Acid Sequence Amino Acid Substitution Binding Sites Brain/metabolism Carrier Proteins/metabolism Cell Line Guanine Nucleotide Exchange Factors/chemistry/metabolism Guanosine Diphosphate/*metabolism Guanosine Triphosphate/*metabolism Humans Insulin/secretion *Intracellular Signaling Peptides and Proteins Kinetics Molecular Sequence Data Mutagenesis, Site-Directed Protein Conformation Protein Isoforms/metabolism Recombinant Fusion Proteins/metabolism Transfection rab3 GTP-Binding Proteins/*chemistry/*metabolism
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