MUTANTS OF RAB3A ANALOGOUS TO ONCOGENIC RAS MUTANTS - SENSITIVITY TO RAB3A-GTPASE ACTIVATING PROTEIN AND RAB3A-GUANINE NUCLEOTIDE RELEASING-FACTOR

Rab3A is a Ras-like GTPase that is believed to function in regulated s ecretion. A GTPase activating protein (GAP) and a guanine nucleotide r eleasing factor (GRF) specific for Rab3A have recently been described. In this study we have described the biochemical activities of Rab3A m utants in codons 31, 81, 135, and 166, which correspond to codons 12, 61, 116, and 146 in Ras. The results demonstrate that simple extrapola tions from the properties of Ras mutants are not valid for all small G TPases. S31V-Rab3A and Q81L-Rab3A had a reduced basal GTPase activity, but surprisingly were sensitive to the effects of Rab3A-GAP and insen sitive to Rab3A-GRF. Q81L-Rab3A and wild-type Rab3A that were bound to a nonhydrolyzable GTP analog had similar affinities for Rab3A-GAP. In vivo, the percent of Q81L-Rab3A (46%) and wild-type Rab3A (43%) compl exed to GTP was similar. These findings indicate that Rab3A-GRF and Ra b3A-GAP interact with residues different from those of previously desc ribed GAPs and GRFs. Both A166V-Rab3A and N135I-Rab3A had increased in trinsic dissociation rates for GDP. However, the dissociation rate for N135I-Rab3A was > 100-fold higher than that for A166V-Rab3A suggestin g that, in vivo, of the two, N135I-Rab3A would more likely be preferen tially in the GTP-bound state.