Amino acid residues in the Ras-like GTPase Rab3A that specify sensitivity to factors that regulate the GTP/GDP cycling of Rab3A.
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.
Burstein
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Department of Pathology, University of Vermont Medical College, Burlington 05405-0068.
Brondyk
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Macara
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D000818:Animals; D001483:Base Sequence; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D016366:Open Reading Frames; D011487:Protein Conformation; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D015183:Restriction Mapping; D020692:rab3 GTP-Binding Proteins; D020703:ras GTPase-Activating Proteins; D020663:ras Guanine Nucleotide Exchange Factors; D020666:ras-GRF1
D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D009419:Nerve Tissue Proteins; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D020663:ras Guanine Nucleotide Exchange Factors; D020666:ras-GRF1; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins