Neurofibromatosis type I (NF1) is a genetic disease of peripheral nervous system that afflicts 1 in 3500 people worldwide, We have recently demonstrated that the NF1 gene product is a member of GAP (GTPase activating protein) protein family which down-regulate ras proteins, guanine nucleotide binding protein involved in regulation of cell growth as well as neoplasia. In this application, we propose to carry out a detailed characterization of the NF1 protein by concentrating on the following experiments. [I] Characterization of NF1 GAP activity and interaction between NF1 and ras proteins (1) The catalytic domain of NF1 will be purified from E. coli or insect cells expressing NF1 and their GAP activity as well will be investigated. (2) Interaction between NF1 and ras proteins will be studied by biochemical experiments. (3) Regions in the catalytic domain responsible for the GAP activity as well as for the interaction with ras proteins will be determined by in vitro mutagenesis of the NF1 protein. A region(s) conferring specificity of target ras proteins will be also be determined. (4) Lipids also appears to be a player in the interaction. Lipid sensitivity of NF1-GAP activity will be addressed by using various lipids and fatty acids. [II] Function of regions flanking the catalytic domain will be investigated by comparing GAP activity of the catalytic domain polypeptide and a polypeptide containing both the catalytic and the flanking regions. [III] NF1 protein in vivo will be characterized by preparing a battery of antibodies. Posttranslational modification, subcellular localization and the presence of any associated proteins will be investigated. [IV] Mutant NF1 protein will be characterized by the purification of known mutant proteins as well as by screening various tissue samples including ones derived from neurofibromatosis patients.
