Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder which manifests clinically with peripheral neurofibromas, cafe-au-lait spots, learning disabilities, and an increased incidence of tumor development. The NF1 gene was identified by positional cloning and its protein product, neurofibromin, determined to be a large cytoplasmic protein involved in the downregulation of p21-ras. In addition, neurofibromin has been shown to be associated with cytoplasmic microtubules and this association may modulate the ability of neurofibromin to downregulate p21-ras. At least three isoforms of NF1, termed type 1 (lacking either the exon 23a or 48a insertions) type 2 (containing the exon 23a insertion) and type 3 (containing the exon 48a insertion) NF1 are generated by the use of alternate exons. Type 2 neurofibromin contains an additional 21 amino acids (exon 23a) inserted into the domain responsible for p2l-ras regulation which has been shown to reduce the ability of this isoform to downregulate p2l-ras in vitro. The expression of type 2 NF1 is induced during in vitro differentiation of Schwann cells and neuroblasts. Type 3 NF1, on the other hand, contains an additional 18 amino acids (exon 48a) inserted into the extreme carboxyl terminus of the protein and is expressed predominantly in muscle. The ability to modulate the function of neurofibromin through changes in isoform expression may represent an additional mechanism for neurofibromin regulation. In order to understand the contributions of these alternatively-spliced isoforms of NF1 to nervous system differentiation. we propose to study (l) the effect of isoform expression during Schwann cell and neuroblast differentiation and (2) the effect of alterations in isoform expression on neurofibromin function. Therefore, to understand the role of neurofibromin in differentiation and development, one must consider the expression of the individual neurofibromin isoforms. Initially, changes in neurofibromin isoform expression incumbent upon in vitro Schwann cell and neuroblast differentiation will be examined using these isoform-specific reagents. Then, these reagents will be used to examine the adult and embryonic expression of neurofibromin isoforms. The effect of isoform expression on Schwann cell and neuroblast proliferation and division shall next be examined by expressing the NF1 isoforms from inducible promoters. In an effort to understand the effect of isoform expression on the known functions of neurofibromin, the ability of these NF1 isoforms to (l) downregulate p2l-ras and (2) interact with cytoplasmic microtubules will be examined. An examination of the differential expression and functional properties of these isoforms will provide additional insights into the role of this important tumor suppressor gene in both health and disease.
