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pubmed:abstractText |
Two cellular factors have been described, Rab3A-GAP (GTPase-activating protein) and Rab3A-GRF (guanine nucleotide releasing factor) which, respectively, accelerate the intrinsic GTPase activity of, or the rate of dissociation of GDP from, the Ras-related GTP-binding protein, p25 Rab3A. Mutational analysis of p25 Rab3A was undertaken to define amino acid residues important for interaction with these factors. Mutations in residues 51-59, which correspond to the effector domain of p21 Ras, completely abolished sensitivity of p25 Rab3A to Rab3A-GRF and decreased the affinity of p25 Rab3A for Rab3A-GRF. Surprisingly, only one mutant in this region was Rab3A-GAP-insensitive, while the others retained partial, complete, or significantly increased GAP responsiveness. Mutations in the first G-domain had only modest effects on intrinsic GTPase activity and little effect on either Rab3A-GRF or Rab3A-GAP interactions. Truncation of 34 residues from the carboxyl terminus had no effect Rab3A-GAP sensitivity but facilitated Rab3A-GRF stimulation. Mutation T36N, analogous to the dominant inhibitory mutation T17N in Ras, which has been hypothesized to sequester an upstream activator of Ras, conferred a 10-fold higher affinity upon p25 Rab3A for Rab3A-GRF.
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