Neurofibromatosis type 1 (NF1), one of the most common inherited diseases, represents a challenge to both clinicians and basic scientists. The protean nature of the disorder and its variable expressivity has made presymptomatic clinical diagnosis difficult and treatment unsatisfactory. The neurofibromatosis gene is thought to behave as a tumor suppressor gene and a better understanding of its role in normal cell biology will provide insights not only into tumor biology but also the mechanisms underlying normal growth and development. Progress in appreciating the function of the NF1 gene will require the identification and characterization of the gene product of the NF1 locus. The work outlined in this grant proposal is directed towards characterizing the NF1 protein and determining its normal function in order to better understand how diseases like NF1 results from abnormalities of the NF1 protein.
The specific aims of this project are directed at characterizing the NF1 gene product: (1) Antibodies will be generated to identify the NF1 protein (NF1 GAP-related protein, NF1GRP). These antibodies will provide reagents to determine functional and structural domains of NF1GRP as well as to define its subcellular and tissue distribution. Proteins which associate with NF1GRP will be isolated by the combined approaches of coimmunoprecipitation using these antibodies and column chromatography using baculovirus-expressed NF1GRP matrices. (2) Analysis of the complete NF1 cDNA sequence has demonstrated sequence similarity between a 400 amino acid region of NF1GRP (catalytic domain) and a family of GTPase-activating proteins. There is nothing known about the remaining 85% of the protein. Site-directed mutagenesis and domain swapping experiments will be undertaken to investigate the role of the catalytic as well as noncatalytic domain regions in NF1GRP function. (3) Recent studies have demonstrated that NF1GRP may be expressed in all tissues. In order to determine what role NF1GRP has in non-neural crest tissues, the Drosophila NF1 homolog will be cloned and studied. Its pattern of expression in adult tissues as well as during the course of Drosophila nervous system development will be investigated. Drosophila provides a unique model system for studying NF1GRP given the relative simplicity of the fruitfly genome, the wealth of information on nervous system development and the ability to determine the impact of NF1GRP on development by direct mutagenesis and through the study of known developmental mutants.
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