IDENTIFICATION OF NEUROFIBROMIN MUTANTS THAT EXHIBIT ALLELE SPECIFICITY OR INCREASED RAS AFFINITY RESULTING IN SUPPRESSION OF ACTIVATED RASALLELES

Neurofibromin plays a critical role in the downregulation of Ras prote ins in neurons and Schwann cells. Thus, the ability of neurofibromin t o interact with Ras is crucial for its function, as mutations in NF1 t hat abolish this interaction fail to maintain function. To investigate the neurofibromin-Ras interaction in a systematic manner, we have car ried out a yeast two-hybrid screen using a mutant of H-ras, H-ras(D92K ), defective for interaction with the GTPase-activated protein-related domain (GRD) of NF1, Two screens of a randomly mutagenized NF1-GRD li brary led to the identification of seven novel NF1 mutants, Characteri zation of the NF1-GRD mutants revealed that one class of mutants are a llele specific for H-ras(D92K). These mutants exhibit increased affini ty for H-ras(D92K) and significantly reduced affinity for wild-type H- ras protein. Furthermore, they do not interact with another H-ras muta nt defective for interaction with GTPase-activating proteins. Another class of mutants are high-affinity mutants which exhibit dramatically increased affinity for both wildtype and mutant forms of Ras, They als o exhibit a striking ability to suppress the heat shock sensitive trai ts of activated RAS2(G19V) in yeast cells. Five mutations cluster with in a region encompassing residues 1391 to 1436 (region II), Three NF1 patient mutations have previously been identified in this region. Two mutations that we identified occur in a region encompassing residues 1 262 to 1276 (region I), Combining high-affinity mutations from both re gions results in even greater affinity for Ras, These results demonstr ate that two distinct regions of NFL-GRD are involved in the Ras inter action and that single amino acid changes can affect NF1's affinity fo r Ras.