This investigator has identified ras mutants, which apparently interact with two separate signaling systems downstream of ras and together are required for optimal proliferative signaling. The proposed studies are an extension of this work. Additional experiments utilizing the yeast two hybrid system will search for new downstream targets of ras. Proteins interacting differentially with ras mutants will be sought in libraries from selected cell populations and will then be tested in binding assays. These and previously identified potential downstream targets of ras will be used to find ras mutants, which bind uniquely to them or sets of downstream targets. The ras mutants and newly identified potential downstream targets will then be tested for biological activity by transfection into a variety of selected cell types, for identification of potential effector-specific biological consequences. In addition, potential complementation between these mutants will be tested. Finally, back mutants in downstream targets, which restore binding to ras mutants will be tested along with these other mutants to directly demonstrate that the two must interact in order to generate a biological signal. One of the mutants identified has been used to induce ruffling following microinjection, in the absence of a strong growth response.
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