Nerve growth factor is critical for the development and maintenance of sympathetic and sensory neurons derived from the neural crest. Recent studies suggest that NGF may also have tropic effects on cholinergic neurons within the central nervous system and may play a role in degenerative diseases such as Alzheimer's disease. NGF has been most thoroughly studied in the mouse submandibular gland where it is present in extremely high levels for reasons that are not known. NGF is produced at much lower levels in target organs of the sympathetic nervous system and in the central nervous system. The form in which NGF is found may differ in sources other than the mouse submandibular gland. NGF is produced as a larger protein precursor which must undergo multiple proteolytic processing steps to produce the 118 amino acid form of NGF that is found in mouse submandibular glands. The proposed study will examine the structure and activity of expressed NGF and precursors and prepare probes to examine processing in other tissues. The cDNA for NGF will be subcloned into expression vectors to produce NGF and specific precursor forms in a wheat germ cell-free system and in E. coli. NGF and precursors will be expressed in wheat germ as a rapid assay system for cahnges in conformation and receptor binding activity. Mutagenized forms of NGF will be screened in an attempt to define the receptor binding site. NGF and precursors will be expressed in E. coli to provide sufficient material for structural studies, to prepare antisera, and study processing events.
