2 DISTINCT REGIONS OF RAS PARTICIPATE IN FUNCTIONAL INTERACTION WITH GDP-GTP EXCHANGERS

We have previously implemented a combined genetic/biochemical approach , for analysis of insertion-deletion mutants, to identify sites of Har vey-Ras participating in the interaction with guanine nucleotide excha ngers, using the yeast Cdc25 as a model exchanger. We showed that posi tions 101-106 may be required for catalyzed exchange. We here present a further improved strategy to define more precisely the residues on R as participating in this interaction. Non-conservative replacements at positions 103 or 105 abolished response to Cdc25 while substitutions at positions 102 or 104 were partially affected. The same substitution s had no effect on coupling to adenylyl cyclase. Since the strategy en ables us to assess Ras functional interaction with both the exchanger and effector simultaneously, we have also examined the effect of subst itutions in the distal part of the switch II region (amino acids 69-78 ). In contrast to other reports, substitutions at positions 69 or 73 p revented Cdc25 response while mutations at position 74 did not prevent this interaction. However, all these substitutions partly affected cy clase activation. These findings establish the crucial role of the 102 -105 region in the catalyzed exchange reaction and suggest that the 69 -74 area would be required for the functional interaction with both ex changers and effector molecules.