Burstein E S, Brondyk W H, Macara I G
Department of Pathology, University of Vermont Medical College, Burlington 05405-0068.
J Biol Chem. 1992 Nov 15;267(32):22715-8.
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.
已发现两种细胞因子，即Rab3A-GAP（GTP酶激活蛋白）和Rab3A-GRF（鸟嘌呤核苷酸释放因子），它们分别加速Ras相关GTP结合蛋白p25 Rab3A的内在GTP酶活性或GDP从其解离的速率。对p25 Rab3A进行了突变分析，以确定与这些因子相互作用的重要氨基酸残基。对应于p21 Ras效应结构域的51-59位残基的突变完全消除了p25 Rab3A对Rab3A-GRF的敏感性，并降低了p25 Rab3A与Rab3A-GRF的亲和力。令人惊讶的是，该区域只有一个突变体对Rab3A-GAP不敏感，而其他突变体则保留了部分、完全或显著增加的GAP反应性。第一个G结构域中的突变对内在GTP酶活性只有适度影响，对Rab3A-GRF或Rab3A-GAP相互作用影响很小。从羧基末端截短34个残基对Rab3A-GAP敏感性没有影响，但促进了Rab3A-GRF刺激。类似于Ras中的显性抑制突变T17N的突变T36N，据推测该突变会隔离Ras的上游激活剂，使p25 Rab3A对Rab3A-GRF的亲和力提高了10倍。
